A novel injectable in situ forming gel based on carboxymethyl hexanoyl chitosan/hyaluronic acid polymer blending for sustained release of berberine

Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions.

Members of the Anoctamin (Ano)/TMEM16A family have recently been identified as essential subunits of the Ca(2+)-activated chloride channel (CaCC). For example, Ano1 is highly expressed in multiple tissues including airway epithelia, where it acts as an apical conduit for transepithelial Cl(-) secretion and helps regulate lung liquid homeostasis and mucus clearance. However, little is known about the oligomerization of this protein in the plasma membrane. Thus, utilizing mCherry- and eGFP-tagged Ano1 constructs, we conducted biochemical and Förster resonance energy transfer (FRET)-based experiments to determine the quaternary structure of Ano1. FRET and co-immunoprecipitation studies revealed that tagged Ano1 subunits directly associated before they reached the plasma membrane. This association was not altered by changes in cytosolic Ca(2+), suggesting that this is a fixed interaction. To determine the oligomeric structure of Ano1, we performed chemical cross-linking, non-denaturing PAGE, and electromobility shift assays, which revealed that Ano1 exists as a dimer. These data are the first to probe the quaternary structure of Ano1. Understanding the oligomeric nature of Ano1 is an essential step in the development of therapeutic drugs that could be useful in the treatment of cystic fibrosis.

Ovarian cancer (OvCa) is the leading cause of gynecologic cancer-related deaths. While the overall response rate to first line therapy is encouraging (~80%), the majority of women develop recurrent disease that is characterized by resistance to chemotherapy. It is believed that cancer stem cells (CSCs) may contribute, in part, to resistance and recurrence of OvCa. To understand the pathways involved in enhancing this stem-like phenotype, we performed RNA sequencing. We identified that the TLR-ILR1 (TIR) pathways are highly activated in cisplatin resistant OvCa and in CSC-enriched 3-dimensional culture models. To further understand the role of the TIR pathway, we mined the Cancer Genome Atlas database and observed that interleukin receptor-associated kinase 1 (IRAK1), a critical mediator of TIR signaling is upregulated in cancer tissues. In addition, the locus surrounding the IRAK1 gene is amplified in 10% of OvCa patients. We confirmed that IRAK1 expression is upregulated in a majority of OvCa samples by immunohistochemistry of a tumor microarray consisting of 100 patient and paired non-cancerous fallopian tube tissues. Furthermore, this upregulation correlated with early cancer onset and shorter overall survival. To study the specific role of IRAK1 in OvCa, we knocked down its expression using specific shRNA. This significantly impaired cancer growth both in vitro in 2-dimension (2D) and 3-dimensional (3D) spheroid cultures, and in vivo in peritoneal disease models. Moreover, IRAK1 knockdown resulted in decreased expression of CSC marker genes, including MYC, ALDH1A1, DCLK1, and KLF4 suggesting a critical role in maintenance of stemness programming. Since IRAK1 is an upstream kinase that is activated by TIR receptors, we were intrigued by mechanisms driving its activation. In this regard, we have observed that low molecular weight hyaluronic acid (LMW HA) is present at high levels (100-200 ng/ml) in malignant ascites following peritoneal metastasis. Treatment of OVCAR8, A2780 and A1847 cells with LMW HA (50-200 ng/ml) induced IRAK1 phosphorylation at 80 ng/ml that was further enhanced at 200 ng/ml. In addition, LMW HA induced stemness and multidrug resistance genes. With additional studies using specific inhibitors, we identified that the increased spheroid formation occurred via a CD44-PKC-IRAK1-STAT3 signaling axis. Finally, using molecular modeling and in silico screening, coupled with Eurofin’s ScanMAX platform, we identified TCS2210 as a novel highly specific IRAK1 inhibitor. Also, TCS2210 abrogated LMW HA induced activation of IRAK1 and STAT3, and CSC marker genes MYC and DCLK1. Moreover, TCS2210 effectively suppressed OvCa cell growth in in vitro 2D and 3D cultures, and in peritoneal disease models alone and in combination with cisplatin. These data, taken together, strongly suggest that IRAK1 is a valid therapeutic target for OvCa. Citation Format: David Standing, Sumedha Gunewardena, Afreen A. Sayed, Michele T. Pritchard, Harsh B. Pathak, Andrew K. Godwin, Shariska Petersen, Dineo Khabele, Jensen A. Roy, Prasad Dandawate, Scott J. Weir, Shrikant Anant. IRAK1: A novel TOLLway to target ovarian cancer [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 5306.

Ovarian cancer (OvCa) is the leading cause of gynecologic cancer-related deaths, due in part to late-stage diagnoses. While the overall response rate to first line therapy is encouraging (~80%), the majority of women develop recurrent disease, characterized by resistance to standard chemotherapy. There is convincing evidence that cancer stem cells (CSCs) have implications in resistance and recurrence of OvCa. To understand the pathways involved in enhancing this stem-like phenotype, we performed RNA sequencing and identified TLR-ILR1 (TIR) pathways as highly activated in cisplatin resistant OvCa and stem-cell populations. Subsequently, using a tumor microarray, we observed that interleukin receptor-associated kinase 1 (IRAK1), a critical mediator of TIR signaling, is upregulated in OvCa patient tissues compared to normal. This expression further correlated with younger diagnosis age and shorter overall survival, suggesting a role in OvCa tumorigenesis. Knockdown of IRAK1 by specific shRNA in OvCa cells, significantly impaired CSC enriched spheroid growth and orthotopic tumor growth in mouse models replicating advanced peritoneal OvCa. RNA sequencing of IRAK1 knocked down cells identified downregulation of CSC related genes including STAT3, MYC, NOTCH1, and NOTCH3. In addition, there was a reduction in the cisplatin efflux transporter ABCC1 and stemness marker gene CD44. Advanced OvCa is often associated (~90% in stage III and IV) with the development of malignant ascites. We demonstrate for the first time that significant amounts of low molecular weight hyaluronic acid fragments (LMW HA) are present in ascites. LMW HA induced IRAK1 dependent activation of non-canonical signaling through PKC-β leading to induction of STAT3 phosphorylation and MYC. This was further accompanied by increased spheroid formation, demonstrating a critical role for IRAK1 in HA-induced stemness. We have further identified a selective IRAK1 inhibitor, TCS2210 (1,2-Dihydro-N-hydroxy-2-oxo-3-(3-phenylpropyl)-6-quinoxalinecarboxamide). TCS2210 abrogated LMW HA induced activation of IRAK1 and expression of stemness genes MYC, NOTCH1, and NOTCH3. Lastly, we found that TCS2210 effectively suppresses OvCa cell growth in vitro and in vivo, synergizing with standard-of-care cisplatin. Our data suggests a role of IRAK1 in OvCa, by enhancing cancer cell growth and stemness. It further suggests that IRAK1 is a putative target for advanced OvCa. Citation Format: David Standing, Prasad Dandawate, Jaimie Johnson, Sumedha Gunewardena, Michele T. Pritchard, Harsh Pathak, Dineo Khabele, Katherine Roby, Andrew Godwin, Roy Jensen, Scott Weir, Shrikant Anant. IRAK1 is a critical mediator of hyaluronic acid induced stemness. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5020.

Hyaluronan (HA, an extracellular matrix glycosaminoglycan) has been reported to have a variety of biological activities, including regulation of inflammation. While high molecular weight HA (HMW HA) is recognized to be anti-inflammatory, the activity of low molecular weight HA (LMW HA, the degradation catabolite of hyaluronidase) in inflammation is less clear. Because of reagent contamination of both hyaluronidase and HA used in many studies, many reported aspects of the associated biology need reinvestigation. There are many reports which have shown that LMW HA is pro-inflammatory; however, some recent publications raised serious doubts that LMW HA and hyaluronidase PH20 are not pro-inflammatory. Endotoxin and other contaminants in the reagents used in previous reports (i.e., bovine testicular hyaluronidase [BTH] Hyal type I-S and IV-S from Sigma) may be responsible for the observed inflammation. Further investigation has shown that the most purified BTH (type VI-S from Sigma) contains no endotoxin, but has substantial level of peptidoglycan, which is also pro-inflammatory. Caution should be taken when conducting studies of inflammation using HA and hyaluronidase that may contain endotoxin and peptidoglycan, as well as other pro-inflammatory contaminants. We have characterized HA affinity to its receptor CD44. While HMW HA binds to CD44 strongly, LMW HA degraded by PH20 hyaluronidase does not bind to CD44 on the cell surface. This may explain the observation that recombinant human hyaluronidase PH20 (rHuPH20) inhibits leukocyte migration upon inflammatory stimulation by interrupting CD44 interaction with HA, mediated by HMW HA. PEGPH20, a pegylated rHuPH20, has the same inhibitory activity on leukocyte transmigration as rHuPH20. Further investigation of leukocyte-inhibiting activity of rHuPH20 may reveal further clinical applications, such as wound healing and arthritis. Another report which is difficult to understand is that the ultra-high molecular weight HA produced by naked mole rat hyaluronan synthase-2 (nmrHAS2) contributes to cancer resistance, probably due to its specific anti-inflammatory activity. In our study, nmrHAS2 is cancer-promoting in human cancer cells, to a similar extent as human HAS2. The proposed cancer resistant activity of nmrHAS2 may apply selectively to its host animal species, the naked mole rat.

Use of distinct green fluorescent protein (GFP) variants permits the study of protein-protein interactions and colocalization in viable transfected cells by fluorescence (Förster) resonance energy transfer (FRET). Flow cytometry is a sensitive method to detect FRET. However, the typical dual-laser methods used in flow cytometric FRET assays are not generally applicable because they require a specialized krypton ultraviolet (UV) laser. The purpose of this work was to develop a flow cytometric method to detect FRET between cyan fluorescent protein (CFP; donor) and yellow fluorescent protein (YFP; acceptor) by using the 458-nm excitation from a single tunable argon-ion laser. FUSE-binding protein (FBP) interacting repressor (FIR) and FBP are c-myc transcription factors and are known to interact physically. To examine their interaction within viable cells, FIR and the binding motif of FBP, the FBP central domain (FBPcd), were fused with CFP and YFP, respectively, and this pair of fluorescently-tagged proteins was used to detect FRET in vivo. Cells transfected with expression plasmids encoding a CFP-FIR fusion protein and YFP as a negative control, a CFP-YFP fusion protein as a positive control, or CFP-FIR and YFP-FBPcd fusion proteins were examined for FRET after excitation with a 458-nm line from a tunable argon-ion laser. FRET was measured as the ratio of YFP:CFP emission or as YFP emission at 564-606 nm. Conventional FRET using the 413-nm UV line from a krypton laser was examined for comparison. Fluorescence signals were separated with a customized optical filter configuration using 530-nm shortpass, 500-nm longpass, and 560-nm shortpass dichroics in addition to 488/30 nm (CFP), 530/30 nm (YFP), and 585/42 nm (FRET) bandpass filters. Further, a laser-scanning confocal microscopic photobleach technique was used to document that FRET occurred by showing that the intensity of donor CFP fluorescence increased after its acceptor YFP was photobleached. Steady-state spectrofluorometry was used to confirm and validate the results detected by flow cytometry. Upon excitation with the 458-nm line of the argon-ion laser, the enhancement of the acceptor YFP signal and the decrease of the CFP signal were easily detected in cells transfected with the CFP-YFP construct or CFP-FIR and YFP-FBPcd. Similarly, FRET was detected under these conditions when the YFP emission was assessed at 564-606 nm. A strong correlation was observed between the increase in the YFP:CFP ratio and the YFP emission detected at 564-606 nm, consistent with the conclusion that FRET was detected comparably by both methods. A conventional flow cytometric krypton UV-laser technique was also used to confirm that FRET occurred with the CFP-YFP fusion protein and from CFP-FIR --> YFP-FBPcd. FRET also was confirmed by a confocal photobleaching technique, in which donor CFP intensity was enhanced after its acceptor YFP was photobleached. The flow cytometric and confocal microscopic results were confirmed by spectrofluorometry. These results demonstrated the feasibility of flow cytometric detection of FRET signals from CFP to YFP by excitation with the 458-nm line from the tunable argon-ion laser. The method was as efficient as excitation with the krypton UV laser and therefore should make FRET a more generally available flow cytometric technique.

Förster resonance energy transfer (FRET) is the direct energy exchange between two-component fluorescent molecules. FRET methods utilize chemically linked molecules or unlinked fluorescent molecules such as fluoresscent protein-protein interactions. FRET is therefore a powerful indicator of molecular proximity, but standardized determination of FRET efficiency is challenged when investigating natural (chemically unlinked) interactions. In this paper, we have examined the interactions of tumor necrosis factor receptor-1 (TNFR1) molecules expressed as recombinant C-terminal fusion proteins of cyan, yellow, or red fluorescent protein (-CFP, -YFP, or -RFP) to evaluate two-molecule chemically unlinked FRET by flow cytometry. We demonstrate three independent FRET pairs of TNFR1 CFP→YFP (FRET-1), YFP→RFP (FRET-2) and CFP→RFP (FRET-3), by comparing TNFR1+TNFR1 with non-interacting TNFR1+CD27 proteins, on both LSR-II and Fortessa X-20 cytometers. We describe genuine FRET activities reflecting TNFR1 homotypic interactions. The FRET events can be visualized during sample acquisition via the use of "spiked" FRET donor cells, together with TNFR1+TNFR1 co-transfected cells, as FRET channel mean fluorescence intensity (MFI) overlays. FRET events can also be indicated by comparing concatenated files of cells expressing either FRET positive events (TNFR1+TNFR1) or FRET negative events (TNFR1+CD27) to generate single-cell scatter plots showing loss of FRET donor brightness. Robust determination of FRET efficiency is then confirmed at the single-cell level by applying matrix calculations based on the measurements of FRET, using donor, acceptor, and FRET fluorescent intensities (I), detector channel emission coefficient (S), fluorescent protein extinction coefficients (ε) and the α factor. In this TNFR1-based system the mean CFP→YFP FRET-1 efficiency is 0.43 (LSR-II) and 0.41 (Fortessa X-20), the mean YFP→RFP FRET-2 efficiency is 0.30 (LSR-II) and 0.29 (Fortessa X-20), and the mean CFP→RFP FRET-3 efficiency is 0.56 (LSR-II) and 0.54 (Fortessa X-20). This study also embraces multi-dimensional clustering using t-SNE, Fit-SNE, UMAP, Tri-Map and PaCMAP to further demonstrate FRET. These approaches establish a robust system for standardized detection of chemically unlinked TNFR1 homotypic interactions with three individual FRET pairs.

An Optically Reconfigurable Förster Resonance Energy Transfer Process for Broadband Switchable Organic Single-Mode Microlasers

The heterotrimeric SecYEG complex comprises a protein-conducting channel in the bacterial cytoplasmic membrane. SecYEG functions together with the motor protein SecA in preprotein translocation. Here, we have addressed the functional oligomeric state of SecYEG when actively engaged in preprotein translocation. We reconstituted functional SecYEG complexes labelled with fluorescent markers into giant unilamellar vesicles at a natively low density. Förster's resonance energy transfer and fluorescence (cross-) correlation spectroscopy with single-molecule sensitivity allowed for independent observations of the SecYEG and preprotein dynamics, as well as complex formation. In the presence of ATP and SecA up to 80% of the SecYEG complexes were loaded with a preprotein translocation intermediate. Neither the interaction with SecA nor preprotein translocation resulted in the formation of SecYEG oligomers, whereas such oligomers can be detected when enforced by crosslinking. These data imply that the SecYEG monomer is sufficient to form a functional translocon in the lipid membrane.

Editor's evaluation: A helicase-tethered ORC flip enables bidirectional helicase loading

Construction of a Homogeneous Enzyme-Free Autocatalytic Nucleic Acid Machinery for High-Performance Intracellular Imaging of MicroRNA

Ratiometric determination of the efficiency of fluorescence or Förster resonance energy transfer (FRET) is one of the most widespread methods for the characterization of protein clustering and conformation. Low photon numbers, often present in pixel-by-pixel determination of FRET efficiency in digital microscopy, result in large uncertainties in the derived FRET parameter. Here, we propose a method based on maximum likelihood estimation (MLE) of FRET efficiency using photon counting detectors to overcome this limitation. Intensities measured in the donor, FRET, and acceptor channels were all assumed to follow Poisson statistics as a result of detector shot noise. The joint probability of photon numbers detected in the donor, FRET, and acceptor channels was derived using an equation describing the relationship between the three measured intensities. The FRET efficiency generating the measured photon numbers with the largest likelihood was determined iteratively providing a single FRET value for all pixels in the calculation. Since as few as 100 pixels are sufficient to provide a maximum likelihood estimate for FRET, biological variability in FRET values can be revealed by performing the analysis for regions of interests in an image. Since the algorithm provides the probability of a combination of donor, FRET, and acceptor intensities observed in each individual pixel given a certain FRET efficiency, outlier pixels with low probabilities could be excluded from the analysis. Simulations carried out with low photon numbers in the presence and absence of outlier pixels revealed that the proposed approach can reliably and reproducibly estimate FRET efficiency. In addition, systematic evaluation of the simulation results showed that the distribution of pixel-by-pixel FRET efficiencies is skewed, and the mean of these FRET values is a biased and unreliable estimate of the FRET efficiency. In the absence of outlier pixels, FRET calculated from summed donor, FRET, and acceptor intensities proved to be as reliable as MLE. We conclude that MLE of FRET outperforms calculations using summed and pixel-by-pixel intensities in biologically relevant situations involving low photon numbers and outlier pixels. © 2014 International Society for Advancement of Cytometry.

The influences of donor and acceptor concentrations on F\"orster resonant energy transfer (FRET) in a separated donor-acceptor quantum dot bilayer structure have been investigated. Donor intra-ensemble energy transfer is shown to have an impact on the donor-acceptor FRET efficiency in the bilayer structure. At high donor concentrations the FRET distance dependence and the acceptor concentration dependence in the separated donor-acceptor layer structure agree well with theories developed for FRET between randomly distributed, homogeneous donor and acceptor ensembles. However, discrepancies between measurement and theory are found at low donor concentrations. A donor concentration study shows that the FRET efficiency decreases with increasing donor concentration even though a donor concentration-independent FRET efficiency is predicted by standard theory. The observed dependence of the FRET efficiency on the donor concentration can be explained within the FRET rate model, for a constant, donor concentration independent FRET rate, by taking into account the concentration dependent donor reference lifetime arising from intra-donor ensemble FRET. This shows that the decrease in the FRET efficiency with increasing donor concentration is not a signature of a change in the donor-acceptor FRET rate, but due to the competition of the donor-acceptor and donor-donor energy transfer for the higher energy donors. As the intra-donor ensemble FRET represents another decay mechanism, the donor quantum yield for the higher energy donors decreases with increasing donor quantum dot (QD) concentration, as can also be seen from the redshift of the donor emission spectrum. Using this concentration dependent donor quantum yield in the calculation of the F\"orster radius, the FRET theory for homogeneous donor and acceptor ensembles can be modified to include the effect of the donor intra-ensemble transfer and to correctly describe the trends and absolute values of the measured FRET efficiencies as a function of the donor and the acceptor concentrations. These results show that in QD systems where intra-donor ensemble FRET is as important as the radiative and nonradiative donor decay mechanisms, the FRET rate rather than the FRET efficiency more appropriately characterizes the donor-acceptor FRET. By fitting with the rate model, FRET rates as high as $(1.2 {\mathrm{ns}}^{)}$ have been determined for the structures presented here.

In vivo quantitative FRET small animal imaging: Intensity versus lifetime-based FRET

Herein, an ionic material (IM) with Förster Resonance Energy Transfer (FRET) characteristics is reported for the first time. The IM is designed by pairing a Nile Blue A cation (NBA+) with an anionic near-infrared (NIR) dye, IR820−, using a facile ion exchange reaction. These two dyes absorb at different wavelength regions. In addition, NBA+ fluorescence emission spectrum overlaps with IR820− absorption spectrum, which is one requirement for the occurrence of the FRET phenomenon. Therefore, the photophysical properties of the IM were studied in detail to investigate the FRET mechanism in IM for potential dye sensitized solar cell (DSSCs) application. Detailed examination of photophysical properties of parent compounds, a mixture of the parent compounds, and the IM revealed that the IM exhibits FRET characteristics, but not the mixture of two dyes. The presence of spectator counterion in the mixture hindered the FRET mechanism while in the IM, both dyes are in close proximity as an ion pair, thus exhibiting FRET. All FRET parameters such as spectral overlap integral, Förster distance, and FRET energy confirm the FRET characteristics of the IM. This article presents a simple synthesis of a compound with FRET properties which can be further used for a variety of applications.