Substantivity of zinc salts used as rinsing solutions and their effect on the inhibition of Streptococcus mutans

The antimicrobial efficacy of zinc (Zn) salts (sulfate and acetate) against Streptococcus mutans (S. mutans) present in the oral cavity was tested in this study. The substantivity of Zn salts was assessed by determining the concentration of Zn in whole, unstimulated saliva and by measuring the magnitude of suppression of salivary S. mutans, 2h after rinsing. The concentration of Zn was measured by atomic absorption spectrometry (AAS) with electrothermal atomization (ET AAS) in saliva sampled before (basal) and 24h after mouth rinsing with different concentrations of Zn (0.1%, 0.5% and 1%) administrated as sulfate and acetate. The estimation of Zn levels in samples collected 30, 60, 90 and 120 min after rinsing was carried out by AAS with flame atomization (FAAS). Immediately after rinsing, the concentration of Zn in saliva sharply increased with respect to the baseline values (0.055+/-0.017 mg/L), followed by a sustained decrease, probably due to clearance of salivary flow or swallowing during sampling. A significant reduction (>87%) in the total mean S. mutans counts was found 2h after rinsing either with sulfate or acetate solutions, as evidence of the high substantivity and effectiveness of the Zn salts tested. A statistically significant inverse relationship (p<0.001 and the Pearson correlation coefficients between -34% and -50%) was found between Zn levels and the respective pH values measured in the samples collected 60 and 120 min after rinsing, sustaining the theory of bacterial glycolysis inhibition.     

Antibiotic resistance and plasmid pattern of enterotoxigenic ST-a strains of Escherichia coli isolated in Puebla, México

Antibiotic susceptibilities of 22 strains of Escherichia coli isolated from children from 0 to 3 years old at the University Hospital of Puebla were determined. Almost all strains were resistant to ampicillin, tetracycline, streptomycin, and kanamycin. Gel electrophoresis of DNA from 10 clinical strains of E. coli revealed a heterogeneous plasmid population. Plasmid DNA, ranging in molecular mass from 1.8 to 120 megadaltons, was demonstrated in 10 strains. Moreover, the frequency of antibiotic transfer ranged from 1.6/10(8) to 2/10, and the simultaneous transfer of the gene encoding heat-stable enterotoxin was also determined. Six out of 10 strains tested were able to cotransfer ST-a as demonstrated by the suckling mouse test. It is possible that antibiotic selective pressure may increase the isolation of enterotoxigenic E. coli strains.     

Structural engineering of the HIV-1 protease molecule with a beta-turn mimic of fixed geometry.

An important goal in the de novo design of enzymes is the control of molecular geometry. To this end, an analog of the protease from human immunodeficiency virus 1 (HIV-1 protease) was prepared by total chemical synthesis, containing a constrained, nonpeptidic type II' beta-turn mimic of predetermined three-dimensional structure. The mimic beta-turn replaced residues Gly16,17 in each subunit of the homodimeric molecule. These residues constitute the central amino acids of two symmetry-related type I' beta-turns in the native, unliganded enzyme. The beta-turn mimic-containing enzyme analog was fully active, possessed the same substrate specificity as the Gly16,17-containing enzyme, and showed enhanced resistance to thermal inactivation. These results indicate that the precise geometry of the beta-turn at residues 15-18 in each subunit is not critical for activity, and that replacement of the native sequence with a rigid beta-turn mimic can lead to enhanced protein stability. Finally, the successful incorporation of a fixed element of secondary structure illustrates the potential of a "molecular kit set" approach to protein design and synthesis.     

Billingen (Lower Arenig/Lower Ordovician) acritarchs from the East European Platform and their palaeobiogeographic significance

Billingen (Lower Arenig/Lower Ordovician) sediments of the St. Petersburg region, northwest Russia and the Leba area, northern Poland of the East European Craton yield acritarch assemblages, which are largely homogenous though displaying minor compositional differences that probably reflect a gradient from inner to outer shelf environments. Comparison with coeval acritarch microflora from the Yangtze Platform, South China, shows an overall similarity between Baltoscandian and South Chinese phytoplankton. The widespread uniformity in the fossil microphytoplankton may be related to the extensive global 'evae' sea-level transgression, which characterized the Billingen time. This suggests that during the Tremadoc through early Arenig times, acritarch assemblages displayed essentially an undifferentiated cold-water and oceanic character along the whole margin of Perigondwana in the South, as well as on the South Chinese and Baltic platforms, at middle latitudes (Mediterranean oceanic Realm). Despite this overall similarity, however, some typical taxa of the high-latitude Mediterranean Province (Arbusculidium, Coryphidium and Striatotheca) occur in South China, but are absent in Baltica. This discrepancy is explained as caused by differences in climatic and physiographic conditions that prevailed at the two palaeocontinents at this time. The inferred pattern of oceanic circulation during the Lower Ordovician is consistent with the palynological evidence of a prevailing warmer climate in Baltica than in South China, although the two palaeocontinents occupied the same palaeolatitudinal position.     

Do the effects of piscivorous largemouth bass cascade to the plankton?

Ecologists have hypothesized that an increase in the biomass of piscivorous fish in lakes will cause a decrease in populations of planktivorous fish, an increase in the size of herbivorous zooplankton and a decrease in the biomass of phytoplankton. Here we present an experimental test of whether the effects of largemouth bass (Micropterus salmoides) cascade to the planktivorous fish, zooplankton and phytoplankton of a 15-ha water storage reservoir. A pilot study indicated that the reservoir was eutrophic with dense populations of planktivorous fish dominated by threadfin shad (Dorosoma petenense). No piscovorous fish were present in the reservoir. We conducted a one-month mesocosm experiment using water and plankton from the reservoir showing that the presence of threadfin shad reduced large-sized zooplankton and increased the productivity and biomass of phytoplankton. To test whether the effects of piscivorous fish could cascade to the plankton, we assessed the effects of the addition of piscivorous largemouth bass on the planktivorous fish, zooplankton and biomass of phytoplankton of the reservoir by monitoring the reservoir during the year before and the two years after largemouth bass were stocked. In the second year after the addition of largemouth bass, the number of planktivorous fish decreased and the relative abundance of threadfin shad declined. Although the abundance of cladocerans increased after the addition of largemouth bass, the average size of zooplankton did not change. We did not detect changes in chlorophyll a, Secchi depth, or concentrations of total phosphorus and total nitrogen as a result of the addition of largemouth bass.     

Comparison of ribosomes from Coxiella burnetii and Escherichia coli by gel electrophoresis, protein synthesis, and immunological techniques.

Ribosomes and postribiosomal supernatant fluid (S-100) were isolated from Coxiella burnetii. The ribosomes functioned in polyuridylic acid-directed polyphenylalanine synthesis in the presence of S-100 from either C. burnetii or Escherichia coli. C. burnetii S-100 promoted translation with E. coli ribosomes. Antisera against E. coli elongation factor G and ribosomal proteins L7/L12 cross-reacted with rickettsial S-100 and ribosomes, respectively. Ribosomal proteins were analyzed by two-dimensional gel electrophoresis.     

A family of leukemia inhibitory factor-binding peptides that can act as antagonists when conjugated to poly(ethylene glycol)

A panel of six na?ve 14-residue random peptide libraries displayed polyvalently on M13 phage was pooled and sorted against human leukemia inhibitory factor (LIF). After four rounds of selection, a single large family of peptides with the consensus sequence XCXXXXG(A/S)(D/E)(W/F)WXCF was found to bind specifically to LIF. Peptides within this family did not bind related members of the interleukin-6 family of cytokines, nor to murine LIF that has 80% sequence identity with human LIF. A representative peptide from this family was synthesized and found to bind to LIF with an affinity of approximately 300 nM. The phage-displayed form of this peptide was able to compete with the LIF receptor alpha chain (LIFR) for binding to LIF; however, the free synthetic peptide was unable to inhibit LIF-LIFR binding or inhibit LIF bioactivity in vitro. Using a panel of human/murine chimeric LIF molecules, the peptide-binding site on LIF was mapped to a groove located between the B and the C helices of the LIF structure, which is distinct from the surfaces involved in binding to receptor. To mimic the effect of the phage particle and convert the free peptide into an antagonist of LIFR binding, a 40 kDa poly(ethylene glycol) (PEG) moiety was conjugated to the synthetic LIF-binding peptide. This PEG-peptide conjugate was found to be both an antagonist of LIF-LIFR binding and of LIF signaling in engineered Ba/F3 cells expressing LIFR and the gp130 coreceptor.     

A fusion protein system for the recombinant production of short disulfide-containing peptides

A recombinant fusion protein system for the production, oxidation, and purification of short peptides containing a single disulfide bond is described. The peptides are initially expressed in Escherichia coli as a fusion to an engineered mutant of the N-terminal SH2 domain of the intracellular phosphatase, SHP-2. This small protein domain confers several important properties which facilitate the production of disulfide-containing peptides: (i) it is expressed at high levels in E. coli; (ii) it can be purified via a hexahistidine tag and reverse-phase HPLC; (iii) it contains no endogenous cysteine residues, allowing the formation of an intrapeptide disulfide bond while still attached to the fusion partner; (iv) it is highly soluble in native buffers, facilitating the production of very hydrophobic peptides and the direct use of fusion products in biochemical assays; (v) it contains a unique methionine residue at the junction of the peptide and fusion partner to facilitate peptide cleavage by treatment with cyanogen bromide (CNBr). This method is useful for producing peptides, which are otherwise difficult to prepare through traditional chemical synthesis approaches, and this has been demonstrated by preparing a number of hydrophobic disulfide-containing peptides derived from phage-display libraries.     

Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative,Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA

Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital Sequencing^TM is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient’s cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing.