Longer Lag Phase Research Articles

Lag Plot: A Novel Method to Determine Viable Bacterial Cell Number via a Single OD Measurement.

Bactericidal activity is a valuable parameter which is considered and measured for antimicrobial compounds. The available standard protocol to evaluate bactericidal activity is based on the direct colony count. Colony counting requires serial dilution, plating, overnight incubation, and direct counting, which is time-and labor-intensive. In regard to eliminate direct plate count, novel techniques were developed based on the real-time growth monitoring which can come with some limitations and drawbacks. These drawbacks encourage us to develop a novel technique with simple procedure to determine viable bacterial cell count. In this procedure, real-time growth monitoring is not required. In fact, after an incubation time, the number of viable bacteria can be determined with a single OD measurement and through an equation. In this regard, four standard bacterial strains, including Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Bacillus subtilis (ATCC 23857), were cultured with descending inoculum densities (1.5 × 107 to 15CFU/mL) and growth curves were drawn. As expected, growth in the samples with lower inoculum densities recorded with longer lag phase. Also, a direct relation was observed between the recorded turbidities and initial cell counts. A logarithmic curve (lag plot) was obtained by plotting the OD, after 12-18h incubation, against initial cell counts. In all examined strains, R2 was calculated in the range of 0.96-0.99 which is acceptable value for coefficient of determination. Equation corresponding to the lag plot was obtained and called lag equation. This equation is applicable to calculate the number of viable cells in unknown samples simply by inoculation, incubation, and single OD measurement. Developed technique can be introduced as a simple substitution for labor- and time-intensive direct colony counting.

Oxidation of Microcystis aeruginosa and Microcystins with Peracetic Acid.

Peracetic acid (PAA) shows potential for use in drinking water treatment as an alternative to prechlorination, such as for mussel control and disinfection by-product precursor destruction, though its impact as a preoxidant during cyanobacterial blooms remains underexplored. Here, Microcystis aeruginosa inactivation and microcystin-LR and -RR release and degradation using PAA were explored. The toxin degradation rates were found to be higher in alkaline conditions than in neutral and acidic conditions. However, all rates were significantly smaller than comparable rates when using free chlorine. The inactivation of M. aeruginosa cells using PAA was faster at acidic pH, showing immediate cell damage and subsequent cell death after 15-60 min of exposure to 10 mg/L PAA. In neutral and alkaline conditions, cell death occurred after a longer lag phase (3-6 h). During cell inactivation, microcystin-LR was released slowly, with <35% of the initial intracellular toxins measured in solution after 12 h of exposure to 10 mg/L PAA. Overall, PAA appears impractically slow for M. aeruginosa cell inactivation or microcystin-LR and -RR destruction in drinking water treatment, but this slow reactivity may also allow it to continue to be applied as a preoxidant for other purposes during cyanobacterial blooms without the risk of toxin release.

A role for the stringent response in ciprofloxacin resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a major cause of nosocomial infections and the leading cause of chronic lung infections in cystic fibrosis and chronic obstructive pulmonary disease patients. Antibiotic treatment remains challenging because P. aeruginosa is resistant to high concentrations of antibiotics and has a remarkable ability to acquire mutations conferring resistance to multiple groups of antimicrobial agents. Here we report that when P. aeruginosa is plated on ciprofloxacin (cipro) plates, the majority of cipro-resistant (ciproR) colonies observed at and after 48 h of incubation carry mutations in genes related to the Stringent Response (SR). Mutations in one of the major SR components, spoT, were present in approximately 40% of the ciproR isolates. Compared to the wild-type strain, most of these isolates had decreased growth rate, longer lag phase and altered intracellular ppGpp content. Also, 75% of all sequenced mutations were insertions and deletions, with short deletions being the most frequently occurring mutation type. We present evidence that most of the observed mutations are induced on the selective plates in a subpopulation of cells that are not instantly killed by cipro. Our results suggests that the SR may be an important contributor to antibiotic resistance acquisition in P. aeruginosa.

Assessment of a Low-Cost Hydrogen Sensor for Detection and Monitoring of Biohydrogen Production during Sugarcane Straw/Vinasse Co-Digestion

In this work, hydrogen production from the co-digestion of sugarcane straw and sugarcane vinasse in the dark fermentation (DF) process was monitored using a cost-effective hydrogen detection system. This system included a sensor of the MQ-8 series, an Arduino Leonardo board, and a computer. For the DF, different concentrations of sugarcane vinasse and volumetric ratios of vinasse/hemicellulose hydrolysate were used together with a thermally pretreated inoculum, while the hydrogen detection system stored the hydrogen concentration data during the fermentation time. The results showed that a higher concentration of vinasse led to higher inhibitors for the DF, resulting in a longer lag phase. Additionally, the hydrogen detection system proved to be a useful tool in monitoring the DF, showcasing a rapid response time, and providing reliable information about the period of adaptation of the inoculum to the substrate. The measurement system was assessed using the error metrics SE, RMSE, and MBE, whose values ranged 0.6 and 5.0% as minimum and maximum values. The CV (1.0–8.0%) and SD (0.79–5.62 ppm) confirmed the sensor’s robustness, while the ANOVA at the 5% significance level affirmed the repeatability of measurements with this instrument. The RMSE values supported the accuracy of the sensor for online measurements (6.08–14.78 ppm). The adoption of this straightforward and affordable method sped up the analysis of hydrogen in secluded regions without incurring the expenses associated with traditional measuring instruments while offering a promising solution for biomass valorization, contributing to the advancement of rural green energy initiatives in remote areas.

Electromagnetic fields effects on microbial growth in cocoa fermentation: A controlled experimental approach using established growth models

Understanding the effects of electromagnetic fields is crucial in the fermentation of cocoa beans, since through precise control of fermentation conditions the sensory and nutritional properties of cocoa beans could be improved. This study aimed to evaluate the effect of oscillating magnetic fields (OMF) on the kinetic growth of the core microbial communities of the Collections Castro Naranjal (CCN 51) cocoa bean. The data was obtained by three different models: Gompertz, Baranyi, and Logistic. The cocoa beans were subjected to different OMF strengths ranging from 0 mT to 80 mT for 1 h using the Helmholtz coil electromagnetic device. The viable microbial populations of lactic acid bacteria (LAB), acetic acid bacteria (AAB), and yeast (Y) were quantified using the colony-forming unit (CFU) counting method. The logistic model appropriately described the growth of LAB and Y under magnetic field exposure. Whereas the Baranyi model was suitable for describing AAB growth. The microbial populations in cocoa beans exposed to magnetic fields showed lower (maximum specific growth rate (μmax), values than untreated controls, with AAB exhibiting the highest average growth rate value at 5 mT and Y having the lowest average maximum growth rate value at 80 mT. The lower maximum specific growth rates and longer lag phases when exposed to magnetic fields compared to controls demonstrate the influence of magnetic fields on microbial growth kinetics.

A stochastic approach for modelling the in-vitro effect of osmotic stress on growth dynamics and persistent cell formation in Listeria monocytogenes

Persistent bacteria (or persisters) can be defined as a microbial subpopulation that, exposed to bactericidal treatment, is killed more slowly than the rest of the population they are part of. They stochastically originate in response to environmental stressors or spontaneously without external signals. When transferred into a fresh medium, persisters can resume active replication although they spend more time adapting to the new conditions remaining in the lag phase longer. They were studied for decades for their ability to survive antibiotic treatments while studies on their formation in food and potential impact on their safety are lacking. The most common food preservation techniques may act as stressors that trigger the formation of persistent bacteria able to survive bactericidal treatments and grow later in foods during storage. This study aimed to investigate a possible relationship between exposure to different salt concentrations (osmotic stress) and the amount of persisters triggered in a strain of Listeria monocytogenes. Furthermore, we described this phenomenon from a mathematical perspective through predictive microbiology models commonly used in the food field. The lag time distribution of a L. monocytogenes ATCC 7644 strain grown in broth with additional 2 %, 4 % and 6 % NaCl was evaluated using the software ScanLag. It uses office scanners to automatically record the colony growth on agar plates and evaluate the frequency distribution of their appearance times (lag phase) by automated image analysis. The same broth cultures were diluted to equalize salt concentration and transferred into a fresh broth to evaluate how the previous salt exposure impacted their growth kinetics. The observed growth curves were reproduced using predictive models in which the mean duration of the lag phase of the whole population took into account the occurrence of persisters with a longer lag phase. The models were solved first using a deterministic approach and then a stochastic one introducing a stochastic term that mimics the variability of lag phase duration due to the persisters occurrence. Results showed that the growth of L. monocytogenes in broth with additional NaCl might trigger the formation of persistent cells whose number increased consistently with salt concentrations. The proposed predictive approach reproduced the observed real curves in strong agreement, especially through the stochastic resolution of the models. Persistence is currently a neglected bacterial defence strategy in the food sector but the persisters' formation during production cannot be excluded; therefore, further insights on the topic are certainly desirable.

Plasticity of growth laws tunes resource allocation strategies in bacteria.

Bacteria like E. coli grow at vastly different rates on different substrates, however, the precise reason for this variability is poorly understood. Different growth rates have been attributed to 'nutrient quality', a key parameter in bacterial growth laws. However, it remains unclear to what extent nutrient quality is rooted in fundamental biochemical constraints like the energy content of nutrients, the protein cost required for their uptake and catabolism, or the capacity of the plasma membrane for nutrient transporters. Here, we show that while nutrient quality is indeed reflected in protein investment in substrate-specific transporters and enzymes, this is not a fundamental limitation on growth rate, at least for certain 'poor' substrates. We show that it is possible to turn mannose, one of the 'poorest' substrates of E. coli, into one of the 'best' substrates by reengineering chromosomal promoters of the mannose transporter and metabolic enzymes required for mannose degradation. This result falls in line with previous observations of more subtle growth rate improvement for many other carbon sources. However, we show that this faster growth rate comes at the cost of diverse cellular capabilities, reflected in longer lag phases, worse starvation survival and lower motility. We show that addition of cAMP to the medium can rescue these phenotypes but imposes a corresponding growth cost. Based on these data, we propose that nutrient quality is largely a self-determined, plastic property that can be modulated by the fraction of proteomic resources devoted to a specific substrate in the much larger proteome sector of catabolically activated genes. Rather than a fundamental biochemical limitation, nutrient quality reflects resource allocation decisions that are shaped by evolution in specific ecological niches and can be quickly adapted if necessary.

Illumination Effects on Bacteriorhodopsin Accumulation in Archaeon Halobacterium Halobium

Highlights Effects of varying illumination conditions, including light intensity, light color, and illumination pattern on archaeon Halobacterium halobium growth and bacteriorhodopsin (BR) accumulation were first evaluated. LED green light was much more energy-efficient than the red and blue lights in this research. If energy consumption was not a concern, LED blue and red lights (or their combinations) were more effective for cell growth and BR accumulation, respectively, with a similar light intensity. Abstract. This study was to understand the effect of LED light intensity, color, and illumination pattern on the growth of and bacteriorhodopsin (BR) accumulation in archaeon Halobacterium halobium. Experimental results showed that archaeon growth and BR content increased with increasing white light intensity. Green and white LED lights were found to be the most effective for archaeon growth and BR accumulation; here, effectiveness is defined based on photons shined on the bioreactor. The 12-h light/12-h dark cycle illumination pattern resulted in longer lag phase but achieved higher final cell growth and BR accumulation than continuous white light or an instant flash of light/dark illumination. The fade pattern that had smooth transitioning among green, blue, and red lights was better than the jump pattern without transitioning. The highest cell dry weight and BR content of H. halobium were 1.84 g l-1 and 11.76 mg l-1, respectively, under 29.85 µmol/m2s LED white light illumination. The green LED light of 1.39 µmol/m2s had the highest energy-specific conversion effectiveness and saved energy consumption by 84 and 87% per cell biomass and BR dry weight, respectively, compared to the worst case of LED white light of 29.85 µmol/m2s. Keywords: Archaeon, Bacteriorhodopsin, Halobacterium halobium, Illumination pattern, LED light, Light color.

Enzymatic degradation of poly(ethylene terephthalate) (PET): Identifying the cause of the hypersensitive enzyme kinetic response to increased PET crystallinity

Plastic pollution poses a significant environmental challenge, with poly(ethylene terephthalate) (PET) being a major contributor due to its extensive use in single use applications such as plastic bottles and other packaging material. Enzymatic degradation of PET offers a promising solution for PET recycling, but the enzyme kinetics in relation to the degree of crystallinity (XC) of the PET substrate are poorly understood. In this study, we investigated the hypersensitive enzyme kinetic response on PET at XC from ∼8.5–12% at 50 °C using the benchmark PET hydrolysing enzyme LCCICCG. We observed a substantial reduction in the maximal enzymatic reaction rate (invVmax) with increasing XC, corresponding to a 3-fold reduction in invVmax when the XC of PET increased from 8.6% to 12.2%. The kinetic analysis revealed that the level of the Mobile Amorphous Fraction (XMAF) was a better descriptor for the enzymatic degradation rate response than XC (or (100%-XC)). By continuous monitoring of the enzymatic reaction progress, we quantified the lag phase prolongation in addition to the steady-state kinetic rates (vss) of the reactions and found that the duration of the lag phase of a reaction could be predicted from the vss and XC by multiple linear regression modeling. The linear correlation between the duration of the lag phase and the vss of the enzymatic PET degradation affirmed that the LCCICCG worked via a random/endo-type enzymatic attack pattern. The longer lag phase at increased XC of PET is proposed to be due to increased substrate entanglement density as well as unproductive enzyme binding to the crystalline regions of PET. The findings enhance our understanding of PET enzymatic degradation kinetics and its dependence on substrate composition, i.e., XMAF and XC.

Microcystin-LR Biodestruction by Autochthonous Microbiota of Different Water Bodies in the North-West of Russia

The ability of the autochthonous microbiota of different water bodies in the North-West of the Russian Federation to degrade the highly toxic microcystin-LR (MC-LR) was shown for the first time. The maximum rate of degradation of MC-LR was noted in water samples from the Sestroretskij Razliv Lake and the Lower Suzdal Lake during the period of mass development of cyanobacteria. In water samples from Lake Ladoga, where no toxic cyanobacterial blooms were previously noted, MC-LR biodegradation proceeded at a much lower rate and with a longer lag phase. The composition of MC-LR biodegradation products indicates the presence in the autochthonous microbiota of the studied objects of microorganisms capable of degrading microcystins both by the mlr mechanism and by the biochemical pathway involving glutathione.

Aquafaba from lentils (Lens culinaris) and common beans (Phaseolus vulgaris): a study of cooking variables and drying process on the techno‐functional and microstructural properties

SummaryThe effect of cooking time and the solid content was analysed in the techno‐functional properties of lentils and common beans aquafaba, and their powders obtained through spray‐drying. Foam drainage kinetic and emulsion destabilisation were successfully modelled showing that lentil aquafaba had better foaming capacity than beans, due to its higher protein content. Moreover, an increase in cooking time results in more liquid incorporated into the foam and a longer lag phase. Bean aquafaba had acceptable emulsifying capacity, attributable to higher soluble carbohydrates. Additionally, microstructure, Fourier Transform Infrared Spectrometry, rehydration capacity, and flowability of aquafaba powders were studied. Microscopy of dried lentil aquafaba showed damaged and collapsed particles, while bean powder consisted of spherical smooth particles. The dominant factor influencing aquafaba functional properties and powder characteristics was the legume type, attributed to their chemical compositions. These findings broaden the insights of alternative sources of aquafaba and enhance the industrial applicability of these residues through highly rehydratable and functional powders.

Label-free quantitative proteomics reveals the antibacterial mechanism of rosemary essential oil against Salmonella enterica serovar Typhimurium

Rosemary essential oil (REO) shows various biological functions and is widely used in the cosmetic and pharmaceutical industries. Salmonella enterica serovar Typhimurium infection treated by antibiotics leads to serious drug resistance and public health hazard . REO as an antibiotic alternative has become the focus of considerable research efforts. However, knowledge regarding the S. Typhimurium growth characteristics and proteomic responses to REO exposure remains unclear. Here, we evaluated the commonalities and differences in chemical compositions of REOs extracted from Salvia rosmarinus cultivars, ‘Algarve’ (AL), ‘Dutch Mill’ (DM), and ‘Majorca Pink’ (MP) using gas chromatography-mass spectrometry. The three REOs displayed distinct chemical composition profiles, and the DM-REO, predominantly composed of 1,8-cineole and α-pinene, had the highest antibacterial activity. Sublethal levels of DM-REO caused irreversible cell membrane damage, longer lag phases, decreased growth rates, and lower maximum optical densities of S. Typhimurium. The underlying molecular regulatory mechanisms of S. Typhimurium under sublethal DM-REO treatment were investigated using label-free quantitative proteomics. Four hundred thirty-six differentially expressed proteins mainly involved in metabolism, ABC transporters , two-component system, quorum sensing , bacterial chemotaxis, and flagellar assembly were identified. The phenotypic observation test showed that DM-REO significantly inhibited S. Typhimurium motility and quorum sensing. These results increase our understanding of the antibacterial mechanism of REO inhibition in S. Typhimurium. • The chemical compositions and antibacterial properties of three REOs were assessed. • The 1/2MIC REO caused longer lag phases and lower growth rates of S. Typhimurium. • The REO was found to damage the cell membrane integrity of S. Typhimurium. • The antibacterial mechanism of REO was explored by Laber-free quantitative proteomics. • The REO inhibited S. Typhimurium motility and quorum sensing.

Promoting the overall energy profit through using the liquid hydrolysate during microwave hydrothermal pretreatment of wheat straw as co-substrate for anaerobic digestion

Liquid hydrolysate (LH) derived from the microwave hydrothermal pretreatment (MHP) of wheat straw (WS) was anaerobically digested together with the solid residual to promote the overall energy profit. Different MHP temperatures (90, 120, 150, 180 °C) and retention times (10, 20, 40 min) were investigated. Increased MHP intensity generated plenty of VFAs (mainly acetate) and phenols in the LH, implying the double-side effect of LH on AD. The highest methane production of 227.92 mL CH4·gVS-1 Raw was obtained with MHP at 120 °C for 10 min, 21.53 % higher than the control. While, MHP at 180 °C for 40 min exhibited 29.02 % lower methane production (113.13 mL CH4·gVS-1 Raw) and 115.86 % longer lag phase (3.13 days) than the control. Butyrate fermentation endowed the treatment groups of 180 °C with resilience from the overload and inhibition. Methanosarcina was largely enriched by the abundant acetate in LH on the early stage of anaerobic digestion (AD), especially when with high MHP intensity. Increased abundance of Methanosaeta and Methanobacterium played a crucial role in maintaining methane production at the middle and later stage. The high number of species and evenness in methanogens community were beneficial for the startup of batch AD. Although negative net energy was obtained, the lower ratio of energy input and output compared with the most researches using the solid residual after MHP as the sole substrate for AD demonstrated the contribution of LH to the overall energy profit.

Mixed culture chain elongation for consumption of acetate and ethanol in anaerobic fermentation: The impact of salt type, dosage and acclimation

Microbial chain elongation is a newly developed carboxylate platform-based bioprocess, which often encounters high salinity stress due to saline feedstock and pH adjustment. In this study, we systematically investigated the effects of salt types (Na+, K+, and NH4+), dosage, and salinity acclimation on microbial chain elongation, and identified the microbial community by high throughput 16S rRNA gene sequencing. The results showed that a high level of Na+ and NH4+ (12.5 g/L of cations) exerted seriously inhibitory effects without chain elongating activity, while K+ had the slightest inhibition only with a little longer lag phase and lower products yield. The chain elongating products yields and the selectivity of caproate decreased with the increasing Na+ concentration, and 8.6 g/L of Na+ was found to be the threshold value for un-acclimated inoculum used for chain elongation. The acclimation to high saline conditions greatly promoted the consumption of acetate and ethanol with a shorter lag phase, and recovered a robust elongating activity for butyrate production. Furthermore, the high throughput 16S rRNA gene sequencing analysis results indicated that six genera, such as Clostridium IV and Clostridium sensu stricto, closely relating chain elongation process were depressed by high salinity, and the salinity acclimation helped to enrich the functional microbes. These findings could provide useful information for engineering microbial chain elongation process under saline conditions.

Verbascoside: An Efficient and Safe Natural Antibacterial Adjuvant for Preventing Bacterial Contamination of Fresh Meat.

Inappropriate and disproportionate antibiotic use contributes immensely to the development of antibiotic resistance in bacterial species associated with food contamination. Therefore, alternative strategies to treat multidrug-resistant (MDR) bacterial infections are urgently needed. In this study, verbascoside was shown to exhibit excellent antibacterial activity and synergistic effects in combination with cell wall synthesis-inhibiting antibiotics, indicating that it can be used as an adjuvant to restore or increase the activity of antibiotics against resistant pathogens. In a mechanistic study, higher concentrations of verbascoside resulted in a longer lag phase and a lower specific exponential-phase growth rate of bacteria. Furthermore, verbascoside exerted its antimicrobial activity through multiple mechanisms, including cell membrane dysfunction, biofilm eradication and changes in cell morphology. The promising antibacterial activity and in vitro safety assessment results suggested that verbascoside can be used as a food additive for fresh meat preservation. Treatment with medium and high doses of verbascoside caused significant bacterial death in meat samples, slowed the spoilage rate, and extended the shelf life. Collectively, verbascoside is expected to be useful as an antibiotic adjuvant to prevent or treat resistant bacteria-related infections and an alternative novel antimicrobial additive in the food industry.

High Cell Density Cultivation of Escherichia coli DH5α in Shake Flasks with a New Formulated Medium

High cell density cultivation necessitates cell division and biomass formation, the mechanisms of which remain poorly understood, especially from the cellular energetics perspective. Specifically, the sensing of energy abundance and the channelling of nutritional energy into biomass formation and cell maintenance remains enigmatic at the sensory, effector and decision levels. Thus, optimization of cell growth remains an iterative trial and error process where the principal parameters are growth medium composition and incubation temperature. In this study, a new semidefined formulated medium was shown to be useful for high cell density cultivation of Escherichia coli DH5α (ATCC 53868). Comprising K2HPO4, 12.54; KH2PO4, 2.31; D-Glucose, 4.0; NH4Cl, 1.0; Yeast extract, 12.0; NaCl, 5.0; MgSO4, 0.24; the medium possessed a high capacity phosphate buffer able to moderate pH fluctuations during cell growth known to be detrimental to biomass formation. With glucose and NH4Cl providing the nutrients for initial growth, followed by a lag phase of 3 hours, a maximal optical density of 12.0 was obtained after 27 hours of cultivation at 37 oC and 230 rpm. Yeast extract provides a secondary source of carbon and nitrogen. Maximal optical density obtained in formulated medium was higher than the 10.1, 4.2, and 3.4 obtained in Tryptic Soy Broth, M9 with 1 g/L of yeast extract, and LB Lennox, respectively. Cultivation of E. coli DH5α in formulated medium with 6 g/L of glucose resulted in a longer lag phase of 8 hours and a longer time (68 hours) to attainment of maximal optical density, which marked the upper limit of glucose concentration beyond which biomass formation would be reduced. Specifically, glucose concentration above 6 g/L markedly reduced biomass formation possibly due to the environmental stress arising from low pH in the culture broth. Glucose concentration below 4 g/L, on the other hand, reduced biomass formation through a smaller pool of nutrients serving as biomass building blocks. Deviation from 1:1 molar ratio between glucose and NH4Cl was not detrimental to biomass formation and growth rates. Collectively, a semi-defined formulated medium could increase optical density of E. coli DH5α beyond that of LB Lennox and Tryptic Soy Broth, and may find use in cultivation of cells for applied microbiology research.

Immobilization of active ammonia-oxidizing archaea in hydrogel beads

Ammonia-oxidizing archaea (AOA) are major players in the nitrogen cycle but their cultivation represents a major challenge due to their slow growth rate and limited tendency to form biofilms. In this study, AOA was embedded in small (~2.5 mm) and large (~4.7 mm) poly(vinyl alcohol) (PVA)—sodium alginate (SA) hydrogel beads cross-linked with four agents (calcium, barium, light, or sulfate) to compare the differences in activity, the diffusivity of nitrogen species (NH4+, NO2−, and NO3−), and polymer leakage in batch systems over time. Sulfate-bound PVA-SA beads were the most stable, releasing the lowest amount of polymer without shrinking. Diffusion coefficients were found to be 2 to 3 times higher in hydrogels than in granules, with ammonium diffusivity being ca. 35% greater than nitrite and nitrate. Despite a longer lag phase in small beads, embedded AOA sustained a high per volume rate of ammonia oxidation compatible with applications in research and wastewater treatment.

Chemo-Radiative Stress of Plasma as a Modulator of Charge-Dependent Nanodiamond Cytotoxicity

Efficient and selective internalization of nanoscale diamonds (also termed nanodiamonds, NDs) by living cells is of fundamental importance for their bionanotechnological applications. The biocompatibility of NDs is well established and has been suggested to arise from the limited membrane perturbation during their cellular translocation. However, the latter may be affected when cells are subjected to external stress. This study shows that the oxidative stress generated by atmospheric pressure cold plasmas (APCP) alters cell sensitivity to NDs, and their cytotoxicity profile. Both positively and negatively charged NDs are nontoxic to cells, here Saccharomyces cerevisiae and human cell lines, i.e., near-normal human mammary epithelial cells (MCF-10A) and breast cancer cells (MDA-MB-468 and T47D), unless the APCP stress is introduced. A brief exposure of the cells to APCP leads to a significant increase in their ND affinity (uptake and/or surface attachment) and intracellular ROS accumulation, particularly for positively charged NDs and both yeast and cancer cells. A concomitant decrease in cell viability and yeast cell growth, reflected by longer lag phases and lower cell density after 24 h of incubation, demonstrates a considerably enhanced ND toxicity to these cells. These results suggest that chemo-radiative stress, such as that produced by plasma, may influence the toxicity of nanoparticles to different cells, with specificity achieved through controlling particle charges. Moreover, since oxidative stress is not only associated with the use of APCP but can arise unintentionally within an organism and/or in the environment, these findings may have broader implications for the use of nontoxic nanoparticles in bionanotechnology in general.

Tyrosinase Inhibition and Kinetic Details of Puerol A Having But-2-Enolide Structure from Amorpha fruticosa.

Puerol A (1) from Amorpha fruticosa showed highly potent inhibition against both monophenolase (IC50 = 2.2 μM) and diphenolase (IC50 = 3.8 μM) of tyrosinase. We tried to obtain a full story of enzyme inhibitory behavior for inhibitor 1 because the butenolide skeleton has never been reported as a tyrosinase inhibitor. Puerol A was proved as a reversible, competitive, simple slow-binding inhibitor, according to the respective parameters; k3 = 0.0279 μM−1 min−1 and k4 = 0.003 min−1. A longer lag-phase and a reduced static-state activity of the enzyme explained that puerol A had a tight formation of the complex with Emet. Dose-dependent inhibition was also confirmed by high-performance liquid chromatography (HPLC) analysis using N-acetyl-l-tyrosine as a substrate, which was completely inhibited at 20 μM. A high binding affinity of 1 to tyrosinase was confirmed by fluorescence quenching analysis. Moreover, puerol A decreased melanin content in the B16 melanoma cell dose-dependently with an IC50 of 11.4 μM.

Is the Arrhenius-correction of biodegradation rates, as recommended through REACH guidance, fit for environmentally relevant conditions? An example from petroleum biodegradation in environmental systems

Biodegradation is a major determinant of chemical persistence in the environment and an important consideration for PBT and environmental risk assessments. It is influenced by several environmental factors including temperature and microbial community structure. According to REACH guidance, a temperature correction based on the Arrhenius equation is recommended for chemical persistence data not performed at the recommended EU mean surface water temperature. Such corrections, however, can lead to overly conservative P/vP assessments. In this paper, the relevance of this temperature correction is assessed for petroleum hydrocarbons, using measured surface water (marine and freshwater) degradation half-time (DT50) and degradation half-life (HL) data compiled from relevant literature. Stringent screening criteria were used to specifically select data from biodegradation tests containing indigenous microbes and conducted at temperatures close to their ambient sampling temperature. As a result, ten independent studies were identified, with 993 data points covering 326 hydrocarbon constituents. These data were derived from tests conducted with natural seawater, or freshwater, at temperatures ranging from 5 to 21 °C. Regressions were performed on the full hydrocarbon dataset and on several individual hydrocarbons. The results were compared to the trend as predicted by the Arrhenius equation and using the activation energy (Ea) as recommend in the REACH Guidance. The comparison shows that the correction recommended in REACH Guidance over predicts the effect of temperature on hydrocarbon biodegradation. These results contrast with temperature manipulated inocula where the test temperature is different from the ambient sampling temperature. In these manipulated systems, the effect of temperature follows the Arrhenius equation more closely. In addition, a more striking effect of temperature on the lag phase was observed with longer lag phases more apparent at lower temperatures. This indicates that the effect of temperature may indeed be even lower when considering hydrocarbon biodegradation without the initial lag phase.