Penicillin allergy that persisted after 66 years: case report.

The most common documented allergy is due to penicillin use, and penicillin allergy is often diagnosed early in childhood. However, fewer than 1% of the approximately 10% of the population with reported penicillin allergy have a true allergy. Antimicrobial stewardship programmes have employed pharmacist-led protocols to rechallenge patients with a documented history of penicillin allergy. There are published data to suggest that patients with a history of penicillin allergy can be successfully rechallenged and desensitised. We report a case of a 74-year-old woman with a documented childhood history of penicillin allergy who was rechallenged with amoxicillin/clavulanate (Augmentin) in the hospital during admission. She was given one trial dose of amoxicillin/clavulanate for the treatment of urinary tract infection to cover organisms detected in the urine culture. Amoxicillin/clavulanate was determined to be the most suitable antibiotic for empirical treatment. Given a documented history of penicillin allergy from over 60 years ago, the likelihood of reactivity was suspected to be low to none. The patient, however, developed an allergic reaction after the one-time oral amoxicillin/clavulanate 875/125 mg dose trial.

The impact of orthokeratology lens wear on binocular vision and accommodation: A short-term prospective study.

PURPOSE: To investigate the effects of short-term orthokeratology (OK) on accommodation and binocular visual function in young adults. METHODS: Twenty-four myopes (18 to 38 years) were fitted with OK lenses in both eyes. Best corrected distance visual acuity (VA), subjective and objective refractions, corneal topography and a series of binocular vision tests were measured at baseline (BL) before lens wear and then repeated after 28 nights of OK. Data from 15 subjects who demonstrated successful OK lens fit are reported. RESULTS: Corneal flattening and hyperopic shifts in spherical equivalent refractive error (all p < 0.001) after 28 nights of OK indicated myopic correction. Improvement in best corrected distance VA was measured after OK (right eye p = 0.021; left eye p = 0.014). Although there was no significant change in mean distance and near phorias and stereoacuity scores after OK compared to BL, there was a significant reduction in standard deviation (SD) and range of data (distance p = 0.01; near p = 0.02; stereoacuity p < 0.001). While there appeared to be an improvement in distance accommodative facility after OK, this failed to reach statistical significance (p = 0.053). Furthermore, there was no change in AC/A gradients with ±1 D and ±2 D lenses after OK compared to BL. CONCLUSIONS: Binocular vision remained unchanged after OK, although variability of phoria and stereoacuity measures reduced. This suggests that OK improves or maintains accommodative and binocular vision function in young adult myopes who achieve good vision with OK. Myopes with phorias outside normal ranges and/or poor distance accommodative facility may benefit most with OK, in binocular and accommodative function.

Adult stem cells in the knifefish cerebellum.

Adult neurogenesis has been described in dozens of brain regions in teleost fish, with the largest number of new neurons being generated in the cerebellum. Here, we characterized the cerebellar neural stem/progenitor cells (NSPCs) in the brown ghost knifefish (Apteronotus leptorhynchus), an established model system of adult neurogenesis. The majority of the new cerebellar cells arise from neurogenic niches located medially, at the interface of the dorsal/ventral molecular layers and the granular layer. NSPCs within these niches give rise to transit-amplifying progenitors which populate the molecular layer, where they continue to proliferate during their migration toward target areas in the granular layer. At any given time, the majority of proliferating cells are located in the molecular layer. Immunohistochemical staining revealed that the stem cell markers Sox2, Meis1/2/3, Islet1, and, to a lesser extent, Pax6, are widely expressed in all regions of the adult cerebellum. A large subpopulation of these NSPCs coexpress S100, GFAP, and/or vimentin, indicating astrocytic identity. This is further supported by the specific effect of the gliotoxin l-methionine sulfoximine, which leads to a targeted decrease in the number of GFAP+ cells that coexpress Sox2 or the proliferation marker PCNA. Pulse-chase analysis of the label size associated with new cells after administration of 5-bromo-2'-deoxyuridine demonstrated that, on average, two additional cell divisions occur after completion of the initial mitotic cycle. Overall numbers of NSPCs in the cerebellum niches increase consistently over time, presumably in parallel with the continuous growth of the brain.

Kinetic cellular phenotypic profiling: prediction, identification, and analysis of bioactive natural products.

Natural products have always been a major source of therapeutic agents; however, the development of traditional herbal products has been currently hampered by the lack of analytic methods suitable for both high-throughput screening and evaluating the mechanism of action. Cellular processes such as proliferation, apoptosis, and toxicity are well-orchestrated in real time. Monitoring these events and their perturbation by natural products can provide high-rich information about cell physiological relevancies being involved. Here, we report a novel cell-based phenotypic profiling strategy that uses electronic impedance readouts for real-time monitoring of cellular responses to traditional Chinese medicines (TCMs). The utility of this approach was used to screen natural herbs that have been historically documented to cure human diseases and that have been classified into seven clusters based on their mechanisms of action. The results suggest that herbal medicines with similar cellular mechanisms produce similar time/dose-dependent cell response profiles (TCRPs). By comparing the TCRPs produced by the Chinese medicinal Cordyceps sinensis with similar TCRPs of chemical compounds, we explored the potential use of herbal TCRPs for predicting cellular mechanisms of action, herbal authentications, and bioactive identification. Additionally, we further compared this novel TCRP technology with high-performance liquid chromatography (HPLC)-based methods for herbal origin-tracing authentication and identification of bioactive ingredients. Together, our findings suggest that using TCRP as an alternative to existing spectroscopic techniques can allow us to analyze natural products in a more convenient and physiologically relevant manner.

Screening and identification of small molecule compounds perturbing mitosis using time-dependent cellular response profiles.

Cellular processes such as cell cycle progression, mitosis, apoptosis, and cell migration are characterized by well-defined events that are modulated as a function of time. Measuring these events in the context of time and its perturbation by small molecule compounds and RNAi can provide mechanistic information about cellular pathways being affected. We have used impedance-based time-dependent cell response profiling (TCRP) to measure and characterize cellular responses to antimitotic compounds or siRNAs. Our findings indicate that small molecule perturbation of mitosis leads to unique TCRP. We have further used this unique TCRP signature to screen 119 595 compound library and identified novel antimitotic compounds based on clustering analysis of the TCRPs. Importantly, 113 of the 117 hit compounds in the TCRP antimitotic cluster were confirmed as antimitotic based on independent assays, thus establishing the robust predictive nature of this profiling approach. In addition, potent and novel agents that induce mitotic arrest either by directly interfering with tubulin polymerization or by other mechanisms were identified. The TCRP approach allows for a practical and unbiased phenotypic profiling and screening tool for small molecule and RNAi perturbation of specific cellular pathways and time resolution of the TCRP approach can serve as a complement for other existing multidimensional profiling approaches.

Natural product derivative Bis(4-fluorobenzyl)trisulfide inhibits tumor growth by modification of beta-tubulin at Cys 12 and suppression of microtubule dynamics.

Bis(4-fluorobenzyl)trisulfide (BFBTS) is a synthetic molecule derived from a bioactive natural product, dibenzyltrisulfide, found in a subtropical shrub, Petiveria allieacea. BFBTS has potent anticancer activities to a broad spectrum of tumor cell lines with IC50 values from high nanomolar to low micromolar and showed equal anticancer potency between tumor cell lines overexpressing multidrug-resistant gene, MDR1 (MCF7/adr line and KBv200 line), and their parental MCF7 line and KB lines. BFBTS inhibited microtubule polymerization dynamics in MCF7 cells, at a low nanomolar concentration of 54 nmol/L, while disrupting microtubule filaments in cells at low micromolar concentration of 1 micromol/L. Tumor cells treated with BFBTS were arrested at G2-M phase, conceivably resulting from BFBTS-mediated antimicrotubule activities. Mass spectrometry studies revealed that BFBTS bound and modified beta-tubulin at residue Cys12, forming beta-tubulin-SS-fluorobenzyl. The binding site differs from known antimicrotubule agents, suggesting that BFBTS functions as a novel antimicrotubule agent. BFBTS at a dose of 25 mg/kg inhibited tumor growth with relative tumor growth rates of 19.91%, 18.5%, and 23.42% in A549 lung cancer, Bcap-37 breast cancer, and SKOV3 ovarian cancer xenografts, respectively. Notably, BFBTS was more potent against MDR1-overexpressing MCF7/adr breast cancer xenografts with a relative tumor growth rate of 12.3% than paclitaxel with a rate of 43.0%. BFBTS displays a novel antimicrotubule agent with potentials for cancer therapeutics.

Submaximal stimulation and f-wave parameters.

PURPOSE: This study evaluates the effect of submaximal stimulation and varying stimulus rates on F-wave parameters. METHODS: F-waves were recorded from the abductor pollicis brevis muscle from 3 normal subjects stimulating at 25%, 50%, and 75% intensity in comparison with supramaximal stimulation based on peak-to-peak compound muscle action potential amplitude. The effect of varying stimulus intensity (0.5, 1.0, and 2.0 Hz) at 30% stimulus intensity was also evaluated. Data were evaluated based on "true" values obtained after 100 stimuli. F-wave parameters studied included latencies (minimal and mean), amplitudes, persistences, durations, chronodispersions, and mean F amplitudes/maximum compound muscle action potential amplitudes. RESULTS: For varying stimulus intensities and rates, the following results were obtained: (1) no meaningful change in F latencies or durations; (2) mean latency values were more reproducible than minimum; (3) amplitudes, persistence, and mean F amplitudes/maximum M-wave amplitude ratios increase linearly with increase in stimulus intensities; (4) chronodispersion increases with increase in stimulus intensity; (5) 20 stimuli appear adequate for true values at supramaximal stimulation but more are needed at submaximal levels; (6) varying stimulus frequency at submaximal stimulation did not meaningfully affect the results. CONCLUSIONS: F-parameters require more stimuli at submaximal stimulation and, except for latencies and durations, would require different normal values than at supramaximal stimulation.

Live cell quality control and utility of real-time cell electronic sensing for assay development.

In this paper we have explored the utility of the real-time cell electronic sensing (RTCES, ACEA Biosciences Inc., San Diego, CA) system for monitoring the quality of live cells in cell-based assays as well as for assay development. We have demonstrated that each cell type displays unique growth kinetic profiles that provide a quantitative account of cell behavior and can be used as a diagnostic tool for cellular quality control. The utility of the specific signature patterns was shown by demonstrating the significant differences in primary cell behavior depending on the supplier. In addition, the RT-CES system was able to differentiate cell behavior depending on the passage stage of the cells. The utility of the RT-CES system as an assay development tool was demonstrated in cytotoxicity assays. The RT-CES system not only provides information regarding the potency of cytotoxic compounds, but in addition relates potency to the rate of the response for each concentration of the compound tested, which is important for understanding the mechanism of compound action. Moreover, real-time display of cytotoxicity data by the RT-CES system allows for calculation of real-time 50% inhibitory concentration (IC50) values or determination of optimal IC(50) value. In summary, the RT-CES system provides high content and information-rich data that are beyond the scope of single-point assays.

Synthesis and anti-tumor evaluation of new trisulfide derivatives.

New bis-aromatic and heterocyclic trisulfide derivatives 5, 7-10 were synthesized by optimizing lead dibenzyl trisulfide natural product (4) to evaluate their anti-tumor activities. Five compounds 5-7, 9, and 10 exhibited potent anti-tumor activities against eight different tumor cell lines with low cytotoxicity against HepG2. Initial SAR was discussed, and MOA of these anti-microtubule agents was suggested based on cell kinetic response patterns observed on RT-CES system.

Dynamic and label-free monitoring of natural killer cell cytotoxic activity using electronic cell sensor arrays.

A microelectronic sensor-based platform, the RT-CES (real time electronic sensing) system, is introduced for label free assessment of natural killer (NK) cell-mediated cytotoxic activity. The RT-CES system was used to dynamically and quantitatively monitor NK-mediated cytotoxic activity towards 8 different adherent target cell lines, including cancer cell lines commonly used in laboratories. The cytotoxic activity monitored by RT-CES system was compared with standard techniques such as MTT measurement and shows good correlation and sensitivity. To test the specificity of the assay, pharmacological agents that inhibit NK cell degranulation and cytotoxic activity were employed and were shown to selectively and dose-dependently inhibit NK-mediated cytotoxic activity toward target cells. In summary, the RT-CES system offers fully automated measurement of cytotoxic activity in real time, which enables large-scale screening of chemical compounds or genes responsible for the regulation of NK-mediated cytotoxic activity.

Real-time monitoring of morphological changes in living cells by electronic cell sensor arrays: an approach to study G protein-coupled receptors.

G protein-coupled receptors (GPCRs) constitute important targets for drug discovery against a wide range of ailments including cancer, inflammatory, and cardiovascular diseases. Efforts are underway to screen selective modulators of GPCRs and also to deorphanize GPCRs with unidentified natural ligands. Most GPCR-based cellular screens depend on labeling or recombinant expression of receptor or reporter proteins, which may not capture the true physiology or pharmacology of the GPCRs. In this paper, we describe a noninvasive and label-free assay for GPCRs that can be used with both engineered and nonengineered cell lines. The assay is based on using cell-electrode impedance to measure minute changes in cellular morphology as a result of ligand-dependent GPCR activation. We have used this technology to assay the functional activation of GPCRs coupled to different signaling pathways and have compared it to standard assays. We have used pharmacological modulators of GPCR signaling pathways to demonstrate the specificity of impedance-based measurements. Our data indicate that cell-electrode impedance measurements offer a convenient, sensitive, and quantitative method for assessing GPCR function. Moreover, the noninvasive nature of the readout offers the added advantage of performing multiple treatments in the same well to study events such as desensitization and receptor cross-talk.

Dynamic monitoring of cell adhesion and spreading on microelectronic sensor arrays.

Cellular interaction with and adhesion on different biological surfaces is a dynamic and integrated process requiring the participation of specialized cell surface receptors, structural proteins, signaling proteins, and the cellular cytoskeleton. In this report, the authors describe a label-free and real-time method for measuring and monitoring cell adhesion on special microplates integrated with electronic cell sensor arrays. These plates were used in conjunction with the real-time cell electronic sensing (RT-CES) system to dynamically and quantitatively monitor the specific interaction of fibroblasts with extracellular matrix (ECM) proteins and compared with standard adhesion techniques. Cell adhesion on ECM-coated cell sensor arrays is dependent on the concentration of ECM proteins coated and is inhibited by agents that disrupt the interaction of ECM with cell surface receptors. Furthermore, the authors demonstrate that the integrity of the actin cytoskeleton is required for productive cell adhesion and spreading on ECM-coated microelectronic sensors. Confirming earlier results, it is shown that interfering with Src expression or activity, via siRNA or small molecule, results in the disruption of adhesion and spreading of Bx PC3 cells. The results indicate that the RT-CES system offers a convenient and quantitative means of assessing the kinetics of cell adhesion in a high-throughput manner.

Label-free, real-time monitoring of IgE-mediated mast cell activation on microelectronic cell sensor arrays.

Immunoglobulin E (IgE)-mediated mast cell activation is involved in the immediate phase of allergic reactions and plays a central role in the onslaught and persistence of allergic diseases. IgE-mediated mast cell activation includes two important events: cell sensitization resulting from IgE binding to Fc (FcepsilonRI) receptor and cell activation triggered by allergen-mediated oligomerization of membrane-bound IgE. Real-time monitoring of these events is needed to dissect the molecular mechanisms underlying IgE-mediated mast cell activation. Existing technologies are limited to label-based end-point assay formats, which detect either early signaling or final phase of mast cell activation. We describe a microelectronic cell sensor-based technology allowing dynamic monitoring of IgE-mediated mast cell sensitization and activation in real-time without any labeling steps. RBL-2H3 mast cells were cultured onto the surface of microelectronic cell sensor arrays integrated into the bottom of microtiter plates, which record electric properties, such as impedance between cell membrane and sensor surface. In the presence of the allergen, dinitrophenyl (DNP)-bovine serum albumin (BSA), anti-DNP IgE-sensitized cells were activated within 5 min and the entire activation process was quantitatively and continuously recorded. Impedance measurements correlate with morphological dynamics and mediator release as measured by beta-hexosaminidase activity, and can be blocked by pharmacological agents, inhibiting IgE-mediated signaling. The assay on microelectronic cell sensor arrays can be scaled up for high-throughput screening of pharmacological inhibitors of IgE-mediated mast cell activation and other cell-based receptor-ligand assays.