Mapping enzyme activity in living systems by real-time mid-infrared photothermal imaging of nitrile chameleons.

Simultaneous spatial mapping of the activity of multiple enzymes in a living system can elucidate their functions in health and disease. However, methods based on monitoring fluorescent substrates are limited. Here, we report the development of nitrile (C≡N)-tagged enzyme activity reporters, named nitrile chameleons, for the peak shift between substrate and product. To image these reporters in real time, we developed a laser-scanning mid-infrared photothermal imaging system capable of imaging the enzymatic substrates and products at a resolution of 300 nm. We show that when combined, these tools can map the activity distribution of different enzymes and measure their relative catalytic efficiency in living systems such as cancer cells, Caenorhabditis elegans, and brain tissues, and can be used to directly visualize caspase-phosphatase interactions during apoptosis. Our method is generally applicable to a broad category of enzymes and will enable new analyses of enzymes in their native context.

Arc/Arg3.1 regulates an endosomal pathway essential for activity-dependent ß-amyloid generation.

Assemblies of ß-amyloid (Aß) peptides are pathological mediators of Alzheimer's Disease (AD) and are produced by the sequential cleavages of amyloid precursor protein (APP) by ß-secretase (BACE1) and γ-secretase. The generation of Aß is coupled to neuronal activity, but the molecular basis is unknown. Here, we report that the immediate early gene Arc is required for activity-dependent generation of Aß. Arc is a postsynaptic protein that recruits endophilin2/3 and dynamin to early/recycling endosomes that traffic AMPA receptors to reduce synaptic strength in both hebbian and non-hebbian forms of plasticity. The Arc-endosome also traffics APP and BACE1, and Arc physically associates with presenilin1 (PS1) to regulate γ-secretase trafficking and confer activity dependence. Genetic deletion of Arc reduces Aß load in a transgenic mouse model of AD. In concert with the finding that patients with AD can express anomalously high levels of Arc, we hypothesize that Arc participates in the pathogenesis of AD.

The innate immunity protein IFITM3 modulates γ-secretase in Alzheimer's disease.

Innate immunity is associated with Alzheimer's disease1, but the influence of immune activation on the production of amyloid-ß is unknown2,3. Here we identify interferon-induced transmembrane protein 3 (IFITM3) as a γ-secretase modulatory protein, and establish a mechanism by which inflammation affects the generation of amyloid-ß. Inflammatory cytokines induce the expression of IFITM3 in neurons and astrocytes, which binds to γ-secretase and upregulates its activity, thereby increasing the production of amyloid-ß. The expression of IFITM3 is increased with ageing and in mouse models that express familial Alzheimer's disease genes. Furthermore, knockout of IFITM3 reduces γ-secretase activity and the formation of amyloid plaques in a transgenic mouse model (5xFAD) of early amyloid deposition. IFITM3 protein is upregulated in tissue samples from a subset of patients with late-onset Alzheimer's disease that exhibit higher γ-secretase activity. The amount of IFITM3 in the γ-secretase complex has a strong and positive correlation with γ-secretase activity in samples from patients with late-onset Alzheimer's disease. These findings reveal a mechanism in which γ-secretase is modulated by neuroinflammation via IFITM3 and the risk of Alzheimer's disease is thereby increased.

Photoacoustic: A Versatile Nongenetic Method for High-Precision Neuromodulation.

High-precision neuromodulation plays a pivotal role in elucidating fundamental principles of neuroscience and treating specific neurological disorders. Optical neuromodulation, enabled by spatial resolution defined by the diffraction limit at the submicrometer scale, is a general strategy to achieve such precision. Optogenetics offers single-neuron spatial resolution with cellular specificity, whereas the requirement of genetic transfection hinders its clinical application. Direct photothermal modulation, an alternative nongenetic optical approach, often associates a large temperature increase with the risk of thermal damage to surrounding tissues.Photoacoustic (also called optoacoustic) neural stimulation is an emerging technology for neural stimulation with the following key features demonstrated. First, the photoacoustic approach demonstrated high efficacy without the need for genetic modification. The generated pulsed ultrasound upon ns laser pulses with energy ranging from a few µJ to tens of µJ is sufficient to activate wild-type neurons. Second, the photoacoustic approach provides sub-100-µm spatial precision. It overcomes the fundamental wave diffraction limit of ultrasound by harnessing the localized ultrasound field generated through light absorption. A spatial precision of 400 µm has been achieved in rodent brains using a fiber-based photoacoustic emitter. Single-cell stimulation in neuronal cultures in vitro and in brain slices ex vivo is achieved using tapered fiber-based photoacoustic emitters. This precision is 10 to 100 times better than that for piezo-based low-frequency ultrasound and is essential to pinpoint a specific region or cell population in a living brain. Third, compared to direct photothermal stimulation via temperature increase, photoacoustic stimulation requires 40 times less laser energy dose to evoke neuron activities and is associated with a minimal temperature increase of less than 1 °C, preventing potential thermal damage to neurons. Fourth, photoacoustics is a versatile approach and can be designed in various platforms aiming at specific applications. Our team has shown the design of fiber-based photoacoustic emitters, photoacoustic nanotransducers, soft biocompatible photoacoustic films, and soft photoacoustic lenses. Since they interact with neurons through ultrasound without the need for direct contact, photoacoustic enables noninvasive transcranial and dura-penetrating brain stimulation without compromising high precision.In this Account, we will first review the basic principles of photoacoustic and discuss the key design elements of PA transducers for neural modulation guided by the principle. We will also highlight how these design goals were achieved from a materials chemistry perspective. The design of different PA interfaces, their unique capability, and their applications in neural systems will be reviewed. In the end, we will discuss the remaining challenges and future perspectives for this technology.

Nanosensor-based monitoring of autophagy-associated lysosomal acidification in vivo.

Autophagy is a cellular process with important functions that drive neurodegenerative diseases and cancers. Lysosomal hyperacidification is a hallmark of autophagy. Lysosomal pH is currently measured by fluorescent probes in cell culture, but existing methods do not allow for quantitative, transient or in vivo measurements. In the present study, we developed near-infrared optical nanosensors using organic color centers (covalent sp3 defects on carbon nanotubes) to measure autophagy-mediated endolysosomal hyperacidification in live cells and in vivo. The nanosensors localize to the lysosomes, where the emission band shifts in response to local pH, enabling spatial, dynamic and quantitative mapping of subtle changes in lysosomal pH. Using the sensor, we observed cellular and intratumoral hyperacidification on administration of mTORC1 and V-ATPase modulators, revealing that lysosomal acidification mirrors the dynamics of S6K dephosphorylation and LC3B lipidation while diverging from p62 degradation. This sensor enables the transient and in vivo monitoring of the autophagy-lysosomal pathway.

HuM195 and its single-chain variable fragment increase Aß phagocytosis in microglia via elimination of CD33 inhibitory signaling.

CD33 is a transmembrane receptor expressed on cells of myeloid lineage and regulates innate immunity. CD33 is a risk factor for Alzheimer's disease (AD) and targeting CD33 has been a promising strategy drug development. However, the mechanism of CD33's action is poorly understood. Here we investigate the mechanism of anti-CD33 antibody HuM195 (Lintuzumab) and its single-chain variable fragment (scFv) and examine their therapeutic potential. Treatment with HuM195 full-length antibody or its scFv increased phagocytosis of ß-amyloid 42 (Aß42) in human microglia and monocytes. This activation of phagocytosis was driven by internalization and degradation of CD33, thereby downregulating its inhibitory signal. HumM195 transiently induced CD33 phosphorylation and its signaling via receptor dimerization. However, this signaling decayed with degradation of CD33. scFv binding to CD33 leads to a degradation of CD33 without detection of the CD33 dimerization and signaling. Moreover, we found that treatments with either HuM195 or scFv promotes the secretion of IL33, a cytokine implicated in microglia reprogramming. Importantly, recombinant IL33 potentiates the uptake of Aß42 in monocytes. Collectively, our findings provide unanticipated mechanistic insight into the role of CD33 signaling in both monocytes and microglia and define a molecular basis for the development of CD33-based therapy of AD.

Notch-RBP-J signaling regulates the transcription factor IRF8 to promote inflammatory macrophage polarization.

Emerging concepts suggest that the functional phenotype of macrophages is regulated by transcription factors that define alternative activation states. We found that RBP-J, the main nuclear transducer of signaling via Notch receptors, augmented Toll-like receptor 4 (TLR4)-induced expression of key mediators of classically activated M1 macrophages and thus of innate immune responses to Listeria monocytogenes. Notch-RBP-J signaling controlled expression of the transcription factor IRF8 that induced downstream M1 macrophage-associated genes. RBP-J promoted the synthesis of IRF8 protein by selectively augmenting kinase IRAK2-dependent signaling via TLR4 to the kinase MNK1 and downstream translation-initiation control through eIF4E. Our results define a signaling network in which signaling via Notch-RBP-J and TLRs is integrated at the level of synthesis of IRF8 protein and identify a mechanism by which heterologous signaling pathways can regulate the TLR-induced inflammatory polarization of macrophages.

Cu(II)-Catalyzed Enantioselective Aza-Friedel-Crafts Reaction of 1-Naphthols and Electron-Rich Phenols with Isatin-Derived Ketimines.

New chiral ligands could be obtained by introducing proline moieties and imidazoline moieties to binaphthyl skeletons. The chiral ligands exhibited balanced rigidity and flexibility which could allow the change of the conformations during the reactions on one hand, and could provide sufficient asymmetric induction on the other. The proline moiety could act as a linker connecting the binaphthyl skeletons and the imidazoline moieties as well as a coordinating group for the central metal, and the electronic and steric properties of the imidazoline groups could be carefully fine-tuned by the use of different substituents. In the presence of Cu(II) catalyst bearing such chiral ligands, aza-Friedel-Crafts reaction of 1-naphthols and electron-rich phenols with isatin-derived ketimines provided the desired products with good to excellent yields and up to 99 % ee. The reactions showed good scalability, and excellent ee could still be obtained when the reaction was carried out in gram-scale.

Supported Biomembrane Systems Incorporating Multiarm Polymers and Bioorthogonal Tethering.

To functionalize interfaces with supported biomembranes and membrane proteins, the challenge is to build stabilized and supported systems that mimic the native lipid microenvironment. Our objective is to control substrate-to-biomembrane spacing and the tethering chemistry so proteoliposomes can be fused and conjugated without perturbation of membrane protein function. Furthermore, the substrates need to exhibit low protein and antibody nonspecific binding to use these systems in assays. We have employed protein orthogonal coupling schemes in concert with multiarm poly(ethylene glycol) (PEG) technology to build supported biomembranes on microspheres. The lipid bilayer structures and tailored substrates of the microsphere-supported biomembranes were analyzed via flow cytometry, confocal fluorescence, and super-resolution imaging microscopy, and the lateral fluidity was quantified using fluorescence recovery after photobleaching (FRAP) techniques. Under these conditions, the 4-arm-PEG20,000-NH2 based configuration gave the most desirable tethering system based on lateral diffusivity and coverage.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway contributes to the defense against bacterial infection in the pea aphid.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling is activated by infections of bacteria, fungi, viruses and parasites and mediated cellular and humoral immune responses. In the pea aphid Acyrthosiphon pisum little is known about the function of JAK/STAT signaling in its immune system. In this study, we first showed that expression of genes in the JAK/STAT signaling, including the receptors Domeless1/2, Janus kinase (JAK) and transcriptional factor Stat92E, is up-regulated upon bacteria Escherichia coli and Staphylococcus aureus and fungus Beauveria bassiana infections. After knockdown of expression of these genes by means of dsRNA injection, the aphids harbored more bacteria and suffered more death after infected with E. coli and S. aureus, but showed no significant change after B. bassiana infection. Our study suggests the JAK/STAT signaling contributes to the defense against bacterial infection in the pea aphid.

Blending Aryl Ketone in Covalent Organic Frameworks to Promote Photoinduced Electron Transfer.

Aryl-ketone derivatives have been acknowledged as promising organic photocatalysts for photosynthesis. However, they are limited by their photostability and have been less explored for photoinduced electron transfer (PET) applications. Herein we demonstrate a novel strategy to cover the shortage of aryl-ketone photocatalysts and control the photoreactivity by implanting symmetric aryl ketones into the conjugated covalent organic frameworks (COFs). To prove the concept, three comparative materials with the same topology and varied electronic structures were built, adopting truxenone knot and functionalized terephthalaldehyde linkers. Spectroscopic investigation and excited carrier dynamics analysis disclosed improvements in the photostability and electronic transfer efficiency as well as the structure-performance relationships toward N-aryl tetrahydroisoquinoline oxidation. This system provides a robust rule of thumb for designing new-generation aryl-ketone photocatalysts.

γ-Secretase fanning the fire of innate immunity.

Innate immunity is the first line of defense against pathogens, alerting the individual cell and surrounding area to respond to this potential invasion. γ-secretase is a transmembrane protease complex that plays an intricate role in nearly every stage of this innate immune response. Through regulation of pattern recognition receptors (PRR) such as TREM2 and RAGE γ-secretase can modulate pathogen recognition. γ-secretase can act on cytokine receptors such as IFNαR2 and CSF1R to dampen their signaling capacity. While γ-secretase-mediated regulated intramembrane proteolysis (RIP) can further moderate innate immune responses through downstream signaling pathways. Furthermore, γ-secretase has also been shown to be regulated by the innate immune system through cytokine signaling and γ-secretase modulatory proteins such as IFITM3 and Hif-1α. This review article gives an overview of how γ-secretase is implicated in innate immunity and the maintenance of its responses through potentially positive and negative feedback loops.

Hypoxia Inducible Factor-1α binds and activates γ-secretase for Aß production under hypoxia and cerebral hypoperfusion.

Hypoxic-ischemic injury has been linked with increased risk for developing Alzheimer's disease (AD). The underlying mechanism of this association is poorly understood. Here, we report distinct roles for hypoxia-inducible factor-1α (Hif-1α) in the regulation of BACE1 and γ-secretase activity, two proteases involved in the production of amyloid-beta (Aß). We have demonstrated that Hif-1α upregulates both BACE1 and γ-secretase activity for Aß production in brain hypoxia-induced either by cerebral hypoperfusion or breathing 10% O2. Hif-1α binds to γ-secretase, which elevates the amount of active γ-secretase complex without affecting the level of individual subunits in hypoxic-ischemic mouse brains. Additionally, the expression of full length Hif-1α increases BACE1 and γ-secretase activity in primary neuronal culture, whereas a transcriptionally incompetent Hif-1α variant only activates γ-secretase. These findings indicate that Hif-1α transcriptionally upregulates BACE1 and nontranscriptionally activates γ-secretase for Aß production in hypoxic-ischemic conditions. Consequently, Hif-1α-mediated Aß production may be an adaptive response to hypoxic-ischemic injury, subsequently leading to increased risk for AD. Preventing the interaction of Hif-1α with γ-secretase may therefore be a promising therapeutic strategy for AD treatment.

New Binaphthyl-Proline-Based Chiral Ligands Bearing Imidazoline Groups: Design, Synthesis, and Their Application in Enantioselective Conjugate Addition of 4-Hydroxycoumarin and Related Nucleophiles to ß,γ-Unsaturated α-Ketoesters.

This paper describes the design and application of new binaphthyl-proline-based chiral ligands bearing imidazoline functional groups. These chiral ligands incorporate the advantages of both the binaphthyl and proline skeletons, they are featured with regulatable electronic and steric properties for the imidazoline functional groups, and form chiral complexes with different metal salts such as cuprous acetate. In the presence of an appropriate amount of a chiral catalyst, enantioselective conjugate addition of 4-hydroxycoumarin or related nucleophiles to different ß,γ-unsaturated α-ketoesters proceeded readily, giving the desired products in high yield (up to 99%) and excellent enantiomeric excess (up to 99%).

Enantioselective Friedel-Crafts Alkylation of Indoles with ß,γ-Unsaturated α-Ketoesters Catalyzed by New Copper(I) Catalysts.

New Cu(I) catalysts are effective in enantioselective Friedel-Crafts alkylation of a variety of indoles with different ß,γ-unsaturated α-ketoesters. A control study shows that such a catalyst system is less sensitive to air, and the reactions can be carried out without special cares such as glovebox operation or moisture/oxygen-free conditions. Preliminary computation results suggest that there exists π-π stacking between the substrate and the catalyst, and such an interaction seems to play a role in stabilizing the reaction intermediate and enhancing the stereoselectivity of the reactions. The desired products can be obtained in up to 98% yield at 99% enantiomeric excess. The same high enantioselectivity can be observed when the reaction is carried in a gram scale, indicating a good scalability of the catalyst system in enantioselective Friedel-Crafts alkylation of different indoles with ß,γ-unsaturated α-ketoesters.

Copper(II)-Catalyzed Enantioselective Aza-Friedel-Crafts Reaction of Indoles with Isatin-Derived N-Boc-Ketimines.

The copper(II)-catalyzed enantioselective aza-Friedel-Crafts reaction of indoles with isatin-derived N-Boc-ketimines was developed by using tunable chiral O-N-N tridentate ligands derived from BINOL and proline. In general, the reaction afforded chiral 3-indolyl-3-aminooxindoles under mild conditions in high yields (83-97%) with excellent ee (69-99%).

Binaphthyl-Proline Hybrid Chiral Ligands: Modular Design, Synthesis, and Enantioswitching in Cu(II)-Catalyzed Enantioselective Henry Reactions.

Chiral O-N-N tridentate ligands were designed from proline and BINOL. Their design strategy and performance were evaluated using a copper(II)-catalyzed asymmetric Henry reaction as a model. The desired ß-nitroalcohols were obtained in up to 94% ee's. Preliminary results suggested that the stereofacial selection of the reactions was mainly controlled by the chiral diamine moiety derived from proline, and matching of the central and axial chiralities was essential for the high stereoselectivity of the reaction. Enantioswitching was observed when an appropriate substituent was introduced to the binaphthyl group. Si-selections were found in reactions using 2a without 3-substituents as chiral ligand, and Re-selections were found with the same high enantioselectivities when 2i bearing the 3-trifluoromethyl group was used as the chiral ligand.

Enantioselective Michael addition of malonates to ß,γ-unsaturated α-ketoesters catalysed by Cu(II) complexes bearing binaphthyl-proline hybrid ligands.

High yields (up to 96%) and high ee (up to 92%) were achieved for chiral copper(II) complex-catalysed enantioselective Michael addition of malonates to ß,γ-unsaturated-α-ketoesters. The chiral ligands took advantage of both the binaphthyl and the proline moieties, and substituents with different electronic and steric features could be tolerated. The reactions could be carried out under mild conditions, and a gram scale reaction could be realised without the loss of yield and enantioselectivity.

Carbon and nitrogen sources consumption by ale and lager yeast strains: a comparative study during fermentation.

The rapid and efficient consumption of carbon and nitrogen sources by brewer's yeast is critical for the fermentation process in the brewing industry. The comparison of the growth characterizations of typical ale and lager yeast, as well as their consumption preference to carbon and nitrogen sources were investigated in this study. Results showed that the ale strain grew faster and had a more extended stationary phase than the lager strain. However, the lager strain was more tolerant to the stressful environment in the later stage of fermentation. Meanwhile, the ale and lager yeast strains possessed varying preferences for metabolizing the specific fermentable sugar or free amino acid involved in the wort medium. The lager strain had a strong capacity to synthesize the extracellular invertase required for hydrolyzing sucrose as well as a strong capability to metabolize glucose and fructose. Furthermore, the lager strain had an advantage in consuming Lys, Arg, Val, and Phe, whereas the ale strain had a higher assimilation rate in consuming Tyr. These findings provide valuable insights into selecting the appropriate brewer's yeast strain based on the wort components for the industrial fermentation process. KEY POINTS: • The lager strain is more tolerant to the stressful environment. • The lager strain has the great capability to synthesize the extracellular invertase. • The assimilation efficiency of free amino acid varies between ale and lager.

Diffusion-Weighted Imaging as a Quantitative Imaging Biomarker for Predicting Proliferation Rate in Hepatocellular Carcinoma: Developing a Radiomics Nomogram.

PURPOSE: This study aimed to explore the predictive performance of diffusion-weighted imaging with apparent diffusion coefficient map in predicting the proliferation rate of hepatocellular carcinoma and to develop a radiomics-based nomogram. METHODS: This was a single-center retrospective study. A total of 110 patients were enrolled. The sample included 38 patients with low Ki67 expression (Ki67 ≤10%) and 72 with high Ki67 expression (Ki67 >10%) as demonstrated by surgical pathology. Patients were randomly divided into either a training (n = 77) or validation (n = 33) cohort. Diffusion-weighted imaging with apparent diffusion coefficient maps was used to extract radiomic features and the signal intensity values of tumor (SI tumor ), normal liver (SI liver ), and background noise (SI background ) from all samples. Subsequently, the clinical model, radiomic model, and fusion model (with clinical data and radiomic signature) were developed and validated. RESULTS: The area under the curve (AUC) of the clinical model for predicting the Ki67 expression including serum α-fetoprotein level ( P = 0.010), age ( P = 0.015), and signal noise ratio ( P = 0.026) was 0.799 and 0.715 in training and validation cohorts, respectively. The AUC of the radiomic model constructed by 9 selected radiomic features was 0.833 and 0.772 in training and validation cohorts, respectively. The AUC of the fusion model containing serum α-fetoprotein level ( P = 0.011), age ( P = 0.019), and rad score ( P < 0.001) was 0.901 and 0.781 in training and validation cohorts, respectively. CONCLUSIONS: Diffusion-weighted imaging as a quantitative imaging biomarker can predict Ki67 expression level in hepatocellular carcinoma across various models.