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Extracellular vesicles (EVs) are mediators of intercellular communication, recently recognised for their clinical applications. Accurate characterisation and quantification of EVs are critical for understanding of their function and clinical relevance. Many platforms utilise fluorescence for EV characterisation, frequently labelling surface proteins to identify EVs. The heterogeneity of EVs and the lack of a universal protein marker encourages the use of generic EV labelling methods, including membrane labelling. Using nano‐flow cytometry, we evaluated six membrane dyes, including MemGlow and CellMask. Evaluation criteria included EV labelling efficacy, non‐specific labelling of very low‐density lipoproteins (VLDLs), brightness and dye aggregation. Significant variation was observed in dye performance, with certain dyes showing poor EV labelling efficacy or high affinity to VLDLs. Importantly, several promising candidates were identified for further investigation. Overall, this study highlights the importance of selecting appropriate membrane dyes for EV staining tailored to the aims of the study and the EV origin. MemGlow and CellMask proved favourable, allowing bright, sensitive staining of EV membranes with minimal aggregation. However, MemGlow showed an affinity to VLDLs, and CellMask requires additional sample handling for optimal labelling. These results contribute to deepening our understanding of EV membrane dyes, allowing for better dye selection and EV identification in future studies. [ABSTRACT FROM AUTHOR]

In this study, a quantitative method based on fluorescein isothiocyanate (FITC)-labelled Hizikia fusiforme polyphenol–polysaccharide complex (HPC) and its purified fractions (PC1, PC4) was used, and its pharmacokinetics and tissue distribution were investigated in mice. The results showed that the FITC-labelled method had good linearity (R2 > 0.99), intra-day and inter-day precision (RSD, %) consistently lower than 15%, recovery (93.19–106.54%), and stability (RSD < 15%), which met the basic criteria for pharmacokinetic studies. The pharmacokinetic and tissue distribution results in mice after administration showed that all three sample groups could enter the blood circulation. and HPC-FITC had a longer half-life (T1/2: 26.92 ± 0.76 h) and mean retention time (MRT0–∞: 36.48 h) due to its larger molecular weight. The three groups of samples could be absorbed by the organism in a short time (0.5 h) mainly in the stomach and intestine; the samples could be detected in the urine after 2 h of administration indicating strong renal uptake, and faecal excretion reached its maximum at 12 h. The samples were also detected in the urine after 2 h of administration. This study provides some theoretical basis for the tissue distribution pattern of polyphenol–polysaccharide complex. [ABSTRACT FROM AUTHOR]

Abstract Extracellular vesicles (EVs) are mediators of intercellular communication, recently recognised for their clinical applications. Accurate characterisation and quantification of EVs are critical for understanding of their function and clinical relevance. Many platforms utilise fluorescence for EV characterisation, frequently labelling surface proteins to identify EVs. The heterogeneity of EVs and the lack of a universal protein marker encourages the use of generic EV labelling methods, including membrane labelling. Using nano‐flow cytometry, we evaluated six membrane dyes, including MemGlow and CellMask. Evaluation criteria included EV labelling efficacy, non‐specific labelling of very low‐density lipoproteins (VLDLs), brightness and dye aggregation. Significant variation was observed in dye performance, with certain dyes showing poor EV labelling efficacy or high affinity to VLDLs. Importantly, several promising candidates were identified for further investigation. Overall, this study highlights the importance of selecting appropriate membrane dyes for EV staining tailored to the aims of the study and the EV origin. MemGlow and CellMask proved favourable, allowing bright, sensitive staining of EV membranes with minimal aggregation. However, MemGlow showed an affinity to VLDLs, and CellMask requires additional sample handling for optimal labelling. These results contribute to deepening our understanding of EV membrane dyes, allowing for better dye selection and EV identification in future studies.

Abstract Background After two decades of extensive microbiome research, the current forefront of scientific exploration involves moving beyond description and classification to uncovering the intricate mechanisms underlying the coalescence of microbial communities. Deciphering microbiome assembly has been technically challenging due to their vast microbial diversity but establishing a synthetic community (SynCom) serves as a key strategy in unravelling this process. Achieving absolute quantification is crucial for establishing causality in assembly dynamics. However, existing approaches are primarily designed to differentiate a specific group of microorganisms within a particular SynCom. Results To address this issue, we have developed the differential fluorescent marking (DFM) strategy, employing three distinguishable fluorescent proteins in single and double combinations. Building on the mini-Tn7 transposon, DFM capitalises on enhanced stability and broad applicability across diverse Proteobacteria species. The various DFM constructions are built using the pTn7-SCOUT plasmid family, enabling modular assembly, and facilitating the interchangeability of expression and antibiotic cassettes in a single reaction. DFM has no detrimental effects on fitness or community assembly dynamics, and through the application of flow cytometry, we successfully differentiated, quantified, and tracked a diverse six-member SynCom under various complex conditions like root rhizosphere showing a different colonisation assembly dynamic between pea and barley roots. Conclusions DFM represents a powerful resource that eliminates dependence on sequencing and/or culturing, thereby opening new avenues for studying microbiome assembly. Video Abstract

Abstract This review presents a comprehensive overview of labelling strategies for endogenous and exogenous extracellular vesicles, that can be utilised both in vitro and in vivo. It covers a broad spectrum of approaches, including fluorescent and bioluminescent labelling, and provides an analysis of their applications, strengths, and limitations. Furthermore, this article presents techniques that use radioactive tracers and contrast agents with the ability to track EVs both spatially and temporally. Emphasis is also placed on endogenous labelling mechanisms, represented by Cre-lox and CRISPR-Cas systems, which are powerful and flexible tools for real-time EV monitoring or tracking their fate in target cells. By summarizing the latest developments across these diverse labelling techniques, this review provides researchers with a reference to select the most appropriate labelling method for their EV based research.

Background: After two decades of extensive microbiome research, the current forefront of scientific exploration involves moving beyond description and classification to uncovering the intricate mechanisms underlying the coalescence of microbial communities. Deciphering microbiome assembly has been technically challenging due to their vast microbial diversity but establishing a synthetic community (SynCom) serves as a key strategy in unravelling this process. Achieving absolute quantification is crucial for establishing causality in assembly dynamics. However, existing approaches are primarily designed to differentiate a specific group of microorganisms within a particular SynCom. Results: To address this issue, we have developed the differential fluorescent marking (DFM) strategy, employing three distinguishable fluorescent proteins in single and double combinations. Building on the mini-Tn7 transposon, DFM capitalises on enhanced stability and broad applicability across diverse Proteobacteria species. The various DFM constructions are built using the pTn7-SCOUT plasmid family, enabling modular assembly, and facilitating the interchangeability of expression and antibiotic cassettes in a single reaction. DFM has no detrimental effects on fitness or community assembly dynamics, and through the application of flow cytometry, we successfully differentiated, quantified, and tracked a diverse six-member SynCom under various complex conditions like root rhizosphere showing a different colonisation assembly dynamic between pea and barley roots. Conclusions: DFM represents a powerful resource that eliminates dependence on sequencing and/or culturing, thereby opening new avenues for studying microbiome assembly. CZiF6AaEp7jbjKaYRmvD15 Video Abstract [ABSTRACT FROM AUTHOR]

This review presents a comprehensive overview of labelling strategies for endogenous and exogenous extracellular vesicles, that can be utilised both in vitro and in vivo. It covers a broad spectrum of approaches, including fluorescent and bioluminescent labelling, and provides an analysis of their applications, strengths, and limitations. Furthermore, this article presents techniques that use radioactive tracers and contrast agents with the ability to track EVs both spatially and temporally. Emphasis is also placed on endogenous labelling mechanisms, represented by Cre-lox and CRISPR-Cas systems, which are powerful and flexible tools for real-time EV monitoring or tracking their fate in target cells. By summarizing the latest developments across these diverse labelling techniques, this review provides researchers with a reference to select the most appropriate labelling method for their EV based research. [ABSTRACT FROM AUTHOR]

We investigated the contamination of the operator and the surrounding environment during wound debridement through simulated operations using fluorescent labelling. On‐site simulated operation assessment was performed before and after the training. Oranges and square towels were used to simulate wounds and the inpatient units, respectively. Fluorescent powder was applied to the surfaces. Operations on oranges simulated bedside debridement, and the postoperative distribution of the fluorescent powder was employed to reflect the contamination of the operator and the surrounding environment. During the pre‐training assessment, contamination was observed in 28 of the 29 trainees. The commonly contaminated parts were the extensor side of the forearm, middle abdomen, upper abdomen, and hands. The right side of the operating area was contaminated in 24 trainees. During the post‐training assessment, contamination was observed in 13 of the 15 trainees. The commonly parts were the hands, extensor side of the forearm, and the lower abdomen. The front, back, left, and right sides of the operating area were contaminated in 12, 9, 11, and 14 trainees, respectively. Contamination of the treatment cart was observed in 5 trainees. Operator and the surrounding environment can be contaminated during wound debridement. Attention should be paid to hand hygiene, wearing and changing of work clothes, and disinfection of the surrounding environment. Moreover, regular training is recommended. [ABSTRACT FROM AUTHOR]

In this study, a quantitative method based on fluorescein isothiocyanate (FITC)-labelled Hizikia fusiforme polyphenol–polysaccharide complex (HPC) and its purified fractions (PC1, PC4) was used, and its pharmacokinetics and tissue distribution were investigated in mice. The results showed that the FITC-labelled method had good linearity (R2 > 0.99), intra-day and inter-day precision (RSD, %) consistently lower than 15%, recovery (93.19–106.54%), and stability (RSD < 15%), which met the basic criteria for pharmacokinetic studies. The pharmacokinetic and tissue distribution results in mice after administration showed that all three sample groups could enter the blood circulation. and HPC-FITC had a longer half-life (T1/2: 26.92 ± 0.76 h) and mean retention time (MRT0–∞: 36.48 h) due to its larger molecular weight. The three groups of samples could be absorbed by the organism in a short time (0.5 h) mainly in the stomach and intestine; the samples could be detected in the urine after 2 h of administration indicating strong renal uptake, and faecal excretion reached its maximum at 12 h. The samples were also detected in the urine after 2 h of administration. This study provides some theoretical basis for the tissue distribution pattern of polyphenol–polysaccharide complex.

During mitosis in metazoan species, the nuclear envelope (NE) undergoes breakdown, and its fragments are absorbed within the membranous network of the endoplasmic reticulum (ER). Past observations by fluorescence microscopy led researchers to think that the NE loses its identity when it is absorbed within the ER membrane. However, in our previous work, we developed a more specific labelling method and found evidence that the NE does not completely lose its identity during mitosis. In the present work, we conduct further experiments, the results of which support the idea that the NE partially retains its identity during mitosis. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), continues to pose a significant global health threat. Identifying new druggable targets is crucial for the advancement of drug development. Equally critical is the development of precise methods for monitoring Mtb to effectively combat this disease. Addressing these needs, our study pinpointed the pore domain (PD) of MtbMmpL3 as a new binding site for virtual screening, which led to the discovery of the small molecule ZY27. To confirm the binding site and action mode of ZY27, we employed cosolvent molecular dynamics (CMD), steered molecular dynamics (SMD), and long timescale molecular dynamics (MD) simulations of 5 μs. These in silico studies verified that ZY27 binds to the PD of MtbMmpL3. In antimicrobial activity tests, ZY27 exhibited potent anti-Mtb activity and high selectivity among mycobacterial species. Whole-genome sequencing of spontaneous ZY27-resistant Mtb variants, complemented by acid-fast staining experiments, confirmed that ZY27 specifically targets MtbMmpL3. Utilizing the ligand-protein binding data, we designed and synthesized two solvatochromic fluorescent probes, 27FP1 and 27FP2, based on ZY27. Further investigations through flow cytometry and confocal microscopy confirmed that these probes specifically label Mtb cells via the MtbMmpL3 binding mechanism. This study provides valuable insights for the design, development, and application of a new generation of diagnostic tools in the form of MtbMmpL3 inhibitors by integrating computational and experimental Approaches. [Display omitted] [ABSTRACT FROM AUTHOR]

Surface modification of silica nanoparticles with organic functional groups while maintaining colloidal stability remains a synthetic challenge. This work aimed to prepare highly dispersed porous hollow organosilica particles (pHOPs) with amino surface modification. The amino-surface modification of pHOPs was carried out with 3-aminopropyl(diethoxy)methylsilane (APDEMS) under various reaction parameters, and the optimal pHOP-NH2 sample was selected and labelled with fluorescein isothiocyanate (FITC) to achieve fluorescent pHOPs (F-HOPs). The prepared pHOPs were thoroughly characterized by transmission electron microscopy, dynamic light scattering, FT-IR, UV-Vis and fluorescence spectroscopies, and microfluidic resistive pulse sensing. The optimal amino surface modification of pHOPs with APDEMS was at pH 10.2, at 60 °C temperature with 10 min reaction time. The positive Zeta potential of pHOP-NH2 in an acidic environment and the appearance of vibrations characteristic to the surface amino groups on the FT-IR spectra prove the successful surface modification. A red-shift in the absorbance spectrum and the appearance of bands characteristic to secondary amines in the FTIR spectrum of F-HOP confirmed the covalent attachment of FITC to pHOP-NH2. This study provides a step-by-step synthetic optimization and characterization of fluorescently labelled organosilica particles to enhance their optical properties and extend their applications. [ABSTRACT FROM AUTHOR]

Fluorescently labelling proteins such as insulin have wide ranging applications in a pharmaceutical research and drug delivery. Human insulin (Actrapid®) was labelled with fluorescein isothiocyanate (FITC) and the synthesised conjugate identified using reverse phase high performance liquid chromatography (RP-HPLC) on a C18 column and a gradient method with mobile phase A containing 0.1% trifluoroacetic acid (TFA) in Millipore water and mobile phase B containing 90% Acetonitrile, 10% Millipore water and 0.1% TFA. Syntheses were carried out at varying reaction times between 4 and 20 h. Mono-labelled FITC-insulin conjugate was successfully synthesised with labelling at the B1 position on the insulin chain using a molar ratio of 2:1 (FITC:insulin) at a reaction time of 18 h and confirmed by electrospray mass spectroscopy. Reactions were studied across a pH range of 7–9.8 and the quantities switch from mono-labelled to di-labelled FITC-insulin conjugates at a reaction time of 2 h (2:1 molar ratio) at pH > 8. The conjugates isolated from the studies had biological activities in comparison to native insulin of 99.5% monoB1, 78% monoA1, 51% diA1B1 and 0.06% triA1B1B29 in HUVEC cells by examining AKT phosphorylation levels. MonoB1 FITC-insulin conjugate was also compared to native insulin by examining cell surface GLUT4 in C2C12 skeletal muscle cells. No significant difference in the cellular response was observed for monoB1 produced in-house compared to native insulin. Therefore mono-labelled FITC-insulin at the B1 position showed similar biological activity as native insulin and can potentially be used for future biomedical applications. [ABSTRACT FROM AUTHOR]

In this study, we report a switchable fluorescence probe based on nitrogen-doped graphene quantum dots (N-GQDs) to detect the presence of tetracycline and L-Cystence. Strong blue emissive N-GQDs were successfully prepared by the hydrothermal treatment of urea and benzophenone, with a quantum yield of 34.9%. Both transmission electron microscopy and atomic force microscope images reveal a nearly monodispersed N-GQDs with spherical morphologies, and provide evidences of 1–3 layered structures of N-GQDs. Meanwhile, the obtained N-GQDs can be utilized for fluorescent labelling due to their superior optical stability and favourable biological compatibility. A strong fluorescence quenching was observed for the presence of tetracycline. The following addition of L-Cysteine can motivate the regeneration of fluorescence. Therefore, a simple strategy for the detection of tetracycline and L-Cystence in natural water was developed due to the relevant sensitivity of N-GQDs nanosensor. [ABSTRACT FROM AUTHOR]

In food industry, Listeria monocytogenes contamination can occur accidentally despite the quality control of raw materials and factory. Decontamination processes or inhibitory effects of ingredients/additives in food products are set up to ensure compliance with hygiene and microbiological criteria. These actions represent stresses for the pathogenic agent, causing fluctuations in its physiological states. Moreover, during these environmental stresses, Listeria monocytogenes can enter in a viable but nonculturable (VBNC) state which is not detected by plate counting but by flow cytometry. This technique coupled with cell staining by fluorescent dyes offers the possibility to assess different physiological states based on different cellular parameters: enzymatic activity, transmembrane integrity, membrane potential, and respiratory activity. In this chapter, we present a method to assess the viability of foodborne pathogens using a double-staining principle based on the assessment of membrane integrity and intracellular esterase activity., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Fluorescent nanoparticles (FNPs) have been widely used in chemistry and medicine for decades, but their employment in biology is relatively recent. Past reviews on FNPs have focused on chemical, physical or medical uses, making the extrapolation to biological applications difficult. In biology, FNPs have largely been used for biosensing and molecular tracking. However, concerns over toxicity in early types of FNPs, such as cadmium‐containing quantum dots (QDs), may have prevented wide adoption. Recent developments, especially in non‐Cd‐containing FNPs, have alleviated toxicity problems, facilitating the use of FNPs for addressing ecological, physiological and molecule‐level processes in biological research. Standardised protocols from synthesis to application and interdisciplinary approaches are critical for establishing FNPs in the biologists' tool kit. Here, we present an introduction to FNPs, summarise their use in biological applications, and discuss technical issues such as data reliability and biocompatibility. We assess whether biological research can benefit from FNPs and suggest ways in which FNPs can be applied to answer questions in biology. We conclude that FNPs have a great potential for studying various biological processes, especially tracking, sensing and imaging in physiology and ecology. [ABSTRACT FROM AUTHOR]

The synthesis of 2′‐deoxycytidine and its 5′‐O‐triphosphate bearing solvatochromic acetophenyl‐thienyl‐aniline fluorophore was developed using the Sonogashira cross‐coupling reaction as the key step. The triphosphate was used for polymerase synthesis of labelled DNA. The labelled nucleotide or DNA exerted weak red fluorescence when excited at 405 nm, but a significant colour change (to yellow or green) and light‐up (up to 20 times) was observed when the DNA probes interacted with proteins or lipids. [ABSTRACT FROM AUTHOR]

Surface modification of silica nanoparticles with organic functional groups while maintaining colloidal stability remains a synthetic challenge. This work aimed to prepare highly dispersed porous hollow organosilica particles (pHOPs) with amino surface modification. The amino-surface modification of pHOPs was carried out with 3-aminopropyl(diethoxy)methylsilane (APDEMS) under various reaction parameters, and the optimal pHOP-NH2 sample was selected and labelled with fluorescein isothiocyanate (FITC) to achieve fluorescent pHOPs (F-HOPs). The prepared pHOPs were thoroughly characterized by transmission electron microscopy, dynamic light scattering, FT-IR, UV-Vis and fluorescence spectroscopies, and microfluidic resistive pulse sensing. The optimal amino surface modification of pHOPs with APDEMS was at pH 10.2, at 60 °C temperature with 10 min reaction time. The positive Zeta potential of pHOP-NH2 in an acidic environment and the appearance of vibrations characteristic to the surface amino groups on the FT-IR spectra prove the successful surface modification. A red-shift in the absorbance spectrum and the appearance of bands characteristic to secondary amines in the FTIR spectrum of F-HOP confirmed the covalent attachment of FITC to pHOP-NH2. This study provides a step-by-step synthetic optimization and characterization of fluorescently labelled organosilica particles to enhance their optical properties and extend their applications.

Two novel, water-soluble, merocyanine fluorophores were readily prepared by microwave-assisted synthesis. Full optical characterization was performed in a series of protic and aprotic solvents, and the dyes displayed fluorescence in the red region with up to a 20-fold decrease in brightness in water, demonstrating a strong environmental sensitivity hereby termed as solvato-fluorogenicity (to distinguish from solvatochromism). Shorter fluorescent lifetimes were also measured in water, which confirmed this character. These dyes were conjugated to a modified polymyxin scaffold that allowed fluorescence “switch-on” upon binding to Gram-negative bacterial membranes, and selective fluorescence detection of bacteria in a wash-free protocol.

Background: Traditionally, scientists studied microbiology through the manner of batch cultures, to conclude the dynamics or outputs by averaging all individuals. However, as the researches go further, the heterogeneities among the individuals have been proven to be crucial for the population dynamics and fates. Results: Due to the limit of technology, single-cell analysis methods were not widely used to decipher the inherent connections between individual cells and populations. Since the early decades of this century, the rapid development of microfluidics, fluorescent labelling, next-generation sequencing, and high-resolution microscopy have speeded up the development of single-cell technologies and further facilitated the applications of these technologies on bacterial analysis. Conclusions: In this review, we summarized the recent processes of single-cell technologies applied in bacterial analysis in terms of intracellular characteristics, cell physiology dynamics, and group behaviors, and discussed how single-cell technologies could be more applicable for future bacterial researches. [ABSTRACT FROM AUTHOR]

Phytophthora capsici is an important oomycete pathogen threatening the vegetable production in China, but very little is known about its population structure. The objective of the present study was to evaluate the genetic diversity of 49 P. capsici isolates obtained from 2007 to 2014 at nine provincial locations in China. Isolates were assessed for mating type, metalaxyl resistance and simple sequence repeat (SSR) genotype. Mating‐type analyses of the isolates showed that both mating types were present in all of the sampled production regions, and the mating‐type frequency in the total Chinese population did not deviate significantly from a 1:1 ratio. Responses of isolates to the fungicide metalaxyl indicated the presence of intermediate resistance to metalaxyl among the field population. A universal fluorescent labelling method was adapted in this study to improve the efficiency of SSR genotyping. Microsatellite genotyping of the isolates using seven SSR markers revealed 44 unique multilocus genotypes. Genetic analyses indicated the existence of two genetic clusters within Chinese P. capsici collection. Clonal reproduction may play a more prominent role in Yunnan Province, but non‐existence of repeated genotypes and existence of both mating types throughout all regions suggest outcrossing and sexual recombination likely play an important role in the overall epidemiology in China. Future studies would include expanded scale sampling at single regions over multiple years to better define the genetic diversity of P. capsici in China. [ABSTRACT FROM AUTHOR]

Highlights • Selective nanochannel modification of track-etched membranes. • Grafting polymerization initiated by thermal activation of remnant peroxides. • Straightforward, homogeneous and reproducible pore modification method. Abstract Track-etched PET membranes (TMs) are one of the first commercially available nanostructured materials. The number, size and shape of pores (or nanochannels) can be carefully tailored at industrial scale. Selective pore modification can be achieved using remnant radicals after the etching process; however, this technique has some limitations. In this work, a simple and straightforward procedure is described for selectively grafting glycidyl methacrylate (GMA) and acrylic acid into the nanochannel walls of track-etched PET membranes. Grafting polymerization was carried out at short and long post-etching time by thermal initiation. The ultra-low amounts of grafting modification were measured by fluorescence labelling and membrane hydrolysis. The presence/absence of polyGMA and polyacrylic acid was confirmed by FTIR spectroscopy and MS-ESI. Peroxide compounds were detected in track-etched membranes and attributed responsibility for the selective grafting polymerization in the nanochannel walls of TMs. This simple and quick procedure will enhance the development of novel functional materials based on this technology. [ABSTRACT FROM AUTHOR]

The infection dynamics and distribution of the ectoparasitic fish monogenean Neobenedenia sp. (Monogenea: Capsalidae) throughout its development was examined on barramundi, Lates calcarifer (Bloch) (Latidae), by labelling transparent, ciliated larvae (oncomiracidia) with a fluorescent dye. Replicate fish were each exposed to approximately 50 fluorescent oncomiracidia and then examined for parasites using an epifluorescence stereomicroscope at 10 time intervals post-exposure (15, 30, 60, 120 min, 24, 48 h, four, eight, 12, and 16 days). Fluorescent labelling revealed that parasites attached underneath and on the surface of the scales of host fish. Parasite infection success was 20% within 15 min, and peaked at 93% two days post-exposure, before gradually declining between four and sixteen days. Differences in parasite distribution on L. calcarifer over time provided strong evidence that Neobenedenia sp. larvae settled opportunistically and then migrated to specific microhabitats. Parasites initially attached (

Fluorescent labelling is a common approach to reveal the molecular details of cellular uptake, internalisation, transport, distribution processes in biological systems. The conjugation with a fluorescent moiety might affect relevant physico-chemical and in vitro transport properties of the bioactive component. A representative set of seven cationic peptides—including cell-penetrating peptides as well as antimicrobial peptides and synthetic derivatives—was selected for our comparative study. Membrane affinity of the peptides and their 5(6)-carboxyfluorescein (Cf) derivatives was determined quantitatively and compared applying Langmuir monolayer of zwitterionic (DPPC) and negatively charged (DPPC + DPPG) lipids as cell membrane models. The interaction with neutral lipid layer is mainly governed by the overall hydrophobicity of the molecule which is remarkably increased by Cf-conjugation for the most hydrophobic Magainin, Melittin and Transportan. A significantly enhanced membrane affinity was detected in negatively charged lipid model monolayer for all of the peptides since the combination of electrostatic and hydrophobic interaction is active in that case. The Cf-conjugation improved the penetration ability of Penetratin and Dhvar4 suggesting that both the highly charged character (Z/n) and the increased hydrophobicity by Cf-conjugation present important contribution to membrane interaction. This effect might also responsible for the observed high in vitro internalisation rate of Penetratin and Dhvar4, while according to in vitro studies they did not cause damage of cell membrane. From the experiments with the given seven cationic peptides, it can be concluded that the Cf-conjugation alters the degree of membrane interaction of such peptides which are moderately hydrophobic and highly charged. [ABSTRACT FROM AUTHOR]

N2-Alkyl-2′-deoxyguanosine triphosphate (N2-alkyl-dGTP) derivatives with methyl, butyl, benzyl, or 4-ethynylbenzyl substituents were prepared and tested as substrates for human DNA polymerases. N2-Benzyl-dGTP was equal to dGTP as a substrate for DNA polymerase κ (pol κ), but was a poor substrate for pols β, δ, η, ι, or ν. In vivo reactivity was evaluated through incubation of N2-4-ethynylbenzyl-dG with wild-type and pol κ deficient mouse embryonic fibroblasts. CuAAC reaction with 5(6)-FAM-azide demonstrated that only cells containing pol κ were able to incorporate N2-4-ethynylbenzyl-dG into the nucleus. This is the first instance of a Y-family-polymerase-specific dNTP, and this method could be used to probe the activity of pol κ in vivo. [ABSTRACT FROM AUTHOR]

[Display omitted] • Bacillus amyloliquefaciens BTSS3, is a marine spore former isolated from deep-sea shark Centroscyllium fabricii. • Whole genome sequencing of BTSS3 was done using the Illumina Hiseq2500 platform. • In-depth automated annotation of CAZymes using dbcan CAZyme database revealed the prescence of enzymes important in complex carbohydrate metabolism. • Functional annotation of genome showed the presence of probiotic genes related to the biosynthesis of bacteriocins, stress resistance, and bile tolerance. • Colonization of fluorescent- labelled Bacillus amyloliquefaciens BTSS3 in zebrafish (Danio rerio) gut confirms its ability to adhere to gut mucosa. Probiotics are microorganisms when administered in adequate amounts, confer health benefits on the host. Many probiotics find application in various industries however, probiotic bacteria linked to marine environments are less explored.Although Bifidobacteria, Lactobacilli , and Streptococcus thermophilus are the most frequently used probiotics, Bacillus spp. have acquired much acceptance in human functional foods due to their increased tolerance and enduring competence in harsh environments like the gastrointestinal (GI) tract. In this study, the 4 Mbp genome sequence of Bacillus amyloliquefaciens strain BTSS3, a marine spore former isolated from deep-sea shark Centroscyllium fabricii , with antimicrobial and probiotic properties was sequenced, assembled, and annotated. Analysis revealed the presence of numerous genes presenting probiotic traits like production of vitamins, secondary metabolites, amino acids, secretory proteins, enzymes and other proteins that allow survival in GI tract as well as adhesion to intestinal mucosa. Adhesion by colonization in the gut was studied in vivo in zebrafish (Danio rerio) using FITC labelled B.amyloliquefaciens BTSS3. Preliminary study revealed the ability of the marine Bacillus to attach to the intestinal mucosa of the fish gut. The genomic data and the in vivo experiment affirms that this marine spore former is a promising probiotic candidate with potential biotechnological applications. [ABSTRACT FROM AUTHOR]

2′-Deoxyadenosine triphosphate (dATP) derivatives bearing diverse substituents (Cl, NH2, CH3, vinyl, ethynyl, and phenyl) at position 2 were prepared and tested as substrates for DNA polymerases. The 2-phenyl-dATP was not a substrate for DNA polymerases, but the dATPs bearing smaller substituents were good substrates in primer-extension experiments, producing DNA substituted in the minor groove. The vinyl-modified DNA was applied in thiol-ene addition and the ethynyl-modified DNA was applied in a CuAAC click reaction to form DNA labelled with fluorescent dyes in the minor groove [ABSTRACT FROM AUTHOR]