Kinetic Lag Research Articles

Effect of the Womersley number on transition to turbulence in pipe flow: An experimental study

The mechanisms driving the transition to turbulence in pulsatile flows are not well understood. Prior studies in this domain have noted the dynamics of this flow regime to depend on the mean Reynolds number, pulsation frequency (i.e., Womersley number), and inflow pulsatile waveform shape. Conflicting findings, particularly regarding the role of the Womersley number on the critical Reynolds number and the development of turbulence, have been reported. The discord has primarily been observed for flows, with Womersley numbers ranging from 4 to 12. Hence, in this work, we use particle image velocimetry to explore the effects of the Womersley number within this 4–12 range on the dynamics of the pulsatile transition. Eighteen test cases were captured using six mean Reynolds numbers (range 800–4200) and five Womersley numbers. Turbulent kinetic energy, turbulence intensity (TI), and phase lag were computed. Our results indicated that the critical Reynolds number was roughly independent of the Womersley number. At high Womersley numbers, the TI trend maintained lower pulsatility, and the flow was observed to mimic a steady transitional flow regime. A plateau of the TI-velocity and TI-acceleration phase lag was observed at a Womersley number of 8, highlighting that this may be the critical value where further increases to the Womersley number do not alter the transition dynamics. Furthermore, this suggests that the phase lag may provide a universal indicator of the specific influence of the Womersley number on transition for a given flow. Overall, these findings elucidate critical details regarding the role of the Womersley number in the transition to turbulence.

Chilled Pacu (Piaractus mesopotamicus) fillets: Modeling Pseudomonas spp. and psychrotrophic bacteria growth and monitoring spoilage indicators by 1H NMR and GC–MS during storage

This study aimed to assess the growth of Pseudomonas spp. and psychrotrophic bacteria in chilled Pacu (Piaractus mesopotamicus), a native South American fish, stored under chilling conditions (0 to 10 °C) through the use of predictive models under isothermal and non-isothermal conditions. Growth kinetic parameters, maximum growth rate (μmax, 1/h), lag time (tLag, h), and (Nmax, Log10 CFU/g) were estimated using the Baranyi and Roberts microbial growth model. Both kinetic parameters, growth rate and lag time, were significantly influenced by temperature (P < 0.05). The square root secondary model was used to describe the bacteria growth as a function of temperature. Secondary models, √μ = 0.016 (T + 10.13) and √μ =0.017 (T + 9.91) presented a linear correlation with R2 values >0.97 and were further validated under non-isothermal conditions. The model's performance was considered acceptable to predict the growth of Pseudomonas spp. and psychrotrophic bacteria in refrigerated Pacu fillets with bias and accuracy factors between 1.24 and 1.49 (fail-safe) and 1.45–1.49, respectively. Fish biomarkers and spoilage indicators were assessed during storage at 0, 4, and 10 °C. Volatile organic compounds, VOCs (1-hexanol, nonanal, octenol, and indicators 2-ethyl-1-hexanol) showed different behavior with storage time (P > 0.05). 1H NMR analysis confirmed increased enzymatic and microbial activity in Pacu fillets stored at 10 °C compared to 0 °C. The developed and validated models obtained in this study can be used as a tool for decision-making on the shelf-life and quality of refrigerated Pacu fillets stored under dynamic conditions from 0 to 10 °C.

Performance of a seed amplification assay for misfolded alpha-synuclein in cerebrospinal fluid and brain tissue in relation to Lewy body disease stage and pathology burden

The development of in vitro seed amplification assays (SAA) detecting misfolded alpha-synuclein (αSyn) in cerebrospinal fluid (CSF) and other tissues has provided a pathology-specific biomarker for Lewy body disease (LBD). However, αSyn SAA diagnostic performance in early pathological stages or low Lewy body (LB) pathology load has only been assessed in small cohorts. Moreover, the relationship between SAA kinetic parameters, the number of αSyn brain seeds and the LB pathology burden assessed by immunohistochemistry has never been systematically investigated. We tested 269 antemortem CSF samples and 138 serially diluted brain homogenates from patients with and without neuropathological evidence of LBD in different stages by the αSyn Real-Time Quaking-Induced Conversion (RT-QuIC) SAA. Moreover, we looked for LB pathology by αSyn immunohistochemistry in a consecutive series of 604 Creutzfeldt–Jakob disease (CJD)-affected brains. αSyn CSF RT-QuIC showed 100% sensitivity in detecting LBD in limbic and neocortical stages. The assay sensitivity was significantly lower in patients in early stages (37.5% in Braak 1 and 2, 73.3% in Braak 3) or with focal pathology (50% in amygdala-predominant). The average number of CSF RT-QuIC positive replicates significantly correlated with LBD stage. Brain homogenate RT-QuIC showed higher sensitivity than immunohistochemistry for the detection of misfolded αSyn. In the latter, the kinetic parameter lag phase (time to reach the positive threshold) strongly correlated with the αSyn seed concentration in serial dilution experiments. Finally, incidental LBD prevalence was 8% in the CJD cohort. The present results indicate that (a) CSF RT-QuIC has high specificity and sufficient sensitivity to detect all patients with LB pathology at Braak stages > 3 and most of those at stage 3; (b) brain deposition of misfolded αSyn precedes the formation of LB and Lewy neurites; (c) αSyn SAA provides “quantitative” information regarding the LB pathology burden, with the lag phase and the number of positive replicates being the most promising variables to be used in the clinical setting.

Insight into the mechanism of action of ORG27569 at the cannabinoid type one receptor utilising a unified mathematical model

Allosteric modulation of CB1 is therapeutically advantageous compared to orthosteric activation as it potentially offers reduced on-target adverse effects. ORG27569 is an allosteric modulator that increases orthosteric agonist binding to CB1 but decreases functional signalling. ORG27569 is characterised by a delay in disinhibition of agonist-induced cAMP inhibition (lag); however, the mechanism behind this kinetic lag is yet to be identified. We aimed to utilise a mathematical model to predict data and design in vitro experiments to elucidate mechanisms behind the unique signalling profile of ORG27569. The established kinetic ternary complex model includes the existence of a transitional state of CB1 bound to ORG27569 and CP55940 and was used to simulate kinetic cAMP data using NONMEM 7.4 and Matlab R2020b. These data were compared with empirical cAMP BRET data in HEK293 cells stably expressing hCB1. The pharmacometric model suggested that the kinetic lag in cAMP disinhibition by ORG27569 is caused by signal amplification in the cAMP assay and can be reduced by decreasing receptor number. This was confirmed experimentally, as reducing receptor number through agonist-induced internalisation resulted in a decreased kinetic lag by ORG27569. ORG27569 was found to have a similar interaction with CP55940 and the high efficacy agonist WIN55,212-2, and was suggested to have lower affinity for CB1 bound by the partial agonist THC compared to CP55940. Allosteric modulators have unique signalling profiles that are often difficult to interrogate exclusively in vitro. We have used a combined mathematical and in vitro approach to prove that ORG27569 causes a delay in disinhibition of agonist-induced cAMP inhibition due to large receptor reserve in this pathway. We also used the pharmacometric model to investigate the common phenomenon of probe dependence, to propose that ORG27569 binds with higher affinity to CB1 bound by high efficacy orthosteric agonists.

Comparison of Different Mechanical Pretreatment Methods for the Anaerobic Digestion of Landscape Management Grass

The aim of this study was to use landscape grass from species-rich orchards for biogas production, thus preserving these very valuable areas for future generations. Since these grass clippings have high lignocellulose content, the substrate has to be pretreated before being fed into the biogas digester. In this study, three different mechanical treatment processes (cross-flow grinder, ball mill and a mounted mower) were investigated and compared with untreated grass clippings. Chemical composition, specific methane yield, degradation kinetics and microscopic images were analyzed. In order to derive recommendations, the harvesting and pretreatment processes were examined in terms of energy demand, additional methane yield, and suitability of the substrate for use in biogas plants, taking into account conservation aspects. Within the pretreatment process, ball milling leads to the highest significant increase in specific methane yield of up to 5.8% and the fastest gas formation kinetics (lag time λBM: 0.01 ± 0.0 d; duration to reach half of total gas production ½M(x)BM: 5.4 ± 0.2 d) compared to the untreated variant (λUT: 1.02 ± 0.2 d; ½M(x)UT: 6.5 ± 0.2 d). A comparison of the energy required for the mechanical disintegration of the substrates with the increased yield of methane during the digestion process shows that the mechanical processing of these substrates appears to be useful. A positive energy balance was achieved for the cross-flow grinder (12.3 kWh tVS−1) and the ball mill (21.4 kWh tVS−1), while the Amazone Grasshopper left a negative balance (−18.3 kWh tVS−1), requiring more energy for substrate pretreatment than was generated as methane surplus. In summary, the pretreatment of landscape management grass is a suitable approach for utilizing agricultural residues efficiently in a biogas plant and thus contributing to sustainable energy production.

Antimicrobial potential of aqueous coffee extracts against pathogens and Lactobacillus species: A food matrix application

Coffee is recognized by its bioactive potential, mainly antioxidant and antimicrobial activity. The aim of this study was to evaluate the antimicrobioal potential of aqueous extracts of Coffea arabica (AR) (roasted) and Coffea canephora [roasted (CR) and green (CG)] against patogens Salmonella Typhimurium and Escherichia coli, and probiotics Lactobacillus plantarum and Lactobacillus rhamnosus. Firstly, the strain's sensitivity were tested by disk diffusion method, followed by the evaluation of growth kinetics (lag phase duration, maximum specific growth rate and logarithmic increase in the population) in culture media and in a food matrix (milk) by the adjustment of Gompertz predictive model. In the step of strain sensivity tests, S. Typhimurium and E. coli were sensitive against all coffee extracts from 0.5% to 5.0%, while the Lactobacilli growth were not affected. In the growth kinetic tests in culture media, roasted coffees extracts reduced logarithmic increase in the population and maximum specific growth rate and improved lag phase for S. Typhimurium; for E. coli the growth was completely inhibited with 2.5% CR and 5.0% AR. The correlations between growth parameters with coffee bioactive contents suggested that melanoidins had the main antimicrobial effect. In the step of food matrix application (growth kinetics in milk), the addition of 5.0% CR reduced S. Typhimurium and E. coli multiplication, while the probiotics L. plantarum and L. rhamnosus where not inhibetd, suggesting a selective effect of coffee extracts as an antimicrobial agent, which may be of great interest for the food industry, especially for probiotic foods application.

Presence and growth prediction of Staphylococcus spp. and Staphylococcus aureus in Minas Frescal cheese, a soft fresh cheese produced in Brazil

Physical-chemical characteristics of Minas Frescal cheese (MFC) favor the growth of Staphylococcus spp. and allow the production of enterotoxins by specific strains. Here, we aimed to characterize the physical-chemical aspects (pH, storage temperature, and salt content) and the presence of Staphylococcus spp. in MFC samples (n = 50) to support a modeling study for the growth by this microorganism. Coagulase-positive staphylococci isolates were obtained and subjected to PCR assays to identify them as Staphylococcus aureus (nuc) and to detect staphylococcal enterotoxin-related genes (sea, seb, sec, sed, see). Staphylococcus aureus growth kinetics (maximum growth rate, Grmax, and lag time) were predicted based on ComBase model and MFC physical-chemical aspects. Mean counts of Staphylococcus spp. ranged from 3.3 to 6.7 log cfu/g, indicating poor hygiene practices during production. Selected isolates (n = 10) were identified as S. aureus, but none presented classical enterotoxin-related genes. pH, temperature, and salt content ranged from 5.80 to 6.62, 5°C to 12°C, and 0.85% to 1.70%, respectively. The Grmax values ranged from 0.012 to 0.419 log cfu/g per h. Independent of the storage temperature, the lowest Grmax values (0.012 to 0.372 log cfu/h) were obtained at pH 5.80 associated with salt content of 1.7%; independent of the pH and salt content, the best temperature to avoid staphylococcal growth was 7.5°C. Hygienic conditions during MFC production must be adopted to avoid staphylococcal contamination, and storage at temperatures lower than 7.5°C can prevent staphylococcal growth and the potential production of enterotoxins.

Controls on eruption style at Rabaul, Papua New Guinea – Insights from microlites, porosity and permeability measurements

Rabaul in Papua-New-Guinea is an extremely active andesitic caldera complex that displays a large spectrum of eruption styles. Since 1878, four sub-plinian (VEI-4) and ten VEI 1–3 (effusive, strombolian, vulcanian) eruptions occurred from Tavurvur and Vulcan, the two main active vents. We study pumiceous tephra, ballistic bombs and a lava flow from five of these eruptions to investigate (1) magma ascent rates and (2) volatile escapement processes during the ascent.We measured total and connected porosities, permeability, and connectivity and related these results with measurements of crystallinity, Microlite Number Density (MND) and Microlite Size Distribution (MSD) of plagioclases and pyroxenes. From the application of existing percolation models, we find that explosive products yield a percolation threshold comprised between 50 and 60 vol% total porosity, while petrophysical parameters of the lava flow and some of the ballistic bombs are explained by bubble collapse driven by surface tension. Sub-plinian products show low phenocryst contents (5–15 vol%), microlites generated by nucleation-driven crystallization or glassy textures due to kinetic crystallization lags. Vulcanian, strombolian and effusive products on the other hand, show medium to high phenocryst contents (15–40 vol%) and microlites that crystallised by growth-dominated processes. MSDs in sub-plinian pumiceous tephra constitute a partial record of drastic magma acceleration. In comparison, MSDs in vulcanian and strombolian ballistic bombs and lava flow show a classic pattern of crystallization at a steady-state due to gradual magma ascent.We find that magma feeding sub-plinian eruptions ascends 2–3 orders of magnitude faster than magma feeding vulcanian/effusive eruptions (≥ 1–100 m/s vs 0.1 m/s). In the case of sub-plinian products, due to the important kinetic crystallization lag, these speeds cannot be estimated using microlite crystallization triggered by decompression as a proxy. Combining petrophysical and textural measurements we suggest that slight changes in initial conditions in the reservoir such as crystallinity or the presence of exsolved volatiles, can have a profound impact on the ascent rate and generate positive or negative feedback effects leading to powerful sub-plinian activity or intermittent vulcanian/ passive effusive activity respectively.

The Seasonality of Submesoscale Energy Production, Content, and Cascade

AbstractSubmesoscale processes in the upper ocean vary seasonally, in tight correspondence with mixed layer thickness variability. Based on a global high‐resolution MITgcm simulation, seasonal evaluation of strong vorticity and spectral analysis of the kinetic energy in the Kuroshio Extension System show the strongest submesoscales occur in March, implying a lag of about a month behind mixed layer thickness maximum in February. An analysis of spectral energy sources and transfers indicates that the seasonality of the submesoscale energy content is a result of the competition between the conversion of available potential energy into submesoscale kinetic energy via a buoyancy production/vertical buoyancy flux associated with mixed layer instability and nonlinear energy transfers to other scales associated with an energy cascade. The buoyancy production is seasonally in phase with the mixed layer depth, but the transfers of energy across scales makes energizing the reservoir of submesoscale kinetic energy lag behind by a month.

Plasma membrane LAT activation precedes vesicular recruitment defining two phases of early T-cell activation

The relative importance of plasma membrane-localized LAT versus vesicular LAT for microcluster formation and T-cell receptor (TCR) activation is unclear. Here, we show the sequence of events in LAT microcluster formation and vesicle delivery, using lattice light sheet microscopy to image a T cell from the earliest point of activation. A kinetic lag occurs between LAT microcluster formation and vesicular pool recruitment to the synapse. Correlative 3D light and electron microscopy show an absence of vesicles at microclusters at early times, but an abundance of vesicles as activation proceeds. Using TIRF-SIM to look at the activated T-cell surface with high resolution, we capture directed vesicle movement between microclusters on microtubules. We propose a model in which cell surface LAT is recruited rapidly and phosphorylated at sites of T-cell activation, while the vesicular pool is subsequently recruited and dynamically interacts with microclusters.

Sequential and Coupled Proton and Electron Transfer Events in the S2 → S3 Transition of Photosynthetic Water Oxidation Revealed by Time-Resolved X-ray Absorption Spectroscopy.

The choreography of electron transfer (ET) and proton transfer (PT) in the S-state cycle at the manganese-calcium (Mn4Ca) complex of photosystem II (PSII) is pivotal for the mechanism of photosynthetic water oxidation. Time-resolved room-temperature X-ray absorption spectroscopy (XAS) at the Mn K-edge was employed to determine the kinetic isotope effect (KIE = τD2O/τH2O) of the four S transitions in a PSII membrane particle preparation in H2O and D2O buffers. We found a small KIE (1.2-1.4) for manganese oxidation by ET from Mn4Ca to the tyrosine radical (YZ•+) in the S0n → S1+ and S1n → S2+ transitions and for manganese reduction by ET from substrate water to manganese ions in the O2-evolving S3n → S0n step, but a larger KIE (∼1.8) for manganese oxidation in the S2n → S3+ step (subscript, number of accumulated oxidizing equivalents; superscript, charge of Mn4Ca). Kinetic lag phases detected in the XAS transients prior to the respective ET steps were assigned to S3+ → S3n (∼150 μs, H2O; ∼380 μs, D2O) and S2+ → S2n (∼25 μs, H2O; ∼120 μs, D2O) steps and attributed to PT events according to their comparatively large KIE (∼2.4, ∼4.5). Our results suggest that proton movements and molecular rearrangements within the hydrogen-bonded network involving Mn4Ca and its bound (substrate) water ligands and the surrounding amino acid/water matrix govern to different extents the rates of all ET steps but affect particularly strongly the S2n → S3+ transition, assigned as proton-coupled electron transfer. Observation of a lag phase in the classical S2 → S3 transition verifies that the associated PT is a prerequisite for subsequent ET, which completes Mn4Ca oxidation to the all-Mn(IV) level.

A comparative study of fibrillation kinetics of two homologous proteins under identical solution condition

Human lysozyme is homologous in the three-dimensional structure to hen lysozyme and the latter is commonly used to understand folding and amyloid aggregation pathway of the former. The fibrillation of the two proteins is known to occur via partial unfolding. A work dedicated to comparing the aggregation-prone conformations and their subsequent conversion into amyloid-like fibrils in an identical condition is not available. This has provided an opportunity to compare the fibrillation behaviors of the two homologous proteins under identical solution condition. In this work, we have shown that the temperature-induced unfolding of the two proteins at pH 1.5 occurred via a three states process. We found that temperature-unfolded states of the two proteins differ in contents of residual secondary and tertiary structures. The temperature-unfolded states of both proteins rapidly converted into well-defined amyloid-like fibrils on stirring at 230 RPM. We further observed that the kinetic parameters, lag time (tlag) and apparent rate constant (kapp) of aggregation of hen lysozyme were markedly enhanced than human lysozyme. Amyloid fibrils formed by the two proteins only slightly differ in their morphology and Tinctorial properties. Therefore, on the basis of our in vitro aggregation and in silico aggregation and α-helical propensities prediction studies, we concluded that the major determinant of acceleration of aggregation of hen lysozyme is the stabilization of amyloidogenic native α-helices in highly dynamics partially-folded state. Comparison of aggregation-prone conformations and their aggregation kinetics parameters also with other protein systems can serve as a useful model to understand the factors promoting amyloid aggregation.

Modeling the Time to Fail of Peach Nectars Formulated by Hurdle Technology

The use of regression with life-data is helpful to observe whether one or more factors affect the failure time (spoilage) of a product, obtaining a model that predicts the time to fail (TTF). TTF models link kinetic (lag time) and probabilistic (growth /no-growth prediction) models for selected formulation/storage conditions. Our objective was to assess the individual and combined effects of pH, aw, and the incorporation of potassium sorbate (KS) or sodium benzoate (BNa) at selected concentrations on the microbial stability of peach nectar during storage at 25°C, in order to model and predict TTF. Peach nectars were formulated with 40% fruit pulp and the necessary sucrose syrup and citric acid to attain aw 0.96, 0.97, or 0.98 and pH 3.0, 3.5, or 4.0; while 0, 500, or 1000ppm of KS or BNa were added. Nectars were stored for 180 days in glass jars at 25°C, and periodically analyzed (standard plate as well as yeast and mould counts). The experimental design and analyses were replicated three times. Storage times that revealed microbial populations higher than 104 CFU/mL and signs of spoilage were registered to model TTF by survival analysis. From the 54 combinations tested, 9 formulations (without antimicrobials) exhibited early spoilage (<5 days). For the combinations formulated with 500ppm of BNa, spoilage was detected after 30 days; much longer spoilage times were observed for nectars with 1000ppm of KS or BNa. In general, KS was more effective than BNa in delaying spoilage when 1000ppm were added. TTF models included individual and interaction effects of the evaluated factors and revealed good agreement among experimental and predicted data (R2>0.90). Survival analysis through TTF models can be used to predict spoilage time under specific factor combinations or to select factor levels for a specific shelf-life of peach nectars.

New experimental data on the human dermal absorption of Simazine and Carbendazim help to refine the assessment of human exposure.

Due to their widespread usage, people are exposed to pesticides on a daily basis. Although these compounds may have adverse effects on their health, there is a gap in the data and the methodology needed to reliably quantify the risks of non-occupational human dermal exposure to pesticides. We used Franz cells and human skin in order to measure the dermal absorption kinetics (steady-state flux, lag time and permeability coefficient) of Carbendazim and Simazine. These parameters were then used to refine the dermal exposure model and a probabilistic simulation was used to quantify risks resulting from exposure to pesticide-polluted waters. The experimentally derived permeability coefficient was 0.0034 cm h(-1) for Carbendazim and 0.0047 cm h(-1) for Simazine. Two scenarios (varying exposure duration and concentration, i.e. environmentally relevant and maximum solubility) were used to quantify the human health risks (hazard quotients) for Carbendazim and Simazine. While no risks were determined in the case of either scenario, the permeability coefficient, which is concentration independent and donor, formulation, compound and membrane specific, may be used in other scenarios and exposure models to quantify more precisely the dermally absorbed dose during exposure to polluted water. To the best of our knowledge, the dermal absorption kinetics parameters defined here are being published for the first time. The usage of experimental permeability parameters in combination with probabilistic risk assessment thus provides a new tool for quantifying the risks of human dermal exposure to pesticides.

Antimicrobial activity of açaí against Listeria innocua

A study was carried out to evaluate antimicrobial activity of açaí against Listeria innocua, as a non-pathogenic surrogate for Listeria monocytogenes, at different temperatures (37, 22, and 10 °C), from a kinetic point of view. With this aim, first the Minimum Inhibitory Concentration (MIC) under optimal growth conditions was established (37 °C), and then the effect of 3 non-inhibitory doses (3, 5, and 7 g/L) at 37, 22, and 10 °C was evaluated on the basis of the kinetic parameters lag time (λ) and maximum specific growth rate (μmax). The Total Phenolic Content (TPC) of the samples tested as a function of açaí concentration was also determined. Results obtained showed that the MIC was 10 g/L, containing 2154.91 ± 126.10 mg of Gallic Acid Equivalents (GAE)/L. At non-inhibitory doses, regardless of temperature, the higher the açaí concentration, the higher the value of λ, and the effect of a variation in açaí concentration on λ was greater at high temperatures. Therefore, the addition of non-inhibitory doses of açaí could provide a means of controlling the growth of L. monocytogenes if a cold chain failure occurs, or if any other reason compromises the microbiological safety of minimally processed foods. Consequently, the present study is important both for the science community and for the food industry because it provides the first mathematical characterization of açaí antimicrobial activity and reveals its potential use to control the concentration and growth of pathogenic bacteria in foods free of non-synthetic additives.

Kinetic investigation of the rate-limiting step of manganese- and iron-lipoxygenases

Lipoxygenases (LOX) oxidize polyunsaturated fatty acids to hydroperoxides, which are generated by proton coupled electron transfer to the metal center with FeIIIOH− or MnIIIOH−. Hydrogen abstraction by FeIIIOH− of soybean LOX-1 (sLOX-1) is associated with a large deuterium kinetic isotope effect (D-KIE). Our goal was to compare the D-KIE and other kinetic parameters at different temperatures of sLOX-1 with 13R-LOX with catalytic manganese (13R-MnLOX). The reaction rate and the D-KIE of sLOX-1 with unlabeled and [11-2H2]18:2n-6 were almost temperature independent with an apparent D-KIE of ∼56 at 30°C, which is in agreement with previous studies. In contrast, the reaction rate of 13R-MnLOX increased 7-fold with temperature (8–50°C), and the apparent D-KIE decreased linearly from ∼38 at 8°C to ∼20 at 50°C. The kinetic lag phase of 13R-MnLOX was consistently extended at low temperatures. The Phe337Ile mutant of 13R-MnLOX, which catalyzes antarafacial hydrogen abstraction and oxygenation in analogy with sLOX-1, retained the large D-KIE and its temperature-dependent reaction rate. The kinetic differences between13R-MnLOX and sLOX-1 may be due to protein dynamics, hydrogen donor–acceptor distances, and to the metal ligands, which may not equalize the 0.7V-gap between the redox potentials of the free metals.

Perturbing the Water Cavity Surrounding the Manganese Cluster by Mutating the Residue D1-Valine 185 Has a Strong Effect on the Water Oxidation Mechanism of Photosystem II

The active site of water oxidation in Photosystem II (PSII) is a Mn4CaO5 cluster that is located in a cavity between the D1 and CP43 protein subunits by which it is coordinated. The remainder of this cavity is filled with water molecules, which serve as a source of substrate and participate in poorly understood hydrogen bond networks that may modulate the function of the Mn4CaO5 cluster. These water molecules interact with the first and second sphere amino acid ligands to the Mn4CaO5 cluster and some water interacts directly with the Mn4CaO5 cluster. Here, the results of mutations to the amino acids that line the walls of several predicted cavities in the immediate vicinity of the Mn4CaO5 cluster were examined in Synechocystis sp. PCC 6803. Of these, mutations of Val185 in the D1 subunit resulted in the most interesting functional alterations. The hydrophobic D1-Val185 occupies a location contacting water molecules that are positioned between the redox active tyrosine (YZ) and the putative proton gate residue, D1-Asp61, and at a position opposite the oxo bridge atom, O5, of the cluster. Mutations of the residue D1-Val185 were produced, with the intention that the substitute residue would extend into the water cavity that includes H2O molecules that interact with the Mn4CaO5 cluster, amino acid ligands of the Mn4CaO5 cluster, YZ and the chloride co-factor of PSII. Three of these mutants, D1-Val185Asn, D1-Val185Thr, and D1-Val185Phe, were able to accumulate significant levels of charge separating PSII and were characterized using polarographic and fluorescent techniques. Of the three substitutions, the phenylalanine substitution was the most severe with a complete inability to evolve oxygen, despite being able to accumulate PSII and to undergo stable charge separation. The threonine substitution had no apparent effect on oxygen evolution other than a 40% reduction in the steady state rate of O2 production compared to the case of wild-type Synechocystis , due to a reduced ability to accumulate PSII centers. The asparagine substitution produced the most complex phenotype with respect to O2 evolution. Although still able to evolve oxygen, D1-Val185Asn does so less efficiently than wild-type PSII, with a higher miss factor than that for the wild type. Most significantly, asparagine substitution dramatically retards the rate of O2 release and results in an extension of the kinetic lag phase prior to O2 release that is highly reminiscent of the effects of mutations produced at D1-Asp61. The observed effects of the D1-Val185Phe and D1-Val185Asn mutations may be due to alterations in the environment of nearby chloride co-factor of PSII and/or alterations in the hydrogen bond network, perhaps impeding the movement of water to a binding site on the metal cluster.

Antimicrobial Potential of Flavoring Ingredients AgainstBacillus cereusin a Milk-Based Beverage

Natural ingredients--cinnamon, cocoa, vanilla, and anise--were assessed based on Bacillus cereus vegetative cell growth inhibition in a mixed liquid whole egg and skim milk beverage (LWE-SM), under different conditions: ingredient concentration (1, 2.5, and 5% [wt/vol]) and incubation temperature (5, 10, and 22 °C). According to the results obtained, ingredients significantly (p<0.05) reduced bacterial growth when supplementing the LWE-SM beverage. B. cereus behavior was mathematically described for each substrate by means of a modified Gompertz equation. Kinetic parameters, lag time, and maximum specific growth rate were obtained. Cinnamon was the most bacteriostatic ingredient and cocoa the most bactericidal one when they were added at 5% (wt/vol) and beverages were incubated at 5 °C. The bactericidal effect of cocoa 5% (wt/vol) reduced final B. cereus log10 counts (log Nf, log10 (colony-forming units/mL)) by 4.10 ± 0.21 log10 cycles at 5 °C.

Stochasticity in Colonial Growth Dynamics of Individual Bacterial Cells

Conventional bacterial growth studies rely on large bacterial populations without considering the individual cells. Individual cells, however, can exhibit marked behavioral heterogeneity. Here, we present experimental observations on the colonial growth of 220 individual cells of Salmonella enterica serotype Typhimurium using time-lapse microscopy videos. We found a highly heterogeneous behavior. Some cells did not grow, showing filamentation or lysis before division. Cells that were able to grow and form microcolonies showed highly diverse growth dynamics. The quality of the videos allowed for counting the cells over time and estimating the kinetic parameters lag time (λ) and maximum specific growth rate (μmax) for each microcolony originating from a single cell. To interpret the observations, the variability of the kinetic parameters was characterized using appropriate probability distributions and introduced to a stochastic model that allows for taking into account heterogeneity using Monte Carlo simulation. The model provides stochastic growth curves demonstrating that growth of single cells or small microbial populations is a pool of events each one of which has its own probability to occur. Simulations of the model illustrated how the apparent variability in population growth gradually decreases with increasing initial population size (N(0)). For bacterial populations with N(0) of >100 cells, the variability is almost eliminated and the system seems to behave deterministically, even though the underlying law is stochastic. We also used the model to demonstrate the effect of the presence and extent of a nongrowing population fraction on the stochastic growth of bacterial populations.

A universal fluorescent acceptor for high-performance liquid chromatography analysis of pro- and eukaryotic polysialyltransferases

Polysialyltransferases (polySTs) play critical roles in diverse biological processes, including neural development, tumorigenesis, and bacterial pathogenesis. Although the bacterial enzymes are presumed to have evolved to provide molecular mimics of the host-specific polysialic acid, no analytical technique is currently available to facilitate a direct comparison of the bacterial and vertebrate enzymes. Here we describe a new fluorescent acceptor, a 1,2-diamino-4,5-methylenedioxybenzene (DMB)-labeled trimer of α2,8-linked sialic acid (DMB–DP3), which primes both pro- and eukaryotic polySTs. High-performance liquid chromatography separation and fluorescence detection (HPLC–FD) of reaction products enabled the sensitive and quantitative detection of polyST activity, even using cell lysates as enzyme source, and revealed product profiles characteristic of each enzyme. Single product resolution afforded by this assay system revealed mechanistic insights into a kinetic lag phase exhibited by the polyST from Neisseria meningitidis serogroup B during chain elongation. DMB–DP3 is the first fluorescent acceptor shown to prime the mammalian polySTs. Moreover, product profiles obtained for the two murine polySTs provided direct biochemical evidence for enzymatic properties that had, until now, only been inferred from the analysis of biological samples. With DMB–DP3, we introduce a universal acceptor that provides an easy, fast, and reliable system for the comprehensive mechanistic and comparative analysis of polySTs.