Rapid Detection of Bacterial Resistance to ß-Lactam Antibiotics with a Relay-Response Chemiluminescence Assay.

Bacterial resistance caused by ß-lactamases has been a major threat to public health around the world, seriously weakening the efficacy of ß-lactam antibiotics, the most widely used therapeutic agents against infectious diseases. To detect the bacterial resistance to ß-lactam antibiotics, particularly specific type of ß-lactam antibiotics, in a rapid manner, we report herein a relay-response chemiluminescence assay. This assay mainly consists of two reagents: a ß-lactam-caged thiophenol and a thiophenol-sensitive chemiluminescence reporter, both of which are synthetically feasible. The selective hydrolysis of ß-lactam by ß-lactamase leads to the releasing of free thiophenol, which then triggers the emission of a chemiluminescence signal in a relay manner. Three thiophenol-caged ß-lactams, structural analogues of cephalothin, cefotaxime, and meropenem, respectively, have been synthesized. And the application of this assay with these analogues of ß-lactam antibiotics allows fast detection of ß-lactamase-expressing resistant bacteria and, more impressively, provides detailed information on the resistant scope of bacteria.

Enzyme-Activatable Near-Infrared Photosensitizer with High Enrichment in Tumor Cells Based on a Multi-Effect Design.

Enzyme-activatable near-infrared (NIR) fluorescent probes and photosensitizers (PSs) have emerged as promising tools for molecular imaging and photodynamic therapy (PDT). However, in living organisms selective retention or even enrichment of these reagents after enzymatic activation at or near sites of interest remains a challenging task. Herein, we integrate non-covalent and covalent retention approaches to introduce a novel "1-to-3" multi-effect strategy-one enzymatic stimulus leads to three types of effects-for the design of an enzyme-activatable NIR probe or PS. Using this strategy, we have constructed an alkaline phosphatase (ALP)-activatable NIR fluorogenic probe and a NIR PS, which proved to be selectively activated by ALP to switch on NIR fluorescence or photosensitizing ability, respectively. Additionally, these reagents showed significant enrichment (over 2000-fold) in ALP-overexpressed tumor cells compared to the culture medium, accompanied by massive depletion of intracellular thiols, the major antioxidants in cells. The investigation of this ALP-activatable NIR PS in an in vivo PDT model resulted in complete suppression of HeLa tumors and full recovery of all tested mice. Encouragingly, even a single administration of this NIR PS was sufficient to completely suppress tumors in mice, demonstrating the high potential of this strategy in biomedical applications.

Stereochemically altered cephalosporins as potent inhibitors of New Delhi metallo-ß-lactamases.

Antibiotic resistance caused by ß-lactamases, particularly metallo-ß-lactamases, has been a major threat to public health globally. New Delhi metallo-ß-lactamase-1 (NDM-1) represents one of the most important metallo-ß-lactamases; the production of NDM-1 in bacterial pathogen significantly reduces the efficacy of ß-lactam antibiotics, including life-saving carbapenems. Herein, we have demonstrated stereochemically altered cephalosporins as potent inhibitors against NDM-1, as well as mutants of NDM. The structure and activity relationship (SAR) study on over twenty cephalosporin analogues discloses the stereochemistry and the substituents on 7-position and 3'-position of cephalosporin are critical to suppress the activity of NDM-1 and the optimal compound 1u exhibited an IC50 of 0.13 µM. Furthermore, a crystal complex of NDM-1 and 1u has been obtained, suggesting this cephalosporin derivative inhibits enzyme activity by the formation of a relatively stable hydrolytic product-NDM-1 intermediate. The discovery in this study may pave the way to turn cephalosporin, a natural substrate of ß-lactamase, into an effective NDM-1 inhibitor to combat antibiotic resistance.

Optimization and Developmental Validation of 38-plex InDel Panel for Ancestry Inference.

OBJECTIVES: The previously established 38-plex InDel system was optimized and its performance was validated according to the Scientific Working Group on DNA Analysis Method (SWGDAM) application guidelines. The ancestry inference accuracy of individuals from East Asian, European, African and mixed populations was verified. METHODS: DNA standard sample 9947A was used as the template to establish the optimal amplification conditions by adjusting primer balance, Mg2+ final concentration and optimizing PCR thermal cycle parameters and amplification volume. The allelic dropout, nonspecific amplification and whether the origin of the inferred samples matched the known information were compared to evaluate the performance of this system. RESULTS: The optimal dosage of this system was 0.125-2 ng DNA template. The results of InDel typing were accurate, the amplification equilibrium was good, and the species specificity was good. This system showed certain tolerance to DNA samples including the inhibitor such as hemoglobin (≤80 µmol/L), indigo (≤40 mmol/L), calcium ion (≤1.0 mmol/L), and humic acid (≤90 ng/µL). The system enabled the direct amplification of DNA from saliva and blood on filter paper, and the results of ethnic inference were accurate. The system successfully detected the mixed DNA sample from two individuals. The test results of the system for common biological materials in practical cases were accurate. CONCLUSIONS: The results of the 38-plex InDel system are accurate and reliable, and the performance of the system meets the requirement of the SWGDAM guidelines. This system can accurately differentiate the ancestry origins of individuals from African, European, East Asian, and Eurasian populations and can be implemented in forensic practice.

Specific detection of IMP-1 ß-lactamase activity using a trans cephalosporin-based fluorogenic probe.

A fluorogenic probe for the specific detection of IMP-1 ß-lactamase activity has been developed. This imaging reagent features a unique trans-acetylamino cephalosporin as an enzymatic recognition moiety, exhibiting excellent selectivity to IMP-1 ß-lactamase over other ß-lactamases, including serine- and metallo-ß-lactamases. The selective activation of the probe by IMP-1 ß-lactamase leads to over 30-fold enhancement in the fluorescence intensity, which allows enzyme activity to be reported with high sensitivity.

Regioselective Difluoromethane sulfonylation and Triflylation of Resorufin Derivatives.

Reported herein is a regioselective difluoromethane sulfonylation or triflylation of resorufin derivatives, which allows easy access to 2-difluoromethane sulfonylated or triflylated resorufin derivatives in good yields. The installation of a difluoromethane sulfonyl group significantly increases the solubility of the chromophore and expands its Stokes shift. A difluoromethane sulfonylated resorufin-based fluorogenic probe proved to be able to image enzyme activity in live cells with a stronger fluorescence signal compared with its resorufin counterpart.

Screening aptamers for serine ß-lactamase-expressing bacteria with Precision-SELEX.

Klebsiella pneumoniae carbapenemase 2 (KPC-2) is a serine ß-lactamase that can hydrolyze almost all ß-lactam antibiotics. The drug resistant problem of bacteria expressing carbapenemases is currently a global problem, therefore, rapid and specific detection of pathogenic bacteria is urgent. In order to obtain an aptamer that can specifically recognize bacteria expressing KPC-2, we have established a method called Precision-SELEX. Precision-SELEX combined protein SELEX and bacterium SELEX. In this method, KPC-2 was used as a target protein, and Escherichia coli expressing KPC-2 (KPC-2 E. coli) was used as a target bacterium. After precision-SELEX, the same aptamer named XK-10 that can recognize KPC-2 and KPC-2 E. coli was obtained while the screening process could be shortened to 4 rounds. Dissociation equilibrium constants were calculated as 0.81 nM by SPR. In addition, XK-10 could specifically bind to KPC-2 E. coli, which was confirmed through flow cytometry and molecular Docking Simulations. The high-content imaging method could detect KPC-2 E. coli. In all, the Precision-SELEX provides an accurate and efficient method to screening aptamers for bacteria.

In Vivo Visualization of γ-Glutamyl Transpeptidase Activity with an Activatable Self-Immobilizing Near-Infrared Probe.

γ-Glutamyl transpeptidase (GGT), a type of cell membrane-bound enzyme, is closely involved in a wide range of physiological and pathological processes, and a large number of fluorogenic probes have been developed to detect the activity of GGT. However, the use of these imaging reagents to visualize GGT activity in vivo is largely limited because of rapid diffusion and clearance of activated fluorophores. Herein, by merging quinone methide and a fluorogenic enzyme substrate, we report an activatable self-immobilizing near-infrared probe for the in vitro and in vivo imaging of GGT activity. This probe is initially fluorescently silent, but the selective activation by GGT is able to significantly increase its fluorescence intensity at 714 nm and covalently anchor activated fluorophores at the site of interest. We have shown that this probe induced a much stronger fluorescence on live GGT-overexpressing cells compared to regular fluorogenic probes and allowed wash-free and real-time imaging of enzyme activity. More importantly, the use of this probe in the imaging of GGT activity in U87MG tumor-bearing mice by i.v. administration indicates that this self-immobilizing reagent is capable of efficiently enhancing its retention at the detection target and thus leads to much improved detection sensitivity compared to regular fluorogenic probes. This study demonstrates the advantage of fluorogenic probes with activatable anchors in the noninvasive imaging of enzyme activity in highly dynamic in vivo systems.

A self-immobilizing near-infrared fluorogenic probe for sensitive imaging of extracellular enzyme activity in vivo.

Reported herein is a self-immobilizing near-infrared fluorogenic probe that can be used to image extracellular enzyme activity in vivo. Using a fluorophore as a quinone methide precursor, this probe covalently anchors at sites of activation and greatly enhances the fluorescence intensity at 710 nm upon enzymatic stimulus, significantly boosting detection sensitivity in a highly dynamic in vivo system.

A novel method combining aptamer-Ag10NPs based microfluidic biochip with bright field imaging for detection of KPC-2-expressing bacteria.

The ß-lactam drugs resistance poses a serious threat to human health throughout the world. Klebsiella pneumoniae carbapenemase 2 (KPC-2) is a carbapenemase that produced in bacteria can hydrolyze carbapenems, which typically considered as the antibiotics of last resort. Therefore, there is an urgent need to quickly and accurately detect whether bacteria express KPC-2. In this paper, a PDMS/glass microfluidic biochip integrated with aptamer-modified Ag10NPs nano-biosensors was developed for rapid, simple and specific pathogenic bacteria detection, more importantly, the biochip was combined with bright field imaging, then the captured bacteria could be observed and counted directly without using extra chemical labeling. KPC-2-expressing Escherichia coli (KPC-2 E.coli) was used as the target bacterium with a detected limit of 102 CFU and capture efficiency exceeded 90%. This method is remarkably specific towards KPC-2 E.coli over other non-resistant bacteria, and pathogen assay only takes ∼1 h to complete in a ready-to-use microfluidic biochip. Furthermore, the effective capture and fast counting of microfluidic biochip system demonstrates its potential for the rapid detection of antibiotic-resistant bacteria.

A minor structure modification serendipitously leads to a highly carbapenemase-specific fluorogenic probe.

Reported herein is a fluorogenic probe for the detection of carbapenemase activity. This reagent features carbapenem as an enzyme recognition motif and a carbon-carbon double bond between carbapenem and the fluorophore, exhibiting high specificity to all carbapenemases, including metallo carbapenemases and serine carbapenemases, over other ß-lactamases.

Late-stage difluoromethylation leading to a self-immobilizing fluorogenic probe for the visualization of enzyme activities in live cells.

Reported herein is a novel p-quinone methide-based self-immobilizing fluorogenic probe for the visualization of ß-galactosidase activities in live cells. This easily prepared imaging reagent massively increases the fluorescence intensity and covalently links to the activation site with high efficiency upon enzymatic trigger.

Highly Efficient Multiple-Labeling Probes for the Visualization of Enzyme Activities.

Quinone methide (QM) as a latent trapping unit has been widely explored in activity-based self-immobilizing reagents. However, further application of this strategy has been largely hampered by the limited labeling efficiency to proteins. In this study, a thorough investigation on the labeling efficiency and the structure of QM-based trapping unit is presented, from which a QM with multiple leaving groups was identified as an optimal trapping unit. An alkaline phosphatase (ALP) immobilizing reagent featured with this multiple-labeling trapping unit exhibited lower nonspecific binding and, remarkably, a significantly higher labeling efficiency over other immobilizing reagents upon enzymatic activation. The utility of this imaging reagent was further demonstrated with the in vitro and in vivo visualization of the ALP activities. Furthermore, the multiple functional trapping unit may find greater value in the other activity-based immobilizing probes.

Highly selective and wash-free visualization of resistant bacteria with a relebactam-derived fluorogenic probe.

Reported herein is a relebactam-derived fluorogenic reagent for covalent labeling of serine ß-lactamases (SBLs), which are the major causes of bacterial resistance to ß-lactam antibiotics. This highly selective imaging reagent generates over 300-fold stronger near-infrared fluorescence signals upon covalently bonding to SBLs, allowing wash-free visualization of live antimicrobial-resistant bacteria.

Copper-mediated direct thiolation of aryl C-H bonds with disulfides.

An efficient copper-mediated ortho-C(sp2)-H thiolation of aromatic amides directed by a novel directing group [4-chloro-2-(1H-pyrazol-1-yl)phenyl]amine has been developed without the need of other additives or oxidants, allowing for an increased usefulness. With the high compatibility of sterically demanding substrates, this reaction is scalable and can tolerate a wide scope of functional groups to provide alkyl and aryl thioethers in good to excellent yields (up to 93%). Furthermore, the protocol has been successfully implemented for the selenylation as well.

A Carbapenem-Based Off-On Fluorescent Probe for Specific Detection of Metallo-ß-Lactamase Activities.

Metallo-ß-lactamase is one of the major clinical threats because this ß-lactam-hydrolyzing enzyme confers significant resistance to most ß-lactam antibiotics, including carbapenems, among bacterial pathogens. Reported herein is a novel fluorogenic sensor for the specific detection of metallo-ß-lactamase activities. This carbapenem-based reagent exhibits excellent selectivity to metallo-ß-lactamase over other serine-ß-lactamases, including serine carbapenemases. The usefulness of this probe was further demonstrated in the rapid identification of metallo-ß-lactamase-expressing pathogenic bacteria.

A carbapenem-based fluorescence assay for the screening of metallo-ß-lactamase inhibitors.

Reported herein is a fluorescence assay for the rapid screening of metallo-ß-lactamase (MBL) inhibitors. This assay employs a fluorogenic carbapenem CPC-1 as substrate and is compatible with all MBLs, including B1, B2 and B3 subclass MBLs. The efficiency of this assay was demonstrated by the rapid inhibition screening of a number of molecules against B2 MBL CphA and 2,3-dimercaprol was identified as a potent CphA inhibitor.

Surface Modified with a Host Defense Peptide-Mimicking ß-Peptide Polymer Kills Bacteria on Contact with High Efficacy.

Methicillin-resistant Staphylococcus aureus (MRSA) has been one of the major nosocomial pathogens to cause frequent and serious infections that are associated with various biomedical surfaces. This study demonstrated that surface modified with host defense peptide-mimicking ß-peptide polymer, has surprisingly high bactericidal activities against Escherichia coli ( E. coli) and MRSA. As surface-tethered ß-peptide polymers cannot move freely to adopt the collaborative interactions with bacterial membrane and are too short to penetrate the cell envelop, we proposed a mode of action by diffusing away the cell membrane-stabilizing divalent ions, Ca2+ and Mg2+. This hypothesis was supported by our study that Ca2+ and Mg2+ supplementation in the assay medium causes up to 80% loss of bacterial killing efficacy and that the addition of divalent ion chelating ethylenediaminetetraacetic acid into the above assay medium leads to significant recovery of the bacterial killing efficacy. In addition to its potent bacterial killing efficacy, the surface-tethered ß-peptide polymer also demonstrated excellent biocompatibility by displaying no hemolysis and supporting mammalian cell adhesion and growth. In conclusion, this study demonstrated the potential of ß-peptide polymer-modified surface in addressing nosocomial infections that are associated with various surfaces in biomedical applications.

A Brønsted acid-promoted asymmetric intramolecular allylic amination of alcohols.

Reported herein is a chiral Brønsted acid-catalyzed asymmetric intramolecular allylic amination reaction, allowing facile access to a range of biologically interesting chiral 2-substituted hydroquinolines in up to 90% yield and with up to 93% ee. Furthermore, a significant effect of an N-protecting group was observed in this asymmetric process.

Organocatalytic Asymmetric Formal [4 + 2] Cycloaddition of in Situ Oxidation-Generated ortho-Quinone Methides and Aldehydes.

An unprecedented chiral secondary amine-catalyzed formal [4 + 2] annulation of aldehydes and oxidation-generated ß-unsubstituted o-QMs is reported. This asymmetric protocol allows direct functionalization of the benzylic C-H bonds and furnishes [4 + 2] cycloadducts, chromanols, with excellent enantioselectivity and in up to 92% yield. The usability of this approach was further demonstrated by the enantioselective synthesis of anticancer Rhinacanthins derivative NKPLS8.