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Accurate classification of tumor cells is of importance for cancer diagnosis and further therapy. In this study, we develop multimolecular marker-activated transmembrane DNA computing systems (MTD). Employing the cell membrane as a native gate, the MTD system enables direct signal output following simple spatial events of "transmembrane" and "in-cell target encounter", bypassing the need of multistep signal conversion. The MTD system comprises two intelligent nanorobots capable of independently sensing three molecular markers (MUC1, EpCAM, and miR-21), resulting in comprehensive analysis. Our AND-AND logic-gated system (MTD AND-AND ) demonstrates exceptional specificity, allowing targeted release of drug-DNA specifically in MCF-7 cells. Furthermore, the transformed OR-AND logic-gated system (MTD OR-AND ) exhibits broader adaptability, facilitating the release of drug-DNA in three positive cancer cell lines (MCF-7, HeLa, and HepG2). Importantly, MTD AND-AND and MTD OR-AND , while possessing distinct personalized therapeutic potential, share the ability of outputting three imaging signals without any intermediate conversion steps. This feature ensures precise classification cross diverse cells (MCF-7, HeLa, HepG2, and MCF-10A), even in mixed populations. This study provides a straightforward yet effective solution to augment the versatility and precision of DNA computing systems, advancing their potential applications in biomedical diagnostic and therapeutic research.

The CRISPR/Cas12a system is a revolutionary genome editing technique that is widely employed in biosensing and molecular diagnostics. However, there are few reports on precisely managing the trans -cleavage activity of Cas12a by simple modification since the traditional methods to manage Cas12a often require difficult and rigorous regulation of core components. Hence, we developed a novel CRISPR/Cas12a regulatory mechanism, named D NA R obots for E nzyme A ctivity M anagement (DREAM), by introducing two simple DNA robots, apurinic/apyrimidinic site (AP site) or nick on target activator. First, we revealed the mechanism of how the DREAM strategy precisely regulated Cas12a through different binding affinities. Second, the DREAM strategy was found to improve the selectivity of Cas12a for identifying base mismatch. Third, a modular biosensor for base excision repair enzymes based on the DREAM strategy was developed by utilizing diversified generation ways of DNA robots, and a multi-signal output platform such as fluorescence, colorimetry, and visual lateral flow strip was constructed. Furthermore, we extended logic sensing circuits to overcome the barrier that Cas12a could not detect simultaneously in a single tube. Overall, the DREAM strategy not only provided new prospects for programmable Cas12a biosensing systems but also enabled portable, specific, and humanized detection with great potential for molecular diagnostics.

SIRT6 is an emerging regulator of longevity. Overexpression of SIRT6 extends the lifespan of mice. Conversely, SIRT6 knockout mice demonstrate severe metabolic defects and a shortened lifespan. The discrepancy between SIRT6's weak in vitro activity and robust in vivo activity has led to the hypothesis that this enzyme can be activated in response to DNA damage in cells. Here, we demonstrate that the deacetylase activity of SIRT6 can be stimulated by DNA strand breaks for synthetic peptide and histone substrates. The mechanism of activation is further explored by using an integrative chemical biology approach. SIRT6 can be preferentially activated by DNA lesions harboring a 5'-phosphate. The N- and C-termini of SIRT6 are strictly required for DNA break-induced activation. Additionally, the defatty-acylase activity of SIRT6 is also sensitive to DNA breaks, although the physiological significance needs further investigation. Collectively, our study sheds important light on the cellular regulation of diverse SIRT6 activities and suggests possible strategies for effective SIRT6 activation., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Tumor-associated macrophages (TAMs) are the most abundant immune cells in solid tumor tissues, which restrict antitumor immunity by releasing tumor-supporting cytokines and attenuating phagocytosis behaviors. In this work, a chimeric peptide engineered bioregulator (ChiP-RS) is constructed for tumor immunotherapy through macrophage polarization and phagocytosis restoration. ChiP-RS is fabricated by utilizing macrophage-targeting chimeric peptide (ChiP) to load Toll-like receptor agonists (R848) and Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 2 (SHP-2) inhibitor (SHP099). Among which, ChiP-RS prefers to be internalized by TAMs, repolarizing M2 macrophages into M1 macrophages to reverse the immunosuppressive microenvironment. In addition, SHP-2 can be downregulated to promote phagocytotic elimination behaviors of M1 macrophages, which will also activate T cell-based antitumor immunity for metastatic tumor therapy. In vitro and in vivo findings demonstrate a superior suppression effect of ChiP-RS against metastatic tumors without systemic side effects. Such a simple but effective nanoplatform provides sophisticated synergism for immunotherapy, which may facilitate the development of translational nanomedicine for metastatic tumor treatment.

The convergence of multivalley bands is originally believed to be beneficial for thermoelectric performance by enhancing the charge conductivity while preserving the Seebeck coefficients, based on the assumption that electron interband or intervalley scattering effects are totally negligible. In this work, we demonstrate that β-Bi with a buckled honeycomb structure experiences a topological transition from a normal insulator to a Z 2 topological insulator induced by spin-orbit coupling, which subsequently increases the band degeneracy and is probably beneficial for enhancement of the thermoelectric power factor for holes. Therefore, strong intervalley scattering can be observed in both band-convergent β- and aw-Bi monolayers. Compared to β-Bi, aw-Bi with a puckered black-phosphorus-like structure possesses high carrier mobilities with 318 cm 2 /(V s) for electrons and 568 cm 2 /(V s) for holes at room temperature. We also unveil extraordinarily strong fourth phonon-phonon interactions in these bismuth monolayers, significantly reducing their lattice thermal conductivities at room temperature, which is generally anomalous in conventional semiconductors. Finally, a high thermoelectric figure of merit ( zT ) can be achieved in both bismuth monolayers, especially for aw-Bi with an n-type zT value of 2.2 at room temperature. Our results suggest that strong fourth phonon-phonon interactions are crucial to a high thermoelectric performance in these materials, and two-dimensional bismuth is probably a promising thermoelectric material due to its enhanced band convergence induced by the topological transition.

The residues of organophosphorus pesticides (OPs) have drawn worldwide increasing attention because of their potential fatal effects on human health and ecological systems. It is of great significance to develop an efficient and portable method for in-field detection of OPs. Herein, a novel core-shell nanocomposite of prussian blue@Fe-covalent organic framework@Au (PB@Fe-COF@Au) was constructed. Fe 2+ and Fe 3+ in PB nanoparticle (PBNP) cores, Fe-porphyrin in COF shells, and AuNPs grown on shells all acted as peroxidase-like catalytic active sites, enabling PB@Fe-COF@Au to possess triplet peroxidase-like activity. A colorimetric, affordable, sensitive, and selective strategy was designed to detect OPs. Compared with previous reports, this sensor realized a wider linear range for chlorpyrifos of 10-800 ng mL -1 with a relatively lower detection limit of 0.61 ng mL -1 , which was attributed to the overlapping triple catalytic sites of PB@Fe-COF@Au and triple response sites to OPs. The assay was successfully employed to detect chlorpyrifos in food and environmental samples. Moreover, to meet the demand of in-field detection for OPs, a spherical hydrogel method based on PB@Fe-COF@Au with visual, portable, and equipment-free features was fabricated. This work provides a new pathway to design and apply effective nanozymes for on-site monitoring of pesticides.

Being the rate-limiting enzyme within the serine biosynthesis pathway, phosphoglycerate dehydrogenase (PHGDH) is abnormally overexpressed in numerous malignant tumor cells and is a promising target for cancer treatment. Here, we report a series of novel PHGDH inhibitors using a focused compound screening and structural optimization approach. The lead compound D8 displayed good enzymatic inhibitory activity (IC 50 = 2.8 ± 0.1 μM), high binding affinity ( K d = 2.33 μM), and sensitivity to the cell lines with the PHGDH gene amplification or overexpression. Furthermore, D8 was proven to restrict the de novo serine synthesis from glucose within MDA-MB-468 cells. X-ray crystallographic analysis, molecular dynamics simulations, and mutagenesis experiments on PHGDH revealed the binding site at D175 inside the NAD + -binding pocket. Finally, D8 exhibited excellent in vivo pharmacokinetic properties ( F = 82.0%) and exerted evident antitumor efficacy in the PC9 xenograft mouse model.

Most oxidative damage on mitochondrial DNA is corrected by the base excision repair (BER) pathway. However, the enzyme that catalyzes the rate-limiting reaction─deoxyribose phosphate (dRP) removal─in the multienzymatic reaction pathway has not been completely determined in mitochondria. Also unclear is how a logical order of enzymatic reactions is ensured. Here, we present structural and enzymatic studies showing that human mitochondrial EXOG (hEXOG) exhibits strong 5'-dRP removal ability. We show that, unlike the canonical dRP lyases that act on a single substrate, hEXOG functions on a variety of abasic sites, including 5'-dRP, its oxidized product deoxyribonolactone (dL), and the stable synthetic analogue tetrahydrofuran (THF). We determined crystal structures of hEXOG complexed with a THF-containing DNA and with a partial gapped DNA to 2.9 and 2.1 Å resolutions, respectively. The structures illustrate that hEXOG uses a controlled 5'-exonuclease activity to cleave the third phosphodiester bond away from the 5'-abasic site. This study provides a structural basis for hEXOG's broad spectrum of substrates. Further, we show that hEXOG can set the order of BER reactions by generating an ideal substrate for the subsequent reaction in BER and inhibit off-pathway reactions.

Spintronics has been recently extended to neuromorphic computing because of its energy efficiency and scalability. However, a biorealistic spintronic neuron with probabilistic "spiking" and a spontaneous reset functionality has not been demonstrated yet. Here, we propose a biorealistic spintronic neuron device based on the heavy metal (HM)/ferromagnet (FM)/antiferromagnet (AFM) spin-orbit torque (SOT) heterostructure. The spintronic neuron can autoreset itself after firing due to the exchange bias of the AFM. The firing process is inherently stochastic because of the competition between the SOT and AFM pinning effects. We also implement a restricted Boltzmann machine (RBM) and stochastic integration multilayer perceptron (SI-MLP) using our proposed neuron. Despite the bit-width limitation, the proposed spintronic model can achieve an accuracy of 97.38% in pattern recognition, which is even higher than the baseline accuracy (96.47%). Our results offer a spintronic device solution to emulate biologically realistic spiking neurons.

Poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors are used in ovarian cancer treatment and have greatly improved the survival rates for homologous recombination repair (HRR)-deficient patients. However, their therapeutic efficacy is limited in HRR-proficient ovarian cancer. Thus, sensitizing HRR-proficient ovarian cancer cells to PARP inhibitors is important in clinical practice. Here, a nanodrug, olaparib-Ga, was designed using self-assembly of the PARP inhibitor olaparib into bovine serum albumin through gallic acid gallium(III) coordination via a convenient and green synthetic method. Compared with olaparib, olaparib-Ga featured an ultrasmall size of 7 nm and led to increased suppression of cell viability, induction of DNA damage, and enhanced cell apoptosis in the SKOV3 and OVCAR3 HRR-proficient ovarian cancer cells in vitro . Further experiments indicated that the olaparib-Ga nanodrug could suppress RRM2 expression, activate the Fe 2+ /ROS/MAPK pathway and HMOX1 signaling, inhibit the PI3K/AKT signaling pathway, and enhance the expression of cleaved-caspase 3 and BAX protein. This, in turn, led to increased cell apoptosis in HRR-proficient ovarian cancer cells. Moreover, olaparib-Ga effectively restrained SKOV3 and OVCAR3 tumor growth and exhibited negligible toxicity in vivo . In conclusion, we propose that olaparib-Ga can act as a promising nanodrug for the treatment of HRR-proficient ovarian cancer.

A 3D printed flexible tactile sensor with graphene-polydimethylsiloxane (PDMS) microspheres for microstructure perception is presented. The structure of the tactile sensor is inspired by the texture of the human finger and is designed to enable the detection of various levels of surface roughness via the processing of tactile signals. The tactile sensor with a unique graphene-PDMS microsphere structure shows excellent comprehensive mechanical properties, including a robust stretching ability (elongation at break of the sensing layer is 70%), excellent sensing ability (short response time of 60 ms), high sensitivity (sensitivity up to 2.4 kPa -1 ), and cycle stability (over 2000 loading cycles). In addition, such versatility and sensitivity allow the electronic skin not only to accurately monitor pressure but also to distinguish various surface topographies with microscale differences, and to detect the action of an air fluid.

A simple, direct fluorescent sensor was developed to simultaneously determine nitric oxide and hydrogen sulfide based on 4-(((3-aminonaphthalen-2-yl)amino)methyl)benzoic acid (DAN-1)-functionalized CdTe/CdS/ZnS quantum dots (QDs@DAN-1). In this sensor, DAN-1 could specifically recognize nitric oxide and yield highly fluorescent naphtho triazole (DAN-1-T). Meanwhile, the fluorescence intensity of the QDs could be quenched by hydrogen sulfide. The QDs and DAN-1-T could be simultaneously excited at 365 nm, and their maximum emission wavelengths were 635 and 440 nm, respectively. Nitric oxide and hydrogen sulfide were simultaneously determined by monitoring two different fluorescence signals. The limits of determination for nitric oxide and hydrogen sulfide were 0.051 and 0.13 μM, respectively. The QDs@DAN-1 sensor was also applied to determine nitric oxide and hydrogen sulfide in human plasma. This sensor may provide a new strategy for investigating the relationship between nitric oxide and hydrogen sulfide and elucidating their roles in related physiological and pathophysiological processes at the same time.

The headgroup (H) stratum (sometimes called the polar region) of membrane bilayers is a relevant yet poorly understood solvation phase for small molecules and macromolecules interacting with the membranes. Solvation of compounds in bilayer strata is characterized experimentally by wide- and small-angle X-ray scattering, neutron diffraction, and various NMR techniques. The quantification is tedious and only available for a limited set of small molecules. Our recently published model of liposome partitioning of small molecules shows that solvation of compounds in the H-stratum of fluid phosphatidylcholine (PC) bilayers correlates well with their solvation in hydrated diacetyl phosphatidylcholine (DAcPC), and solvation in the core (C) depends in a similar way on that in n-hexadecane. These two correlations became a basis for a model describing the location of compounds in the H- and C-strata and at the connecting interface as a nonlinear function of the fragment solvation characteristics of the compounds. In this study, refractivity of hydrated DAcPC phases with varying water contents was measured and polarity was determined using the steady-state fluorescence of indole and Nile Red. The results were compared with the published data obtained by other techniques for PC bilayers in liposomes or on solid supports. The demonstrated qualitative agreement, as well as the polarity and refractivity dependencies on the DAcPC concentration, supports the suitability of hydrated DAcPC as the H-stratum surrogate. Interestingly, depending on hydrations typical for the H-strata of fluid PC bilayers, the dielectric constant could decrease significantly from 31.0 to 7.3 for 16 and 8 water molecules per headgroup, respectively. Although additional experiments are needed for confirmation, this observation could help set proper dielectric constant magnitudes in continuum-based computational models of accumulation and crossing of the PC bilayers with varying hydration levels thanks to the temperature or the structure of fatty acid chains.

Enzymatic stereodivergent synthesis to access all possible product stereoisomers bearing multiple stereocenters is relatively undeveloped, although enzymes are being increasingly used in both academic and industrial areas. When two stereocenters and thus four stereoisomeric products are involved, obtaining stereodivergent enzyme mutants for individually accessing all four stereoisomers would be ideal. Although significant success has been achieved in directed evolution of enzymes in general, stereodivergent engineering of one enzyme into four highly stereocomplementary variants for obtaining the full complement of stereoisomers bearing multiple stereocenters remains a challenge. Using Candida antarctica lipase B (CALB) as a model, we report the protein engineering of this enzyme into four highly stereocomplementary variants needed for obtaining all four stereoisomers in transesterification reactions between racemic acids and racemic alcohols in organic solvents. By generating and screening less than 25 variants of each isomer, we achieved >90% selectivity for all of the four possible stereoisomers in the model reaction. This difficult feat was accomplished by developing a strategy dubbed "focused rational iterative site-specific mutagenesis" (FRISM) at sites lining the enzyme's binding pocket. The accumulation of single mutations by iterative site-specific mutagenesis using a restricted set of rationally chosen amino acids allows the formation of ultrasmall mutant libraries requiring minimal screening for stereoselectivity. The crystal structure of all stereodivergent CALB variants, flanked by MD simulations, uncovered the source of selectivity.

In this work, a new magnetic, ratiometric fluorescent nanoprobe has been designed and fabricated by encapsulating Fe 3 O 4 magnetic nanoparticles (MNPs) and dual-emissive carbon dots into the cavities of metal-organic frameworks (MOFs). This one-pot method combined hybrid characteristics of MOFs with multiple properties of the encapsulated functional materials. The MOF-based nanoprobe possessed the advantages of MOFs (strong adsorption ability, accumulating the analytes), Fe 3 O 4 MNPs (magnetic separation), and ratiometric sensors (eliminating the variabilities caused by the instability of the instruments and environment). The MOF-based nanoprobe was dispersible and stable in aqueous solution, and the nanoprobe was applied to HClO sensing. This work will provide a promising strategy for design and synthesis of novel MOF-based composite materials.

Sirtuins consume stoichiometric amounts of nicotinamide adenine dinucleotide (NAD + ) to remove an acetyl group from lysine residues. These enzymes have been implicated in regulating various cellular events and have also been suggested to mediate the beneficial effects of calorie restriction (CR). However, controversies on sirtuin biology also peaked during the past few years because of conflicting results from different research groups. This is partly because these enzymes have been discovered recently and the intricate interaction loops between sirtuins and other proteins make the characterization of them extremely difficult. Current molecular biology and proteomics techniques report protein abundance rather than active sirtuin content. Innovative chemical tools that can directly probe the functional state of sirtuins are desperately needed. We have obtained a set of powerful activity-based chemical probes that are capable of assessing the active content of sirtuins in model systems. These probes consist of a chemical "warhead" that binds to the active site of active enzyme and a handle that can be used for the visualization of these enzymes by fluorescence. In complex native proteome, the probes can selectively "highlight" the active sirtuin components. Furthermore, these probes were also able to probe the dynamic change of sirtuin activity in response to cellular stimuli. These chemical probes and the labeling strategies will provide transformative technology to allow the direct linking of sirtuin activity to distinct physiological processes. They will create new opportunities to investigate how sirtuins provide health benefits in adapting cells to environmental cues and provide critical information to dissect sirtuin regulatory networks.

A multifunctional nanoplatform, which generally integrates biosensing, imaging diagnosis, and therapeutic functions into a single nanoconstruct, has great important significance for biomedicine and nanoscience. Here, we developed a core-shell-shell multifunctional polydopamine (PDA) modified upconversion nanoplatform for intracellular tumor-related mRNAs detection and near-infrared (NIR) light triggered photodynamic and photothermal synergistic therapy (PDT-PTT). The nanoplatform was constructed by loading a silica shell on the hydrophobic upconversion nanoparticles (UCNPs) with hydrophilic photosensitizer methylene blue (MB) entrapped in it, and then modifying PDA shells through an in situ self-polymerization process, thus yielding a core-shell-shell nanoconstruct UCNP@SiO 2 -MB@PDA. By taking advantages of preferential binding properties of PDA for single-stranded DNA over double-stranded DNA and the excellent quenching property of PDA, a UCNP@SiO 2 -MB@PDA-hairpin DNA (hpDNA) nanoprobe was developed through adsorption of fluorescently labeled hpDNA on PDA shells for sensing intracellular tumor-related mRNAs and discriminating cancer cells from normal cells. In addition, the fluorescence resonance energy transfer from the upconversion fluorescence (UCF) emission at 655 nm of the UCNPs to the photosensitizer MB molecules could be employed for PDT. Moreover, due to the strong NIR absorption and high photothermal conversion efficiency of PDA, the UCF emission at 800 nm of the UCNPs could be used for PTT. We demonstrated that the UCNP@SiO 2 -MB@PDA irradiated with NIR light had considerable PDT-PTT effect. These results revealed that the developed multifunctional nanoplatform provided promising applications in future oncotherapy by integrating cancer diagnosis and synergistic therapy.

In this work, a new kind of N, Cu codoped carbon dots (N/Cu-CDs) was prepared via a facile one-pot hydrothermal method by using citric acid monohydrate, copper acetate monohydrate and diethylenetriamine. The prepared N/Cu-CDs with a high quantum yield (50.1%) showed excitation-independent emission at 460 nm. The structure and fluorescence properties of N/Cu-CDs were characterized by high-resolution transmission electron microscopy, fluorescence spectrofluorometer, FT-IR spectrometer, UV-visible spectrophotometer and X-ray photoelectron spectroscopy. N/Cu-CDs were applied to establishing a ratiometric fluorescence probe toward H 2 O 2 based on the inner filter effect (IFE) between N/Cu-CDs and DAP (2,3-diaminophenazine, the oxidative product of o-phenylenediamine (OPD)), and provided a ratiometric fluorescence universal platform for detection of the metabolites participating in H 2 O 2 -generation reactions (cholesterol and xanthine). The proposed method was demonstrated to be ultrasensitive and highly selective for cholesterol and xanthine assay with detection limits of 0.03 and 0.10 μM, respectively. The fluorescence probe built was applied to the determination of cholesterol and xanthine in human serum with satisfactory results.

Bufadienolides are the major pharmacologic constituents of traditional Chinese medicine Chan'su, which is frequently used clinically for cancer treatment in China. Motivated by reducing or avoiding the cardiac toxicity of bufadienolides, we have designed, synthesized, and evaluated the fibroblast activation protein α (FAPα) activated tripeptide arenobufagin prodrugs with the purpose of improving the safety of arenobufagin (a representative bufadienolide). Among these FAPα-activated prodrugs, 3f exhibited the best hydrolytic efficiency by recombinant human FAPα (rhFAPα) and was activated in tumors. The LD 50 of 3f was 6.5-fold higher than that of arenobufagin. We also observed that there are nonapparent changes in echocardiography, pathological section of cardiac muscle, and the lactate dehydrogenase activities (LDH) in 3f-treatment tumor-bearing mice, even when the dose reached 3 times the amount of parent drug arenobufagin that was used. Compound 3f also exhibits significant antitumor activity in vitro and in vivo. The improved safety profile and favorable anticancer properties of 3f warrant further studies of the potential clinical implications. Our study suggests that FAPα prodrug strategy is an effective approach for successful increasing the therapeutic window of bufadienolides.

The acetylated headgroup of the most abundant mammalian phospholipid, 1,2-diacetyl-3- sn -phosphatidyl choline (DAcPC), has several important applications in research. For instance, it can be dissolved in the same amount of water as in the fluid PC bilayer, to create a surrogate of a PC headgroup stratum, for studying solvation of small molecules and the influence of their structure on the process. In contrast to PC derivatives with longer acyl chains, DAcPC does not self-aggregate, rendering the aqueous solution homogeneous and suitable for simplified analyses of interactions of molecules with the headgroups. Several studies have been published where DAcPC was used in a crudely purified form. Here we describe a one-step preparation of DAcPC from commercially available bulk chemicals and purification of the product by crystallization and washing. The process gives a good yield and is easily scalable. The availability of enantiopure, crystalline DAcPC could open the door to more extensive biochemical, pharmacological, and nutritional studies of this interesting chemical., Competing Interests: Notes. The authors declare no competing financial interest.

Blood glucose monitoring has attracted extensive attention because diabetes mellitus is a worldwide public health problem. Here, we reported an upconversion fluorescence detection method based on manganese dioxide (MnO2)-nanosheet-modified upconversion nanoparticles (UCNPs) for rapid, sensitive detection of glucose levels in human serum and whole blood. In this strategy, MnO2 nanosheets on the UCNP surface serve as a quencher. UCNP fluorescence can make a recovery by the addition of H2O2, which can reduce MnO2 to Mn(2+), and the glucose can thus be monitored based on the enzymatic conversion of glucose by glucose oxidase to generate H2O2. Because of the nonautofluorescent assays offered by UCNPs, the developed method has been applied to monitor glucose levels in human serum and whole blood samples with satisfactory results. The proposed approach holds great potential for diabetes mellitus research and clinical diagnosis. Meanwhile, this nanosystem is also generalizable and can be easily expanded to the detection of various H2O2-involved analytes.

Phospholipase D (PLD) is a critical component of intracellular signal transduction and has been implicated in many important biological processes. It has been observed that there are abnormalities in PLD expression in many human cancers, and PLD is thus recognized as a potential diagnostic biomarker as well as a target for drug discovery. We report for the first time a phospholipid-modified nanoprobe for ratiometric upconversion fluorescence (UCF) sensing and bioimaging of PLD activity. The nanoprobe can be synthesized by a facile one-step self-assembly of a phospholipid monolayer composed of poly(ethylene glycol) (PEG)ylated phospholipid and rhodamine B-labeled phospholipid on the surface of upconversion nanoparticles (UCNPs) NaYF4: 20%Yb, 2%Er. The fluorescence resonance energy transfer (FRET) process from the UCF emission at 540 nm of the UCNPs to the absorbance of the rhodamine B occurs in the nanoprobe. The PLD-mediated hydrolysis of the phosphodiester bond makes rhodamine B apart from the UCNP surface, leading to the inhibition of FRET. Using the unaffected UCF emission at 655 nm as an internal standard, the nanoprobe can be used for ratiometric UCF detection of PLD activity with high sensitivity and selectivity. The PLD activity in cell lysates is also determined by the nanoprobe, confirming that PLD activity in a breast cancer cell is at least 7-fold higher than in normal cell. Moreover, the nanoprobe has been successfully applied to monitoring PLD activity in living cells by UCF bioimaging. The results reveal that the nanoprobe provides a simple, sensitive, and robust platform for point-of-care diagnostics and drug screening in biomedical applications.

A novel nanocomplex displaying single-excitation and dual-emission fluorescent properties has been developed through a crown-like assembly of dye-encapsulated silica particles decorated with satellite AuNCs for live cell imaging of highly reactive oxygen species (hROS), including •OH, ClO(-) and ONOO(-). The design of this nanocomplex is based on our new finding that the strong fluorescence of AuNCs can be sensitively and selectively quenched by these hROS. The nanocomplex is demonstrated to have excellent biocompatibility, high intracellular delivery efficiency, and stability for long-time observations. The results reveal that the nanocomplex provides a sensitive sensor for rapid imaging of hROS signaling with high selectivity and contrast.

Nicotinamidases are metabolic enzymes that hydrolyze nicotinamide to nicotinic acid. These enzymes are widely distributed across biology, with examples found encoded in the genomes of Mycobacteria, Archaea, Eubacteria, Protozoa, yeast, and invertebrates, but there are none found in mammals. Although recent structural work has improved our understanding of these enzymes, their catalytic mechanism is still not well understood. Recent data show that nicotinamidases are required for the growth and virulence of several pathogenic microbes. The enzymes of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans regulate life span in their respective organisms, consistent with proposed roles in the regulation of NAD(+) metabolism and organismal aging. In this work, the steady state kinetic parameters of nicotinamidase enzymes from C. elegans, Sa. cerevisiae, Streptococcus pneumoniae (a pathogen responsible for human pneumonia), Borrelia burgdorferi (the pathogen that causes Lyme disease), and Plasmodium falciparum (responsible for most human malaria) are reported. Nicotinamidases are generally efficient catalysts with steady state k(cat) values typically exceeding 1 s(-1). The K(m) values for nicotinamide are low and in the range of 2 -110 μM. Nicotinaldehyde was determined to be a potent competitive inhibitor of these enzymes, binding in the low micromolar to low nanomolar range for all nicotinamidases tested. A variety of nicotinaldehyde derivatives were synthesized and evaluated as inhibitors in kinetic assays. Inhibitions are consistent with reaction of the universally conserved catalytic Cys on each enzyme with the aldehyde carbonyl carbon to form a thiohemiacetal complex that is stabilized by a conserved oxyanion hole. The S. pneumoniae nicotinamidase can catalyze exchange of (18)O into the carboxy oxygens of nicotinic acid with H(2)(18)O. The collected data, along with kinetic analysis of several mutants, allowed us to propose a catalytic mechanism that explains nicotinamidase and nicotinic acid (18)O exchange chemistry for the S. pneumoniae enzyme involving key catalytic residues, a catalytic transition metal ion, and the intermediacy of a thioester intermediate.

Nicotinamidases are salvage enzymes that convert nicotinamide to nicotinic acid. These enzymes are essential for the recycling of nicotinamide into NAD(+) in most prokaryotes and most single-cell and multicellular eukaryotes, but not in mammals. The significance of these enzymes for nicotinamide salvage and for NAD(+) homeostasis has stimulated interest in nicotinamidases as possible antibiotic targets. Nicotinamidases are also regulators of intracellular nicotinamide concentrations, thereby regulating signaling of downstream NAD(+)-consuming enzymes, such as the NAD(+)-dependent deacetylases (sirtuins). Here, we report several high-resolution crystal structures of the nicotinamidase from Streptococcus pneumoniae (SpNic) in unliganded and ligand-bound forms. The structure of the C136S mutant in complex with nicotinamide provides details about substrate binding, while a trapped nicotinoyl thioester in a complex with SpNic reveals the structure of the proposed thioester reaction intermediate. Examination of the active site of SpNic reveals several important features, including a metal ion that coordinates the substrate and the catalytically relevant water molecule and an oxyanion hole that both orients the substrate and offsets the negative charge that builds up during catalysis. Structures of this enzyme with bound nicotinaldehyde inhibitors elucidate the mechanism of inhibition and provide further details about the catalytic mechanism. In addition, we provide a biochemical analysis of the identity and role of the metal ion that orients the ligand in the active site and activates the water molecule responsible for hydrolysis of the substrate. These data provide structural evidence of several proposed reaction intermediates and allow for a more complete understanding of the catalytic mechanism of this enzyme.

Sirtuins are protein-modifying enzymes distributed throughout all forms of life. These enzymes bind NAD(+), a universal metabolite, and react it with acetyllysine residues to effect deacetylation of protein side chains. This NAD(+)-dependent deacetylation reaction has been observed for sirtuin enzymes derived from archaeal, eubacterial, yeast, metazoan, and mammalian species, suggesting conserved chemical mechanisms for these enzymes. The first chemical step of deacetylation is the reaction of NAD(+) with an acetyllysine residue which forms an enzyme-bound ADPR-peptidylimidate intermediate and nicotinamide. In this manuscript, the transition state for the ADP-ribosylation of acetyllysine is solved for an Archaeoglobus fulgidus sirtuin (Af2Sir2). Kinetic isotope effects (KIEs) were obtained by the competitive substrate method and were [1(N)-(15)N] = 1.024(2), [1'(N)-(14)C] = 1.014(4), [1'(N)-(3)H] = 1.300(3), [2'(N)-(3)H] = 1.099(5), [4'(N)-(3)H] = 0.997(2), [5'(N)-(3)H] = 1.020(5), [4'(N)-(18)O] = 0.984(5). KIEs were calculated for candidate transition state structures using computational methods (Gaussian 03 and ISOEFF 98) in order to match computed and experimentally determined KIEs to solve the transition state. The results indicate that the enzyme stabilizes a highly dissociated oxocarbenium ionlike transition state with very low bond orders to the leaving group nicotinamide and the nucleophile acetyllysine. A concerted yet highly asynchronous substitution mechanism forms the ADPR-peptidylimidate intermediate of the sirtuin deacetylation reaction.

Methods to construct 2'-deoxy-2'-fluoro nucleosides have undergone limited improvement in the last 20 years in spite of the substantially increased value of these compounds as pharmaceuticals and as tools for studying biological processes. We herein describe a consolidated approach to synthesize precursors to these commercially and scientifically valuable compounds via diastereocontrolled fluorination of the readily available precursor 2-deoxy-d-ribonolactone. With employment of appropriate sterically bulky silyl protecting groups at the 3 and 5 positions, controlled electrophilic fluorination of the Li-ribonolactone enolate by N-fluorodibenzenesulfonamide yielded the corresponding 2-deoxy-2-fluoroarabinolactone in high isolated yield (72%). The protected 2-deoxy-2,2-difluororibonolactone was obtained similarly in high yield from a second round of electrophilic fluorination (two steps, 51% from protected ribonolactone starting material). Accomplishment of the difficult ribofluorination of the lactone was achieved by the directive effects of a diastereoselectively installed alpha-trimethylsilyl group. Electrophilic fluorination of a protected 2-deoxy-2-trimethylsilylarabinolactone via enolate generation provided the protected 2-deoxy-2-fluororibolactone as the exclusive fluorinated product. The reaction also yielded the starting material, the desilylated protected 2-deoxyribonolactone, which was recycled to provide a 38% chemical yield of the fluorinated product (versus initial protected ribonolactone) after consecutive silylation and fluorination cycles. Using our fluorinated sugar precursors, we prepared the 2'-fluoroarabino-, 2'-fluororibo-, and 2',2'-difluoronicotinamide adenine dinucleotides (NAD(+)) of potential biological interest. These syntheses provide the most consolidated and efficient methods for production of sugar precursors of 2'-deoxy-2'-fluoronucleosides and have the advantage of utilizing an air-stable electrophilic fluorinating agent. The fluorinated NAD(+)s are anticipated to be useful for studying a variety of cellular metabolic and signaling processes.

Sirtuins are NAD (+)-dependent enzymes that deacetylate a variety of cellular proteins and in some cases catalyze protein ADP-ribosyl transfer. The catalytic mechanism of deacetylation is proposed to involve an ADPR-peptidylimidate, whereas the mechanism of ADP-ribosyl transfer to proteins is undetermined. Herein we characterize a Plasmodium falciparum sirtuin that catalyzes deacetylation of histone peptide sequences. Interestingly, the enzyme can also hydrolyze NAD (+). Two mechanisms of hydrolysis were identified and characterized. One is independent of acetyllysine substrate and produces alpha-stereochemistry as established by reaction of methanol which forms alpha-1- O-methyl-ADPR. This reaction is insensitive to nicotinamide inhibition. The second solvolytic mechanism is dependent on acetylated peptide and is proposed to involve the imidate to generate beta-stereochemistry. Stereochemistry was established by isolation of beta-1- O-methyl-ADPR when methanol was added as a cosolvent. This solvolytic reaction was inhibited by nicotinamide, suggesting that nicotinamide and solvent compete for the imidate. These findings establish new reactions of wildtype sirtuins and suggest possible mechanisms for ADP-ribosylation to proteins. These findings also illustrate the potential utility of nicotinamide as a probe for mechanisms of sirtuin-catalyzed ADP-ribosyl transfer.