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ABSTRACT The lack of effective treatment options against Pseudomonas aeruginosa is one of the main contributors to the silent pandemic. Many antibiotics are ineffective against resistant isolates due to poor target site penetration, efflux, or β-lactamase hydrolysis. Critical insights to design optimized antimicrobial therapies and support translational drug development are needed. In the present work, we analyzed the periplasmic drug uptake and binding to PBPs of 11 structurally different β-lactams and 4 β-lactamase inhibitors (BLIs) in P. aeruginosa PAO1. The contribution of the most prevalent β-lactam resistance mechanisms to MIC and periplasmic target attainment was also assessed. Bacterial cultures (6.5 log10 CFU/mL) were exposed to 1/2× PAO1 MIC of each antibiotic for 30 min. Unbound PBPs were labeled with Bocillin FL and analyzed using a FluorImager. Imipenem extensively inactivated all targets. Cephalosporins preferentially targeted PBP1a and PBP3. Aztreonam and amdinocillin bound exclusively to PBP3 and to PBP2 and PBP4, respectively. Penicillins bound preferentially to PBP1a, PBP1b, and PBP3. BLIs displayed poor PBP occupancy. Inactivation of oprD elicited a notable reduction of imipenem target attainment, and it was to a lesser extent in the other carbapenems. Improved PBP occupancy was observed for the main targets of the widely used antipseudomonal penicillins, cephalosporins, meropenem, aztreonam, and amdinocillin upon oprM inactivation, in line with MIC changes. AmpC constitutive hyperexpression caused a substantial PBP occupancy reduction for the penicillins, cephalosporins, and aztreonam. Data obtained in this work will support the rational design of optimized β-lactam-based combination therapies against resistant P. aeruginosa infections. IMPORTANCE The growing problem of antibiotic resistance in Gram-negative pathogens is linked to three key aspects, (i) the progressive worldwide epidemic spread of multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) Gram-negative strains, (ii) a decrease in the number of effective new antibiotics against multiresistant isolates, and (iii) the lack of mechanistically informed combinations and dosing strategies. Our combined efforts should focus not only on the development of new antimicrobial agents but the adequate administration of these in combination with other agents currently available in the clinic. Our work determined the effectiveness of these compounds in the clinically relevant bacteria Pseudomonas aeruginosa at the molecular level, assessing the net influx rate and their ability to access their targets and achieve bacterial killing without generating resistance. The data generated in this work will be helpful for translational drug development.

Evaluation of mitochondrial function in aerobic cells is crucial for understanding the conditions that can potentially compromise their physiology. From the fields of Toxicology to Oncology, various approaches involving freshly isolated fractions of mitochondria, permeabilized cells, intact cells, or animal tissues have been employed to investigate metabolism through oxygen consumption.Several techniques are available for measuring oxygen consumption in liquids. These include polarography with oxygen electrodes, which can employ chemical, electrochemical, or optical detection methods, as well as the use of fluorescent or luminescent probes. In this chapter, we will review the concepts previously discussed in earlier editions, focusing on Clark-type electrodes for electrochemical detection. Additionally, we will explore other approaches that involve intact cells and tissue explants with minimal plasmatic membrane alterations. These techniques provide an integrated view of Glycolysis, Krebs Cycle and Oxidative Phosphorylation. Despite being a classical and cost-effective system, this elegant technique continues to amaze us with its versatility and generation of reliable data., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Macronutrients of pulses or cereals are stored in the cotyledon or endosperm cells with protection from intact cell walls. However, pulses and cereals are generally processed into fine particles during food production. For example, after milling, the macronutrients enclosed in the intact cells are released and are easily accessible to digestive enzymes in the gastrointestinal tract, leading to high metabolic responses. Therefore, studies on the health effects of intact cells and developing an alternative ingredient with a higher proportion of intact cells are areas of emerging interest. In this review, we highlighted the smallest unit of whole grain, an individual cell, as "nutritional capsules" and elucidated the structure–function of the nutritional capsules, followed by isolation techniques, as a potential novel functional ingredient and food. The polysaccharides' monomeric composition, secondary structure, and interactions determine the cell wall properties including the cell detachment during isolation and isolated cell properties. The intact cellular structure is retained after mild food processing and digestion, thereby, contributing to a lower extent/rate of digestion of entrapped macronutrients. Furthermore, the excursed intact capsules in the colonic environment modulate the population and diversity of microbiota, favouring the increased production of the short‐chain fatty acids (SCFAs). The structural schematic model of Type‐I and Type‐II cells is developed together with the schematics of the cell wall isolation process. The review provides a critical summary of the recent trends in intact plant cells as a functional‐nutritional food. It paves the way for the industrial production of intact cells as a novel food ingredient. [ABSTRACT FROM AUTHOR]

Mitochondria are organelles with significant cellular functions, especially cellular bioenergetics and apoptosis. They are structural and functional elements of cell respiration with the electron transport system (ETS), whose role is to provide adenosine triphosphate (ATP), used as a source of chemical energy. The Krebs cycle and fatty acid oxidation take place within mitochondria. Other metabolic pathways and cycles include some steps inside and outside the mitochondria (e.g., the urea cycle, steroid biosynthesis, heme biosynthesis, and cardiolipin synthesis). Dysfunction of mitochondria plays a critical role in the pathophysiology of a variety of diseases including degenerative diseases, aging, and cancer, etc. Nowadays the interest of the mitochondrial respiratory function is still increasing due to their importance in the physiology and pathophysiology of an organism. Neuroblastoma cell line SH-SY5Y is widely used as an in vitro model in neurodegenerative diseases, where mitochondrial dysfunction is considered as a key mechanism in pathophysiology of neurodegenerative disorders. This paper gives first insight into the mitochondrial respiration and characterization of SH-SY5Y cells, with basic information of respiration in different coupling control states including ROUTINE, LEAK, and maximal electron transport (ET) capacity. [ABSTRACT FROM AUTHOR]

Starch is the major stored carbohydrate in grains and legumes. Apart from nutritional functionality, starch has multiple industrial applications. Starch is synthesised in plant cells along with proteins, lipids, and polyphenols. These macromolecules interact both in planta as well as during downstream processing, e.g., extraction of starch. Whilst the interaction of starch with protein, lipids, and other hydrocolloids during processing is widely reported, the in planta interactions and their effect on food and nutritional functionality is mostly overlooked. This review provides an overview of mechanisms of interaction of starch with protein, lipids, and polyphenols in planta and the effect of these interactions on food processing as well as human nutrition. The interaction of starch with other macronutrients as well as polyphenols are described at the granular level as well as at the cellular level and presented in a schematic model (Fig. 1). The granular level interactions such as with surface and internal protein and lipids associated with granules, extra-granular storage proteins and formation of amylose-lipid and polyphenol complexes primarily affect the processing functionality of starch, whereas cellular level interactions and encapsulation enhance nutritional functionality of starch in terms of lowering the metabolic responses from energy dense nutrients. Thus, consideration of in planta interactions among macronutrients as well as with cell walls is important during processing, in both selection of ingredients as well as the formulation of foods. Image 1 • Starch interacts with proteins, lipids, phytochemicals and cell walls in planta. • In planta interactions affect the functional and nutritional properties of starch. • Intact cellular structures conserve interactions and nutritional functionality. [ABSTRACT FROM AUTHOR]

Highlights: • BAC filters are able to mitigate a sudden change in feed water quality. • To minimize the growth potential, sufficient nutrients in the feed were required. • The addition of phosphate resulted in the lowest DOC concentration in the effluent. • The influence of intact cells in the feed of BAC filters was shown to be limited. Abstract Biological activated carbon (BAC) filtration is an important treatment step in the production of drinking water especially if drinking water is produced from surface water. The performance and processes within a BAC filter have been of interest for researchers since the 1980's, mainly because of its ability to remove natural organic matter known as disinfection precursors. A malfunction of one of the pre-treatment steps might affect the feed water quality into the BAC filters. The main objective of this study was to determine the immediate response of the BAC filters to a rapid change in feed water quality. It was shown that with the studied setup it was possible to compare the effect of different pre-treatment steps and subsequent different water qualities on the performance of the BAC filters on the long term adaptation. However, especially the immediate response was not studied in detail before. All filters were able to mitigate a sudden change in feed water quality, either through improved adsorption or increased activity of the biomass on the filter. As a result of this resilience against sudden changes, it is therefore concluded that there is no direct need for very stringent on-line monitoring and continuous adjustments of the feed water quality of the BAC filters. The addition of phosphate resulted in the lowest dissolved organic carbon (DOC) concentration in the effluent of the BAC filters. In this study the influence of intact cells in the feed water on the performance of the BAC filters was shown to be limited. [ABSTRACT FROM AUTHOR]

This work describes the first electrochemical immunosensor reported for the determination of IL-13 receptor Rα2 (IL-13Rα2), an emerging relevant biomarker in metastatic colon cancer. The approach involves the formation of sandwich immunocomplexes using specific capture (CAb) and biotinylated detector antibodies (BDAb) further labeled with an streptavidin-horseradish peroxidase (Strep-HRP) polymer, onto carboxylic acid-modified magnetic microbeads (HOOC-MBs). Amperometric detection at disposable carbon screen-printed electrodes (SPCEs) using the (H 2 O 2 )/hydroquinone (HQ) system was employed to monitor the affinity reactions. The developed immunosensor exhibits a linear calibration plot over the 3.9–100 ng mL −1 concentration range, a LOD of 1.2 ng mL −1 and excellent selectivity against other non-target proteins. The amperometric immunosensor was applied successfully to quantify for the first time the IL-13Rα2 expression in raw lysates of colon cancer cells and to discriminate the metastatic potential of intact cells through recognition of this target extracellular receptor. In comparison with the commercial Enzyme-Linked ImmunoSorbent Assay (ELISA) kit involving the same immunoreagents, the immunosensor provides a similar LOD in a half-time for the assay. [ABSTRACT FROM AUTHOR]

Limiting the rate and extent of starch digestion is a major target for increasing the nutritional value of cereal-based foods. One mechanism that could be exploited is the ability of intact cell walls to protect intracellular starches from enzyme hydrolysis, but the extent to which this mechanism is valid for cereal endosperm cells is not well understood. This study aimed to isolate individual intact cellular structures from cereals, viz. wheat and sorghum, in order to elucidate the effect of intactness of cell walls on enzymic hydrolysis of entrapped starch. Intact cells were isolated from dry milled flour obtained using three grinding rolls coupled with a wet sieving technique using selected sieves having varying apertures. The intact cellular structure in wheat and sorghum hindered the hydrolysis of entrapped starch as observed from the lower extent of digestion (9 and 7%) compared to deliberately broken cells (19 and 17% under the same conditions). The extent of digestion was markedly increased once the intact cells were cooked (33 and 26% for wheat and sorghum cooked cells), but this was less than half the digestion extent of non-encapsulated cooked starches (77 and 62% respectively). Microscopic observations coupled with fluorescence labelling of enzyme, cell walls and starch suggest a) wheat and sorghum cell walls are effective barriers for access of amylase; and b) both an extensive protein matrix (particularly in sorghum) and non-catalytic binding of amylase on cell wall surfaces can limit the amylolysis of starch within intact cells. Furthermore, the presence of incompletely gelatinised starch inside cooked intact cells, suggests limited swelling of granules trapped inside the cells. This study shows how preservation of cellular matrices in cereal-based foods could be beneficial for increasing the amount of enzyme resistant starch in cereals with added nutritional benefits. [ABSTRACT FROM AUTHOR]

Upon high light excitation in photosynthetic bacteria, various triplet states of pigments can accumulate leading to harmful effects. Here, the generation and lifetime of flash-induced carotenoid triplets (3Car) have been studied by observation of the quenching of bacteriochlorophyll (BChl) fluorescence in different strains of photosynthetic bacteria including Rvx. gelatinosus (anaerobic and semianaerobic), Rsp. rubrum, Thio. roseopersicina, Rba. sphaeroides 2.4.1 and carotenoid- and cytochrome-deficient mutants Rba. sphaeroides Ga, R-26, and cycA, respectively. The following results were obtained: (1) 3Car quenching is observed during and not exclusively after the photochemical rise of the fluorescence yield of BChl indicating that the charge separation in the reaction center (RC) and the carotenoid triplet formation are not consecutive but parallel processes. (2) The photoprotective function of 3Car is not limited to the RC only and can be described by a model in which the carotenoids are distributed in the lake of the BChl pigments. (3) The observed lifetime of 3Car in intact cells is the weighted average of the lifetimes of the carotenoids with various numbers of conjugated double bonds in the bacterial strain. (4) The lifetime of 3Car measured in the light is significantly shorter (1-2 μs) than that measured in the dark (2-10 μs). The difference reveals the importance of the dynamics of 3Car before relaxation. The results will be discussed not only in terms of energy levels of the 3Car but also in terms of the kinetics of transitions among different sublevels in the excited triplet state of the carotenoid. [ABSTRACT FROM AUTHOR]

The kinetics of bacteriochlorophyll fluorescence in intact cells of the purple nonsulfur bacterium Rhodobacter sphaeroides were measured under continuous and pulsed actinic laser diode (808 nm wavelength and maximum 2 W light power) illumination on the micro- and millisecond timescale. The fluorescence induction curve was interpreted in terms of a combination of photochemical and triplet fluorescence quenchers and was demonstrated to be a reflection of redox changes and electron carrier dynamics. By adjustment of the conditions of single and multiple turnovers of the reaction center, we obtained 11 ms-1 and 120 μs-1 for the rate constants of cytochrome c23+ detachment and cyclic electron flow, respectively. The effects of cytochrome c2 deletion and chemical treatments of the bacteria and the advantages of the fluorescence induction study on the operation of the electron transport chain in vivo were discussed. [ABSTRACT FROM AUTHOR]

The membrane-associated c-type cytochromes ( c-Cyts) have been well known as the key enzymes mediating extracellular electron transfer to terminal electron acceptors, resulting in biogeochemical elemental transformation, contaminant degradation, and nutrient cycling. Although c-Cyts-mediated metal reduction or oxidation have been mainly investigated with the purified proteins of metal reducing/oxidizing bacteria, the in vivo behavior of c-Cyts is still unclear, given the difficulty in measuring the proteins of intact cells. Fortunately, the in situ spectroscopy would be ideal for measuring the reaction kinetics of c-Cyts in intact cells under noninvasive physiological conditions. It can also help the establishment of kinetic/thermodynamic models of extracellular electron transfer processes, which are essential to understand the electron transfer mechanisms at the molecular scale. This review briefly summarizes the current advances in spectral methods for examining the c-Cyts in intact cells of dissimilatory metal reducing bacteria and Fe(II)-oxidizing bacteria. [ABSTRACT FROM AUTHOR]

Mercury adsorption on the cell surface and intracellular uptake by bacteria represent the key first step in the production and accumulation of highly toxic mercury in living organisms. In this work, the biophysical characteristics of mercury bioaccumulation are studied in intact cells of photosynthetic bacteria by use of analytical (dithizone) assay and physiological photosynthetic markers (pigment content, fluorescence induction, and membrane potential) to determine the amount of mercury ions bound to the cell surface and taken up by the cell. It is shown that the Hg(II) uptake mechanism (1) has two kinetically distinguishable components, (2) includes co-opted influx through heavy metal transporters since the slow component is inhibited by Ca channel blockers, (3) shows complex pH dependence demonstrating the competition of ligand binding of Hg(II) ions with H ions (low pH) and high tendency of complex formation of Hg(II) with hydroxyl ions (high pH), and (4) is not a passive but an energy-dependent process as evidenced by light activation and inhibition by protonophore. Photosynthetic bacteria can accumulate Hg(II) in amounts much (about 10) greater than their own masses by well-defined strong and weak binding sites with equilibrium binding constants in the range of 1 (μM) and 1 (mM), respectively. The strong binding sites are attributed to sulfhydryl groups as the uptake is blocked by use of sulfhydryl modifying agents and their number is much (two orders of magnitude) smaller than the number of weak binding sites. Biofilms developed by some bacteria (e.g., Rvx. gelatinosus) increase the mercury binding capacity further by a factor of about five. Photosynthetic bacteria in the light act as a sponge of Hg(II) and can be potentially used for biomonitoring and bioremediation of mercury-contaminated aqueous cultures. [ABSTRACT FROM AUTHOR]

Because of its high protein content, Jack bean (Canavalia ensiformis) is a promising alternative protein source. However, the utilization of Jack bean is limited due to the long cooking time to achieve palatable softness. We hypothesize that the cooking time may influence protein and starch digestibility. In this study, we characterized seven Jack bean collections with different optimal cooking times in terms of their proximate composition, microstructure and protein and starch digestibility. Kidney bean was included as a reference for microstructure and protein and starch digestibility. Proximate composition showed that Jack bean collections have a protein content ranging from 28.8 to 39.3%, a starch content ranging from 31 to 41%, a fiber content from 15.4 to 24.6%, and a concanavalin A content in the range 35-51 mg/g dry cotyledon. Particle sizes ranging between 125 and 250 µm were chosen as a representative sample of the whole bean to characterize microstructure and digestibility of the seven collections. Confocal laser microscopy (CLSM) revealed that Jack bean cells have an oval shape and contain starch granules embedded in a protein matrix similar to kidney bean cells. The diameter of Jack bean cells was measured by image analysis of CLSM micrographs and ranged from 103 to 123 µm, while the diameter of starch granules was 31-38 µm, comparatively larger than that of the kidney bean starch granules. Isolated intact cells were used to determine the starch and protein digestibility in the Jack beans collections. The digestion kinetics of starch followed a logistic model, whereas the digestion kinetics of protein followed a fractional conversion model. We found no correlation between optimal cooking time and kinetic parameters of protein and starch digestibility, implying that optimal cooking time is not predictive of protein and starch digestibility. In addition, we tested the effect of reduced cooking times on protein and starch digestibility on one Jack bean collection. The result showed that reducing cooking time significantly reduces starch digestibility, but not protein digestibility. The present study contributes to our understanding of the effect of food processing on protein and starch digestibility in legumes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

It is becoming increasingly apparent that mitochondria dysfunction plays an important role in pathogenesis of Huntington’s disease (HD) but the underlying mechanism is still elusive. Thus, there is a still need for further studies concerning the upstream events in the mitochondria dysfunction that could contribute to cell death observed in HD. Taking into account the fundamental role of the voltage-dependent anion-selective channel (VDAC) in mitochondria functioning it is reasonable to consider the channel as a crucial element in HD etiology. Therefore, we applied inducible PC12 cell model of HD to determine the relationship between the effect of expression of wild type and mutant huntingtin (Htt and mHtt, respectively) on cell survival and mitochondria functioning in intact cells under conditions of undergoing cell divisions. Because after 48h of Htt and mHtt expression differences in mitochondria functioning co-occurred with differences in the cell viability we decided to estimate the effect of Htt and mHtt expression lasted for 48h on VDAC functioning. Therefore we isolated VDAC from the cells and tested the preparations by black lipid membrane (BLM) system. We observed that the expression of mHtt, but not Htt, resulted in changes of the open state conductance and voltage-dependence when compared to control cells cultured in the absence of the expression. Importantly, for all the VDAC preparations we observed a dominant quantitative content of VDAC1 and the quantitative relationships between VDAC isoforms were not changed by Htt and mHtt expression. Thus, Htt and mHtt-mediated functional changes of VDAC, being predominantly VDAC1, which occur shortly after these protein appearance in cells may result in differences concerning mitochondria functioning and viability of cells expressing Htt and mHtt. The assumption is important for better understanding of cytotoxicity as well as cytoprotection mechanisms of potential clinical application.

The β-lactam antibiotics have been successfully used for decades to combat susceptible Pseudomonas aeruginosa, which has a notoriously difficult to penetrate outer membrane (OM). However, there is a dearth of data on target site penetration and covalent binding of penicillin-binding proteins (PBP) for β-lactams and β-lactamase inhibitors in intact bacteria. We aimed to determine the time course of PBP binding in intact and lysed cells and estimate the target site penetration and PBP access for 15 compounds in P. aeruginosa PAO1. All β-lactams (at 2 × MIC) considerably bound PBPs 1 to 4 in lysed bacteria. However, PBP binding in intact bacteria was substantially attenuated for slow but not for rapid penetrating β-lactams. Imipenem yielded 1.5 ± 0.11 log 10 killing at 1h compared to <0.5 log 10 killing for all other drugs. Relative to imipenem, the rate of net influx and PBP access was ~ 2-fold slower for doripenem and meropenem, 7.6-fold for avibactam, 14-fold for ceftazidime, 45-fold for cefepime, 50-fold for sulbactam, 72-fold for ertapenem, ~ 249-fold for piperacillin and aztreonam, 358-fold for tazobactam, ~547-fold for carbenicillin and ticarcillin, and 1,019-fold for cefoxitin. At 2 × MIC, the extent of PBP5/6 binding was highly correlated ( r 2 = 0.96) with the rate of net influx and PBP access, suggesting that PBP5/6 acted as a decoy target that should be avoided by slowly penetrating, future β-lactams. This first comprehensive assessment of the time course of PBP binding in intact and lysed P. aeruginosa explained why only imipenem killed rapidly. The developed novel covalent binding assay in intact bacteria accounts for all expressed resistance mechanisms., Competing Interests: The authors declare no conflict of interest.

It is becoming increasingly apparent that mitochondria dysfunction plays an important role in the pathogenesis of Huntington's disease (HD), but the underlying mechanism is still elusive. Thus, there is a still need for further studies concerning the upstream events in the mitochondria dysfunction that could contribute to cell death observed in HD. Taking into account the fundamental role of the voltage-dependent anion-selective channel (VDAC) in mitochondria functioning, it is reasonable to consider the channel as a crucial element in HD etiology. Therefore, we applied inducible PC12 cell model of HD to determine the relationship between the effect of expression of wild type and mutant huntingtin (Htt and mHtt, respectively) on cell survival and mitochondria functioning in intact cells under conditions of undergoing cell divisions. Because after 48 h of Htt and mHtt expression differences in mitochondria functioning co-occurred with differences in the cell viability, we decided to estimate the effect of Htt and mHtt expression lasted for 48 h on VDAC functioning. Therefore, we isolated VDAC from the cells and tested the preparations by black lipid membrane system. We observed that the expression of mHtt, but not Htt, resulted in changes of the open state conductance and voltage-dependence when compared to control cells cultured in the absence of the expression. Importantly, for all the VDAC preparations, we observed a dominant quantitative content of VDAC1, and the quantitative relationships between VDAC isoforms were not changed by Htt and mHtt expression. Thus, Htt and mHtt-mediated functional changes of VDAC, being predominantly VDAC1, which occur shortly after these protein appearances in cells, may result in differences concerning mitochondria functioning and viability of cells expressing Htt and mHtt. The assumption is important for better understanding of cytotoxicity as well as cytoprotection mechanisms of potential clinical application. [ABSTRACT FROM AUTHOR]

It is widely recognized that the outer membrane c-type cytochromes (OM c-Cyts) of metal-reducing bacteria play a key role in microbial metal reduction processes. However, the in situ redox status of OM c-Cyts during microbial metal reduction processes remain poorly understood. In this study, diffuse-transmission UV/Vis spectroscopy is used to investigate the in situ spectral reaction of Cr(VI) reduction by c-Cyts in intact Shewanella oneidensis MR-1 cells under different incubation conditions. The reduced c-Cyts decreased transiently at the beginning and then recovered gradually over time. The Cr(VI) reduction rates decreased with increasing initial Cr(VI) concentrations, and Cr(III) was identified as a reduced product. The presence of Cr(III) substantially inhibited Cr(VI) reduction and the recovery of reduced c-Cyts, indicating that Cr(III) might inhibit cell growth. Cr(VI) reduction rates increased with increasing cell density. The highest Cr(VI) reduction rate and fastest recovery of c-Cyts were obtained at pH 7.0 and 30℃C, with sodium lactate serving as an electron donor. The presence of O2 strongly inhibited Cr(VI) reduction, suggesting that O2 might compete with Cr(VI) as an electron acceptor in cells. This study provides a case of directly examining in vivo reaction properties of an outer-membrane enzyme during microbial metal reduction processes under non-invasive physiological conditions. [ABSTRACT FROM AUTHOR]

Heightened sensitivity of forensic DNA techniques has led to an increased variety of samples tested, often yielding complex DNA mixtures, in turn making the interpretation of profiling results more complicated. Currently, there is no prescribed upstream laboratory method to separate complex DNA mixtures by their contributors; therefore, a method is needed that could reduce mixtures into their component parts. Various cell sorting applications have the potential to be this method, if intact cells can be reliably obtained from forensic samples. Here, the effects of elution buffer and swab substrate on the recovery of intact, human, white blood cells from dried blood samples were evaluated. Approximately 328,000 cells per swab were deposited onto cotton, flocked, and dissolvable swabs. The whole-cell elution of the dried samples was evaluated with water, phosphate buffered saline, and AutoMACS® elution buffers. We demonstrate that AutoMACS® buffer is superior for the elution of intact cells, compared to phosphate buffered saline and water. When swab type was considered, the highest yield of intact cells resulted from flocked swabs, as opposed to cotton or dissolvable swabs. [ABSTRACT FROM AUTHOR]

The lack of effective treatment options against Pseudomonas aeruginosa is one of the main contributors to the silent pandemic. Many antibiotics are ineffective against resistant isolates due to poor target site penetration, efflux, or β-lactamase hydrolysis. Critical insights to design optimized antimicrobial therapies and support translational drug development are needed. In the present work, we analyzed the periplasmic drug uptake and binding to PBPs of 11 structurally different β-lactams and 4 β-lactamase inhibitors (BLIs) in P. aeruginosa PAO1. The contribution of the most prevalent β-lactam resistance mechanisms to MIC and periplasmic target attainment was also assessed. Bacterial cultures (6.5 log 10 CFU/mL) were exposed to 1/2× PAO1 MIC of each antibiotic for 30 min. Unbound PBPs were labeled with Bocillin FL and analyzed using a FluorImager. Imipenem extensively inactivated all targets. Cephalosporins preferentially targeted PBP1a and PBP3. Aztreonam and amdinocillin bound exclusively to PBP3 and to PBP2 and PBP4, respectively. Penicillins bound preferentially to PBP1a, PBP1b, and PBP3. BLIs displayed poor PBP occupancy. Inactivation of oprD elicited a notable reduction of imipenem target attainment, and it was to a lesser extent in the other carbapenems. Improved PBP occupancy was observed for the main targets of the widely used antipseudomonal penicillins, cephalosporins, meropenem, aztreonam, and amdinocillin upon oprM inactivation, in line with MIC changes. AmpC constitutive hyperexpression caused a substantial PBP occupancy reduction for the penicillins, cephalosporins, and aztreonam. Data obtained in this work will support the rational design of optimized β-lactam-based combination therapies against resistant P. aeruginosa infections. IMPORTANCE The growing problem of antibiotic resistance in Gram-negative pathogens is linked to three key aspects, (i) the progressive worldwide epidemic spread of multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) Gram-negative strains, (ii) a decrease in the number of effective new antibiotics against multiresistant isolates, and (iii) the lack of mechanistically informed combinations and dosing strategies. Our combined efforts should focus not only on the development of new antimicrobial agents but the adequate administration of these in combination with other agents currently available in the clinic. Our work determined the effectiveness of these compounds in the clinically relevant bacteria Pseudomonas aeruginosa at the molecular level, assessing the net influx rate and their ability to access their targets and achieve bacterial killing without generating resistance. The data generated in this work will be helpful for translational drug development.

• Biofilm thickness increased along the feed water path due to temperature gradient. • Diversity of EPS fluorescence peaks decreased with the increase in feed temperature. • Larger proportion of thermophilic bacteria colonized membranes at 55°C and 65°C. • Intact cells in biofilms were dominant over the damaged cells. • Biofilm maturation alleviated bacterial passage to permeate side. Biofouling is a hurdle of seawater desalination that increases water costs and energy consumption. In membrane distillation (MD), biofouling development is complicated due to the temperature effect that adversely affects microbial growth. Given the high relevance of MD to regions with abundant warm seawater, it is essential to explore the biofouling propensity of microbial communities with higher tolerance to elevated temperature conditions. This study presents a comprehensive analysis of the spatial and temporal biofilm distribution and associated membrane fouling during direct contact MD (DCMD) of the Red Sea water. We found that structure and composition of the biofilm layer played a significant role in the extent of permeate flux decline, and biofilms that built up at 45°C had lower bacterial concentration but higher extracellular polymeric substances (EPS) content as compared to biofilms that formed at 55 °C and 65°C. Pore wetting and bacterial passage to the permeate side were initially observed but slowed down as operating time increased. Intact cells in biofilms dominated over the damaged cells at any tested condition emphasizing the high adaptivity of the Red Sea microbial communities to elevated feed temperatures. A comparison of microbial abundance revealed a difference in bacterial distribution between the feed and biofilm samples. A shift in the biofilm microbial community and colonization of the membrane surface with thermophilic bacteria with the feed temperature increase was observed. The results of this study improve our understanding of biofouling propensity in MD that utilizes temperature-resilient feed waters. [Display omitted]. [ABSTRACT FROM AUTHOR]

Reduction of starch digestibility in starchy foods is beneficial for lowering the risks for major non-communicable diseases. Preserving cell integrity is known to delay starch digestibility in flour but its effect in bread is not clear. In this study, the effect of increasing particle size on in vitro starch digestibility of durum wheat flour, dough, and bread was investigated. Cell integrity was retained during bread processing for medium (1000 µm-1800 µm), and large (>1800 µm) flour, whereas in small one cell walls were mostly damaged (<350 µm). In vitro starch digestibility of flour decreased increasing particle size, but no difference was found in dough. In bread, instead, a modest decrease of starch digestibility for the bread made by large particle was observed, likely due to its dense structure. In conclusion, a high particle size could limit starch digestibility in durum wheat flour but not in bread., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The understanding of protein aggregation is a central issue in different fields of protein science, from the heterologous protein production in biotechnology to amyloid aggregation in several neurodegenerative and systemic diseases. To this goal, it became more and more evident the crucial relevance of studying protein aggregation in the complex cellular environment, since it allows to take into account the cellular components affecting protein aggregation, such as chaperones, proteases, and molecular crowding. Here, we discuss the use of several biochemical and biophysical approaches that can be employed to monitor protein aggregation within intact cells, focusing in particular on bacteria that are widely employed as microbial cell factories. [ABSTRACT FROM AUTHOR]

The sensitivity of intact cells of purple photosynthetic bacterium Rhodobacter sphaeroides wild type to low level (<100 μM) of mercury (Hg) contamination was evaluated by absorption and fluorescence spectroscopies of the bacteriochlorophyll-protein complexes. All assays related to the function of the reaction center (RC) protein (induction of the bacteriochlorophyll fluorescence, delayed fluorescence and light-induced oxidation and reduction of the bacteriochlorophyll dimer and energization of the photosynthetic membrane) showed prompt and later effects of the mercury ions. The damage expressed by decrease of the magnitude and changes of rates of the electron transfer kinetics followed complex (spatial and temporal) pattern according to the different Hg sensitivities of the electron transport (donor/acceptor) sites including the reduced bound and free cytochrome c and the primary reduced quinone. In contrast to the RC, the light harvesting system and the bc complex demonstrated much higher resistance against the mercury pollution. The 850 and 875 nm components of the peripheral and core complexes were particularly insensitive to the mercury(II) ions. The concentration of the photoactive RCs and the connectivity of the photosynthetic units decreased upon mercury treatment. The degree of inhibition of the photosynthetic apparatus was always higher when the cells were kept in the light than in the dark indicating the importance of metabolism in active transport of the mercury ions from outside to the intracytoplasmic membrane. Any of the tests applied in this study can be used for detection of changes in photosynthetic bacteria at the early stages of the action of toxicants. [ABSTRACT FROM AUTHOR]

The implication of the cannabinoid receptor 1 (CB receptor) in several pathophysiological states has sparked the development of selective antagonists. Here we compare binding of the antagonists [H]-AZ12491187, [H]-taranabant and [H]-rimonabant to intact human embryonic kidney cells stably expressing recombinant human CB receptors (CB1r cells). Unlabelled ligands decreased the total binding of the three radioligands with closely the same order of potency: i.e. AZ12288553 ∼ AZ12491187 ∼ taranabant > rimonabant. Nondisplaceable (i.e. nonspecific) binding to the CB1r cells was the same as total binding to the wells containing untransfected cells and it was more pronounced for [H]-AZ12491187 and [H]-rimonabant than for [H]-taranabant. [H]-Rimonabant and (to a lesser extent) [H]-AZ12491187 were also prone to bind nonspecifically to the walls of the wells. Compared to the other radioligands, [H]-rimonabant displayed lower potency for the CB receptors in saturation binding studies and faster association and dissociation in kinetic experiments. When dissociated, the three radioligands also showed prominent rebinding to the cells in medium only. This could be relieved by the presence of excess of unlabelled ligand and of bovine serum albumin (BSA) but a combination thereof was most efficient. The long 'residence time' of AZ12491187 at the CB receptor (because of slow dissociation and prominent rebinding) and its pronounced incorporation into the membranes of the cells could contribute to long-lasting in vivo CB receptor blockade. [ABSTRACT FROM AUTHOR]

A pump and probe fluorometer with a laser diode as single light source has been constructed for measurement of fast induction and relaxation of the fluorescence yield in intact cells, chromatophores and isolated reaction centers of photosynthetic bacteria. The time resolution of the fluorometer is limited by the repetition time of the probing flashes to 20 μs. The apparatus offers high sensitivity, excellent performance and can become a versatile device for a range of demanding applications. Some of them are demonstrated here including fast and easy investigation of the (1) organization and redox state of the photosynthetic apparatus of the intact cells of different bacterial strains and mutants and (2) electron transfer reactions on donor and acceptor sides of isolated reaction centers. The compact design of the mechanics, optics, electronics, and data processing makes the device easy to use as outdoor instrument or to integrate into larger measuring systems. [ABSTRACT FROM AUTHOR]

D2-type dopamine receptors are major recognition sites for antipsychotic drugs. There are two splice variants: D2S and D2L with an additional 29 amino acid sequence in the third intracellular loop. Only little comparative information is hitherto available about their pharmacological properties and none of these studies dealt with intact cell systems. This prompted us to investigate the binding properties of [3H]-raclopride, a hydrophilic benzamide, and [3H]-spiperone, a highly hydrophobic butyrophenone, to intact CHO cells expressing recombinant human D2L-receptors. Presently, we have repeated and extended this experimental approach to the human D2S-receptors in the same cell system. Except for a slower dissociation of [3H]-spiperone from D2S, the binding properties of these and other antagonists were not significantly different for both isoforms ( P > 0.05). The very slow dissociation of the atypical antipsychotic clozapine was surprising in light of its low affinity. Two experiments pointed out the existence of non-competitive interactions between raclopride and spiperone for D2S as well as D2L (A. Packeu, J. P. De Backer & G. Vauquelin, in preparation). Alongside the different physicochemical properties of these ligands, this finding fits with a model wherein the hydrophilic raclopride approaches the D2L-receptor from the aqueous phase, while the hydrophobic spiperone approaches the receptor by lateral diffusion between the membrane lipids. These different modes of approach could imply the existence of topologically distinct ligand binding sites at D2-receptors. [ABSTRACT FROM AUTHOR]

We recently investigated the binding properties of the antagonists [3H]-raclopride and [3H]-spiperone to intact Chinese hamster ovary cells expressing recombinant human D2long-dopamine receptors (CHO-D2L cells). Compared with saturation binding with [3H]-raclopride, raclopride reduced [3H]-spiperone binding with to low potency in competition binding experiments. The present findings illustrate the ability of spiperone to inhibit [3H]-raclopride binding non-competitively. While raclopride only decreases the apparent KD of [3H]-raclopride in saturation binding experiments, spiperone only decreases the number of sites to which [3H]-raclopride binds with high affinity. Also, while the IC50 of raclopride depends on the concentration of [3H]-raclopride in competition experiments, this is not the case for spiperone. Kinetic studies reveal that the binding of raclopride at its high affinity sites does not affect the association of subsequently added [3H]-spiperone nor the rebinding of freshly dissociated [3H]-spiperone to the same or surrounding receptors. Yet, spiperone does not affect the dissociation rate of [3H]-raclopride and raclopride does not affect the (genuine) dissociation rate of [3H]-spiperone. The easiest way to interpret the present findings in molecular terms is to assume that D2L-receptors or their dimeric complexes possess two distinct binding sites: one with high affinity/accessibility for [3H]-raclopride and the other one with high affinity/accessibility for [3H]-spiperone. The ability of bound spiperone to inhibit high affinity raclopride binding while the reverse is not the case suggests for the occurrence of non-reciprocal allosteric interactions. These new findings could point at the occurrence of allosteric interactions between different classes of D2-receptor antagonists. [ABSTRACT FROM AUTHOR]

Abstract: NAD(P)H oxidoreductases of the outer mitochondrial membrane (OMM) are able to activate various xenobiotics and stimulate the production of reactive oxygen species and the opening of the mitochondrial permeability transition pore. However, the role of these systems in the cell damage by xenobiotics and chemotherapeutic drugs is poorly understood because the methods for the selective assessment of their activity have not been elaborated and specific inhibitors are unknown. Here we propose a method for the semiquantitative assessment of the activity of NAD(P)H oxidoreductases of the OMM in intact and permeabilized cells that is based on the flow cytometry detection of dimethylbiacridene, a fluorescent product of two-electron reduction of lucigenin. The method uses the structural feature of mitochondrial organization: the proximity of the sites of one-electron reduction of lucigenin to cation radical (NAD(P)H oxidoreductases of the OMM) to the sites of its subsequent oxidation (cytochrome c oxidase). The inhibition of cytochrome c oxidase by cyanide selectively activates the dimethylbiacridene formation by oxidoreductases of the OMM but not by other cellular oxidoreductases. The proposed protocol allows one to assess the lucigenin reductase (two-electron) activity of NAD(P)H oxidoreductases of the OMM and to compare it with the activity of other cellular systems that can be used for the analysis of the role of these systems in the cell damage by xenobiotics and antitumor drugs. [Copyright &y& Elsevier]

The present study compared the anti-oxidative ability of intact cells and intracellular extracts of two lactic acid bacterial strains, Bifidobacterium longum and Lactobacillus delbrueckii ssp. bulgaricus. Results showed that both intact cells and intracellular extracts of 109 cells of B. longuin and L. delbrueckii ssp. bulgaricus had the ability to scavengeα-diphenyl-β-picrylhydrazyl (DPPH) free radical by 70.4-75.1%, to inhibit liposome peroxidation by 25-31%, and to decrease significantly the malondialdehyde (MDA) production in Intestine 407 cells. The effect of intact cells and intracellular extracts of these two bacterial strains on the oxidation of low density lipoprotein (LDL) isolated from cerebrovascular accident (CVA) patients and healthy subjects was also compared. Oxidation of LDL was monitored by measuring the lag time for the formation of conjugated dienes in isolated LDL particles. When LDL was treated respectively with 109 intact cells of B. longum and L. deibrueckli ssp. bulgaricus, the lag time of oxidation of LDL was prolonged significantly. The extent of inhibition was greater on LDL isolated from healthy subjects than from CVA patients. When LDL from either CVA patients or healthy subjects was treated with intracellular extracts of 109 cells of B. longum and L. delbrueckii ssp. bulgaricus, respectively, the copper-mediated oxidation was extensively inhibited with a lag time exceeding 180 mm. Results from this study show a greater inhibitory effect on LDL oxidation exerted by the intracellular extract than the intact cells, suggesting the presence of effective inhibitory factors in the intracellular extract. [ABSTRACT FROM AUTHOR]

Tremella fuciformis is one of higher basidiomycetes. Its basidiospore can reproduce yeast-like conidia, also called the blastospore by budding. The yeast-like conidia of T. fuciformis is monokaryotic and easy to culture by submerged fermentation similar to yeast. So it is a good recipient cell for exogenous gene expression. In this study, two expression vectors pGlg-gfp containing gpd-Gl promoter and gfp gene and pGlg-hph containing gpd-Gl promoter and hph gene were constructed. The lowest sensitive concentration of hygromycin for the blastospore was determined on three types of media. Our experiments showed that the lowest sensitive concentration of hygromycin for the blastospore was 5 μg/mL on MA medium. The intact blastospores were transformed with the expression vector pGlg-hph by electroporation. The putative transformants were obtained by the MA selective medium. Experimental results showed that the most effective parameters for the electroporation of intact blastospores were obtained by using STM buffer, 1.0×108 cells/mL of blastospores, 200 μL in transformation volume, 6 μg plasmid, 2.0 kV/cm of electric pulse voltage, stillness culturing on MB liquid medium for 48 h after electroporation. In these transformation conditions, the efficiency reached 277 colonies/μg DNA. Co-transformation of plasmid pGlg-gfp and pGlg-hph with ratio of 1:1 was performed by electroporation with the optimal parameters. The putative co-transformants were obtained by the MA selective medium. Eight randomly selected colonies from the vast putative co-transformants were analyzed by PCR detection and Southern blotting. The experiments showed that the gfp was integrated into the genomes of three transformants. The co-transformation efficiency was 37.5%. Green fluorescence was observed under laser scanning confocal microscope in these gfp positive transformants. This indicates that the exogenous gfp can be expressed effectively in the yeast-like conidia of T. fuciformis. [ABSTRACT FROM AUTHOR]

Abstract: D2-dopamine receptors mediate most of the physiological actions of dopamine and are important recognition sites for antipsychotic drugs. Earlier binding studies were predominantly done with broken cell preparations with the tritiated D2-receptor antagonists [3H]-raclopride, a hydrophilic benzamide, and [3H]-spiperone, a highly hydrophobic butyrophenone. Here we compared [3H]-raclopride and [3H]-spiperone binding properties in intact Chinese Hamster Ovary cells stably expressing recombinant human D2L-receptors. Specific binding of both radioligands occurred to a comparable number of sites. In contrast to the rapid dissociation of [3H]-raclopride in both medium only and in the presence of an excess of unlabelled ligand [3H]-spiperone dissociation was only observed in the latter condition, and it was still slower than in broken cell preparations. However, this could not explain the pronounced difference in the potency of some unlabelled ligands to compete with both radioligands. To integrate these new findings, a model is proposed in which raclopride approaches the receptor from the aqueous phase, while spiperone approaches the receptor by lateral diffusion within the membrane. [Copyright &y& Elsevier]

Capillary electrophoresis (CE) of erythrocytes from different sources under various conditions is reported in this paper. It was found that erythrocyte samples from sheep, duck, and human showed characteristic and reproducible elution peaks, and that the retention times of A-, B-, AB-, and O-type erythrocytes from human blood were distinctively different; even subtle differences, among individuals with the same blood type could be detected by CE. A strictly linear correlation was obtained between the peak area and the amount of human erythrocyte over a range of 4.8×102–1.9×104 cells (r=0.999), indicating that CE could be used for rapid and accurate quantification of erythrocytes. Using this CE protocol, the decrease of the surface electrical charge of erythrocyte during storage was confirmed. Therefore, this work demonstrated that CE could be a useful alternative for characterizing and quantifying erythrocytes or other cells. [Copyright &y& Elsevier]

Here we describe for the first time a cell-based scintillation proximity assay using membrane soluble scintillants (MSS). MSS have a scintillant `head'' group (2,5-diphenyloxazole) attached to a lipophilic `tail.'' MSS do not scintillate in an aqueous environment in the presence of a radioactive source: however, in a non-aqueous environment, such as a lipid bilayer (e.g., liposome or cell membrane), scintillation does occur. MSS can be incorporated into liposomes. When these MSS-containing liposomes are fused with the plasma membranes of cells in culture the MSS are incorporated into the cell membrane. Radiolabelled molecules in close proximity to the cell membrane will then elicit a scintillation signal. This system has been used to successfully monitor [14C]methionine uptake in HeLa cells and may be used in radiochemical and radioligand binding assays either in vivo or on microsomal preparations obtained from tissues. This new scintillation proximity technology could be readily adapted for high-throughput screening. [Copyright &y& Elsevier]

A sensitive method for measuring the circular dichroism (CD) of living HeLa cells in the visible-near infrared (IR) region is developed. The changes in CD spectra from 250 to 780 nm of HeLa cell suspension after the first and second irradiation at 820 nm in dose 9 J/cm2 are investigated. The CD spectrum of the intact cells is well structured and characterized by a positive signal in the UV (250-290 nm) and visible-near IR (500-780 nm) regions as well as by a negative signal in 300-450 nm region. Distinct maxima in the visible-near IR region are recorded at 566, 634, 680, 712, and 741 nm. As a rule, the peak circular dichroism signals decrease in the irradiated cells except of the area 750-770 nm. Peak positions (except the peak at 680 nm) shift as a rule to the long-wavelength direction. The most remarkable changes in peak positions as well as in CD signals are recorded in the region 750-770 nm: an appearance of the new peak at 767 nm after the first irradiation and its shift to 752 nm after the second irradiation. The peaks at 712 and 741 nm disappear after the irradiation. A new peak appears at 601 nm. It is assumed that the changes in the degree of oxidation of the chromophores of cytochrome c oxidase caused by the irradiation are accompanied by conformational changes in their vicinity. It can be suggested that these changes are occurring in CuB environment [ABSTRACT FROM PUBLISHER]

In the present study we demonstrate that the 4‐amino analogue of tetrahydrobiopterin, 2,4‐diamino‐5,6,7,8‐tetrahydro‐6‐(l‐erythro‐1,2‐dihydroxypropyl)pteridine (4‐amino‐H4biopterin) binds with high affinity to recombinant endothelial NO synthase and concomitantly inhibits enzyme activity [IC50 = 14.8 ± 7.5 µm in the presence of added 5,6,7,8‐tetrahydro‐l‐erythrobiopterin (H4biopterin) 10 µm] as efficiently as previously shown for inducible NO synthase [Mayer, B., Wu, C.Q., Gorren, A.C.F., Pfeiffer, S., Schmidt, K., Clark, P., Stuehr, D.J. & Werner, E.R. (1997) Biochemistry 36, 8422–8427]. In cultured porcine endothelial cells, however, 4‐amino‐H4biopterin was less effective in inhibiting NO formation (IC50 = 420 ± 36 µm) as compared with inhibition of the inducible isoform in murine fibroblasts (IC50 = 15 ± 4.9 µm) and in human DLD‐1 adenocarcinoma cells (IC50 = 55 ± 10.3 µm). In all cells investigated, the inhibitory effect of 4‐amino‐H4biopterin was markedly enhanced by depletion of intracellular H4biopterin and could be overcome by increasing intracellular H4biopterin concentrations. Endothelial cells contained lower amounts of H4biopterin [5.2 ± 0.3 pmol·(mg protein)-1] than fibroblasts [19.4 ± 2.7 pmol·(mg protein)-1] and DLD‐1 cells [8.3 ± 1.1 pmol·(mg protein)-1], so that the selectivity of 4‐amino‐H4biopterin towards inducible NO synthase was not explained by differences in the H4biopterin levels. Because 4‐amino‐H4biopterin did not suppress expression of NO synthase in cytokine‐treated cells, we suggest that... [ABSTRACT FROM AUTHOR]

Intact cells and resealed ghosts of a homogeneous reticulocyte population isolated from the blood of phenylhydrazine-treated rats bound β-adrenergic ligands reversibly, stereospecifically and with high affinity. Maximal specific binding capacity under control conditions (37° C), corresponded to 9.9 ± 0.8 fmol/μl cells (∼ 600 sites/cell) and was similar in intact cells and ghosts. Pretreatment with metabolic inhibitors decreased the density of binding sites in intact cells to 6.48 ± 1.1 fmol/μl cells, but had no effect in ghosts. Incubation at 1°C reduced specific binding in paired experiments by 68 and 44% in intact cells and ghosts, respectively. Rewarming to 37°C increased specific binding in cells and ghosts by 270 and 190%, respectively. A temperature shift from 1 to 37°C reduced the K value for the antagonist (-)timolol from 8.6 ± 1.5 to 1.1 ± 0.3 nmol/l in intact cells, while no significant reduction in K was observed with ghosts. Under all conditions the receptor population was homogeneous with respect to antagonist affinity but inhomogenous with respect to agonist affinity. Low affinity agonist binding sites predominated in native cells and in GTP- loaded resealed ghosts (apparent K values 447 and 680 nmol/l, respectively). High affinity binding sites predominated in both preparations at 1°C ( K 29 and 14 nmol/l, respectively). β-Adrenoceptor sites in starved cells and ghosts at 37°C showed intermediate apparent K values. GTP had no effect on antagonist affinity or on the density of β-adrenoceptors. The results suggest that intact metabolizing cells can regulate β-adrenoceptor density by mechanisms which are not shared by ghost cells. The fractional contribution of high and low affinity states of the receptor to the overall binding of agonists seemed to be determined largely by the intracellular GTP concentration. [ABSTRACT FROM AUTHOR]

Transient expression of the β-glucuronidase (GUS) gene has been studied in leaf-derived embryogenic callus of sweetpotato Ipomoea batatas L. (Lam.) by electroporation. The influence of several factors including electric field strength, buffer composition, time course of transient GUS gene expression, DNA concentration, enzyme, and polyethylene glycol (PEG) treatment was examined on GUS gene expression (number of blue spots). Maximum GUS gene expression (an average of 90 blue spots/fifty mg fresh weight callus tissue) was observed after 48 h when callus pieces were preincubated with electroporation (EPR) buffer for 1 h, followed by electroporation with a single electric pulse of 500 V/cm discharged from a 960-μF capacitor in the presence of 20 μg DNA/ml and 8.3 μl NaCl (3 M). Changing the electroporation buffer conductivity (by varying the buffer composition with low-high salt concentrations), had only slight effect on the number of blue spots. Similarly, the time course study of GUS gene expression revealed that GUS activity could be detected 12 h after electroporation with a maximum activity after 72 h (112 blue spots). Increasing the amount of DNA from 5 to 50 μg/ml in the EPR buffer had a slight effect on the expression frequency (from 20–110 blue spots, and 112 blue spots with 20 μg/ml). The number of blue spots was increased by enzymatic wounding of callus pieces for 10 min and by addition of 200 μl PEG 4000 (15%) before electroporation. These results suggest that intact cell electroporation can be used for producing transgenic sweetpotato tissue. [ABSTRACT FROM AUTHOR]

Semi-quantitative and direct determination of labelled sulphydryl groups on the surface of intact erythrocytes has been accomplished for the first time with surface-enhanced resonance Raman scattering (SERRS). The method, which involves the use of citrate-reduced silver colloids, is sensitive and selective. A 10 M effective concentration of picomole quantities of sulphydryl groups was determined in the presence of the normally overwhelming signal from haemoglobin. This seminal study suggests that SERRS may be applied to other in situ, site-directed labelling experiments. [ABSTRACT FROM AUTHOR]

The adenine nucleotide pools and the NADH pool were compared in intact Nitrobacter winogradskyi cells grown under different conditions. The NADH pool was highest in nitrite-grown cells (22.0 nmol/mg N), less high in acetategrown cells (15.1 nmol/mg N),and lowest in pyruvate-grown cells (11.9 nmol/mg N). The adenine nucleotide pools and the NADH pool were determined after the transition from anaerobic to aerobic conditions. In both autotrophically and heterotrophically grown cells the ATP pool decreased within the first second after the addition of oxygen and then increased. In cells grown with nitrite or acetate the NADH pool increased the first second after the addition of oxygen then decreased below the initial value. In pyruvate-grown cells the changes in the NADH pool were less obvious. In the presence of rotenone autotrophic cells were able to generate ATP, but the reverse energy-dependent electron transport was inhibited. Consequently, NADH was not synthesized. N,N′-dicyclohexylcarbodiimide an inhibitor of ATPase, prevented both ATP and NADH generation. [ABSTRACT FROM AUTHOR]

Factors influencing the transient expression of introduced foreign DNA in electroporated protoplasts and intact cells of sugar beet were determined by assaying for the activity of chloramphenicol acetyltransferase (CAT), using a rectangular pulse generating system. Extractable CAT activity depended upon 1) applied plasmid DNA concentration, 2) protoplast density, 3) the interaction between pulse field strength, duration, number, time interval between pulses and the resultant effect on culture viability, and 4) the physiological state of the protoplasts. Mesophyll protoplasts were more susceptible to damage by electroporation, and were more specific in their requirement for electroporations which allowed CAT expression, than were protoplasts derived from suspension culture cells. CAT activity was also demonstrated, at low levels, after electroporation of intact suspension culture cells, and could be increased by pectinase treatment of the cells before electroporation. [ABSTRACT FROM AUTHOR]

Nuclear magnetic resonance (NMR) spectroscopy allows the determination of atomic-level information on intermolecular interactions, molecular structure, and molecular dynamics in the cellular environment. This may be broadly divided into studies focused on obtaining detailed molecular information in the intracellular context ("in-cell") or those focused on characterizing molecules or events at the cell surface ("on-cell"). In this review, we outline some key NMR techniques applied for on-cell NMR studies through both solution- and solid-state NMR and survey studies that have used these techniques to uncover key information. In particular, we focus on the application of on-cell NMR spectroscopy to characterize ligand interactions with cell surface membrane proteins such as G-protein coupled receptors (GPCRs) and receptor tyrosine kinases. These techniques allow for quantification of binding affinities, competitive binding assays, delineation of ligands involved in binding, ligand bound-state conformational determination, evaluation of receptor structuring and dynamics, and inference of distance constraints characteristic of the ligand-receptor bound state. Interestingly, it is possible to avoid the barriers of production and purification of membrane proteins while obtaining directly physiologically relevant information through on-cell NMR. We also provide a brief survey of the applicability of on-cell NMR approaches to other classes of cell surface molecules.

Innovative iron-based nanoparticles were synthesized, characterized and tested for the first time for harvesting single and mixed algal culture from real wastewater. The tailor-made magnetic nanoparticles (MNPs; Fe-MNP-I and Fe-MNP-II) achieved a percentage algae harvesting efficiency (%AHE) higher than 95% using a concentration of MNPs (C MNP) of 25 ± 0.3 (std. dev = 0.08) mg.L−1, mixing speed (M speed) of 120 ± 2 (std. dev = 0.10) rpm, short contact time (C t) of 7 ± 0.1 (std. dev = 0.05) min and separation time (SP t) of 3 ± 0.1 (std. dev = 0.09) min. The optimum operational conditions for harvesting of Chlorella vulgaris (C.v) were determined at (C MNP = 40 ± 0.4 (std. dev = 0.5) g MNPs.L−1, SP t = 2.5 ± 0.4 (std. dev = 0.1) min, M speed = 145 ± 3 (std. dev = 1.50) rpm and C t = 5 ± 0.3 (std. dev = 0.10) min using surface response methodology. Langmuir model describes better the adsorption behavior of algae-Fe-MNP-I system, while both Langmuir and Freundlich fit well the adsorption behavior of algae-Fe-MNP-II. The maximum adsorption capacity of Spirulina platensis (SP.PL) (18.27 ± 0.07 (std. dev = 0.19) mg DWC.mg particles −1) was higher than that for Chlorella vulgaris (C.v) (11.52 ± 0.01 (std. dev = 0.34) mg DWC.mg particles −1) and mixed algal culture (M.X) (17.20 ± 0.07 (std. dev = 0.54) mg DWC.mg particles −1) over Fe-MNP-I. Zeta potential measurements revealed that the adsorption mechanism between MNPs and algal strains is controlled by electrostatic interaction. The synthesized MNPs were recycled 10 times using alkaline-ultrasonic regeneration procedure. Unlabelled Image • Innovative nanoparticles were prepared and use to harvest single and mixed algae cells. • Algae harvesting efficiency of Chlorella vulgaris reached above 95% within 5 min. • Nanoparticles can be reused up to 10 times without significant efficiency loss. • Surface response methodology was used to identified optimum harvesting conditions. • Magnetic separation provides great potential to save time and energy during algal harvesting. [ABSTRACT FROM AUTHOR]