Chemoenzymatic synthesis and utilization of a SAM analog with an isomorphic nucleobase.

SalL, an enzyme that catalyzes the synthesis of SAM from l-methionine and 5'-chloro-5'-deoxyoadenosine, is shown to accept 5'-chloro-5'-deoxythienoadenosine as a substrate and facilitate the synthesis of a synthetic SAM analog with an unnatural nucleobase. This synthetic cofactor is demonstrated to replace SAM in the DNA methylation reaction with M.TaqI.

Nanoparticle-mediated thermal Cancer therapies: Strategies to improve clinical translatability.

Despite significant advances, cancer remains a leading global cause of death. Current therapies often fail due to incomplete tumor removal and nonspecific targeting, spurring interest in alternative treatments. Hyperthermia, which uses elevated temperatures to kill cancer cells or boost their sensitivity to radio/chemotherapy, has emerged as a promising alternative. Recent advancements employ nanoparticles (NPs) as heat mediators for selective cancer cell destruction, minimizing damage to healthy tissues. This approach, known as NP hyperthermia, falls into two categories: photothermal therapies (PTT) and magnetothermal therapies (MTT). PTT utilizes NPs that convert light to heat, while MTT uses magnetic NPs activated by alternating magnetic fields (AMF), both achieving localized tumor damage. These methods offer advantages like precise targeting, minimal invasiveness, and reduced systemic toxicity. However, the efficacy of NP hyperthermia depends on many factors, in particular, the NP properties, the tumor microenvironment (TME), and TME-NP interactions. Optimizing this treatment requires accurate heat monitoring strategies, such as nanothermometry and biologically relevant screening models that can better mimic the physiological features of the tumor in the human body. This review explores the state-of-the-art in NP-mediated cancer hyperthermia, discussing available nanomaterials, their strengths and weaknesses, characterization methods, and future directions. Our particular focus lies in preclinical NP screening techniques, providing an updated perspective on their efficacy and relevance in the journey towards clinical trials.

Behavior of Escherichia coli in a heterogeneous gelatin-dextran mixture.

In a gelatin-dextran mixture, changing the (relative and/or absolute) concentration of the components leads to the formation of different microstructures. Confocal laser scanning microscopy illustrated that the nature of the microstructure determines the location and morphology of Escherichia coli colonies. Observations indicate that bacterial growth preferentially occurs in the dextran phase, regardless of the microstructure.

Aggregatibacter actinomycetemcomitans adhesion inhibited in a flow cell.

INTRODUCTION: Microbial interactions are considered important in the adhesion process of pathogenic bacteria in the oral cavity. This study addressed the hypothesis that a streptococcal biofilm influences the hard tissue colonization by the periodontopathogen Aggregatibacter actinomycetemcomitans under hydrodynamic conditions. METHODS: The colonization of a green-fluorescent-protein-labelled A. actinomycetemcomitans strain on surfaces coated with a streptococcal biofilm, was monitored in real time using a confocal laser scanning microscope-mounted flow cell. Culture and quantitative polymerase chain reaction data were obtained in parallel from a Modified Robbins Device. RESULTS: Colonization of A. actinomycetemcomitans was inhibited by the four tested streptococci (Streptococcus sanguinis, Streptococcus cristatus, Streptococcus salivarius, and Streptococcus mitis). The most inhibiting species was S. sanguinis. CONCLUSION: These results confirmed the hypothesis that some bacterial species influence A. actinomycetemcomitans colonization of hard surfaces in vitro under hydrodynamic conditions.

Colonization of hard and soft surfaces by Aggregatibacter actinomycetemcomitans under hydrodynamic conditions.

INTRODUCTION: Oral bacteria must attach to hard and soft tissues to colonize the oral cavity in the presence of a variety of forces caused by shear and flow. In vitro models mimicking this dynamic process are indispensable to study factors that might interfere with the first step towards infection. For extrapolation purposes the comparability between the dynamics of colonization on hard vs. soft surfaces needs to be evaluated. METHODS: The colonization of glass and epithelial cell surfaces by the periodontal pathogen Aggregatibacter actinomycetemcomitans was followed in time with two flow cell models: a modified Robbins device (MRD) and an in situ image analysis system. RESULTS: The number of A. actinomycetemcomitans recovered from the soft surfaces in the MRD experiments was higher than on glass. The amount of bacteria on the hard surfaces kept increasing with time, while on soft surfaces saturation was reached. The microscope-mounted flow cell allowed real-time in situ monitoring of the colonization process of both surfaces. CONCLUSION: These experimental models may have a great contribution to make in the development of new treatment approaches for periodontal diseases. Colonization by A. actinomycetemcomitans could be studied under flow conditions and its dynamics showed important surface-dependent characteristics.

A novel method for in situ synthesis of SERS-active gold nanostars on polydimethylsiloxane film.

A novel one-step in situ synthesis of gold nanostars (AuNSs) on a pre-cured polydimethylsiloxane (PDMS) film is proposed for the fabrication of highly sensitive surface-enhanced Raman scattering (SERS) substrates. Plasmonic activity of the substrates was investigated by collecting SERS maps of 4-mercaptobenzoic acid (4-MBA). The applicability of these flexible substrates is further demonstrated by SERS-based pesticide detection on fruit skin.

Use of Dual Marker Transposons to Identify New Symbiosis Genes in Rhizobium.

Rhizobium etli elicits nitrogen-fixing nodules on the roots of Phaseolus vulgaris. Using a composite dual-marker mini-Tn5 transposon carrying combinations of a constitutively expressed gfp gene and a promoterless gusA gene, we identified novel genes required for an efficient symbiosis. The induction of the gusA gene was used to determine the expression level of the different target genes under conditions partly mimicking the symbiotic environment ex planta. The green fluorescence was used to localize the bacteria in infection threads or inside the plant cells. Among the identified R. etli mutants, several produced a Nod- phenotype, whereas others were Fix- or displayed a reduced acetylene reduction activity during symbiosis. Partial sequence analysis of the mutated genes allowed us to classify them as nodulation genes, nitrogen fixation genes, genes possessing various enzymatic functions previously not yet associated with symbiosis, and genes displaying no similarity to any other sequence in the database. This methodology can be used to screen large numbers of mutants in the search for novel genes important for Rhizobium-legume symbiosis, and may be adapted to study other plant-bacterium interactions.

A new analysis method of single molecule fluorescence using series of photon arrival times: theory and experiment.

Up to now, single molecule fluorescence experiments were performed by dividing the time into a set of intervals and to observe the number of fluorescence photons arriving in each interval. It is obvious that the detected photons carry less information than the arrival times of the photons themselves. From the arrival times, one can still calculate the number of photons in any user-defined interval; whereas, when only the number of photons in an interval are recorded, information about their positions in time is lost. Therefore, we present a new analysis method of single molecule fluorescence data based on the positions in time of the detected fluorescence photons. We derive mathematically different statistical characteristics describing the single molecule fluorescence experiment assuming an immobilized molecule. The theory of point processes using the generating functionals formalism is ideally suited for a consistent description, linking the statistical characteristics of the excitation and detected photons to the statistical characteristics of the single motionless molecule. We then use computer-generated data sets mimicking the single molecule fluorescence experiment to explore the parametric estimation of mono- and bi-exponential single molecule impulse response functions (SMIRFs) via the following statistical characteristics: the probability density distributions (pdd) of the single and first photocount time positions in a user-defined detection interval, the probability distribution of the number of photocounts per user-defined detection interval, the time correlation function and the pdd of the time interval between two consecutive photocounts. It is shown that all of the above characteristics ensure a satisfactory recovery of the decay time of mono-exponential SMIRFs for a broad range of excitation intensities and widths of user-defined detection intervals. For bi-exponential SMIRFs, the selection of the experimental conditions is more critical and dependent on the detection procedure. At lower excitation intensities it is advantageous to use the pdds of the single and first photocount time occurrences in the user-defined detection interval. To show the practical usefulness of the new analysis method, series of photon arrival times from immobilized single molecules of DiI and rhodamine 6G were analyzed to estimate triplet lifetimes and intersystem crossing yields.

Triplet states as non-radiative traps in multichromophoric entities: single molecule spectroscopy of an artificial and natural antenna system.

Energy transfer in antenna systems, ordered arrays of chromophores, is one of the key steps in the photosynthetic process. The photophysical processes taking place in such multichromophoric systems, even at the single molecule level, are complicated and not yet fully understood. Instead of directly studying individual antenna systems, we have chosen to focus first on systems for which the amount of chromophores and the interactions among the chromophores can be varied in a systematic way. Dendrimers with a controlled number of chromophores at the rim fulfill those requirements perfectly. A detailed photophysical study of a second-generation dendrimer, containing eight peryleneimide chromophores at the rim, was performed 'J. Am. Chem. Soc., 122 (2000) 9278'. One of the most intriguing findings was the presence of collective on/off jumps in the fluorescence intensity traces of the dendrimers. This phenomenon can be explained by assuming a simultaneous presence of both a radiative trap (energetically lowest chromophoric site) and a non-radiative trap (triplet state of one chromophore) within one individual dendrimer. It was shown that an analogue scheme could explain the collective on/off jumps in the fluorescence intensity traces of the photosynthetic pigment B-phycoerythrin (B-PE) (Porphyridium cruentum). The different values of the triplet lifetime that could be recovered for a fluorescence intensity trace of B-PE were correlated with different intensity levels in the trace, suggesting different chromophores acting as a trap as function of time.

Fluorescence Detection from Single Dendrimers with Multiple Chromophores.

The differences in the fluorescence behavior of a polyphenylene dendrimer with eight peryleneimides chromophores (1) and a single hexaphenylperyleneimide chromophore have been investigated at a single-molecule level through the combination of ultrasensitive fluorescence detection and microscopy.

Absolute determination of photoluminescence quantum efficiency using an integrating sphere setup.

An integrating sphere-based setup to obtain a quick and reliable determination of the internal quantum efficiency of strongly scattering luminescent materials is presented. In literature, two distinct but similar measurement procedures are frequently mentioned: a "two measurement" and a "three measurement" approach. Both methods are evaluated by applying the rigorous integrating sphere theory. It was found that both measurement procedures are valid. Additionally, the two methods are compared with respect to the uncertainty budget of the obtained values of the quantum efficiency. An inter-laboratory validation using the two distinct procedures was performed. The conclusions from the theoretical study were confirmed by the experimental data.

A non-invasive fluorescent staining procedure allows Confocal Laser Scanning Microscopy based imaging of Mycobacterium in multispecies biofilms colonizing and degrading polycyclic aromatic hydrocarbons.

To study the micro scale interactions of Mycobacterium with bacteria belonging to other genera by means of Confocal Laser Scanning Microscopy (CLSM), a procedure was developed to non-invasively and fluorescently stain Mycobacterium without compromising the signal produced by commonly used fluorescent reporter genes. The procedure makes use of the commercial non-specific nucleic acid stain Syto62 and was optimized to efficiently stain Mycobacterium cells in suspensions and biofilms. The staining procedure was found non-invasive towards overall cell viability, biofilm architecture and fluorescence signals emitted by other organisms expressing the fluorescent reporter genes gfp and dsRed. The procedure was successfully applied to visualize the comportment of the PAH-degrading Mycobacterium sp. VM552 in triple species biofilms containing, in addition to strain VM552, the GFP labeled PAH-degrading Sphingomonas sp. LH128-GFP and DsRed-labeled Pseudomonas putida OUS82(RF), and colonizing a glass substrate coated with phenanthrene crystals in flow chambers. CLSM imaging and subsequent appropriate image processing of the biofilms show that the comportment of strain Mycobacterium sp. VM552 was largely affected by the presence of the other organisms. The data support the value of the staining procedure to study ecological questions about micro scale behavior and niche occupation of Mycobacterium in multi-species systems.

Nanopatterned monolayers of an adsorbed chromophore.

A simple lift-off process was developed to rapidly fabricate nanopatterned photofunctional surfaces. Dye molecules of a perylene derivative (PDID) were adsorbed irreversibly on clean silicon through the holes of an electron-beam lithographied polymer mask. The subsequent removal of the mask in a proper solvent results in PDID nanosized regions of width as small as 30 nm for stripes and of diameter as small as 120 nm for dots. Numerical analyses of atomic force microscopy and laser-scanning confocal microscopy images show that the dye molecules are confined to the regions defined by the lithographic process, with the integrated fluorescence intensity being essentially proportional to the size of the nanofeatures. This demonstrates that a simple organic lift-off process compatible with clean-room technology, and not involving any chemical step, is able to produce photofunctional nanopatterned surfaces, even though the dye is not chemically bonded to the silicon surface.

Fluorescence lifetime fluctuations of single molecules probe the local environment of oligomers around the glass transition temperature.

Single molecule fluorescence experiments have been performed on a BODIPY-based dye embedded in oligo(styrene) matrices to probe the density fluctuations and the relaxation dynamics of chain segments surrounding the dye molecules. The time-dependent fluorescence lifetime of the BODIPY probe was recorded as an observable for the local density fluctuations. At room temperature, the mean fraction of holes surrounding the probes is shown to be unaffected by the molecular weight in the glassy state. In contrast, the free volume increases significantly in the supercooled regime. These observations are discussed in the framework of the entropic theories of the glass transition.

Energy dissipation in multichromophoric single dendrimers.

Single-molecule spectroscopy of well-chosen dendritic multichromophoric systems allows investigation of fundamental photophysical processes such as energy or electron transfer in much greater detail than the respective ensemble measurements. In dendrimers with multiple chromophores, energy hopping and transfer to the chromophore with the energetically lowest S(1) state was observed. If more than one chromophore is in an excited state in one molecule, annihilation, either singlet-triplet or singlet-singlet, can occur. In the latter case, a higher singlet state is populated opening new deactivation pathways. In the presence of an electron donor, reversible electron transfer could be observed, and the rate constants of forward and backward electron transfer were established. The value of these rate constants fluctuates time-correlated with the rotational motion of the dendrimer arms and the mobility of the embedding matrix.

Fluorescence study of a field-induced director reorientation in a liquid crystalline polyacrylate.

The electric field-induced director reorientation is investigated by fluorescence spectroscopy and turbidimetry. The dynamics of this reorientation are studied as a function of temperature, applied voltage, and frequency.

Fluorescence study of field-induced director reorientations in low mass liquid crystalline compounds.

The Frederickz transition and the dynamic scattering deformation in 4-alkyloxy-4'-cyanobiphenyl compounds are investigated by fluorescence spectroscopy and turbidimetry. The main characteristics of the Frederickz deformation, reversibility and threshold voltage, in the nematic and the smectic A phase are investigated by means of fluorescence spectroscopy. Furthermore, the dynamics of the Frederickz transition are studied as a function of temperature and applied voltage. The results obtained with both techniques are compared and correlate well.