Tetramethyl Rhodamine Research Articles

Nucleic acid-functionalized upconversion luminescence biosensor based on strand displacement-mediated signal amplification for the detection of trivalent chromium ions

BackgroundRapid industrial development has generated serious pollution, including the presence of toxic and harmful heavy metal ions. Among them, trivalent chromium ion (Cr3+) is a very important element that poses a threat to life and health in our industrial wastewater pollution. Thus, it is important to develop efficient fluorescence methods for Cr3+ detection. In this study, an upconversion luminescence biosensor for detecting Cr3+ was constructed based on a DNAzyme, strand displacement reaction (SDR), and DNA-functionalized upconversion nanoparticles (UCNPs). ResultsThe sulfonate-rich poly (sodium 4-styrene sulfonate) (PSS) was modified onto the surface of UCNPs, forming UCNPs@PSS. Then, NH2-Capture probe DNA (NH2-Cp) was further modified onto the UCNPs@PSS surface through sulfonylation, resulting in UCNPs@PSS@NH2-Cp. The DNAzyme activated by Cr3+ triggered the release of the primer probe (Pp), which initiated the SDR system cycle, thereby releasing a tetramethylrhodamine (TAMRA)-modified signal probe (TAMRA-Sp). Finally, UCNPs@PSS@NH2-Cp bound to TAMRA-Sp through complementary base pairing, causing UCNPs and TAMRA to approach each other. Because of the luminescence resonance energy transfer (LRET) mechanism, the upconversion luminescence (UCL) signal of the UCNPs was quenched by TAMRA, enabling the detection of Cr3+ by the change of I585/I545 ratio. This biosensor has good stability, selectivity, and sensitivity, with a linear range of 0.5–75 nM and a detection limit of 0.135 nM for Cr3+. Significance and noveltyFirstly, based on LRET between UCNPs and TAMRA, the quantitative analysis of Cr3+ is achieved through the changes of ratio fluorescence. Secondly, the specificity of the biosensor is improved by utilizing the specific recognition of DNA enzymes. Thirdly, the signal amplification technology of the SDR cycle greatly improves the sensitivity of biosensor. This biosensor will be useful for future environmental safety monitoring and biopsy of biological fluids.

Fluorophore-Labeled Pyrrolones Targeting the Intracellular Allosteric Binding Site of the Chemokine Receptor CCR1.

In this study, we describe the structure-based development of the first fluorescent ligands targeting the intracellular allosteric binding site (IABS) of the CC chemokine receptor type 1 (CCR1), a G protein-coupled receptor (GPCR) that has been pursued as a drug target in inflammation and immune diseases. Starting from previously reported intracellular allosteric modulators of CCR1, tetramethylrhodamine (TAMRA)-labeled ligands were designed, synthesized, and tested for their suitability as fluorescent tracers to probe binding to the IABS of CCR1. In the course of these studies, we developed LT166 (12) as a highly versatile fluorescent CCR1 ligand, enabling cell-free as well as cellular NanoBRET-based binding studies in a nonradioactive and high-throughput manner. Besides the detection of intracellular allosteric ligands by direct competition with 12, we were also able to monitor the binding of extracellular antagonists due to their positive cooperative binding with 12. Thereby, we provide a straightforward and nonradioactive method to easily distinguish between ligands binding to the IABS of CCR1 and extracellular negative modulators. Further, we applied 12 for the identification of novel chemotypes for intracellular CCR1 inhibition that feature high binding selectivity for CCR1 over CCR2. For one of the newly identified intracellular CCR1 ligands (i.e., 23), we were able to show CCR1 over CCR2 selectivity also on a functional level and demonstrated that this compound inhibits basal β-arrestin recruitment to CCR1, thereby acting as an inverse agonist. Thus, our fluorescent CCR1 ligand 12 represents a highly promising tool for future studies of CCR1-targeted pharmacology and drug discovery.

FRETting about CRISPR-Cas Assays: Dual-Channel Reporting Lowers Detection Limits and Times-to-Result.

Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated Protein (CRISPR-Cas) systems have evolved several mechanisms to specifically target foreign DNA. These properties have made them attractive as biosensors. The primary drawback associated with contemporary CRISPR-Cas biosensors is their weak signaling capacity, which is typically compensated for by coupling the CRISPR-Cas systems to nucleic acid amplification. An alternative strategy to improve signaling capacity is to engineer the reporter, i.e., design new signal-generating substrates for Cas proteins. Unfortunately, due to their reliance on custom synthesis, most of these engineered reporter substrates are inaccessible to many researchers. Herein, we investigate a substrate based on a fluorescein (FAM)-tetramethylrhodamine (TAMRA) Förster resonant energy-transfer (FRET) pair that functions as a seamless "drop-in" replacement for existing reporters, without the need to change any other aspect of a CRISPR-Cas12a-based assay. The reporter is readily available and employs FRET to produce two signals upon cleavage by Cas12a. The use of both signals in a ratiometric manner provides for improved assay performance and a decreased time-to-result for several CRISPR-Cas12a assays when compared to a traditional FAM-Black Hole Quencher (BHQ) quench-based reporter. We comprehensively characterize this reporter to better understand the reasons for the improved signaling capacity and benchmark it against the current standard CRISPR-Cas reporter. Finally, to showcase the real-world utility of the reporter, we employ it in a Recombinase Polymerase Amplification (RPA)-CRISPR-Cas12a DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) assay to detect Human papillomavirus in patient-derived samples.

Solasonine promotes apoptosis of non-small cell lung cancer cells by regulating the Bcl-2/Bax/caspase-3 pathway

To investigate the effect of solasonine, an active component of Solanum nigrum, on proliferation and apoptosis of non-small cell lung cancer PC9 cells. PC9 cells were treated with 2, 5, 10, 15, 20, or 25 μmol/L solasonine, and the changes in cell proliferation were examined using CCK-8 assay. Tetramethyl rhodamine ethyl ester (TMRE) was used to detect the changes in mitochondrial membrane potential, and caspase-3/7 detection kit and GreenNucTM caspase-3/Annexin V-mCherry kit for live cell were used to analyze the changes in caspase-3 of the cells. Annexin V-FITC/PI double staining was employed to analyze the apoptosis rate of the cells. The effect of PTEN inhibitors on solasonine-induced cell apoptosis was examined by detecting apoptosis-related protein expressions using Western blotting. Solasonine treatment for 24, 48, and 72 h significantly lowered the viability of PC9 cells. The cells treated with solasonine for 24 h showed significantly decreased mitochondrial membrane potential and increased cell apoptosis with enhanced caspase-3/7 and caspase-3 activities and expression of cleaved caspase-3. Solasonine treatment significantly decreased phosphorylation levels of PI3K and Akt, increased the protein expressions of PTEN and Bax, and lowered the expression of Bcl-2 protein in the cells. Solasonine inhibits proliferation and induces apoptosis of PC9 cells by regulating the Bcl-2/Bax/caspase-3 pathway and its upstream proteins.

LncRNA SNHG1 enhances cartilage regeneration by modulating chondrogenic differentiation and angiogenesis potentials of JBMMSCs via mitochondrial function regulation

BackgroundCartilage is a kind of avascular tissue, and it is difficult to repair itself when it is damaged. In this study, we investigated the regulation of chondrogenic differentiation and vascular formation in human jaw bone marrow mesenchymal stem cells (h-JBMMSCs) by the long-chain noncoding RNA small nucleolar RNA host gene 1 (SNHG1) during cartilage tissue regeneration.MethodsJBMMSCs were isolated from the jaws via the adherent method. The effects of lncRNA SNHG1 on the chondrogenic differentiation of JBMMSCs in vitro were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), Pellet experiment, Alcian blue staining, Masson’s trichrome staining, and modified Sirius red staining. RT-qPCR, matrix gel tube formation, and coculture experiments were used to determine the effect of lncRNA SNHG1 on the angiogenesis in JBMMSCs in vitro. A model of knee cartilage defects in New Zealand rabbits and a model of subcutaneous matrix rubber suppositories in nude mice were constructed for in vivo experiments. Changes in mitochondrial function were detected via RT-qPCR, dihydroethidium (DHE) staining, MitoSOX staining, tetramethyl rhodamine methyl ester (TMRM) staining, and adenosine triphosphate (ATP) detection. Western blotting was used to detect the phosphorylation level of signal transducer and activator of transcription 3 (STAT3).ResultsAlcian blue staining, Masson’s trichrome staining, and modified Sirius Red staining showed that lncRNA SNHG1 promoted chondrogenic differentiation. The lncRNA SNHG1 promoted angiogenesis in vitro and the formation of microvessels in vivo. The lncRNA SNHG1 promoted the repair and regeneration of rabbit knee cartilage tissue. Western blot and alcian blue staining showed that the JAK inhibitor reduced the increase of STAT3 phosphorylation level and staining deepening caused by SNHG1. Mitochondrial correlation analysis revealed that the lncRNA SNHG1 led to a decrease in reactive oxygen species (ROS) levels, an increase in mitochondrial membrane potential and an increase in ATP levels. Alcian blue staining showed that the ROS inhibitor significantly alleviated the decrease in blue fluorescence caused by SNHG1 knockdown.ConclusionsThe lncRNA SNHG1 promotes chondrogenic differentiation and angiogenesis of JBMMSCs. The lncRNA SNHG1 regulates the phosphorylation of STAT3, reduces the level of ROS, regulates mitochondrial energy metabolism, and ultimately promotes cartilage regeneration.

Alkyl anchor–modified artificial viral capsid budding outside-to-inside and inside-to-outside giant vesicles

ABSTRACT The budding of human immunodeficiency virus from an infected host cell is induced by the modification of structural proteins bearing long-chain fatty acids, followed by their anchoring to the cell membrane. Although many model budding systems using giant unilamellar vesicles (GUVs) induced by various stimuli have been developed, constructing an artificial viral budding system of GUVs using only synthesized molecules remains challenging. Herein, we report the construction of an artificial viral capsid budding system from a lipid bilayer of GUV. The C-terminus of the β-annulus peptide was modified using an octyl chain as an alkyl anchor via a disulfide bond. The self-assembly of the β-annulus peptide with an octyl chain formed an artificial viral capsid aggregate. The fluorescence imaging and transmission electron microscopy observations revealed that the addition of the tetramethylrhodamine (TMR)-labeled octyl chain–bearing β-annulus peptide to the outer aqueous phase of GUV induced the budding of the capsid-encapsulated daughter vesicle outside-to-inside the mother GUV. Conversely, the encapsulation of the TMR-labeled octyl chain–bearing β-annulus peptide in the inner aqueous phase of GUV induced the budding of the capsid-encapsulated daughter vesicle inside-to-outside the mother GUV. Contrarily, the addition of the TMR-labeled β-annulus peptide to GUV barely induced budding. It was demonstrated that the higher the membrane fluidity of GUV, the more likely budding would be induced by the addition of the alkyl anchor–modified artificial viral capsid. The simple virus-mimicking material developed in this study, which buds off through membrane anchoring, can provide physicochemical insights into the mechanisms of natural viral budding from cells.

Leptin Induces Evidence of Placental Mitochondrial Dysfunction in a Mouse Model of Preeclampsia

Preeclampsia (PE) affects 5-10% of pregnancies worldwide that increase maternal and fetal morbidity and mortality. PE is often characterized by hypertension, endothelial dysfunction, and restriction of intrauterine fetal growth. Our lab established that mid-gestation leptin infusion in pregnant mice induces a model of PE, notably reducing fetal growth in late gestation. Clinical studies indicate that PE patients demonstrate elevated levels of leptin compared to healthy pregnancies as well as present with evidence of placental mitochondrial dysfunction. Therefore, we hypothesize that mid-late gestation leptin infusion induces placental mitochondrial dysfunction in pregnant mouse placenta. We infused timed-pregnant C57/Bl6 mice with saline (sham) or leptin (0.9 mg/kg/day) via s.c. osmotic minipump from gestation day (GD)11-18 (n=4-8). We collected placentas on GD18 for analysis. We isolate mitochondria from mouse placenta via differential centrifugation, after douncing pulverized tissue. From the isolated mitochondria, we assessed morphology via electron microscopy (EM) and performed amplex red assay for reactive oxygen species (ROS) measurements and tetramethyl rhodamine methyl ester (TMRM) dye was used to measure membrane potential. Pairwise comparisons were by student’s t-test. Our data indicates that leptin decreased membrane potential in placenta mitochondria compared to the sham placentas (1466±16.42 sham vs 1353±32.48 leptin A.U./mg of protein, **p<0.01). In comparison, there were no differences in H2O2 production between the sham and leptin-infused placental mitochondrial isolations (7381±125.2 leptin vs 7311±180.3 sham A.U./mg of protein). EM analysis of one sham and one leptin placenta indicated an increase in mitophagy in the leptin-infused placenta slices as well as decreased mitochondrial circularity, suggesting that leptin increases mitochondrial death. In summary, these data indicate that leptin induces mitochondrial dysfunction which in turn may contribute to placental dysfunction and reduced uteroplacental fetal growth in PE. 1R01HL169576, 4R00HL146948. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Activatable theranostic nanoprobes for Fluorescence/MR imaging and microenvironment remodeling of early cartilage degeneration

Osteoarthritis (OA) is a degenerative joint disease of the knee joint, suffering from the limited therapeutic effect and serious side effects. Unfortunately, there are currently rarely available materials to fundamentally delay and monitor OA progression. The OA microenvironment, including excessive inflammation, matrix metalloproteinases (MMPs), oxidative stress, and reactive oxygen species (ROS), play a crucial role in the pathogenesis of OA. Utilizing and remodeling OA microenvironment may be an effective strategy for the diagnosis and treatment of OA. This work developed an intelligent responsive nanoprobe, Gd-HMPB@SMT@MMP (GHPSM) by encapsulating S-methylisothiourea (SMT) loaded mesoporous Prussian Blue (Gd-HMPB) with Tetramethyl Rhodamine (TAMRA) labeled MMP-13 peptide (MMP-13-TAMRA) for this aim. GHPSM displayed enhanced accumulation in OA cartilage site due to the decrease in the content of negatively charged aminoglycans (GAGs) in early degenerative articular cartilage and activated fluorescence signal as well as SMT controlled release due to the cleavage of MMP-13-TAMRA from GHPSM by overexpressed MMP-13 matrix protease in OA microenvironment. Furthermore, integrating the function of SMT and PB nanozyme, GHPSM reshaped the OA microenvironment by reducing the release of ROS and inflammatory factors (TNF-α, IL-6, iNOS), increasing the expression of intermediate structural proteins (COL2 and Aggrescan), thereby reducing cartilage degradation and chondrocyte apoptosis, ultimately delaying the development of OA. The progressions of early OA disease after different treatments were successfully tracked based on fluorescence/MR imaging with X-ray imaging of knee joint for comparation. These findings highlight the potentials of GHPSM in monitoring the status of cartilage in real-time and delay OA progression by reshaping the OA microenvironment, providing novel ideas for future research in OA diagnosis and treatment.

Active targeting of nanotherapeutics using power cavitation imaging with a linear array transducer

Power cavitation imaging (PCI) is an emerging strategy for monitoring blood–brain barrier (BBB) opening, enabling spatial discrimination of cavitation intensity through power Doppler-analogous processing of passive cavitation images. Single-element cavitation detectors, while beneficial in signal monitoring for cavitation controllers, provide limited spatial information, conferring the need for an image-guided approach to ensure accurate cavitation localization and regulation. This study aims to evaluate the capability of PCI to spatially correlate real-time acoustic cavitation emissions with mechanical bioeffects as a predictor of P-selectin-targeted nanocarrier delivery. Preliminary sonoporation experiments were performed in vitro with brain tissue derived mouse endothelial cells and 3 kDa tetramethylrhodamine (TMR)-dextran. A focused ultrasound transducer (0.5 MHz, 5000 cycles/pulse, 5 Hz PRF, 30 second treatment duration) was used to sonicate cells in small volume (25 μl) cell suspension. Homogenous distribution of TMR-dextran in the cytosol and nucleus was observed via fluorescence microscopy at lower pressures (7% ± 2% fluorescent cells), while higher pressures had no significant differences above control (3% ± 2% vs 1% nontreated) due to microbubble destruction. Future experiments will correlate sonoporation with P-selectin expression and validate a linear array PCI system against contrast-enhanced MRI as a guide for P-selectin-targeted nanocarrier delivery in mouse models of medulloblastoma.

Netrin1-carrying magnetic microspheres with magnetic field activate neurotrophin factors to guide neuronal outgrowth in vitro and in vivo

Nerve injuries often result in the failure of functional synapses connections, primarily due to the absence of neurotrophic factors and guidance at the injury sites. In response to this challenge, an effective approach involving magnetic microspheres carrying axonal guidance cue (Netrin1) was presented. These microspheres were prepared using Fe3O4 and PLGA via electrospray. Netrin1 was incorporated into structural particles that could be oriented in a magnetic field and released at the injury sites. In this study, oriented and longer axons were observed in the cortical neurons after 10 μg/mL magnetic microspheres (MMSs) treatment. Under an applied magnetic field of 5 mT, Netrin1-carrying magnetic microspheres (N@MMSs) demonstrated a more significant increase in cell viability, mitochondrial membrane potential, mRNA expressions of BDNF and NGF compared to MMSs. These findings were assessed using the cell counting kit-8, tetramethyl rhodamine fluorescence, and real time PCR assay, respectively. Furthermore, N@MMSs demonstrated significant protection against glutamate-induced neuronal oxidative toxicity, as evaluated by mitochondrial respiratory function and glycolysis stress using the Seahorse Extracellular Flux analyzer. Subsequently, N@MMSs and a magnetic field were applied to adult mice after sciatic nerve injury. Behavior analysiss, including the open field test, indicated that N@MMSs typically increased motor activity under the magnetic field and significantly decreased paw licking latency on the 14th day. Finally, both BDNF and NGF expressions increased compared to the injury group. In conclusion, N@MMSs show promise as alternative particles for nerve regeneration.

A comparative analysis of gastrointestinal tract barrier function and immune markers in gilt vs. sow progeny at birth and weaning.

Progeny born to primiparous sows (gilt progeny; GP) have lower birth, weaning and slaughter weights than sow progeny (SP). GP also have reduced gastrointestinal tract (GIT) development, as evidenced by lower organ weights. Therefore, the aim of this experiment was to quantify changes in GIT barrier function that occur in birth and weaning, representing two major challenges to the young piglet. The effects of parity (GP vs. SP) in GIT barrier integrity function were quantified at four timepoints: birth (~0h), 24h after birth (24h), 1-d preweaning (PrW), and 1-d postweaning (PoW) in commercially reared piglets. Due to inherent differences between newborn and weanling pigs, the results were analyzed in two cohorts, birth (0 vs. 24h, n = 31) and weaning (PrW vs. PoW, n = 40). Samples of the stomach, jejunum, ileum, and colon were excised after euthanasia and barrier integrity was quantified by measuring transepithelial resistance (TER), macromolecular permeability, the abundance of inflammatory proteins (IL-8, IL-1β, and TNF-α) and tight junction proteins (claudin-2 and -3). Papp was characterized using a dual tracer approach comprising 4 KDa fluorescein isothiocyanate (FD4) and 150kDa tetramethyl rhodamine isothiocyanate (T150)-labeled dextrans. Characteristic effects of the initiation of feeding and weaning were observed on the GIT with the initiation of feeding, such as increasing TER and reducing Papp at 24h, consistent with mucosal growth (P = 0.058) This was accompanied by increased cytokine abundance as evidenced by elevations in TNF-α and IL-1β. However, GP had increased IL-8 abundance (P = 0.011 and 0.063 for jejunum and ileum respectively) at birth than 24h overall. In the weaning cohort, jejunal and ileal permeability to FD4 was higher in GP (P = 0.05 and 0.022, respectively) while only higher ileal T150 was observed in GP (P = 0.032). Ileal claudin-2 abundance tended to be higher in SP overall (P = 0.063), but GP ileal claudin-2 expression was upregulated weaning while no change was observed in SP (P = 0.043). Finally, other than a higher jejunal TNF-α abundance observed in SP (P = 0.016), no other effect of parity was observed on inflammatory markers in the weaning cohort. The results from this study indicate that the GIT of GP have poorer adaptation to early life events, with the response to weaning, being more challenging which is likely to contribute to poorer postweaning growth.

Palladium (II) compounds containing oximes as promising antitumor agents for the treatment of osteosarcoma: An in vitro and in vivo comparative study with cisplatin

Drug resistance, evasion of cell death and metastasis are factors that contribute to the low cure rate and disease-free survival in osteosarcomas (OS). In this study, we demonstrated that a new class of oxime-containing organometallic complexes called Pd-BPO (O3) and Pd-BMO (O4) are more cytotoxic than cisplatin (CDDP) for SaOS-2 and U2OS cells using the MTT assay. Annexin-FITC/7-AAD staining demonstrated a greater potential for palladium-oxime complexes to induce death in SaOS-2 cells than CDDP, an event confirmed using the pan-caspase inhibitor Z-VAD-FMK. Compared to CDDP, only palladium-oxime complexes eradicated the clonogenicity of SaOS-2 cells after 7 days of treatment. The involvement of the lysosome-mitochondria axis in the cell death-inducing properties of the complexes was also evaluated. Using LysoTracker Red to label the acidic organelles of SaOS-2 cells treated with the O3 and O4 complexes, a decrease in the fluorescence intensity of this probe was observed in relation to CDDP and the control. Lysosomal membrane permeabilization (LMP) was also induced by the O3 and O4 complexes in an assay using acridine orange (A/O). The greater efficiency of the complexes in depolarizing the mitochondrial membrane compared to SaOS-2 cells treated with CDDP was also observed using TMRE (tetramethyl rhodamine, ethyl ester). For in vivo studies, C. elegans was used and demonstrated that both complexes reduce body bends and pharyngeal pumping after 24 h of treatment to the same extent as CDDP. We conclude that both palladium-oxime complexes are more effective than CDDP in inducing tumor cell death. The toxicity of these complexes to C. elegans was like that induced by CDDP. These results encourage preclinical studies aimed at developing more effective drugs for the treatment of osteosarcoma (OS). Furthermore, we propose palladium-oxime complexes as a new class of antineoplastic agents.

Microscale thermophoresis sensor for Cd2+ using aptamer with tetramethylrhodamine label at an internal site

Rapid sensitive detection of toxic cadmium ion (Cd2+) is demanded in environmental analysis and food safety. Aptamer against Cd2+ becomes attractive recognition element for Cd2+ sensors. The emerging microscale thermophoresis (MST) technique brings new power for optical biosensors construction by employing fluorescently labeled ligand, which measures fluorescence response of samples inside capillaries to localized infrared (IR) laser heating. Herein, we reported a MST aptasensor for Cd2+ by using the aptamer with tetramethylrhodamine (TMR) label at an internal site, and demonstrated that TMR position had large influence on MST responses to Cd2+. The 15-mer aptamer with a TMR conjugated on the internal 12th thymine (T) generated a much larger MST signal change upon Cd2+ binding than the aptamer with TMR labeled at terminals. By utilizing the optimal aptamer probe with TMR labeled at the internal T site, we achieved detection of Cd2+ with a detection limit about 0.5 nM, and the MST aptasensor allowed to detect Cd2+ in real water samples. This work is helpful for developing sensitive aptamer MST sensors for wide applications.

Myrislignan targets extracellular signal-regulated kinase (ERK) and modulates mitochondrial function to dampen osteoclastogenesis and ovariectomy-induced osteoporosis

BackgroundActivated osteoclasts cause excessive bone resorption, and disrupt bone homeostasis, leading to osteoporosis. The extracellular signal-regulated kinase (ERK) signaling is the classical pathway related to osteoclast differentiation, and mitochondrial reactive oxygen species are closely associated with the differentiation of osteoclasts. Myrislignan (MRL), a natural product derived from nutmeg, has multiple pharmacological activities; however, its therapeutic effect on osteoporosis is unclear. Here, we investigated whether MRL could inhibit osteoclastogenesis and bone mass loss in an ovariectomy mouse model by suppressing mitochondrial function and ERK signaling.MethodsTartrate-resistant and phosphatase (TRAP) and bone resorption assays were performed to observe the effect of MRL on osteoclastogenesis of bone marrow macrophages. MitoSOX RED and tetramethyl rhodamine methyl ester (TMRM) staining was performed to evaluate the inhibitory effect of MRL on mitochondria. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was performed to detect whether MRL suppressed the expression of osteoclast-specific genes. The impact of MRL on the protein involved in the mitogen-activated protein kinase (MAPK) and nuclear factor-κB pathways was evaluated using western blotting. In addition, a specific ERK agonist LM22B-10, was used to revalidate the inhibitory effect of MRL on ERK. Finally, we established an ovariectomy mouse model to assess the therapeutic effect of MRL on osteoporosis in vivo.ResultsMRL inhibited osteoclast differentiation and the associated bone resorption, by significantly decreasing osteoclastic gene expression. Mechanistically, MRL inhibited the phosphorylation of ERK by suppressing the mitochondrial function, thereby downregulating the nuclear factor of activated T cells 1 (NFATc1) signaling. LM22B-10 treatment further verified the targeted inhibition effect of MRL on ERK. Microscopic computed tomographic and histologic analyses of the tibial tissue sections indicated that ovariectomized mice had lower bone mass and higher expression of ERK compared with normal controls. However, MRL treatment significantly reversed these effects, indicating the anti-osteoporosis effect of MRL.ConclusionWe report for the first time that MRL inhibits ERK signaling by suppressing mitochondrial function, thereby ameliorating ovariectomy-induced osteoporosis. Our findings can provide a basis for the development of a novel therapeutic strategy for osteoporosis.

Human Hematopoietic Stem Cells Have Altered Mitochondrial Activity after Stem Cell Transplantation

Hematopoietic stem cells (HSCs) give rise to the entire blood system. With their high regenerative potential, they are sometimes the only curative option for many malignancies. The success of HSCT depends on both the quantity of HSCs and on their ability to produce life-long blood. However, their regenerative potential is not unlimited. Transplanted HSCs sustain injury that alters their subsequent ability to generate blood cells. This leads to ineffective hematopoiesis and can ultimately lead to graft failure. Injury to HSCs is also a risk factor for secondary neoplasms, including leukemia. Our goal is to elucidate the mechanisms behind post-transplant HSC decline. Mitochondria are critically important for HSC function (Filippi Exp Hematol 2021). During regeneration of the bone marrow, HSCs become activated and undergo mitochondrial reprogramming, including reorganization of the mitochondrial network and enhanced mitochondrial activity (Filippi et al Blood 2019, Ito et al Nat Rev Mol Cell Biol 2014). However, after transplantation, HSCs accumulate mitochondria that are abnormal in organization and activity. These abnormal mitochondria lead to decline of HSC function and ineffective hematopoiesis (Hinge et al Cell Stem Cell 2020). While these findings have been replicated in murine models, it has not been studied within the human system. Our hypothesis is that after transplantation, human HSCs have altered mitochondria leading to ineffective hematopoiesis. We examined changes in HSC function using post-transplant samples of patient's bone marrow and a xenotransplant system (human CD34+ peripheral blood stem cells transplanted into sublethally irradiated immunodeficient NOD/SCID/IL-2 receptor-γ null mice). We first measured mitochondrial membrane potential using tetramethyl rhodamine ethyl ester (TMRE) dye and analyzed via flow cytometry. Three to six months after transplant in the xenotransplant model, we found that the mitochondrial membrane potential of huCD34+CD38-CD90+ HSCs was similar to the non-transplanted HSCs. However, huCD34+CD38+ progenitors exhibited a drastic 3-fold increase in mitochondrial membrane potential in comparison to the non-transplanted, huCD34+CD38+ progenitor cells. The mitochondrial membrane potential of CD34+CD38+ progenitors from transplanted bone marrow patient samples was also higher than non-transplanted CD34+CD38+bone marrow control cells. To understand if alterations in mitochondrial activity affect HSC differentiation, we placed xenotransplanted HSCs into in vitro culture. Post-transplanted huCD34+ cells failed to expand within 6 days of in vitro culture (serum-free culture with self-renewing cytokines, SCF+TPO+FLT3) compared to huCD34+ control. In subsequent differentiation assays, post-transplanted huCD34+ cultures gave rise to myeloid cells only. This contrasts with control huCD34+ cultures that produced myeloid, erythroid, and megakaryocytic cells. Interestingly, if the post-transplanted HSCs were given serum and cytokines for differentiation after only 3 days in self-renewing conditions, the cultured cells expanded similarly to non-transplanted HSCs and generated myeloid, erythroid, and megakaryocytic cells. This suggests that transplanted HSCs have decreased proliferation and differentiation ability in comparison to non-transplanted HSCs and respond differently to cytokines in vitro. This could account for ineffective hematopoiesis observed in some post-transplant patients. In conclusion, this study indicates that both mitochondrial activity and in-vitro growth change in HSCs after transplantation, despite re-establishing quiescence. This indicates that human hematopoietic stem and progenitor cells incur injury after transplantation. These findings are clinically important as the changes in mitochondrial activity in HSCs may play a role in post-transplant hematopoietic stem cell decline. Future single cell RNA sequencing analyses will provide mechanistic information regarding post-transplant changes in metabolism and potential targets for improving HSC function in transplant patients.

Drug discovery for heart failure targeting myosin-binding protein C

Cardiac MyBP-C (cMyBP-C) interacts with actin and myosin to fine-tune cardiac muscle contractility. Phosphorylation of cMyBP-C, which reduces the binding of cMyBP-C to actin and myosin, is often decreased in patients with heart failure (HF) and is cardioprotective in model systems of HF. Therefore, cMyBP-C is a potential target for HF drugs that mimic its phosphorylation and/or perturb its interactions with actin or myosin. We labeled actin with fluorescein-5-maleimide (FMAL) and the C0-C2 fragment of cMyBP-C (cC0-C2) with tetramethylrhodamine (TMR). We performed two complementary high-throughput screens (HTS) on an FDA-approved drug library, to discover small molecules that specifically bind to cMyBP-C and affect its interactions with actin or myosin, using fluorescence lifetime (FLT) detection. We first excited FMAL and detected its FLT, to measure changes in fluorescence resonance energy transfer (FRET) from FMAL (donor) to TMR (acceptor), indicating binding. Using the same samples, we then excited TMR directly, using a longer wavelength laser, to detect the effects of compounds on the environmentally sensitive FLT of TMR, to identify compounds that bind directly to cC0-C2. Secondary assays, performed on selected modulators with the most promising effects in the primary HTS assays, characterized the specificity of these compounds for phosphorylated versus unphosphorylated cC0-C2 and for cC0-C2 versus C1-C2 of fast skeletal muscle (fC1-C2). A subset of identified compounds modulated ATPase activity in cardiac and/or skeletal myofibrils. These assays establish the feasibility of the discovery of small-molecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF.

A Cascade Signal Amplification Strategy for the Ultrasensitive Fluorescence Detection of Cu2+ via λ-Exonuclease-Assisted Target Recycling with Mismatched Catalytic Hairpin Assembly.

Herein, an ultrasensitive DNAzyme-based fluorescence biosensor for detecting Cu2+ was designed using the cascade signal amplification strategy, coupling λ-exonuclease-assisted target recycling and mismatched catalytic hairpin assembly (MCHA). In the designed detection system, the target, Cu2+, can activate the Cu2+-dependent DNAzyme to cause a cleavage reaction, releasing ssDNA (tDNA). Then, tDNA binds to hairpin DNA (H0) with an overhanging 5'-phosphorylated terminus to form dsDNA with a blunt 5'-phosphorylated terminus, which activates the dsDNA to be digested by λ-Exo and releases tDNA along with another ssDNA (iDNA). Subsequently, the iDNA initiates MCHA, which can restore the fluorescence of carboxyfluorescein (FAM) previously quenched by tetramethylrhodamine (TAMRA), resulting in a strong fluorescent signal. Furthermore, MCHA efficiently improves the signal-to-noise ratio of the detection system. More importantly, tDNA recycling can be achieved with the λ-Exo digestion reaction to release more iDNA, efficiently amplifying the fluorescent signal and further improving the sensitivity to Cu2+ with a detection limit of 60 fM. The practical application of the developed biosensor was also demonstrated by detecting Cu2+ in real samples, proving it to be an excellent analytical strategy for the ultrasensitive quantification of heavy metal ions in environmental water sources.

Lipidomic analysis identifies long-chain acylcarnitine as a target for ischemic stroke

IntroductionLipid metabolism dysfunction is widely involved in the pathological process of acute ischemic stroke (AIS). The coordination of lipid metabolism between neurons and astrocytes is of great significance. However, the full scope of lipid dynamic changes and the function of key lipids during AIS remain unknown. Hence, identifying lipid alterations and characterizing their key roles in AIS is of great importance. MethodsUntargeted and targeted lipidomic analyses were applied to profile lipid changes in the ischemic penumbra and peripheral blood of transient middle cerebral artery occlusion (tMCAO) mice as well as the peripheral blood of AIS patients. Infarct volume and neurological deficits were assessed after tMCAO. The cell viability and dendritic complexity of primary neurons were evaluated by CCK8 assay and Sholl analysis. Seahorse, MitoTracker Green, tetramethyl rhodamine methyl ester (TMRM), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and MitoSOX were used as markers of mitochondrial health. Fluorescent and isotopic free fatty acid (FFA) pulse-chase assays were used to track FFA flux in astrocytes. ResultsLong-chain acylcarnitines (LCACs) were the lipids with the most dramatic changes in the ischemic penumbra and peripheral blood of tMCAO mice. LCACs were significantly elevated on admission in AIS patients and associated with poor outcomes in AIS patients. Increasing LCACs through a bolus administration of palmitoylcarnitine amplified stroke injury, while decreasing LCACs by overexpressing carnitine palmitoyltransferase 2 (CPT2) ameliorated stroke injury. Palmitoylcarnitine aggravated astrocytic mitochondrial damage after OGD/R, while CPT2 overexpression in astrocytes ameliorated cocultured neuron viability. Further study revealed that astrocytes stimulated by OGD/R liberated FFAs from lipid droplets into mitochondria to form LCACs, resulting in mitochondrial damage and lowered astrocytic metabolic support and thereby aggravated neuronal damage. ConclusionLCACs could accumulate and damage neurons by inducing astrocytic mitochondrial dysfunction in AIS. LCACs play a crucial role in the pathology of AIS and are novel promising diagnostic and prognostic biomarkers for AIS.

Anthelmintic nitazoxanide protects against experimental pulmonary fibrosis.

Nitazoxanide is a therapeutic anthelmintic drug. Our previous studies found that nitazoxanide and its metabolite tizoxanide activated adenosine 5'-monophosphate-activated protein kinase (AMPK) and inhibited signal transducer and activator of transcription 3 (STAT3) signals. As AMPK activation and/or STAT3 inhibition are targets for treating pulmonary fibrosis, we hypothesized that nitazoxanide would be effective in experimental pulmonary fibrosis. The mitochondrial oxygen consumption rate of cells was measured by using the high-resolution respirometry system Oxygraph-2K. The mitochondrial membrane potential of cells was evaluated by tetramethyl rhodamine methyl ester (TMRM) staining. The target protein levels were measured by using western blotting. The mice pulmonary fibrosis model was established through intratracheal instillation of bleomycin. The examination of the lung tissues changes were carried out using haematoxylin and eosin (H&E), and Masson staining. Nitazoxanide and tizoxanide activated AMPK and inhibited STAT3 signalling in human lung fibroblast cells (MRC-5 cells). Nitazoxanide and tizoxanide inhibited transforming growth factor-β1 (TGF-β1)-induced proliferation and migration of MRC-5 cells, collagen-I and α-smooth muscle cell actin (α-SMA) expression, and collagen-I secretion from MRC-5 cells. Nitazoxanide and tizoxanide inhibited epithelial-mesenchymal transition (EMT) and inhibited TGF-β1-induced Smad2/3 activation in mouse lung epithelial cells (MLE-12 cells). Oral administration of nitazoxanide reduced the bleomycin-induced mice pulmonary fibrosis and, in the established bleomycin-induced mice, pulmonary fibrosis. Delayed nitazoxanide treatment attenuated the fibrosis progression. Nitazoxanide improves the bleomycin-induced pulmonary fibrosis in mice, suggesting a potential application of nitazoxanide for pulmonary fibrosis treatment in the clinic.

Fluorescent Ligands Enable Target Engagement Studies for the Intracellular Allosteric Binding Site of the Chemokine Receptor CXCR2.

Herein, we report the structure-based development of fluorescent ligands targeting the intracellular allosteric binding site (IABS) of CXC chemokine receptor 2 (CXCR2), a G protein-coupled receptor (GPCR) that has been pursued as a drug target in oncology and inflammation. Starting from the cocrystallized intracellular CXCR2 antagonist 00767013 (1), tetramethylrhodamine (TAMRA)-labeled CXCR2 ligands were designed, synthesized, and tested for their suitability as fluorescent reporters to probe binding to the IABS of CXCR2. By means of these studies, we developed Mz438 (9a) as a high-affinity and selective fluorescent CXCR2 ligand, enabling cell-free as well as cellular NanoBRET-based binding studies in a nonisotopic and high-throughput manner. Further, we show that 9a can be used as a tool to visualize intracellular target engagement for CXCR2 via fluorescence microscopy. Thus, our small-molecule-based fluorescent CXCR2 ligand 9a represents a promising tool for future studies of CXCR2 pharmacology.