Fluorescence-based methods for the detection of pressure-induced spore germination and inactivation

The application of high pressure (HP) provides an opportunity for the non-thermal preservation of high-quality foods, whereas highly resistant bacterial endospores play an important role. It is known that the germination of spores can be initiated by the application of HP. Moreover, the resistance properties of spores are highly dependent on their physiological states, which are passed through during the germination. To distinguish between different physiological states and to detect the amount of germinated spores after HP treatments, two fluorescence-based methods were applied. A flow cytometric method using a double staining with SYTO 16 as an indicator for germination and propidium iodide as an indicator for membrane damage was used to detect different physiological states of the spores. During the first step of germination, the spore-specific dipicolinic acid (DPA) is released [P. Setlow, Spore germination, Curr. Opin. Microbiol. 6 (2003), pp. 550–556]. DPA reacts with added terbium to form a distinctive fluorescent complex. After measuring the fluorescence intensity at 270 nm excitation wavelength in a fluorescence spectrophotometer, the amount of germinated spores can be determined. Spores of Bacillus subtilis were treated at pressures from 150 to 600 MPa and temperatures from 37 °C to 60 °C in 0.05 M ACES buffer solution (pH 7) for dwell times of up to 2 h. During the HP treatments, inactivation up to 2log ₁₀ cycles and thermal sensitive populations up to 4log ₁₀ cycles could be detected by plate counts. With an increasing number of thermal sensitive spores, an increased proportion of spores in germinated states was detected by flow cytometry. Also the released amount of DPA increased during the dwell times. Moreover, a clear pressure-temperature-time-dependency was shown by screening different conditions. The fluorescence-based measurement of the released DPA can provide the opportunity of an online monitoring of the germination of spores under HP inside the HP vessel. Implementation can be done using diamond anvil cells, units with inspection glasses or by inserting an optical fiber into the HP vessel. The analytical methods used can help to understand the complex mechanism of germination and inactivation of bacterial spores. Due to its universal, process-independent character, the application of these methods is feasible for established and emerging technologies.     

High pressure inactivation of Clostridium botulinum type E endospores in model emulsion systems

Clostridium botulinum type E is a cold-tolerant, neurotoxigenic, endospore-forming organism, primarily associated with aquatic environments. High pressure thermal (HPT) processing presents a promising tool to enhance food safety and stability. The effect of fat on HPT inactivation of C. botulinum type E spores was investigated using an emulsion model system. The distribution of spores in oil-in-water (O/W) emulsions and their HPT (300–750?MPa, 45–75?°C, 10?min) inactivation was determined as a function of emulsion fat content (30–70% (v/v) soybean oil in buffer). Approximately 26% and 74% of the spores were located at the oil–buffer interface and the continuous phase, respectively. Spore inactivation in emulsion systems decreased with increasing oil contents, which suggests that the fat content of food plays an important role in the protection of C. botulinum type E endospores against HPT treatments. These results can be helpful for future safety considerations.     

Decrease in optical density as a results of germination of Alicyclobacillus acidoterrestris spores under high hydrostatic pressure

Alicyclobacillus acidoterrestris is a spore-forming bacterium, causing spoilage of juices. The spores of these bacteria have the ability to survive in the typical conditions used for thermal pasteurization. Therefore, the use of other techniques such as high hydrostatic pressure is considered for their inactivation. The effect of hydrostatic pressure of 200–500 MPa, at temperatures 4–50 °C for 15 min, on the dynamics of germination of A. acidoterrestris spores in apple juice and pH 4 buffer was studied. To estimate the share of germinated spores, the method of determining the optical density at a wavelength of 660 nm (OD₆₆₀) was used. Parameters of hydrostatic pressure treatment used in this work affected the dynamics of germination of A. acidoterrestris spores in apple juice, and the temperature had the greatest effect. The results indicate that nutrients present in apple juice can promote the germination of A. acidoterrestris spores.     

Development of Aptamer Beacons for Rapid Presumptive Detection of <Emphasis Type="Italic">Bacillus</Emphasis> Spores

A library of 92 DNA aptamer sequences was developed against Bacillus anthracis (nonpathogenic Sterne strain) spores and anthrose sugar immobilized on magnetic beads. The selected DNA sequences were studied for similarities and potential binding pockets between the B. anthracis spore and anthrose aptamers. Several recurring loop structures were identified and tested for their potential to act as aptamer beacons when labeled with TYE 665 dye on their 5′ ends and Iowa Black quencher on their 3′ ends. Of these candidate sequences, two beacons designated BAS-6F and BAS-6R emerged which gave strong fluorescence responses at high spore concentrations (greater than 30,000 spores/ml). These aptamer beacons also detect B. cereus and B. thuringiensis spores with greater fluorescence intensity, but do not strongly detect vegetative cells from an array of other bacterial species. BAS-6F and 6R are also not capable of detecting pure anthrose, thereby probably ruling that epitope out as a spore surface target for these particular beacons. While not extremely sensitive, the BAS-6F and 6R aptamer beacons are potentially valuable for rapid presumptive detection of anthrax or Bacillus spores in suspect powders or bioterrorist activity where spore concentrations are anticipated to be high. The sequence similarities of these beacons to other published Bacillus spore aptamers are also discussed.     

Application of Fluorescent Nanocrystals (q-dots) for the Detection of Pathogenic Bacteria by Flow-Cytometry

Fluorescent semiconductor nanocrystals (q-dots) benefit from practical features such as high fluorescence intensity, broad excitation band and emission diameter dependency. These unique spectroscopic characterizations make q-dots excellent candidates for new fluorescent labels in multi-chromatic analysis, such as Flow-Cytometry (FCM). In this work we shall present new possibilities of multi-labeling and multiplex analysis of pathogenic bacteria, by Flow-Cytometry (FCM) analysis and new specific IgG—q-dots conjugates. We have prepared specific conjugates against B. anthracis spores (q-dots585-IgGαB. anthracis and q-dots655-IgGαB.anthracis). These conjugates enabled us to achieve double staining of B. anthracis spores which improve the FCM analysis specificity versus control Bacillus spores. Moreover, multiplexed analysis of B. anthracis spores and Y. pestis bacteria was achieved by using specific antibodies labeled with different q-dots to obtain: q-dots585-IgGαB. anthracis and q-dots655-IgGαY.pestis, each characterized by its own emission peak as a marker. Specific and sensitive multiplex analysis for both pathogens has been achieved, down to 10³ bacteria per ml in the sample.     

Inactivation of Staphylococcus aureus using high hydrostatic pressure

The aim of this work was to develop a high-pressure decontamination and sterilization process for pharmaceutical treatments as was developed in food processing in the late eighties. The lack of normalized biological indicators able to validate sterilizing treatments under high pressure led us to select representative pathogenic strains from flora and the European Pharmacopoeia. We selected the following four bacterial strains: Candida albicans (ATCC 10231), Psuedomonas aeruginosa (ATCC 9027), spores of Aspergillus niger (ATCC 16404) and Staphylococcus aureus (ATCC 6538).

This present study is focussed on S. aureus. Successive pressurization and depressurization cycles appeared to be more efficient than a continuous high-pressure treatment. Importantly, these pressure conditions, temperature and process duration are perfectly compatible with current industrial plants. These results show that HHP technology is a new alternative to inactivate pathogenic strains in accordance with pharmaceutical requirements.     

Interspecific allometry of bone dimensions: A review of the theoretical models

A fascinating problem in biological scaling is the variation of long-bone length (or diameter) Y with body mass M in mammals, birds, and other vertebrates. It turns out that Y and M are related by a power law, namely Y=Y₀M^b, where Y₀ is a constant and b is the so-called allometric exponent. The origin of these power laws is still unclear because, in general, biological laws do not follow from physical ones in a simple manner.Here we make a historical review of this subject, summarizing the main experimental papers as well as discussing the main theoretical proposals. Long-bone allometry seems to be determined by the need to resist the particular loads applied to each bone in each taxon. Experimental measurements of in vivo stresses have found that mammalian long bones are subjected mainly to compression and bending, while avian wing-bones and reptilian limb-bones suffer a high degree of torsion. A recent model, based on the hypothesis that mammalian long-bone allometry is determined by compressive and bending loads, was able elucidate several aspects of mammalian limb-bone scaling. However, an explanation for avian and reptilian long-bone allometry is still missing.     

Nanomechanical analysis of Clostridium tyrobutyricum spores

In this work we report on the measurement of the Young modulus of the external surface of Clostridium tyrobutyricum spores in air with an atomic force microscope. The Young modulus can be reliably measured despite the strong tip-spore adhesion forces and the need to immobilize the spores due to their slipping on most substrates. Moreover, we investigate the disturbing factors and consider some practical aspects that influence the measurements of elastic properties of biological objects with the atomic force microscopy indentation techniques.     

The variability of times to detect growth from individual Clostridium botulinum type E endospores is differentially affected by high pressure treatments

High pressure thermal (HPT) processing is a candidate technology for the production of safe and stable food. However, little is known about the effect of HPT or high hydrostatic pressure (HHP) treatments at ambient temperature on the variability of times to detect growth from individual spores. We investigated this effect by treating Clostridium botulinum type E spores with HHP (200–600?MPa, 20°C) and HPT (600?MPa, 80°C and 800?MPa, 60°C). Our results indicate that the mean detection times increase and the frequency distribution shifts toward longer times when HHP treatment intensity is increased. HPT treatments result in a highly scattered distribution. In contrast, pressure levels ≤300?MPa decrease detection times and heterogeneity of their distribution, which could lead to an increase in the potential risk originating from C. botulinum type E spores. Data provided here could help to refine risk assessment regarding this important food intoxicator.     

Moderate hydrostatic pressure–temperature combinations for inactivation of Bacillus subtilis spores

We report the effect of using moderate hydrostatic pressure, 40–140?MPa, at moderate temperature (38–58°C) to inactivate Bacillus subtilis spores in McIlvaine's citric phosphate buffer at pH 6. We have investigated several parameters: pressure applied, holding time, pressure cycling, and temperature. The kinetics of spore inactivation is reported. The results show that spore inactivation is exponentially proportional to the time the sample is exposed to pressure. Spore germination and inactivation occur at the hydrostatic pressures/temperature combinations we explored. Cycling the pressure while keeping the total time at high pressure constant does not significantly increase spore inactivation. We show that temperature increases spore inactivation at two different rates; a slow rate below 33°C, and at a more rapid rate at higher temperatures. Increasing pressure leads to an increase in spore inactivation below 95?MPa; however, further increases in pressure give a similar rate kill. The time dependence of the effect of pressure is consistent with the first-order model (R²?>?0.9). The thermal resistance values (Z_T) of B. subtilis spores are 30°C, 37°C, and 40°C at 60, 80, 100?MPa. The increase in Z_T value at higher pressures indicates lower temperature sensitivity. The pressure resistance values (Z_P) are 125, 125 and 143?MPa at 38°C, 48°C, and 58°C. These Z_P values are lower than those reported for B. subtilis spores in the literature, which indicates higher sensitivity at pressures less than about 140?MPa. We show that at temperatures <60°C, B. subtilis spores are inactivated at pressures below 100?MPa. This finding could have implications for the design of the sterilization equipment.     

Element‐specific microtomographic imaging of Drosophila brain stained with high‐Z probes

An application of X‐ray microtomography to the Drosophila adult brain stained with colloidal gold and a platinum compound is described. The transparency of biological tissue to hard X‐rays enables tomographic visualization of the three‐dimensional structure of tissue entrails. Each high‐Z element was visualized as a three‐dimensional structure from the difference absorption coefficient image at the corresponding L_III absorption edge. The cortex of the optic lobe was selectively visualized by the specific adsorption of colloidal gold. The entire structure revealed by the platinum impregnation allowed the anatomical assignment of the gold‐stained structures. Selective staining and specific visualization of biological tissues at micrometer resolution should elucidate the three‐dimensional cellular organization essential for the understanding and application of biological microstructures.     

A diversity-oriented synthesis of 3-(2-amino-1,6-dihydro-6-oxo-pyrimidin-5-yl)propanoic esters

The synthesis of dimethyl 2-(methoxymethylene) pentanedioates by an unusual Michael addition of 3,3-dimethoxypropionate to α, β-unsaturated esters is described. These new intermediates can subsequently be converted to methyl 3-(2-amino-1,6-dihydro-6-oxo-pyrimidin-5-yl)propanoates upon treatment with guanidine carbonate. The resulting pyrimidine derivatives are open-chain analogues of pyrido[2,3-d]pyrimidines with interesting biological activities.     

Backscatter effects of surfaces composed of dry biological particles

We present the backscattering of particulate surfaces consisting of dry biological particles using two laboratory photopolarimeters that measure intensity and degree of linear polarization in a phase-angle range 0.2-60°. We measure scattering properties from three samples composed of dry biological particles, Bacillus subtilis var. niger (BG) spores and samples of fungi Aspergillus terreus and Sporisorium cruentum spores. We find that the surfaces display a prominent brightness opposition effect and significant negative polarization near backscattering angles. The brightness and polarimetric phase curves are different for B. subtilis and the fungi.     

Entropy and enthalpy calculations from perturbation and integration thermodynamics methods using molecular dynamics simulations: applications to the calculation of hydration and association thermodynamic properties

Calculation of association thermodynamic properties using molecular simulation is essential in computational chemistry. In the case of good agreement with the experimental thermodynamic binding properties, this type of calculation may complement experimental works by providing a microscopic view of the association process. Whereas the calculation of the free energy of association is nowadays well controlled, the calculation of the enthalpy and entropy of association remains difficult in most cases, especially as the association involves hosts and guests of biological interest. A novel method for calculating the entropy change from a molecular dynamics simulation is described. Within the theoretical framework, we discuss the different approximations leading to the final stage of the operational expressions of ΔG and ΔH in the NpT ensemble and we establish an expression for ΔS using the Free Energy Perturbation (FEP) formalism in this statistical ensemble. Finally, we illustrate the theoretical considerations by calculations of the hydration entropy changes between cations of different masses and charges. We extend the study by calculating the changes in the thermodynamic properties of association of inorganic cations with a macrocycle of biological interest.     

Mutagenic effect of accelerated heavy ions on bacterial cells

The heavy ion accelerators of the Joint Institute for Nuclear Research were used to study the regularities and mechanisms of formation of different types of mutations in prokaryote cells. The induction of direct (lac⁻, ton B⁻, col B) mutations for Esherichia coli cells and reverse his⁻ → His⁺ mutations of Salmonella typhimurium, Bacillus subtilis cells under the action of radiation in a wide range of linear energy transfer (LET) was studied. The regularities of formation of gene and structural (tonB trp-) mutations for Esherichia coli bacteria under the action of accelerated heavy ions were studied. It was demonstrated that the rate of gene mutations as a function of the dose under the action of Γ rays and accelerated heavy ions is described by linear-quadratic functions. For structural mutations, linear “dose-effect” dependences are typical. The quadratic character of mutagenesis dose curves is determined by the “interaction” of two independent “hitting” events in the course of SOS repair of genetic structures. The conclusion made was that gene mutations under the action of accelerated heavy ions are induced by δ electron regions of charged particle tracks. The methods of SOS chromotest, SOS lux test, and λ prophage induction were used to study the regularities of SOS response of cells under the action of radiations in a wide LET range. The following proposition was substantiated: the molecular basis for formation of gene mutations are cluster single-strand DNA breaks, and that for structural mutations, double-strand DNA breaks. It was found out that the LET dependence of the relative biological efficiency of accelerated ions is described by curves with a local maximum. It was demonstrated that the biological efficiency of ionizing radiations with different physical characteristics on cells with different genotype, estimated by the lethal action, induction of gene and deletion mutations, precision excision of transposons, is determined by the specific features of energy transfer of the radiations that affect the character of induced DNA damage, and the efficiency inducible and constitutive cell repair systems. The growth of relative biological efficiency of heavy charged particles is determined by the growth of the damage yield of the DNA participating in the formation of radiation-induced effects, and higher efficiency of inducible repair systems. It was established that the LET value (L _max) for which the maximum (according to the applied irradiation criteria) coefficients of relative biological efficiency are observed varies depending on the character of the registered radiation induced effect. It was demonstrated that for gene mutations and induction of precision excision of mobile elements the values of L _max are realized in a LET range of ≈20 keV/μm. For lethal effects of irradiation and induction of deletion mutations the value of L _max is ≈ 100 and 50 keV/μm, respectively. The differences in the L _max for the studied radiation gene effectis are determined by the different type of DNA damage participating in the mutation process. A molecular model of the formation of gene mutations in Escherichia coli cells under the action of ionizing radiation was proposed. Basic DNA radiation damage and main repair ways were considered in the framework of this model. The basis is the idea of the decisive role of mutagenic, error-prone, branch of SOS repair in fixing premutation DNA damage into point mutations. It was demonstrated that the central mechanism in this process is the formation of an inducible multi-enzymatic complex including the DNA polymerase V (Umu C), RecA-protease, SSB proteins, subunits of DNA polymerase III, performing erroneous DNA synthesis on the damaged matrix. A mathematical model of induction of gene mutations under ultraviolet cell irradiation was developed based on the molecular model.     

Pressure-assisted thermal sterilization of soup

The overall efficiency of an existing scale-up pressure-assisted thermal sterilization (PATS) unit was investigated with regards to inactivation of Geobacillus stearothermophilus spores suspended in pumpkin soup. The PATS unit is a double pipe heat exchanger in which the soup is pumped into its inner high pressure tube and constrained by two high pressure valves, while steam is continuously passed through the annular region to heat the content. The technology is based on pressure generation by thermal expansion of the liquid in an enclosure. In this work, the addition of an air line to push the treated liquid food out of the existing PATS unit has improved the overall quality of the treated samples, as evidenced by achieving higher log reduction of the spores. Compared with thermal processing, the application of PATS shows the potential for lowering the thermal treatment temperature, offering improved food quality.     

Fluorescence Assay Based on Aptamer-Quantum Dot Binding to <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Spores

A novel assay was developed for the detection of Bacillus thuringiensis (BT) spores. The assay is based on the fluorescence observed after binding an aptamer-quantum dot conjugate to BT spores. The in vitro selection and amplification technique called SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used in order to identify the DNA aptamer sequence specific for BT. The 60 base aptamer was then coupled to fluorescent zinc sulfide-capped, cadmium selenide quantum dots (QD). The assay is semi-quantitative, specific and can detect BT at concentrations of about 1,000 colony forming units/ml.     

基于delta-P1近似模型的空间分辨漫反射一阶散射参量灵敏度研究

以delta-P1近似光学模型为基础, 推导了双点源近似下空间分辨漫反射一阶散射参量μ_s'灵敏度的解析式, 并进行了数值分析和比较. 研究表明, 与混合漫射近似模型和漫射近似模型相比, delta-P1 近似模型能更好地描述强散射较强吸收情况下近光源区域生物组织漫反射光子的分布, 且在有效反照率a'>0.83时, 获得最佳优化距离ρ_opt, ρ_opt 随μ_s' 的增大而减小, 且在距光源约2.7—4个输运平均自由程处μ_s'的变化对测量吸收的影响最小. 这项研究对于优化传感器几何结构以及生物组织光学参量的测量具有重要意义.     

Auger electron emission in proton-induced interactions in living matter: A TILDA-V Monte Carlo tracking

The present work focuses on studying the contribution of the Auger electron emission in proton-induced interactions in biological matter. The Monte Carlo track-structure code, TILDA-V, was then used for modeling the protons beams of 10 keV to 100 MeV in biological matter, namely, water vapor and hydrated DNA. The main ionizing processes are described by means of an extensive set of ab initio differential and total cross sections computed within a quantum-mechanical CDW-EIS approximation.