Quantum Dot-Fab' Conjugates as Compact Immunolabels for Microtubule Imaging and Cell Classification.

Antibodies and their conjugates of fluorescent labels are widely applied in life sciences research and clinical pathology. Among diverse label types, compact quantum dots (QDs) provide advantages of multispectral multiplexing, bright signals in the deep red and infrared, and low steric hindrance. However, QD-antibody conjugates have random orientation of the antigen-binding domain which may interfere with labeling and are large (20-30 nm) and heterogeneous, which limits penetration into biospecimens.

CHCHD2 mediates glioblastoma cell proliferation, mitochondrial metabolism, hypoxia‑induced invasion and therapeutic resistance.

Glioblastoma (GBM) is the most common and malignant primary brain tumor affecting adults and remains incurable. The mitochondrial coiled‑coil‑helix‑coiled‑coil‑helix domain‑containing protein 2 (CHCHD2) has been demonstrated to mediate mitochondrial respiration, nuclear gene expression and cell migration; however, evidence of this in GBM is lacking. In the present study, it was hypothesized that CHCHD2 may play a functional role in U87 GBM cells expressing the constitutively active epidermal growth factor receptor variant III (EGFRvIII).

Antibody Self-Assembly Maximizes Cytoplasmic Immunostaining Accuracy of Compact Quantum Dots.

Antibody conjugates of quantum dots (QDs) are expected to transform immunofluorescence staining by expanding multiplexed analysis and improving target quantification. Recently, a new generation of small QDs coated with multidentate polymers has improved QD labeling density in diverse biospecimens, but new challenges prevent their routine use. In particular, these QDs exhibit nonspecific binding to fixed cell nuclei and their antibody conjugates have random attachment orientations.

From δ-aminolevulinic acid to chlorophylls and every step in between: in memory of Constantin (Tino) A. Rebeiz, 1936-2019.

Constantin A. (Tino) Rebeiz, a pioneer in the field of chlorophyll biosynthesis, and a longtime member of the University of Illinois community of plant biologists, passed away on July 25, 2019. He came to the USA at a time that was difficult for members of minority groups to be in academia.

Airyscan super-resolution microscopy of mitochondrial morphology and dynamics in living tumor cells.

Mitochondrial morphology is regulated by continuous fusion-and-fission events that are essential for maintaining normal function. Despite the prominence of mitochondrial function in energy generation and cell signaling, understanding of processes of fusion and fission dynamics has been hampered by the lack of high-resolution optical systems that accommodate live-cell imaging. We have examined different confocal modalities in terms of resolution and signal-to-noise ratio (SNR) in a point scanning confocal microscope with Airyscan super-resolution (AS-SR).

Distinct responses of compartmentalized glutathione redox potentials to pharmacologic quinones targeting NQO1.

Deoxynyboquinone (DNQ), a potent novel quinone-based antineoplastic agent, selectively kills solid cancers with overexpressed cytosolic NAD(P)H:quinone oxidoreductase-1 (NQO1) via excessive ROS production. A genetically encoded redox-sensitive probe was used to monitor intraorganellar glutathione redox potentials (E) as a direct indicator of cellular oxidative stress following chemotherapeutic administration. Beta-lapachone (β-lap) and DNQ-induced spatiotemporal redox responses were monitored in human lung A549 and pancreatic MIA-PaCa-2 adenocarcinoma cells incubated with or without dicumarol and ES936, potent NQO1 inhibitors.

Thiol-based antioxidants elicit mitochondrial oxidation via respiratory complex III.

Excessive oxidation is widely accepted as a precursor to deleterious cellular function. On the other hand, an awareness of the role of reductive stress as a similar pathological insult is emerging. Here we report early dynamic changes in compartmentalized glutathione (GSH) redox potentials in living cells in response to exogenously supplied thiol-based antioxidants.

Inhibition of glutathione synthesis distinctly alters mitochondrial and cytosolic redox poise.

The glutathione couple GSH/GSSG is the most abundant cellular redox buffer and is not at equilibrium among intracellular compartments. Perturbation of glutathione poise has been associated with tumorigenesis; however, due to analytical limitations, the underlying mechanisms behind this relationship are poorly understood. In this regard, we have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real-time glutathione redox potentials in the cytosol and mitochondrial matrix of tumorigenic and non-tumorigenic cells.

Transient light-induced intracellular oxidation revealed by redox biosensor.

We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30s were implemented.

Förster resonance energy transfer-based sensor targeting endoplasmic reticulum reveals highly oxidative environment.

The glutathione thiol/disulfide couple is the major redox buffer in the endoplasmic reticulum (ER); however, mechanisms by which it contributes to the tightly regulated redox environment of this intracellular organelle are poorly understood. The recent development of genetically encoded, ratiometric, single green fluorescent protein-based redox-sensitive (roGFP) sensors adjusted for more oxidative environments enables non-invasive measurement of the ER redox environment in living cells. In turn, Förster resonance energy transfer (FRET) sensors based on two fluorophore probes represent an alternative strategy for ratiometric signal acquisition.

Development of a high-dynamic range, GFP-based FRET probe sensitive to oxidative microenvironments.

We report the optimization of a novel redox-sensitive probe with enhanced dynamic range and an exceptionally well-positioned oxidative midpoint redox potential. The present work characterizes factors that contribute to the improved Förster resonance energy transfer (FRET) performance of this green fluorescent protein (GFP)-based redox sensor. The α-helical linker, which separates the FRET donor and acceptor, has been extended in the new probe and leads to a decreased FRET efficiency in the linker's reduced, 'FRET-off' state.

Imaging in real-time with FRET the redox response of tumorigenic cells to glutathione perturbations in a microscale flow.

Despite the potential benefits of selective redox-modulating strategies for cancer therapy, an efficacious methodology for testing therapies remains elusive because of the difficulty in measuring intracellular redox potentials over time. In this report, we have incorporated a new FRET-based biosensor to follow in real time redox-sensitive processes in cells transformed to be tumorigenic and cultured in a microfluidic channel. A microfluidic network was used to control micro-scale flow near the cells and at the same time deliver drugs exogenously.

Engineering redox-sensitive linkers for genetically encoded FRET-based biosensors.

The ability to sense intracellular or intraorganellar reduction/oxidation conditions would provide a powerful tool for studying normal cell proliferation, differentiation, and apoptosis. Genetically encoded biosensors enable monitoring of the intracellular redox environment. We report the development of chimeric polypeptides useful as redox-sensitive linkers in conjunction with Förster resonance energy transfer (FRET).

Chloroplast biogenesis 92: In situ screening for divinyl chlorophyll(ide) a reductase mutants by spectrofluorometry.

Chlorophyll biosynthetic heterogeneity is rooted mainly in parallel divinyl (DV) and monovinyl (MV) biosynthetic routes interconnected by 4-vinyl reductases (4VRs) that convert DV tetrapyrroles to MV tetrapyrroles by conversion of the vinyl group at position 4 of the macrocycle to ethyl. What is not clear at this stage is whether the various 4VR activities are catalyzed by one enzyme of broad specificity or by a family of enzymes encoded by one gene or multiple genes with each enzyme having narrow specificity. Additional research is needed to identify the various regulatory components of 4-vinyl reduction.

Chloroplast biogenesis 89: development of analytical tools for probing the biosynthetic topography of photosynthetic membranes by determination of resonance excitation energy transfer distances separating metabolic tetrapyrrole donors from chlorophyll a acceptors.

The thorough understanding of photosynthetic membrane assembly requires a deeper knowledge of the coordination and regulation of the chlorophyll (Chl) and thylakoid apoprotein biosynthetic pathways. As a working hypothesis we have recently proposed three different Chl-thylakoid apoprotein biosynthesis models: a single-branched Chl biosynthetic pathway (SBP)-single location model, a SBP-multilocation model, and a multibranched Chl biosynthetic pathway (MBP)-sublocation model. The detection of resonance excitation energy transfer between tetrapyrrole precursors of Chl, and several Chl-protein complexes, has made it possible to test the validity of the proposed Chl-thylakoid apoprotein biosynthesis models by resonance excitation energy transfer determinations.

Chloroplast biogenesis 88. Protochlorophyllide b occurs in green but not in etiolated plants.

It has recently been reported that protochlorophyllide (Pchlide) b is an abundant pigment in barley etioplasts but is rather unstable, as it is rapidly converted to Pchlide a by 7-formyl reductase during pigment extraction with conventional 80% acetone (Reinbothe, S., Pollmann, S., and Reinbothe, C.

Chloroplast biogenesis 87: Evidence of resonance excitation energy transfer between tetrapyrrole intermediates of the chlorophyll biosynthetic pathway and chlorophyll a.

The thorough understanding of photosynthetic membrane assembly requires a deeper knowledge of the coordination of chlorophyll (Chl) and thylakoid apoprotein biosynthesis. As a working model for future investigations, we have proposed three Chl-thylakoid apoprotein biosynthesis models, namely, a single-branched Chl biosynthetic pathway (SBP) single-location model, an SBP multilocation model and a multibranched Chl biosynthetic pathway (MBP) sublocation model. Rejection or validation of these models can be probed by determination of resonance excitation energy transfer between various tetrapyrrole intermediates of the Chl biosynthetic pathway and various thylakoid Chl-protein complexes.