bluuebunny/crossref_metadata_2025 huggingface dataset:
The mitochondrial genome of the hexactinellid sponge Aphrocallistes vastus: Evidence for programmed translational frameshifting
Authors: Rosengarten Rafael D , Sperling Erik A , Moreno Maria A , Leys Sally P , Dellaporta Stephen L
Published: January 2008

Background

Mitochondrial genomes (mtDNA) of numerous sponges have been sequenced as part of an ongoing effort to resolve the class-level phylogeny of the Porifera, as well as to place the various lower metazoan groups on the animal-kingdom tree. Most recently, the partial mtDNA of two glass sponges, class Hexactinellida, were reported. While previous phylogenetic estimations based on these data remain uncertain due to insufficient taxon sampling and accelerated rates of evolution, the mtDNA molecules themselves reveal interesting traits that may be unique to hexactinellids. Here we determined the first complete mitochondrial genome of a hexactinellid sponge, Aphrocallistes vastus, and compared it to published poriferan mtDNAs to further describe characteristics specific to hexactinellid and other sponge mitochondrial genomes.

Results

The A. vastus mtDNA consisted of a 17,427 base pair circular molecule containing thirteen protein-coding genes, divergent large and small subunit ribosomal RNAs, and a reduced set of 18 tRNAs. The A. vastus mtDNA showed a typical hexactinellid nucleotide composition and shared a large synteny with the other sequenced glass sponge mtDNAs. It also contained an unidentified open reading frame and large intergenic space region. Two frameshifts, in the cox3 and nad6 genes, were not corrected by RNA editing, but rather possessed identical shift sites marked by the extremely rare tryptophan codon (UGG) followed by the common glycine codon (GGA) in the +1 frame.

Conclusion

Hexactinellid mtDNAs have shown similar trends in gene content, nucleotide composition, and codon usage, and have retained a large gene syntenty. Analysis of the mtDNA of A. vastus has provided evidence diagnostic for +1 programmed translational frameshifting, a phenomenon disparately reported throughout the animal kingdom, but present in the hexactinellid mtDNAs that have been sequenced to date.
Insertion Sequences show diverse recent activities in Cyanobacteria and Archaea
Authors: Zhou Fengfeng , Olman Victor , Xu Ying
Published: January 2008

Background

Mobile genetic elements (MGEs) play an essential role in genome rearrangement and evolution, and are widely used as an important genetic tool.

Results

In this article, we present genetic maps of recently active Insertion Sequence (IS) elements, the simplest form of MGEs, for all sequenced cyanobacteria and archaea, predicted based on the previously identified ~1,500 IS elements. Our predicted IS maps are consistent with the NCBI annotations of the IS elements. By linking the predicted IS elements to various characteristics of the organisms under study and the organism's living conditions, we found that (a) the activities of IS elements heavily depend on the environments where the host organisms live; (b) the number of recently active IS elements in a genome tends to increase with the genome size; (c) the flanking regions of the recently active IS elements are significantly enriched with genes encoding DNA binding factors, transporters and enzymes; and (d) IS movements show no tendency to disrupt operonic structures.

Conclusion

This is the first genome-scale maps of IS elements with detailed structural information on the sequence level. These genetic maps of recently active IS elements and the several interesting observations would help to improve our understanding of how IS elements proliferate and how they are involved in the evolution of the host genomes.
Aerial Dissemination of Clostridium difficilespores
Authors: Roberts Katherine , Smith Caroline F , Snelling Anna M , Kerr Kevin G , Banfield Kathleen R , Sleigh P Andrew , Beggs Clive B
Published: January 2008

Background

Clostridium difficile-associated diarrhoea (CDAD) is a frequently occurring healthcare-associated infection, which is responsible for significant morbidity and mortality amongst elderly patients in healthcare facilities. Environmental contamination is known to play an important contributory role in the spread of CDAD and it is suspected that contamination might be occurring as a result of aerial dissemination of C. difficile spores. However previous studies have failed to isolate C. difficile from air in hospitals. In an attempt to clarify this issue we undertook a short controlled pilot study in an elderly care ward with the aim of culturing C. difficile from the air.

Methods

In a survey undertaken during February (two days) 2006 and March (two days) 2007, air samples were collected using a portable cyclone sampler and surface samples collected using contact plates in a UK hospital. Sampling took place in a six bedded elderly care bay (Study) during February 2006 and in March 2007 both the study bay and a four bedded orthopaedic bay (Control). Particulate material from the air was collected in Ringer's solution, alcohol shocked and plated out in triplicate onto Brazier's CCEY agar without egg yolk, but supplemented with 5 mg/L of lysozyme. After incubation, the identity of isolates was confirmed by standard techniques. Ribotyping and REP-PCR fingerprinting were used to further characterise isolates.

Results

On both days in February 2006, C. difficile was cultured from the air with 23 samples yielding the bacterium (mean counts 53 – 426 cfu/m3 of air). One representative isolate from each of these was characterized further. Of the 23 isolates, 22 were ribotype 001 and were indistinguishable on REP-PCR typing. C. difficile was not cultured from the air or surfaces of either hospital bay during the two days in March 2007.

Conclusion

This pilot study produced clear evidence of sporadic aerial dissemination of spores of a clone of C. difficile, a finding which may help to explain why CDAD is so persistent within hospitals and difficult to eradicate. Although preliminary, the findings reinforce concerns that current C. difficile control measures may be inadequate and suggest that improved ward ventilation may help to reduce the spread of CDAD in healthcare facilities.
Systematic screening for unsafe driving due to medical conditions: Still debatable
Authors: Leproust Sandy , Lagarde Emmanuel , Salmi L Rachid
Published: January 2008

Background

Assessing people's ability to drive has become a public health concern in most industrialized countries. Although age itself is not a predictive factor of an increased risk for dangerous driving, the prevalence of medical conditions that may impair driving increases with age. Because the implementation of a screening for unsafe driving due to medical conditions is a public health issue, its usefulness should be judged using standardised criteria already proposed for screening for chronic disease. The aim of this paper is to propose standardised criteria suitable to assess the scientific validity of screening for unsafe driving due to medical conditions, and identify potential issues to be clarified before screening can be implemented and effective.

Discussion

Using criteria developed for screening for chronic diseases and published studies on driving with medical conditions, we specify six criteria to judge the opportunity of screening for unsafe driving due to medical conditions. This adaptation was needed because of the complexity of the natural history of medical conditions and their potential consequences on driving and road safety. We then illustrate that published studies pleading for or against screening for unsafe driving due to medical conditions fail to provide the needed documentation. Individual criteria were mentioned in 3 to 72% of 36 papers pleading for or against screening. Quantitative estimates of relevant indicators were provided in at most 42% of papers, and some data, such as the definition of an appropriate unsafe driving period were never provided.

The standardised framework described in this paper provides a template for assessing the effectiveness (or lack of effectiveness) of proposed measures for screening for unsafe driving due to medical conditions. Even if most criteria were mentioned in the published literature pleading for or against such a screening, the failure to find quantitative and evidence-based estimates of relevant indicators provides useful insight for further research.
Constitutive overexpression of a novel 21 kDa protein by Hodgkin Lymphoma and Aggressive Non-Hodgkin Lymphomas
Authors: Zhou Minglong , Fadlelmola Faisal M , Cohn Jason B , Skinnider Brian , Gascoyne Randy D , Banerjee Diponkar
Published: January 2008

Background

CD30, a 120 kDa surface phosphorylated protein is a member of tumour necrosis/nerve growth factor receptor (TNF/NGFR) family and constitutively expressed by Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin lymphoma (HL) and the neoplastic cells of Anaplastic Large Cell Lymphoma (ALCL). A disease-specific protein marker is yet to be identified in Hodgkin lymphoma cells. In order to define HL-specific biomarkers, novel murine monoclonal antibodies were developed in our laboratory.

Results

Murine monoclonal antibodies (mabs) were raised against the B3 sub clone of HL-derived cell line KM-H2. Two of these mabs (clone R23.1 mab and clone R24.1 mab) are IgG1 class antibodies that recognize a 21 kDa protein present at the cell membrane and in the cytoplasm in HL-derived cell lines. Clone R24.1 mab recognizes a formalin-resistant epitope and labels HRS cells in tissue samples from patients with HL of the classical type, ALCL, and subsets of T and B cell aggressive Non-Hodgkin Lymphomas (NHL). The antigen recognized by the clone R23.1 mab and clone R24.1 mab does not share epitopes with CD30 cluster regions A, B, or C, and, unlike CD30, is not expressed by phytohemagglutinin (PHA) activated T cells.

Conclusion

The 21 kDa protein detected by clone R23.1 and clone R24.1 mabs is a novel membrane-associated protein that may be a potential marker for the diagnosis and targeted therapy of HL and aggressive T and B cell NHL.
Multiple independent origins of a protease inhibitor resistance mutation in salvage therapy patients
Authors: Kapoor Amit , Shapiro Beth , Shafer Robert W , Shulman Nancy , Rhee Soo-Yon , Delwart Eric L
Published: January 2008

Background

Combination anti-viral therapies have reduced treatment failure rates by requiring multiple specific mutations to be selected on the same viral genome to impart high-level drug resistance. To determine if the common protease inhibitor resistance mutation L90M is only selected once or repeatedly on different HIV genetic backbones during the course of failed anti-viral therapies we analyzed a linked region of the viral genome during the evolution of multi-drug resistance.

Results

Using L90M allele specific PCR we amplified and sequenced gag-pro regions linked to very early L90M containing HIV variants prior to their emergence and detection as dominant viruses in 15 failed salvage therapy patients. The early minority L90M linked sequences were then compared to those of the later L90M viruses that came to dominate the plasma quasispecies. Using Bayesian evolutionary analysis sampling trees the emergence of L90M containing viruses was seen to take place on multiple occasion in 5 patients, only once for 2 patients and an undetermined number of time for the remaining 8 patients.

Conclusion

These results indicate that early L90M mutants can frequently be displaced by viruses carrying independently selected L90M mutations rather than by descendents of the earlier mutants.
Multiple-locus variable number of tandem repeat analysis (MLVA) of Irish verocytotoxigenic Escherichia coliO157 from feedlot cattle: uncovering strain dissemination routes
Authors: Murphy Mary , Minihan Donal , Buckley James F , O'Mahony Micheál , Whyte Paul , Fanning Séamus
Published: January 2008

Background

The identification of the routes of dissemination of Escherichia coli (E. coli) O157 through a cohort of cattle is a critical step to control this pathogen at farm level. The aim of this study was to identify potential routes of dissemination of E. coli O157 using Multiple-Locus Variable number of tandem repeat Analysis (MLVA).

Results

Thirty-eight environmental and sixteen cattle faecal isolates, which were detected in four adjacent pens over a four-month period were sub-typed. MLVA could separate these isolates into broadly defined clusters consisting of twelve MLVA types. Strain diversity was observed within pens, individual cattle and the environment.

Conclusion

Application of MLVA is a broadly useful and convenient tool when applied to uncover the dissemination of E. coli O157 in the environment and in supporting improved on-farm management of this important pathogen. These data identified diverse strain types based on amplification of VNTR markers in each case.
Genomic chart guiding embryonic stem cell cardiopoiesis
Authors: Faustino Randolph S , Behfar Atta , Perez-Terzic Carmen , Terzic Andre
Published: January 2008

Background

Embryonic stem cells possess a pluripotent transcriptional background with the developmental capacity for distinct cell fates. Simultaneous expression of genetic elements for multiple outcomes obscures cascades relevant to specific cell phenotypes. To map molecular patterns critical to cardiogenesis, we interrogated gene expression in stem cells undergoing guided differentiation, and defined a genomic paradigm responsible for confinement of pluripotency.

Results

Functional annotation analysis of the transcriptome of differentiating embryonic stem cells exposed downregulated components of DNA replication, recombination and repair machinery, cell cycling, cancer mechanisms, and RNA post-translational modifications. Concomitantly, cardiovascular development, cell-to-cell signaling, cell development and cell movement were upregulated. These simultaneous gene ontology rearrangements engaged a repertoire switch that specified lineage development. Bioinformatic integration of genomic and gene ontology data further unmasked canonical signaling cascades prioritized within discrete phases of cardiopoiesis. Examination of gene relationships revealed a non-stochastic network anchored by integrin, WNT/β-catenin, transforming growth factor β and vascular endothelial growth factor pathways, validated by manipulation of selected cascades that promoted or restrained cardiogenic yield. Moreover, candidate genes within anchor pathways acted as nodes that organized correlated expression profiles into functional clusters, which collectively orchestrated and secured an overall cardiogenic theme.

Conclusion

The present systems biology approach reveals a dynamically integrated and tractable gene network fundamental to embryonic stem cell specification, and represents an initial step towards resolution of a genomic cardiopoietic atlas.
Aging in the absence of TLR2 is associated with reduced IFN-γ responses in the large intestine and increased severity of induced colitis
Authors: Albert Eric J , Marshall Jean S
Published: January 2008

Age-associated changes in immune function and their implications for intestinal inflammation are poorly understood. Defects in innate immunity have been shown to enhance intestinal inflammation and have been demonstrated upon aging. This study aimed to determine the consequences of aging in the presence and absence of TLR2 on intestinal inflammation. Young and aged (>60 weeks), control C57Bl/6 and TLR2-deficient (TLR2−/−) mice were examined. The cecum and mid-colon were analyzed for tissue damage, cytokine profiles, and trefoil factor 3 (TFF3) expression at baseline or after 5 days of treatment with dextran sodium sulfate (DSS) and 5 or 13 days recovery. Untreated, aged TLR2−/− mice had no significant intestinal inflammation but had reduced colonic IFN-γ and IL-10 compared with younger mice. Aged TLR2−/− mice developed more severe colitis than other groups, as indicated by histological examination and overall weight loss. There were significant increases in colonic IFN-γ following DSS treatment in young but not in aged mice. TFF3 was substantially reduced in the cecum and increased in the colon of aged but not younger TLR2−/− mice following DSS treatment. These results demonstrate that even upon aging, TLR2-deficient animals did not develop intestinal disease. However, they failed to respond appropriately to an inflammatory insult, and the consequences of this were most severe in aged animals. Cytokine and TFF3 changes associated with aging may contribute to more severe intestinal inflammation.
Interplay between the Endocannabinoid System and GnRH-I in the Forebrain of the Anuran Amphibian Rana esculenta
Authors: Meccariello Rosaria , Franzoni Maria Fosca , Chianese Rosanna , Cottone Erika , Scarpa Donatella , Donna Daniela , Cobellis Gilda , Guastalla Alda , Pierantoni Riccardo , Fasano Silvia
Published: January 2008

The morphofunctional relationship between the endocannabinoid system and GnRH activity in the regulation of reproduction has poorly been investigated in vertebrates. Due to the anatomical features of lower vertebrate brain, in the present paper, we chose the frog Rana esculenta (anuran amphibian) as a suitable model to better investigate such aspects of the reproductive physiology. By using double-labeling immunofluorescence aided with a laser-scanning confocal microscope, we found a subpopulation of the frog hypothalamic GnRH neurons endowed with CB1 cannabinoid receptors. By means of semiquantitative RT-PCR assay, we have shown that, during the annual sexual cycle, GnRH-I mRNA (formerly known as mammalian GnRH) and CB1 mRNA have opposite expression profiles in the brain. In particular, this occurs in telencephalon and diencephalon, the areas mainly involved in GnRH release and control of the reproduction. Furthermore, we found that the endocannabinoid anandamide is able to inhibit GnRH-I mRNA synthesis; buserelin (a GnRH agonist), in turn, inhibits the synthesis of GnRH-I mRNA and induces an increase of CB1 transcription. Our observations point out the occurrence of a morphofunctional anatomical basis to explain a reciprocal relationship between the endocannabinoid system and GnRH neuronal activity.
Microribonucleic Acid-21 Increases Aldosterone Secretion and Proliferation in H295R Human Adrenocortical Cells
Authors: Romero Damian G. , Plonczynski Maria W. , Carvajal Cristian A. , Gomez-Sanchez Elise P. , Gomez-Sanchez Celso E.
Published: January 2008

MicroRNAs (miRNAs) are endogenous small noncoding RNAs that decrease the expression levels of specific genes by translational repression, sequestration, and degradation of their mRNAs. Angiotensin II is an important modulator of adrenal zona glomerulosa cell physiology, including steroidogenesis and proliferation among many other physiological processes. Because each miRNA may regulate the expression levels of multiple genes, thereby resembling the transcription regulatory networks triggered by transcription factors, we hypothesize that specific miRNAs may be involved in angiotensin II-mediated adrenocortical cell physiology. The human adrenocortical cell line H295R is the only adrenal cell line available with a steroid secretion pattern and regulation similar to freshly isolated adrenocortical cells. We screened for miRNAs regulated by angiotensin II in H295R cells and found that miRNA-21 expression levels were specifically modulated by angiotensin II. Angiotensin II time dependently increased miRNA-21 expression reaching a 4.4-fold induction after 24 h. Angiotensin II-mediated miRNA-21 expression resulted in biologically active miRNA-21, determined using a fusion mRNA reporter system carrying miRNA-21 target sequences in its 3′ untranslated region. Up-regulation of miRNA-21 intracellular levels increased aldosterone secretion but not cortisol. Elevation of miRNA-21 levels also increased cell proliferation in H295R cells. In summary, miRNA-21 is an endogenously expressed miRNA in human adrenal cells. miRNA-21 expression is up-regulated by angiotensin II, and its overexpression caused an increase in aldosterone secretion and cell proliferation. Alterations in miRNA-21 expression levels or function may be involved in dysregulation of angiotensin II signaling and abnormal aldosterone secretion by adrenal glands in humans.
Welcoming β-Catenin to the Gonadotropin-Releasing Hormone Transcriptional Network in Gonadotropes
Authors: Salisbury Travis B. , Binder April K. , Nilson John H.
Published: January 2008

GnRH binds its G-coupled protein receptor, GnRHR, on pituitary gonadotropes and stimulates transcription of Cga, Lhb, and Fshb. These three genes encode two heterodimeric glycoprotein hormones, LH and FSH, that act as gonadotropins by regulating gametogenesis and steroidogenesis in both the testes and ovary. GnRH also regulates transcription of Gnrhr. Thus, regulated expression of Cga, Lhb, Fshb, and Gnrhr provides a genomic signature unique to functional gonadotropes. Steadily increasing evidence now indicates that GnRH regulates transcription of its four signature genes indirectly through a hierarchical transcriptional network that includes distinct subclasses of DNA-binding proteins that comprise the immediate early gene (IEG) family. These IEGs, in turn, confer hormonal responsiveness to the four signature genes. Although the IEGs confer responsiveness to GnRH, they cannot act alone. Instead, additional DNA-binding proteins, including the orphan nuclear receptor steroidogenic factor 1, act permissively to allow the four signature genes to respond to GnRH-induced changes in IEG levels. Emerging new findings now indicate that β-catenin, a transcriptional coactivator and member of the canonical WNT signaling pathway, also plays an essential role in transducing the GnRH signal by interacting with multiple DNA-binding proteins in gonadotropes. Herein we propose that these interactions with β-catenin define a multicomponent transcriptional network required for regulated expression of the four signature genes of the gonadotrope, Cga, Lhb, Fshb, and Gnrhr.
Hd3a and RFT1 are essential for flowering in rice
Authors: Komiya Reina , Ikegami Akiko , Tamaki Shojiro , Yokoi Shuji , Shimamoto Ko
Published: January 2008

RICE FLOWERING LOCUS T 1 (RFT1/FT-L3) is the closest homologue of Heading date 3a (Hd3a), which is thought to encode a mobile flowering signal and promote floral transition under short-day(SD) conditions. RFT1 is located only 11.5 kb from Hd3a on chromosome 6. Although RFT1 RNAi plants flowered normally, double RFT1-Hd3a RNAi plants did not flower up to 300 days after sowing (DAS), indicating that Hd3a and RFT1 are essential for flowering in rice. RFT1 expression was very low in wild-type plants, but there was a marked increase in RFT1 expression by 70 DAS in Hd3a RNAi plants, which flowered 90 DAS. H3K9 acetylation around the transcription initiation site of the RFT1 locus had increased by 70 DAS but not at 35 DAS. In the absence of Hd3a and RFT1expression, transcription of OsMADS14 and OsMADS15, two rice orthologues of Arabidopsis APETALA1, was strongly reduced, suggesting that they act downstream of Hd3a and RFT1. These results indicate that Hd3a and RFT1 act as floral activators under SD conditions, and that RFT1 expression is partly regulated by chromatin modification.
Cux2(Cutl2) integrates neural progenitor development with cell-cycle progression during spinal cord neurogenesis
Authors: Iulianella Angelo , Sharma Madhulika , Durnin Michael , Vanden Heuvel Greg B. , Trainor Paul A.
Published: January 2008

Neurogenesis requires the coordination of neural progenitor proliferation and differentiation with cell-cycle regulation. However, the mechanisms coordinating these distinct cellular activities are poorly understood. Here we demonstrate for the first time that a Cut-like homeodomain transcription factor family member, Cux2 (Cutl2), regulates cell-cycle progression and development of neural progenitors. Cux2loss-of-function mouse mutants exhibit smaller spinal cords with deficits in neural progenitor development as well as in neuroblast and interneuron differentiation. These defects correlate with reduced cell-cycle progression of neural progenitors coupled with diminished Neurod and p27Kip1activity. Conversely, in Cux2 gain-of-function transgenic mice, the spinal cord is enlarged in association with enhanced neuroblast formation and neuronal differentiation, particularly with respect to interneurons. Furthermore, Cux2 overexpression induces high levels of Neurod and p27Kip1. Mechanistically, we discovered through chromatin immunoprecipitation assays that Cux2 binds both the Neurodand p27Kip1 promoters in vivo, indicating that these interactions are direct. Our results therefore show that Cux2functions at multiple levels during spinal cord neurogenesis. Cux2initially influences cell-cycle progression in neural progenitors but subsequently makes additional inputs through Neurod and p27Kip1 to regulate neuroblast formation, cell-cycle exit and cell-fate determination. Thus our work defines novel roles for Cux2 as a transcription factor that integrates cell-cycle progression with neural progenitor development during spinal cord neurogenesis.
Myo2p, a class V myosin in budding yeast, associates with a large ribonucleic acid–protein complex that contains mRNAs and subunits of the RNA-processing body
Authors: Chang Wakam , Zaarour Rania F. , Reck-Peterson Samara , Rinn John , Singer Robert H. , Snyder Michael , Novick Peter , Mooseker Mark S.
Published: January 2008

Myo2p is an essential class V myosin in budding yeast with several identified functions in organelle trafficking and spindle orientation. The present study demonstrates that Myo2p is a component of a large RNA-containing complex (Myo2p–RNP) that is distinct from polysomes based on sedimentation analysis and lack of ribosomal subunits in the Myo2p–RNP. Microarray analysis of RNAs that coimmunoprecipitate with Myo2p revealed the presence of a large number of mRNAs in this complex. The Myo2p–RNA complex is in part composed of the RNA processing body (P-body) based on coprecipitation with P-body protein subunits and partial colocalization of Myo2p with P-bodies. P-body disassembly is delayed in the motor mutant, myo2-66, indicating that Myo2p may facilitate the release of mRNAs from the P-body.
High Diagnostic Accuracy of Antigen Microarray for Sensitive Detection of Hepatitis C Virus Infection
Authors: Kwon Jung-ah , Lee Hyeseon , Lee Kap N o , Chae Kwangchun , Lee Seram , Lee Dong-ki , Kim Soyoun
Published: January 2008

Background: Hepatitis C virus (HCV) can be transmitted through blood transfusion. Screening ELISA, the most widely used method for HCV diagnosis, sometimes yields false-positive and false-negative results, so a confirmatory test is used. This secondary testing is labor-intensive and expensive, and thus is impractical for massive blood bank screening. Therefore, a new massive screening method with high accuracy is needed for sensitive and specific detection of HCV.

Methods: With sol-gel material, we designed novel antigen microarray in 96-well plates for HCV detection. Each individual well was spotted with 4 different HCV antigens. We used this new system to test 154 patient serum samples previously tested for HCV by ELISA (87 HCV positive and 67 HCV negative) (HCV EIA3.0, ABBOTT). We assessed the detection limit of our microarray system with the use of serial 10-fold dilutions of an HCV-positive sample.

Results: Our microarray assay was reproducible and displayed higher diagnostic accuracy (specificity) (98.78%) than did the ELISA (81.71%). Our method yielded significantly fewer false-positive results than did the ELISA. The detection limit of our assay was 1000 times more sensitive than that of the ELISA. In addition, we found this novel assay technology to be compatible with the currently employed automated methods used for ELISA.

Conclusion: We successfully applied the sol-gel–based protein microarray technology to a screening assay for HCV diagnosis with confirmatory test-level accuracy. This new, inexpensive method will improve the specificity and sensitivity of massive sample diagnosis.
High-Resolution Melting Analysis of the spa Repeat Region of Staphylococcus aureus
Authors: Stephens Alex J , Inman-Bamber John , Giffard Philip M , Huygens Flavia
Published: January 2008

Background: The staphylococcal protein A (spa) locus of Staphylococcus aureus contains a complex repeat structure and is commonly used for single-locus sequence-based genotyping. The real-time PCR platform supports genotyping methods that are single step and closed tube and potentially can be carried out simultaneously with diagnosis. We describe here a method for genotyping S. aureus using high-resolution melting (HRM) analysis of the spa polymorphic region X.

Methods: The conventional PCR spa assay was modified and optimized for the Rotor-Gene 6000 instrument (Corbett Life Science). HRM analysis on the Corbett Rotor-Gene 6000 instrument was used to test 22 known spa sequences obtained from 44 diverse methicillin-resistant S. aureus (MRSA) isolates. Criteria for calling pairs of melting curves “same” or “different” were developed empirically by converting the data to difference graph format with one curve defined as the control. HRM curve comparison between runs was done to determine the portability of the method. The assay performance was assessed by genotyping uncharacterized isolates, carrying out blind trials, and comparing HRM profiles from different runs.

Results: HRM analysis of 44 diverse MRSA isolates generated 20 profiles from 22 spa sequence types. The 2 unresolved HRM spa types differed by only 1 bp. Two blind trials demonstrated complete reproducibility with respect to calling the different spa types. Interrun comparisons of HRM curves were successfully developed, indicating the robustness of the method.

Conclusion: Analysis of the spa locus by HRM resolves spa sequence variants. This single- and closed-tube single-step method for S. aureus genotyping can be easily combined with the interrogation of other genetic markers.
Rapid Quantitative Method Using Spin Columns to Measure Porphobilinogen in Urine
Authors: Roshal Mikhail , Turgeon Jeanne , Rainey Petrie M
Published: January 2008

Background: Large increases of urinary porphobilinogen (PBG) indicate acute porphyria, which may be due to acute intermittent porphyria, variegate porphyria, or hereditary coproporphyria. These conditions are relatively rare but share symptoms with more common conditions, such as acute surgical abdomen, and often must be ruled out rapidly. Reported quantitative methods for PBG measurement are time-consuming and inconvenient. We developed a rapid quantitative method that uses resin-packed spin columns to measure PBG in urine.

Method: We applied urine to anion exchange resin in a spin column, then performed centrifugal separation and washing. PBG was eluted in 1 mol/L acetic acid and reacted with Ehrlich’s reagent. After 5 min, we measured absorbance at 525, 555, and 585 nm. PBG concentration (mg/L) was calculated as 88 (A555 − ½(A525 + A585)).

Results: The reportable PBG concentration range was 0.2–15 mg/L. Between-day (total) imprecision (CV) was 8.4% at 1.2 mg/L and 3.5% at 4.4 mg/L. Comparison with our established method (x) yielded a Deming regression equation: y = 1.04x − 0.01 mg/L (R2 = 0.98; Sy,x = 0.87 mg/L). No interference was noted from urobilinogen or highly colored urine specimens.

Conclusions: This method for PBG measurement is more rapid and precise than other methods. This test can serve as a quick screening test and facilitates batch analysis for routine quantitative testing.
The IFCC Reference Measurement System for HbA1c: A 6-Year Progress Report
Authors: Weykamp Cas , John W Garry , Mosca Andrea , Hoshino Tadao , Little Randie , Jeppsson Jan-Olof , Goodall Ian , Miedema Kor , Myers Gary , Reinauer Hans , Sacks David B , Slingerland Robbert , Siebelder Carla
Published: January 2008

Background: The IFCC Reference Measurement System for hemoglobin (Hb)A1c (IFCC-RM) has been developed within the framework of metrologic traceability and is embedded in a network of 14 reference laboratories. This paper describes the outcome of 12 intercomparison studies (periodic evaluations to control essential elements of the IFCC-RM).

Methods: Each study included: unknown samples (to test individual network laboratories); known samples (controls); recently manufactured calibrators (to check calculated assigned value); stored calibrators (to test stability) and a calibration-set (to calibrate the IFCC-RM). The unknown samples are measured by use of the IFCC-RM and the designated comparison methods [DCMs; the National Glycohemoglobin Standardization Program (NGSP) in the US, Japanese Diabetes Society/Japanese Society for Clinical Chemistry (JDS/JSCC) in Japan, and Mono-S in Sweden] are used to investigate the stability of the Master Equation (ME), the relationship between IFCC-RM and DCMs.

Results: A total of 105 IFCC-RM data sets were evaluated: 95 were approved, 5 were not, and for 5 no data were submitted. Trend analysis of the MEs, expressed as change in percentage HbA1c per year, revealed 0.000% (NGSP, not significant), −0.030%, (JDS/JSCC; significant) and −0.016% (Mono-S; not significant). Evaluation of long-term performance revealed no systematic change over time; 2 laboratories showed significant bias, 1 poor reproducibility. The mean HbA1c determined by laboratories performing mass spectrometry (MS) was the same as the mean determined by laboratories using capillary electrophoresis (CE), but the reproducibility at laboratories using CE was better. One batch of new calibrators was not approved. All stored calibrators were stable.

Conclusion: A sound reference system is in place to ensure continuity and stability of the analytical anchor for HbA1c.
Sensitivity functions and their uses in inverse problems
Authors: Banks H. T. , Dediu S. , Ernstberger S. L.
Published: January 2008

In this note we present a critical review of the some of the positive features as well as some of the shortcomings of the generalized sensitivity functions (GSF) of Thomaseth–Cobelli in comparison to traditional sensitivity functions (TSF). We do this from a computational perspective of ordinary least squares estimation or inverse problems using two illustrative examples: the Verhulst–Pearl logistic growth model and a recently developed agricultural production network model. Because GSF provide information on the relevance of data measurements for the identification of certain parameters in a typical parameter estimation problems, we argue that they provide the basis for new tools for investigators in design of inverse problem studies.
An inverse problem for the non-selfadjoint matrix Sturm–Liouville equation on the half-line
Authors: Freiling G. , Yurko V.
Published: January 2008

An inverse spectral problem is studied for the non-selfadjoint matrix Sturm–Liouville differential equation on the half-line. We give a formulation of the inverse problem, prove the corresponding uniqueness theorem and provide a constructive procedure for the solution of the inverse problem by the method of spectral mappings. The obtained results are natural generalizations of the classical results in inverse problem theory for scalar Sturm–Liouville operators.
Coordinated Changes of Mitochondrial Biogenesis and Antioxidant Enzymes During Osteogenic Differentiation of Human Mesenchymal Stem Cells
Authors: Chen Chien-Tsun , Shih Yu-Ru V. , Kuo Tom K. , Lee Oscar K. , Wei Yau-Huei
Published: January 2008

The multidifferentiation ability of mesenchymal stem cells holds great promise for cell therapy. Numerous studies have focused on the establishment of differentiation protocols, whereas little attention has been paid to the metabolic changes during the differentiation process. Mitochondria, the powerhouse of mammalian cells, vary in their number and function in different cell types with different energy demands, but how these variations are associated with cell differentiation remains elusive. In this study, we investigated the changes of mitochondrial biogenesis and bioenergetic function using human mesenchymal stem cells (hMSCs) because of their well-defined differentiation potentials. Upon osteogenic induction, the copy number of mitochondrial DNA, protein subunits of the respiratory enzymes, oxygen consumption rate, and intracellular ATP content were increased, indicating the upregulation of aerobic mitochondrial metabolism. On the other hand, undifferentiated hMSCs showed higher levels of glycolytic enzymes and lactate production rate, suggesting that hMSCs rely more on glycolysis for energy supply in comparison with hMSC-differentiated osteoblasts. In addition, we observed a dramatic decrease of intracellular reactive oxygen species (ROS) as a consequence of upregulation of two antioxidant enzymes, manganese-dependent superoxide dismutase and catalase. Finally, we found that exogenous H2O2 and mitochondrial inhibitors could retard the osteogenic differentiation. These findings suggested an energy production transition from glycolysis to oxidative phosphorylation in hMSCs upon osteogenic induction. Meanwhile, antioxidant enzymes were concurrently upregulated to prevent the accumulation of intracellular ROS. Together, our findings suggest that coordinated regulation of mitochondrial biogenesis and antioxidant enzymes occurs synergistically during osteogenic differentiation of hMSCs.

Disclosure of potential conflicts of interest is found at the end of this article.
Comparison of Reporter Gene and Iron Particle Labeling for Tracking Fate of Human Embryonic Stem Cells and Differentiated Endothelial Cells in Living Subjects
Authors: Li Zongjin , Suzuki Yoriyasu , Huang Mei , Cao Feng , Xie Xiaoyan , Connolly Andrew J. , Yang Phillip C. , Wu Joseph C.
Published: January 2008

Human embryonic stem (hES) cells are pluripotent stem cells capable of self-renewal and differentiation into virtually all cell types. Thus, they hold tremendous potential as cell sources for regenerative therapies. The concurrent development of accurate, sensitive, and noninvasive technologies capable of monitoring hES cells engraftment in vivo can greatly expedite basic research prior to future clinical translation. In this study, hES cells were stably transduced with a lentiviral vector carrying a novel double-fusion reporter gene that consists of firefly luciferase and enhanced green fluorescence protein. Reporter gene expression had no adverse effects on cell viability, proliferation, or differentiation to endothelial cells (human embryonic stem cell-derived endothelial cells [hESC-ECs]). To compare the two popular imaging modalities, hES cells and hESC-ECs were then colabeled with superparamagnetic iron oxide particles before transplantation into murine hind limbs. Longitudinal magnetic resonance (MR) imaging showed persistent MR signals in both cell populations that lasted up to 4 weeks. By contrast, bioluminescence imaging indicated divergent signal patterns for hES cells and hESC-ECs. In particular, hESC-ECs showed significant bioluminescence signals at day 2, which decreased progressively over the following 4 weeks, whereas bioluminescence signals from undifferentiated hES cells increased dramatically during the same period. Post-mortem histology and immunohistochemistry confirmed teratoma formation after injection of undifferentiated hES cells but not hESC-ECs. From these data taken together, we concluded that reporter gene is a better marker for monitoring cell viability, whereas iron particle labeling is a better marker for high-resolution detection of cell location by MR. Furthermore, transplantation of predifferentiated rather than undifferentiated hES cells would be more suited for avoiding teratoma formation.

Disclosure of potential conflicts of interest is found at the end of this article.
Loading tests on compacted soil, bottom-ash and lime layers
Authors: Consoli N. C. , Thomé A. , Donato M. , Graham J.
Published: January 2008

This paper addresses the interpretation of loading tests bearing on a layered system formed by a compacted soil, coal bottom-ash and carbide lime top-layer overlying a compressible residual soil stratum. Load–settlement behaviour is observed from tests carried out using circular steel plates ranging from 0.30 to 0.60 m diameter on the top of a 0.15–0.60 m thick artificially cemented layer. Field data demonstrate the effectiveness of compacted soil–ash–lime layers in increasing bearing capacity and reducing foundation settlements where shallow foundations are used on weak residual soils. The paper also stresses the need to express test results in terms of dimensionless variables in plots of normalised applied pressure against settlement-to-diameter ratio. The efficiency of existing analytical solutions for layered cohesive-frictional soils in determining the bearing capacity of footings on processed cemented soil overlying a weakly bonded residual soil with high void ratio is evaluated.
CFA pile behaviour in very stiff lodgement till
Authors: Farrell E. R. , Lawler M. L.
Published: January 2008

The design of CFA piles in the stiff/hard lodgement till that underlies most of Dublin is usually based on published experience from similar piles in other soils that lack the high density and stiffness of this till. The results of load tests on two instrumented test piles are presented, one 450 mm in diameter and 12·3 m long and the other 600 mm in diameter and 11 m long, which were loaded to 3·15 MN and 4·5 MN respectively. A 1 m long instrumented dummy pile was formed at the same time as the prototype and was tested in the laboratory to confirm the calibrations for use in interpreting the instrumentation. The instrumented piles showed that high average shaft resistances of 230 kPa and 270 kPa were mobilised, which were of similar magnitude to those recorded on an instrumented driven pile in this lodgement till that was reported previously. The pile load tests indicate an estimated Nc value of about 7 for the bearing capacity factor in ultimate end resistance, which is less than the value of 9 normally used and considerably less than a value of about 55 estimated from the tests on the driven pile. The results also indicate a change in the load–pile-head deflection curves with time, which could be related to disturbance of the ground caused by the construction process.
The Influence of Soil Fertility on Rhizosphere Effects in Northern Hardwood Forest Soils
Authors: Phillips Richard P. , Fahey Timothy J.
Published: January 2008

Enhanced levels of microbial biomass and activity in the rhizosphere arise from labile C released from roots but the factors that mediate such “rhizosphere effects” are poorly understood. We hypothesized that the magnitude of rhizosphere effects would be reduced by increases in soil fertility, consistent with plant C allocation theory, which predicts decreased C flux to roots in fertile soils. Replicate plots of sugar maple (Acer saccharumMarshall) and northern red oak (Quercus rubraL.) at the Turkey Hill Plantations, New York, and yellow birch (Betula allenghaniensisBritton) at the Hubbard Brook Experimental Forest, New Hampshire, were fertilized from 2001 to 2003. In fall of 2003, rhizosphere and bulk soils were sampled from the organic horizon of control and fertilized plots and analyzed for microbial biomass and nutrient transformation rates. In general, fertilization reduced microbial biomass and activity in plots of all three species, and the magnitude of such effects was generally greater in rhizosphere than in bulk soil. In red oak soils, fertilization reduced rhizosphere effects on microbial biomass, net N mineralization rates, and phosphatase enzyme activity (P= 0.026, 0.091, and 0.061, respectively). In contrast, fertilization only reduced rhizosphere effects on microbial biomass in sugar maple soils (P= 0.019). In yellow birch soils, fertilization decreased rhizosphere effects on microbial biomass (P= 0.015). These results suggest that soil fertility may mediate the degree to which roots affect microbial activity in forest soils, possibly due to fertilizer‐induced shifts in belowground C supply.
Contamination of spices with moulds potential producers of sterigmatocystine
Authors: Kocic-Tanackov Suncica , Dimic Gordana , Karalic Dragana
Published: January 2008

The presence of moulds, potential producers of sterigmatocystine, was investigated in 20 samples of spices (cinnamon, caraway, marjoram and clove). The following five species were isolated from the samples: Aspergillus versicolor, Aspergillus sydowii, Aspergillus ustus, Emericella nidulans and Eurotium herbariorum. E. herbariorum was present in all the investigated spice samples, accounting for 7.6% of total mycopopulation in caraway and 42.9% in cloves. A. versicolor was detected in cinnamon, caraway and marjoram, accounting for 0.4% of total mycopopulation in caraway and 1.5% in cinnamon; A. ustus accounted for 0.1% of total mycopopulation in caraway and 0.4% in marjoram, while A. sydowii accounted for 18.9% of total mycopopulation in marjoram and 7.1% in clove. E. nidulans was a contaminant of caraway (0.01%) and marjoram (0.4%). .
Kombucha fermentation on raw extracts of different cultivars of Jerusalem artichoke
Authors: Loncar Eva , Malbasa Radomir , Kolarov Ljiljana
Published: January 2008

Kombucha is a symbiosis between yeasts and acetic bacteria. It usually grows on sweetened black tea, but cultivation is possible on many other substrates. Jerusalem artichoke tubers extract is one of them. Tubers are suitable for the dietetic nutrition because of the low monosaccharide content and presence of some polyfructan ingredients which act as prebiotic. Five different cultivars of Jerusalem artichoke were used for the preparation of substrates for kombucha fermentation. The aim of this paper was the investigation of the influence of different Jerusalem artichoke cultivars on metabolic activity of kombucha. Composition of carbohydrates was followed using thin-layer chromatography and pH, reducing sugars content and yield of biomass were measured. Most of the samples with Jerusalem artichoke tubers extract contained fructose, probably small amount of glucose, fructo-oligosaccharides with different degree of polymerization and, inulin. Considering TLC chromatograms, Jerusalem artichoke cultivar did not affect significantly the composition of oligosaccharides in the fermentative liquid, as only minor differences were observed.
Influence of the heat treatment on the color of ground pepper (Capsicum annuum)
Authors: Vracar Ljubo , Tepic Aleksandra , Vujicic Biserka , Solaja Srdjan
Published: January 2008

Red pepper (Capsicum annuum L.) is one of the most important vegetables in the world. The main ground pepper quality attributes are extractable color, surface color, qualitative and quantitative carotenoid content. In this work, the influence of heat treatment on ground pepper quality was investigated. Microbiological status was examined in non-sterilized and sterilized ground pepper. Color changes were assessed by measuring the extractable color (ASTA) and surface color, using a photocolorimeter. The obtained results showed that at the end of experiment, non-sterilized samples had higher color values in comparison to the sterilized ones. Also, color deterioration was heightened at room temperature.