One-Step Detection and Classification of Bacterial Carbapenemases in 10 Minutes Using Fluorescence Identification of ß-Lactamase Activity.

Rapid and accurate diagnosis of bacterial carbapenemases remains a major challenge for clinical laboratories. A novel assay was developed here using fluorescence identification of ß-lactamase activity (FIBA) to permit rapid detection and classification of bacterial carbapenemases. By mixing a fluorogenic ß-lactamase substrate, ß-LEAF (ß-lactamase enzyme-activated fluorophore), with bacterial isolates plus the respective inhibitor (imipenem for noncarbapenemase ß-lactamases, clavulanic acid for type A carbapenemases, and EDTA for type B carbapenemases), objective results with 95% to 100% sensitivity and specificity were generated in 10 min. FIBA is ≈$1/test and consists of only a single mixing step. Given the combination of rapidity, accuracy, low cost, and simplicity, this novel carbapenemase detection and classification assay is well positioned to be applied in clinical microbiology laboratories to provide guidance for the choice of proper treatment and control of globally prevalent carbapenemase-positive infections.

Novel Rapid Test for Detecting Carbapenemase.

We developed a carbapenemase test based on the ability of imipenem to inhibit noncarbapenemase ß-lactamases. The test uses bacterial isolates with a fluorescent ß-lactamase substrate, producing objective results with 100% sensitivity and specificity in 10 minutes. The assay is inexpensive and consists of only 1 mixing step.

Selective treatment and monitoring of disseminated cancer micrometastases in vivo using dual-function, activatable immunoconjugates.

Drug-resistant micrometastases that escape standard therapies often go undetected until the emergence of lethal recurrent disease. Here, we show that it is possible to treat microscopic tumors selectively using an activatable immunoconjugate. The immunoconjugate is composed of self-quenching, near-infrared chromophores loaded onto a cancer cell-targeting antibody. Chromophore phototoxicity and fluorescence are activated by lysosomal proteolysis, and light, after cancer cell internalization, enabling tumor-confined photocytotoxicity and resolution of individual micrometastases. This unique approach not only introduces a therapeutic strategy to help destroy residual drug-resistant cells but also provides a sensitive imaging method to monitor micrometastatic disease in common sites of recurrence. Using fluorescence microendoscopy to monitor immunoconjugate activation and micrometastatic disease, we demonstrate these concepts of "tumor-targeted, activatable photoimmunotherapy" in a mouse model of peritoneal carcinomatosis. By introducing targeted activation to enhance tumor selectively in complex anatomical sites, this study offers prospects for catching early recurrent micrometastases and for treating occult disease.

Simultaneous delivery of cytotoxic and biologic therapeutics using nanophotoactivatable liposomes enhances treatment efficacy in a mouse model of pancreatic cancer.

A lack of intracellular delivery systems has limited the use of biologics such as monoclonal antibodies (mAb) that abrogate molecular signaling pathways activated to promote escape from cancer treatment. We hypothesized that intracellular co-delivery of the photocytotoxic chromophore benzoporphyrin derivative monoacid A (BPD) and the anti-VEGF mAb bevacizumab in a nanophotoactivatable liposome (nanoPAL) might enhance the efficacy of photodynamic therapy (PDT) combined with suppression of VEGF-mediated signaling pathways. As a proof-of-concept we found that nanoPAL-PDT induced enhanced extra- and intracellular bevacizumab delivery and enhanced acute cytotoxicity in vitro. In an in vivo subcutaneous mouse model of pancreatic ductal adenocarcinoma, nanoPAL-PDT achieved significantly enhanced tumor reduction. We attribute this to the optimal incorporation of insoluble BPD into the lipid bilayer, enhancing photocytotoxicity, and the simultaneous spatiotemporal delivery of bevacizumab, ensuring efficient neutralization of the rapid but transient burst of VEGF following PDT. From the Clinical Editor: Most patients with pancreatic ductal adenocarcinoma (PDAC) by the time present the disease it is very advanced, which unavoidably translates to poor survival. For these patients, use of traditional chemotherapy often becomes ineffective due to tumor resistance to drugs. Photodynamic therapy (PDT) can be an effective modality against chemo-resistant cancers. In this article, the authors investigated the co-delivery of a photocytotoxic agent and anti-VEGF mAb using liposomes. This combination was shown to results in enhanced tumor killing. This method should be applicable to other combination of treatments.

Torque-induced slip of the rotary motor F1-ATPase.

F(1)-ATPase plays an essential role in cellular metabolism by linking rotational motion to ATP hydrolysis/synthesis. We measure the torque profile of F(1) in both ATP hydrolysis and synthesis directions using a novel magnetic nanorod assay. F(1) is found to decouple ATP synthesis from rotary motion at a surprisingly low torque. This low-torque slip mechanism protects the enzyme from excessive load and may play a broader biological role by reducing the production of reactive oxygen species.

REAP (Rapid Elimination of Active Plasmodium): A photodynamic strategy exploiting intrinsic kinetics of the parasite to combat severe malaria.

Plasmodium falciparum, the causative organism of Malaria is a mosquito-borne parasitic disease which infects red blood cells (RBCs), where it multiplies rapidly and goes through different stages of its life cycle. When the parasite load exceeds >3% in the blood, malaria transforms into severe malaria which requires immediate attention as death occurs within hours to days. The increase in people traveling to malaria-endemic areas and resistance/partial resistance to most known antimalarial drugs has put the current management scheme in jeopardy. To improve the patient outcome at this point, the physician may opt to perform exchange transfusions from another individual as an adjunct therapy to reduce parasitized RBCs, but the strategy has many drawbacks, including chances of infection. These limitations can be mitigated if the patient's own blood is withdrawn/extracted, sterilized from the parasitic load and then re-transfused almost similar to what is done in extracorporeal blood treatment for sepsis, poisoning and graft versus host disease. Thus, in the present study a light-based photochemical approach, Photodynamic Therapy (PDT) built on delta-aminolevulinic acid-protoporphyrin IX (ALA-PpIX) synthesis is exploited. This modality was effective at destruction of both resistant and susceptible strains of parasites, including at a high load mimicking severe drug resistant malaria. The current findings have set the stage for concept of an ALA-PpIX based PDT platform, "the REAP (Rapid Elimination of Active Plasmodium) strategy". This approach provides an additional tool towards the defense against multi-drug resistant severe malaria, and other intracellular blood pathogens, dependent on heme-synthesis.

Simultaneous sizing and electrophoretic mobility measurement of sub-micron particles using Brownian motion.

The size and surface chemistry of micron scale particles are of fundamental importance in studies of biology and air particulate pollution. However, typical electrophoretic measurements of these and other sub-micron scale particles (300 nm-1 µm) cannot resolve size information within heterogeneous mixtures unambiguously. Using optical microscopy, we monitor electrophoretic motion together with the Brownian velocity fluctuations - using the latter to measure size by either the Green-Kubo relation or by calibration from known size standards. Particle diameters are resolved to ±12% with 95% confidence. Strikingly, the size resolution improves as the particle size decreases due to the increased Brownian motion. The sizing ability of the Brownian assessed electrophoresis method described here complements the electrophoretic mobility resolution of the traditional CE.

Photodynamic inactivation of bacterial carbapenemases restores bacterial carbapenem susceptibility and enhances carbapenem antibiotic effectiveness.

The global emergence of carbapenemases in bacterial pathogens has rendered many life-threatening infections untreatable. Even though using carbapenemase inhibitors are a proven strategy in the battle against bacterial carbapenem resistance, developing inhibitors that could universally inactivate all bacterial carbapenemases is extremely challenging given the large diversity and the continuous evolution of bacterial carbapenemases. Antimicrobial photodynamic therapy (aPDT), an upcoming antimicrobial therapy, is demonstrated here for the first time to be a generalized approach to impair the bacterial carbapenemases without being limited by the molecular identities of the carbapenemases. In addition, aPDT is shown to prevent carbapenem antibiotic degradation, thereby enhancing the efficacy of carbapenem antibiotic against the carbapenemase-producing pathogens. Besides the enzyme activity impairment, aPDT was documented here to be genetically toxic for bacteria, and thus radically damage the carbapenemase genetic determinants in bacteria and prevent the transmission of carbapenemases among pathogens. By leveraging the universal carbapenemase-inactivating property of aPDT, it may be possible to make the incurable infections caused by the bacterial carbapenemases susceptible to carbapenem again.

Multichannel correlation improves the noise tolerance of real-time hyperspectral microimage mosaicking.

Live-subject microscopies, including microendoscopy and other related technologies, offer promise for basic biology research as well as the optical biopsy of disease in the clinic. However, cellular resolution generally comes with the trade-off of a microscopic field-of-view. Microimage mosaicking enables stitching many small scenes together to aid visualization, quantitative interpretation, and mapping of microscale features, for example, to guide surgical intervention. The development of hyperspectral and multispectral systems for biomedical applications provides motivation for adapting mosaicking algorithms to process a number of simultaneous spectral channels. We present an algorithm that mosaics multichannel video by correlating channels of consecutive frames as a basis for efficiently calculating image alignments. We characterize the noise tolerance of the algorithm by using simulated video with known ground-truth alignments to quantify mosaicking accuracy and speed, showing that multiplexed molecular imaging enhances mosaic accuracy by leveraging observations of distinct molecular constituents to inform frame alignment. A simple mathematical model is introduced to characterize the noise suppression provided by a given group of spectral channels, thus predicting the performance of selected subsets of data channels in order to balance mosaic computation accuracy and speed. The characteristic noise tolerance of a given number of channels is shown to improve through selection of an optimal subset of channels that maximizes this model. We also demonstrate that the multichannel algorithm produces higher quality mosaics than the analogous single-channel methods in an empirical test case. To compensate for the increased data rate of hyperspectral video compared to single-channel systems, we employ parallel processing via GPUs to alleviate computational bottlenecks and to achieve real-time mosaicking even for video-rate multichannel systems anticipated in the future. This implementation paves the way for real-time multichannel mosaicking to accompany next-generation hyperspectral and multispectral video microscopy.

Guiding Empiric Treatment for Serious Bacterial Infections via Point of Care [Formula: see text]-Lactamase Characterization.

Fever is one of the most common symptoms of illness in infants and represents a clinical challenge due to the potential for serious bacterial infection. As delayed treatment for these infections has been correlated with increased morbidity and mortality, broad-spectrum [Formula: see text]-lactam antibiotics are often prescribed while waiting for microbiological lab results (1-3 days). However, the spread of antibiotic resistance via the [Formula: see text]-lactamase enzyme, which can destroy [Formula: see text]-lactam antibiotics, has confounded this paradigm; empiric antibiotic regimens are increasingly unable to cover all potential bacterial pathogens, leaving some infants effectively untreated until the pathogen is characterized. This can lead to lifelong sequela or death. Here, we introduce a fluorescent, microfluidic assay that can characterize [Formula: see text]-lactamase derived antibiotic susceptibility in 20 min with a sensitivity suitable for direct human specimens. The protocol is extensible, and the antibiotic spectrum investigated can be feasibly adapted for the pathogens of regional relevance. This new assay fills an important need by providing the clinician with hitherto unavailable point of care information for treatment guidance in an inexpensive and simple diagnostic format.

Microscale receiver operating characteristic analysis of micrometastasis recognition using activatable fluorescent probes indicates leukocyte imaging as a critical factor to enhance accuracy.

Molecular-targeted probes are emerging with applications for optical biopsy of cancer. An underexplored potential clinical use of these probes is to monitor residual cancer micrometastases that escape cytoreductive surgery and chemotherapy. Here, we show that leukocytes, or white blood cells, residing in nontumor tissues--as well as those infiltrating micrometastatic lesions--uptake cancer cell-targeted, activatable immunoconjugates nonspecifically, which limits the accuracy and resolution of micrometastasis recognition using these probes. Receiver operating characteristic analysis of freshly excised tissues from a mouse model of peritoneal carcinomatosis suggests that dual-color imaging, adding an immunostain for leukocytes, offers promise for enabling accurate recognition of single cancer cells. Our results indicate that leukocyte identification improves micrometastasis recognition sensitivity and specificity from 92 to 93%--for multicellular metastases >20 to 30 µm in size--to 98 to 99.9% for resolving metastases as small as a single cell.

Rapid, low-cost fluorescent assay of ß-lactamase-derived antibiotic resistance and related antibiotic susceptibility.

Antibiotic resistance (AR) is increasingly prevalent in low and middle income countries (LMICs), but the extent of the problem is poorly understood. This lack of knowledge is a critical deficiency, leaving local health authorities essentially blind to AR outbreaks and crippling their ability to provide effective treatment guidelines. The crux of the problem is the lack of microbiology laboratory capacity available in LMICs. To address this unmet need, we demonstrate a rapid and simple test of ß -lactamase resistance (the most common form of AR) that uses a modified ß -lactam structure decorated with two fluorophores quenched due to their close proximity. When the ß -lactam core is cleaved by ß -lactamase, the fluorophores dequench, allowing assay speeds of 20 min to be obtained with a simple, streamlined protocol. Furthermore, by testing in competition with antibiotics, the ß -lactamase-associated antibiotic susceptibility can also be extracted. This assay can be easily implemented into standard lab work flows to provide near real-time information of ß -lactamase resistance, both for epidemiological purposes as well as individualized patient care.

PDT dose parameters impact tumoricidal durability and cell death pathways in a 3D ovarian cancer model.

The successful implementation of photodynamic therapy (PDT)-based regimens depends on an improved understanding of the dosimetric and biological factors that govern therapeutic variability. Here, the kinetics of tumor destruction and regrowth are characterized by systematically varying benzoporphyrin derivative (BPD)-light combinations to achieve fixed PDT doses (M × J cm(-2)). Three endpoints were used to evaluate treatment response: (1) Viability evaluated every 24 h for 5 days post-PDT; (2) Photobleaching assessed immediately post-PDT; and (3) Caspase-3 activation determined 24 h post-PDT. The specific BPD-light parameters used to construct a given PDT dose significantly impact not only acute cytotoxic efficacy, but also treatment durability. For each dose, PDT with 0.25 µM BPD produces the most significant and sustained reduction in normalized viability compared to 1 and 10 µM BPD. Percent photobleaching correlates with normalized viability for a range of PDT doses achieved within BPD concentrations. To produce a cytotoxic response with 10 µM BPD that is comparable to 0.25 and 1 µM BPD a reduction in irradiance from 150 to 0.5 mW cm(-2) is required. Activated caspase-3 does not correlate with normalized viability. The parameter-dependent durability of outcomes within fixed PDT doses provides opportunities for treatment customization and improved therapeutic planning.

Rapid morphological characterization of isolated mitochondria using Brownian motion.

Mitochondrial morphology has been associated with numerous pathologies including cancer, diabetes, obesity and heart disease. However, the connection is poorly understood-in part due to the difficulty of characterizing the morphology. This impedes the use of morphology as a tool for disease detection/monitoring. Here, we use the Brownian motion of isolated mitochondria to characterize their size and shape in a high throughput fashion. By using treadmill exercise training, mitochondria from heart and gastrocnemius of Balb/c mice were modulated in size and used to investigate the protocol. Consistent with previous reports, the heart mitochondria of untrained mice increased 5% in diameter immediately after a single bout of moderate exercise (1.091 ± 0.004 µm) as compared to completely sedentary controls (1.040 ± 0.022 µm). In addition, no change was observed in the size of gastrocnemius mitochondria (1.025 ± 0.018 µm), which was also in agreement with previous studies. The method was also successfully applied to smaller Saccharomyces cerevisiae mitochondria.

Nonlinear dielectric spectroscopy as an indirect probe of metabolic activity in thylakoid membrane.

Nonlinear dielectric spectroscopy (NDS) is a non-invasive probe of cellular metabolic activity with potential application in the development of whole-cell biosensors. However, the mechanism of NDS interaction with metabolic membrane proteins is poorly understood, partly due to the inherent complexity of single cell organisms. Here we use the light-activated electron transport chain of spinach thylakoid membrane as a model system to study how NDS interacts with metabolic activity. We find protein modification, as opposed to membrane pump activity, to be the dominant source of NDS signal change in this system. Potential mechanisms for such protein modifications include reactive oxygen species generation and light-activated phosphorylation.

Nonlinear impedance of whole cells near an electrode as a probe of mitochondrial activity.

By simultaneously measuring the bulk media and electrode interface voltages of a yeast (Saccharomyces cerevisiae) suspension subjected to an AC voltage, a yeast-dependent nonlinear response was found only near the current injection electrodes. Computer simulation of yeast near a current injection electrode found an enhanced voltage drop across the yeast near the electrode due to slowed charging of the electrode interfacial capacitance. This voltage drop is sufficient to induce conformation change in membrane proteins. Disruption of the mitochondrial electron transport chain is found to significantly change the measured nonlinear current response, suggesting nonlinear impedance can be used as a non-invasive probe of cellular metabolic activity.

Nonlinear dielectric spectroscopy for label-free detection of respiratory activity in whole cells.

We report on a novel electromagnetic biosensing technique for detecting respiratory activity in whole cells suspended in aqueous solution. Application of a pure sinusoidal voltage between two outer electrodes applies an oscillatory electric field to the aqueous cell suspension at frequencies in the range of one to several kHz. The fundamental and higher order harmonic responses are measured across two inner electrodes using a dynamic signal analyzer. Aqueous suspensions of S. cerevisiae (budding yeast), with both active and inactive mitochondrial electron transport (respiratory) chains are employed for this study. We find that the measured third harmonic for certain frequency ranges shows significant temporal changes in actively respiring yeast, while little significant changes are observed in yeast with suppressed respiratory activity, i.e. mutant yeast strains or yeast in the presence of respiratory inhibitors. The method holds potential for further development to detect respiratory activity in live tissue in vitro and perhaps in vivo for clinical applications.