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Lactate accumulation in the tumor microenvironment was shown to be closely related to tumor growth and immune escape, and suppression of lactate production by inhibiting lactate dehydrogenase A (LDHA) has been pursued as a potential novel antitumor strategy. However, only a few potent LDHA inhibitors have been developed and most of them did not show potent antitumor effects in vivo. To this end, we designed new LDHA inhibitors and obtained a novel potent LDHA inhibitor, ML-05. ML-05 inhibited cellular lactate production and tumor cell proliferation, which was associated with inhibition of ATP production and induction of reactive oxygen species and G

A photo-caged STING agonist featuring a tumor-targeting carbonic anhydrase warhead was designed and synthesized for photo-controllable activation of STING signaling.

Although Bcl-xL has been shown to retrotranslocate Bax from mitochondria to cytosol, other studies have found that Bcl-xL also stabilizes the mitochondrial localization of Bax. It is still unclear what causes the difference in Bcl-xL-regulated Bax localization. Bad, a BH3-only protein with a high affinity for Bcl-xL, may play an important role in Bcl-xL-regulated Bax shuttling. Here, we found that Bcl-xL enhanced both translocalization and retrotranslocation of mitochondrial Bax, as evidenced by quantitative co-localization, western blots and fluorescence loss in photobleaching (FLIP) analyses. Notably, Bad knockdown prevented Bcl-xL-mediated Bax retrotranslocation, indicating Bad was essential for this process. Quantitative fluorescence resonance energy transfer (FRET) imaging in living cells and co-immunoprecipitation analyses showed that the interaction of Bcl-xL with Bad was stronger than that with Bax. The Bad mimetic ABT-737 dissociated Bax from Bcl-xL on isolated mitochondria, suggesting that mitochondrial Bax was directly liberated to cytosol due to Bad binding to Bcl-xL. In addition, MK-2206, an Akt inhibitor, decreased Bad phosphorylation while increasing cytosolic Bax proportion. Our data firmly demonstrate a notion that Bad binds to mitochondrial Bcl-xL to release Bax, resulting in retrotranslocation of Bax to cytosol, and that the amount of Bad involved is regulated by Akt signaling.

Myeloid cell leukemia-1 (Mcl-1) is involved in the regulation of mitochondrial fission and fusion. This report aims to explore whether Mcl-1 can interact with mitochondrial fission factor (Mff) and regulate Mff-mediated mitochondrial fragmentation and apoptosis. Fluorescence images of living cells coexpressing YFP-Mff and CFP-Mcl-1 showed that Mcl-1 markedly inhibited Mff-mediated mitochondrial fragmentation and apoptosis, suggesting that Mcl-1 played a key role in inhibiting mitochondrial fission. The cells coexpressing YFP-Mff and CFP-Mcl-1 exhibited consistent fluorescence resonance energy transfer (FRET) efficiency with that of the cells coexpressing CFP-Mcl-1 and YFP, demonstrating that Mcl-1 did not directly bind to Mff on mitochondria. Collectively, Mcl-1 inhibits Mff-mediated mitochondrial fission and apoptosis not via directly binding to Mff on mitochondria.

BackgroundDrug-target interactions (DTIs) prediction becomes more and more important for accelerating drug research and drug repositioning. Drug-target interaction network is a typical model for DTIs prediction. As many different types of relationships exist between drug and target, drug-target interaction network can be used for modeling drug-target interaction relationship. Recent works on drug-target interaction network are mostly concentrate on drug node or target node and neglecting the relationships between drug-target.ResultsWe propose a novel prediction method for modeling the relationship between drug and target independently. Firstly, we use different level relationships of drugs and targets to construct feature of drug-target interaction. Then, we use line graph to model drug-target interaction. After that, we introduce graph transformer network to predict drug-target interaction.ConclusionsThis method introduces a line graph to model the relationship between drug and target. After transforming drug-target interactions from links to nodes, a graph transformer network is used to accomplish the task of predicting drug-target interactions.

Current chimeric antigen receptor (CAR)-T manufacturing utilizes viral vectors and extensive ex vivo expansion at large central facilities leading to an exhausted CAR-T phenotype, high costs and long vein-to-vein times. While allogeneic CAR-T can reduce delays in patient treatment, they require extensive manipulation of donor cells, severe lymphodepletion and demonstrate short persistence limiting their therapeutic window. The UltraCAR-T® platform is designed to overcome these limitations by utilizing non-viral multigene delivery and a rapid, decentralized manufacturing process without ex vivo activation or expansion of T cells. Patient’s own T cells are collected and manufactured at the medical center and re-infused one day after gene transfer. Here we describe the next generation UltraCAR-T platform that addresses the inhibitory tumor microenvironment by incorporating a novel mechanism for intrinsic downregulation of one or more checkpoint inhibitor (CPI) genes. Our design achieves intrinsic CPI blockade without gene editing and is aimed at avoiding systemic toxicity and the high cost of combining CPI antibodies. Next generation UltraCAR-T cells simultaneously express CAR, membrane bound IL-15 (mbIL15), kill switch, and incorporate intrinsic CPI blockade. To illustrate the ability of this platform, we designed exemplary non-viral transposons to generate UltraCAR-T cells against multiple tumor targets incorporating intrinsic blockade of either one (PD-1) or two (PD-1 and TIGIT) CPI genes. Healthy donor T cells were transfected using the UltraPorator™ electroporation system to manufacture UltraCAR-T cells without ex vivo activation or expansion. The co-expression of CAR, mbIL15 and kill switch was confirmed by flow cytometry and western blot. Activated UltraCAR-T showed significant reduction in CPI gene expression compared to control CAR-T cells lacking the CPI blockade and did not show unintended off-target activity. Downregulation of CPI gene(s) on UltraCAR-T enhanced cytotoxicity and inflammatory cytokine production, especially at low effector to target (E:T) cell ratios, when co-cultured with PD-L1+/CD155+ tumor cells. Single-cell cytokine proteomics showed significant increase in polyfunctionality of UltraCAR-T with intrinsic downregulation of CPI gene(s). In vivo, a single infusion of receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific UltraCAR-T with intrinsic PD-1 blockade resulted in rapid expansion, an increase in preferred T cell memory (TSCM/TCM) populations, and significantly improved overall survival of ROR1+ PD-L1+ tumor bearing mice. These preclinical data highlight the improved efficacy of incorporating intrinsic CPI blockade in UltraCAR-T cells using non-viral gene delivery and an established rapid, decentralized manufacturing process. Citation Format: Tim Chan, Cheryl Bolinger, Sean Scott, Mengyan Du, Carol Poortman, Byron Koenitzer, Taranjit Athwal, Lindsey Shepard, R. Daniel Slone, Shourik Dutta, Steven Zilko, James M. Dunleavey, Giorgio Zenere, Jacques Plummer, Bernward Klocke, Christian Zinser, Shamim Ahmad, Douglas E. Brough, Rutul R. Shah, Helen Sabzevari. Incorporation of intrinsic checkpoint blockade enhances functionality of multigenic autologous UltraCAR-T® cells manufactured using non-viral gene delivery and rapid manufacturing process [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2821.

We here used fluorescence imaging to explore the effect of co-overexpression of Mcl-1 and Bak/BH3-only proteins on mitochondrial morphology. The cells co-expressing CFP-Mcl-1 and YFP-Bak/BimL/Puma/tBid showed co-localization of Mcl-1 with Bak/Puma/BimL/tBid and also showed the inhibitory action of Mcl-1 on the Bak-, BimL-, Puma- or tBid-mediated cell death. Co-expression of Mcl-1 and Bak but not BH3-only proteins induced time-dependent mitochondrial swelling. Fluorescence resonance energy transfer (FRET) imaging proved the direct binding of Mcl-1 to Bak, BimL, Puma and tBid, respectively. In addition, Mcl-1 prevented Bak oligomerization by retrotranslocating Bak from mitochondria into cytoplasm. Moreover, Mcl-1-Bak complex exhibited a good co-localization with mitochondria, and co-expression of Mcl-1 and Bak for more than 24 h not only induced mitochondrial swelling but also impaired mitochondrial membrane potential. Collectively, co-expression of Mcl-1 and Bak but not BH3-only proteins significantly induced mitochondrial swelling and subsequent loss of mitochondrial membrane potential.

Bcl-2 family proteins, as central players of apoptotic program, participate in the regulation of mitochondrial network. Here, quantitative live-cell Fluorescence Resonance Energy Transfer (FRET) two-hybrid assay was used to confirm the homo-/hetero-oligomerization of mitofusins (MFN2 and MFN1), and also demonstrate the binding of MFN2 to MFN1 with 1:1 stoichiometry. FRET two-hybrid assay for living cells co-expressing CFP-labeled Bcl-XL (anti-apoptotic Bcl-2 family protein) and YFP-labeled MFN2/MFN1 demonstrate the binding of MFN2/MFN1 to Bcl-XL with 1:1 stoichiometry. Neither MFN2 nor MFN1 bind with monomeric Bax in healthy cells, but both MFN2 and MFN1 combine with punctate Bax (pro-apoptotic Bcl-2 family protein) during apoptosis. Oligomerized Bak (pro-apoptotic Bcl-2 family protein) can only associates with MFN1 but not MFN2. Moreover, co-expression of Bcl-XL and MFN2/MFN1 exhibited the same anti-apoptotic ability as the expression of Bcl-XL alone to staurosporine-induced apoptosis, indicating the full anti-apoptotic ability of Bcl-XL complexed with MFN2 or MFN1. However, knockdown of MFN2 but not MFN1 reduced mitochondrial aggregation induced by overexpressed Bcl-XL, indicating that MFN2 but not MFN1 mediates Bcl-XL-induced mitochondrial aggregation.

We recently developed two quantitative fluorescence resonance energy transfer (FRET) measurement methods based on spectral unmixing of emission spectra (IIem-spFRET) and excitation-emission spectra (ExEm-spFRET), respectively. We here evaluated robustness of the two methods by implementing them on a self-assembled quantitative FRET measurement system using the cells expressing different constructs. For the cells with larger signal-to-noise (S/N) ratio (>9), the two methods obtained consistent FRET efficiency (E) values and total concentration ratio (RC) values of acceptor to donor for all constructs; for the cells with 3 9, the two methods obtained consistent FRET efficiency (E) values and total concentration ratio (RC) values of acceptor to donor for all constructs; for the cells with 3 < S/N < 9, IIem-spFRET obtained bigger RC values than the expected value for VCV construct; for the cells with S/N < 3, although IIem-spFRET method obtained inaccurate E and RC values for VCV construct, the two methods also obtained consistent E and RC values for all other constructs. Collectively, our experimental results demonstrate that both ExEm-spFRET and IIem-spFRET methods are very applicable for live-cell FRET measurement, and ExEm-spFRET has superior robustness especially for the cells high E and low S/N ratio.

Quantum yield ratio (QA /QD ) and absorption ratio (KA /KD ) in all excitation wavelengths used between acceptor and donor are indispensable to quantitative fluorescence resonance energy transfer (FRET) measurement based on linearly unmixing excitation-emission spectra (ExEm-spFRET). We here describe an approach to simultaneously measure QA /QD and KA /KD values by linearly unmixing the excitation-emission spectra of at least two different donor-acceptor tandem constructs with unknown FRET efficiency. To measure the QA /QD and KA /KD values of Venus (V) to Cerulean (C), we used a wide-field fluorescence microscope to image living HepG2 cells separately expressing each of four different C-V tandem constructs at different emission wavelengths with 435 nm and 470 nm excitation respectively to obtain the corresponding excitation-emission spectrum (SDA ). Every SDA was linearly unmixed into the contributions (weights) of three excitation-emission spectra of donor (WD ) and acceptor (WA ) as well as donor-acceptor sensitisation (WS ). Plot of WS /WD versus WA /WD for the four C-V plasmids from at least 40 cells indicated a linear relationship with 1.865 of absolute intercept (QA /QD ) and 0.273 of the reciprocal of slope (KA /KD ), which was validated by quantitative FRET measurements adopting 1.865 of QA /QD and 0.273 of KA /KD for C32V, C5V, CVC and VCV constructs respectively in living HepG2 cells.

Traditional methods for chimeric antigen receptor (CAR) T manufacturing utilize viral vectors, ex vivo activation and expansion of T cells to achieve clinically relevant cell numbers, which leads to an exhausted T cell phenotype, high manufacturing costs, and treatment delays. The UltraCAR-T platform is designed to overcome these limitations using our advanced non-viral gene delivery system and a rapid, overnight manufacturing process (Blood 2019 134 (Supplement_1):2660; Blood 2020 136 (Supplement 1):17); Cancer Research 2020 80 (16Suppl):6593). UltraCAR-T cells, which express antigen specific CAR, membrane-bound IL-15 (mbIL15), and kill switch genes, are manufactured at the medical center's cGMP facility using autologous T cells and administered back to the patient only one day after gene transfer. UltraCAR-T cells are currently under clinical investigation for hematological (NCT03927261) and solid tumors (NCT03907527). Here we describe the advancement of the UltraCAR-T platform to address the inhibitory tumor microenvironment by incorporating intrinsic checkpoint blockade without the need for complex and expensive gene editing techniques. PRGN-3007, based on the next generation of the UltraCAR-T platform, is engineered to simultaneously express CAR for targeting receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is overexpressed on many hematological and solid tumors; mbIL15 for enhanced in vivo expansion and persistence; kill switch for improved safety profile; and a novel mechanism for the intrinsic blockade of PD-1 gene expression. This approach of intrinsic blockade of PD-1 expression, only on UltraCAR-T cells, is aimed to avoid systemic toxicity and high cost of checkpoint inhibitors by eliminating the need for combination treatment. PRGN-3007 is manufactured using the already established rapid and streamlined UltraCAR-T manufacturing process. PRGN-3007 was generated using multiple healthy donor T cells using multi-cistronic non-viral vector and the overnight manufacturing process. The co-expression of CAR, mbIL15 and kill switch transgenes was confirmed by flow cytometry, western blotting, and qPCR. Furthermore, PRGN-3007 showed significant reduction in PD-1 expression on UltraCAR-T cells compared to ROR1 CAR-T cells lacking PD-1 blockade (Control ROR1 CAR-T). The downregulation of PD-1 expression on PRGN-3007 resulted in enhanced ROR1-specific cytotoxicity and release of inflammatory cytokines upon co-culture with various ROR1 + PD-L1 + hematological and solid tumor cells compared to Control ROR1 CAR-T, especially at low effector to target cell ratios. Single-cell cytokine proteomics showed that the downregulation of PD-1 expression on PRGN-3007 resulted in a significantly higher number of polyfunctional CAR-T cells compared to Control ROR1 CAR-T. Expression of mbIL15 on UltraCAR-T, with or without downregulation of PD-1 expression, resulted in robust expansion in presence of ROR1 antigen, lack of autonomous expansion in absence of ROR1, and durable persistence even in absence of exogenous cytokines in vitro. Furthermore, PRGN-3007 was selectively and effectively eliminated by the kill switch activator antibody treatment. A single administration of PRGN-3007, only one day after gene transfer, effectively reduced tumor burden and significantly improved overall survival (p In summary, these preclinical data highlight the overall safety and improved efficacy of incorporating intrinsic downregulation of PD-1 expression on UltraCAR-T cells using non-viral gene delivery and the established rapid, decentralized manufacturing process. These data provide a strong rationale for the evaluation of PRGN-3007 for the treatment of ROR1 + malignancies. Figure: Overall survival in in an established model of mantle cell lymphoma in NSG mice. Tumor cells were engrafted in mice on Day 0 and treatments were administered on Day 8. Data shown is from 8 mice/group at the start of the study. * p Figure 1 Figure 1. Disclosures Chan: Precigen, Inc: Current Employment, Current equity holder in publicly-traded company. Scott: Precigen, Inc: Current Employment. Du: Precigen, Inc: Current Employment. Bolinger: Precigen, Inc: Current Employment. Poortman: Precigen, Inc: Current Employment. Shepard: Precigen, Inc: Current Employment. Koenitzer: Precigen, Inc: Current Employment. Govekung: Precigen, Inc: Current Employment. Sailor: Precigen, Inc: Current Employment. Johnson: Precigen, Inc: Current Employment. Plummer: Precigen, Inc: Current Employment. Zilko: Precigen, Inc: Current Employment. Dutta: Precigen, Inc: Current Employment. Kunchithapautham: Precigen, Inc: Current Employment. Athwal: Precigen, Inc: Current Employment. Klocke: Precigen, Inc: Current Employment. Zinser: Precigen, Inc: Current Employment. Abdeladhim: Precigen, Inc: Current Employment. Ahmad: Precigen, Inc: Current Employment; Kite, A Gilead Company: Ended employment in the past 24 months. Metenou: Precigen, Inc: Current Employment. Semnani: Precigen, Inc: Current Employment. Brough: Precigen, Inc: Current Employment, Current equity holder in publicly-traded company. Shah: Precigen: Current Employment, Current equity holder in publicly-traded company. Sabzevari: Precigen: Current Employment, Current equity holder in publicly-traded company; Kinnate BioPharma: Membership on an entity's Board of Directors or advisory committees; Compass Therapeutics: Current equity holder in publicly-traded company.

Simultaneous spectral unmixing of excitation and emission spectra (ExEm unmixing) has inherent ability resolving spectral crosstalks, two key issues of quantitative fluorescence resonance energy transfer (FRET) measurement, of both the excitation and emission spectra between donor and acceptor without additional corrections. We here set up a filter-based multichannel wide-field microscope for ExEm unmixing-based FRET imaging (m-ExEm-spFRET) containing a constant system correction factor (fsc ) for a stable system. We performed m-ExEm-spFRET with four- and two-wavelength excitation respectively on our system to quantitatively image single living cells expressing FRET tandem constructs, and obtained accurate FRET efficiency (E) and concentration ratio of acceptor to donor (RC ). We also performed m-ExEm-spFRET imaging for single living cells coexpressing CFP-Bax and YFP-Bax, and found that the E values were about 0 for control cells and about 28% for staurosporin-treated cells when RC were larger than 1, indicating that staurosporin induced significant oligomerisation.

Only a small fraction of the antigens expressed by malaria parasites have been evaluated as vaccine candidates. A successful malaria subunit vaccine will likely require multiple antigenic targets to achieve broad protection with high protective efficacy. Here we describe protective efficacy of a novel antigen, Plasmodium yoelii (Py) E140 (PyE140), evaluated against P. yoelii challenge of mice. Vaccines targeting PyE140 reproducibly induced up to 100% sterile protection in both inbred and outbred murine challenge models. Although PyE140 immunization induced high frequency and multifunctional CD8+ T cell responses, as well as CD4+ T cell responses, protection was mediated by PyE140 antibodies acting against blood stage parasites. Protection in mice was long-lasting with up to 100% sterile protection at twelve weeks post-immunization and durable high titer anti-PyE140 antibodies. The E140 antigen is expressed in all Plasmodium species, is highly conserved in both P. falciparum lab-adapted strains and endemic circulating parasites, and is thus a promising lead vaccine candidate for future evaluation against human malaria parasite species.

This report aims to explore how Bcl-xL, a Bcl-2 family protein, regulates PINK1/Parkin-dependent mitophagy. Compared with the Hela cells expressing Parkin alone, co-expression of Bcl-xL significantly inhibited CCCP (Carbonyl cyanide 3- chlorophenylhydrazone)-induced mitochondrial Parkin accumulation and mitophagy. Western blotting analysis illustrated that over-expressed Bcl-xL inhibited CCCP-induced decrease of mitochondrial proteins in Parkin over-expressed cells. Fluorescence loss in photobleaching (FLIP) analyses demonstrated that Bcl-xL inhibited the CCCP-induced translocation of Parkin into mitochondria not by retrotranslocating Parkin from mitochondria to cytoplasm. Fluorescence resonance energy transfer (FRET) imaging revealed in Hela cells that Bcl-xL physically bound with Parkin to form oligomer in cytoplasm, and that Bcl-xL also directly interacted with PINK1 on mitochondria. analysis for HEK293 T cells verified that endogenous Bcl-xL interacted with both endogenous Parkin and PINK1. Collectively, Bcl-xL inhibits PINK1/Parkin- dependent mitophagy by preventing the accumulation of Parkin on mitochondria via two regulation ways: directly binds to Parkin in cytoplasm to prevent the translocation of Parkin from cytoplasm to mitochondria and directly binds to PINK1 on mitochondria to inhibit the Parkin from cytoplasm to mitochondria by PINK1.

Simultaneous linear unmixing of excitation-emission spectra (ExEm-unmixing)-based fluorescence resonance energy transfer (FRET) two-hybrid assay method, named as ExEm-FRET two-hybrid assay, was developed for evaluating the stoichiometric ratio of macromolecular complexes in living cells. Linear unmixing of the excitation-emission spectra (SDA) of cells obtains the weight factors of donor (WD), acceptor (WA) and acceptor sensitization (WS), yielding ED and EA (donor- and acceptor-centric FRET efficiency) images. ExEm-FRET two-hybrid assay employs pixel-to-pixel titration curves of ED/EA versus the free acceptor (Ca)/donor (Cd) concentration deduced from the three weight factors to obtain EA,max and ED,max (the maximal EA and ED), thus yielding the stoichiometric ratio (EA,max/ED,max) of donor-tagged protein to acceptor-tagged protein.

Bax oligomerization is essential for triggering mitochondrial outer membrane permeabilization (MOMP) in many apoptotic programs. However, it is controversial whether Bax dimer is sufficient to trigger MOMP. In this report, multiple Gaussian function-based FRET analysis (Multi-Gaussian FRET analysis) was used to dissect the dimerization and then tetramerization of Bax in relation to MOMP. Multi-Gaussian FRET analysis on the time-lapse FRET images of single living cells co-expressing CFP-Bax and YFP-Bax revealed that formation of mitochondrial Bax homodimers preceded MOMP within 3 min and Bax dimer transformed into tetramer within 6 min concomitantly with complete MOMP within 10 min, providing direct evidence in support of the sufficient ability of Bax dimers to trigger MOMP at least in natural cells.

3-cube-based Förster resonance energy transfer (FRET) microscopy, a sensitized acceptor FRET quantification method, has been widely used to visualize dynamic protein-protein interaction in living cells. Determining the FRET sensitized-quenching transition factor (G factor) of a particular donor-acceptor pair and optical system is crucial for 3-cube FRET quantification. We here improved the acceptor photobleaching-based G factor determination method (termed as mPb-G) and the two-plasmid-based G factor determination method (termed as mTP-G) for rapid and reliable measurement of the G factor. mTP-G method determines G factor by simultaneously detecting three images of cells exclusively expressing each of two tandem constructs with multiple donors and multiple acceptors. This method circumvents switchover of the cells exclusively expressing each of the two constructs. mPb-G method images G factor by detecting three images of cells expressing a donor-acceptor tandem FRET construct before and after partially photobleaching acceptor. We performed the two methods on our dual-channel wide-field FRET microscope to obtain reliable G factor, and also measured the FRET efficiency and acceptor-to-donor concentration ratio of tandem constructs with different acceptor-donor stoichiometries in living HepG2 cells. mTP-G and mPb-G methods provide two simple and reliable tools for determining the G factor, in turn, quantitatively measuring FRET signal and monitoring dynamic biochemical processes in living cells.

BCL-XL, an anti-apoptotic BCL-2 family protein, potently inhibits BAK oligomerization and the formation of toxic mitochondrial pores in response to cellular stress. This report aims to explore which form of mitochondrial monomeric and oligomerized BAK can be retrotranslocated by BCL-XL. Fluorescence imaging of living cells co-expressing CFP-BCL-XL and YFP-BAK showed that BCL-XL markedly inhibited mitochondrial BAK oligomerization and resulted in partial cytosolic BAK distribution. Live-cell fluorescence resonance energy transfer (FRET) analyses showed that BAK auto-oligomerized on mitochondria and BCL-XL physically sequestrated monomeric BAK to prevent BAK oligomerization. Fluorescence loss in photobleaching (FLIP) analyses showed that BCL-XL retrotranslocated the monomeric BAK from mitochondria into cytosol, whereas monomeric BAK reduced the retrotranslocation rate of BCL-XL. Live-cell time-lapse imaging and FLIP experiments in living cells with BAK oligomers displayed that BCL-XL did not depolymerize or retrotranslocate the oligomerized BAK. Collectively, BCL-XL retrotranslocates monomeric instead of oligomerized BAK from mitochondria into cytosol.

Acceptor-sensitized quantitative Forster resonance energy transfer (FRET) measurement (E-FRET) is mainly impeded by donor emission crosstalk and acceptor direct excitation crosstalk. In this paper, we develop a novel E-FRET approach (Lux-E-FRET) based on linear unmixing (Lux) of the fluorescence intensity ratio between two detection channels with each excitation of two different wavelengths. The two detection channels need not to selectively collect the emission of donor or acceptor, and the excitation wavelengths need not to selectively excite donor or acceptor. For a tandem FRET sensor, Lux-E-FRET only needs single excitation wavelength. We performed Lux-E-FRET measurements on our dual-channel wide-field fluorescence microscope for FRET constructs in living cells, and obtained consistent FRET efficiencies with those measured by other methods. Collectively, Lux-E-FRET completely overcomes all spectral crosstalks and thus is applicable to the donor-acceptor pair with larger spectral overlapping.

Simultaneous spectral unmixing of excitation and emission spectra (ExEm unmixing) has the inherent ability to resolve donor emission, fluorescence resonance energy transfer (FRET)-sensitized acceptor emission and directly excited acceptor emission. We here develop an ExEm unmixing-based quantitative FRET measurement method (EES-FRET) independent of excitation intensity and detector parameter setting. The ratio factor (rK), predetermined using a donor-acceptor tandem construct, of total acceptor absorption to total donor absorption in excitation wavelengths used is introduced for determining the concentration ratio of acceptor to donor. We implemented EES-FRET method on a wide-field microscope to image living cells expressing tandem FRET constructs with different donor-acceptor stoichiometry.

The spontaneous excitation-emission (ExEm) spectrum is introduced to the quantitative mExEm-spFRET methodology we recently developed as a spectral unmixing component for quantitative fluorescence resonance energy transfer measurement, named as SPEES-FRET method. The spectral fingerprints of both donor and acceptor were measured in HepG2 cells with low autofluorescence separately expressing donor and acceptor, and the spontaneous spectral fingerprint of HEK293 cells with strong autofluoresence was measured from blank cells. SPEES-FRET was performed on improved spectrometer-microscope system to measure the FRET efficiency (E) and concentration ratio (R C ) of acceptor to donor vales of FRET tandem plasmids in HEK293 cells, and obtained stable and consistent results with the expected values. Moreover, SPEES-FRET always obtained stable results for the bright and dim cells coexpressing Cerulean and Venus or Cyan Fluorescent Protein (CFP)-Bax and Yellow fluorescent protein (YFP)-Bax, and the E values between CFP-Bax and YFP-Bax were 0.02 for healthy cells and 0.14 for the staurosporine (STS)-treated apoptotic cells. Collectively, SPEES-FRET has very strong robustness against cellular autofluorescence, and thus is applicable to quantitative evaluation on the protein-protein interaction in living cells with strong autofluoresence.

We here introduce a fluorescence resonance energy transfer (FRET) two-hybrid assay method to measure the maximal donor(D)- and acceptor(A)-centric FRET efficiency (ED,max and EA,max) of the D-A complex and its stoichiometry by linearly fitting the donor-centric FRET efficiency (ED) to the acceptor-to-donor concentration ratio (RC) and acceptor-centric FRET efficiency (EA) to 1/RC, respectively. We performed this method on a wide-field fluorescence microscope for living HepG2 cells co-expressing FRET tandem constructs and free donor/acceptor and obtained correct ED, EA, and stoichiometry values of those tandem constructs. Evaluation on the binding of Bad with Bcl-XL in Hela cells showed that Bad interacted strongly with Bcl-XL to form a Bad-Bcl-XL complex on mitochondria, and one Bad interacted mainly with one Bcl-XL molecule in healthy cells, while with multiple (maybe 2) Bcl-XL molecules in apoptotic cells.

The present study aims to explore the neuro-protective effects of purified Sparassis crispa polysaccharides against L-glutamic acid (L-Glu)-induced differentiated PC12 (DPC12) cell damages and its underlying mechanisms. The Sparassis crispa water extract was purified by a DEAE-52 cellulose anion exchange column and a Sepharose G-100 column. A fraction with a molecular weight of 75 kDa and a diameter of 88.9 nm, entitled SCWEA, was obtained. SCWEA was identified with a triple helix with (1→3)-linked Rha in the backbone, and (1→2) linkages and (1→6) linkages in the side bone. Our results indicated that the pre-treatment of DPC12 cells with SCWEA prior to L-Glu exposure effectively reversed the reduction on cell viability (by 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay) and reduced L-Glu-induced apoptosis (by Hoechst staining). SCWEA decreased the accumulation of intracellular reactive oxygen species, blocked Ca(2+) influx and prevented depolarization of the mitochondrial membrane potential in DPC12 cells. Furthermore, SCWEA normalized expression of anti-apoptotic proteins in L-Glu-explored DPC12 cells. These results suggested that SCWEA protects against L-Glu-induced neuronal apoptosis in DPC12 cells and may be a promising candidate for treatment against neurodegenerative disease.

Based on our recently developed quantitative fluorescence resonance energy transfer (FRET) measurement method using simultaneous spectral unmixing of excitation and emission spectra (ExEm-spFRET), we here set up an improved spectrometer-microscope (SM) for implementing modified ExEm-spFRET (mExEm-spFRET), in which a system correction factor (fsc) is introduced. Our SM system is very stable for at least six months. Implementation of mExEm-spFRET with four or two excitation wavelengths on SM for single living cells expressing different FRET constructs obtained consistent FRET efficiency (E) and acceptor-donor concentration ratio (Rc) values. We also performed mExEm-spFRET measurement for single living cells coexpressing cyan fluorescent protein (CFP)-Bax and yellow fluorescent protein (YFP)-Bax and found that the E values between CFP-Bax and YFP-Bax were very low (2.2%) and independent of Rc for control cells, indicating that Bax did not exist as homooligomer in healthy cells, but positively proportional to Rc in the case of Rc1 and kept constant value (25%) when Rc1 for staurosporine (STS)-treated cells, demonstrating that all Bax formed homooligomer after STS treatment for 6 h.