Probe's Ability Research Articles

Utilising a 1,8-naphthalimide probe for the ratiometric fluorescent visualisation of caspase-3.

The development of selective and sensitive probes for monitoring caspase-3 activity-a critical enzyme involved in apoptosis-remains an area of significant interest in biomedical research. Herein, we report the synthesis and characterisation of a novel ratiometric fluorescent probe, Ac-DEVD-PABC-Naph, designed to detect caspase-3 activity. The probe utilises a 1,8-naphthalimide fluorophore covalently linked to a peptide sequence via a self-immolative p-aminobenzyl alcohol (PABA) linker. Upon enzymatic cleavage by caspase-3, the probe undergoes spontaneous degradation, releasing the free naphthalimide fluorophore, resulting in a ratiometric change in fluorescence emission. Spectroscopic studies revealed a time-dependent ratiometric fluorescent response, demonstrating the probe's ability to visualise caspase-3 activity with high sensitivity. Enzyme kinetics such as K m (Michaelis constant), k cat (turnover number), and LOD (Limit of Detection) were obtained, suggesting that the probe possesses comparable kinetic data to other probes in literature, but with the added benefits of ratiometric detection. Selectivity studies also demonstrated the probe's specificity for caspase-3 over other endogenous species and enzymes. Ac-DEVD-PABC-Naph may be a promising tool for the quantitative detection and fluorescent visualisation of caspase-3 activity in biological systems, with potential applications in apoptosis research and drug development.

Synthesis and characterization of 64Cu-labeled Geldanamycin derivative for imaging HSP90 expression in breast cancer

Heat shock protein 90 (HSP90) plays a crucial role in cancer cell growth and metastasis by stabilizing overexpressed signaling proteins. Inhibiting HSP90 has emerged as a promising anti-cancer strategy. In this study, we aimed to develop and characterize a HSP90-targeted molecular imaging probe, [64Cu]Cu-DOTA-BDA-GM, based on a specific HSP90 inhibitor, geldanamycin (GM), for PET imaging of cancers. GM is modified at the C-17 position with 1,4-butane-diamine (BDA) and linked to 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for 64Cu radiolabeling. We evaluated the probe's specific binding to HSP90-expressing cells using Chinese hamster ovary (CHO) cells and breast cancer cells including MDA-MB-231, MDA-MB-435S, MCF7, and KR-BR-3 cell lines. A competition study with non-radioactive GM-BDA yielded an IC50 value of 1.35 ± 0.14 nM, underscoring the probe's affinity for HSP90. In xenograft models of MDA-MB-231 breast cancer, [64Cu]Cu-DOTA-BDA-GM showcased targeted tumor localization, with significant radioactivity observed up to 18 h post-injection. Blocking studies using unlabeled GM-BDA and treatment with the anticancer drug Vorinostat (SAHA), which can affect the expression and activity of numerous proteins, such as HSPs, confirmed the specificity and sensitivity of the probe in cancer targeting. Additionally, PET/CT imaging in a lung metastasis mouse model revealed increased lung uptake of [64Cu]Cu-DOTA-BDA-GM in metastatic sites, significantly higher than in non-metastatic lungs, illustrating the probe's ability to detect metastatic breast cancer. In conclusion, [64Cu]Cu-DOTA-BDA-GM represents a sensitive and specific approach for identifying HSP90 expression in breast cancer and metastases, offering promising implications for clinical diagnosis and monitoring.

A polydiacetylene (PDA) based dual-mode optical sensor for the ppb level selective detection of biogenic polyamines

Biogenic polyamines (BPAs), specifically spermine and spermidine, play vital roles in cellular physiology, influencing processes like cell growth and proliferation. Beyond their cellular implications, these amines serve as biomarkers for cancer and aging-related diseases. This study presents a dual-output optical sensor for BPAs using polydiacetylene (PDA). The sensor utilizes alendronate's phosphate groups as recognition units for BPAs in liquid and solid phases. Ammonium groups of BPAs interact with negatively charged alendronate-modified PDA (PDAPH) liposomes causing a color change from blue to red accompanied by a gradual increase in fluorescence output at λmax 622 nm. The sensor exhibits high sensitivity with low detection limits of 4.3 ppb and 5.1 ppb for spermine and spermidine in colorimetric analysis, and lower limits of 1.6 and 1.4 ppb in fluorimetric measurements respectively, demonstrating its dual output ability. PDAPH liposomes also respond to cadaverine and putrescine indicating the probe's ability to detect key BPAs. The sensor shows minimal responses to interfering species, confirming its selective affinity towards BPAs. Practical validation in real water, urine, and serum samples demonstrates the sensor's efficiency. Immobilization of PDAPH liposomes into sodium alginate results in the creation of PDA beads. These beads exhibit a discernible color change when exposed to BPA concentrations exceeding 0.3 μM with a sufficiently low detection limit of 11.7 ppb in the solid phase. These findings highlight the efficiency of the PDAPH liposome sensor for accurate and reliable biogenic polyamine detection.

Probing the Competition between Duplex, G-Quadruplex and i-Motif Structures of the Oncogenic c-Myc DNA Promoter Region.

Development of probe systems that provide unique spectral signatures for duplex, G-quadruplex (GQ) and i-motif (iM) structures is very important to understand the relative propensity of a G-rich-C-rich promoter region to form these structures. Here, we devise a platform using a combination of two environment-sensitive nucleoside analogs namely, 5-fluorobenzofuran-modified 2'-deoxyuridine (FBF-dU) and 5-fluoro-2'-deoxyuridine (F-dU) to study the structures adopted by a promoter region of the c-Myc oncogene. FBF-dU serves as a dual-purpose probe containing a fluorescent and 19 F NMR label. When incorporated into the C-rich sequence, it reports the formation of different iMs via changes in its fluorescence properties and 19 F signal. F-dU incorporated into the G-rich ON reports the formation of a GQ structure whose 19 F signal is clearly different from the signals obtained for iMs. Rewardingly, the labeled ONs when mixed with respective complementary strands allows us to determine the relative population of different structures formed by the c-Myc promoter by the virtue of the probe's ability to produce distinct and resolved 19 F signatures for different structures. Our results indicate that at physiological pH and temperature the c-Myc promoter forms duplex, random coil and GQ structures, and does not form an iM. Whereas at acidic pH, the mixture largely forms iM and GQ structures. Taken together, our system will complement existing tools and provide unprecedented insights on the population equilibrium and dynamics of nucleic acid structures under different conditions.

Precise design and synthesis of an AIE-active glutathione fluorescent probe and its exogenous biological imaging

Real-time and accurate detection of intracellular GSH content is crucial for clinical diagnosis, disease treatment, and pathogen detection. Creating fluorescent probes that possess aggregation induced emission (AIE) and precisely detect GSH remains a formidable challenge despite the widespread reporting of numerous fluorescent probes for GSH recognition. This may due to the absence of effective strategies. Herein, using a "turn off - turn on" strategy, a novel fluorescent probe DPND with AIE characteristic has been devised and synthesized. It can detect GSH selectively and uninhibitedly in the presence of various ions. The limit of detection of DPND-Cu2+ for GSH was calculated to be 1.17 μM, demonstrating the probe's ability to detect GSH quantitatively. More crucially, the probe exhibits low toxicity and solid biocompatibility while recognizing GSH in HeLa cells and mices, indicating that it may be a promising candidate for in vivo detection of GSH. Consequently, this work offers the potential to precisely tailor molecular recognition of GSH in addition to providing ongoing exploration of approaches for monitoring GSH.

An activatable NIR‐II fluorescent probe for tracking heavy‐metal ion and high‐level salt‐induced oxidative stress in plant sprouts

AbstractHumans and plants have become enfolded and inseparable. Abiotic stresses in particular oxidative stress caused by heavy‐metal ions or high‐level salt contamination deleteriously impact plants’ growth process and have become a major threat to sustaining food security. Sprouting is the first step in plants’ growth process. When plant sprouts endure oxidative stress induced by toxic heavy‐metal ions or high‐level salt, accelerated generation of reactive oxygen species (e.g., H2O2) occurs inside plant sprouts; hence in‐situ H2O2 in plant sprouts could serve as the in‐vivo biomarker for tracking the oxidative stress in plant sprouts. Herein, we design an activatable probe CT‐XA‐H2O2 to track the oxidative stress in plant sprouts via in vivo NIR‐II fluorescent imaging. In CT‐XA‐H2O2, cyano‐thiazole acts as the electron‐accepting moiety and xanthane‐aminodiphenyl as the electron‐donating moiety, and dioxaborolane as the biomarker‐responsive unit and fluorescence quencher. The probe CT‐XA‐H2O2 shows weak fluorescent emission. When H2O2 is present, the dioxaborolane in the probe is cleaved, consequently, the dye CT‐XA‐OH is generated and brings about significant fluorescent signals for detecting and imaging the in‐situ biomarker. Moreover, the aminodiphenyl group endues the chromophore (the activated probe) with aggregation‐induced emission characteristics, which ensures stronger fluorescence in the aggregated state in the aqueous milieu. The probe CT‐XA‐H2O2 has been employed in the Cd2+‐ion or high‐level salt (NaCl) induced oxidative stress models of soybean sprouts and peanut sprouts, and the experimental results evidently reveal the probe's ability for in‐situ biomarker‐activatable in‐vivo detection and imaging in the plants' sprouts.

A Side-Viewing Endoscopic Probe With Distal Micro Rotary Scanner for Multimodal Luminal Imaging and Analysis

This paper reports a novel micro electromagnetic actuator serving as the distal rotary scanner of an endoscopic probe developed for side-viewing luminal tissue screening and analysis enabled through different endoscopic modalities. The micro rotary optical scanner, for the first time, offers stepwise, low-speed, and high-speed rotational motions to be compatible with endoscopic optical coherence tomography (OCT) and Raman spectroscopy. In comparison with preceding designs, this rotary scanner is shown to provide up to 125× higher revolution speeds per power via a ~50% narrower body while limiting its operation temperature within a biologically safe level for in-vivo scanning purposes. A preliminary experiment on human skin tissues is performed using the prototyped side-viewing OCT endoscopic probe equipped with the developed distal scanner. The results demonstrate the probe's ability for real-time 360° imaging of live tissue with both high speed and high resolution. These results pave the path for further testing on human internal lumens.

Exploring the simultaneous biothiols-differentiating detecting feature of a BODIPY chemosensor with DFT/TDDFT

The simultaneous biothiols-differentiating fluorescence detecting feature of a boron dipyrromethene (BODIPY) derivative (Probe1) has been investigated through DFT/TDDFT calculations. Two decay pathways are revealed for the fluorescence sensing process, and the probe's ability to simultaneously and fluorescently differentiate cysteine (Cys), homocysteine (Hcy), sulfureted hydrogen (H2S), glutathione (GSH) is associated with the different excited-state properties of Probe1 and the biothiols-induced reaction products: the S1 state of Probe1 and the GSH/H2S-induced reaction product NBD-S-GSH/NBD-S is non-emissive, with complete/obvious charge separation; whereas, the S1 state of Cys/Hcy-induced reaction product NBD-S-Cys/NBD-S-Hcy as well as a common product P1 from all biothiols-induced reactions is a bright emissive state. The energetic and charge features of the excited states suggest a donor photoinduced electron transfer mechanism for the fluorescence quenching. It is also found that the intramolecular charge separation character of S1 state accounts for some of spectral behaviors, such as the signal intensity changes and the spectral shift, which can also be utilized in the simultaneously differentiating sensing of biothiols.

Glycoprotein fluorescent speed sensing by newly-synthesized boronic complex probe and chip supramolecular electrophoresis

In this work, we proposed the concept of fluorescent speed sensing (FSS) of glycoprotein in chip supramolecular electrophoresis (CSE). To achieve a stable FSS run, we firstly synthesized a novel boronic acid-based diben-zoxanthene receptor (Rp3) by a simple one-pot reaction and the complex probe of F1@Rp3 containing Rp3 and fluorescein (F1), and performed a series of experiments to assess the complex probe's ability for recognition of saccharides and glycoproteins. Secondly, we revealed the mechanism of 'balance electromigration of supramolecular complex probe' in FSS and developed the relevant theory of FSS via a series of theoretical derivation. The FSS theory showed that the moving speed of F1 was a function of glycoprotein content, indicating a novel model of glycoprotein sensing. To demonstrate the validity and utility of developed model and theory, IgG and ovalbumin were selected as model glycoproteins for the relevant experiments. The results verified the validity of FSS concept to glycoprotein assay with good accuracy (less than 6.33 % difference from standard HPLC method), stability (less than 6.04 % RSD) and linearity (higher than R2 = 0.99) as well as fair sensitivity (8.48 ng). Finally, the FSS was successfully used for the analysis of HbA1c (a key biomarker in diabetes blood). The developed FSS concept and complex probe have great potential for glycoprotein sensing, particularly for the diagnosis of diabetes.

Selection of PAUT probes for submarine pressure hull integrity assessment

Submarine pressure hulls must withstand high hydraulic pressure and be free of defects. To improve the precision of defect detection, we herein examined different probes for optimal defect assessment by applying the Phased Array Ultrasonic Testing (PAUT) method. Two sets of probe design parameters were selected by considering pressure hull characteristics and analyzed through modeling. PAUT probes were applied, and defect assessment results were compared based on ultrasonic signals of various simulated defects in specimens designed to be the same as actual pressure hulls. The final selected design parameters for the submarine probe, which were designed to minimize the grating lobe of wave interference effect and improve the ultrasonic resolution of pressure hull welds, were identified through the experiment. The improvement in the probe's ability to detect defects in a pressure hull was verified. Furthermore, the accuracy of defect length measurement was improved, enhancing the applicability of the technique.

Electrically Small Dipole Antenna Probe for Quasistatic Electric Field Measurements in Transcranial Magnetic Stimulation.

The present paper designs, constructs, and tests an electrically small dipole antenna probe for the measurement of electric field distributions with the ultimate purpose to directly measure electric fields induced by a transcranial magnetic stimulation (TMS) coil. Its unique features include applicability to measurements in both air and conducting medium, high spatial resolution, large frequency band from 100 Hz to 300 KHz, efficient feedline isolation via a printed Dyson balun, and accurate mitigation of noise. Prior work in this area is thoroughly reviewed. The proposed probe design is realized in hardware; implementation details and design tradeoffs are described. Test data are presented for the measurement of a constant wave capacitor electric field, demonstrating the probe's ability to properly measure electric fields caused by a charge distribution. Test data are also presented for the measurement of a constant wave solenoidal electric field, demonstrating the probe's ability to measure electric fields caused by Faraday's law of induction. Those are the primary fields for the transcranial magnetic stimulation. Further steps necessary for the application of this probe as an instrument for TMS coil design are discussed.

Which probes can report intrinsic dynamic heterogeneity of a glass forming liquid?

Using extrinsic probes to study a host system relies on the probes' ability to accurately report the host properties under study. Probes have long been used to characterize dynamic heterogeneity, the phenomenon in which a liquid near its glass transition exhibits distinct dynamics as a function of time and position, with molecules within nanometers of each other exhibiting dynamics that may vary by orders of magnitude. The spatial and temporal characteristics of dynamic heterogeneity demand the selection of probes using stringent criteria on their size and dynamics. In this report, we study the dynamic heterogeneity of the prototypical molecular glass former o-terphenyl by investigating single molecule rotation of two perylene dicarboximide probe molecules that differ in size and comparing this to results obtained previously with the probe BODIPY268. It is found that a probe's ability to accurately report dynamic heterogeneity in o-terphenyl depends on whether the reported distribution of dynamics overlaps with the intrinsic dynamics of the host, which is naturally related to the width of the intrinsic dynamics and the magnitude of dynamical shift in probe dynamics relative to the host. We show that a probe that rotates ≈15 times more slowly than the intrinsic dynamics of the host o-terphenyl senses the slowest ≈5% of the full dynamic heterogeneity whereas one that rotates ≈65 times more slowly than the host fails to report dynamic heterogeneity of the host.

Temperature Dependence of CN and SCN IR Absorptions Facilitates Their Interpretation and Use as Probes of Proteins

Cyano and thiocyano groups have received attention as IR probes of local protein electrostatics or solvation, due to their strong absorptions and the ability to site specifically incorporate them within proteins. However, interpreting their spectra requires knowing whether they engage in hydrogen bonds (H-bonds). Existing methods for the detection of such H-bonding interactions are based on structural analysis or correlations between IR and NMR signals and are labor intensive and possibly ambiguous. Here, using model systems we show that the absorption frequency of both probes is linearly correlated with temperature and that the slope of the resulting line (frequency-temperature line slope or FTLS) reflects the nature of the probe's microenvironment, including whether or not the probe is engaged in H-bonds. We then show that the same linear dependence is observed with p-cyano phenylalanine, cyanylated cysteine, or cyanylated homocysteine incorporated at different positions within the N-terminal Src homology 3 domain of the murine adapter protein Crk-II. The FTLSs indicate that p-cyano phenylalanine incorporated at two positions is engaged in strong H-bonding, while it is involved in weaker H-bonding at a third position. In contrast, the FTLS of the cyanylated cysteine or cyanylated homocysteine absorptions indicates that they do not engage in H-bonding at either a buried or surface exposed position. While the differences likely reflect side chain flexibility and the probe's ability to avoid solvent, the data suggest that the temperature dependence of the absorption provides a simple method to gauge the probe's environment, including the presence of H-bonding.

Silk-Backed Structural Optimization of High-Density Flexible Intracortical Neural Probes

Many chronic neuroscience studies require neural probes that can reliably record with a large number of electrodes in a densely configured array. Previous works have shown that adverse tissue reaction can be significantly reduced as probe shanks are scaled down toward subcellular dimensions. In addition, flexible probes can mitigate shear stress-induced tissue damage due to micromotion. However, both size reduction and flexibility compromise probe's ability to penetrate the pia mater, especially when many electrodes are distributed across multiple probe shanks. In this paper, we present a method to lithographically pattern a biodegradable silk coating that provides temporary mechanical stiffness for the surgical insertion of flexible probes without any conventional design constraints on the probe size, shape, or material. After insertion, the silk is completely dissolved in the tissue, only leaving the flexible minimum-geometry probes inside the brain. We validated the design by successfully inserting silk-backed 64-channel parylene probes into the motor cortex of Long-Evans rats (n = 6) and recorded in vivo neural activity for six weeks.

Design and characterization of a novel multimodal fiber-optic probe and spectroscopy system for skin cancer applications.

The design and characterization of an instrument combining Raman, fluorescence, and reflectance spectroscopic modalities is presented. Instrument development has targeted skin cancer applications as a novel fiber-optic probe has been specially designed to interrogate cutaneous lesions. The instrument is modular and both its software and hardware components are described in depth. Characterization of the fiber-optic probe is also presented, which details the probe's ability to measure diagnostically important parameters such as intrinsic fluorescence and absorption and reduced scattering coefficients along with critical performance metrics such as high Raman signal-to-noise ratios at clinically practical exposure times. Validation results using liquid phantoms show that the probe and system can extract absorption and scattering coefficients with less than 10% error. As the goal is to use the instrument for the clinical early detection of skin cancer, preliminary clinical data are also presented, which indicates our system's ability to measure physiological quantities such as relative collagen and nicotinamide adenine dinucleotide concentration, oxygen saturation, blood volume fraction, and mean vessel diameter.

A blunt sampling probe developed for an in situ leak test of HEPA filters in high-level biosafety laboratory

High-efficiency air filtration is an important means of preventing harmful aerosol overflow in biosafety laboratories. For convenience in the in situ scanning leak test of a high-efficiency particulate air (HEPA) filter to ensure the filter's intactness, a cylindrical blunt sampling probe is developed. The sampling inlet is of the slot type and the inlet's length equal the width of the HEPA filter. Thus, the entire downstream face of the HEPA filter may be scanned. To evaluate the blunt sampling probe's ability to detect leaks, the scanning leak test performed on a HEPA filter unit was compared with a test using the thin-walled rectangular sampling probe that is recommended by relevant standards. Based on the comparison, the leak penetration determined using the blunt sampling probe was slightly lower than that determined using the reference sampling probe. Additionally, differences were observed according to the location that was tested. However, the blunt sampling probe could detect leaks through the comprehensive analysis of the local penetration of most-penetrating particle size (MPPS) and non-MPPS particles.

Abstract 5285: Novel antibody-based probes for the detection and study of pre-malignant breast cancer cells

Abstract Breast cancer is the most prevalent cancer in women. Every year in the US over 192,000 new cases are diagnosed with ∼62,000 of these women presenting at clinics with stage 0 ductal carcinoma in situ (DCIS), of whom 12-15% will develop an invasive tumor within 10 years. Currently there is no reliable method of identifying these women causing high morbidity due to over-treatment and high mortality from under-treatment. Recently our team discovered a population of variant human mammary epithelial cells (vHMECs) which show the correct phenotype for progression to malignancy. Through gene profiling we identified a specific cell surface antigen (SA) for targeting vHMECs, allowing the development of imaging probes able to provide information on the behavior of these cells in vivo as well as forming the basis of a clinically useful probe for early detection and prevention of breast cancer. We based our imaging probes on a proprietary antibody (anti-SA) specific to the cell surface antigen identified during our gene profiling studies. Using a combination of fluorescence microscopy and flow cytometry we optimized its labeling with a near-IR fluorophore, Alexafluor-680 (AF-680), to produce an optical probe that retained its high affinity for vHMECs. Following successful in vitro evaluation of the probe it was tested in vivo. Nude mice were implanted in one mammary fat pad with luciferase-transduced vHMECs (2.5e5-1e6) and in the contra-lateral pad with an equal number of a SA-ve cell line. Bioluminescence imaging was used to confirm successful implantation and 48 hours after cell implantation the probe (∼1.5 nM based on AF680) was administered via tail vein injection. The mice were then imaged using reflectance and tomographic techniques. To further assess the specificity of the probe xenograft models where mice were implanted with both basal (MDA-MB-231, SUM-159-PT, MDA-MD-468; all SA+ve) and luminal (SKBR3, SUM-52-PE, MDA-MB-453; all SA-ve) breast cancer cell lines in the contra-lateral fat pads were used to ascertain the probe's ability to discriminate between these two breast cancer sub-types. The probe effectively labeled vHMECs in vitro and in vivo. The presence of the vHMECs was clearly discerned upon surgical exposure of the mammary fat pad 24 hours after probe injection. In contrast the signal from the SA-ve implant was 40% lower. When tested in mice implanted with both basal and luminal breast cancer cell lines, the fluorescence signal measured from the basal cells was significantly higher (195% higher; student's t-test, one tailed, p=0.0029). Our optical probe is able to selectively label SA+ve cells in vivo, fulfilling our initial goal of developing an imaging probe to facilitate the study of vHMECs. In addition our results indicate that it can be used to discriminate the more aggressive basal breast cancer sub-type from the luminal sub-type, pointing to its future clinical utility in personalized breast cancer diagnosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5285. doi:10.1158/1538-7445.AM2011-5285

Fiber-based probe for electrooptic sampling

A fiber-based electrooptic probe that can be incorporated into existing electrooptic sampling systems is presented. The probe is made by attaching an electrooptic crystal to the end of a standard optical fiber and thinned to a thickness of 1.6 mum using selective wet etching. Measurements of the electric field over a coplanar waveguide structure verify the probe's ability to sense electric fields. The probe can be accurately placed a few micrometers over a test point by monitoring the power reflected back into the probe off of a conductor on a circuit under test

Suitability of the Plant Root Simulator Probe for Use in the Mojave Desert

To find a quick, relatively inexpensive measure for soil chemistry in the Mojave Desert (MD), we evaluated the use of the ion‐exchange resin membrane (IEM) plant root simulator (PRS) probe. Tests were conducted along a floodplain of the Virgin River in Nevada. Probes were buried at 15 and 40 cm. Probes were left in place for three time intervals (30, 60, and 90 d) in two seasonal periods (wetter [WP] and drier [DP], 2004), which were delineated according to the amount of precipitation and soil temperature and moisture. The sampling design was replicated in three pits during the WP and DP. Soil moisture and soil temperature were monitored at 25, 50, 75, and 100 cm. The probes were able to detect differences in ion sorption between the two burial depths, although differences were not always statistically significant. Ion sorption onto the probes generally increased from Month 1 to 3, but the result was not linear. The sorption of some ions fluctuated during the three‐month period, with ions desorbing and readsorbing or ion chemistry decreasing over the course of the study. Soil moisture and temperature did not appear to affect the probe's ability to detect differences across depths or season. Based on results from this experiment, we conclude that the burial time required for assessing relative differences in ion chemistries at our sites is one month or less and that the PRS probe may be useful for detecting relative differences in ion chemistries among other soils in the MD.

Monitoring Caspase Activity in Living Cells Using Fluorescent Proteins and Flow Cytometry

A molecular probe was developed to monitor caspase activity in living cells by flow cytometry. It consists of CFP and YFP with a peptide linker containing two caspase-cleavage sites (LEVD). Its expression resulted in intense fluorescence resonance energy transfer (FRET), whereas cleavage of this linker by caspases eliminated FRET because of physical separation of the CFP and YFP moieties. Using flow cytometry, cells expressing this probe exhibited two patterns, strong FRET and diminished or absent FRET. The appearance of diminished FRET was inhibited by a pan-caspase inhibitor z-VAD or D->A mutations in the LEVD sequence and was markedly increased by apoptosis-inducing agents, etoposide and camptothecin, or overexpression of a caspase 8-red fluorescent protein fusion protein. Importantly, this probe's ability to monitor caspase activity was comparable with results obtained with fluorogenic substrates or fluorochrome-labeled inhibitors of caspases. Specific caspase inhibitors indicated the probe was highly sensitive to cleavage by caspase 6 and 8, less sensitive to caspase 4, and resistant to other caspases. Activation of caspase 8 by Fas engagement markedly increased the probe's cleavage, whereas treatment of caspase 8-deficient cells with anti-Fas did not increase cleavage. However, staurosporine induced cleavage of the probe in caspase 8-deficient cells by a mechanism that was inhibited by overexpression of bcl-x. Taken together, the data indicate that this caspase-sensitive probe can be used to monitor the basal and apoptosis-related activities of caspases, including an initiator caspase, caspase 8, and effector caspases, such as caspase 6.