Amyloid-like Self-Assembly of a Cellular Compartment.

Most vertebrate oocytes contain a Balbiani body, a large, non-membrane-bound compartment packed with RNA, mitochondria, and other organelles. Little is known about this compartment, though it specifies germline identity in many non-mammalian vertebrates. We show Xvelo, a disordered protein with an N-terminal prion-like domain, is an abundant constituent of Xenopus Balbiani bodies. Disruption of the prion-like domain of Xvelo, or substitution with a prion-like domain from an unrelated protein, interferes with its incorporation into Balbiani bodies in vivo. Recombinant Xvelo forms amyloid-like networks in vitro. Amyloid-like assemblies of Xvelo recruit both RNA and mitochondria in binding assays. We propose that Xenopus Balbiani bodies form by amyloid-like assembly of Xvelo, accompanied by co-recruitment of mitochondria and RNA. Prion-like domains are found in germ plasm organizing proteins in other species, suggesting that Balbiani body formation by amyloid-like assembly could be a conserved mechanism that helps oocytes function as long-lived germ cells.

Symmetry breaking of fluorophore binding to a G-quadruplex generates an RNA aptamer with picomolar KD.

Fluorogenic RNA aptamer tags with high affinity enable RNA purification and imaging. The G-quadruplex (G4) based Mango (M) series of aptamers were selected to bind a thiazole orange based (TO1-Biotin) ligand. Using a chemical biology and reselection approach, we have produced a MII.2 aptamer-ligand complex with a remarkable set of properties: Its unprecedented KD of 45 pM, formaldehyde resistance (8% v/v), temperature stability and ligand photo-recycling properties are all unusual to find simultaneously within a small RNA tag. Crystal structures demonstrate how MII.2, which differs from MII by a single A23U mutation, and modification of the TO1-Biotin ligand to TO1-6A-Biotin achieves these results. MII binds TO1-Biotin heterogeneously via a G4 surface that is surrounded by a stadium of five adenosines. Breaking this pseudo-rotational symmetry results in a highly cooperative and homogeneous ligand binding pocket: A22 of the G4 stadium stacks on the G4 binding surface while the TO1-6A-Biotin ligand completely fills the remaining three quadrants of the G4 ligand binding face. Similar optimization attempts with MIII.1, which already binds TO1-Biotin in a homogeneous manner, did not produce such marked improvements. We use the novel features of the MII.2 complex to demonstrate a powerful optically-based RNA purification system.

Artificial RNA tags that tightly bind fluorogenic ligands have many RNA imaging and RNA-protein biomolecular purification applications. Here, we report and structurally characterize a very small (20-nt) biologically compatible G-quadruplex based aptamer that can be inserted into commonly found GNRA tetraloops. This aptamer binds its fluorogenic ligand with an unprecedented picomolar binding affinity and is very stable against thermal and chemical insults. As the ligand can be modified to include biotin, this RNA tag can also be bound to streptavidin magnetic beads. After washing, tagged RNA can be cleanly eluted by exposing the beads to intense green light, which photobleaches the bound fluorogenic ligand, triggering the release of the bound RNA complex.

Efficacy of host cell serine protease inhibitor MM3122 against SARS-CoV-2 for treatment and prevention of COVID-19.

We developed a novel class of peptidomimetic inhibitors targeting several host cell human serine proteases, including transmembrane protease serine 2 (TMPRSS2), matriptase, and hepsin. TMPRSS2 is a membrane-associated protease that is highly expressed in the upper and lower respiratory tracts and is utilized by SARS-CoV-2 and other viruses to proteolytically process their glycoproteins, enabling host cell entry, replication, and dissemination of new virus particles. We have previously shown that compound MM3122 exhibited subnanomolar potency against all three proteases and displayed potent antiviral effects against SARS-CoV-2 in a cell viability assay. Herein, we demonstrate that MM3122 potently inhibits viral replication in human lung epithelial cells and is also effective against the EG.5.1 variant of SARS-CoV-2. Furthermore, we evaluated MM3122 in a mouse model of COVID-19 and demonstrated that MM3122 administered intraperitoneally (IP) before (prophylactic) or after (therapeutic) SARS-CoV-2 infection had significant protective effects against weight loss and lung congestion and reduced pathology. Amelioration of COVID-19 disease was associated with a reduction in proinflammatory cytokine and chemokine production after SARS-CoV-2 infection. Prophylactic, but not therapeutic, administration of MM3122 also reduced virus titers in the lungs of SARS-CoV-2-infected mice. Therefore, MM3122 is a promising lead candidate small-molecule drug for the treatment and prevention of infections caused by SARS-CoV-2 and other coronaviruses. IMPORTANCE: SARS-CoV-2 and other emerging RNA coronaviruses are a present and future threat in causing widespread endemic and pandemic infection and disease. In this paper, we have shown that the novel host cell protease inhibitor, MM3122, blocks SARS-CoV-2 viral replication and is efficacious as both a prophylactic and a therapeutic drug for the treatment of COVID-19 given intraperitoneally in mice. Targeting host proteins and pathways in antiviral therapy is an underexplored area of research, but this approach promises to avoid drug resistance by the virus, which is common in current antiviral treatments.

Temperature-dependent intracellular crystallization of firefly luciferase in mammalian cells is suppressed by D-luciferin and stabilizing inhibitors.

Firefly luciferase (Fluc) from Photinus pyralis is one of the most widely used reporter proteins in biomedical research. Despite its widespread use, Fluc's protein phase transition behaviors and phase separation characteristics have not received much attention. Current research uncovers Fluc's intrinsic property to phase separate in mammalian cells upon a simple cell culture temperature change. Specifically, Fluc spontaneously produced needle-shaped crystal-like inclusion bodies upon temperature shift to the hypothermic temperatures ranging from 25 °C to 31 °C. The crystal-like inclusion bodies were not associated with or surrounded by membranous organelles and were likely built from the cytosolic pool of Fluc. Furthermore, the crystal-like inclusion formation was suppressed when cells were cultured in the presence of D-luciferin and its synthetic analog, as well as the benzothiazole family of so-called stabilizing inhibitors. These two classes of compounds inhibited intracellular Fluc crystallization by different modes of action as they had contrasting effects on steady-state luciferase protein accumulation levels. This study suggests that, under substrate insufficient conditions, the excess Fluc phase separates into a crystal-like state that can modulate intracellular soluble enzyme availability and protein turnover rate.

Cavity hairpin ThT-light nucleic acid switches: the construction of label- and enzyme-free sensing and imaging platforms.

Thioflavin T (ThT) is a classical fluorescent dye gaining prominence in current research regarding nucleic acid conformations (NACs). However, most NACs with the ability to excite ThT fluorescent are unique or form in demanding conditions, limiting the extensiveness and depth of ThT application in sensing and imaging. Therefore, this study proposed CGG-AAA mismatched cavity hairpin ThT-light nucleic acid switches (CHTLNAS) with excellent fluorescence excitation over 500-fold higher than spontaneous, 17∼20-fold higher than ssDNA and 2.5∼5-fold higher than complementary duplex. Based on the excellent fluorescence excitation, convenient conformation formation, good sequence programmability, and flexible allosteric ability (known as the Worm-crack pod mechanism mediated by the target), it achieved the label- and enzyme-free detection of tetracycline (TET) and berberine (BB) at the pM level within 10 min. Moreover, it was found enable to realize the sensitive tracking of intracellular carriers at the nM level of ThT entry concentration, and prolongated its cell nuclear-entry time of ThT over 8 h, overcoming the non-specific high background signal interference of ThT in the nuclear region, and expanding the diversified application of ThT in cell biology research. Therefore, CHTLNAS is a more universal, practical tool than G-quadruplex or other kinds of NACs for ThT development and utilization in sensing and imaging platforms.

Structural control of fibrin bioactivity by mechanical deformation.

Fibrin is the fibrous protein network that comprises blood clots; it is uniquely capable of bearing very large tensile strains (up to 200%) due to multiscale force accommodation mechanisms. Fibrin is also a biochemical scaffold for numerous enzymes and blood factors. The biomechanics and biochemistry of fibrin have been independently studied. However, comparatively little is known about how fibrin biomechanics and biochemistry are coupled: how does fibrin deformation influence its biochemistry? In this study, we show that mechanically induced protein structural changes in fibrin affect fibrin biochemistry. We find that tensile deformation of fibrin leads to molecular structural transitions of α-helices to ß-sheets, which reduced binding of tissue plasminogen activator (tPA), an enzyme that initiates fibrin lysis. Moreover, binding of tPA and Thioflavin T, a commonly used ß-sheet marker, were mutually exclusive, further demonstrating the mechano-chemical control of fibrin biochemistry. Finally, we demonstrate that structural changes in fibrin suppressed the biological activity of platelets on mechanically strained fibrin due to reduced αIIbß3 integrin binding. Our work shows that mechanical strain regulates fibrin molecular structure and biological activity in an elegant mechano-chemical feedback loop, which possibly extends to other fibrous biopolymers.

CSF-1 maintains pathogenic but not homeostatic myeloid cells in the central nervous system during autoimmune neuroinflammation.

The receptor for colony stimulating factor 1 (CSF-1R) is important for the survival and function of myeloid cells that mediate pathology during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). CSF-1 and IL-34, the ligands of CSF-1R, have similar bioactivities but distinct tissue and context-dependent expression patterns, suggesting that they have different roles. This could be the case in EAE, given that CSF-1 expression is up-regulated in the CNS, while IL-34 remains constitutively expressed. We found that targeting CSF-1 with neutralizing antibody halted ongoing EAE, with efficacy superior to CSF-1R inhibitor BLZ945, whereas IL-34 neutralization had no effect, suggesting that pathogenic myeloid cells were maintained by CSF-1. Both anti­CSF-1 and BLZ945 treatment greatly reduced the number of monocyte-derived cells and microglia in the CNS. However, anti­CSF-1 selectively depleted inflammatory microglia and monocytes in inflamed CNS areas, whereas BLZ945 depleted virtually all myeloid cells, including quiescent microglia, throughout the CNS. Anti­CSF-1 treatment reduced the size of demyelinated lesions and microglial activation in the gray matter. Lastly, we found that bone marrow­derived immune cells were the major mediators of CSF-1R­dependent pathology, while microglia played a lesser role. Our findings suggest that targeting CSF-1 could be effective in ameliorating MS pathology, while preserving the homeostatic functions of myeloid cells, thereby minimizing risks associated with ablation of CSF-1R­dependent cells.

The Flt3-inhibitor quizartinib augments apoptosis and promotes maladaptive remodeling after myocardial infarction in mice.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) targeting fms-like tyrosine kinase 3 (Flt3) such as quizartinib were specifically designed for acute myeloid leukemia treatment, but also multi-targeting TKIs applied to solid tumor patients inhibit Flt3. Flt3 is expressed in the heart and its activation is cytoprotective in myocardial infarction (MI) in mice. OBJECTIVES: We sought to test whether Flt3-targeting TKI treatment aggravates cardiac injury after MI. METHODS AND RESULTS: Compared to vehicle, quizartinib (10 mg/kg/day, gavage) did not alter cardiac dimensions or function in healthy mice after four weeks of therapy. Pretreated mice were randomly assigned to MI or sham surgery while receiving quizartinib or vehicle for one more week. Quizartinib did not aggravate the decline in ejection fraction, but significantly enhanced ventricular dilatation one week after infarction. In addition, apoptotic cell death was significantly increased in the myocardium of quizartinib-treated compared to vehicle-treated mice. In vitro, quizartinib dose-dependently decreased cell viability in neonatal rat ventricular myocytes and in H9c2 cells, and increased apoptosis as assessed in the latter. Together with H2O2, quizartinib potentiated the phosphorylation of the pro-apoptotic mitogen activated protein kinase p38 and augmented H2O2-induced cell death and apoptosis beyond additive degree. Pretreatment with a p38 inhibitor abolished apoptosis under quizartinib and H2O2. CONCLUSION: Quizartinib potentiates apoptosis and promotes maladaptive remodeling after MI in mice at least in part via a p38-dependent mechanism. These findings are consistent with the multi-hit hypothesis of cardiotoxicity and make cardiac monitoring in patients with ischemic heart disease under Flt3- or multi-targeting TKIs advisable.

Reprogrammed mRNA translation drives resistance to therapeutic targeting of ribosome biogenesis.

Elevated ribosome biogenesis in oncogene-driven cancers is commonly targeted by DNA-damaging cytotoxic drugs. Our previous first-in-human trial of CX-5461, a novel, less genotoxic agent that specifically inhibits ribosome biogenesis via suppression of RNA polymerase I (Pol I) transcription, revealed single-agent efficacy in refractory blood cancers. Despite this clinical response, patients were not cured. In parallel, we demonstrated a marked improvement in the in vivo efficacy of CX-5461 in combination with PI3K/AKT/mTORC1 pathway inhibitors. Here, we reveal the molecular basis for this improved efficacy observed in vivo, which is associated with specific suppression of translation of mRNAs encoding regulators of cellular metabolism. Importantly, acquired resistance to this cotreatment is driven by translational rewiring that results in dysregulated cellular metabolism and induction of a cAMP-dependent pathway critical for the survival of blood cancers including lymphoma and acute myeloid leukemia. Our studies thus identify key molecular mechanisms underpinning the response of blood cancers to selective inhibition of ribosome biogenesis and define metabolic vulnerabilities that will facilitate the rational design of more effective regimens for Pol I-directed therapies.

Iodine-125 seed inhibits proliferation and promotes apoptosis of cholangiocarcinoma cells by inducing the ROS/p53 axis.

With advances in radioactive particle implantation in clinical practice, Iodine-125 (125I) seed brachytherapy has emerged as a promising treatment for cholangiocarcinoma (CCA), showing good prognosis; however, the underlying molecular mechanism of the therapeutic effect of 125I seed is unclear. To study the effects of 125I seed on the proliferation and apoptosis of CCA cells. CCA cell lines, RBE and HCCC-9810, were treated with reactive oxygen species (ROS) scavenger acetylcysteine (NAC) or the p53 functional inhibitor, pifithrin-α hydrobromide (PFTα). Cell counting kit-8 (CCK-8) assay, 5-bromo-2-deoxy-uridine (BrdU) staining, and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry assay were performed to test the radiation-sensitivity of 125I seed toward CCA cells at different radiation doses (0.4 mCi and 0.8 mCi). 2,7-dichlorofluorescein diacetate (DCF-DA) assay, real-time quantitative polymerase chain reaction (RT-qPCR), and western blot analysis were performed to assess the effect of 125I seed on the ROS/p53 axis. A dose-dependent inhibitory effect of 125I seeds on the proliferation of CCA cells was observed. The 125I seed promoted apoptosis of CCA cells and induced the activation of the ROS/p53 pathway in a dose-dependent manner. NAC or PFTα treatment effectively reversed the stimulatory effect of 125I seed on the proliferation of CCA cells. NAC or PFTα suppressed apoptosis and p53 protein expression induced by the 125I seed. 125I seed can inhibit cell growth mainly through the apoptotic pathway. The mechanism may involve the activation of p53 and its downstream apoptotic pathway by up-regulating the level of ROS in cells.

Quizartinib plus chemotherapy in newly diagnosed patients with FLT3-internal-tandem-duplication-positive acute myeloid leukaemia (QuANTUM-First): a randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: Patients with acute myeloid leukaemia (AML) positive for internal tandem duplication (ITD) mutations of FLT3 have poor outcomes. Quizartinib, an oral, highly potent, selective, type 2 FLT3 inhibitor, plus chemotherapy showed antitumour activity with an acceptable safety profile in patients with FLT3-ITD-positive newly diagnosed AML. The aim of the study was to compare the effect of quizartinib versus placebo on overall survival in patients with FLT3-ITD-positive newly diagnosed AML aged 18-75 years. METHODS: We conducted a randomised, double-blind, placebo-controlled, phase 3 trial comparing quizartinib and placebo in combination with chemotherapy in induction and consolidation, followed by quizartinib or placebo single-agent continuation, in patients with FLT3-ITD-positive newly diagnosed AML at 193 hospitals and clinics in 26 countries in Europe; North America; and Asia, Australia, and South America. Patients aged 18-75 years were eligible. Patients were randomly assigned (1:1) to the quizartinib group or the placebo group by an independent biostatistician through an interactive web and voice response system, stratified by region, age, and white blood cell count at diagnosis. Patients, investigators, funders, and contract research organisations were masked to treatments assigned. Induction therapy comprised a standard 7 + 3 induction regimen of cytarabine 100 mg/m2 per day (or 200 mg/m2 per day allowed if institutional or local standard) by continuous intravenous infusion from day 1 to day 7 and anthracycline (daunorubicin 60 mg/m2 per day or idarubicin 12 mg/m2 per day) by intravenous infusion on days 1, 2, and 3, then quizartinib 40 mg orally or placebo once per day, starting on day 8, for 14 days. Patients with complete remission or complete remission with incomplete neutrophil or platelet recovery received standard consolidation with high-dose cytarabine plus quizartinib (40 mg per day orally) or placebo, allogeneic haematopoietic cell transplantation (allo-HCT), or both as consolidation therapy, followed by continuation of single-agent quizartinib or placebo for up to 3 years. The primary outcome was overall survival, defined as time from randomisation until death from any cause and assessed in the intention-to-treat population. Safety was evaluated in all patients who received at least one dose of quizartinib or placebo. This study is registered with ClinicalTrials.gov (NCT02668653). FINDINGS: Between Sept 27, 2016, and Aug 14, 2019, 3468 patients with AML were screened and 539 patients (294 [55%] male patients and 245 [45%] female patients) with FLT3-ITD-positive AML were included and randomly assigned to the quizartinib group (n=268) or placebo group (n=271). 148 (55%) of 268 patients in the quizartinib group and 168 (62%) of 271 patients in the placebo group discontinued the study, primarily because of death (133 [90%] of 148 in the quizartinib group vs 158 [94%] of 168 in the placebo group) or withdrawal of consent (13 [9%] of 148 in the quizartinib group vs 9 [5%] of 168 in the placebo group). Median age was 56 years (range 20-75, IQR 46·0-65·0). At a median follow-up of 39·2 months (IQR 31·9-45·8), median overall survival was 31·9 months (95% CI 21·0-not estimable) for quizartinib versus 15·1 months (13·2-26·2) for placebo (hazard ratio 0·78, 95% CI 0·62-0·98, p=0·032). Similar proportions of patients in the quizartinib and placebo groups had at least one adverse event (264 [100%] of 265 in the quizartinib group and 265 [99%] of 268 in the placebo group) and one grade 3 or higher adverse event (244 [92%] of 265 in the quizartinib group and 240 [90%] of 268 in the placebo group). The most common grade 3 or 4 adverse events were febrile neutropenia, hypokalaemia, and pneumonia in both groups and neutropenia in the quizartinib group. INTERPRETATION: The addition of quizartinib to standard chemotherapy with or without allo-HCT, followed by continuation monotherapy for up to 3 years, resulted in improved overall survival in adults aged 18-75 years with FLT3-ITD-positive newly diagnosed AML. Based on the results from the QuANTUM-First trial, quizartinib provides a new, effective, and generally well tolerated treatment option for adult patients with FLT3-ITD-positive newly diagnosed AML. FUNDING: Daiichi Sankyo.

A Randomized, Controlled Trial of the Pan-PPAR Agonist Lanifibranor in NASH.

BACKGROUND: Management of nonalcoholic steatohepatitis (NASH) is an unmet clinical need. Lanifibranor is a pan-PPAR (peroxisome proliferator-activated receptor) agonist that modulates key metabolic, inflammatory, and fibrogenic pathways in the pathogenesis of NASH. METHODS: In this phase 2b, double-blind, randomized, placebo-controlled trial, patients with noncirrhotic, highly active NASH were randomly assigned in a 1:1:1 ratio to receive 1200 mg or 800 mg of lanifibranor or placebo once daily for 24 weeks. The primary end point was a decrease of at least 2 points in the SAF-A score (the activity part of the Steatosis, Activity, Fibrosis [SAF] scoring system that incorporates scores for ballooning and inflammation) without worsening of fibrosis; SAF-A scores range from 0 to 4, with higher scores indicating more-severe disease activity. Secondary end points included resolution of NASH and regression of fibrosis. RESULTS: A total of 247 patients underwent randomization, of whom 103 (42%) had type 2 diabetes mellitus and 188 (76%) had significant (moderate) or advanced fibrosis. The percentage of patients who had a decrease of at least 2 points in the SAF-A score without worsening of fibrosis was significantly higher among those who received the 1200-mg dose, but not among those who received the 800-mg dose, of lanifibranor than among those who received placebo (1200-mg dose vs. placebo, 55% vs. 33%, P = 0.007; 800-mg dose vs. placebo, 48% vs. 33%, P = 0.07). The results favored both the 1200-mg and 800-mg doses of lanifibranor over placebo for resolution of NASH without worsening of fibrosis (49% and 39%, respectively, vs. 22%), improvement in fibrosis stage of at least 1 without worsening of NASH (48% and 34%, respectively, vs. 29%), and resolution of NASH plus improvement in fibrosis stage of at least 1 (35% and 25%, respectively, vs. 9%). Liver enzyme levels decreased and the levels of the majority of lipid, inflammatory, and fibrosis biomarkers improved in the lanifibranor groups. The dropout rate for adverse events was less than 5% and was similar across the trial groups. Diarrhea, nausea, peripheral edema, anemia, and weight gain occurred more frequently with lanifibranor than with placebo. CONCLUSIONS: In this phase 2b trial involving patients with active NASH, the percentage of patients who had a decrease of at least 2 points in the SAF-A score without worsening of fibrosis was significantly higher with the 1200-mg dose of lanifibranor than with placebo. These findings support further assessment of lanifibranor in phase 3 trials. (Funded by Inventiva Pharma; NATIVE ClinicalTrials.gov number, NCT03008070.).

Prediction of the Feasibility of Using the ≪Gold Standard≫ Thioflavin T to Detect Amyloid Fibril in Acidic Media.

Ordered protein aggregates, amyloid fibrils, form toxic plaques in the human body in amyloidosis and neurodegenerative diseases and provide adaptive benefits to pathogens and to reduce the nutritional value of legumes. To identify the amyloidogenic properties of proteins and study the processes of amyloid fibril formation and degradation, the cationic dye thioflavin T (ThT) is the most commonly used. However, its use in acidic environments that induce amyloid formation in vitro can sometimes lead to misinterpretation of experimental results due to electrostatic repulsion. In this work, we show that calculating the net charge per residue of amyloidogenic proteins or peptides is a simple and effective approach for predicting whether their fibrils will interact with ThT at acidic pH. In particular, it was shown that at pH 2, proteins and peptides with a net charge per residue > +0.18 are virtually unstained by this fluorescent probe. The applicability of the proposed approach was demonstrated by predicting and experimentally confirming the absence of ThT interaction with amyloids formed from green fluorescent (sfGFP) and odorant-binding (bOBP) proteins, whose fibrillogenesis was first carried out in an acidic environment. Correct experimental evidence that the inability to detect these fibrils under acidic conditions is precisely because of the lack of dye binding to amyloids (and not their specific structure or the low fluorescence quantum yield of the bound dye) and that the number of ThT molecules associated with fibrils increases with decreasing acidity of the medium was obtained by using the equilibrium microdialysis approach.

New G-Triplex DNA Dramatically Activates the Fluorescence of Thioflavin T and Acts as a Turn-On Fluorescent Sensor for Uracil-DNA Glycosylase Activity Detection.

G-triplexes are G-rich oligonucleotides composed of three G-tracts and have absorbed much attention due to their potential biological functions and attractive performance in biosensing. Through the optimization of loop compositions, DNA lengths, and 5'-flanking bases of G-rich sequences, a new stable G-triplex sequence with 14 bases (G3-F15) was discovered to dramatically activate the fluorescence of Thioflavin T (ThT), a water-soluble fluorogenic dye. The fluorescence enhancement of ThT after binding with G3-F15 reached 3200 times, which was the strongest one by far among all of the G-rich sequences. The conformations of G3-F15 and G3-F15/ThT were studied by circular dichroism. The thermal stability measurements indicated that G3-F15 was a highly stable G-triplex structure. The conformations of G3-F15 and G3-F15/ThT in the presence of different metal cations were studied thoroughly by fluorescent spectroscopy, circular dichroism, and nuclear magnetic resonance. Furthermore, using the G3-F15/ThT complex as a fluorescent probe, a robust and simple turn-on fluorescent sensor for uracil-DNA glycosylase activity was developed. This study proposes a new systematic strategy to explore new functional G-rich sequences and their ligands, which will promote their applications in diagnosis, therapy, and biosensing.

Ultrasensitive Fluorescent Microsensors Based on Aptamers Modified with SYBR Green I for Visual Quantitative Detection of Organophosphate Pesticides.

Organophosphate pesticides (OPs) are widely utilized in agricultural production, and the residues threaten public health and environmental safety due to their toxicity. Herein, a novel and simple DNA aptamer-based sensor has been fabricated for the rapid, visual, and quantitative detection of profenofos and isocarbophos. The proposed DNA aptamers with a G-quadruplex spatial structure could be recognized by SYBR Green I (SG-I), resulting in strong green fluorescence emitted by SG-I. The DNA aptamers exhibit a higher specific binding ability to target OP molecules through aromatic ring stacking, disrupting the interaction between SG-I and DNA aptamers to induce green fluorescence quenching. Meanwhile, the fluorescence wavelength of G-quadruplex fluorescence emission peaks changes, accompanied by an obvious fluorescence variation from green to blue. SG-I-modified aptasensor without any additive reference fluorescence units for use in multicolor fluorescence assay for selective monitoring of OPs was first developed. The developed aptasensor provides a favorable linear range from 0 to 200 nM, with a low detection limit of 2.48 and 3.01 nM for profenofos and isocarbophos, respectively. Moreover, it offers high selectivity and stability in real sample detection with high recoveries. Then, a self-designed portable smartphone sensing platform was successfully used for quantitative result outputs, demonstrating experience in designing a neotype sensing strategy for point-of-care pesticide monitoring.

Coordination Synergistic-Induced J-Aggregation Enhanced Fluorescent Performance of HBT-Excimers and Imaging Applications.

Developing a novel strategy to improve the optical performances of fluorescent probes is a vital factor in elevating its practical application; viz., novel biocompatible fluorescent probes with excellent multifunctions exhibited unparalleled advantages in probing functions of intracellular molecules to elucidate intracellular events in living systems. Herein, we have successfully constructed a new strategy that aggregation and coordination synergistically induce (2-hydroxylphenyl-benzothiazole) HBT derivatives to form excimers with large red-shifted fluorescence and application for insight into stress-response zinc fluctuations in living systems. We have synthesized four HBT-based derivatives and deeply investigated the response mechanism by fluorescent spectral studies, demonstrating that probes 3 and 4 showcased large red shifts in emission wavelength due to J-aggregation. More interestingly, the fluorescence of probe 4 was significantly enhanced in the presence of a zinc ion, suggesting that zinc coordination synergistically induced J-aggregation. Probe 4 was successfully applied to image zinc fluctuations in different models of living systems, proving that this probe is a powerful tool to unveil the relationship between invasive stress and diseases by monitoring endogenous zinc fluctuations.

Field Detection of Uranyl in Coastal Water of China Using a Portable Device via DNA Photocleavage.

The urgent need for field detection of uranium in seawater is 2-fold: to provide prompt guidance for uranium extraction and to prevent human exposure to nuclear radiation. However, current methods for this purpose are largely hindered by bulky instrumentation, high costs of developed materials, and severe matrix interferences, which limit their further application in the field. Herein, we demonstrated a portable and label-free strategy for the field detection of uranyl in seawater based on the efficient photocleavage of DNA. Further experiments confirmed the generation of ultraviolet (UV) light-induced reactive oxygen species (ROS), such as O2•- and •OH, which fragmented oligomeric DNA in the presence of uranyl and UV light. Detailed studies showed that DNA significantly enhances uranyl absorption in the UV-visible region, leading to the generation of more ROS. A fluorescence system for the selective detection of uranyl in seawater was established by immobilizing two complementary oligonucleotides with the fluorescent dye SYBR Green I. The strategy of UV-induced photocleavage offers high selectivity, excellent interference immunity, and high sensitivity for uranyl, with a detection limit of 6.8 nM. Additionally, the fluorescence can be visually detected using a 3D-printed miniaturized device integrated with a smartphone. This method has been successfully applied to the on-site detection of uranyl in seawater in 18 Chinese coastal cities and along the coast of Hainan Island within 3 min for a single sample. The sample testing and field analysis results indicate that this strategy has promising potential for real-time monitoring of trace uranyl in China's coastal waters. It is expected to be utilized for the rapid assessment of nuclear contamination and nuclear engineering construction.

Distinct binding modes of a benzothiazole derivative confer structural bases for increasing ERK2 or p38α MAPK selectivity.

Mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 2 (ERK2) and p38α MAP kinase (p38α MAPK), regulate various cellular responses. ERK2 is a drug target for treating many diseases, such as cancer, whereas p38α has attracted much attention as a promising drug target for treating inflammatory disorders. ERK2 is a critical off-target for p38α MAPK and vice versa. In this study, an allosteric ERK2 inhibitor with a benzothiazole moiety (compound 1) displayed comparable inhibitory activity against p38α MAPK. Crystal structures of these MAPKs showed that compound 1 bound to the allosteric site of ERK2 and p38α MAPK in distinct manners. Compound 1 formed a covalent bond with Cys162 of p38α MAPK, whereas this covalent bond was absent in the ERK2 complex even though the corresponding cysteine is conserved in ERK2. Structural dissection combined with computational simulations indicated that an amino acid difference in the allosteric site is responsible for the distinct binding modes of compound 1 with ERK2 and p38α MAPK. These structural insights underline the feasibility of developing highly selective and potent ERK2 and p38α MAPK inhibitors.

Effects of solid lipid nanocarrier containing methyl urolithin A by coating folate-bound chitosan and evaluation of its anti-cancer activity.

BACKGROUND: Nanotechnology-based drug delivery systems have received much attention over the past decade. In the present study, we synthesized Methyl Urolithin A-loaded solid lipid nanoparticles decorated with the folic acid-linked chitosan layer called MuSCF-NPs and investigated their effects on cancer cells. METHODS: MuSCF-NPs were prepared using a high-pressure homogenization method and characterized using FTIR, FESEM, DLS, and zeta potential methods. Drug encapsulation was assessed by spectrophotometry and its cytotoxic effect on various cancer cells (MDA-MB231, MCF-7, PANC, AGS, and HepG2) by the MTT method. Antioxidant activity was assessed by the ABTS and DPPH methods, followed by expression of genes involved in oxidative stress and apoptosis by qPCR and flow cytometry. RESULTS: The results showed the formation of monodisperse and stable round nanoparticles with a size of 84.8 nm. The drug loading efficiency in MuSCF-NPs was reported to be 88.6%. MuSCF-NPs exhibited selective cytotoxicity against MDA-MB231 cells (IC50 = 40 µg/mL). Molecular analysis showed a significant increase in the expression of Caspases 3, 8, and 9, indicating that apoptosis was occurring in the treated cells. Moreover, flow cytometry results showed that the treated cells were arrested in his SubG1 phase, confirming the pro-apoptotic effect of the nanoparticles. The results indicate a high antioxidant effect of the nanoparticles with IC50 values ​​of 45 µg/mL and 1500 µg/mL against ABTS and DPPH, respectively. The reduction of catalase gene expression confirmed the pro-oxidant effect of nanoparticles in cancer cells treated at concentrations of 20 and 40 µg/mL. CONCLUSIONS: Therefore, our findings suggest that the MuSCF-NPs are suitable candidates, especially for breast cancer preclinical studies.

High-Throughput Screening of pH-Dependent ß-sheet Self-Assembling Peptide.

pH-dependent peptide biomaterials hold tremendous potential for cell delivery and tissue engineering. However, identification of responsive self-assembling sequences with specified secondary structure remains a challenge. In this work, An experimental procedure based on the one-bead one-compound (OBOC) combinatorial library is developed to rapidly screen self-assembling ß-sheet peptides at neutral aqueous solution (pH 7.5) and disassemble at weak acidic condition (pH 6.5). Using the hydrophobic fluorescent molecule thioflavin T (ThT) as a probe, resin beads displaying self-assembling peptides show fluorescence under pH 7.5 due to the insertion of ThT into the hydrophobic domain, and are further cultured in pH 6.5 solution. The beads with extinguished fluorescence are selected. Three heptapeptides are identified that can self-assemble into nanofibers or nanoparticles at pH 7.5 and disassemble at pH 6.5. P1 (LVEFRHY) shows a rapid acid response and morphology transformation with pH modulation. Changes in the charges of histidine and hydrophobic phenyl motif of phenylalanine may play important roles in the formation of pH-responsive ß-sheet nanofiber. This high-throughput screening method provides an efficient way to identify pH-dependent ß-sheet self-assembling peptide and gain insights into structural design of such nanomaterials.