Evaluation of DNA extraction methods for Bacillus anthracis spores isolated from spiked food samples

Aims

Nine commercial DNA extraction kits were evaluated for the isolation of DNA from 10‐fold serial dilutions of Bacillus anthracis spores using quantitative real‐time PCR (qPCR). The three kits determined by qPCR to yield the most sensitive and consistent detection (Epicenter MasterPure Gram Positive; MoBio PowerFood; ABI PrepSeq) were subsequently tested for their ability to isolate DNA from trace amounts of B. anthracis spores (approx. 6·5 × 10¹ and 1·3 × 10² CFU in 25 ml or 50 g of food sample) spiked into complex food samples including apple juice, ham, whole milk and bagged salad and recovered with immunomagnetic separation (IMS).

Methods and Results

The MasterPure kit effectively and consistently isolated DNA from low amounts of B. anthracis spores captured from food samples. Detection was achieved from apple juice, ham, whole milk and bagged salad from as few as 65 ± 14, 68 ± 8, 66 ± 4 and 52 ± 16 CFU, respectively, and IMS samples were demonstrated to be free of PCR inhibitors.

Conclusions

Detection of B. anthracis spores isolated from food by IMS differs substantially between commercial DNA extraction kits; however, sensitive results can be obtained with the MasterPure Gram Positive kit.

Significance and Impact of the Study

The extraction protocol identified herein combined with IMS is novel for B. anthracis and allows detection of low levels of B. anthracis spores from contaminated food samples.     

Loop‐mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidian Enterocytozoon hepatopenaei in penaeid shrimp

Aims

Enterocytozoon hepatopenaei is an emerging microsporidian parasite that has been linked to recent losses caused by white faeces syndrome (WFS) in cultivated giant or black tiger shrimp Penaeus (Penaeus) monodon and whiteleg shrimp Penaeus (Litopenaeus) vannamei in Asia. To more accurately assess its impact on shrimp production and to determine reservoir carriers for control measures, our objective was to establish a loop‐mediated isothermal amplification (LAMP) assay combined with colorimetric nanogold (AuNP) for rapid, sensitive and inexpensive detection of this parasite.

Methods and Results

A set of six specific primers was designed to successfully detect the SSU rRNA gene of E. hepatopenaei by a LAMP reaction of 45 min at 65°C combined with visual detection of the amplification product via hybridization at 65°C for 5 min with a ssDNA‐labelled nanogold probe, followed by salt‐induced AuNP aggregation (total assay time, approximately 50 min). This method gave similar results to LAMP followed by electrophoresis or spectrophotometric detection, and it was more sensitive (0·02 fg total DNA) than a conventional nested PCR (0·2 fg total DNA). The new method gave negative results with shrimp DNA templates extracted from diseased shrimp containing other pathogens, indicating that the LAMP‐AuNP assay was specific for E. hepatopenaei.

Conclusions

Without sacrificing sensitivity or specificity, the new LAMP‐AuNP assay significantly reduced the time, ease and cost for molecular detection of E. hepatopenaei in shrimp.

Significance and Impact of the study

The new method employs simple, inexpensive equipment and involves simple steps making it applicable for small field laboratories. Wider application of the method to screen broodstock before use in a hatchery, to screen postlarvae before stocking shrimp ponds, to test for natural carriers and to monitor shrimp in rearing ponds would help to assess and reduce the negative impact of this parasite in shrimp farming.     

Encapsulated boron as an osteoinductive agent for bone scaffolds

The aim of this study was to develop boron (B)-releasing polymeric scaffold to promote regeneration of bone tissue. Boric acid-doped chitosan nanoparticles with a diameter of approx. 175 nm were produced by tripolyphosphate (TPP)-initiated ionic gelation process. The nanoparticles strongly attached via electrostatic interactions into chitosan scaffolds produced by freeze-drying with approx. 100 μm pore diameter. According to the ICP-OES results, following first 5 h initial burst release, fast release of B from scaffolds was observed for 24 h incubation period in conditioned medium. Then, slow release of B was performed over 120 h. The results of the cell culture studies proved that the encapsulated boron within the scaffolds can be used as an osteoinductive agent by showing its positive effects on the proliferation and differentiation of MC3T3-E1 preosteoblastic cells.     

Does a preceding hand wash and drying time after surgical hand disinfection influence the efficacy of a propanol-based hand rub?

Background  

Recently, a propanol-based hand rub has been described to exceed the efficacy requirements of the European standard EN 12791 in only 1.5 min significantly. But the effect of a 1 min preceding hand wash and the effect of one additional minute for evaporation of the alcohol after its application on the efficacy after a 1.5 min application time has never been studied.     

Catesbeianin-1, a novel antimicrobial peptide isolated from the skin of <Emphasis Type="Italic">Lithobates catesbeianus</Emphasis> (American bullfrog)

Objectives

To identify and characterize a novel antimicrobial peptide, catesbeianin-1.

Results

Catesbeianin-1 is 25 amino acids long and is α-helical, cationic and amphipathic. It had antimicrobial activity against Gram-positive and Gram-negative bacteria. It was resistant against trypsin and pepsin. Catesbeianin-1 exhibited moderate hemolytic activity (approx 8%) at 100 μg/ml, and its HC₅₀ (50% hemolytic concentration) was 300 μg/ml. Its cytotoxicity was approx 10–20% at 100 μg/ml, and its CC₅₀ (50% cytotoxic concentration) was >100 μg/ml. The LD₅₀ of catesbeianin-1 in mice was 80 mg/kg. At 3.1 µg/ml, catesbeianin-1 significantly inhibited the growth of methicillin-resistant Staphylococcus aureus.

Conclusions

A new antimicrobial peptide from the skin of Lithobates catesbeianus (American bullfrog) may represent a template for the development of novel antimicrobial agents.

     

EGFR inhibitor C225 increases the radiosensitivity of human lung squamous cancer cells

Background  

The purpose of the present study is to investigate the direct biological effects of the epidermal growth factor receptor (EGFR) inhibitor C225 on the radiosensitivity of human lung squamous cancer cell-H520. H520 cells were treated with different dosage of ⁶⁰Co γ ray irradiation (1.953 Gy/min) in the presence or absence of C225. The cellular proliferation, colony forming capacity, apoptosis, the cell cycle distribution as well as caspase-3 were analyzed in vitro.     

Analysis of the lambdoid prophage element e14 in the E. coli K-12 genome

Background  

Many sequenced bacterial genomes harbor phage-like elements or cryptic prophages. These elements have been implicated in pathogenesis, serotype conversion and phage immunity. The e14 element is a defective lambdoid prophage element present at 25 min in the E. coli K-12 genome. This prophage encodes important functional genes such as lit (T4 exclusion), mcrA (modified cytosine restriction activity) and pin (recombinase).     

Endurance training of respiratory muscles improves cycling performance in fit young cyclists

Background  

Whether or not isolated endurance training of the respiratory muscles improves whole-body endurance exercise performance is controversial, with some studies reporting enhancements of 50 % or more, and others reporting no change. Twenty fit (VO₂ max 56.0 ml/kg/min), experienced cyclists were randomly assigned to three groups. The experimental group (n = 10) trained their respiratory muscles via 20, 45 min sessions of hyperpnea. The placebo group (n = 4) underwent "sham" training (20, 5 min sessions), and the control group (n = 6) did no training.     

Metabolites produced by probiotic Lactobacilli rapidly increase glucose uptake by Caco-2 cells

Background  

Although probiotic bacteria and their metabolites alter enterocyte gene expression, rapid, non-genomic responses have not been examined. The present study measured accumulation of tracer (2 μM) glucose by Caco-2 cells after exposure for 10 min or less to a chemically defined medium (CDM) with different monosaccharides before and after anaerobic culture of probiotic Lactobacilli.     

Toxicity assessment of common organic solvents using a biosensor based on algal photosynthetic activity measurement

The acute toxicities of common organic solvents (e.g., methanol, ethanol, isopropanol, acetone, acetonitrile, and dimethylformamide) were evaluated using a biosensor based on microalgal photosynthesis measurement. The biosensor was air-tight, with no headspace, preventing volatile organic toxicants from escaping into the environment as well as partitioning from the aqueous phase into the headspace until equilibrium was reached. Both the incubating and exposure times were set at 10 min. It was observed that only 2 h was needed to obtain complete dose-related inhibition of photosynthetic activity. The results showed that all the tested organic solvents inhibited algal photosynthesis with EC₅₀ ranging between 589 and 2,570 mM. The inhibition of these solvents was in the order: isopropanol > acetone > acetonitrile > ethanol > dimethylformamide > methanol. The quantitative structure-activity relationship (QSAR) between toxicity data and partition coefficient of the examined compounds could be modeled as follows: ${\text{log}}_{{10}} {\text{EC}}_{{50}} \;{\left( {\mu {\text{M}}} \right)} = - 0.6428\;{\text{log}}\;P + 5.76\;{\left( {{\text{R}}^{2} \approx 0.88} \right)}The acute toxicities of common organic solvents (e.g., methanol, ethanol, isopropanol, acetone, acetonitrile, and dimethylformamide) were evaluated using a biosensor based on microalgal photosynthesis measurement. The biosensor was air-tight, with no headspace, preventing volatile organic toxicants from escaping into the environment as well as partitioning from the aqueous phase into the headspace until equilibrium was reached. Both the incubating and exposure times were set at 10 min. It was observed that only 2 h was needed to obtain complete dose-related inhibition of photosynthetic activity. The results showed that all the tested organic solvents inhibited algal photosynthesis with EC₅₀ ranging between 589 and 2,570 mM. The inhibition of these solvents was in the order: isopropanol > acetone > acetonitrile > ethanol > dimethylformamide > methanol. The quantitative structure-activity relationship (QSAR) between toxicity data and partition coefficient of the examined compounds could be modeled as follows: \textlog₁₀ \textEC₅₀   ( m\textM ) = - 0.6428  \textlog  P + 5.76  ( \textR² ? 0.88 ){\text{log}}_{{10}} {\text{EC}}_{{50}} \;{\left( {\mu {\text{M}}} \right)} = - 0.6428\;{\text{log}}\;P + 5.76\;{\left( {{\text{R}}^{2} \approx 0.88} \right)}. This indicates that the photosynthetic activity of the microalga Pseudokirchneriella subcapitata is highly dependent on the hydrophobicity of these commonly used organic solvents.     

Flow cytometry and GISH reveal mixed ploidy populations and Spartina nonaploids with genomes of S. alterniflora and S. maritima origin

Background

The genus Spartina exhibits extensive hybridization and includes classic examples of recent speciation by allopolyploidy. In the UK there are two hexaploid species, S. maritima and S. alterniflora, as well as the homoploid hybrid S. × townsendii (2n = 60) and a derived allododecaploid S. anglica (2n = 120, 122, 124); the latter two are considered to have originated in Hythe, southern England at the end of the 19th century.

Methods

Genomic in situ hybridization (GISH) and flow cytometry were used to characterize the genomic composition and distribution of these species and their ploidy levels at Eling Marchwood and Hythe, both near Southampton, southern England.

Key Results

GISH identified approx. 60 chromosomes each of S. maritima and S. alterniflora origin in S. anglica and 62 chromosomes from S. alterniflora and 30 chromosomes from S. maritima in a nonaploid individual from Eling Marchwood, UK. GISH and flow cytometry also revealed that most (94 %) individuals examined at Hythe were hexaploid (the remaining two individuals (6 %) were dodedcaploid; n = 34), whereas hexaploid (approx. 36 % of plants), nonaploid (approx. 27 %) and dodecaploid (approx. 36 %) individuals were found at Eling Marchwood (n = 22).

Conclusions

Nonaploid individuals indicate the potential for introgression between hexaploid and dodecaploid species, complicating the picture of polyploid-induced speciation within the genus. Despite the aggressive ecological habit of S. anglica, it has not out-competed S. × townsendii at Hythe (homoploid hybrids at a frequency of 94 %, n = 34), despite >100 years of coexistence. The success of GISH opens up the potential for future studies of polyploid-induced genome restructuring in this genus.     

Fluxes and compartmentation of 3-O-methyl-D-glucose in Riccia fluitans

In thalli of the aquatic liverwort, Riccia fluitans, 3-O-methyl-D-glucose (3-OMG) is not metabolized. Intracellular compartmentation, accumulation and transmembrane fluxes of 3-OMG have been determined by compartmental analysis. A novel set of equations has been derived to extend this method to non-steady-state conditions of constant but unequal unidirectional fluxes. Efflux kinetics with 3-OMG and L-glucose revealed two intracellular flux compartments, presumably cytoplasm and vacuole; an additional quickly exchanging compartment (half-time approx. 1 min) has been assigned to the apoplast. With 1 mM 3-OMG given externally, cytoplasmic 3-OMG concentration (c _c) attains a quasi-steady state of about 10 mM lasting for >100 h, whereas the presumed vacuolar concentration (c _v) rises steadily, but does not reach flux equilibrium even after two weeks (c _v=46 mM). After 24 h incubation with 0.03 mM 3-OMG, c _c=1 mM approx., and c _v=3 mM approx., thus indicating accumulation by active hexose transport at both the plasmalemma and tonoplast. External D-glucose, but no D-mannitol, competitively inhibits 3-OMG uptake (cis-inhibition) and stimulates 3-OMG efflux at the plasmalemma by a factor up to 2.5. This trans-stimulation saturates half-maximally at 1.5 mM D-glucose. It clearly indicates a hexose carrier in the plasmalemma with one substrate-binding site for D-glocose and 3-OMG, alternately exposed to the cytoplasmic and outside compartment. The extent of the measured trans-stimulation can only be explained, if in the transport cycle the translocation of the empty substrate-binding site across the plasmalemma is rate-limiting.     

Blood pressures,heart rate and locomotor activity during salt loading and angiotensin II infusion in protease-activated receptor 2 (PAR<Subscript>2</Subscript>) knockout mice

Background  

In this study we used radiotelemetry to measure hemodynamic variables and locomotor activity in conscious unrestrained male Protease-Activated Receptor 2 (PAR-2) knockout mice in order to provide a detailed assessment of their blood pressure phenotype. In addition we tested for an influence of PAR-2 on salt-sensitivity (8% versus 0.5% NaCl diet, 2.5 weeks) and angiotensin II-induced hypertension (1 μg Ile⁵-angiotensin II/kg/min versus 0.25 μl/h saline, 2 weeks).     

Whole-body cryostimulation increases parasympathetic outflow and decreases core body temperature

The cardiovascular, autonomic and thermal response to whole-body cryostimulation exposure are not completely known. Thus the aim of this study was to evaluate objectively and noninvasively autonomic and thermal reactions observed after short exposure to very low temperatures. We examined 25 healthy men with mean age 30.1±3.7 years and comparable anthropomorphical characteristic. Each subject was exposed to cryotherapeutic temperatures in a cryogenic chamber for 3 min (approx. −120 °C). The cardiovascular and autonomic parameters were measured noninvasively with Task Force^® Monitor. The changes in core body temperature were determined with the Vital Sense^® telemetric measurement system. Results show that 3 min to cryotherapeutic temperatures causes significant changes in autonomic balance which are induced by peripheral and central blood volume changes. Cryostimulation also induced changes in core body temperature, maximum drop of core temperature was observed 50–60 min after the stimulation. Autonomic and thermal reactions to cryostimulation were observed up to 6 h after the exposure and were not harmful for examined subjects.     

Quinine sulfate and bacterial invasion

Background

As many patients who receive antimalarial drugs for treatment of noninfectious, inflammatory diseases are also immunosuppressed and might have a concomitant bacterial infection, we studied the effectiveness of these drugs against bacterial infections, to find out whether they could protect against (and even treat) such conditions and obviate the need for an additional antibiotic drug.

Methods

Effect of QS on bacterial growth: Escherichia coli (E. coli) HB101 pRI203 were cultured overnight at 37°C in TSB and inoculated (approx 1 × 10⁷ cells /ml) in MEM in the presence of QS at various concentrations (0, 50 and 100 μM).The effect of QS at concentration of 50 and 100 μM on the entry process of E. coli HB101 pRI203 into HeLa cells was studied under different experimental conditions: 1. QS was incubated with 3 × 10⁵ HeLa cells for 60 min at 37°C prior to infection. 2. QS was added to HeLa cell monolayers during the infection period.

Results

QS showed no antibacterial activity after 24 h of incubation.The invasive efficiency of the bacteria was significantly inhibited at a dose-dependent manner, when QS was added to HeLa cells for 60 min at 37°C prior to infection (condition 1), and to a lesser extent when added during the period of infection (condition 2).

Conclusions

Although the antimalarials are generally regarded as being inactive against most extracellular bacterial species, our results indicate that QS significantly inhibited the internalization/invasion efficacy of E. coli in the host cells.

     

Adaptive phenotypic plasticity of Pseudoroegneria spicata: response of stomatal density, leaf area and biomass to changes in water supply and increased temperature

Background and Aims

Changes in rainfall and temperature brought about through climate change may affect plant species distribution and community composition of grasslands. The primary objective of this study was to test how manipulation of water and temperature would influence the plasticity of stomatal density and leaf area of bluebunch wheatgrass, Pseudoroegneria spicata. It was hypothesized that: (1) an increased water supply will increase biomass and leaf area and decrease stomatal density, while a reduced water supply will cause the opposite effect; (2) an increase in temperature will reduce biomass and leaf area and increase stomatal density; and (3) the combinations of water and temperature treatments can be aligned along a stress gradient and that stomatal density will be highest at high stress.

Methods

The three water supply treatments were (1) ambient, (2) increased approx. 30 % more than ambient through weekly watering and (3) decreased approx. 30 % less than ambient by rain shades. The two temperature treatments were (1) ambient and (2) increased approx. 1–3 °C by using open-top chambers. At the end of the second experimental growing season, above-ground biomass was harvested, oven-dried and weighed, tillers from bluebunch wheatgrass plants sampled, and the abaxial stomatal density and leaf area of tillers were measured.

Key Results

The first hypothesis was partially supported – reducing water supply increased stomatal density, but increasing water supply reduced leaf area. The second hypothesis was rejected. Finally, the third hypothesis could not be fully supported – rather than a linear response there appears to be a parabolic stomatal density response to stress.

Conclusions

Overall, the abaxial stomatal density and leaf area of bluebunch wheatgrass were plastic in their response to water and temperature manipulations. Although bluebunch wheatgrass has the potential to adapt to changing climate, the grass is limited in its ability to respond to a combination of reduced water and increased temperature.Key words: Bluebunch wheatgrass, Pseudoroegneria spicata, biomass, climate change, grassland, open top chamber, rain shade, stomata     

Telomere length determined by the fluorescence in situ hybridisation distinguishes malignant and benign cells in cytological specimens

Background

Telomeres are tandem repeats of TTAGGG at the end of eukaryotic chromosomes that play a key role in preventing chromosomal instability. The aim of the present study is to determine telomere length using fluorescence in situ hybridisation (FISH) on cytological specimens.

Methods

Aspiration samples (n = 41) were smeared on glass slides and used for FISH.

Results

Telomere signal intensity was significantly lower in positive cases (cases with malignancy, n = 25) as compared to negative cases (cases without malignancy, n = 16), and the same was observed for centromere intensity. The difference in DAPI intensity was not statistically significant. The ratio of telomere to centromere intensity did not show a significant difference between positive and negative cases. There was no statistical difference in the signal intensities of aspiration samples from ascites or pleural effusion (n = 23) and endoscopic ultrasound‐guided FNA samples from the pancreas (n = 18).

Conclusions

The present study revealed that telomere length can be used as an indicator to distinguish malignant and benign cells in cytological specimens. This novel approach may help improve diagnosis for cancer patients.     

Effect of lactoperoxidase on the antimicrobial effectiveness of the thiocyanate hydrogen peroxide combination in a quantitative suspension test

Background  

The positive antimicrobial effects of increasing concentrations of thiocyanate (SCN-) and H₂O₂ on the human peroxidase defence system are well known. However, little is known about the quantitative efficacy of the human peroxidase thiocyanate H₂O₂ system regarding Streptococcus mutans and sanguinis, as well as Candida albicans. The aim of this study was to evaluate the effect of the enzyme lactoperoxidase on the bactericidal and fungicidal effectiveness of a thiocyanate-H₂O₂ combination above the physiological saliva level. To evaluate the optimal effectiveness curve, the exposure times were restricted to 1, 3, 5, and 15 min.     

Past vegetation dynamics in the Yellowstone region highlight the vulnerability of mountain systems to climate change

Aim

Reconstruct the long‐term ecosystem dynamics of the region across an elevational gradient as they relate to climate and local controls. In particular, we (1) describe the dominant conifers' history; (2) assess changes in vegetation composition and distribution; and (3) note periods of abrupt change versus stability as means of better understanding vegetation responses to environmental variability.

Location

Greater Yellowstone Ecosystem (GYE; USA).

Time period

16.5 ka bp ‐present.

Major taxa studied

Juniperus, Picea, Abies, Pinus, Pseudotsuga.

Methods

The vegetation reconstruction was developed from 15 pollen records. Results were interpreted based on modern pollen–vegetation relationships estimated from a suite of regression‐based approaches.

Results

Calibrated pollen data suggest that late‐glacial vegetation, dominated by shrubs and Juniperus, lacks a modern counterpart in the area. Picea, Abies and Pinus expanded at 16 ka bp in association with postglacial warming and co‐occurred in mixed‐conifer parkland/forest after 12 ka bp . This association along with Pinus contorta forest, which was present after 9 ka bp , has persisted with little change at middle and high elevations to the present day. This stability contrasts with the dynamic history of plant communities at low elevations, where shifts between parkland, steppe and forest over the last 8,000 years were likely driven by variations in effective moisture and fire.

Main conclusions

The postglacial vegetation history of the GYE highlights the dynamic nature of mountain ecosystems and informs on their vulnerability to future climate change: (1) most of the conifers have been present in the area for >12,000 years and survived climate change by adjusting their elevational ranges; (2) some plant associations have exhibited stability over millennia as a result of nonclimatic controls; and (3) present‐day forest cover is elevationally more compressed than at any time in history, probably due to the legacy of the Medieval Climate Anomaly and the Little Ice Age.     

A new method for the reconstitution of the anion transport system of the human erythrocyte membrane

Summary The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent Triton X-100. It was incorporated into spherical lipid bilayers by the following procedure: (1) Dry phosphatidylcholine was suspended in the protein solution. Octylglucopyranoside was added until the milky suspension became clear. (2) The sample was dialyzed overnight against detergentfree buffer. (3) Residual Triton X-100 was removed from the opalescent vesicle suspension by sucrose density gradient centrifugation and subsequent dialysis. Sulfate efflux from the vesicles was studied, under exchange conditions, using a filtration method. Three vesicle subpopulations could be distinguished by analyzing the time course of the efflux. One was nearly impermeable to sulfate, and efflux from another was due to leaks. The largest subpopulation, however, showed transport characteristics very similar to those of the anion transport system of the intact erythrocyte membrane: transport numbers (at 30°C) close to 20 sulfate molecules per band 3 and min, an activation energy of approx. 140 kJ/mol, a pH maximum at pH 6.2, saturation of the sulfate flux at sulfate concentrations around 100mm, inhibition of the flux by H₂DIDS and flufenamate (approx.K _l-values at 30°C: 0.1 and 0.7 m, respectively), and right-side-out orientation of the transport protein (as judged from the inhibition of sulfate efflux by up to 98% by externally added H₂DIDS). Thus, the system represents, for the first time, a reconstitution of all the major properties of the sulfate transport across the erythrocyte membrane.