The two faces of microorganisms in traditional brewing and the implications for no- and low-alcohol beers.

The production of alcoholic beverages is intrinsically linked to microbial activity. This is because microbes such as yeast are associated with the production of ethanol and key sensorial compounds that produce desirable qualities in fermented products. However, the brewing industry and other related sectors face a step-change in practice, primarily due to the growth in sales of no- and low-alcohol (NoLo) alternatives to traditional alcoholic products. Here we review the involvement of microbes across the brewing process, including both their positive contributions and their negative (spoilage) effects. We also discuss the opportunities for exploiting microbes for NoLo beer production, as well as the spoilage risks associated with these products. For the latter, we highlight differences in composition and process conditions between traditional and NoLo beers and discuss how these may impact the microbial ecosystem of each product stream in relation to microbiological stability and final beer quality.

Rapid Rule Out of Culture-Negative Bloodstream Infections by Use of a Novel Approach to Universal Detection of Bacteria and Fungi.

BACKGROUND: Currently it can take up to 5 days to rule out bloodstream infection. With the low yield of blood cultures (approximately 10%), a significant number of patients are potentially exposed to inappropriate therapy that can lead to adverse events. More rapid rule out can accelerate deescalation or cessation of antimicrobial therapy, improving patient outcomes. METHODS: A method is described, termed enzymatic template generation and amplification (ETGA), that universally and sensitively detects DNA polymerase activity liberated from viable bacteria and fungi isolated from blood culture samples as a measure of bloodstream infection. ETGA was applied in a diagnostic test format to identify negative blood cultures after an overnight incubation. Performance data for a prototype (Cognitor) and automated (Magnitor) version of the test are presented. RESULTS: The Cognitor manual assay displayed analytical reactivity for a panel of the 20 most prevalent causes of bloodstream infection, with a detection range of 28-9050 CFU/mL. Validation with 1457 clinical blood cultures showed a negative predictive value of 99.0% compared to blood culture incubation for 5 days. Magnitor showed an improved detection range of 1-67 CFU/mL, allowing for detection of bacteria-supplemented blood cultures after 2-8 h incubation, and Candida albicans-supplemented blood cultures at 16-22 h, 5-15 h faster than blood culture. Removing an aliquot from a blood culture bottle and replacing the bottle into the incubator was shown not to result in contaminating organisms being introduced. CONCLUSIONS: The described method displays excellent breadth and detection for microbial cells and demonstrates the capability of confirming negative blood cultures after an overnight incubation in a blood culture instrument.

A method to analyze, sort, and retain viability of obligate anaerobic microorganisms from complex microbial communities.

A high speed flow cytometric cell sorter was modified to maintain a controlled anaerobic environment. This technology enabled coupling of the precise high-throughput analytical and cell separation capabilities of flow cytometry to the assessment of cell viability of evolved lineages of obligate anaerobic organisms from cocultures.

Three-dimensional molecular imaging with photothermal optical coherence tomography.

Optical coherence tomography (OCT) is a three-dimensional optical imaging technique that has been successfully implemented in ophthalmology for imaging the human retina, and in studying animal models of disease. OCT can nondestructively visualize structural features in tissue at cellular-level resolution, and can exploit contrast agents to achieve molecular contrast. Photothermal OCT relies on the heat-producing capabilities of antibody-conjugated gold nanoparticles to achieve molecular contrast. A pump laser at the nanoparticle resonance wavelength is used to heat the nanoparticles in the sample, and the resulting changes in the index of refraction around the nanoparticles are detected by phase-sensitive OCT. Lock-in detection of the pump beam amplitude-modulated frequency and the detector frequency allow for high-sensitivity images of molecular targets. This approach is attractive for nondestructive three-dimensional molecular imaging deep (approximately 2 mm) within biological samples. The protocols described here achieve a sensitivity of 14 parts per million (weight/weight) nanoparticles in the sample, which is sufficient to differentiate EGFR (epidermal growth factor receptor)-overexpressing cells from minimally expressing cells in three-dimensional cell constructs.

Hyperspectral molecular imaging of multiple receptors using immunolabeled plasmonic nanoparticles.

This work presents simultaneous imaging and detection of three different cell receptors using three types of plasmonic nanoparticles (NPs). The size, shape, and composition-dependent scattering profiles of these NPs allow for a system of multiple distinct molecular markers using a single optical source. With this goal in mind, tags consisting of anti-epidermal growth factor receptor gold nanorods, anti-insulin-like growth factor 1-R silver nanospheres, and human epidermal growth factor receptor 2Ab gold nanospheres were developed to monitor the expression of receptors commonly overexpressed by cancer cells. These labels were chosen because they scatter strongly in distinct spectral windows. A hyperspectral darkfield microspectroscopy system was developed to record the scattering spectra of cells labeled with these molecular tags. Simultaneous monitoring of multiple tags may lead to applications such as profiling of cell line immunophenotype and investigation of receptor signaling pathways. Single, dual, and triple tag experiments were performed to analyze NP tag specificity as well as their interactions. Distinct resonance peaks were observed in these studies, showing the ability to characterize cell lines using conjugated NPs. However, interpreting shifts in these peaks due to changes in a cellular dielectric environment may be complicated by plasmon coupling between NPs bound to proximal receptors and other coupling mechanisms due to the receptors themselves.

Monitoring of receptor dimerization using plasmonic coupling of gold nanoparticles.

The dimerization of receptors on the cell membrane is an important step in the activation of cell signaling pathways. Several methods exist for observing receptor dimerization, including coimmunoprecipitation, chemical cross-linking, and fluorescence resonance energy transfer (FRET). These techniques are limited in that only FRET is appropriate for live cells, but even that method suffers from photobleaching and bleed-through effects. In this study, we implement an alternative method for the targeting of HER-2 homodimer formation based on the plasmonic coupling of gold nanoparticles functionalized with HER-2 Ab. In the presented studies, SK-BR-3 cells, known to overexpress HER-2, are labeled with these nanoparticles and receptor colocalization is observed using plasmonic coupling. HER-2 targeted nanoparticles bound to these cells exhibit a peak resonance that is significantly red-shifted relative to those bound to similar receptors on A549 cells, which have significantly lower levels of HER-2 expression. This significant red shift indicates plasmonic coupling is occurring and points to a new avenue for assessing dimerization by monitoring their colocalization. To determine that dimerization is occurring, the refractive index of the nanoenvironment of the labels is assessed using a theoretical analysis based on the Mie coated sphere model. The results indicate scattering by single, isolated nanoparticles for the low HER-2 expressing A549 cell line, but the scattering observed for the HER-2 overexpressing SK-BR-3 cell line may only be explained by plasmonic-coupling of proximal nanoparticle pairs. To validate the conformation of nanoparticles bound to HER-2 receptors undergoing dimerization, discrete dipole approximation (DDA) models are used to assess spectra of scattering by coupled nanoparticles. Comparison of the experimental results with theoretical models indicates that NP dimers are formed for the labeling of SK-BR-3 cells, suggesting that receptor dimerization has been observed.

Polarization mapping of nanoparticle plasmonic coupling.

We propose the use of polarization mapping as a tool to better separate the effects of plasmonic coupling from the local refractive index for molecular imaging and biosensing using gold nanoparticles. Polarization mapping allows identification of the orthogonal excitation mode when the particle dimer orientation is unknown, as may be the case when using plasmonic nanoparticles for cell labeling. This information can be used to sense relative changes in the dielectric environment, or for absolute dielectric sensing with additional a priori interparticle distance information. First, the theoretical scattering by nanoparticle pairs is modeled under parallel and orthogonal polarization orientations and increasing interparticle separation. Second, polarization mapping of substrate bound nanoparticles using dark-field microspectroscopy is investigated as a method to isolate the individual plasmonic coupling modes associated with a pair of nanoparticles without reorientation of the sample. The results of this study provide useful insight toward potential avenues for monitoring distances using plasmonic nanoparticles and sensing the local refractive index using nanoparticle pairs when the pair orientation is not known, as may be the case when using nanoparticles for cell receptor labeling.

Plasmonic flow cytometry by immunolabeled nanorods.

Fluorescence-based flow cytometry measures multiple cellular characteristics, including levels of receptor expression, by assessing the fluorescence intensity from a population of cells whose cell surface receptors are bound by a fluorescently labeled antibody or ligand for that receptor. Functionalized noble metal nanoparticles provide a complementary method of receptor labeling based on plasmonics for population analysis by flow cytometry. The potential benefits of using plasmonic nanoparticles to label cell surface receptors in flow cytometry include scattering intensity from a single particle that is equivalent to fluorescence intensity of 105 fluorescein molecules, biocompatibility and low cytotoxicity, and nonquenching optical properties. The large spectral tunability of nanorods also provides convenient access to plasmonic markers with peak surface plasmon resonances ranging from 600 to 2,200 nm, unlike gold nanosphere markers that are limited to visible wavelengths. Gold nanorod-based plasmonic flow cytometry is demonstrated herein by comparing the scattering of cells bound to anti-epidermal growth factor receptor (EGFR)-conjugated nanorods to the emission of cells bound to anti-EGFR-conjugated fluorescent labels. EGFR-expressing cells exhibited a statistically significant six-fold increase in scattering when labeled with anti-EGFR-conjugated nanorods compared with labeling with IgG1-conjugated nanorods. Large scattering intensities were observed despite using a 1,000-fold lower concentration of nanorod-conjugated antibody relative to the fluorescently labeled antibody.

Molecular imaging and quantitative measurement of epidermal growth factor receptor expression in live cancer cells using immunolabeled gold nanoparticles.

OBJECTIVE: The goal of this study was to assess whether immunolabeled nanoparticle biomarkers are comparable to fluorescent marker imaging in measuring epidermal growth factor receptor (EGFR) expression. MATERIALS AND METHODS: EGFR expression was quantified using both imaging methods in four cell lines: A431 human epidermoid carcinoma cells, which are known to have high EGFR expression; two cell lines with lower EGFR expression (270-GBM human glioblastoma xenograft cells and H2224 human glioblastoma xenograft cells); and MDA-MB-453 breast carcinoma cells, which do not express EGFR. To enhance contrast of the nanoparticle biomarkers, a darkfield microspectroscopy system was used that includes a custom epi-illumination light train. RESULTS: Nanoparticle-bound cells were clearly distinguished from control cells not bound to nanoparticles in that they showed a significant increase in detected intensity under darkfield illumination due to nanoparticle scattering. The average nanoparticle-scattering intensity for A431 cells was 41.5 counts per cell compared with 24.7 for 270-GBM cells, 8.77 for H2224 cells, and 0.44 for MDA-MB-453 cells. The average fluorescence intensity for A431 cells was 35.3 counts per cell compared with 28.7 for 270-GBM cells, 5.91 for H2224 cells, and 2.07 for MDA-MB-453 cells. A plot of fluorescence intensity versus nanoparticle-scattering intensity for all four cell lines showed that the data agree with a linear relationship given by the following equation: NP = 1.0691 x FL - 0.3873, where NP is the nanoparticle-scattering intensity and FL is the fluorescence intensity. The covariance of the data with the trend line was R(2) = 0.9409. The average peak wavelength of nanoparticle scattering was 570.93 nm for A431 cells, 565.26 nm for 270-GBM cells, and 562.70 nm for H2224 cells (with no clear peaks observed for MDA-MB-453 cells). This spectral trend shows that nanoparticle scattering may reveal additional information about their nanoenvironment via refractive index sensitivity. CONCLUSION: Immunolabeled nanoparticles can quantify receptor expression with performance comparable to fluorescence markers and show promise to better characterize receptor expression via their refractive index sensitivity.

Photothermal optical coherence tomography of epidermal growth factor receptor in live cells using immunotargeted gold nanospheres.

Molecular imaging is a powerful tool for investigating disease processes and potential therapies in both in vivo and in vitro systems. However, high resolution molecular imaging has been limited to relatively shallow penetration depths that can be accessed with microscopy. Optical coherence tomography (OCT) is an optical analogue to ultrasound with relatively good penetration depth (1-2 mm) and resolution (approximately 1-10 microm). We have developed and characterized photothermal OCT as a molecular contrast mechanism that allows for high resolution molecular imaging at deeper penetration depths than microscopy. Our photothermal system consists of an amplitude-modulated heating beam that spatially overlaps with the focused spot of the sample arm of a spectral-domain OCT microscope. Validation experiments in tissuelike phantoms containing gold nanospheres that absorb at 532 nm revealed a sensitivity of 14 ppm nanospheres (weight/weight) in a tissuelike environment. The nanospheres were then conjugated to anti-EGFR, and molecular targeting was confirmed in cells that overexpress EGFR (MDA-MB-468) and cells that express low levels of EGFR (MDA-MB-435). Molecular imaging in three-dimensional tissue constructs was confirmed with a significantly lower photothermal signal (p<0.0001) from the constructs composed of cells that express low levels of EGFR compared to the overexpressing cell constructs (300% signal increase). This technique could potentially augment confocal and multiphoton microscopy as a method for deep-tissue, depth-resolved molecular imaging with relatively high resolution and target sensitivity, without photobleaching or cytotoxicity.

Molecular imaging of epidermal growth factor receptor in live cells with refractive index sensitivity using dark-field microspectroscopy and immunotargeted nanoparticles.

We present a study using plasmonic nanoparticles (NPs) to image epidermal growth factor receptor (EGFR) in live cells. Through detailed analysis of the NP scattering spectra, we determine the intracellular refractive index (RI) within attoliter volumes inside of the living cells. Molecular imaging is demonstrated using anti-EGFR labeled gold nanospheres delivered to cancer cells that overexpress EGFR, with targeted binding confirmed by appropriate control experiments. RI determination is achieved by measurement of the bound NPs' scattering spectra, acquired using a precision dark-field microspectroscopy system and through careful characterization of the NP properties throughout the immuno-labeling procedure. To demonstrate the effect of receptor-mediated uptake, the data are compared to similar spectral measurements using antibody-free NPs, taken up by the cells through nonspecific mechanisms. In these experiments, NP aggregation introduces interparticle effects in the scattering spectra, suggesting that EGFR-mediated internalization of NPs may provide an advantage for maintaining NP isolation upon uptake. The results of this study show the potential utility of dark-field microspectroscopy and labeled NPs for live cell imaging. By demonstrating RI sensitivity over nanometer length scales, this study also presents a potential new avenue for assessing the structure and dynamics of live cells.

Photocatalytic oxidation of soot by P25 TiO2 films.

The photomineralisation of soot by P25 titania films is studied using FT-IR and the process shown to involve the oxidation of carbon to CO2 exclusively. The efficiency of this process is low, however, with a formal quantum efficiency of 1.1 x 10(-4) molecules of carbon oxidized per incident photon of UVA light. The cause of this low efficiency is attributed largely to the less than intimate contact between the fibrous soot layer and the surface of the photocatalyst.

Phosphate availability regulates biosynthesis of two antibiotics, prodigiosin and carbapenem, in Serratia via both quorum-sensing-dependent and -independent pathways.

Serratia sp. ATCC 39006 produces two secondary metabolite antibiotics, 1-carbapen-2-em-3-carboxylic acid (Car) and the red pigment, prodigiosin (Pig). We have previously reported that production of Pig and Car is controlled by N-acyl homoserine lactone (N-AHL) quorum sensing, with synthesis of N-AHLs directed by the LuxI homologue SmaI, and is also regulated by Rap, a member of the SlyA family. We now describe further characterization of the SmaI quorum-sensing system and its connection with other regulatory mechanisms. We show that the genes responsible for biosynthesis of Pig, pigA-O, are transcribed as a single polycistronic message in an N-AHL-dependent manner. The smaR gene, transcribed convergently with smaI and predicted to encode the LuxR homologue partner of SmaI, was shown to possess a negative regulatory function, which is uncommon among the LuxR-type transcriptional regulators. SmaR represses transcription of both the pig and car gene clusters in the absence of N-AHLs. Specifically, we show that SmaIR exerts its effect on car gene expression via transcriptional control of carR, encoding a pheromone-independent LuxR homologue. Transcriptional activation of the pig and car gene clusters also requires a functional Rap protein, but Rap dependency can be bypassed by secondary mutations. Transduction of these suppressor mutations into wild-type backgrounds confers a hyper-Pig phenotype. Multiple mutations cluster in a region upstream of the pigA gene, suggesting this region may represent a repressor target site. Two mutations mapped to genes encoding pstS and pstA homologues, which are parts of a high-affinity phosphate transport system (Pst) in Escherichia coli. Disruption of pstS mimicked phosphate limitation and caused concomitant hyper-production of Pig and Car, which was mediated, in part, through increased transcription of the smaI gene. The Pst and SmaIR systems define distinct, yet overlapping, regulatory circuits which form part of a complex regulatory network controlling the production of secondary metabolites in Serratia ATCC 39006.