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Mitchison, T. J. "Polewards microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence." Journal of Cell Biology 109, nr 2 (1.08.1989): 637–52. http://dx.doi.org/10.1083/jcb.109.2.637.
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I have synthesized a novel derivative of carboxyfluorescein that is nonfluorescent, but can be converted to a fluorescent form by exposure to 365-nm light. This photoactivable, fluorescent probe was covalently attached to tubulin and microinjected into mitotic tissue culture cells, where it incorporated into functional spindles. To generate a fluorescent bar across the mitotic spindle, metaphase cells were irradiated with a slit microbeam. This bar decreased in intensity over the first minute, presumably due to turnover of nonkinetochore microtubules. The remaining fluorescent zones, now presumably restricted to kinetochore microtubules, moved polewards at 0.3-0.7 microns/min. This result provides strong evidence for polewards flux in kinetochore microtubules. In conjunction with earlier biotin-tubulin incorporation experiments (Mitchison, T. J., L. Evans, E. Schulze, and M. Kirschner. 1986. Cell. 45:515-527), I conclude that microtubules polymerize at kinetochores and depolymerize near the poles throughout metaphase. The significance of this observation for spindle structure and function is discussed. Local photoactivation of fluorescence should be a generally useful method for following molecular dynamics inside living cells.
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Chen, Jie, Liangliang Zhu, Heyi Xie, Jing Zhang, Yongsheng Mao, Zhenhong Huang, Bo Shi i Su Chen. "Microfluidic assembly of uniform fluorescent microbeads from quantum-dot-loaded fluorine-containing microemulsion". Polymer International 63, nr 11 (14.04.2014): 1953–58. http://dx.doi.org/10.1002/pi.4737.
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Santamarina, Sofía C., Daniel A. Heredia, Andrés M. Durantini i Edgardo N. Durantini. "Porphyrin Polymers Bearing N,N′-Ethylene Crosslinkers as Photosensitizers against Bacteria". Polymers 14, nr 22 (15.11.2022): 4936. http://dx.doi.org/10.3390/polym14224936.
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The appearance of microbes resistant to antibiotics requires the development of alternative therapies for the treatment of infectious diseases. In this work two polymers, PTPPF16-EDA and PZnTPPF16-EDA, were synthesized by the nucleophilic aromatic substitution of 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin and its Zn(II) complex with ethylenediamine, respectively. In these structures, the tetrapyrrolic macrocycles were N,N′-ethylene crosslinked, which gives them greater mobility. The absorption spectra of the polymers showed a bathochromic shift of the Soret band of ~10 nm with respect to the monomers. This effect was also found in the red fluorescence emission peaks. Furthermore, both polymeric materials produced singlet molecular oxygen with high quantum yields. In addition, they were capable of generating superoxide anion radicals. Photodynamic inactivation sensitized by these polymers was tested in Staphylococcus aureus and Escherichia coli bacteria. A decrease in cell viability greater than 7 log (99.9999%) was observed in S. aureus incubated with 0.5 μM photosensitizer upon 30 min of irradiation. Under these conditions, a low inactivation of E. coli (0.5 log) was found. However, when the cells were treated with KI, the elimination of the Gram-negative bacteria was achieved. Therefore, these polymeric structures are interesting antimicrobial photosensitizing materials for the inactivation of pathogens.
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Qiao, Tian, Soohyun Kim, Wonmok Lee i Hyunjung Lee. "Biodegradable and Porous Poly(lactic-co-glycolic acid) Microbeads for In vitro Evaluation of Negatively Charged Fluorescent Bacteria". Macromolecular Research 27, nr 3 (marzec 2019): 321–26. http://dx.doi.org/10.1007/s13233-019-7104-6.
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Erktan, Amandine, Matthias C. Rillig, Andrea Carminati, Alexandre Jousset i Stefan Scheu. "Protists and collembolans alter microbial community composition, C dynamics and soil aggregation in simplified consumer–prey systems". Biogeosciences 17, nr 20 (17.10.2020): 4961–80. http://dx.doi.org/10.5194/bg-17-4961-2020.
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Abstract. Microbes play an essential role in soil functioning including biogeochemical cycling and soil aggregate formation. Yet, a major challenge is to link microbes to higher trophic levels and assess consequences for soil functioning. Here, we aimed to assess how microbial consumers modify microbial community composition (PLFA markers), as well as C dynamics (microbial C use, SOC concentration and CO2 emission) and soil aggregation. We rebuilt two simplified soil consumer–prey systems: a bacterial-based system comprising amoebae (Acanthamoeba castellanii) feeding on a microbial community dominated by the free-living bacterium Pseudomonas fluorescens and a fungal-based system comprising collembolans (Heteromurus nitidus) grazing on a microbial community dominated by the saprotrophic fungus Chaetomium globosum. The amoeba A. castellanii did not affect microbial biomass and composition, but it enhanced the formation of soil aggregates and tended to reduce their stability. Presumably, the dominance of P. fluorescens, able to produce antibiotic toxins in response to the attack by A. castellanii, was the main cause of the unchanged microbial community composition, and the release of bacterial extracellular compounds, such as long-chained polymeric substances or proteases, in reaction to predation was responsible for the changes in soil aggregation as a side effect. In the fungal system, collembolans significantly modified microbial community composition via consumptive and non-consumptive effects including the transport of microbes on the body surface. As expected, fungal biomass promoted soil aggregation and was reduced in the presence of H. nitidus. Remarkably, we also found an unexpected contribution of changes in bacterial community composition to soil aggregation. In both the bacterial and fungal systems, bacterial and fungal communities mainly consumed C from soil organic matter (rather than the litter added). Increased fungal biomass was associated with an increased capture of C from added litter, and the presence of collembolans levelled off this effect. Neither amoebae nor collembolans altered SOC concentrations and CO2 production. Overall, the results demonstrated that trophic interactions are important for achieving a mechanistic understanding of biological contributions to soil aggregation and may occur without major changes in C dynamics and with or without changes in the composition of the microbial community.
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Cánovas, Verónica, Salvador Garcia-Chumillas, Fuensanta Monzó, Lorena Simó-Cabrera, Carmen Fernández-Ayuso, Carmen Pire i Rosa María Martínez-Espinosa. "Analysis of Polyhydroxyalkanoates Granules in Haloferax mediterranei by Double-Fluorescence Staining with Nile Red and SYBR Green by Confocal Fluorescence Microscopy". Polymers 13, nr 10 (14.05.2021): 1582. http://dx.doi.org/10.3390/polym13101582.
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Haloferaxmediterranei is a haloarchaeon of high interest in biotechnology because it produces and mobilizes intracellular polyhydroxyalkanoate (PHA) granules during growth under stress conditions (limitation of phosphorous in the culture media), among other interesting metabolites (enzymes, carotenoids, etc.). The capability of PHA production by microbes can be monitored with the use of staining-based methods. However, the staining of haloarchaea cells is a challenging task; firstly, due to the high ionic strength of the medium, which is inappropriate for most of dyes, and secondly, due to the low permeability of the haloarchaea S-layer to macromolecules. In this work, Haloferax mediterranei is used as a halophilic archaeon model to describe an optimized protocol for the visualization and analysis of intracellular PHA granules in living cells. The method is based on double-fluorescence staining using Nile red and SYBR Green by confocal fluorescence microscopy. Thanks to this method, the capability of PHA production by new haloarchaea isolates could be easily monitored.
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Pleva, Pavel, Lucie Bartošová, Daniela Máčalová, Ludmila Zálešáková, Jana Sedlaříková i Magda Janalíková. "Biofilm Formation Reduction by Eugenol and Thymol on Biodegradable Food Packaging Material". Foods 11, nr 1 (21.12.2021): 2. http://dx.doi.org/10.3390/foods11010002.
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Biofilm is a structured community of microorganisms adhering to surfaces of various polymeric materials used in food packaging. Microbes in the biofilm may affect food quality. However, the presence of biofilm can ensure biodegradation of discarded packaging. This work aims to evaluate a biofilm formation on the selected biodegradable polymer films: poly (lactic acid) (PLA), poly (butylene adipate-co-terephthalate) (PBAT), and poly (butylene succinate) (PBS) by selected bacterial strains; collection strains of Escherichiacoli, Staphylococcusaureus; and Bacillus pumilus, Bacillussubtilis, Bacillustequilensis, and Stenotrophomonasmaltophilia isolated from dairy products. Three different methods for biofilm evaluation were performed: the Christensen method, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and fluorescence microscopy. High biofilm formation was confirmed on the control PBS film, whereas low biofilm formation ability was observed on the PLA polymer sample. Furthermore, the films with incorporated antimicrobial compounds (thymol or eugenol) were also prepared. Antimicrobial activity and also reduction in biofilm formation on enriched polymer films were determined. Therefore, they were all proved to be antimicrobial and effective in reducing biofilm formation. These films can be used to prepare novel active food packaging for the dairy industry to prevent biofilm formation and enhance food quality and safety in the future.
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Yu, Runlan, Hongsheng Chai, Zhaojing Yu, Xueling Wu, Yuandong Liu, Li Shen, Jiaokun Li i in. "Behavior and Mechanism of Cesium Biosorption from Aqueous Solution by Living Synechococcus PCC7002". Microorganisms 8, nr 4 (30.03.2020): 491. http://dx.doi.org/10.3390/microorganisms8040491.
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Many efforts have focused on the adsorption of metals from contaminated water by microbes. Synechococcus PCC7002, a major marine cyanobacteria, is widely applied to remove metals from the ocean’s photic zone. However, its ability to adsorb cesium (Cs) nuclides has received little attention. In this study, the biosorption behavior of Cs(I) from ultrapure distilled water by living Synechococcus PCC7002 was investigated based on kinetic and isotherm studies, and the biosorption mechanism was characterized by Fourier-transform infrared spectroscopy, Raman spectroscopy, scanning electron microscopy, transmission electron microscopy, energy-dispersive X-ray spectrometry, and three-dimensional excitation emission matrix fluorescence spectroscopy. Synechococcus PCC7002 showed extremely high tolerance to Cs ions and its minimal inhibitory concentration was 8.6 g/L. Extracellular polymeric substances (EPS) in Synechococcus PCC7002 played a vital role in this tolerance. The biosorption of Cs by Synechococcus PCC7002 conformed to a Freundlich-type isotherm model and pseudo-second-order kinetics. The binding of Cs(I) was primarily attributed to the extracellular proteins in EPS, with the amino, hydroxyl, and phosphate groups on the cell walls contributing to Cs adsorption. The biosorption of Cs involved two mechanisms: Passive adsorption on the cell surface at low Cs concentrations and active intracellular adsorption at high Cs concentrations. The results demonstrate that the behavior and mechanism of Cs adsorption by Synechococcus PCC7002 differ based on the Cs ions concentration.
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Han, Qingqing, Qingqing Wang, Aiping Gao, Xuefei Ge, Rong Wan i Xinhua Cao. "Fluorescent Quinoline-Based Supramolecular Gel for Selective and Ratiometric Sensing Zinc Ion with Multi-Modes". Gels 8, nr 10 (21.09.2022): 605. http://dx.doi.org/10.3390/gels8100605.
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A gelator 1 containing functional quinoline and Schiff base groups that could form organogels in DMF, DMSO, acetone, ethanol and 1,4-dioxane was designed and synthesized. The self-assembly process of geator 1 was characterized by field emission scanning electron microscopy (FESEM), UV-vis absorption spectroscopy, fluorescence emission spectroscopy, Fourier transform infrared spectroscopy(FTIR), X-ray powder diffraction (XRD) and water contact angle. Under non-covalent interactions, gelator 1 self-assembled into microbelts and nanofiber structures with different surface wettability. Weak fluorescence was emitted from the solution and gel state of 1. Interestingly, gelator 1 exhibited good selectivity and sensitivity towards Zn2+ in solution and gel states along with its emission enhancement and change. The emission intensity at 423 nm of solution 1 in 1,4-dioxane was slightly enhanced, and a new emission peak appeared at 545 nm along with its intensity sequentially strengthened in the titration process. The obvious ratiometric detection process was presented with a limit of detection (LOD) of 5.51 μM. The detection mechanism was revealed by a theoretical calculation and NMR titration experiment, which was that Zn2+ induced the transition from trans- to cis- of molecule 1 and further coordinated with 1. This study will introduce a new method for the construction of functional self-assembly gel sensors for the detection of Zn2+.
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Klueglein, Nicole, Fabian Zeitvogel, York-Dieter Stierhof, Matthias Floetenmeyer, Kurt O. Konhauser, Andreas Kappler i Martin Obst. "Potential Role of Nitrite for Abiotic Fe(II) Oxidation and Cell Encrustation during Nitrate Reduction by Denitrifying Bacteria". Applied and Environmental Microbiology 80, nr 3 (22.11.2013): 1051–61. http://dx.doi.org/10.1128/aem.03277-13.
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ABSTRACTMicroorganisms have been observed to oxidize Fe(II) at neutral pH under anoxic and microoxic conditions. While most of the mixotrophic nitrate-reducing Fe(II)-oxidizing bacteria become encrusted with Fe(III)-rich minerals, photoautotrophic and microaerophilic Fe(II) oxidizers avoid cell encrustation. The Fe(II) oxidation mechanisms and the reasons for encrustation remain largely unresolved. Here we used cultivation-based methods and electron microscopy to compare two previously described nitrate-reducing Fe(II) oxidizers (Acidovoraxsp. strain BoFeN1 andPseudogulbenkianiasp. strain 2002) and two heterotrophic nitrate reducers (Paracoccus denitrificansATCC 19367 andP. denitrificansPd 1222). All four strains oxidized ∼8 mM Fe(II) within 5 days in the presence of 5 mM acetate and accumulated nitrite (maximum concentrations of 0.8 to 1.0 mM) in the culture media. Iron(III) minerals, mainly goethite, formed and precipitated extracellularly in close proximity to the cell surface. Interestingly, mineral formation was also observed within the periplasm and cytoplasm; intracellular mineralization is expected to be physiologically disadvantageous, yet acetate consumption continued to be observed even at an advanced stage of Fe(II) oxidation. Extracellular polymeric substances (EPS) were detected by lectin staining with fluorescence microscopy, particularly in the presence of Fe(II), suggesting that EPS production is a response to Fe(II) toxicity or a strategy to decrease encrustation. Based on the data presented here, we propose a nitrite-driven, indirect mechanism of cell encrustation whereby nitrite forms during heterotrophic denitrification and abiotically oxidizes Fe(II). This work adds to the known assemblage of Fe(II)-oxidizing bacteria in nature and complicates our ability to delineate microbial Fe(II) oxidation in ancient microbes preserved as fossils in the geological record.
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Chang, Shun-Hsien, Ying-Ju Chen, Hsiang-Jung Tseng, Hsin-I. Hsiao, Huey-Jine Chai, Kuo-Chung Shang, Chorng-Liang Pan i Guo-Jane Tsai. "Antibacterial Activity of Chitosan–Polylactate Fabricated Plastic Film and Its Application on the Preservation of Fish Fillet". Polymers 13, nr 5 (25.02.2021): 696. http://dx.doi.org/10.3390/polym13050696.
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This research prepared chitosan–PLA plastic films by extrusion, analyzed the physical and mechanical properties and antibacterial activity of the fabricated plastic films, and used them to preserve grouper fillet. We added chitosan (220 kDa, 93% DD) in the weight ratio of 0.5–2% into the PLA to prepare the chitosan–PLA films. With the increasing chitosan dosage, both the water vapor transmission rate and moisture content of chitosan–PLA films increased. Among the three doses of chitosan (0.5%, 1%, and 2%) added to PLA, 0.5% chitosan–PLA film had the highest antibacterial activity. This plastic film had an inhibitory efficiency of over 95% against Escherichia coli, Pseudomonas fluorescens, and Staphylococcus aureus. The action of covering the fish fillet with 0.5% chitosan–PLA film significantly reduced several microbes’ counting (i.e., mesophiles, psychrophiles, coliforms, Pseudomonas, Aeromonas, and Vibrio) and total volatile basic nitrogen (TVBN) value in the grouper fillets stored at 4 °C. Thus, such action prolongs the fish fillets’ shelf life to up to at least nine days, and this 0.5% chitosan–PLA film shows promising potential for preserving refrigerated fish.
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Kumar, Parveen, Joshua Tamayo, Ruei-Feng Shiu, Wei-Chun Chin i Arvind Gopinath. "Size-Dependent Diffusion and Dispersion of Particles in Mucin". Polymers 15, nr 15 (29.07.2023): 3241. http://dx.doi.org/10.3390/polym15153241.
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Mucus, composed significantly of glycosylated mucins, is a soft and rheologically complex material that lines respiratory, reproductive, and gastrointestinal tracts in mammals. Mucus may present as a gel, as a highly viscous fluid, or as a viscoelastic fluid. Mucus acts as a barrier to the transport of harmful microbes and inhaled atmospheric pollutants to underlying cellular tissue. Studies on mucin gels have provided critical insights into the chemistry of the gels, their swelling kinetics, and the diffusion and permeability of molecular constituents such as water. The transport and dispersion of micron and sub-micron particles in mucin gels and solutions, however, differs from the motion of small molecules since the much larger tracers may interact with microstructure of the mucin network. Here, using brightfield and fluorescence microscopy, high-speed particle tracking, and passive microrheology, we study the thermally driven stochastic movement of 0.5–5.0 μm tracer particles in 10% mucin solutions at neutral pH, and in 10% mucin mixed with industrially relevant dust; specifically, unmodified limestone rock dust, modified limestone, and crystalline silica. Particle trajectories are used to calculate mean square displacements and the displacement probability distributions; these are then used to assess tracer diffusion and transport. Complex moduli are concomitantly extracted using established microrheology techniques. We find that under the conditions analyzed, the reconstituted mucin behaves as a weak viscoelastic fluid rather than as a viscoelastic gel. For small- to moderately sized tracers with a diameter of lessthan 2 μm, we find that effective diffusion coefficients follow the classical Stokes–Einstein relationship. Tracer diffusivity in dust-laden mucin is surprisingly larger than in bare mucin. Probability distributions of mean squared displacements suggest that heterogeneity, transient trapping, and electrostatic interactions impact dispersion and overall transport, especially for larger tracers. Our results motivate further exploration of physiochemical and rheological mechanisms mediating particle transport in mucin solutions and gels.
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Senarat, Setthapong, Sarun Tuntarawongsa, Nutdanai Lertsuphotvanit, Catleya Rojviriya, Thawatchai Phaechamud i Takron Chantadee. "Levofloxacin HCl-Loaded Eudragit L-Based Solvent Exchange-Induced In Situ Forming Gel Using Monopropylene Glycol as a Solvent for Periodontitis Treatment". Gels 9, nr 7 (18.07.2023): 583. http://dx.doi.org/10.3390/gels9070583.
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Solvent exchange-induced in situ forming gel (ISG) is currently an appealing dosage form for periodontitis treatment via localized injection into the periodontal pocket. This study aims to apply Eudragit L and Eudragit S as matrix components of ISG by using monopropylene glycol as a solvent for loading levofloxacin HCl for periodontitis treatment. The influence of Eudragit concentration was investigated in terms of apparent viscosity, rheological behavior, injectability, gel-forming behavior, and mechanical properties. Eudragit L-based formulation presented less viscosity, was easier to inject, and could form more gel than Eudragit S-based ISG. Levofloxacin HCl-loading diminished the viscosity of Eudragit L-based formulation but did not significantly change the gel formation ability. Higher polymer loading increased viscosity, force-work of injectability, and hardness. SEM photographs and µCT images revealed their scaffold formation, which had a denser topographic structure and less porosity attained owing to higher polymer loading and less in vitro degradation. By tracking with fluorescence dyes, the interface interaction study revealed crucial information such as solvent movement ability and matrix formation of ISG. They prolonged the drug release for 14 days with fickian drug diffusion kinetics and increased the release amount above the MIC against test microbes. The 1% levofloxacin HCl and 15% Eudragit L dissolved in monopropylene glycol (LLM15) was a promising ISG because of its appropriate viscosity (3674.54 ± 188.03 cP) with Newtonian flow, acceptable gel formation and injectability (21.08 ± 1.38 N), hardness (33.81 ± 2.3 N) and prolonged drug release with efficient antimicrobial activities against S. aureus (ATCC 6538, 6532, and 25923), methicillin-resistant S. aureus (MRSA) (S. aureus ATCC 4430), E. coli ATCC 8739, C. albicans ATCC 10231, P. gingivalis ATCC 33277, and A. actinomycetemcomitans ATCC 29522; thus, it is the potential ISG formulation for periodontitis treatment by localized periodontal pocket injection.
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Macedonia, Mary C., Julia L. Drewes, Nicholas O. Markham, Alan J. Simmons, Joseph T. Roland, Paige N. Vega, Cherie’ R. Scurrah i in. "Clinically adaptable polymer enables simultaneous spatial analysis of colonic tissues and biofilms". npj Biofilms and Microbiomes 6, nr 1 (24.09.2020). http://dx.doi.org/10.1038/s41522-020-00143-x.
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Abstract Microbial influences on host cells depend upon the identities of the microbes, their spatial localization, and the responses they invoke on specific host cell populations. Multimodal analyses of both microbes and host cells in a spatially resolved fashion would enable studies into these complex interactions in native tissue environments, potentially in clinical specimens. While techniques to preserve each of the microbial and host cell compartments have been used to examine tissues and microbes separately, we endeavored to develop approaches to simultaneously analyze both compartments. Herein, we established an original method for mucus preservation using Poloxamer 407 (also known as Pluronic F-127), a thermoreversible polymer with mucus-adhesive characteristics. We demonstrate that this approach can preserve spatially-defined compartments of the mucus bi-layer in the colon and the bacterial communities within, compared with their marked absence when tissues were processed with traditional formalin-fixed paraffin-embedded (FFPE) pipelines. Additionally, antigens for antibody staining of host cells were preserved and signal intensity for 16S rRNA fluorescence in situ hybridization (FISH) was enhanced in poloxamer-fixed samples. This in turn enabled us to integrate multimodal analysis using a modified multiplex immunofluorescence (MxIF) protocol. Importantly, we have formulated Poloxamer 407 to polymerize and cross-link at room temperature for use in clinical workflows. These results suggest that the fixative formulation of Poloxamer 407 can be integrated into biospecimen collection pipelines for simultaneous analysis of microbes and host cells.
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Demir, Zehra, Pinar Ozmen i Bunyamin Karagoz. "Selective detection of Fe (III) via fluorescence turn-on mechanism with Rhodamine tethered poly(vinyl amine) microbeads". Polymer Bulletin, 7.10.2021. http://dx.doi.org/10.1007/s00289-021-03930-4.
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Wang, Yinghe, Haina Qi, Yunrui Xie, Hong Shao, Liu Yang, Dawei Sun, Qianli Ma, Wensheng Yu i Xiangting Dong. "Design and one-pot direct electrospinning construction of high-performance magnetic@conductive@fluorescent tri-coaxial microbelts and array". Polymer Testing, listopad 2022, 107857. http://dx.doi.org/10.1016/j.polymertesting.2022.107857.
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Ghosh, Sreejita, Dibyajit Lahiri, Moupriya Nag, Tanmay Sarkar, Siddhartha Pati, Hisham Atan Edinur, Manoj Kumar, Muhammad R. A. Mohd Zain i Rina Rani Ray. "Precision targeting of food biofilm-forming genes by microbial scissors: CRISPR-Cas as an effective modulator". Frontiers in Microbiology 13 (9.08.2022). http://dx.doi.org/10.3389/fmicb.2022.964848.
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The abrupt emergence of antimicrobial resistant (AMR) bacterial strains has been recognized as one of the biggest public health threats affecting the human race and food processing industries. One of the causes for the emergence of AMR is the ability of the microorganisms to form biofilm as a defense strategy that restricts the penetration of antimicrobial agents into bacterial cells. About 80% of human diseases are caused by biofilm-associated sessile microbes. Bacterial biofilm formation involves a cascade of genes that are regulated via the mechanism of quorum sensing (QS) and signaling pathways that control the production of the extracellular polymeric matrix (EPS), responsible for the three-dimensional architecture of the biofilm. Another defense strategy utilized commonly by various bacteria includes clustered regularly interspaced short palindromic repeats interference (CRISPRi) system that prevents the bacterial cell from viral invasion. Since multigenic signaling pathways and controlling systems are involved in each and every step of biofilm formation, the CRISPRi system can be adopted as an effective strategy to target the genomic system involved in biofilm formation. Overall, this technology enables site-specific integration of genes into the host enabling the development of paratransgenic control strategies to interfere with pathogenic bacterial strains. CRISPR-RNA-guided Cas9 endonuclease, being a promising genome editing tool, can be effectively programmed to re-sensitize the bacteria by targeting AMR-encoding plasmid genes involved in biofilm formation and virulence to revert bacterial resistance to antibiotics. CRISPRi-facilitated silencing of genes encoding regulatory proteins associated with biofilm production is considered by researchers as a dependable approach for editing gene networks in various biofilm-forming bacteria either by inactivating biofilm-forming genes or by integrating genes corresponding to antibiotic resistance or fluorescent markers into the host genome for better analysis of its functions both in vitro and in vivo or by editing genes to stop the secretion of toxins as harmful metabolites in food industries, thereby upgrading the human health status.
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Karunatilaka, Krishanthi S., Elizabeth A. Cameron, Eric C. Martens, Nicole M. Koropatkin i Julie S. Biteen. "Superresolution Imaging Captures Carbohydrate Utilization Dynamics in Human Gut Symbionts". mBio 5, nr 6 (11.11.2014). http://dx.doi.org/10.1128/mbio.02172-14.
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ABSTRACTGut microbes play a key role in human health and nutrition by catabolizing a wide variety of glycans via enzymatic activities that are not encoded in the human genome. The ability to recognize and process carbohydrates strongly influences the structure of the gut microbial community. While the effects of diet on the microbiota are well documented, little is known about the molecular processes driving metabolism. To provide mechanistic insight into carbohydrate catabolism in gut symbionts, we studied starch processing in real time in the modelBacteroides thetaiotaomicronstarch utilization system (Sus) by single-molecule fluorescence. Although previous studies have explored Sus protein structure and function, the transient interactions, assembly, and collaboration of these outer membrane proteins have not yet been elucidated in live cells. Our live-cell superresolution imaging reveals that the polymeric starch substrate dynamically recruits Sus proteins, serving as an external scaffold for bacterial membrane assembly of the Sus complex, which may promote efficient capturing and degradation of starch. Furthermore, by simultaneously localizing multiple Sus outer membrane proteins on theB. thetaiotaomicroncell surface, we have characterized the dynamics and stoichiometry of starch-induced Sus complex assembly on the molecular scale. Finally, based on Sus protein knockout strains, we have discerned the mechanism of starch-induced Sus complex assembly in live anaerobic cells with nanometer-scale resolution. Our insights into the starch-induced outer membrane protein assembly central to this conserved nutrient uptake mechanism pave the way for the development of dietary or pharmaceutical therapies to controlBacteroidetesin the intestinal tract to enhance human health and treat disease.IMPORTANCEIn this study, we used nanometer-scale superresolution imaging to reveal dynamic interactions between the proteins involved in starch processing by the prominent human gut symbiontBacteroides thetaiotaomicronin real time in live cells. These results represent the first working model of starch utilization system (Sus) complex assembly and function during glycan catabolism and are likely to describe aspects of how other Sus-like systems function in human gutBacteroidetes. Our results provide unique mechanistic insights into a glycan catabolism strategy that is prevalent within the human gut microbial community. Proper understanding of this conserved nutrient uptake mechanism is essential for the development of dietary or pharmaceutical therapies to control intestinal tract microbial populations, to enhance human health, and to treat disease.