PLOS ONE Alerts: New Articles

Synthetic Bone Substitute Engineered with Amniotic Epithelial Cells Enhances Bone Regeneration after Maxillary Sinus Augmentation

2013-05-17T21:00:00Z

by Barbara Barboni, Carlo Mangano, Luca Valbonetti, Giuseppe Marruchella, Paolo Berardinelli, Alessandra Martelli, Aurelio Muttini, Annunziata Mauro, Rossella Bedini, Maura Turriani, Raffaella Pecci, Delia Nardinocchi, Vincenzo Luca Zizzari, Stefano Tetè, Adriano Piattelli, Mauro Mattioli

Background

Evidence has been provided that a cell-based therapy combined with the use of bioactive materials may significantly improve bone regeneration prior to dental implant, although the identification of an ideal source of progenitor/stem cells remains to be determined.

Aim

In the present research, the bone regenerative property of an emerging source of progenitor cells, the amniotic epithelial cells (AEC), loaded on a calcium-phosphate synthetic bone substitute, made by direct rapid prototyping (rPT) technique, was evaluated in an animal study.

Material And Methods

Two blocks of synthetic bone substitute (∼0.14 cm³), alone or engineered with 1×10⁶ ovine AEC (oAEC), were grafted bilaterally into maxillary sinuses of six adult sheep, an animal model chosen for its high translational value in dentistry. The sheep were then randomly divided into two groups and sacrificed at 45 and 90 days post implantation (p.i.). Tissue regeneration was evaluated in the sinus explants by micro-computer tomography (micro-CT), morphological, morphometric and biochemical analyses.

Results And Conclusions

The obtained data suggest that scaffold integration and bone deposition are positively influenced by allotransplantated oAEC. Sinus explants derived from sheep grafted with oAEC engineered scaffolds displayed a reduced fibrotic reaction, a limited inflammatory response and an accelerated process of angiogenesis. In addition, the presence of oAEC significantly stimulated osteogenesis either by enhancing bone deposition or making more extent the foci of bone nucleation. Besides the modulatory role played by oAEC in the crucial events successfully guiding tissue regeneration (angiogenesis, vascular endothelial growth factor expression and inflammation), data provided herein show that oAEC were also able to directly participate in the process of bone deposition, as suggested by the presence of oAEC entrapped within the newly deposited osteoid matrix and by their ability to switch-on the expression of a specific bone-related protein (osteocalcin, OCN) when transplanted into host tissues.

miR-219-5p Inhibits Receptor Tyrosine Kinase Pathway by Targeting EGFR in Glioblastoma

2013-05-17T21:00:00Z

by Soumya Alige Mahabala Rao, Arivazhagan Arimappamagan, Paritosh Pandey, Vani Santosh, Alangar Sathyaranjandas Hegde, Bangalore Ashwathnarayanara Chandramouli, Kumaravel Somasundaram

Glioblastoma is one of the common types of primary brain tumors with a median survival of 12–15 months. The receptor tyrosine kinase (RTK) pathway is known to be deregulated in 88% of the patients with glioblastoma. 45% of GBM patients show amplifications and activating mutations in EGFR gene leading to the upregulation of the pathway. In the present study, we demonstrate that a brain specific miRNA, miR-219-5p, repressed EGFR by directly binding to its 3′-UTR. The expression of miR-219-5p was downregulated in glioblastoma and the overexpression of miR-219-5p in glioma cell lines inhibited the proliferation, anchorage independent growth and migration. In addition, miR-219-5p inhibited MAPK and PI3K pathways in glioma cell lines in concordance with its ability to target EGFR. The inhibitory effect of miR-219-5p on MAPK and PI3K pathways and glioma cell migration could be rescued by the overexpression of wild type EGFR and vIII mutant of EGFR (both lacking 3′-UTR and thus being insensitive to miR-219-5p) suggesting that the inhibitory effects of miR-219-5p were indeed because of its ability to target EGFR. We also found significant negative correlation between miR-219-5p levels and total as well as phosphorylated forms of EGFR in glioblastoma patient samples. This indicated that the downregulation of miR-219-5p in glioblastoma patients contribute to the increased activity of the RTK pathway by the upregulation of EGFR. Thus, we have identified and characterized miR-219-5p as the RTK regulating novel tumor suppressor miRNA in glioblastoma.

DRAM1 Regulates Autophagy Flux through Lysosomes

2013-05-17T21:00:00Z

by Xing-Ding Zhang, Lin Qi, Jun-Chao Wu, Zheng-Hong Qin

We have previously reported that the mitochondria inhibitor 3-nitropropionic acid (3-NP), induces the expression of DNA damage-regulated autophagy modulator1 (DRAM1) and activation of autophagy in rat striatum. Although the role of DRAM1 in autophagy has been previously characterized, the detailed mechanism by which DRAM1 regulates autophagy activity has not been fully understood. The present study investigated the role of DRAM1 in regulating autophagy flux. In A549 cells expressing wilt-type TP53, 3-NP increased the protein levels of DRAM1 and LC3-II, whereas decreased the levels of SQSTM1 (sequestosome 1). The increase in LC3-II and decrease in SQSTM1 were blocked by the autophagy inhibitor 3-methyl-adenine. Lack of TP53 or knock-down of TP53 in cells impaired the induction of DRAM1. Knock-down of DRAM1 with siRNA significantly reduced 3-NP-induced upregulation of LC3-II and downregulation of SQSTM1, indicating DRAM1 contributes to autophagy activation. Knock-down of DRAM1 robustly decreased rate of disappearance of induced autophagosomes, increased RFP-LC3 fluorescence dots and decreased the decline of LC3-II after withdraw of rapamycin, indicating DRAM1 promotes autophagy flux. DRAM1 siRNA inhibited lysosomal V-ATPase and acidification of lysosomes. As a result, DRAM1 siRNA reduced activation of lysosomal cathepsin D. Similar to DRAM1 siRNA, lysosomal inhibitors E64d and chloroquine also inhibited clearance of autophagosomes and activation of lysosomal cathapsin D after 3-NP treatment. These data suggest that DRAM1 plays important roles in autophagy activation induced by mitochondria dysfunction. DRAM1 affects autophagy through argument of lysosomal acidification, fusion of lysosomes with autophagosomes and clearance of autophagosomes.

Does Ecophysiology Determine Invasion Success? A Comparison between the Invasive Boatman Trichocorixa verticalis verticalis and the Native Sigara lateralis (Hemiptera, Corixidae) in South-West Spain

2013-05-17T21:00:00Z

by Cristina Coccia, Piero Calosi, Luz Boyero, Andy J. Green, David T. Bilton

Background

Trichocorixa verticalis verticalis, a native of North America, is the only alien corixid identified in Europe. First detected in 1997 in southern Portugal, it has spread into south-west Spain including Doñana National Park. Its impact on native taxa in the same area is unclear, but it is the dominant species in several permanent, saline wetlands.

Methodology/Principal Findings

We investigated whether the ecophysiology of this alien species favours its spread in the Iberian Peninsula and its relative success in saline areas. We compared physiological responses to heating (Critical Thermal maximum), cooling (Critical Thermal minimum) and freezing (Super Cooling Point) in the native Sigara lateralis and introduced T. v. verticalis acclimated to different temperatures and salinities. The larger S. lateralis generally outperformed T. v. verticalis and appeared to possess a broader thermal tolerance range. In both taxa, CT_max was highest in animals exposed to a combination of high conductivities and relatively low acclimation temperatures. However, CT_max was generally higher in T. v. verticalis and lower in S. lateralis when acclimated at higher temperatures. CT_min were lower (greater tolerance to cold) after acclimation to high conductivities in T. v. verticalis, and following acclimation to low conductivities in S. lateralis. Both acclimation temperature and conductivity influenced corixids' freezing tolerance; however, only in T. v. verticalis did SCP decrease after exposure to both high temperature and conductivity. T. v. verticalis showed a higher range of mean responses over all treatments.

Conclusions

Whilst the native S. lateralis may have a broader thermal range, the alien species performs particularly well at higher salinities and temperatures and this ability may facilitate its invasion in Mediterranean areas. The greater plasticity of T. v. verticalis may further facilitate its spread in the future, as it may be more able to respond to climate shifts than the native species.

Paricalcitol Attenuates 4-Hydroxy-2-Hexenal-Induced Inflammation and Epithelial-Mesenchymal Transition in Human Renal Proximal Tubular Epithelial Cells

2013-05-17T21:00:00Z

by Chang Seong Kim, Soo Yeon Joo, Ko Eun Lee, Joon Seok Choi, Eun Hui Bae, Seong Kwon Ma, Suhn Hee Kim, JongUn Lee, Soo Wan Kim

4-Hydroxy-2-hexenal (HHE), the aldehyde product of lipid peroxidation, may be responsible for the pathogenesis of progressive renal disease. Recently, paricalcitol (19-nor-1,25-dihydroxyvitamin D2) was shown to be renoprotective through its anti-inflammatory and antifibrotic effects in various experimental nephropathy models. In this study, we investigated the effects of paricalcitol on inflammation and epithelial-mesenchymal transition (EMT) after HHE-induced renal tubular epithelial cell injury. To investigate the molecular mechanisms underlying HHE-induced renal tubular cell injury, the human proximal tubular epithelial (HK-2) cells cultured with 10 µM HHE in the presence or absence of paricalcitol. In HK-2 cells, paricalcitol attenuated the HHE-induced expression of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase, and prevented nuclear factor-κB (NF-κB) activation. The expression of the inflammatory proteins inducible nitric oxide synthase and cyclooxygenase-2 was attenuated by paricalcitol pretreatment. In addition, HHE increased the expression of the transforming growth factor (TGF)-β/Smad signaling proteins and fibrotic proteins, such as α-smooth muscle actin and connective tissue growth factor; this inducible expression was suppressed by pretreatment with paricalcitol. Treatment with HHE resulted in the activation of the β-catenin signaling pathway, and paricalcitol pretreatment reduced the expression of β-catenin in HHE-treated HK-2 cells. Coimmunoprecipitation shows that paricalcitol induced vitamin D receptor (VDR)/β-catenin complex formation in HK-2 cells. Also immunofluorescence staining revealed that co-localization of VDR and β-catenin in the nuclei. ICG-001, an inhibitor of β-catenin, decreased the expression of TGF-β1 and attenuated HHE-induced tubular EMT. These results show that paricalcitol attenuated HHE-induced renal tubular cell injury by suppressing inflammation and EMT process through inhibition of the NF-κB, TGF-β/Smad, and β-catenin signaling pathways.

Prediction of Response to Treatment by Gene Expression Profiling of Peripheral Blood in Patients with Microscopic Polyangiitis

2013-05-17T21:00:00Z

by Akihiro Ishizu, Utano Tomaru, Taichi Murai, Tomohiro Yamamoto, Tatsuya Atsumi, Takashi Yoshiki, Wako Yumura, Kunihiro Yamagata, Hidehiro Yamada, Shunichi Kumagai, Manae S. Kurokawa, Machi Suka, Hirofumi Makino, Shoichi Ozaki, for JMAAV

The JMAAV study was an open-labeled prospective clinical trial, which proposed severity-based treatment protocols for patients with microscopic polyangiitis (MPA). The results suggest that the proposed protocols are useful (remission rate: 89.4%), but are also indicative of relapse or patient demise regardless of the treatment (recurrence rate: 19.0%; mortality rate: 10.6%). The aim of this study is to develop the method to predict response to the treatment in patients with MPA. In the present study, transcriptome analysis was performed using peripheral blood from patients enrolled in the JMAAV study before and 1-week after the beginning of treatment. The gene expression profile before treatment was not directly related to the response to the treatment. However, when the samples from 9 patients with good response (persistent remission for 18 months) were examined, the expression of 88 genes was significantly altered by the treatment. Thirty statistically reliable genes were selected, and then the alteration of expression by the treatment was examined among 22 patients, including 17 with good response, which was defined as persistent remission for 18 months and 5 with poor response, which was defined as relapse after remission or no remission. Discrimination analysis between the alteration of expression of the 30 genes by the treatment and the response identified a combination of 16 genes as the most valuable gene set to predict the response to the treatment. This preliminary study identified IRF7, IFIT1, IFIT5, OASL, CLC, GBP-1, PSMB9, HERC5, CCR1, CD36, MS4A4A, BIRC4BP, PLSCR1, DEFA1/DEFA3, DEFA4, and COL9A2 as the important genes that can predict the response to the treatment in patients with MPA at an early point during the therapy.

HACEK Infective Endocarditis: Characteristics and Outcomes from a Large, Multi-National Cohort

2013-05-17T21:00:00Z

by Stephen T. Chambers, David Murdoch, Arthur Morris, David Holland, Paul Pappas, Manel Almela, Nuria Fernández-Hidalgo, Benito Almirante, Emilio Bouza, Davide Forno, Ana del Rio, Margaret M. Hannan, John Harkness, Zeina A. Kanafani, Tahaniyat Lalani, Selwyn Lang, Nigel Raymond, Kerry Read, Tatiana Vinogradova, Christopher W. Woods, Dannah Wray, G. Ralph Corey, Vivian H. Chu, International Collaboration on Endocarditis Prospective Cohort Study (ICE-PCS) Investigators

The HACEK organisms (Haemophilus species, Aggregatibacter species, Cardiobacterium hominis, Eikenella corrodens, and Kingella species) are rare causes of infective endocarditis (IE). The objective of this study is to describe the clinical characteristics and outcomes of patients with HACEK endocarditis (HE) in a large multi-national cohort. Patients hospitalized with definite or possible infective endocarditis by the International Collaboration on Endocarditis Prospective Cohort Study in 64 hospitals from 28 countries were included and characteristics of HE patients compared with IE due to other pathogens. Of 5591 patients enrolled, 77 (1.4%) had HE. HE was associated with a younger age (47 vs. 61 years; p<0.001), a higher prevalence of immunologic/vascular manifestations (32% vs. 20%; p<0.008) and stroke (25% vs. 17% p = 0.05) but a lower prevalence of congestive heart failure (15% vs. 30%; p = 0.004), death in-hospital (4% vs. 18%; p = 0.001) or after 1 year follow-up (6% vs. 20%; p = 0.01) than IE due to other pathogens (n = 5514). On multivariable analysis, stroke was associated with mitral valve vegetations (OR 3.60; CI 1.34–9.65; p<0.01) and younger age (OR 0.62; CI 0.49–0.90; p<0.01). The overall outcome of HE was excellent with the in-hospital mortality (4%) significantly better than for non-HE (18%; p<0.001). Prosthetic valve endocarditis was more common in HE (35%) than non-HE (24%). The outcome of prosthetic valve and native valve HE was excellent whether treated medically or with surgery. Current treatment is very successful for the management of both native valve prosthetic valve HE but further studies are needed to determine why HE has a predilection for younger people and to cause stroke. The small number of patients and observational design limit inferences on treatment strategies. Self selection of study sites limits epidemiological inferences.

Phonotactic Diversity Predicts the Time Depth of the World’s Language Families

2013-05-17T21:00:00Z

by Taraka Rama

The ASJP (Automated Similarity Judgment Program) described an automated, lexical similarity-based method for dating the world’s language groups using 52 archaeological, epigraphic and historical calibration date points. The present paper describes a new automated dating method, based on phonotactic diversity. Unlike ASJP, our method does not require any information on the internal classification of a language group. Also, the method can use all the available word lists for a language and its dialects eschewing the debate on ‘language’ vs. ‘dialect’. We further combine these dates and provide a new baseline which, to our knowledge, is the best one. We make a systematic comparison of our method, ASJP’s dating procedure, and combined dates. We predict time depths for world’s language families and sub-families using this new baseline. Finally, we explain our results in the model of language change given by Nettle.

Error Properties of Argos Satellite Telemetry Locations Using Least Squares and Kalman Filtering

2013-05-17T21:00:00Z

by Janice D. Boyd, Donald J. Brightsmith

Study of animal movements is key for understanding their ecology and facilitating their conservation. The Argos satellite system is a valuable tool for tracking species which move long distances, inhabit remote areas, and are otherwise difficult to track with traditional VHF telemetry and are not suitable for GPS systems. Previous research has raised doubts about the magnitude of position errors quoted by the satellite service provider CLS. In addition, no peer-reviewed publications have evaluated the usefulness of the CLS supplied error ellipses nor the accuracy of the new Kalman filtering (KF) processing method. Using transmitters hung from towers and trees in southeastern Peru, we show the Argos error ellipses generally contain <25% of the true locations and therefore do not adequately describe the true location errors. We also find that KF processing does not significantly increase location accuracy. The errors for both LS and KF processing methods were found to be lognormally distributed, which has important repercussions for error calculation, statistical analysis, and data interpretation. In brief, “good” positions (location codes 3, 2, 1, A) are accurate to about 2 km, while 0 and B locations are accurate to about 5–10 km. However, due to the lognormal distribution of the errors, larger outliers are to be expected in all location codes and need to be accounted for in the user’s data processing. We evaluate five different empirical error estimates and find that 68% lognormal error ellipses provided the most useful error estimates. Longitude errors are larger than latitude errors by a factor of 2 to 3, supporting the use of elliptical error ellipses. Numerous studies over the past 15 years have also found fault with the CLS-claimed error estimates yet CLS has failed to correct their misleading information. We hope this will be reversed in the near future.

Expression Profile of microRNAs Regulating Proliferation and Differentiation in Mouse Adult Cardiac Stem Cells

2013-05-17T21:00:00Z

by Luis Brás-Rosário, Alex Matsuda, Ana Isabel Pinheiro, Rui Gardner, Telma Lopes, Andreia Amaral, Margarida Gama-Carvalho

The identification of cardiac cells with stem cell properties changed the paradigm of the heart as a post mitotic organ. These cells proliferate and differentiate into cardiomyocytes, endothelial and vascular smooth muscle cells, providing for cardiac cell homeostasis and regeneration. microRNAs are master switches controlling proliferation and differentiation, in particular regulating stem cell biology and cardiac development. Modulation of microRNAs -regulated gene expression networks holds the potential to control cell fate and proliferation, with predictable biotechnologic and therapeutic applications. To obtain insights into the regulatory networks active in cardiac stem cells, we characterized the expression profile of 95 microRNAs with reported functions in stem cell and tissue differentiation in mouse cardiac stem cells, and compared it to that of mouse embryonic heart and mesenchymal stem cells. The most highly expressed microRNAs identified in cardiac stem cells are known to target key genes involved in the control of cell proliferation and adhesion, vascular function and cardiomyocyte differentiation. We report a subset of differentially expressed microRNAs that are proposed to act as regulators of differentiation and proliferation of adult cardiac stem cells, providing novel insights into active gene expression networks regulating their biological properties.

Human Papillomavirus Type 6 and 11 Genetic Variants Found in 71 Oral and Anogenital Epithelial Samples from Australia

2013-05-17T21:00:00Z

by Jennifer A. Danielewski, Suzanne M. Garland, Jenny McCloskey, Richard J. Hillman, Sepehr N. Tabrizi

Genetic variation of 49 human papillomavirus (HPV) 6 and 22 HPV11 isolates from recurrent respiratory papillomatosis (RRP) (n = 17), genital warts (n = 43), anal cancer (n = 6) and cervical neoplasia cells (n = 5), was determined by sequencing the long control region (LCR) and the E6 and E7 genes. Comparative analysis of genetic variability was examined to determine whether different disease states resulting from HPV6 or HPV11 infection cluster into distinct variant groups. Sequence variation analysis of HPV6 revealed that isolates cluster into variants within previously described HPV6 lineages, with the majority (65%) clustering to HPV6 sublineage B1 across the three genomic regions examined. Overall 72 HPV6 and 25 HPV11 single nucleotide variations, insertions and deletions were observed within samples examined. In addition, missense alterations were observed in the E6/E7 genes for 6 HPV6 and 5 HPV11 variants. No nucleotide variations were identified in any isolates at the four E2 binding sites for HPV6 or HPV11, nor were any isolates found to be identical to the HPV6 lineage A or HPV11 sublineage A1 reference genomes. Overall, a high degree of sequence conservation was observed between isolates across each of the regions investigated for both HPV6 and HPV11. Genetic variants identified a slight association with HPV6 and anogenital lesions (p = 0.04). This study provides important information on the genetic diversity of circulating HPV 6 and HPV11 variants within the Australian population and supports the observation that the majority of HPV6 isolates cluster to the HPV6 sublineage B1 with anogenital lesions demonstrating an association with this sublineage (p = 0.02). Comparative analysis of Australian isolates for both HPV6 and HPV11 to those from other geographical regions based on the LCR revealed a high degree of sequence similarity throughout the world, confirming previous observations that there are no geographically specific variants for these HPV types.

Group Behavioural Responses of Atlantic Salmon (Salmo salar L.) to Light, Infrasound and Sound Stimuli

2013-05-17T21:00:00Z

by Samantha Bui, Frode Oppedal, Øyvind J. Korsøen, Damien Sonny, Tim Dempster

Understanding species-specific flight behaviours is essential in developing methods of guiding fish spatially, and requires knowledge on how groups of fish respond to aversive stimuli. By harnessing their natural behaviours, the use of physical manipulation or other potentially harmful procedures can be minimised. We examined the reactions of sea-caged groups of 50 salmon (1331±364 g) to short-term exposure to visual or acoustic stimuli. In light experiments, fish were exposed to one of three intensities of blue LED light (high, medium and low) or no light (control). Sound experiments included exposure to infrasound (12 Hz), a surface disturbance event, the combination of infrasound and surface disturbance, or no stimuli. Groups that experienced light, infrasound, and the combination of infrasound and surface disturbance treatments, elicited a marked change in vertical distribution, where fish dived to the bottom of the sea-cage for the duration of the stimulus. Light treatments, but not sound, also reduced the total echo-signal strength (indicative of swim bladder volume) after exposure to light, compared to pre-stimulus levels. Groups in infrasound and combination treatments showed increased swimming activity during stimulus application, with swimming speeds tripled compared to that of controls. In all light and sound treatments, fish returned to their pre-stimulus swimming depths and speeds once exposure had ceased. This work establishes consistent, short-term avoidance responses to these stimuli, and provides a basis for methods to guide fish for aquaculture applications, or create avoidance barriers for conservation purposes. In doing so, we can achieve the manipulation of group position with minimal welfare impacts, to create more sustainable practices.

A Universal Pedicle Screw and V-Rod System for Lumbar Isthmic Spondylolysis: A Retrospective Analysis of 21 Cases

2013-05-17T21:00:00Z

by Xiong-sheng Chen, Sheng-yuan Zhou, Lian-shun Jia, Xiao-min Gu, Lei Fang, Wei Zhu

Objective

To investigate the surgical outcome of a universal pedicle screw-V rod system and isthmic bone grafting for isthmic spondylolysis.

Methods

Twenty-four patients with isthmic spondylolysis at L5 and grade 0–I spondylolisthesis (Meyerding classification) received isthmic bone graft and stabilization using the universal pedicle screw-V rod system. Back pain was evaluated using the visual analog scale (VAS) and time to bone healing, improvement in spondylolisthesis and intervertebral space height at L5/S1 and L4/L5 were assessed.

Results

Twenty-one patients were followed up for 24 months and included in the analysis. Back pain was markedly improved at 3 months postoperatively with a statistical difference in VAS scores compared with preoperative VAS scores (P<0.001). The VAS scores were 0 to 3 at 6 months postoperatively in all patients and no back pain was reported in all patients except 2 patients who complained of back pain after prolonged sitting. X-ray examination showed a bone graft healing time of 3 to 12 months. Grade I spondylolisthesis improved to grade 0 in 4 patients and no noticeable change was observed in the remaining 17 cases. The intervertebral space height at L5/S1 was statistically increased (P<0.05) while no statistically significant change was seen at L4/L5. There was no statistically significant difference in the ROM of the intervertebral disks of L5/S1 and L4/5 before and after surgery.

Conclusions

The universal pedicle screw-V rod system and isthmic bone grafting directly repairs isthmic spondylolysis and reduces back pain, prevents anterior displacement of the diseased segment and maintains intervertebral space height, thus offering a promising alternative to current approaches for isthmic spondylolysis.

Trophic Dynamics of Deep-Sea Megabenthos Are Mediated by Surface Productivity

2013-05-17T21:00:00Z

by Samuele Tecchio, Dick van Oevelen, Karline Soetaert, Joan Navarro, Eva Ramírez-Llodra

Most deep-sea benthic ecosystems are food limited and, in the majority of cases, are driven by the organic matter falling from the surface or advected downslope. Species may adapt to this scarceness by applying a wide variety of responses, such as feeding specialisation, niche width variation, and reduction in metabolic rates. The Mediterranean Sea hosts a gradient of food availability at the deep seafloor over its wide longitudinal transect. In the Mediterranean, broad regional studies on trophic habits are almost absent, and the response of deep-sea benthos to different trophic conditions is still speculative. Here, we show that both primary and secondary production processes taking place at surface layers are key drivers of deep-sea food web structuring. By employing an innovative statistical tool, we interpreted bulk-tissue δ¹³C and δ¹⁵N isotope ratios in benthic megafauna, and associated surface and mesopelagic components from the 3 basins of the Mediterranean Sea at 3 different depths (1200, 2000, and 3000 m). The trophic niche width and the amplitude of primary carbon sources were positively correlated with both primary and secondary surface production indicators. Moreover, mesopelagic organic matter utilization processes showed an intermediate position between surface and deep benthic components. These results shed light on the understanding of deep-sea ecosystems functioning and, at the same time, they demand further investigation.

Tobacco Smoking Leads to Extensive Genome-Wide Changes in DNA Methylation

2013-05-17T21:00:00Z

by Sonja Zeilinger, Brigitte Kühnel, Norman Klopp, Hansjörg Baurecht, Anja Kleinschmidt, Christian Gieger, Stephan Weidinger, Eva Lattka, Jerzy Adamski, Annette Peters, Konstantin Strauch, Melanie Waldenberger, Thomas Illig

Environmental factors such as tobacco smoking may have long-lasting effects on DNA methylation patterns, which might lead to changes in gene expression and in a broader context to the development or progression of various diseases. We conducted an epigenome-wide association study (EWAs) comparing current, former and never smokers from 1793 participants of the population-based KORA F4 panel, with replication in 479 participants from the KORA F3 panel, carried out by the 450K BeadChip with genomic DNA obtained from whole blood. We observed wide-spread differences in the degree of site-specific methylation (with p-values ranging from 9.31E-08 to 2.54E-182) as a function of tobacco smoking in each of the 22 autosomes, with the percent of variance explained by smoking ranging from 1.31 to 41.02. Depending on cessation time and pack-years, methylation levels in former smokers were found to be close to the ones seen in never smokers. In addition, methylation-specific protein binding patterns were observed for cg05575921 within AHRR, which had the highest level of detectable changes in DNA methylation associated with tobacco smoking (–24.40% methylation; p = 2.54E-182), suggesting a regulatory role for gene expression. The results of our study confirm the broad effect of tobacco smoking on the human organism, but also show that quitting tobacco smoking presumably allows regaining the DNA methylation state of never smokers.

Meta-Analysis: Melatonin for the Treatment of Primary Sleep Disorders

2013-05-17T21:00:00Z

by Eduardo Ferracioli-Oda, Ahmad Qawasmi, Michael H. Bloch

Study Objectives

To investigate the efficacy of melatonin compared to placebo in improving sleep parameters in patients with primary sleep disorders.

Design

PubMed was searched for randomized, placebo-controlled trials examining the effects of melatonin for the treatment of primary sleep disorders. Primary outcomes examined were improvement in sleep latency, sleep quality and total sleep time. Meta-regression was performed to examine the influence of dose and duration of melatonin on reported efficacy.

Participants

Adults and children diagnosed with primary sleep disorders.

Interventions

Melatonin compared to placebo.

Results

Nineteen studies involving 1683 subjects were included in this meta-analysis. Melatonin demonstrated significant efficacy in reducing sleep latency (weighted mean difference (WMD) = 7.06 minutes [95% CI 4.37 to 9.75], Z = 5.15, p<0.001) and increasing total sleep time (WMD = 8.25 minutes [95% CI 1.74 to 14.75], Z = 2.48, p = 0.013). Trials with longer duration and using higher doses of melatonin demonstrated greater effects on decreasing sleep latency and increasing total sleep time. Overall sleep quality was significantly improved in subjects taking melatonin (standardized mean difference = 0.22 [95% CI: 0.12 to 0.32], Z = 4.52, p<0.001) compared to placebo. No significant effects of trial duration and melatonin dose were observed on sleep quality.

Conclusion

This meta-analysis demonstrates that melatonin decreases sleep onset latency, increases total sleep time and improves overall sleep quality. The effects of melatonin on sleep are modest but do not appear to dissipate with continued melatonin use. Although the absolute benefit of melatonin compared to placebo is smaller than other pharmacological treatments for insomnia, melatonin may have a role in the treatment of insomnia given its relatively benign side-effect profile compared to these agents.

Integration of 1H NMR and UPLC-Q-TOF/MS for a Comprehensive Urinary Metabonomics Study on a Rat Model of Depression Induced by Chronic Unpredictable Mild Stress

2013-05-17T21:00:00Z

by Hong-mei Jia, Yu-fei Feng, Yue-tao Liu, Xing Chang, Lin Chen, Hong-wu Zhang, Gang Ding, Zhong-mei Zou

Depression is a type of complex psychiatric disorder with long-term, recurrent bouts, and its etiology remains largely unknown. Here, an integrated approach utilizing ¹H NMR and UPLC-Q-TOF/MS together was firstly used for a comprehensive urinary metabonomics study on chronic unpredictable mild stress (CUMS) treated rats. More than twenty-nine metabolic pathways were disturbed after CUMS treatment and thirty-six potential biomarkers were identified by using two complementary analytical technologies. Among the identified biomarkers, nineteen (10, 11, 16, 17, 21–25, and 27–36) were firstly reported as potential biomarkers of CUMS-induced depression. Obviously, this paper presented a comprehensive map of the metabolic pathways perturbed by CUMS and expanded on the multitude of potential biomarkers that have been previously reported in the CUMS model. Four metabolic pathways, including valine, leucine and isoleucine biosynthesis; phenylalanine, tyrosine and tryptophan biosynthesis; tryptophan metabolism; synthesis and degradation of ketone bodies had the deepest influence in the pathophysiologic process of depression. Fifteen potential biomarkers (1–2, 4–6, 15, 18, 20–23, 27, 32, 35–36) involved in the above four metabolic pathways might become the screening criteria in clinical diagnosis and predict the development of depression. Moreover, the results of Western blot analysis of aromatic L-amino acid decarboxylase (DDC) and indoleamine 2, 3-dioxygenase (IDO) in the hippocampus of CUMS-treated rats indicated that depletion of 5-HT and tryptophan, production of 5-MT and altered expression of DDC and IDO together played a key role in the initiation and progression of depression. In addition, none of the potential biomarkers were detected by NMR and LC-MS simultaneously which indicated the complementary of the two kinds of detection technologies. Therefore, the integration of ¹H NMR and UPLC-Q-TOF/MS in metabonomics study provided an approach to identify the comprehensive potential depression-related biomarkers and helpful in further understanding the underlying molecular mechanisms of depression through the disturbance of metabolic pathways.

A Simple High-Content Cell Cycle Assay Reveals Frequent Discrepancies between Cell Number and ATP and MTS Proliferation Assays

2013-05-17T21:00:00Z

by Grace Ka Yan Chan, Tracy L. Kleinheinz, David Peterson, John G. Moffat

In order to efficiently characterize both antiproliferative potency and mechanism of action of small molecules targeting the cell cycle, we developed a high-throughput image-based assay to determine cell number and cell cycle phase distribution. Using this we profiled the effects of experimental and approved anti-cancer agents with a range mechanisms of action on a set of cell lines, comparing direct cell counting versus two metabolism-based cell viability/proliferation assay formats, ATP-dependent bioluminescence, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reduction, and a whole-well DNA-binding dye fluorescence assay. We show that, depending on compound mechanisms of action, the metabolism-based proxy assays are frequently prone to 1) significant underestimation of compound potency and efficacy, and 2) non-monotonic dose-response curves due to concentration-dependent phenotypic ‘switching’. In particular, potency and efficacy of DNA synthesis-targeting agents such as gemcitabine and etoposide could be profoundly underestimated by ATP and MTS-reduction assays. In the same image-based assay we showed that drug-induced increases in ATP content were associated with increased cell size and proportionate increases in mitochondrial content and respiratory flux concomitant with cell cycle arrest. Therefore, differences in compound mechanism of action and cell line-specific responses can yield significantly misleading results when using ATP or tetrazolium-reduction assays as a proxy for cell number when screening compounds for antiproliferative activity or profiling panels of cell lines for drug sensitivity.

Broadening of the T-Cell Repertoire to HIV-1 Gag p24 by Vaccination of HLA-A2/DR Transgenic Mice with Overlapping Peptides in the CAF05 Adjuvant

2013-05-17T21:00:00Z

by Karen S. Korsholm, Ingrid Karlsson, Sheila T. Tang, Lea Brandt, Else Marie Agger, Claus Aagaard, Peter Andersen, Anders Fomsgaard

Induction of broad T-cell immune responses is regarded as critical for vaccines against the human immunodeficiency virus type 1 (HIV-1) which exhibit high diversity and, therefore, focus has been on inducing cytotoxic CD8 T-cell responses against the more conserved parts of the virus, such as the Gag protein. Herein, we have used the p24 protein which contains a range of conserved T-cell epitopes. We demonstrate that a vaccine of HIV-1 subtype B consensus group-specific antigen (Gag) p24 protein with the CD8-inducing liposomal cationic adjuvant formulation (CAF) 05, induces both CD4 and CD8 T-cell responses in CB6F1 mice. The adjuvanted vaccine also induced functional antigen-specific cytotoxicity in vivo. Furthermore, we found that when fragmenting the Gag p24 protein into overlapping Gag p24 peptides, a broader T-cell epitope specificity was induced in the humanized human leukocyte antigen (HLA)-A2/DR-transgenic mouse model. Thus, combining overlapping Gag p24 peptides with CAF05 appears to be a promising and simple strategy for inducing broader T-cell responses to multiple conserved epitopes which will be relevant for both prophylactic and therapeutic HIV-1 vaccines.

Burying Dogs in Ancient Cis-Baikal, Siberia: Temporal Trends and Relationships with Human Diet and Subsistence Practices

2013-05-17T21:00:00Z

by Robert J. Losey, Sandra Garvie-Lok, Jennifer A. Leonard, M. Anne Katzenberg, Mietje Germonpré, Tatiana Nomokonova, Mikhail V. Sablin, Olga I. Goriunova, Natalia E. Berdnikova, Nikolai A. Savel’ev

The first objective of this study is to examine temporal patterns in ancient dog burials in the Lake Baikal region of Eastern Siberia. The second objective is to determine if the practice of dog burial here can be correlated with patterns in human subsistence practices, in particular a reliance on terrestrial mammals. Direct radiocarbon dating of a suite of the region’s dog remains indicates that these animals were given burial only during periods in which human burials were common. Dog burials of any kind were most common during the Early Neolithic (∼7–8000 B.P.), and rare during all other time periods. Further, only foraging groups seem to have buried canids in this region, as pastoralist habitation sites and cemeteries generally lack dog interments, with the exception of sacrificed animals. Stable carbon and nitrogen isotope data indicate that dogs were only buried where and when human diets were relatively rich in aquatic foods, which here most likely included river and lake fish and Baikal seal (Phoca sibirica). Generally, human and dog diets appear to have been similar across the study subregions, and this is important for interpreting their radiocarbon dates, and comparing them to those obtained on the region’s human remains, both of which likely carry a freshwater old carbon bias. Slight offsets were observed in the isotope values of dogs and humans in our samples, particularly where both have diets rich in aquatic fauna. This may result from dietary differences between people and their dogs, perhaps due to consuming fish of different sizes, or even different tissues from the same aquatic fauna. This paper also provides a first glimpse of the DNA of ancient canids in Northeast Asia.

Structural and Functional Analysis of the N-terminal Domain of the Streptococcus gordonii Adhesin Sgo0707

2013-05-17T21:00:00Z

by Åsa Nylander, Gunnel Svensäter, Dilani B. Senadheera, Dennis G. Cvitkovitch, Julia R. Davies, Karina Persson

The commensal Streptococcus gordonii expresses numerous surface adhesins with which it interacts with other microorganisms, host cells and salivary proteins to initiate dental plaque formation. However, this Gram-positive bacterium can also spread to non-oral sites such as the heart valves and cause infective endocarditis. One of its surface adhesins, Sgo0707, is a large protein composed of a non-repetitive N-terminal region followed by several C-terminal repeat domains and a cell wall sorting motif. Here we present the crystal structure of the Sgo0707 N-terminal domains, refined to 2.1 Å resolution. The model consists of two domains, N1 and N2. The largest domain, N1, comprises a putative binding cleft with a single cysteine located in its centre and exhibits an unexpected structural similarity to the variable domains of the streptococcal Antigen I/II adhesins. The N2-domain has an IgG-like fold commonly found among Gram-positive surface adhesins. Binding studies performed on S. gordonii wild-type and a Sgo0707 deficient mutant show that the Sgo0707 adhesin is involved in binding to type-1 collagen and to oral keratinocytes.

Sleep Loss and the Inflammatory Response in Mice Under Chronic Environmental Circadian Disruption

2013-05-17T21:00:00Z

by Allison J. Brager, J. Christopher Ehlen, Oscar Castanon-Cervantes, Divya Natarajan, Patrick Delisser, Alec J. Davidson, Ketema N. Paul

Shift work and trans-time zone travel lead to insufficient sleep and numerous pathologies. Here, we examined sleep/wake dynamics during chronic exposure to environmental circadian disruption (ECD), and if chronic partial sleep loss associated with ECD influences the induction of shift-related inflammatory disorder. Sleep and wakefulness were telemetrically recorded across three months of ECD, in which the dark-phase of a light-dark cycle was advanced weekly by 6 h. A three month regimen of ECD caused a temporary reorganization of sleep (NREM and REM) and wake processes across each week, resulting in an approximately 10% net loss of sleep each week relative to baseline levels. A separate group of mice were subjected to ECD or a regimen of imposed wakefulness (IW) aimed to mimic sleep amounts under ECD for one month. Fos-immunoreactivity (IR) was quantified in sleep-wake regulatory areas: the nucleus accumbens (NAc), basal forebrain (BF), and medial preoptic area (MnPO). To assess the inflammatory response, trunk blood was treated with lipopolysaccharide (LPS) and subsequent release of IL-6 was measured. Fos-IR was greatest in the NAc, BF, and MnPO of mice subjected to IW. The inflammatory response to LPS was elevated in mice subjected to ECD, but not mice subjected to IW. Thus, the net sleep loss that occurs under ECD is not associated with a pathological immune response.

Information Filtering via a Scaling-Based Function

2013-05-17T21:00:00Z

by Tian Qiu, Zi-Ke Zhang, Guang Chen

Finding a universal description of the algorithm optimization is one of the key challenges in personalized recommendation. In this article, for the first time, we introduce a scaling-based algorithm (SCL) independent of recommendation list length based on a hybrid algorithm of heat conduction and mass diffusion, by finding out the scaling function for the tunable parameter and object average degree. The optimal value of the tunable parameter can be abstracted from the scaling function, which is heterogeneous for the individual object. Experimental results obtained from three real datasets, Netflix, MovieLens and RYM, show that the SCL is highly accurate in recommendation. More importantly, compared with a number of excellent algorithms, including the mass diffusion method, the original hybrid method, and even an improved version of the hybrid method, the SCL algorithm remarkably promotes the personalized recommendation in three other aspects: solving the accuracy-diversity dilemma, presenting a high novelty, and solving the key challenge of cold start problem.

Genome-Wide Association Study Reveals Constant and Specific Loci for Hematological Traits at Three Time Stages in a White Duroc × Erhualian F2 Resource Population

2013-05-17T21:00:00Z

by Zhiyan Zhang, Yuan Hong, Jun Gao, Shijun Xiao, Junwu Ma, Wanchang Zhang, Jun Ren, Lusheng Huang

Hematological traits are important indicators of immune function and have been commonly examined as biomarkers of disease and disease severity in humans. Pig is an ideal biomedical model for human diseases due to its high degree of similarity with human physiological characteristics. Here, we conducted genome-wide association studies (GWAS) for 18 hematological traits at three growth stages (days 18, 46 and 240) in a White Duroc × Erhualian F₂ intercross. In total, we identified 38 genome-wide significant regions containing 185 genome-wide significant SNPs by single-marker GWAS or LONG-GWAS. The significant regions are distributed on pig chromosomes (SSC) 1, 4, 5, 7, 8, 10, 11, 12, 13, 17 and 18, and most of significant SNPs reside on SSC7 and SSC8. Of the 38 significant regions, 7 show constant effects on hematological traits across the whole life stages, and 6 regions have time-specific effects on the measured traits at early or late stages. The most prominent locus is the genomic region between 32.36 and 84.49 Mb on SSC8 that is associated with multiple erythroid traits. The KIT gene in this region appears to be a promising candidate gene. The findings improve our understanding of the genetic architecture of hematological traits in pigs. Further investigations are warranted to characterize the responsible gene(s) and causal variant(s) especially for the major loci on SSC7 and SSC8.

Using High Angular Resolution Diffusion Imaging Data to Discriminate Cortical Regions

2013-05-17T21:00:00Z

by Zoltan Nagy, Daniel C. Alexander, David L. Thomas, Nikolaus Weiskopf, Martin I. Sereno

Brodmann’s 100–year–old summary map has been widely used for cortical localization in neuroscience. There is a pressing need to update this map using non–invasive, high–resolution and reproducible data, in a way that captures individual variability. We demonstrate here that standard HARDI data has sufficiently diverse directional variation among grey matter regions to inform parcellation into distinct functional regions, and that this variation is reproducible across scans. This characterization of the signal variation as non–random and reproducible is the critical condition for successful cortical parcellation using HARDI data. This paper is a first step towards an individual cortex–wide map of grey matter microstructure, The gray/white matter and pial boundaries were identified on the high–resolution structural MRI images. Two HARDI data sets were collected from each individual and aligned with the corresponding structural image. At each vertex point on the surface tessellation, the diffusion–weighted signal was extracted from each image in the HARDI data set at a point, half way between gray/white matter and pial boundaries. We then derived several features of the HARDI profile with respect to the local cortical normal direction, as well as several fully orientationally invariant features. These features were taken as a fingerprint of the underlying grey matter tissue, and used to distinguish separate cortical areas. A support–vector machine classifier, trained on three distinct areas in repeat 1 achieved 80–82% correct classification of the same three areas in the unseen data from repeat 2 in three volunteers. Though gray matter anisotropy has been mostly overlooked hitherto, this approach may eventually form the foundation of a new cortical parcellation method in living humans. Our approach allows for further studies on the consistency of HARDI based parcellation across subjects and comparison with independent microstructural measures such as ex–vivo histology.

Genotyping-by-Sequencing (GBS): A Novel, Efficient and Cost-Effective Genotyping Method for Cattle Using Next-Generation Sequencing

2013-05-17T21:00:00Z

by Marcos De Donato, Sunday O. Peters, Sharon E. Mitchell, Tanveer Hussain, Ikhide G. Imumorin

High-throughput genotyping methods have increased the analytical power to study complex traits but high cost has remained a barrier for large scale use in animal improvement. We have adapted genotyping-by-sequencing (GBS) used in plants for genotyping 47 animals representing 7 taurine and indicine breeds of cattle from the US and Africa. Genomic DNA was digested with different enzymes, ligated to adapters containing one of 48 unique bar codes and sequenced by the Illumina HiSeq 2000. PstI was the best enzyme producing 1.4 million unique reads per animal and initially identifying a total of 63,697 SNPs. After removal of SNPs with call rates of less than 70%, 51,414 SNPs were detected throughout all autosomes with an average distance of 48.1 kb, and 1,143 SNPs on the X chromosome at an average distance of 130.3 kb, as well as 191 on unmapped contigs. If we consider only the SNPs with call rates of 90% and over, we identified 39,751 on autosomes, 850 on the X chromosome and 124 on unmapped contigs. Of these SNPs, 28,843 were not tightly linked to other SNPs. Average marker density per autosome was highly correlated with chromosome size (coefficient of correlation = −0.798, r² = 0.637) with higher density in smaller chromosomes. Average SNP call rate was 86.5% for all loci, with 53.0% of the loci having call rates >90% and the average minor allele frequency being 0.212. Average observed heterozygosity ranged from 0.046–0.294 among individuals, and from 0.064–0.197 among breeds, with Brangus showing the highest diversity as expected. GBS technique is novel, flexible, sufficiently high-throughput, and capable of providing acceptable marker density for genomic selection or genome-wide association studies at roughly one third of the cost of currently available genotyping technologies.

A Texture Based Pattern Recognition Approach to Distinguish Melanoma from Non-Melanoma Cells in Histopathological Tissue Microarray Sections

2013-05-17T21:00:00Z

by Elton Rexhepaj, Margrét Agnarsdóttir, Julia Bergman, Per-Henrik Edqvist, Michael Bergqvist, Mathias Uhlén, William M. Gallagher, Emma Lundberg, Fredrik Ponten

Aims

Immunohistochemistry is a routine practice in clinical cancer diagnostics and also an established technology for tissue-based research regarding biomarker discovery efforts. Tedious manual assessment of immunohistochemically stained tissue needs to be fully automated to take full advantage of the potential for high throughput analyses enabled by tissue microarrays and digital pathology. Such automated tools also need to be reproducible for different experimental conditions and biomarker targets. In this study we present a novel supervised melanoma specific pattern recognition approach that is fully automated and quantitative.

Methods and Results

Melanoma samples were immunostained for the melanocyte specific target, Melan-A. Images representing immunostained melanoma tissue were then digitally processed to segment regions of interest, highlighting Melan-A positive and negative areas. Color deconvolution was applied to each region of interest to separate the channel containing the immunohistochemistry signal from the hematoxylin counterstaining channel. A support vector machine melanoma classification model was learned from a discovery melanoma patient cohort (n = 264) and subsequently validated on an independent cohort of melanoma patient tissue sample images (n = 157).

Conclusion

Here we propose a novel method that takes advantage of utilizing an immuhistochemical marker highlighting melanocytes to fully automate the learning of a general melanoma cell classification model. The presented method can be applied on any protein of interest and thus provides a tool for quantification of immunohistochemistry-based protein expression in melanoma.

Primary Cilia: The Chemical Antenna Regulating Human Adipose-Derived Stem Cell Osteogenesis

2013-05-17T21:00:00Z

by Josephine C. Bodle, Candace D. Rubenstein, Michelle E. Phillips, Susan H. Bernacki, Jie Qi, Albert J. Banes, Elizabeth G. Loboa

Adipose-derived stem cells (ASC) are multipotent stem cells that show great potential as a cell source for osteogenic tissue replacements and it is critical to understand the underlying mechanisms of lineage specification. Here we explore the role of primary cilia in human ASC (hASC) differentiation. This study focuses on the chemosensitivity of the primary cilium and the action of its associated proteins: polycystin-1 (PC1), polycystin-2 (PC2) and intraflagellar transport protein-88 (IFT88), in hASC osteogenesis. To elucidate cilia-mediated mechanisms of hASC differentiation, siRNA knockdown of PC1, PC2 and IFT88 was performed to disrupt cilia-associated protein function. Immunostaining of the primary cilium structure indicated phenotypic-dependent changes in cilia morphology. hASC cultured in osteogenic differentiation media yielded cilia of a more elongated conformation than those cultured in expansion media, indicating cilia-sensitivity to the chemical environment and a relationship between the cilium structure and phenotypic determination. Abrogation of PC1, PC2 and IFT88 effected changes in both hASC proliferation and differentiation activity, as measured through proliferative activity, expression of osteogenic gene markers, calcium accretion and endogenous alkaline phosphatase activity. Results indicated that IFT88 may be an early mediator of the hASC differentiation process with its knockdown increasing hASC proliferation and decreasing Runx2, alkaline phosphatase and BMP-2 mRNA expression. PC1 and PC2 knockdown affected later osteogenic gene and end-product expression. PC1 knockdown resulted in downregulation of alkaline phosphatase and osteocalcin gene expression, diminished calcium accretion and reduced alkaline phosphatase enzymatic activity. Taken together our results indicate that the structure of the primary cilium is intimately associated with the process of hASC osteogenic differentiation and that its associated proteins are critical players in this process. Elucidating the dynamic role of the primary cilium and its associated proteins will help advance the application of hASC in generating autologous tissue engineered therapies in critical defect bone injuries.

The Human Gut Chip “HuGChip”, an Explorative Phylogenetic Microarray for Determining Gut Microbiome Diversity at Family Level

2013-05-17T21:00:00Z

by William Tottey, Jeremie Denonfoux, Faouzi Jaziri, Nicolas Parisot, Mohiedine Missaoui, David Hill, Guillaume Borrel, Eric Peyretaillade, Monique Alric, Hugh M. B. Harris, Ian B. Jeffery, Marcus J. Claesson, Paul W. O'Toole, Pierre Peyret, Jean-François Brugère

Evaluating the composition of the human gut microbiota greatly facilitates studies on its role in human pathophysiology, and is heavily reliant on culture-independent molecular methods. A microarray designated the Human Gut Chip (HuGChip) was developed to analyze and compare human gut microbiota samples. The PhylArray software was used to design specific and sensitive probes. The DNA chip was composed of 4,441 probes (2,442 specific and 1,919 explorative probes) targeting 66 bacterial families. A mock community composed of 16S rRNA gene sequences from intestinal species was used to define the threshold criteria to be used to analyze complex samples. This was then experimentally verified with three human faecal samples and results were compared (i) with pyrosequencing of the V4 hypervariable region of the 16S rRNA gene, (ii) metagenomic data, and (iii) qPCR analysis of three phyla. When compared at both the phylum and the family level, high Pearson's correlation coefficients were obtained between data from all methods. The HuGChip development and validation showed that it is not only able to assess the known human gut microbiota but could also detect unknown species with the explorative probes to reveal the large number of bacterial sequences not yet described in the human gut microbiota, overcoming the main inconvenience encountered when developing microarrays.

The Effect of Extracellular pH Changes on Intracellular pH and Nitric Oxide Concentration in Endothelial and Smooth Muscle Cells from Rat Aorta

2013-05-17T21:00:00Z

by Verena K. Capellini, Carolina B. A. Restini, Lusiane M. Bendhack, Paulo R. B. Evora, Andréa C. Celotto

Aims

It has been known for more than a century that pH changes can alter vascular tone. However, there is no consensus about the effects of pH changes on vascular response. In this study, we investigated the effects of extracellular pH (pH_o) changes on intracellular pH (pH_i) and intracellular nitric oxide concentration ([NO]_i) in freshly isolated endothelial cells and cross sections from rat aorta.

Main Methods

The HCl was used to reduce the pH_o from 7.4 to 7.0 and from 7.4 to 6.5; the NaOH was used to increase the pH_o from 7.4 to 8.0 and from 7.4 to 8.5. The fluorescent dyes 5-(and-6)-carboxy SNARF-1, acetoxymethyl ester, acetate (SNARF-1) and diaminofluorescein-FM diacetate (DAF-FM DA) were employed to measure the pH_i and [NO]_i, respectively. The fluorescence intensity was measured in freshly isolated endothelial cells by flow cytometry and in freshly obtained aorta cross sections by confocal microscopy.

Key Findings

The endothelial and vascular smooth muscle pH_i was increased at pH_o 8.5. The extracellular acidification did not change the endothelial pH_i, but the smooth muscle pH_i was reduced at pH_o 7.0. At pH_o 8.5 and pH_o 6.5, the endothelial [NO]_i was increased. Both extracellular alkalinization and acidification increased the vascular smooth muscle [NO]_i.

Significance

Not all changes in pH_o did result in pH_i changes, but disruption of acid-base balance in both directions induced NO synthesis in the endothelium and/or vascular smooth muscle.