Clinical Evaluation of a Chairside Amalgam Separator to Meet Environmental Protection Agency Dental Wastewater Regulatory Compliance.

OBJECTIVES: The aim of this study was to evaluate the performance of a commercially available chairside amalgam separator (CAS) in a clinical setting in which a relatively high number of amalgam restorations are placed. Performance parameters investigated included service life, amalgam collected, mercury concentrations in effluent, and solids retention efficiency. METHODS AND MATERIALS: CASs were tested per International Organization of Standardization (ISO) 11143:2008 prior to installation in a military dental treatment facility and after removal from service (n=4) in order to confirm compliance with the recently enacted United States Environmental Protection Agency (EPA) Effluent Limitations Guidelines and Standards for the Dental Category. During the units' time in service, biweekly effluent grab samples were collected from the high-volume evacuation system of each chair (n=6) and analyzed for total mercury concentration by inductively coupled plasma mass spectrometry (ICP-MS). The mean total accumulated solids at the end of service life (n=6) was determined for potential design optimization. The service life expectancy in a military dental treatment facility was determined in terms of calendar and workdays. Procedural data were collected to determine the daily mean number of amalgam surfaces placed during the service life of each chairside amalgam separator (n=9). RESULTS: The CAS evaluated met minimum EPA compliance requirements when used in a military dental treatment facility. The solids removal efficiency at the end of service life was 99.82% ± 0.14% (n=4). The mean service life (n=8) was 131.6 ± 45.1 calendar days (67.1±37.6 workdays). Effluent mercury concentrations ranged from 0.05 to 11.93 mg/L. Total solids accumulated in each CAS (n=6) at the end of service life was 195.4 ± 63.4 g. The mean number of amalgam surfaces placed per workday during the service life span of each CAS was 8.4 ± 1.4.

Effect of Tooth-structure Thickness on Light Attenuation and Depth of Cure.

Newer bulk-fill composites claim a greater depth of cure than conventional resin-based composites. To facilitate complete curing, the manufacturer of SonicFill (Kerr) recommends curing from the occlusal, as well as the buccal and lingual, surfaces of the tooth. The purpose of this study was to quantify the degree of curing light attenuation as it passes through natural tooth structure, and how this attenuation affects the depth of cure of different posterior resin composites. Ten noncarious extracted mandibular third molars were sectioned to produce 5-mm-thick pieces of buccal tooth structure. Sanding 0.5-mm increments from the flattened surface produced 4.5-, 4.0-, 3.5-, 3.0-, 2.5-, 2.0-, and finally 1.5-mm-thick sections. A Bluephase G2 (Ivoclar) curing light with an 8-mm-diameter light guide set on high for 20 seconds was used for measurement of irradiance as it passed through different thicknesses of tooth structure and air. The average irradiance (mW/cm(2)) was measured with a MARC-RC Resin Calibrator with a 4-mm-diameter sensor (BlueLight Analytics). To measure depth of cure of a conventional hybrid composite (Herculite Ultra; Kerr) vs a bulk-fill hybrid composite (SonicFill) through varying thicknesses of tooth structure, composites were cured in a 4-mm-diameter × 10.25-mm-long split mold according to International Organization for Standardization 4049. A mean and standard deviation was determined per group. Data were analyzed with a one-way analysis of variance (ANOVA)/Tukey test and two-way ANOVA/Tukey test (α=0.05). One-way ANOVA/Tukey found a significant decrease in irradiance based on thickness of tooth structure or distance through air (p<0.001). Two-way ANOVA/Tukey found a significant decrease in depth of cure based on thickness of tooth structure (p<0.001) and on composite type (p<0.001) with no significant interaction (p=0.623). SonicFill had a significantly greater depth of cure than Herculite Ultra.

A serum factor that recalcifies demineralized bone is conserved in bony fish and sharks but is not found in invertebrates.

We investigated the evolutionary origin of a serum activity that induces calcification within a type I collagen matrix, an activity previously described in rat and bovine serum. Serum was obtained from vertebrates with calcified tissues (bony fish and shark), vertebrates without calcified tissues (lamprey and hagfish), and three invertebrates (marine worm, crab, and sea urchin). Serum from the bony fish and shark proved to contain a potent nucleator of collagen calcification; like the previously described calcifying activity in rat serum, the fish and shark activities are both able to recalcify a demineralized rat tibia when tested in Dulbecco's modified Eagle medium containing as little as 1.5% of the respective serum and have an apparent molecular weight of 50-150 kDa. No calcifying activity could be detected in any of several experimental tests of invertebrate or hagfish serum. Weak calcifying activity could be detected in lamprey serum, but calcification was restricted to the growth plate of the decalcified tibia, with no detectable calcification in the type I collagen of the midshaft. These studies reveal a correlation between the evolutionary timing of the appearance of calcified tissues in vertebrates and the appearance of the serum activity that initiates calcification within collagen and, therefore, support the hypothesis that this serum activity may play a role in normal calcification of bone.

The genome of the social amoeba Dictyostelium discoideum.

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.

Mineralization of decalcified bone occurs under cell culture conditions and requires bovine serum but not cells.

The purpose of this study was to develop an in vitro model system for bone matrix mineralization in the absence of cells. For this model, we utilized EDTA-decalcified new-born rat tibias with the cartilaginous ends intact, allowing us to visually determine the specificity of mineralization within the bone. Our results show that supplementation of DMEM culture medium with 10mM beta-glycerophosphate and 15% fetal bovine serum (FBS) results in non-physiological mineral percipitation in the tibia because of the generation of supraphysiological (5mM) levels of inorganic phosphate in the medium. The same medium supplemented only with inorganic phosphate to a final concentration of 2mM failed to mineralize a decalcified tibia matrix. However, additional supplementation of this medium with as little as 5% FBS resulted in mineralization of those regions of the type I collagen where mineral was found prior to decalcification, with no evidence for mineralization in the cartilage at the bone ends or in the periosteum. Analysis of the mineral by Fourier-transform infrared spectroscopy and powder X-ray diffraction shows that tibias that have been decalcified and then remineralized contain an apatitic mineral that is strikingly similar to the mineral in normal bone. Tendon, a type I collagen matrix not normally mineralized in vivo, also mineralizes when incubated in DMEM containing 2mM Pi and as little as 1.5% FBS, but not when incubated in DMEM without serum. These data indicate that serum contains a nucleator of type I collagen matrix mineralization, and that mineralization of type I collagen under cell culture conditions requires serum but not living cells.

The complete genome sequence and analysis of Corynebacterium diphtheriae NCTC13129.

Corynebacterium diphtheriae is a Gram-positive, non-spore forming, non-motile, pleomorphic rod belonging to the genus Corynebacterium and the actinomycete group of organisms. The organism produces a potent bacteriophage-encoded protein exotoxin, diphtheria toxin (DT), which causes the symptoms of diphtheria. This potentially fatal infectious disease is controlled in many developed countries by an effective immunisation programme. However, the disease has made a dramatic return in recent years, in particular within the Eastern European region. The largest, and still on-going, outbreak since the advent of mass immunisation started within Russia and the newly independent states of the former Soviet Union in the 1990s. We have sequenced the genome of a UK clinical isolate (biotype gravis strain NCTC13129), representative of the clone responsible for this outbreak. The genome consists of a single circular chromosome of 2 488 635 bp, with no plasmids. It provides evidence that recent acquisition of pathogenicity factors goes beyond the toxin itself, and includes iron-uptake systems, adhesins and fimbrial proteins. This is in contrast to Corynebacterium's nearest sequenced pathogenic relative, Mycobacterium tuberculosis, where there is little evidence of recent horizontal DNA acquisition. The genome itself shows an unusually extreme large-scale compositional bias, being noticeably higher in G+C near the origin than at the terminus.

Sequence of Plasmodium falciparum chromosomes 1, 3-9 and 13.

Since the sequencing of the first two chromosomes of the malaria parasite, Plasmodium falciparum, there has been a concerted effort to sequence and assemble the entire genome of this organism. Here we report the sequence of chromosomes 1, 3-9 and 13 of P. falciparum clone 3D7--these chromosomes account for approximately 55% of the total genome. We describe the methods used to map, sequence and annotate these chromosomes. By comparing our assemblies with the optical map, we indicate the completeness of the resulting sequence. During annotation, we assign Gene Ontology terms to the predicted gene products, and observe clustering of some malaria-specific terms to specific chromosomes. We identify a highly conserved sequence element found in the intergenic region of internal var genes that is not associated with their telomeric counterparts.

The genome sequence of Schizosaccharomyces pombe.

We have sequenced and annotated the genome of fission yeast (Schizosaccharomyces pombe), which contains the smallest number of protein-coding genes yet recorded for a eukaryote: 4,824. The centromeres are between 35 and 110 kilobases (kb) and contain related repeats including a highly conserved 1.8-kb element. Regions upstream of genes are longer than in budding yeast (Saccharomyces cerevisiae), possibly reflecting more-extended control regions. Some 43% of the genes contain introns, of which there are 4,730. Fifty genes have significant similarity with human disease genes; half of these are cancer related. We identify highly conserved genes important for eukaryotic cell organization including those required for the cytoskeleton, compartmentation, cell-cycle control, proteolysis, protein phosphorylation and RNA splicing. These genes may have originated with the appearance of eukaryotic life. Few similarly conserved genes that are important for multicellular organization were identified, suggesting that the transition from prokaryotes to eukaryotes required more new genes than did the transition from unicellular to multicellular organization.

Genome sequence of Yersinia pestis, the causative agent of plague.

The Gram-negative bacterium Yersinia pestis is the causative agent of the systemic invasive infectious disease classically referred to as plague, and has been responsible for three human pandemics: the Justinian plague (sixth to eighth centuries), the Black Death (fourteenth to nineteenth centuries) and modern plague (nineteenth century to the present day). The recent identification of strains resistant to multiple drugs and the potential use of Y. pestis as an agent of biological warfare mean that plague still poses a threat to human health. Here we report the complete genome sequence of Y. pestis strain CO92, consisting of a 4.65-megabase (Mb) chromosome and three plasmids of 96.2 kilobases (kb), 70.3 kb and 9.6 kb. The genome is unusually rich in insertion sequences and displays anomalies in GC base-composition bias, indicating frequent intragenomic recombination. Many genes seem to have been acquired from other bacteria and viruses (including adhesins, secretion systems and insecticidal toxins). The genome contains around 150 pseudogenes, many of which are remnants of a redundant enteropathogenic lifestyle. The evidence of ongoing genome fluidity, expansion and decay suggests Y. pestis is a pathogen that has undergone large-scale genetic flux and provides a unique insight into the ways in which new and highly virulent pathogens evolve.

Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18.

Salmonella enterica serovar Typhi (S. typhi) is the aetiological agent of typhoid fever, a serious invasive bacterial disease of humans with an annual global burden of approximately 16 million cases, leading to 600,000 fatalities. Many S. enterica serovars actively invade the mucosal surface of the intestine but are normally contained in healthy individuals by the local immune defence mechanisms. However, S. typhi has evolved the ability to spread to the deeper tissues of humans, including liver, spleen and bone marrow. Here we have sequenced the 4,809,037-base pair (bp) genome of a S. typhi (CT18) that is resistant to multiple drugs, revealing the presence of hundreds of insertions and deletions compared with the Escherichia coli genome, ranging in size from single genes to large islands. Notably, the genome sequence identifies over two hundred pseudogenes, several corresponding to genes that are known to contribute to virulence in Salmonella typhimurium. This genetic degradation may contribute to the human-restricted host range for S. typhi. CT18 harbours a 218,150-bp multiple-drug-resistance incH1 plasmid (pHCM1), and a 106,516-bp cryptic plasmid (pHCM2), which shows recent common ancestry with a virulence plasmid of Yersinia pestis.

Salmonella enterica serovar Typhi possesses a unique repertoire of fimbrial gene sequences.

Salmonella enterica serotype Typhi differs from nontyphoidal Salmonella serotypes by its strict host adaptation to humans and higher primates. Since fimbriae have been implicated in host adaptation, we investigated whether the serotype Typhi genome contains fimbrial operons which are unique to this pathogen or restricted to typhoidal Salmonella serotypes. This study established for the first time the total number of fimbrial operons present in an individual Salmonella serotype. The serotype Typhi CT18 genome, which has been sequenced by the Typhi Sequencing Group at the Sanger Centre, contained a type IV fimbrial operon, an orthologue of the agf operon, and 12 putative fimbrial operons of the chaperone-usher assembly class. In addition to sef, fim, saf, and tcf, which had been described previously in serotype Typhi, we identified eight new putative chaperone-usher-dependent fimbrial operons, which were termed bcf, sta, stb, ste, std, stc, stg, and sth. Hybridization analysis performed with 16 strains of Salmonella reference collection C and 22 strains of Salmonella reference collection B showed that all eight putative fimbrial operons of serotype Typhi were also present in a number of nontyphoidal Salmonella serotypes. Thus, a simple correlation between host range and the presence of a single fimbrial operon seems at present unlikely. However, the serotype Typhi genome differed from that of all other Salmonella serotypes investigated in that it contained a unique combination of putative fimbrial operons.

Massive gene decay in the leprosy bacillus.

Leprosy, a chronic human neurological disease, results from infection with the obligate intracellular pathogen Mycobacterium leprae, a close relative of the tubercle bacillus. Mycobacterium leprae has the longest doubling time of all known bacteria and has thwarted every effort at culture in the laboratory. Comparing the 3.27-megabase (Mb) genome sequence of an armadillo-derived Indian isolate of the leprosy bacillus with that of Mycobacterium tuberculosis (4.41 Mb) provides clear explanations for these properties and reveals an extreme case of reductive evolution. Less than half of the genome contains functional genes but pseudogenes, with intact counterparts in M. tuberculosis, abound. Genome downsizing and the current mosaic arrangement appear to have resulted from extensive recombination events between dispersed repetitive sequences. Gene deletion and decay have eliminated many important metabolic activities including siderophore production, part of the oxidative and most of the microaerophilic and anaerobic respiratory chains, and numerous catabolic systems and their regulatory circuits.

Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491.

Neisseria meningitidis causes bacterial meningitis and is therefore responsible for considerable morbidity and mortality in both the developed and the developing world. Meningococci are opportunistic pathogens that colonize the nasopharynges and oropharynges of asymptomatic carriers. For reasons that are still mostly unknown, they occasionally gain access to the blood, and subsequently to the cerebrospinal fluid, to cause septicaemia and meningitis. N. meningitidis strains are divided into a number of serogroups on the basis of the immunochemistry of their capsular polysaccharides; serogroup A strains are responsible for major epidemics and pandemics of meningococcal disease, and therefore most of the morbidity and mortality associated with this disease. Here we have determined the complete genome sequence of a serogroup A strain of Neisseria meningitidis, Z2491. The sequence is 2,184,406 base pairs in length, with an overall G+C content of 51.8%, and contains 2,121 predicted coding sequences. The most notable feature of the genome is the presence of many hundreds of repetitive elements, ranging from short repeats, positioned either singly or in large multiple arrays, to insertion sequences and gene duplications of one kilobase or more. Many of these repeats appear to be involved in genome fluidity and antigenic variation in this important human pathogen.

The complete nucleotide sequence of chromosome 3 of Plasmodium falciparum.

Analysis of Plasmodium falciparum chromosome 3, and comparison with chromosome 2, highlights novel features of chromosome organization and gene structure. The sub-telomeric regions of chromosome 3 show a conserved order of features, including repetitive DNA sequences, members of multigene families involved in pathogenesis and antigenic variation, a number of conserved pseudogenes, and several genes of unknown function. A putative centromere has been identified that has a core region of about 2 kilobases with an extremely high (adenine + thymidine) composition and arrays of tandem repeats. We have predicted 215 protein-coding genes and two transfer RNA genes in the 1,060,106-base-pair chromosome sequence. The predicted protein-coding genes can be divided into three main classes: 52.6% are not spliced, 45.1% have a large exon with short additional 5' or 3' exons, and 2.3% have a multiple exon structure more typical of higher eukaryotes.

Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence.

Countless millions of people have died from tuberculosis, a chronic infectious disease caused by the tubercle bacillus. The complete genome sequence of the best-characterized strain of Mycobacterium tuberculosis, H37Rv, has been determined and analysed in order to improve our understanding of the biology of this slow-growing pathogen and to help the conception of new prophylactic and therapeutic interventions. The genome comprises 4,411,529 base pairs, contains around 4,000 genes, and has a very high guanine + cytosine content that is reflected in the biased amino-acid content of the proteins. M. tuberculosis differs radically from other bacteria in that a very large portion of its coding capacity is devoted to the production of enzymes involved in lipogenesis and lipolysis, and to two new families of glycine-rich proteins with a repetitive structure that may represent a source of antigenic variation.

The nucleotide sequence of Saccharomyces cerevisiae chromosome IX.

Large-scale systematic sequencing has generally depended on the availability of an ordered library of large-insert bacterial or viral genomic clones for the organism under study. The generation of these large insert libraries, and the location of each clone on a genome map, is a laborious and time-consuming process. In an effort to overcome these problems, several groups have successfully demonstrated the viability of the whole-genome random 'shotgun' method in large-scale sequencing of both viruses and prokaryotes. Here we report the sequence of Saccharomyces cerevisiae chromosome IX, determined in part by a whole-chromosome 'shotgun', and describe the particular difficulties encountered in the random 'shotgun' sequencing of an entire eukaryotic chromosome. Analysis of this sequence shows that chromosome IX contains 221 open reading frames (ORFs), of which approximately 30% have been sequenced previously. This chromosome shows features typical of a small Saccharomyces cerevisiae chromosome.

The nucleotide sequence of Saccharomyces cerevisiae chromosome XIII.

Systematic sequencing of the genome of Saccharomyces cerevisiae has revealed thousands of new predicted genes and allowed analysis of long-range features of chromosomal organization. Generally, genes and predicted genes seem to be distributed evenly throughout the genome, having no overall preference for DNA strand. Apart from the smaller chromosomes, which can have substantially lower gene density in their telomeric regions, there is a consistent average of one open reading frame (ORF) approximately every two kilobases. However, one of the most surprising findings for a eukaryote with approximately 6,000 genes was the amount of apparent redundancy in its genome. This redundancy occurs both between individual ORFs and over more extensive chromosome regions, which have been duplicated preserving gene order and orientation. Here we report the entire nucleotide sequence of chromosome XIII, the sixth-largest S. cerevisiae chromosome, and demonstrate that its features and organization are consistent with those observed for other S. cerevisiae chromosomes. Analysis revealed 459 ORFs, 284 have not been identified previously. Both intra- and interchromosomal duplications of regions of this chromosome have occurred.

Observations on autoregulation in skeletal muscle: the effects of arterial hypoxia.

Autoregulation of blood flow in skeletal muscle, as manifested by steady-state resistance changes, has been shown to be present in the low range of perfusion pressure but has been demonstrated by some observers to be lacking at higher perfusion pressures. Transient responses have often been neglected or observed only qualitatively in analyses of autoregulation. The present study was undertaken (1) to determine the relative importance of steady-state and transient responses in flow in demonstrating autoregulation of blood flow over a broad range of perfusion pressures, (2) to establish a means of quantitating autoregulation, and (3) to observe the effect of hypoxia on autoregulation. In isolated, perfused canine gracilis muscle, perfusion pressure was increased and subsequently returned to baseline (9.7 +/- 0.13 kPa [73 +/- 1 mmHg]) during perfusion with normally oxygenated blood (PO2 = 9.3-13.3 kPa [70-100 mmHg]), and mildly (PO2 = 6.1-9.2 kPa [46-69 mmHg]), moderately (PO2 = 4.5-6.0 kPa [34-45 mmHg]), or severely (PO2 = 2.7-4.4kPa [20-33 mmHg]) hypoxic blood. Consistent with other studies canine gracilis muscle was often found to possess passive vascular responses when only steady-state parameters were considered. However, quantitation of the transient response in flow with step increases in perfusion pressure demonstrated substantial transient responses under conditions of normal oxygenation, and progressive attenuation of flow transients with increasing hypoxia.