Heat shock potentiates aminoglycosides against gram-negative bacteria by enhancing antibiotic uptake, protein aggregation, and ROS.

The potentiation of antibiotics is a promising strategy for combatting antibiotic-resistant/tolerant bacteria. Herein, we report that a 5-min sublethal heat shock enhances the bactericidal actions of aminoglycoside antibiotics by six orders of magnitude against both exponential- and stationary-phase Escherichia coli. This combined treatment also effectively kills various E. coli persisters, E. coli clinical isolates, and numerous gram-negative but not gram-positive bacteria and enables aminoglycosides at 5% of minimum inhibitory concentrations to eradicate multidrug-resistant pathogens Acinetobacter baumannii and Klebsiella pneumoniae. Mechanistically, the potentiation is achieved comprehensively by heat shock-enhanced proton motive force that thus promotes the bacterial uptake of aminoglycosides, as well as by increasing irreversible protein aggregation and reactive oxygen species that further augment the downstream lethality of aminoglycosides. Consistently, protonophores, chemical chaperones, antioxidants, and anaerobic culturing abolish heat shock-enhanced aminoglycoside lethality. We also demonstrate as a proof of concept that infrared irradiation- or photothermal nanosphere-induced thermal treatments potentiate aminoglycoside killing of Pseudomonas aeruginosa in a mouse acute skin wound model. Our study advances the understanding of the mechanism of actions of aminoglycosides and demonstrates a high potential for thermal ablation in curing bacterial infections when combined with aminoglycosides.

Selective targeting of TET catalytic domain promotes somatic cell reprogramming.

Ten-eleven translocation (TET) family enzymes (TET1, TET2, and TET3) oxidize 5-methylcytosine (5mC) and generate 5-hydroxymethylcytosine (5hmC) marks on the genome. Each TET protein also interacts with specific binding partners and partly plays their role independent of catalytic activity. Although the basic role of TET enzymes is well established now, the molecular mechanism and specific contribution of their catalytic and noncatalytic domains remain elusive. Here, by combining in silico and biochemical screening strategy, we have identified a small molecule compound, C35, as a first-in-class TET inhibitor that specifically blocks their catalytic activities. Using this inhibitor, we explored the enzymatic function of TET proteins during somatic cell reprogramming. Interestingly, we found that C35-mediated TET inactivation increased the efficiency of somatic cell programming without affecting TET complexes. Using high-throughput mRNA sequencing, we found that by targeting 5hmC repressive marks in the promoter regions, C35-mediated TET inhibition activates the transcription of the BMP-SMAD-ID signaling pathway, which may be responsible for promoting somatic cell reprogramming. These results suggest that C35 is an important tool for inducing somatic cell reprogramming, as well as for dissecting the other biological functions of TET enzymatic activities without affecting their other nonenzymatic roles.

Elevated IL-23 in skin promotes IL-23 derived Th17 responses in leprosy patients.

Leprosy is an infectious disease caused by non-cultivable bacteria Mycobacterium leprae. Th17 cells play vital roles during pathogenesis of leprosy reactions and IL-23 is involved in Th17 cell differentiation. Although previous studies have reported the participation of IL-23 in leprosy patients in peripheral blood, the role of this cytokine in skin has not yet been described for the disease. In this study, we first evaluated IL-23 expression in the skin of patients with leprosy. Data showed that in keratinocytes, endothelial cells, and macrophages, IL-23 expression was markedly higher in patients compared to that in the normal skin controls. Also, leprosy patients presented higher percentage of IL-17A-producing IL-23R + CD4 T cells than healthy donors. IL-23R blocking induced markedly downregulated IL-17A secretion in leprosy patients but not in healthy donors. Furthermore, TGF-ß expression was significantly elevated after IL-23R blocking. Overall, this study establishes that Th17 cells produce IL-17A in an IL-23 dependent manner in the skin of leprosy patients and provides more focused treatment strategies for Mycobacterium leprae.

Broadband emission Gd3Sc2Al3O12:Ce3+ transparent ceramics with a high color rendering index for high-power white LEDs/LDs.

The discovery of single structure Ce3+ doped garnet transparent ceramics (TCs) with a broad full width at half maximum (FWHM) is essential to realize a high CRI for high-power white light emitting diodes (LEDs) and laser diodes (LDs). In this work, by utilizing the ion substitution engineering strategy, pure phase Gd3Sc2Al3O12:Ce3+ (GSAG:Ce) TC with a broad FWHM of 132.4 nm and a high CRI value of 80.7 was fabricated through the vacuum sintering technique for the first time. The optimized in-line transmittance of TCs was 58.4% @ 800 nm. Notably, the GSAG:Ce TCs exhibited a remarkable red shift from 546 nm to 582 nm, with a high internal quantum efficiency (IQE) of 46.91%. The degraded thermal stability in Ce:GSAG TCs was observed compared with that of Ce:YAG TC, owing to the narrowed band gap of GSAG. Additionally, remote excitation white LEDs/LDs were constructed by combining GSAG:Ce TCs with blue LED chips or laser sources. A tunable color hue from yellow to shinning white was achieved in white LEDs, whereas the acquired CRI and CCT of the white LDs were 69.5 and 7766 K, respectively. This work provides a new perspective to develop TCs with high CRI for their real applications in high-power white LEDs/LDs.

Synthesis and evaluation of 18F-PTTCO-Cys40-Exendin-4 for PET imaging of ectopic insulinomas in rodents.

A major limitation in the development of radiolabeled Exendin-4 analogues (short half-life isotopes) is an inability to efficiently and rapidly separate final products from precursors. This is important as lack of purity in the final product decreases probe efficiency. The purpose of this study was to develop a method to prepare the high-purity imaging reagent [18F] PTTCO-Cys40-Exendin-4. To accomplish this, magnetic TCO-beads were incubated with the crude product to remove unlabeled Exendin-4. In rodents pre-treatment with purified [18F] PTTCO-Cys40-Exendin-4 (~1.85 MBq) allowed precise microPET imaging of ectopic insulinomas. Moreover, analogue uptake was successfully blocked by administering non-labelled "cold" Exendin-4. Biodistribution data revealed that [18F] PTTCO-Cys40-Exendin-4 accumulated specifically in GLP-1R-enriched insulinomas in mice, confirming results obtained using miroPET. Investigation of [18F] PTTCO-Cys40-Exendin-4 as a tracer to image portal vein-transplanted pancreatic islets is proceeding in animals.

A Platform To Enhance Quantitative Single Molecule Localization Microscopy.

Quantitative single molecule localization microscopy (qSMLM) is a powerful approach to study in situ protein organization. However, uncertainty regarding the photophysical properties of fluorescent reporters can bias the interpretation of detected localizations and subsequent quantification. Furthermore, strategies to efficiently detect endogenous proteins are often constrained by label heterogeneity and reporter size. Here, a new surface assay for molecular isolation (SAMI) was developed for qSMLM and used to characterize photophysical properties of fluorescent proteins and dyes. SAMI-qSMLM afforded robust quantification. To efficiently detect endogenous proteins, we used fluorescent ligands that bind to a specific site on engineered antibody fragments. Both the density and nano-organization of membrane-bound epidermal growth factor receptors (EGFR, HER2, and HER3) were determined by a combination of SAMI, antibody engineering, and pair-correlation analysis. In breast cancer cell lines, we detected distinct differences in receptor density and nano-organization upon treatment with therapeutic agents. This new platform can improve molecular quantification and can be developed to study the local protein environment of intact cells.

Template-Catalyzed, Disulfide Conjugation of Monoclonal Antibodies Using a Natural Amino Acid Tag.

The high specificity and favorable pharmacological properties of monoclonal antibodies (mAbs) have prompted significant interest in re-engineering this class of molecules to add novel functionalities for enhanced therapeutic and diagnostic potential. Here, we used the high affinity, meditope-Fab interaction to template and drive the rapid, efficient, and stable site-specific formation of a disulfide bond. We demonstrate that this template-catalyzed strategy provides a consistent and reproducible means to conjugate fluorescent dyes, cytotoxins, or "click" chemistry handles to meditope-enabled mAbs (memAbs) and memFabs. More importantly, we demonstrate this covalent functionalization is achievable using natural amino acids only, opening up the opportunity to genetically encode cysteine meditope "tags" to biologics. As proof of principle, genetically encoded, cysteine meditope tags were added to the N- and/or C-termini of fluorescent proteins, nanobodies, and affibodies, each expressed in bacteria, purified to homogeneity, and efficiently conjugated to different memAbs and meFabs. We further show that multiple T-cell and Her2-targeting bispecific molecules using this strategy potently activate T-cell signaling pathways in vitro. Finally, the resulting products are highly stable as evidenced by serum stability assays (>14 d at 37 °C) and in vivo imaging of tumor xenographs. Collectively, the platform offers the opportunity to build and exchange an array of functional moieties, including protein biologics, among any cysteine memAb or Fab to rapidly create, test, and optimize stable, multifunctional biologics.

Gold nanoparticles as a platform for creating a multivalent poly-SUMO chain inhibitor that also augments ionizing radiation.

Protein-protein interactions mediated by ubiquitin-like (Ubl) modifications occur as mono-Ubl or poly-Ubl chains. Proteins that regulate poly-SUMO (small ubiquitin-like modifier) chain conjugates play important roles in cellular response to DNA damage, such as those caused by cancer radiation therapy. Additionally, high atomic number metals, such as gold, preferentially absorb much more X-ray energy than soft tissues, and thus augment the effect of ionizing radiation when delivered to cells. In this study, we demonstrate that conjugation of a weak SUMO-2/3 ligand to gold nanoparticles facilitated selective multivalent interactions with poly-SUMO-2/3 chains leading to efficient inhibition of poly-SUMO-chain-mediated protein-protein interactions. The ligand-gold particle conjugate significantly sensitized cancer cells to radiation but was not toxic to normal cells. This study demonstrates a viable approach for selective targeting of poly-Ubl chains through multivalent interactions created by nanoparticles that can be chosen based on their properties, such as abilities to augment radiation effects.

Spectroscopic and statistical physics elucidation for Cu(II) remediation using magnetic bio adsorbent based on Fe3O4-chitosan-graphene oxide.

Efficient harnessing of heavy metal pollution is an urgent environmental task. Herein, magnetic bio adsorbent (MB) based on Fe3O4-chitosan-graphene oxide composite was fabricated via one step co-precipitation for adsorptive remediation of Cu(II). Remediation efficiency was evaluated by batch adsorption, meanwhile adsorption mechanism was elucidated by spectroscopic tests (XPS, UV-Vis absorption and fluorescent emission spectra), statistical physics formalism, isotherm and kinetic fittings. Results show, MB reaches adsorption percent and quantity of 87.61 % and 350.43 mg·g-1 for Cu(II) in 30 min. By virtue of paramagnetism, MB can be readily recovered with a permanent magnet for easy regeneration and cyclic use, thereby retaining adsorption quantity 279.99 mg·g-1 at the fifth cycle. The Freundlich and pseudo second order model satisfactorily describes the adsorption, designating chemical interaction as the rate limiting step. Statistical physics calculation suggests two points. (1) Multi-ionic adsorption mechanism with exothermic, spontaneous and energy lowering feature. (2) Density of adsorption sites increases with temperature, resulting in improved adsorption capacity. Spectroscopic analysis confirms the involvement of CO, CO, -NH2 in Cu(II) uptake via electron donation. These explorations contribute with novel theoretical illumination for understanding both the thermodynamic feature and atomic scale mechanism of common pollutants adsorption by bio adsorbent like Fe3O4-chitosan-graphene oxide.

Multifunctional cross-sensitive magnetic alginate-chitosan-polyethylene oxide nanofiber sensor for human-machine interaction.

Flexible nanofiber membranes are compelling materials for the development of functional multi-mode sensors; however, their essential features such as high cross-sensitivity, reliable stability and signal discrimination capability have rarely been realized simultaneously in one sensor. Here, a novel multi-mode sensor with a nanofiber membrane structure based on multiple interpenetrating networks of bidisperse magnetic particles, sodium alginate (SA), chitosan (CHI) in conjunction with polyethylene oxide hydrogels was prepared in a controllable electrospinning technology. Specifically, the morphology distributions of nanofibers could be regulated by the crosslinking degree of the interpenetrating networks and the spinning process parameters. The incorporation of SA and CHI endowed the sensor with desirable flexibility, ideal biocompatibility and skin-friendly property. Besides, the assembled sensors not only displayed preferable magnetic sensitivity of 0.34 T-1 and reliable stability, but also exhibited favorable cross-sensitivity, quick response time, and long-term durability for over 5000 cycles under various mechanical stimuli. Importantly, the multi-mode stimuli could be discriminated via producing opposite electrical signals. Furthermore, based on the signal distinguishability of the sensor, a wearable Morse code translation system assisted by the machine learning algorithm was demonstrated, enabling a high recognizing accuracy (>99.1 %) for input letters and numbers information. Due to the excellent multifunctional sensing characteristics, we believe that the sensor will have a high potential in wearable soft electronics and human-machine interactions.

Naltrexone blocks alcohol-induced effects on kappa-opioid receptors in the plasma membrane.

Naltrexone (NTX), a homologue of the opiate antidote naloxone, is an orally active long-acting mu-opioid receptor (MOP) antagonist used in the treatment of opiate dependence. NTX is also found to relieve craving for alcohol and is one of the few FDA-approved drugs for alcohol use disorder (AUD). Reports that NTX blocks the actions of endogenous opioids released by alcohol are not convincing, suggesting that NTX interferes with alcohol actions by affecting opioid receptors. MOP and kappa-opioid receptor (KOP) are structurally related but functionally different. MOP is mainly located in interneurons activated by enkephalins while KOP is located in longer projections activated by dynorphins. While the actions of NTX on MOP are well established, the interaction with KOP and addiction is not well understood. We used sensitive fluorescence-based methods to study the influence of alcohol on KOP and the interaction between KOP and NTX. Here we report that alcohol interacts with KOP and its environment in the plasma membrane. These interactions are affected by NTX and are exerted both on KOP directly and on the plasma membrane (lipid) structures ("off-target"). The actions of NTX are stereospecific. Selective KOP antagonists, recently in early clinical trials for major depressive disorder, block the receptor but do not show the full action profile of NTX. The therapeutic effect of NTX treatment in AUD may be due to direct actions on KOP and the receptor environment.

The protective role of tissue-resident interleukin 17A-producing gamma delta T cells in Mycobacterium leprae infection.

Mycobacterium leprae is a kind of disease-causing bacteria and results in leprosy in human. Gamma delta (γδ) T cell is a T-cell subset that is presented in both human dermis and epidermis. These cells bridge innate and adaptive immune responses and play critical roles in regulating anti-microbial defense, wound healing, and skin inflammation. Here, we investigated skin resident γδ T cells in patients with leprosy. Our data showed that γδ T cells significantly accumulated in skin lesions of leprosy patients with tuberculoid (TT) form. IL-23 can predominantly stimulate dermal γδ T cells to produce interleukin 17 (IL-17), a cytokine which may lead to disease protection. These γδ T cells expressed a specific set of surface molecules, and majority of these cells were Vδ1+. Also, IL-23 can stimulate the expansion of dermal γδ T cells expansion. Moreover, our results revealed that the transcription factor RORγt was responsible for IL-17A expression in leprosy lesion. Therefore, these data indicated that IL-23-responsive dermal γδ T cells were the major resource of IL-17A production in the skin and could be a potential target in the treatment of leprosy.

The m6A reader IGF2BP2 regulates glutamine metabolism and represents a therapeutic target in acute myeloid leukemia.

N6-Methyladenosine (m6A) modification and its modulators play critical roles and show promise as therapeutic targets in human cancers, including acute myeloid leukemia (AML). IGF2BP2 was recently reported as an m6A binding protein that enhances mRNA stability and translation. However, its function in AML remains largely elusive. Here we report the oncogenic role and the therapeutic targeting of IGF2BP2 in AML. High expression of IGF2BP2 is observed in AML and associates with unfavorable prognosis. IGF2BP2 promotes AML development and self-renewal of leukemia stem/initiation cells by regulating expression of critical targets (e.g., MYC, GPT2, and SLC1A5) in the glutamine metabolism pathways in an m6A-dependent manner. Inhibiting IGF2BP2 with our recently identified small-molecule compound (CWI1-2) shows promising anti-leukemia effects in vitro and in vivo. Collectively, our results reveal a role of IGF2BP2 and m6A modification in amino acid metabolism and highlight the potential of targeting IGF2BP2 as a promising therapeutic strategy in AML.

Asymmetric synthesis of the core structure of leucosceptroids A-D.

The asymmetric synthesis of the core structure of leucosceptroids A-D has been achieved. The key steps of the synthesis includes the formation of the cis-2,5-disubstituted THF ring by TPAP catalytic oxidative cyclization followed by a highly diastereoselective intramolecular Diels-Alder reaction to fashion the fused tricyclic hydrindane ring system.

Synthesis and anticancer activities of ageladine A and structural analogs.

A series of ageladine A analogs that include 2-aminoimidazo[4,5-c]azepines (seven-membered rings) and 2-amino-3H-imidazo[4,5-c]pyridine (six-membered rings) derivatives were synthesized and evaluated for their anticancer effects against several human cancer cell lines and MMP-2 inhibition in vitro. Only compounds possessing the aromatic azepine (seven-membered ring) core showed anticancer activity with IC(50) values in the low micromolar range.

Cetuximab-Triptolide Conjugate Suppresses the Growth of EGFR-Overexpressing Lung Cancers through Targeting RNA Polymerase II.

To overcome poor pharmacokinetics and toxicity of triptolide (TPL), a natural compound that exhibits potent anticancer activities, we developed a novel antibody-drug conjugate (ADC) to specifically deliver TPL to epidermal growth factor receptor (EGFR)-overexpressing non-small cell lung cancer (NSCLC) and others. The ADC (Cet-TPL) is made by conjugation of TPL to lysine residues of cetuximab (Cet), a clinically available anti-EGFR monoclonal antibody. Studies of antitumor efficacy demonstrated that Cet-TPL drastically suppressed in vitro proliferation and in vivo growth of these EGFR-overexpressing cancers, including NSCLC A549 and H1299 cells and two patient-derived xenografts, and head and neck squamous carcinoma UM-SCC6 cell, while it did not inhibit the proliferation and growth of NSCLC H520 that rarely expresses EGFR. Furthermore, immunofluorescence analysis revealed that Cet-TPL was effectively internalized and transported into lysosomes of EGFR-overexpressing cells. Cet-TPL effectively led to degradation of RNA polymerase II (Pol II) and demethylation of histone H3 lysines, and significantly induced apoptosis in these EGFR-overexpressing cancers. Compared with TPL, Cet, or their combination, Cet-TPL displayed higher target-specific cytotoxicity against EGFR-expressing cancers and much lower in vivo toxicity. In addition, Cet-TPL efficiently suppressed the activated EGFR pathway in UM-SCC6 cancer cells. Taken together, Cet-TPL represents a potent targeting therapeutic agent against EGFR-overexpressing NSCLC and others.

Live Mycobacterium leprae inhibits autophagy and apoptosis of infected macrophages and prevents engulfment of host cell by phagocytes.

Previous studies demonstrated that live Mycobacterium leprae (M. leprae) infection promoted macrophage differentiation toward the M2 type, with elevated interleukin (IL)-10 production. The underlying mechanism is not entirely clear. In this study, we treated macrophages with primary M. leprae strains isolated from both lepromatous leprosy (L-lep) and tuberculoid leprosy (T-lep) patients. We found that infection by live M. leprae, regardless of the primary strain, resulted in M2 skewing in the infected macrophage. This skewing was associated with downregulated IRGM expression, a core organizer protein in the autophagy assembly and reduced autophagosome formation, and with lower annexin V staining and lower caspase 3 and caspase 9 activity. Moreover, live M. leprae-infected macrophages prevented efficient phagocytosis by uninfected bystander macrophages. As a result, the phagocytes secreted less pro-inflammatory cytokines, and preferentially primed anti-inflammatory T cell responses. Together, these results suggested that live M. leprae could employ a strain-independent mechanism to suppress inflammation, possibly involving the inhibition of autophagy and apoptosis in the infected macrophages.

Mechanically interlocked functionalization of monoclonal antibodies.

Because monoclonal antibodies (mAbs) have exceptional specificity and favorable pharmacology, substantial efforts have been made to functionalize them, either with potent cytotoxins, biologics, radionuclides, or fluorescent groups for therapeutic benefit and/or use as theranostic agents. To exploit our recently discovered meditope-Fab interaction as an alternative means to efficiently functionalize mAbs, we used insights from the structure to enhance the affinity and lifetime of the interaction by four orders of magnitude. To further extend the lifetime of the complex, we created a mechanical bond by incorporating an azide on the meditope, threading the azide through the Fab, and using click chemistry to add a steric group. The mechanically interlocked, meditope-Fab complex retains antigen specificity and is capable of imaging tumors in mice. These studies indicate it is possible to "snap" functionality onto mAbs, opening the possibility of rapidly creating unique combinations of mAbs with an array of cytotoxins, biologics, and imaging agents.