Linking Cell Health and Reactive Oxygen Species from Secondary Organic Aerosols Exposure.

Oxidative stress is a possible mechanism by which ambient fine particulate matter (PM) exerts adverse biological effects. While multiple biological effects and reactive oxygen species (ROS) production have been observed upon PM exposure, whether the biological effects are ROS-mediated remains unclear. Secondary organic aerosols (SOA) constitute a major fraction of fine PM and can contribute substantially to its toxicity. In this work, we measured three types of cell responses (mitochondrial membrane potential (MMP), caspase 3/7 activity, and ROS) and investigated their associations upon exposure to SOA formed from anthropogenic (naphthalene) and biogenic (α-pinene) precursors. MMP and caspase 3/7 activity (an early indicator of apoptosis) are key indicators of cell health, and changes of them could occur downstream of ROS-mediated pathways. We observed a significant increase in caspase 3/7 activity after SOA exposure, suggesting that apoptosis is an important pathway of cell death induced by SOA. We further found strong associations between a decrease in MMP and increase in caspase 3/7 activity with an increase in cellular ROS level. These results suggest that cell health is largely dependent on the cellular ROS level, highlighting oxidative stress as a key mechanism for biological effects from SOA exposure. Linear regression analyses reveal greater changes of the three cellular responses with increasing carbon oxidation state (OSc) of SOA, suggesting that SOA are more toxic when they are more oxidized. Overall, our work provides critical insights into the associations between cell health and ROS level upon SOA exposure and proposes that OSc could be a suitable proxy to assess the overall SOA toxicity.

Unveiling Antioxidant and Antiproliferative Effects of Prosopis juliflora Leaves against Human Prostate Cancer LNCaP Cells.

Herbal medications or formulations are regularly recommended by clinicians as a potential therapeutic method for a variety of human ailments, including cancer. Although Prosopis juliflora extracts have shown promise in anticancer activity, the effects on prostate cancer and the accompanying molecular mechanisms of action are still unexplored. This research aims at the antioxidant, antiproliferative, and apoptosis-inducing properties of Prosopis juliflora methanolic leaves extract in human prostate cancer LNCaP cells. The antioxidant ability of the extract was assessed using the DPPH (2, 2-diphenyl-2-picrylhydrazyl) and two additional reducing power tests. Antitumor activity was determined using MTT cell viability tests and LDH cytotoxicity assays. The probable mechanism of apoptotic cell death was further investigated utilizing a caspase-3 activation assay and qRT-PCR mRNA expression investigations of apoptotic-related genes. The results revealed that the methanol extract of Prosopis juliflora leaves contains alkaloids, flavonoids, tannins, glycosides, and phenols, all of which have substantial antioxidant activity. In vitro anticancer tests demonstrated that extract therapy resulted in a dose-dependent reduction in cell viability of LNCaP prostate cancer cells, but normal HaCaT cells showed no cytotoxic effects. Furthermore, plant extract therapy increased caspase-3 activation and mRNA expression of apoptotic-related genes, suggesting that this could be a mechanism for cancer cell growth suppression. The significance of Prosopis juliflora as a source of new antioxidant compounds against prostate cancer was emphasized in the current study. However, more study is needed to demonstrate the efficacy of Prosopis juliflora leaves extract in the treatment of prostate cancer.

Modifying a bacterial tyrosinase zymogen for use in protease activity assays.

Current clinical laboratory assays are not sufficient for determining the activity of many specific human proteases yet. In this study, we developed a general approach that enables the determination of activities of caspase-3 based on the proteolytic activation of the engineered zymogen of the recombinant tyrosinase from Verrucomicrobium spinosum (Vs-tyrosinase) by detecting the diphenolase activity in an increase in absorbance at 475 nm. Here, we designed three different zymogen constructs of Vs-tyrosinase, including RSL-pre-pro-TYR, Pre-pro-TYR, and Pro-TYR. The active domain was fused to the reactive site loop (RSL) of α1-proteinase inhibitor and/or its own signal peptide (pre) and/or its own C-terminal domain (pro) via a linker containing a specific caspase-3 cleavage site. Further studies revealed that both RSL peptide and TAT signal peptide were able to inhibit tyrosinase diphenolase activity, in which RSL-pre-pro-TYR had the lowest background signals. Therefore, a specific protease activity such as caspase-3 could be detected when a suitable zymogen was established. Our results could provide a new way to directly detect the activities of key human proteases, for instance, to monitor the efficacy and safety of tumor therapy by determining the activity of apoptosis-related caspase-3 in patients. KEY POINTS: • RSL inhibited the activity of Verrucomicrobium spinosum tyrosinase. • N-pre and C-terminal domain exerted stronger dual inhibition on the Vs-tyrosinase. • The activity of caspase-3 could be measured by the zymogen activation system.

Electrochemical determination of caspase-3 using signal amplification by HeLa cells modified with silver nanoparticles.

An electrochemical sensor capable of quantitative determination of caspase-3 activities was developed. A thiolated peptide whose sequence contained a caspase-3 cleaved site and a cell penetration sequence was preimmobilized onto an electrode. The quantification of caspase-3 was accomplished after cell penetration and the subsequent adsorption of silver nanoparticles (AgNPs). The oxidation current of AgNPs was found to be inversely proportional to the concentration of caspase-3 between 0.02 and 0.2 U/mL. A detection limit of 0.02 U/mL for caspase-3 was achieved due to the large number of positively charged AgNPs adsorbed onto the negatively charged cells. The proof of concept was demonstrated by monitoring the cleavage of surface-confined peptide substrates by caspase-3 in cell lysates. The current sensor could be extended to detect cells by replacing the surface-confined peptide with aptamers that recognize cells. Thus, the use of a cell as a matrix for AgNPs shows excellent potential for constructing electrochemical sensors and provides a useful alternative for sensor development in the future. Cells modified with silver nanoparticles were utilized as the electrochemical readout of an electrochemical assay.

Xanthohumol-Induced Rat Glioma C6 Cells Death by Triggering Mitochondrial Stress.

AIM: To investigate the underlying mechanisms of xanthohumol (XN) on the proliferation inhibition and death of C6 glioma cells. METHODS: To determine the effects of XN on C6 cells, cell proliferation and mortality after XN treatment were assessed by SRB assay and trypan blue assay respectively. Apoptotic rates were evaluated by flowcytometry after Annexin V-FITC/PI double staining. The influence of XN on the activity of caspase-3 was determined by Western blot (WB); and nuclear transposition of apoptosis-inducing factor (AIF) was tested by immunocytochemistry and WB. By MitoSOXTM staining, the mitochondrial ROS were detected. Mitochondrial function was also tested by MTT assay (content of succinic dehydrogenase), flow cytometry (mitochondrial membrane potential (MMP)-JC-1 staining; mitochondrial abundance-mito-Tracker green), immunofluorescence (MMP-JC-1 staining; mitochondrial morphology-mito-Tracker green), WB (mitochondrial fusion-fission protein-OPA1, mfn2, and DRP1; mitophagy-related proteins-Pink1, Parkin, LC3B, and P62), and high-performance liquid chromatography (HPLC) (energy charge). Finally, mitochondrial protein homeostasis of C6 cells after XN treatment with and without LONP1 inhibitor bortezomib was investigated by trypan blue assay (proliferative activity and mortality) and WB (mitochondrial protease LONP1). All cell morphology images were taken by a Leica Microsystems microscope. RESULTS: XN could lead to proliferation inhibition and death of C6 cells in a time- and dose-dependent manner and induce apoptosis of C6 cells through the AIF pathway. After long incubation of XN, mitochondria of C6 cells were seriously impaired, and mitochondria had a diffuse morphology and mitochondrial ROS were increased. The content of succinic dehydrogenase per cell was significantly decreased after XN insults of 24, 48, and 72 h. The energy charge was weakened after XN insult of 24 h. Furthermore, the MMP and mitochondrial abundance were significantly decreased; the protein expression levels of OPA1, mfn2, and DRP1 were down-regulated; and the protein expression levels of Pink1, Parkin, LC3B-II/LC3B-I, and p62 were up-regulated in long XN incubation times (24, 48, and 72 h). XN incubation with bortezomib for 48 h resulted in lower proliferative activity and higher mortality of C6 cells and caused the cell to have visible vacuoles. Moreover, the protein expression levels of LONP1 was up-regulated gradually as XN treatment time increased. CONCLUSION: These data supported that XN could induce AIF pathway apoptosis of the rat glioma C6 cells by affecting the mitochondria.

Effects of hyperbaric oxygen pretreatment on brain antioxidant capacity in rats with decompression sickness.

OBJECTIVE: Decompression sickness (DCS) causes serious brain hypoxic-ischemic injury. This experiment was designed to observe whether hyperbaric oxygen (HBO2) pretreatment played a neuroprotective effect in decompression sickness rat models and to explore the mechanism of protective effects. METHODS: Sprague-Dawley (SD) male rats were pretreated with HBO2 and then underwent decompression to establish the DCS rat model. Antioxidant capacities were evaluated by detecting peroxides (GPx), superoxide dismutase (SOD), catalase (CAT) activity and malondialdehyde (MDA) content in brains. The levels of metal elements manganese (Mn), zinc (Zn), iron (Fe) and magnesium (Mg) in brain tissues were assessed by flame atomic absorption spectrometry. Necrosis and apoptosis of neurons were assessed by H-E staining and immunohistochemical staining. RESULTS: HBO2 pretreatment reduced the degree of necrosis and apoptosis in brain tissues of decompression sickness rat models. In addition, HBO2 pretreatment increased GPx, SOD and CAT activities and reduced MDA accumulation. It also increased the content of Mn, Zn, Fe and Mg in brain tissue, which are all related to free radical metabolism. CONCLUSION: These results suggested that HBO2 pretreatment has protective effects on brain injury of rats with decompression sickness. The mechanism of the protective effects may be related to reducing oxidative damage by affecting metal elements in vivo.

Bioorthogonal "Labeling after Recognition" Affording an FRET-Based Luminescent Probe for Detecting and Imaging Caspase-3 via Photoluminescence Lifetime Imaging.

Bis-labeling with a luminescent energy donor/acceptor pair onto biological substrates affords probes which give FRET readouts for the detection of interaction partners. However, the covalently bound luminophores bring about steric hindrance and nonspecific interaction, which probably perturb the biological recognition. Herein, we designed a highly sensitive and specific "labeling after recognition" sensing approach, where luminophore labeling occurred after the biological recognition. Taking the cutting enzyme caspase-3 as an example, we demonstrated the detection of its catalytic activity in solution and apoptotic cells using the tetrapeptide motif Asp-Glu-Val-Asp (DEVD) as the cleavable substrate, and an iridium(III) complex and a rhodamine derivative as the energy donor/acceptor pair. The DEVD tetrapeptide was modified with an azide and a GK-norbornylene groups at the amino and carboxyl terminuses, respectively, which allowed donor/acceptor bis-labeling via two independent catalysis-free bioorthogonal reactions. The phosphorescence lifetime of the iridium(III) complex was quenched upon bis-labeling owing to the intracellular FRET to the rhodamine derivative, and significantly elongated upon the peptide being catalytically cleaved by caspase-3. Interestingly, the sensitivity and efficiency of the lifetime responses were much higher in the "labeling after recognition" sensing approach. Molecular docking analysis showed that the steric hindrance and nonspecific interactions partially inhibited the biological recognition of the DEVD substrate by caspase-3. The imaging of the catalytic activity of caspase-3 in apoptotic cells was demonstrated via photoluminescence lifetime imaging microscopy. Lifetime analysis not only confirmed the occurrence of intracellular bioorthogonal bis-labeling and catalytic cleavage, but also showed the extent to which the two dynamic processes occurred.

Fast Activation and Tracing of Caspase-3 Involved Cell Apoptosis by Combined Electrostimulation and Smart Signal-Amplified SERS Nanoprobes.

Caspase-3 is considered as one of the key proteases that can spontaneously regulate the life activities of cells, and its activation (usually is a slow process) will execute the apoptosis process of cells. Rapid activation of caspase-3 on demand in living-cells is therefore highly desired toward precise cancer therapy but it is still a key challenge. Herein, we applied electrostimulus (ES) to achieve fast activation of caspase-3 and trigger cell apoptosis, and developed a smart magnetic-plasmonic assembly nanoprobes (A-nanoprobes) to real-time trace cellular caspase-3 activation at the single cell level. The designer core-satellite A-nanoprobe, working specific to the activated caspase-3 via a disassembly tactic, provides strong "hot spots" to improve the sensitivity and therefore enables SERS sensing of cellular caspase-3 upon activated by ES. Single-cell analysis revealed that the ES can rapidly activate the apoptosis pathway of caspase-3 on demand to make the DNA fragmentation and ultimately induce the cell apoptosis. Such method and nanoplatform were further used to monitor ES-triggered caspase-3 activation in cell apoptosis process of different cell types, revealing that more caspase-3 will be activated for cancerous cells than normal cells during the ES to induce cells apoptosis. This strategy and platform are promising for detecting cellular caspase-3 and other enzymes in the process of cancer diagnosis and treatments.

Single-Cell Analysis of Signaling Proteins Provides Insights into Proapoptotic Properties of Anticancer Drugs.

Single-cell DNA analysis technology has provided unprecedented insights into many physiological and pathological processes. In contrast, technologies that allow protein analysis in single cells have lagged behind. Herein, a method called single-cell Plasmonic ImmunoSandwich Assay (scPISA) that is capable of measuring signaling proteins and protein complexes in single living cells is described. scPISA is straightforward, comprising specific in-cell extraction and ultrasensitive plasmonic detection. It is applied to evaluate the efficacy and kinetics of cytotoxic drugs. It reveals that different drugs exhibit distinct proapoptotic properties at the single-cell level. A set of new parameters is thus proposed for comprehensive and quantitative evaluation of the efficacy of anticancer drugs. It discloses that metformin can dramatically enhance the overall anticancer efficacy when combined with actinomycin D, although it itself is significantly less effective. Furthermore, scPISA reveals that survivin interacts with cytochrome C and caspase-3 in a dynamic fashion in single cells during continuous drug treatment. As compared with conventional assays, scPISA exhibits several significant advantages, such as ultrahigh sensitivity, single-cell resolution, fast speed, and so on. Therefore, this approach may provide a powerful tool for wide, important applications from basic research to clinical applications, particularly precision medicine.

Experimental keratitis in rats caused by Acanthamoeba griffini: A kinetic histopathological study.

The aim of this study was to evaluate the inflammation process that resulted from the inoculation of Wistar Rats with Acanthamoeba griffini, a virulent T3 Acanthamoeba genotype that produces keratitis. Haematoxylin and eosin, periodic acid stain, immunohistochemistry and morphometry were used to analyse tissues from rats of an Acanthamoeba keratitis (AK) model. Two weeks after inoculating the rats with A griffini trophozoites, the thickness of the stroma had diminished, followed by an increase in thickness at 4 weeks. At the latter time, an abundance of inflammatory infiltrate cells was observed, some found to express IL-1ß, IL-10 and/or caspase 3. Intercellular adhesion molecule-1 was expressed in corneal blood vessels amid the abundant vascularization characteristic of the development of AK. Through an immunohistochemical technique, trophozoites were detected at 2 and 4 weeks post-inoculation. By 8 weeks, there were a low number of trophozoites and cysts and the corneas of infected rats were similar in thickness to those of the controls. Thus, the rats were capable of healing experimental AK in the present rat model. Diverse immunological mechanisms regulated the inflammatory process in acute AK induced by A griffini in a murine model.

Radiopharmacological evaluation of a caspase-3 responsive probe with optimized pharmacokinetics for PET imaging of tumor apoptosis.

Early evaluation of the therapy efficiency can promote the development of anti-tumor drugs and optimization of the treatment method. Caspase-3 is a key biomarker for early apoptosis. Detection of caspase-3 activity is essential for quick assessment of the curative effect. We have reported a PET probe that could image drug-induced tumor apoptosis in vivo. However, high liver uptake limits its application. In order to optimize the pharmacokinetics of the previous probe, we introduced a hydrophilic peptide sequence to minimize liver uptake. The structure of the new probe was confirmed by mass spectrometry and nuclear magnetic resonance. This probe was able to cross the cell membrane freely and could be converted into a dimer through the condensation reaction of 2-cyano-6-aminobenzothiazole (CBT) and cysteine in response to intracellular activated caspase-3 and glutathione (GSH). The hydrophobic dimers further self-assembled into nanoparticles, which could enhance the probe aggregation in apoptotic tumor tissues. In vivo experiments showed that the tumor uptake of the new probe was higher than that of the previous probe, while the liver uptake of the new probe was significantly reduced. The new probe might be promising in imaging apoptotic tumors with suitable pharmacokinetics.

Combination effect of exercise training and eugenol supplementation on the hippocampus apoptosis induced by chlorpyrifos.

The aim of this study was to investigate the combination effect of exercise training and eugenol supplementation on the hippocampus apoptosis induced by CPF. 64 adult male albino rats were randomly selected and devided into eight groups of eight including: control, exercise (EXE), chlorpyrifos (CPF), Control + Oil (Co + Oil), Control + DMSO (Co + DMSO), chlorpyrifos + eugenol (CPF + Sup), chlorpyrifos + exercise (CPF + Exe) and, chlorpyrifos + exercise + eugenol (CPF + Exe + Eu). Four experimental groups received intraperitoneal injection (5 days a week) of 3.0 mg/kg body weight CPF in DMSO for 6 consecutive weeks. The exercise groups performed aerobic 5 days per week over 4 weeks. Eugenol were administered by gavage. Finally, the animals were sacrificed using CO2 gas (a half of the rats were anesthetized with ketamine and xylazine and then perfused) to evaluate hippocampus histology and parameters. The results of this study showed that CPF injection significantly decreased BDNF, AChE and ATP in CA1 area of the hippocampus (p ˂ 0.05). Also, CA1 apoptosis by tunnel assay, it was found that CPF receiving groups with different dosage, showed a significant increase compared to other groups, which was confirmed by increasing cytochrome C and procaspase-3 in CPF groups (p ˂ 0.05). The result of this study show that 4 weeks of exercise training and eugenol supplementation does not improve the destructive effects of CPF in CA1 area of the hippocampus. As a result, it is recommended that future studies longer periods for treatment with exercise and eugenol supplementation.

Galectin-3 mediates survival and apoptosis pathways during Trypanosoma cruzi-host cell interplay.

Neglected tropical diseases, such as Chagas disease caused by the protozoa Trypanosoma cruzi, affect millions of people worldwide but lack effective treatments that are accessible to the entire population, especially patients with the debilitating chronic phase. The recognition of host cells, invasion and its intracellular replicative success are essential stages for progression of the parasite life cycle and the development of Chagas disease. It is predicted that programmed cell death pathways (apoptosis) would be activated in infected cells, either via autocrine secretion or mediated by cytotoxic immune cells. This process should play a key role in resolving infections by hindering the evolutionary success of the parasite. In this research, we performed assays to investigate the role of the lectin galectin-3 (Gal3) in parasite-host signaling pathways. Using cells with endogenous levels of Gal3 compared to Gal3-deficient cells (induced by RNA interference), we demonstrated that T. cruzi mediated the survival pathways and the subverted apoptosis through Gal3 promoting a pro-survival state in infected cells. Infected Gal3-depleted cells showed increased activation of caspase 3 and pro-apoptotic targets, such as poly (ADP-ribose) polymerase (PARP), and lower accumulation of anti-apoptotic proteins, such as c-IAP1, survivin and XIAP. During the early stages of infection, Gal3 translocates from the cytoplasm to the nucleus and must act in survival pathways. In a murine model of experimental infection, Gal3 knockout macrophages showed lower infectivity and viability. In vivo infection revealed a lower parasitemia and longer survival and an increased spleen cellularity in Gal3 knockout mice with consequences on the percentage of T lymphocytes (CD4+ CD11b+) and macrophages. In addition, cytokines such as IL-2, IL-4, IL-6 and TNF-α are increased in Gal3 knockout mice when compared to wild type genotype. These data demonstrate a Gal3-mediated complex interplay in the host cell, keeping infected cells alive long enough for infection and intracellular proliferation of new parasites. However, a continuous knowledge of these signaling pathways should contribute to a better understanding the mechanisms of cell death subversion that are promoted by protozoans in the pathophysiology of neglected diseases such as Chagas disease.

Unimolecular Chemo-fluoro-luminescent Reporter for Crosstalk-Free Duplex Imaging of Hepatotoxicity.

Real-time multiplex imaging is imperative to biology and diagnosis but remains challenging for optical modality. Herein, a unimolecular chemo-fluoro-luminescent reporter (CFR) is synthesized for duplex imaging of drug-induced hepatotoxicity (DIH), a long-term medical concern. CFR simultaneously detects superoxide anion (O2•-) and caspase-3 (casp3) through respective activation of its independent chemiluminescence and near-infrared fluorescence channels. Such a crosstalk-free duplex imaging capability of CFR enables longitudinal measurement of two correlated biomolecular events (oxidative stress and cellular apoptosis) during the progression of DIH, identifying O2•- as an earlier biomarker for detection of DIH both in vitro and in vivo. Moreover, CFR detects DIH 17.5 h earlier than histological changes. Thus, our study not only develops a sensitive optical reporter for early detection of DIH but also provides a general molecular design strategy for duplex imaging.

Quantification of Low Copy Number Proteins in Single Cells.

We have developed an ultrasensitive and highly selective method to quantify low copy number intracellular proteins in a single cell using a low-cost laser-induced fluorescence (LIF) detector and a BV605 fluorescent probe. Active caspase3 proteins in cells were labeled by corresponding antibody-BV605 fluorescent binding, and a cell was injected into a 20 cm × 50 µm i.d. capillary column, followed by in situ lysis and capillary electrophoresis (CE)-LIF analysis. About seven active caspase3 protein molecules in a detection volume of 91 pL could be detected. In our method, cross-bounding proteins other than active caspase3 could be separated and distinguished by differences of retention time. By using Si photodiode assembly as a fluorescent detector instead of PMT, the dynamic range of the LIF is over 4 orders of magnitude. In this experiment, we found that the number of active caspase3 molecules in 98 single Jurkat cells were from 629 to 12171, reflecting significant heterogeneity among the cells although they were from the same batch. For extended application, it could also be applied to quantify other types of low copy number proteins in a single cell as long as the corresponding antibodies are provided. This high-sensitive method could also be a promising tool for earlier cancer diagnosis and related disease pathway research which is relevant to low copy number proteins. In addition, this low-cost system could also be easily expanded to an array system for high-throughput quantitation of low copy proteins in single cells.

Photoelectrochemical Approach to Apoptosis Evaluation via Multi-Functional Peptide- and Electrostatic Attraction-Guided Excitonic Response.

The excitonic response between nanomaterials is distance-dependent, and thus, interparticle distance is a key factor in fabricating diverse photoelectrochemical (PEC) systems. Current studies focus on DNA-mediated regulation of interparticle distance. However, limited by high demands of base-pairing and flexibility of DNA, it is hard for DNA to achieve precise regulation, especially in a short distance. To pursue better PEC performances in bioanalyses, alternative biological materials should be explored to replace DNA as new "distance controllers". In this work, a peptide with three functional sequences is designed to control interparticle distance between positive-charged Au nanoparticles ((+) AuNPs) and negative-charged CdTe quantum dots ((-) CdTe QDs). Relying on the function of binding sequence, (+) AuNPs and (-) CdTe QDs may be separated to a certain distance by the multifunctional peptide. In this case, the excitonic response is relatively weak, and an evident PEC response can be observed. Because it contains the substrate sequence of caspase-3, the peptide is cleaved in the presence of caspase-3. As a result, without the support of intact peptide, electrostatic attraction plays a dominant role, leading to the aggregation of oppositely charged AuNPs and CdTe QDs, which strengthens the excitonic response and attenuates the PEC response. On the basis of these principles, a novel PEC approach was fabricated to sensitively quantify caspase-3. Meanwhile, caspase-3 in staurosporine-treated A549 cells are also determined by the approach, and the obtained results agree well with the fluorescent intensity of confocal images, manifesting that the proposed PEC method can monitor apoptosis in a label-free strategy. Overall, the study reveals the capability of peptides in controlling interparticle distance of nanomaterials, which may accelerate the development of peptide-based PEC analytical methods.

Probiotic mixture of Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 attenuates hippocampal apoptosis induced by lipopolysaccharide in rats.

In recent years, the beneficial impact of targeted gut microbiota manipulation in various neurological disorders has become more evident. Therefore, probiotics have been considered as a promising approach to modulate brain gene expression and neuronal pathways even in some neurodegenerative diseases. The purpose of this study was to determine the effect of probiotic biotherapy with combination of Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 on the expression levels of proteins critical to neuronal apoptosis in hippocampus of lipopolysaccharide (LPS)-exposed rats. Four groups of animals (Control, LPS, Probiotic + LPS, and Probiotic) were treated with maltodextrin (placebo) or probiotic (109 CFU/ml/rat) for 2 weeks by gavage. On the 15th day, a single intraperitoneal dose of saline or LPS (1 mg/kg) was injected and 4 h later, protein assessment was performed by western blotting in hippocampal tissues. LPS significantly increased the Bax, Bax/Bcl-2 ratio, and cleaved caspase-3 expression along with decreased the Bcl-2 and procaspase-3 protein levels. However, probiotic pretreatment (L. helveticus R0052 + B. longum R0175) significantly downregulated the Bax and Bax/Bcl-2 ratio accompanied with upregulated Bcl-2 expression. Prophylactic treatment with these bacteria also attenuated LPS-induced caspase-3 activation by remarkably increasing the expression of procaspase-3 while reducing the level of cleaved caspase-3 in target tissues. Our data indicate that probiotic formulation (L. helveticus R0052 + B. longum R0175) alleviated hippocampal apoptosis induced by LPS in rats via the gut-brain axis and suggest that this probiotic could play a beneficial role in some neurodegenerative conditions.

AuNP-peptide probe for caspase-3 detection in living cells by SERS.

Colloidal nanoparticles can be used as surface-enhanced Raman scattering (SERS) substrates because the very close spacing between particles existing in these colloidal systems is beneficial for the generation of extremely strong and highly spatially localized electric field enhancements. Herein, a caspase-3-specified peptide was used as a molecular cross-linker to engineer gold nanoparticle (AuNP) junctions in a controllable manner. The peptide was designed with a sequence of CCALNNPFFDVED (Cys-Cys-Ala-Leu-Asn-Asn-Pro-Phe-Phe-Asp-Val-Glu-Asp) or CCALNNKYDDVED (Cys-Cys-Ala-Leu-Asn-Asn-Lys-Tyr-Asp-Asp-Val-Glu-Asp), where the CALNN (Cys-Ala-Leu-Asn-Asn) fragment helps to stabilize AuNP suspension in aqueous media and the sequence of DVED (Asp-Glu-Val-Asp) can be cleaved by caspase-3. In addition, the PFF (Pro-Phe-Phe) or KYD (Lys-Tyr-Asp) was exposed and interacted via the hydrophobic or alternate negative and positive electro-interaction in the presence of caspase-3, inducing the aggregation of colloidal Au-peptides accompanied with the enhancement of SERS. It can be observed that the SERS-enhanced signals were correlated with the caspase-3 concentrations and the limit of detection can reach 1.5 ng mL-1. Finally, this caspase-3-specified AuNP-peptide probe has been found to be a promising candidate for its application in the analysis of caspase-3 in living cells.

Sensitive detection of caspase-3 enzymatic activities and inhibitor screening by mass spectrometry with dual maleimide labelling quantitation.

There is a great need to develop sensitive and specific methods for quantitative analysis of caspase-3 activities in cell apoptosis. Herein, we report a new method for sensitive detection of caspase-3 enzyme activities and inhibitor screening based on dual maleimide (DuMal) labeling quantitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Evaluation of caspase-3 activities is performed using MS analysis of the enzymatic product of the peptide probe, which fuses a caspase-3 cleavable peptide segment (DEVD) and a quantifiable "ID tag" (a peptide segment of FRGLRGFKC labeled by maleimide). The DuMal labeling technique features non-isotopic tagging, rapid reactions, and reproducible quantitation. We have achieved quantitative analysis of the enzyme activities with a limit of detection (LOD) and limit of quantitation (LOQ) of caspase-3 down to 0.11 nM and 0.29 nM respectively and a proof-of-concept demonstration of its inhibitor screening. Our method has further been tested for caspase-3 activities in a Parkinson's disease cellular model, suggesting a useful tool for protease activity research.

Dual-channel surface plasmon resonance monitoring of intracellular levels of the p53-MDM2 complex and caspase-3 induced by MDM2 antagonist Nutlin-3.

MDM2 can mediate the degradation of tumor suppressor p53 through an autoregulatory feedback loop, in which MDM2 abolishes wild-type p53 function and accelerates malignant transformation. However, the incorporation of MDM2 antagonist Nutlin-3 could reactivate the transcriptional activity of p53, up-regulate caspase-3, and induce apoptosis. In this work, the simultaneous and label-free monitoring of p53-MDM2 complex and caspase-3 levels in cancer cells before and after Nutlin-3 treatment was conducted using dual-channel surface plasmon resonance (SPR). The p53-MDM2 complex was captured in one fluidic channel covered with consensus double-stranded (ds)-DNA, while the other channel was pre-immobilized with caspase-3-specific biotinylated DEVD-containing peptides. To amplify the SPR signals, the attachment of streptavidin (SA)-conjugated anti-MDM2 antibody in both channels was achieved. The signal diversity before and after Nutlin-3 treatment is indicative of the difference in the levels of the intracellular p53-MDM2 complex and caspase-3. The limit of detection for p53-MDM2 and caspase-3 down to 4.54 pM and 0.03 ng mL-1, respectively, was attained. Upon treatment with Nutlin-3, MCF-7 cancer cells with wild-type p53 showed decreased expression of the p53-MDM2 complex and an increased caspase-3 level, while MDA-MB-231 cancer cells with mutant p53 exhibited an elevated caspase-3 level and unchanged p53-MDM2 complex expression. The apoptosis of MCF-7 and MDA-MB-231 cancer cells upon Nutlin-3 treatment follows a p53-dependent and a p53-independent pathway, respectively. The proposed method is sensitive, selective and label-free, holding great promise for assaying intracellular p53-MDM2 complex and caspase-3 levels and differentiating Nutlin-3-mediated p53-dependent or p53-independent apoptotic pathways.