Dithiothreitol-based protein equalisation in the context of multiple myeloma: Enhancing proteomic analysis and therapeutic insights.

In this study, we employed the dithiothreitol-based protein equalisation technique and analytical proteomics to better understand myeloma diseases by comparing the proteomes of pellets and supernatants formed upon application of DTT on serum samples. The number of unique proteins found in pellets was 252 for healthy individuals and 223 for multiple myeloma patients. The comparison of these proteomes showed 97 dysregulated proteins. The unique proteins found for supernatants were 264 for healthy individuals and 235 for multiple myeloma patients. The comparison of these proteomes showed 87 dysregulated proteins. The analytical proteomic comparison of both groups of dysregulated proteins is translated into parallel dysregulated pathways, including chaperone-mediated autophagy and protein folding, addressing potential therapeutic interventions. Future research endeavours in personalised medicine should prioritize refining analytical proteomic methodologies using serum dithiothreitol-based protein equalisation to explore innovative therapeutic strategies. We highlight the advanced insights gained from this analytical strategy in studying multiple myeloma, emphasising its complex nature and the critical role of personalised, targeted analytical techniques in enhancing therapeutic efficacy in personalised medicine.

Kinetics of ascorbate and dithiothreitol oxidation by soluble copper, iron, and manganese, and 1,4-naphthoquinone: Influence of the species concentration and the type of fluid.

An alternative metric to account for particulate matter (PM) composition-based toxicity is the ability of PM-species to generate reactive oxygen species (ROS) and deplete antioxidants, the so-called oxidative potential (OP). Acellular OP assays are the most used worldwide, mainly those based on ascorbic acid (AA) and dithiothreitol (DTT) depletion; OP values are calculated from AA/DTT concentration over time kinetic curves. Since a great variability in OP-DTT and OP-AA values can be found in the literature, the understanding of those factors affecting the kinetic rate of AA and DTT oxidation in the presence of PM-bound species will improve the interpretation of OP values. In this work, a kinetic study of the oxidation rate of AA and DTT driven by species usually found in PM (transition metals and naphthoquinone (NQ)) was carried out. In particular, the influence of the concentration of Cu(II), Fe(II), Fe(III), Mn(II), Mn(III), and 1,4-NQ, and the type of fluid used in the assay (phosphate buffer (PB), phosphate buffer saline (PBS) and artificial lysosomal fluid (ALF)) is analysed and discussed. The reaction orders with respect to the AA/DTT and the active compound, and the kinetic rate constants were also determined. The results show great variability in OP values among the studied species depending on the fluid used; the OP values were mostly higher in PB0.05 M, followed by PBS1x and ALF. Moreover, different species concentration-responses for OP-DTT/OP-AA were obtained. These differences were explained by the different reaction orders and kinetic rate constants obtained for each active compound in each fluid.

Effect of the drug cyclophosphamide on the activity of porcine kidney betaine aldehyde dehydrogenase.

The enzyme betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) catalyzes the synthesis of glycine betaine (GB), an osmolyte and osmoprotectant. Also, it participates in several metabolic pathways in humans. All BADHs known have cysteine in the active site involved in the aldehyde binding, whereas the porcine kidney enzyme (pkBADH) also has a neighborhood cysteine, both sensitive to oxidation. The antineoplastic and immuno-suppressant pre-drug cyclophosphamide (CTX), and its bioactivation products, have two highly oxidating chlorine atoms. This work aimed to analyze the effect of CTX in the activity of porcine kidney betaine aldehyde dehydrogenase. PkBADH was incubated with varying CTX concentration (0 to 2.0 mM) at 25 °C and lost 50 % of its activity with 2.0 mM CTX. The presence of the coenzyme NAD+ (0.5 mM) decreased 95% the activity in 2.0 mM CTX. The substrate betaine aldehyde (0.05 and 0.4 mM, and the products NADH (0.1-0.5 mM) and GB (1 and 10 mM) did not have an effect on the enzyme inactivation by CTX. The reducing agents, dithiothreitol and ß-mercaptoethanol, reverted the pkBADH inactivation, but reduced glutathione (GSH) was unable to restore the enzyme activity. Molecular docking showed that CTX could enter at the enzyme active site, where its chlorine atoms may interact with the catalytic and the neighboring cysteines. The results obtained show that CTX inactivates the pkBADH due to oxidation of the catalytic cysteine or because it oxidizes catalytic and neighborhood cysteine, forming a disulfide bridge with a concomitant decrease in the activity of the enzyme.

NanoTPOT: Enhanced Sample Preparation for Quantitative Nanoproteomic Analysis.

With the ever-growing need for protein-level understanding in pathological research, proteomics researchers thrive to examine detailed proteome dynamics using crucial, yet often limited, primary and clinical samples. Aside from mass spectrometer instrumentation advancement, a single-tube-based high-throughput sample processing workflow is imperative to ensure sensitive, quantitative, and reproducible protein analysis for these increasingly sophisticated studies. Leveraging the benefits of an acid-cleavable detergent, RapiGest SF Surfactant (Waters Corporation), we developed and optimized a nanoproteomic workflow that we termed Nanogram TMT Processing in One Tube (NanoTPOT). Through the assessment of proteolytic digestion, tandem mass tag (TMT) labeling, online and offline fractionation strategies, our optimized workflow effectively eliminated the need for sample desalting and enabled compatible sample processing for mass spectrometry analysis. We further applied the NanoTPOT workflow to examine cellular response to stress caused by dithiothreitol in HeLa cells, where we identified and quantified 6935 and 5474 proteins in TMT 10-plex experiments with one microgram of lysate protein and 2000 sorted HeLa cells (roughly half microgram lysate protein) in each channel, respectively. Our workflow has been proven to be an effective alternative for current nanoproteomic sample processing to minimize sample loss in biological and clinical applications.

Structure-function analyses of alkylhydroperoxidase D from Streptococcus pneumoniae reveal an unusual three-cysteine active site architecture.

During aerobic growth, the Gram-positive facultative anaerobe and opportunistic human pathogen Streptococcus pneumoniae generates large amounts of hydrogen peroxide that can accumulate to millimolar concentrations. The mechanism by which this catalase-negative bacterium can withstand endogenous hydrogen peroxide is incompletely understood. The enzyme alkylhydroperoxidase D (AhpD) has been shown to contribute to pneumococcal virulence and oxidative stress responses in vivo We demonstrate here that SpAhpD exhibits weak thiol-dependent peroxidase activity and, unlike the previously reported Mycobacterium tuberculosis AhpC/D system, SpAhpD does not mediate electron transfer to SpAhpC. A 2.3-Å resolution crystal structure revealed several unusual structural features, including a three-cysteine active site architecture that is buried in a deep pocket, in contrast to the two-cysteine active site found in other AhpD enzymes. All single-cysteine SpAhpD variants remained partially active, and LC-MS/MS analyses revealed that the third cysteine, Cys-163, formed disulfide bonds with either of two cysteines in the canonical Cys-78-X-X-Cys-81 motif. We observed that SpAhpD formed a dimeric quaternary structure both in the crystal and in solution, and that the highly conserved Asn-76 of the AhpD core motif is important for SpAhpD folding. In summary, SpAhpD is a weak peroxidase and does not transfer electrons to AhpC, and therefore does not fit existing models of bacterial AhpD antioxidant defense mechanisms. We propose that it is unlikely that SpAhpD removes peroxides either directly or via AhpC, and that SpAhpD cysteine oxidation may act as a redox switch or mediate electron transfer with other thiol proteins.

Thioredoxin interacting protein (Txnip) forms redox sensitive high molecular weight nucleoprotein complexes.

Thioredoxin interacting protein (Txnip) is an α-arrestin protein that regulates pleiotropic biological responses. Txnip acts as a cancer suppressor and is a critical regulator of energy metabolism. To investigate molecular mechanisms involving Txnip, we searched for its protein binding partners using tandem affinity purification and proteomics analyses and identified several viable candidates, including HSP90, HSP70, and Prp31. We showed, by native PAGE, that Txnip is involved in the formation of high molecular weight complexes (1000-1300 kDa) in the nuclear fraction of cells treated with glucose and bortezomib. DTT treatment partly dissolved these high molecular weight complexes, suggesting that Txnip forms redox sensitive high-order nucleoprotein complexes. RNAse treatment slightly decreased the complex and RNA-seq showed differential expression of RNAs in the complex between Txnip protein overexpressing and control cells, indicating the involvement of RNAs in the complex. These results collectively provide a model whereby Txnip exerts its functions through multiple binding partners, forming transient higher-order complexes to regulate other signaling molecules.

Sequentially dynamic polymeric micelles with detachable PEGylation for enhanced chemotherapeutic efficacy.

To achieve enhanced cancer therapy, a sequentially dynamic polymeric drug delivery system (ortho ester-linked PEGylated poly(disulfide)s-based micelle-doxorubicin (PS-g-OEMPEG-DOX)) is successfully constructed. The PEGylated micelle can keep stable in sodium dodecyl sulfate (SDS) solution at pH 7.4, but be prone to DePEGylation and dynamic size changes via the hydrolysis of ortho ester linkages in side chains at tumoral extracellular pH value (6.5). Moreover, the micelle can rapidly release DOX via the cleavage of poly(disulfide)s in backbone at intracellular reductive milieu (10 mmol/L of dithiothreitol (DTT)). The dynamic micelle with detachable PEGylation achieves the stable blood circulation, improved cellular uptake and cytotoxicity, stronger in vitro penetration and inhibition of tumoral multicellular spheroids, and significant in vivo tumor accumulation and inhibition while decreasing side effects. Thus, the sequentially dynamic polymeric micelle with detachable PEGylation can be considered as a promising and effective drug delivery system in cancer therapy.

Quantification and 13C-Tracer analysis of total reduced glutathione by HPLC-QTOFMS/MS.

Glutathione is an essential intra- and extracellular antioxidant. The level of glutathione in the body is highly related to different disease states and is a useful indicator of disease risk and oxidative stress status. We have developed a sensitive, selective, and comprehensive LC-MS/MS method for the absolute quantification and 13C-tracer analysis of total glutathione using dithiothreitol for the reduction of glutathione disulfide. The limit of detection (LOD) was 0.01 µM, while the lower limit of quantification (LLOQ) was 0.78 µM, with the linear (R = 0.9997) range extending up to 100 µM. The intra-run and inter-run coefficients of variation of 2.49% and 2.04%, respectively, attest to high repeatability. Mean (±SD) recoveries of three different concentrations (low, medium, high) of GSH spiked into aliquots of HCT116 cells prior to cell extraction were 108.9% (±2.1), 100.8% (±8.3), and 99.9% (±7.1), respectively. Finally, using a 20 Da wide Q1 window in MRM mode, we were able to detect and relatively quantify all isotopic labeling states of GSH extracted from HCT116 cells fed with either 13C-labeled glucose or glutamine.

Novel adenosine-derived inhibitors of Cryptosporidium parvum inosine 5'-monophosphate dehydrogenase.

We have found cyclophane-type adenosine derivatives having p-quinone amide moieties (1 and 2) as weak inhibitors of Cryptosporidium parvum inosine 5'-monophosphate dehydrogenase (CpIMPDH) from the Hokkaido University Chemical Library via the luciferase-based high-throughput screening. To obtain more potent inhibitors, we synthesized four new derivatives free from cyclophane rings (3-6). The N-H derivatives 3 and 5 showed more potent activities (24.4 and 11.1 µM, respectively) in the presence of dithiothreitol (DTT), whereas the N-methyl derivative 4 indicated more potent activity (2.1 µM) without DTT. Conformational analysis of compounds 3 and 4 suggested that N-H amide 3 binds to IMP-binding site in the DTT mediated manner.

A biodegradable poly(amido amine) based on the antimicrobial polymer polyhexamethylene biguanide for efficient and safe gene delivery.

Inspired by the excellent membrane affinity of antimicrobial polymers, we synthesized a novel biodegradable poly(amino amine) polymer with pendent side chains that mimic the widely used biocide polyhexamethylene biguanide (PHMB) for gene delivery. Michael addition polymerization was utilized to form the polymer scaffold between N,N'-cystaminebisacrylamide (CBA) and N-Boc-1,6-diaminohexane (Boc-DAH) followed by N-Boc deprotection. Then the exposed primary amino groups were partly (about 75%) transformed into biguanide by an addition reaction with dicyandiamide to obtain the final product CBA-DAH-biguanide (CBA-DAH-BG). The polymer CBA-DAH-BG was able to condense plasmid DNA (pDNA) into nano-sized (<200 nm), positively-charged (>35 mV) polyplexes that were well resistant to heparin and DNase I. Rapid DNA release was observed in the presence of dithiothreitol (DTT), indicating that CBA-DAH-BG was equipped with biodegradability by the cleavage of disulfide bonds, which was helpful for unpacking DNA and decreasing cytotoxicity. CBA-DAH-BG/pDNA polyplexes were characterized by efficient cellular uptake efficacy, extremely low cytotoxicity, and high transfection efficiency in two cell lines (i.e., NIH/3T3 and U87 MG), compared to 25 kDa polyethyleneimine (PEI) and the intermediate product CBA-DAH that were both devoid of biguanide groups. Of note, clathrin-mediated endocytosis and lipid rafts played an important role in the internalization of the polyplexes. Taken together, this strategy described herein may represent an innovative avenue for the design of more advanced nonviral gene vectors with high transfection efficiency and biocompatibility.

l-Dehydroascorbic acid recycled by thiols efficiently scavenges non-thermal plasma-induced hydroxyl radicals.

Recent development in electronics has enabled the use of non-thermal plasma (NTP) to strictly direct oxidative stress in a defined location at near-physiological temperature. In preclinical studies or human clinical trials, NTP promotes blood coagulation, wound healing with disinfection, and selective killing of cancer cells. Although these biological effects of NTP have been widely explored, the stoichiometric quantitation of free radicals in liquid phase has not been performed in the presence of biocompatible reducing agents, which may modify the final biological effects of NTP. Here we quantitated hydroxyl radicals, a major reactive oxygen species generated after NTP exposure, by electron paramagnetic resonance (EPR) spectroscopy using two distinct spin-trapping probes, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO), in the presence of thiols or antioxidants. l-Ascorbic acid (AsA) at 25-50 µM concentrations (physiological concentration in the serum) significantly scavenged these hydroxyl radicals, whereas dithiothreitol (DTT), reduced glutathione (GSH), and N-acetyl-cysteine (NAC) as thiols were required in millimolar concentrations to perform scavenging activities. l-Dehydroascorbic acid (DHA), an oxidized form of AsA, necessitated the presence of 25-50 µM DTT or sub-millimolar concentrations of GSH and NAC for the scavenging of hydroxyl radicals and failed to scavenge hydroxyl radicals by itself. These results suggest that the redox cycling of AsA/DHA via thiols and cellular AsA metabolism are important processes to be considered while applying NTP to cells and tissues. Further studies are warranted to elucidate the interaction between other reactive species generated by NTP and biomolecules to promote biological and medical applications of NTP.

Identification of an acetonitrile addition impurity formed during peptide disulfide bond reduction using dithiothreitol and Tris(2-carboxyethyl)phosphine.

Identification and localization of modifications in peptides containing multiple disulfide bonds is challenging due to inefficient fragmentation in mass spectrometry (MS) analysis. In cases where MS fragmentation techniques such as electron capture dissociation (ECD), electron transfer dissociation (ETD), and ultraviolet photodissociation (UVPD) fail to achieve efficient fragmentation, off-line disulfide bond reduction techniques are typically employed prior to MS analysis. Some commonly used reducing agents include dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP). In this work, we describe the detection and identification of an unexpected impurity that formed during the reduction of Peptide A, containing multiple disulfide bonds, while using DTT or TCEP as reducing agents and acetonitrile as a co-solvent. The DTT reduced products were found to be a mixture of the expected linear Peptide A (fully reduced) and an unknown product (>50%) with a mass corresponding to linear Peptide A plus 41 Da ([reduced-M + 41]). A series of experiments were subsequently performed to investigate the identity and origin of this impurity. Disulfide bond reduction with DTT was performed in aqueous mixtures containing acetonitrile, methanol, and deuterated acetonitrile; and with TCEP in aqueous mixtures containing acetonitrile. Additionally, glycine amino acid was used as a surrogate to investigate the mechanism. The liquid chromatography-mass spectrometry (LCMSMS) results demonstrated that the [reduced-M + 41] impurity was an acetonitrile addition on the peptide's N-terminal glycine. The corresponding impurity [M + 41] was also found in the native Peptide A (non-reduced), suggesting that small amounts of this impurity may also be generated during the synthesis in the upstream process steps. By understanding the formation of this process-related impurity [M + 41], one could potentially reduce or eliminate its presence in Peptide A through chemical controls. Finally, this observation provides caution against using acetonitrile as a co-solvent during DTT- or TCEP-promoted reduction of peptides with an uncapped N-terminus primary amine.

Dimeric Prodrug Self-Delivery Nanoparticles with Enhanced Drug Loading and Bioreduction Responsiveness for Targeted Cancer Therapy.

Efficient drug accumulation in tumor cells is essential for cancer therapy. Herein, we developed dimeric prodrug self-delivery nanoparticles (NPs) with enhanced drug loading and bioreduction responsiveness for triple negative breast cancer (TNBC) therapy. Specially designed camptothecin dimeric prodrug (CPTD) containing a disulfide bond was constructed to realize intracellular redox potential controlled drug release. Direct conjugation of hydrophobic CPTD to poly(ethylene glycol) PEG5000, a prodrug-based amphiphilic CPTD-PEG5000 co-polymer was synthesized, which could encapsulate parental CPTD prodrug spontaneously and form ultrastable NPs due to the highly analogous structure. Such dimeric prodrug self-delivery nanoparticles showed ultrahigh stability with critical micelle concentration as low as 0.75 µg/mL and remained intact during endocytosis. In addition, neurotensin (NT), a 13 amino acid ligand, was further modified on the nanoparticles for triple negative breast cancer (TNBC) targeting. Optimized NT-CPTD NPs showed improved pharmacokinetics profile and increased drug accumulation in TNBC lesions than free CPT, which largely reduced the systemic toxicity and presented an improved anticancer efficacy in vivo. In summary, with advantages of extremely high drug loading capacity, tumor microenvironmental redox responsiveness, and targeted TNBC accumulation, NT-CPTD NPs showed their potential for effective triple negative breast cancer therapy.

Distinct effects of daratumumab on indirect and direct antiglobulin tests: a new method employing 0.01 mol/L dithiothreitol for negating the daratumumab interference with preserving K antigenicity (Osaka method).

BACKGROUND: There is an increasing demand for daratumumab (DARA), an immunoglobulin (Ig)G1κ monoclonal antibody (MoAb) that recognizes CD38, to manage relapsed or refractory multiple myeloma (MM) patients. However, DARA leads to positive and panreactive agglutination reactions in indirect antiglobulin tests (IATs) in vitro (the DARA interference). In addition, effects of DARA on red blood cells (RBCs) in vivo remains elusive. STUDY DESIGN AND METHODS: To develop a new method to negate the DARA interference, the effects of various concentrations of dithiothreitol (DTT) on RBC CD38 and Kell antigenicity in combination with an automatic blood cell washing centrifuge were compared with the AABB standard procedure in parallel. Moreover, direct antiglobulin tests (DATs) for RBCs in DARA-treated MM patients were examined. RESULTS: A quantity of 0.01 mol/L DTT as well as the AABB procedure (equivalent to 0.15 mol/L DTT in our procedure) markedly reduced the reactivity of phycoerythrin-mouse anti-CD38 MoAb and DARA with RBCs. In sharp contrast to the AABB procedure, 0.01 mol/L DTT partially preserved K antigenicity and allowed the determination of phenotype of K antigen even in the presence of the DARA interference. In contrast, DAT for RBCs obtained from MM patients showed a weak positive or negative reaction. Immunoblotting further indicated that DARA induced loss of CD38 in vivo. CONCLUSION: A simple and reliable method to negate the DARA interference with partially preserving Kell antigenicity is proposed (Osaka method). CD38 antigenicity is susceptible to 0.01 mol/L DTT treatment even in the presence of DARA. Our data also demonstrate distinct effects of DARA on IAT in vitro and DAT in vivo.

Improved in situ seeding of 3D printed scaffolds using cell-releasing hydrogels.

The design of tissue engineered scaffolds based on polymerized high internal phase emulsions (polyHIPEs) has emerged as a promising bone grafting strategy. We previously reported the ability to 3D print emulsion inks to better mimic the structure and mechanical properties of native bone while precisely matching defect geometry. In the current study, redox-initiated hydrogel carriers were investigated for in situ delivery of human mesenchymal stem cells (hMSCs) utilizing the biodegradable macromer, poly(ethylene glycol)-dithiothreitol. Hydrogel carrier properties including network formation time, sol-gel fraction, and swelling ratio were modulated to achieve rapid cure without external stimuli and a target cell-release period of 5-7 days. These in situ carriers enabled improved distribution of hMSCs in 3D printed polyHIPE grafts over standard suspension seeding. Additionally, carrier-loaded polyHIPEs supported sustained cell viability and osteogenic differentiation of hMSCs post-release. In summary, these findings demonstrate the potential of this in situ curing hydrogel carrier to enhance the cell distribution and retention of hMSCs in bone grafts. Although initially focused on improving bone regeneration, the ability to encapsulate cells in a hydrogel carrier without relying on external stimuli that can be attenuated in large grafts or tissues is expected to have a wide range of applications in tissue engineering.

Selective Extraction and Antioxidant Properties of Thiol-Containing Peptides in Soy Glycinine Hydrolysates.

A highly selective procedure to extract thiol-containing peptides (TCPs) from complicated soy glycinin hydrolysates (SGHs) was described. This procedure included the reduction of disulfide bonds by 1,4-dithiothreitol (DTT) and enrichment of TCPs through Thiopropyl-Sephrose 6B covalent chromatography. TCPs were confirmed using a strategy based on mass shift after differential alkylation of sulfhydryl groups with iodoacetamide and N-ethylmaleimide by matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS). The antioxidant activities of TCPs were evaluated using chemical assays. DTT reduction increased the concentration of sulfhydryl groups from 1.8 µmol/g to 113.8 µmol/g. The efficiency of the extraction was improved by optimizing the loading of sample, extraction and desorption time and the content of desorption reagent. Both of the adsorption and desorption process were found to fit a pseudo-second order model. MALDI-TOF-MS showed that 36 of the 45 extracted peptides were TCPs. The EC50 of TCPs for DPPH, hydroxyl radical, and superoxide anion radical was 0.1, 1.49 and 0.084 mg/mL, respectively. The reducing power of TCPs (0.2 mg/mL) was of 0.375. These results suggest that the combination of DTT reduction and Thiopropyl-Sepharose 6B covalent chromatograph was a successful pathway to extract TCPs from SGHs and the TCPs could be used as potential antioxidants.

Giant Plasma Membrane Vesicles: An Experimental Tool for Probing the Effects of Drugs and Other Conditions on Membrane Domain Stability.

Giant plasma membrane vesicles (GPMVs) are isolated directly from living cells and provide an alternative to vesicles constructed of synthetic or purified lipids as an experimental model system for use in a wide range of assays. GPMVs capture much of the compositional protein and lipid complexity of intact cell plasma membranes, are filled with cytoplasm, and are free from contamination with membranes from internal organelles. GPMVs often exhibit a miscibility transition below the growth temperature of their parent cells. GPMVs labeled with a fluorescent protein or lipid analog appear uniform on the micron-scale when imaged above the miscibility transition temperature, and separate into coexisting liquid domains with differing membrane compositions and physical properties below this temperature. The presence of this miscibility transition in isolated GPMVs suggests that a similar phase-like heterogeneity occurs in intact plasma membranes under growth conditions, albeit on smaller length scales. In this context, GPMVs provide a simple and controlled experimental system to explore how drugs and other environmental conditions alter the composition and stability of phase-like domains in intact cell membranes. This chapter describes methods to generate and isolate GPMVs from adherent mammalian cells and to interrogate their miscibility transition temperatures using fluorescence microscopy.

Fabrication of dual stimuli-responsive multicompartmental drug carriers for tumor-selective drug release.

There has been increasing attention to the development of multi-stimuli-responsive drug carriers for precisely controlled drug release at target disease areas. In this study, pH- and redox-responsive hybrid drug carriers were fabricated by using both ketal-based acid-cleavable precursors and disulfide-based reducible precursors via stop-flow lithography. pH- and redox-sensitive drug release of the dual stimuli-responsive hybrid particles was confirmed, demonstrating their feasibility for selective and efficient drug release into tumor tissues in acidic and highly reductive environments. It was also found that the drug release rate of the particles was fine-tuned by modulating monomer compositions in the precursor. Importantly, the dual stimuli-responsive hybrid particles exhibited synergistic, controlled drug release in complex stimuli (both pH and redox stimuli) environments. To achieve tumor-selective combination chemotherapy, multicompartmental drug carriers consist of an acid-degradable compartment and a reducible compartment, which can separately encapsulate individual model drugs in each of the compartments. The multicompartmental particles exhibited independent drug release upon exposure to the corresponding stimulus. The dual stimuli-responsive, multicompartmental particles are effective drug carriers for tumor-selective release of a drug cocktail, leading to synergistic combination chemotherapy.

Reduced-HMGB1 suppresses poly(I:C)-induced inflammation in keratinocytes.

BACKGROUND: High mobility group box 1 (HMGB1) is a nuclear protein that stabilizes DNA and facilitates gene transcription. Additionally, cell stress or death induces the release of HMGB1 outside the cell membrane, where HMGB1 functions as an alarmin, causing an inflammatory response in combination with other cytokines, damage-associated molecular patterns (DAMPs), and pathogen-associated molecular patterns (PAMPs). OBJECTIVE: To evaluate the effect of reduced-HMGB1 (previously termed chemoattractive-HMGB1) on polyinosine-polycytidylic acid [poly(I:C)]-induced inflammation in normal human keratinocytes (NHKs). METHODS: We focused on downstream components of the poly(I:C)-Toll-like receptor 3 (TLR3), retinoic acid-inducible gene-I (RIG-I), and melanoma differentiation-associated protein 5 (MDA5) pathways, including IκBα, nuclear factor (NF)-κB p65, mitogen-activated protein kinase (MAPK), and interferon regulatory factor 3 (IRF3), and assessed whether these pathways are involved in the suppression of poly(I:C)-induced inflammation in NHKs by HMGB1. An immunoprecipitation was performed to know whether HMGB1 could bind to poly(I:C), and immunofluorescence staining and flow cytometric analysis were performed to check whether reduced-HMGB interferes with cellular uptake of poly(I:C) translocation (possibly by endocytosis). RESULTS: Application of exogenous HMGB1 before, but not after, exerted a suppressive effect on poly(I:C)-induced inflammation in NHKs. In addition, reduced-HMGB1, but not disulfide-HMGB1, exerted a suppressive effect on poly(I:C)-induced inflammation in NHKs, suggesting the importance of the redox status of exogenous HMGB1. Pre-treatment with reduced-HMGB1 inhibited the phosphorylation of IκBα, NF-κB p65, and IRF3 induced by poly(I:C) stimulation in NHKs; however, phosphorylation of p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) was unaffected. Disulfide-HMGB1 formed a complex with poly(I:C), as did reduced- and oxidized-HMGB1, albeit to a lesser extent. Immunofluorescence staining and flow cytometric analysis indicated that reduced-HMGB interferes with cellular uptake of poly(I:C) translocation (possibly by endocytosis). CONCLUSION: These findings suggest that pre-treatment with reduced-HMGB1 ameliorates poly(I:C)-mediated inflammation in NHKs.

Dithiothreitol-based protein equalization technology to unravel biomarkers for bladder cancer.

This study aimed to assess the benefits of dithiothreitol (DTT)-based sample treatment for protein equalization to assess potential biomarkers for bladder cancer. The proteome of plasma samples of patients with bladder carcinoma, patients with lower urinary tract symptoms (LUTS) and healthy volunteers, was equalized with dithiothreitol (DTT) and compared. The equalized proteomes were interrogated using two-dimensional gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry. Six proteins, namely serum albumin, gelsolin, fibrinogen gamma chain, Ig alpha-1 chain C region, Ig alpha-2 chain C region and haptoglobin, were found dysregulated in at least 70% of bladder cancer patients when compared with a pool of healthy individuals. One protein, serum albumin, was found overexpressed in 70% of the patients when the equalized proteome of the healthy pool was compared with the equalized proteome of the LUTS patients. The pathways modified by the proteins differentially expressed were analyzed using Cytoscape. The method here presented is fast, cheap, of easy application and it matches the analytical minimalism rules as outlined by Halls. Orthogonal validation was done using western-blot. Overall, DTT-based protein equalization is a promising methodology in bladder cancer research.