A methanotrophic bacterium to enable methane removal for climate mitigation.

The rapid increase of the potent greenhouse gas methane in the atmosphere creates great urgency to develop and deploy technologies for methane mitigation. One approach to removing methane is to use bacteria for which methane is their carbon and energy source (methanotrophs). Such bacteria naturally convert methane to CO and biomass, a value-added product and a cobenefit of methane removal.

Therapeutic Targeting of Stromal-Tumor HGF-MET Signaling in an Organotypic Triple-Negative Breast Tumor Model.

Unlabelled: The tumor microenvironment (TME) promotes proliferation, drug resistance, and invasiveness of cancer cells. Therapeutic targeting of the TME is an attractive strategy to improve outcomes for patients, particularly in aggressive cancers such as triple-negative breast cancer (TNBC) that have a rich stroma and limited targeted therapies. However, lack of preclinical human tumor models for mechanistic understanding of tumor-stromal interactions has been an impediment to identify effective treatments against the TME.

In vivo quantification of polyhydroxybutyrate (PHB) in the alphaproteobacterial methanotroph, Methylocystis sp. Rockwell.

Methane is a common industrial by-product that can be used as feedstock for production of the biopolymer polyhydroxybutyrate (PHB) by alphaproteobacterial methanotrophs. In vivo assessment of PHB production would shed light on the biosynthesis process and guide design of improved production strategies, but it is currently difficult to perform efficiently. In this study, the alphaproteobacterial methanotroph Methylocystis sp.

Osmoregulatory Role of Betaine and Betaine/γ-Aminobutyric Acid Transporter 1 in Post-Traumatic Syringomyelia.

Syringomyelia (SM) is primarily characterized by the formation of a fluid-filled cyst that forms in the parenchyma of the spinal cord following injury or other pathology. Recent omics studies in animal models have identified dysregulation of solute carriers, channels, transporters, and small molecules associated with osmolyte regulation during syrinx formation/expansion in the spinal cord. However, their connections to syringomyelia etiology are poorly understood.

NIR-emitting styryl dyes with large Stokes' shifts for imaging application: From cellular plasma membrane, mitochondria to Zebrafish neuromast.

Near-infrared (NIR) emitting probes with very large Stokes' shifts play a crucial role in bioimaging applications, as the optical signals in this region exhibit high signal to background ratio and allow deeper tissue penetration. Herein we illustrate NIR-emitting probe with very large Stokes' shifts (Δλ ≈ 260 - 272 nm) by integrating the excited-state intramolecular proton transfer (ESIPT) unit 2-(2'-hydroxyphenyl)benzoxazole (HBO) into a pyridinium derived cyanine. The ESIPT not only enhances the Stokes' shifts but also improves the quantum efficiency of the probe (ф = 0.

Organotypic breast tumor model elucidates dynamic remodeling of tumor microenvironment.

Fibroblasts are a critical component of tumor microenvironments and associate with cancer cells physically and biochemically during different stages of the disease. Existing cell culture models to study interactions between fibroblasts and cancer cells lack native tumor architecture or scalability. We developed a scalable organotypic model by robotically encapsulating a triple negative breast cancer (TNBC) cell spheroid within a natural extracellular matrix containing dispersed fibroblasts.

Synthesis of highly selective lysosomal markers by coupling 2-(2'-hydroxyphenyl)benzothiazole (HBT) with benzothiazolium cyanine (Cy): the impact of substituents on selectivity and optical properties.

HBT-Cy 1 has been previously reported as a highly selective fluorescent probe for lysosome visualization in live cells. To further investigate the role of the structural components of HBT-Cy in lysosome selectivity, cyanine based fluorescent probe series (2-5) have been synthesized in good yields by connecting benzothiazolium cyanine (Cy) with 2-hydroxyphenylbenzothiazole (HBT) via a meta phenylene ring. Probes 2-5 exhibited exceptional photophysical properties including bright red-emission (λ≈ 630-650 nm), a large Stokes shift (Δλ > 130 nm) and high fluorescence quantum yields (φ≈ 0.

A NIR-emitting cyanine with large Stokes shifts for live cell imaging: large impact of the phenol group on emission.

There are a limited number of near-infrared (NIR) emitting (λem = 700-900 nm) molecular probes for imaging applications. A NIR-emitting probe that exhibits emission at ∼800 nm with a large Stokes shift was synthesized and found to exhibit excellent selectivity towards mitochondria for live-cell imaging. The photophysical properties were attributed to an excited "cyanine structure" via intramolecular charge transfer (ICT) involving a phenol group.

NIR-Emitting Hemicyanines with Large Stokes' Shifts for Live Cell Imaging: from Lysosome to Mitochondria Selectivity by Substituent Effect.

Lysosome imaging without perturbing intracellular activity remains challenging, as the current commercial lysosome probes contain weakly basic amino groups that could perturb lysosome pH. Herein, we illustrate NIR-emitting dyes 2 and 3 (λ ≈ 700 nm) with very large Stokes' shifts (Δλ = 231-246 nm), attributing to the presence of a 2-hydroxyphenyl(benzo[d]oxazol) (HBO) unit that undergoes excited-state intramolecular proton transfer (ESIPT). The structures of and also contain a hemicyanine unit with benzothiazolium and indolium as a terminal group, respectively.

Fluorescence-based analysis of the intracytoplasmic membranes of type I methanotrophs.

Most methanotrophic bacteria maintain intracytoplasmic membranes which house the methane-oxidizing enzyme, particulate methane monooxygenase. Previous studies have primarily used transmission electron microscopy or cryo-electron microscopy to look at the structure of these membranes or lipid extraction methods to determine the per cent of cell dry weight composed of lipids. We show an alternative approach using lipophilic membrane probes and other fluorescent dyes to assess the extent of intracytoplasmic membrane formation in living cells.

An NIR emitting styryl dye with large Stokes shift to enable co-staining study on zebrafish neuromast hair cells.

Hearing loss is a significant public health problem, and the "loss of sensory hair cells" is one of two leading causes in humans. Advanced imaging reagents are desirable for understanding the role of the surrounding support cells in the loss or regeneration of the hair cells. A styryl dye was found to exhibit NIR emission (λ ≈ 684 nm) with a very large Stokes shift (Δν ≈ 9190 cm), due to the incorporation of excited state intramolecular proton transfer (ESIPT) mechanism.

A Fluorescent Flavonoid for Lysosome Imaging: the Effect of Substituents on Selectivity and Optical Properties.

Lysosome selective bright orange-red emitting flavonoid (2) was synthesized by attaching a strong donor (NPh) group into flavonoid skeleton. As a result of efficient intra molecular charge transfer due to the strong donor group, a significant bathochromic shift was observed from the emission of 2b (with a -NPh group, λ ≈ 590 nm), in comparison that of 1b (with a -NMe group, λ ≈ 519 nm). The role of the substituent effect towards ICT was further studied by low temperature spectral analysis.

A fluorescent flavonoid for lysosome detection in live cells under "wash free" conditions.

Lysosomes are vital organelles in living cells, which have acidic environments (pH 4.0-5.0) where macrobiomolecules and malfunctioning organelles are broken down into monomers by hydrolase activity.

NIR-emitting benzothiazolium cyanines with an enhanced stokes shift for mitochondria imaging in live cells.

A series of benzothiazolium-based hemicyanines (3a-3f) have been synthesized. Evaluation of their photophysical properties shows that they exhibit improved photophysical characteristics. In comparison with the available commercial MitoTrackers, the new probes revealed an enhanced Stokes shift (Δλ ∼ 80 nm) and minimized aggregation for increased sensitivity.

Fluorescent flavonoids for endoplasmic reticulum cell imaging.

Visualization of subcellular organelles in vivo is critical for basic biomedical research and clinical applications. Two new flavonoids with an amide substituent were synthesized and characterized. The flavonoids were nearly non-fluorescent in aqueous environment, but exhibited two emission peaks (one at 495-536 nm and the other at 570-587 nm) in organic solvents, which were assigned to the excited normal (N) and tautomer (T) emission.

An NIR-emitting lysosome-targeting probe with large Stokes shift via coupling cyanine and excited-state intramolecular proton transfer.

An NIR-emitting probe (λ ∼ 700 nm) with a large Stokes shift (Δλ ≈ 234 nm) is synthesized by using excited-state intramolecular proton transfer (ESIPT). The phenolic proton, which controls ESIPT, acts as a switch to give strong fluorescence at pH ≈ 5. The probe can selectively show lysosome organelles, therefore leading to a lysosome probe without exhibiting "an alkalinizing effect".

Synthesis, characterization, in vitro SAR and in vivo evaluation of N,N'bisnaphthylmethyl 2-alkyl substituted imidazolium salts against NSCLC.

Alkyl- and N,N'-bisnaphthyl-substituted imidazolium salts were tested in vitro for their anti-cancer activity against four non-small cell lung cancer cell lines (NCI-H460, NCI-H1975, HCC827, A549). All compounds had potent anticancer activity with 2 having IC values in the nanomolar range for three of the four cell lines, a 17-fold increase in activity against NCI-H1975 cells when compared to cisplatin. Compounds 1-4 also showed high anti-cancer activity against nine NSCLC cell lines in the NCI-60 human tumor cell line screen.

Global Molecular Analyses of Methane Metabolism in Methanotrophic Alphaproteobacterium, Methylosinus trichosporium OB3b. Part II. Metabolomics and 13C-Labeling Study.

In this work we use metabolomics and (13)C-labeling data to refine central metabolic pathways for methane utilization in Methylosinus trichosporium OB3b, a model alphaproteobacterial methanotrophic bacterium. We demonstrate here that similar to non-methane utilizing methylotrophic alphaproteobacteria the core metabolism of the microbe is represented by several tightly connected metabolic cycles, such as the serine pathway, the ethylmalonyl-CoA (EMC) pathway, and the citric acid (TCA) cycle. Both in silico estimations and stable isotope labeling experiments combined with single cell (NanoSIMS) and bulk biomass analyses indicate that a significantly larger portion of the cell carbon (over 60%) is derived from CO2 in this methanotroph.

Single cell methods for methane oxidation analysis.

Respiration is widely used for evaluation of the metabolic capabilities or physiological state of the microbial culture. This chapter describes novel approaches for characterization of respiration at a single cell level: (1) flow cytometry-based redox sensing (FCRS) of actively metabolizing microbes; (2) respiration response imaging (RRI) for real-time detection of substrate stimulated redox responses of individual cells; (3) respiration detection system: microobservation chamber (RDS: MC), a single cell analysis system for carrying out the physiological and genomic profiling of cells capable of respiring C(1) compounds. The techniques are suitable for description of physiological heterogeneity among cells in a single microbial population and could be used to characterize distribution of methylotrophic ability among microbial cells in the natural environmental samples.

The role of physiological heterogeneity in microbial population behavior.

As the ability to analyze individual cells in microbial populations expands, it is becoming apparent that isogenic microbial populations contain substantial cell-to-cell differences in physiological parameters such as growth rate, resistance to stress and regulatory circuit output. Subpopulations exist that are manyfold different in these parameters from the population average, and these differences arise by stochastic processes. Such differences can dramatically affect the response of cells to perturbations, especially stress, which in turn dictates overall population response.

Cytoplasmic protein mobility in osmotically stressed Escherichia coli.

Facile diffusion of globular proteins within a cytoplasm that is dense with biopolymers is essential to normal cellular biochemical activity and growth. Remarkably, Escherichia coli grows in minimal medium over a wide range of external osmolalities (0.03 to 1.

Methods of changing biopolymer volume fraction and cytoplasmic solute concentrations for in vivo biophysical studies.

In vitro changes in polymer volume fraction (macromolecular crowding) and changes in solute or salt concentration typically have large effects on protein and nucleic acid processes (e.g., folding, binding, assembly, precipitation, crystallization).

Crowding and confinement effects on protein diffusion in vivo.

The first in vivo measurements of a protein diffusion coefficient versus cytoplasmic biopolymer volume fraction are presented. Fluorescence recovery after photobleaching yields the effective diffusion coefficient on a 1-mum-length scale of green fluorescent protein within the cytoplasm of Escherichia coli grown in rich medium. Resuspension into hyperosmotic buffer lacking K+ and nutrients extracts cytoplasmic water, systematically increasing mean biopolymer volume fraction, , and thus the severity of possible crowding, binding, and confinement effects.

Cytoplasmic expression of nm23 predicts the potential for cerebral metastasis in patients with primary cutaneous melanoma.

The nm23 gene is a putative tumour and metastasis suppressor gene. A number of studies have found that reduction of its expression is associated with increased metastatic potential in several human malignancies. Similarly, clinical studies have shown correlation between reduced nm23 protein expression and a poor prognosis.

Differential expression of the nm23 protein in the progression of oesophageal adenocarcinoma.

Aim: Some studies have shown that abnormalities of the nm23 gene or its expression may be important in tumour dissemination, suggesting that the gene may have metastasis suppressing activity. This study set out to determine if nm23 protein expression is altered with progression and dissemination in oesophageal adenocarcinoma.

Methods: Paraffin-embedded, archival tissues of surgical resection specimens of oesophageal adenocarcinoma (n=46), some of which were accompanied by tissue from areas with high-grade dysplasia (n=24) and from metastasis in regional lymph nodes (n=16) were studied.