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We present a seed-mediated synthesis method for producing silver nanoplates (AgNPTs) with customizable size and thickness, ensuring high yield and precise optical properties. This approach leverages ethylenediaminetetraacetic acid (EDTA) as a key component in the synthesis process, utilizing small single-crystal silver seeds. The interaction between Ag + ions and EDTA at varying pH levels dynamically regulates silver complexation and reduction kinetics during seed overgrowth, leading to the formation of truncated nanoplates with superior optical responses. By adjusting the pH within the range of 8-10.5, we can manipulate the growth of the nanoplates, enabling a flexible optical response ranging from 519 to 1006 nm due to changes in their size and thickness. Additionally, nanoplate overgrowth extends plasmon resonance up to approximately 2000 nm. The incorporation of Adenosine 5' monophosphate (AMP) not only enhances nanoplate stability but also allows for precise thickness adjustment independent of growth kinetics. This method provides a systematic approach to tailor nanoplate morphology and optical properties with unprecedented precision. The role of EDTA is attributed to its complexation ability with Ag + and its assistance in facet evolution, supported by density functional theory (DFT) simulations of surface energies modified by EDTA adsorption. Furthermore, DFT calculations confirm that AMP can further modify the surface energies of different facets, enabling precise thickness control., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Monitoring Hg 2+ levels in aqueous environments is crucial to assess the potential methylmercury contamination via bacterial conversion, however, existing methods often require extensive sample treatment and expensive equipment. To mitigate this issue, this study examines the synthesis and application of three naphthalimide-based compounds, with significant fluorescent and solvatochromic behavior (C1, C2, and C3). Compounds C1 and C2 demonstrated a strong affinity for Hg 2+ metal ions, with C2 showing selectivity and a strong antibacterial profile, particularly against S. aureus (MIC 50 (C2) = 0.01 µg/mL). Moreover, these compounds were incorporated into three polymeric matrices, namely polyvinyl chloride (PVC), poly (methyl methacrylate-co-methacrylic acid) (PMMMA), and Starch, allowing for the development of solid-support sensors/surfaces with a strong antibacterial profile, highlighting the inherent dual-functionality of the compounds. Interestingly, the C2-doped Starch biopolymer detected low concentrations of Hg 2+ ions, such as 23 nM in tap water (value within the WHO standards for drinking water), through a rapid spectroscopic evaluation without sample treatment. This biopolymer was generated via a sustainable, green-chemistry-oriented, temperature-dependent water/Starch synthetic route, without the addition of plasticizers and any associated ecotoxicity. The study used sustainable methods for environmental monitoring and antibacterial applications, advancing material science to offer effective, accessible, and eco-friendly solutions for detecting and mitigating mercury pollution and bacterial contaminations, enhancing environmental and health safety., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Biothiols, such as cysteine (Cys), glutathione (GSH), and homocysteine (Hcy), play crucial roles in various physiological processes and serve as biomarkers for oxidative stress and redox homeostasis. Their structural similarities, however, pose significant challenges in selective detection and quantification, limiting the availability of suitable probes. Here, we report the design and synthesis of a novel ratiometric fluorescent sensor based on a seleno-BODIPY (Se-BODIPY) derivative, enabling rapid discrimination and quantification of Cys, Hcy, and GSH with low detection limits (Cys = 0.8 μM, Hcy = 20.4 μM, and GSH = 35.9 μM) via fluorescence. The probe exhibits high selectivity towards these biothiols over 11 amino acids, operating through dual-mode detection (absorption and emission spectra) with a visible color change from blue to orange (Cys/Hcy) or pink (GSH) in a turn-on fluorescence process. Notably, the distinct reaction mechanisms between Se-BODIPY and GSH versus Cys/Hcy lead to a more prominent blue shift for Cys/Hcy, facilitating their differentiation. Kinetic studies further differentiate Cys from Hcy, with the BODIPY reacting much faster with Cys than the latter. The effectiveness of the sensor was demonstrated in quantifying biothiols in urine samples, providing a non-invasive method with high recovery rates. Additionally, its incorporation into paper strips allows detection of biothiols in water samples via visible and UV light-induced color changes, indicating its potential for solid-state detection without organic solvents.

Four functionalized BODIPY derivatives (BDP1 to BDP4) were synthesized and their optical properties investigated both in solution and when incorporated into a solid matrix. Recognizing the versatility of BODIPY derivatives and the increasing interest in developing new luminescent organic dyes embedded in polymers, the BODIPY derivatives were dispersed into two types of polymeric matrices: Poly(methyl methacrylate) (PMMA) and Thermoplastic Polyurethane (TPU), both as films and microparticles. This resulted in eight new BODIPY-doped polymer films and eight types of BODIPY-doped polymeric microparticles for use in aqueous solutions. The integration of the BODIPY dyes into the polymeric matrices combines the unique properties of the polymer films, such as porosity, flexibility, and elasticity, with the excellent photophysical characteristics of the BODIPYs. Importantly, the dispersion minimized issues such as aggregation-caused quenching commonly observed in solid-state luminescent materials. The thermometric responses of all polymer films were evaluated by studying their solid-state emission spectra in the 25-200 °C temperature range. The reversibility of these temperature-induced changes was also assessed, revealing excellent recovery of luminescence. These promising results suggest these materials could have applications as fluorescent thermometric sensors. Furthermore, we explored the potential of the brominated (BDP3) and chalcogenated (BDP4) BODIPY derivatives as ammonia sensors. The two derivatives produced yellow fluorescent products upon interaction with the analyte. Kinetic studies using solid-state emission spectra of BDP4@TPU and BDP4@PMMA showed significant differences in reaction rates (minutes for BDP4@TPU and hours in the case of BDP4@PMMA) attributable to the higher permeability of TPU when compared with PMMA. Detection and quantification of ammonia concentration were conducted by means of simple photographic analysis, measuring the "R" (red) and "G" (green) components of RGB color parameters. The results from the photographic method correlated well with the results from fluorimetric spectroscopy studies. The photographic analysis is straightforward, portable, and does not require expensive equipment. Finally, we successfully applied polymeric microparticles doped with BODIPYs to detect ammonia in water, demonstrating their effectiveness without the need for organic solvents. This highlights their potential for environmental monitoring and other applications requiring sensitive and selective detection methods., Competing Interests: Declarations Ethical approval No ethical approval was necessary. Confllict of interest The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Purpose: T1 bladder cancer is known for its high progression and recurrence rates. Identifying aggressive tumours at the non-muscle-invasive stage is crucial to allow early interventions and subsequently increase patient survival. This study aimed to investigate the potential of the cubilin/myeloperoxidase (CUBN/MPO) ratio as a high-grade T1 bladder cancer biomarker., Methods: Urine samples were collected from 30 patients who underwent transurethral resection of the tumour with high-grade T1 bladder cancer (June 2015 to December 2019) before surgery. The urinary proteome was analysed using high-resolution mass spectrometry and the CUBN/MPO ratio was calculated. The primary outcome was the recurrence during the follow-up (around 31.5 months after resection). Univariate Cox regression and Kaplan-Meier curves were used for data analysis., Results: Patients with a low CUBN/MPO ratio exhibited upregulated MPO and/or downregulated CUBN. This group of patients had a higher incidence of disease recurrence and progression. Low CUBN/MPO ratio was significantly associated with a higher likelihood of recurrence, progression, and death. It is worth noting that this study was exploratory and conducted on a small sample size, so further research is needed to validate these findings in larger cohorts., Conclusion: This study highlights the potential of the CUBN/MPO ratio as a prognostic biomarker for high-grade T1 bladder cancer., (© 2024. The Author(s).)

The global increase in multidrug-resistant bacteria poses a challenge to public health and requires the development of new antibacterial materials. In this study, we examined the bactericidal properties of mesoporous silica-coated silver nanoparticles, varying the core sizes (ca. 28 nm and 51 nm). We also investigated gold nanoparticles (ca. 26 nm) coated with mesoporous silica as possible inert metal cores. To investigate the modification of antimicrobial activity after the surface charge change, we used silver nanoparticles with a silver core of 28 nm coated with a mesoporous shell (ca. 16 nm) and functionalized with a terminal amine group. Furthermore, we developed a facile method to create mesoporous silica-coated silver nanoparticles (Ag@mSiO 2 ) doped films using polyurethane (IROGRAN ® ) as a polymer matrix via solution casting. The antibacterial effects of silver nanoparticles with different core sizes were analyzed against Gram-negative and Gram-positive bacteria relevant to the healthcare and food industry. The results demonstrated that gold nanoparticles were inert, while silver nanoparticles exhibited antibacterial effects against Gram-negative ( Escherichia coli and Salmonella enterica subsp. enterica serovar Choleraesuis) and Gram-positive ( Bacillus cereus ) strains. In particular, the larger Ag@mSiO 2 nanoparticles showed a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of 18 µg/mL in the Salmonella strain. Furthermore, upon terminal amine functionalization, reversing the surface charge to positive values, there was a significant increase in the antibacterial activity of the NPs compared to their negative counterparts. Finally, the antimicrobial properties of the nanoparticle-doped polyurethane films revealed a substantial improvement in antibacterial efficacy. This study provides valuable information on the potential of mesoporous silica-coated silver nanoparticles and their applications in fighting multidrug-resistant bacteria, especially in the healthcare and food industries.

The need for non-invasive therapies capable of conserving drug efficiency and stability while having specific targetability against colorectal cancer (CRC), has made nanoparticles preferable vehicles and principal building blocks for the development of complex and multi-action anti-tumoral approaches. For that purpose, we herein report the production of a combinatory anti-tumoral nanotherapy using the production of a new targeting towards CRC lines. To do so, Magneto-fluorescent NANO3 nanoparticles were used as nanocarriers for a combination of the drugs doxorubicin (DOX) and ofloxacin (OFLO). NANO3 nanoparticles' surface was modified with two different targeting agents, a newly synthesized (anti-CA IX acetazolamide derivative (AZM-SH)) and a commercially available (anti-epidermal growth factor receptor (EGFR), Cetuximab). The cytotoxicity revealed that only DOX-containing nanosystems showed significant and even competitive cytotoxicity when compared to that of free DOX. Interestingly, surface modification with AZM-SH promoted an increased cellular uptake in the HCT116 cell line, surpassing even those functionalized with Cetuximab. The results show that the new target has high potential to be used as a nanotherapy agent for CRC cells, surpassing commercial targets. As a proof-of-concept, an oral administration form of NANO3 systems was successfully combined with Eudragit ® enteric coating and studied under extreme conditions.

The rising of multidrug-resistant bacteria and their associated proliferation as harmful microorganisms boosts the creation of new antibacterial surfaces and biomaterials with applications ranging from health to food packing. Herein, low-cost antibacterial PVA:PVP copolymers containing cyanine derivatives (1, 2, and 3) and their respective Cu 2+ complexes are successfully obtained and tested against Gram-negative and Gram-positive bacteria. The possible application in food packing is addressed by covering the surface of typical paper mockups with the doped polymers. All dye-doped polymers present a broad-spectrum antibacterial effect against Gram-positive bacteria, especially for Bacillus cereus (B. cereus), Staphylococcus aureus (S. aureus), and methicillin-resistant S. aureus (MRSA) strains, with PVA:PVP@3 and PVA:PVP@3-Cu being the most effective. Moreover, polymers containing cyanine derivatives present interesting inhibition effects against Pseudomonas aeruginosa (P. aeruginosa), where the production of its characteristic blue/green virulent pigment is not observed. Of the coated paper mockups, PVA:PVP:paper@2 and PVA:PVP:paper@2-Cu are most effective against B. cereus and S. aureus, while PVA:PVP:paper@3 and PVA:PVP:paper@3-Cu are most effective against the MRSA strain. In these formulations, direct contact inhibition mechanisms appear to be more significant than diffusional mechanisms, due to cyanine release hindrance, making them very interesting and versatile platforms for medical and food applications., (© 2022 Wiley-VCH GmbH.)

The use of nanoparticles in multiple industries has raised concerned voices about the assessment of their toxicity/antimicrobial activity and the development of standardized handling protocols. Issues emerge during the antimicrobial assaying of multiple cargo, colorimetric, colloidal nanoformulations, as standard protocols often rely on visual evaluations, or optical density (OD) measurements, leading to high variance inhibitory concentrations (MIC). Thus, a fast, luminescence-based assay for the effective assessment of the antimicrobial activity of nanoparticles is herein reported, using the bioluminescence of an in-house E. coli ATCC ® 8739 TM construct with the pMV306G13 + Lux plasmid ( E. coli Lux). The new strain's sensitivity to ofloxacin as a standard antibiotic was confirmed, and the methodology robustness verified against multiple nanoparticles and colorimetric drugs. The reduction of incubation from 24 to only 8 h, and the sole use of luminescence (LUX 490 ) to accurately determine and distinguish MIC 50 and MIC 90 , are two main advantages of the method. By discarding OD measurements, one can avoid turbidity and color interferences when calculating bacterial growth. This approach is an important tool that contributes to the standardization of methods, reducing samples' background interference and focusing on luminescence as a direct probe for bacterial metabolic activity, growth and, most importantly, the correct assessment of nanomaterials' antimicrobial activity.

In this work, the successful preparation and characterization of gold nanorods (AuNRs) coated with a mesoporous silica shell (AuNRs@Simes) was achieved. Conjugation with methylene blue (MB) as a model drug using ultrasound-stimulated loading has been explored for further application in light-mediated antibacterial studies. Lyophilization of this conjugated nanosystem was analyzed using trehalose (TRH) as a cryogenic protector. The obtained stable dry formulation shows potent antimicrobial activity against Gram-negative ( Escherichia coli ) and Gram-positive ( Staphylococcus aureus ) bacteria after a simple post-treatment irradiation method with a red laser during a short time period.

Non-viral gene delivery using exogenous microRNAs is a potential strategy for fighting cancers with poor prognosis and which lack specific therapies, such as triple-negative breast cancer (TNBC). Herein we report the synthesis of six nontoxic electrostatic polymeric nanocapsules (P1 to P6) for microRNA delivery in TNBC cells. 1H Nuclear Magnetic Resonance (NMR) spectroscopy and Scanning Electron Microscopy (SEM) were used to characterize the nanopolyplexes, synthesized with Poly(L-Lysine) and hyaluronic acid (Ha). Studies on the activity of the ternary HA/PLI/miRNA-34 nanopolyplexes towards TNBC cell line MDA-MB-231 were conducted. The nanopolyplexes mediated intracellular restoration of tumor suppressor miR34a was evaluated by using Western blotting to quantify the expression level of the Bcl-2 protein. The results suggest that the P5, with a ratio PLI/Ha of 0.05, was the most promising for the delivery of miR-34a into TNBC cells; the P5 nanocapsules were able to reduce Bcl-2 expression at a protein level, and had an effect in the overall cell viability after 24 h treatment.

•Special Issue on 4th IC 3 EM 2020.•Science, Techniques, Surveillance.•The importance of Science.•Fluorescent dyes., Competing Interests: The authors declares any conflict of interest., (© 2020 Elsevier Ltd. All rights reserved.)

Aim: Report our results of biomarker discovery in formalin-fixed paraffin-embedded (FFPE) nasopharyngeal carcinoma (NPC) via proteomic analysis., Background: Nasopharyngeal carcinoma (NPC) is a rare cancer in Western countries. Proteomic analysis have already been reported as a useful tool to provide biomarkers. Formalin-fixed paraffin-embedded (FFPE) samples, despite largely underused, can provide invaluable information for biomarker research via proteomic analysis., Methods: FFPE samples of NPC were submitted to protein extraction followed by FASP-digestion and label-free quantitative mass spectrometry (MS). Patients' received concurrent chemoradiation with or without adjuvant chemotherapy as per Intergroup 0099 trial. IMRT was delivered following the RTOG0615 specifications. Toxicity was scored using the CTCAE 4.03 tables. Survival was estimated using Kaplan-Meier curves. Log-rank was used to detect differences. KEGG ontology graphics were generated., Results: 28 FFPE samples from NPC patients were used. Patients were: 79% male, 97% Caucasians, 86% WHO type 3, 40% T1, 10% T2, 25% T3, and 25% T4. With a median follow up of 37 months, local control was 83 (T1, 100% T2, T3 and T4), overall survival was 84%, and six patients developed distant metastases. All five patients that died were due to metastatic disease. Tumor samples contained a median of 75% of tumor material. We found Epstein-Barr (EBV) and Herpes simplex (HSV) viruses' related proteins significantly present in early-stage primary NPC (T1 and T2, p  < 0.01). A pool of 10 proteins was statistically up-regulated in the metastatic group of patients ( p  < 0.01). Median survival from this M1 group was <1 year ( p  < 0.001)., Conclusions: FFPE samples yielded adequate material for MS analysis. We found EBV and HSV related proteins on early-stage NPC, and proteomic profiling associated with distant metastases, potential candidates of disease biomarkers. Validation is needed., (© 2020 Greater Poland Cancer Centre. Published by Elsevier B.V. All rights reserved.)

We propose a new high-throughput ultrafast method for large-scale proteomics approaches by speeding up the classic filter aided sample preparation protocol, FASP, from overnight to 2.5 h. Thirty-six samples can be treated in 2.5 h, and the method is scalable to 96-well plate-based pipelines. After a modification of the FASP-tube, the steps of protein reduction, protein alkylation, and protein digestion of complex proteomes are done in just 5.25 min, each one under the effects of an ultrasonic field (7 cycles: 30 s on and 15 s off). The new method was compared to the standard overnight digestion FASP protocol, and no statistical differences were found for more than 92.4%, 92%, and 93.3% of the proteins identified by studying the proteome of E. coli , mouse brain, and mouse liver tissue samples, respectively. Furthermore, the successful relative label-free quantification of four spiked proteins in E. coli samples, BSA, β-lactoglobulin, α-casein, and α-lactalbumin, was achieved, using either the ultrasonic-based FASP protocol or the classic overnight one. The new US-FASP method matches the analytical minimalism rules as time, cost, sample requirement, reagent consumption, energy requirements, and production of waste products are reduced to a minimum while maintaining high sample throughput in a robust manner as all of the advantages of the filter aided sample preparation protocol are maintained.

A novel analytical approach is proposed to discriminate between solid biopsies of chromophobe renal cell carcinoma (chRCC) and renal oncocytoma (RO). The method comprises the following steps: (i) ultrasonic extraction of proteins from solid biopsies, (ii) protein depletion with acetonitrile, (iii) ultrasonic assisted in-solution digestion using magnetic nanoparticle with immobilized trypsin, (iv) C18 tip-based preconcentration of peptides, (v) sequential extraction of the peptides with ACN, (vi) MALDI-snapshot of the extracts and (vii) investigation of the extract containing the most discriminating features using high resolution mass spectrometry. With this approach we have been able to differentially cluster renal oncocytoma and chromophobe renal cell carcinoma and identified 18 proteins specific to chromophobe and seven unique to renal oncocytoma. Chromophobes express proteins associated with ATP function (ATP5I & 5E; VATE1 & G2; ADT2), glycolysis (PGK1) and neuromedin whilst oncocytomas express ATP5H, ATPA, DEPD7 and TRIPB thyroid receptor interacting protein., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We present a method to preserve and process urine proteins for proteomic analysis in a filter aided sample preparation (FASP) format. The method combines concentration of urine proteins in ultrafiltration devices, their thermal stabilization, allowing long term storage of the samples, and filter aided sample preparation. Proteomes of four different urines were preserved during 48 h and 6 months using (i) the classic freeze preservation at -20 °C, (ii) snap-heated freeze-free preservation at laboratory temperature (20 °C) and (iii) snap-heated preservation at -60 °C. The three storage methods can effetely preserve the urine proteome for at least 6 months without significant alterations. Abundances of more than 500 proteins and specially 24 selected -cleared or -approved protein assayed in serum or plasma were found similar within the three preservation methods assessed. The new method here proposed dramatically simplifies the conditions for preserving the urine proteome for biobanks in terms of space and storage, including lowering the risks of sample degradation caused by misfunction of the freezer. Furthermore, the shipping of large number of samples can be made without the need of freezing. The application of the FASP format to isolate and preserve the proteins facilitates long-term storage and processing of proteome of urine samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The spatial distribution of chemical elements in different types of samples is an important field in several research areas such as biology, paleontology or biomedicine, among others. Elemental distribution imaging by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is an effective technique for qualitative and quantitative imaging due to its high spatial resolution and sensitivity. By applying this technique, vast amounts of raw data are generated to obtain high-quality images, essentially making the use of specific LA-ICP-MS imaging software that can process such data absolutely mandatory. Since existing solutions are usually commercial or hard-to-use for average users, this work introduces LA-iMageS, an open-source, free-to-use multiplatform application for fast and automatic generation of high-quality elemental distribution bioimages from LA-ICP-MS data in the PerkinElmer Elan XL format, whose results can be directly exported to external applications for further analysis. A key strength of LA-iMageS is its substantial added value for users, with particular regard to the customization of the elemental distribution bioimages, which allows, among other features, the ability to change color maps, increase image resolution or toggle between 2D and 3D visualizations.

Sequential chemical depletion of serum coupled to C18 sequential extraction of peptides as a rapid tool for human serum multiple profiling is herein presented. The methodology comprises depletion with DTT and then with ACN; the extract thus obtained is then summited to fast protein digestion using ultrasonic energy. The pool of peptides is subsequently concentrated using C18-based Zip-tips and the peptides are sequentially extracted using different concentrations of ACN. Each extract is mass-spectrometry profiled with MALDI. The different spectra thus obtained are then successfully used for classification purposes. A total of 40 people, comprising 20 healthy and 20 non-healthy donors, were successfully classified using this method, with an excellent q-value<0.05. The proposed method is cheap as it entails few chemicals, DTT and ACN, simple in terms of handling, and fast. In addition, the methodology is of broad application as it can be used for any study applied to serum samples or other complex biological fluids., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: The field of biomarker discovery, development and application has been the subject of intense interest and activity, especially with the recent emergence of new technologies, such as proteomics-based approaches. In proteomics, search for biomarkers in biological fluids such as human serum is a challenging issue, mainly due to the high dynamic range of proteins present in these types of samples. Methods for reducing the content of most highly abundant proteins have been developed, including immunodepletion or protein equalization. In this work, we report for the first time the combination of a chemical sequential depletion method based in two protein precipitations with acetonitrile and DTT, with a subsequent two-dimensional difference in-gel electrophoresis (2D-DIGE) analysis for the search of osteoarthritis (OA) biomarkers in human serum. The depletion method proposed is non-expensive, of easy implementation and allows fast sample throughput., Results: Following this workflow, we have compared sample pools of human serum obtained from 20 OA patients and 20 healthy controls. The DIGE study led to the identification of 16 protein forms (corresponding to 14 different proteins) that were significantly (p < 0.05) altered in OA when compared to controls (8 increased and 7 decreased). Immunoblot analyses confirmed for the first time the increase of an isoform of Haptoglobin beta chain (HPT) in sera from OA patients., Conclusions: Altogether, these data demonstrate the utility of the proposed chemical sequential depletion for the analysis of sera in protein biomarker discovery approaches, exhibit the usefulness of quantitative 2D gel-based strategies for the characterization of disease-specific patterns of protein modifications, and also provide a list of OA biomarker candidates for validation.

In this work three methods to diminish the content of most highly abundant proteins in human serum have been studied and compared. Protein depletion with ACN or DTT and protein equalization with the ProteoMiner(™) (PM) have been assessed by 1-D gel electrophoresis and MS. After treatment 5, 18 and 9 major proteins within the 20 most abundant proteins in serum were identified for the ACN, DTT and PM methods, respectively. The ACN method was efficient for depleting high molecular weight proteins, over 75 KDa, resulting in 10±4% (n=3) of the total protein content remaining in the depleted serum. In addition, 75% of the proteins belonging to the group of the 20 most abundant proteins were not detected, making this depletion strategy a cheap alternative to expensive commercial tools regularly used for removing high abundance proteins from serum. The ACN extract was found rich in apolipoproteins. The dithithreitol method promotes the precipitation of proteins rich in disulfide bonds, mainly albumin, with 73±7% (n=3) of the total protein content remaining in the depleted serum, which was found rich in immunoglobulins. The PM method compresses the dynamic range of the serum proteins, rendering an extract containing 16±2% (n=3) of the total initial protein content. The extract was found to be rich in both apolipoproteins and immunoglobulins. As a general rule the DTT and PM methods provide a compression of the dynamic range of serum protein concentrations while the ACN method allows an effective depletion of the protein fraction above 72 KDa., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this paper we present two case studies of Proteomics applications development using the AIBench framework, a Java desktop application framework mainly focused in scientific software development. The applications presented in this work are Decision Peptide-Driven, for rapid and accurate protein quantification, and Bacterial Identification, for Tuberculosis biomarker search and diagnosis. Both tools work with mass spectrometry data, specifically with MALDI-TOF spectra, minimizing the time required to process and analyze the experimental data., (Copyright 2011 The Author(s). Published by Journal of Integrative Bioinformatics.)

A simple, fast and miniaturized ultrasonic probe assisted protocol for acid extraction of trace and major elements from street dust samples, prior to final determination by ICP-MS, has been optimized and validated by standard reference materials and applied during an urban air quality monitoring campaign. 15 mg samples were treated for 3 min with 1 mL concentrated HNO(3)-HCl (1:3, v/v) extracting solution, by a 1mm diameter titanium sonotrode connected to a 200 W and 24 kHz ultrasonic device at 80% amplitude. After centrifugation, the extracts were assayed by ICP-MS with good recoveries for the certified elements. Statistical analysis of real sample results by cluster analysis allowed the correct grouping of the samples according to the influence of traffic and construction/demolition activities. Correlation of street dust and PM10 elemental profiles showed the potential applicability of the proposed analytical protocol as a simple and effective way for urban air quality monitoring., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In the present work we report on a novel and fast protocol for accurate bottom-up protein quantification that overcomes the drawbacks of in-gel digestion and MALDI analysis, while maintaining their benefits. It relies on the following steps: (i) gel electrophoresis separation of proteins, (ii) fast in-gel protein digestion with trypsin, (iii) (18)O-labeling through the decoupled method, (iv) quantification through selected peptides previously chosen using the (18)O inverse labeling approach and that, finally, (v) it takes advantage of software specifically developed to select the peptides that will drive the quantification of the protein in an automated mode. We have accurately quantified the following six proteins: glycogen phosphorylase, BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor, and α-lactalbumin. As a case study we have quantified the protein vitellogenin in plasma of Cyprinus carpio exposed to high levels of estrogens. The proposed new protocol was validated against the traditional ELISA method; both were found to provide comparable results (non-parametric Mann-Whitney U-test).

A rapid sample treatment procedure for the gas chromatography/mass spectrometry (GC/MS) determination of anabolic steroids in human urine has been developed. The new procedure makes use of ultrasonic energy to reduce reaction times and increase the overall sensitivity. The following variables affecting the performance of the ultrasonic treatment were optimised: (i) time, (ii) device, (iii) frequency, and (iv) temperature. It was found that, under an ultrasonic field, the hydrolysis of conjugated steroids with beta-glucuronidase from Escherichia coli K12 was possible with a treatment time of 10 min. The accuracy and precision of the ultrasonic method were found to be in agreement with those achieved with the conventional thermal conductivity procedure (Student's t-test; p = 0.05, n = 10). After the enzymatic hydrolysis, the derivatisation of the target compounds with trimethylsilyl (TMS) reagent, methyl-N-trimethylsilyltrifluoroacetamide (MSTFA)/NH(4)I/dithioerythritol (DTE) (1000:2:4, v/w/w), was also accelerated using ultrasonic energy. In order to test the applicability of the use of ultrasonic energy in the acceleration of the derivatisation reaction with TMS, the classic method of thermal conductivity was applied for comparative purposes to a pool of 35 androgenic anabolic steroids (AAS) and/or their metabolites. The results demonstrated that after 3 min of sonication in a Sonoreactor device (50% amplitude), 19 of the 35 compounds studied showed similar reaction yield to those obtained with the classic procedure requiring 30 min (Student's t-test; p = 0.05, n = 5); 13 increased to higher silylation yields; and for the steroids 1-testosterone, danazol and etiocholanolone-D5, the same results were obtained using a sonication time of 5 min.The overall applicability of the ultrasonic-based sample treatment method is shown by the analysis of five urine samples. The results are similar to those achieved by the routine procedure. The new method is fast, robust, and allows high sample throughput., (2010 John Wiley & Sons, Ltd.)

New data on sample preparation and matrix selection for the fast screening of androgenic anabolic steroids (AAS) by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) is presented. The rapid screening of 15 steroids included in the World Anti-Doping Agency (WADA) prohibited list using MALDI was evaluated. Nine organic and two inorganic matrices were assessed in order to determine the best matrix for steroid identification in terms of ionisation yield and interference by characteristic matrix ions. The best results were achieved for the organic matrices 2-(4-hydroxyphenylazo)benzoic acid (HABA) and trans-3-indoleacrylic acid (IAA). Good signals for all the steroids studied were obtained for concentrations as low as 0.010 and 0.050 microg/mL on the MALDI sample plate for the HABA and IAA matrices, respectively. For these two matrices, the sensitivity achieved by MALDI is comparable with the sensitivity achieved by gas chromatography/mass spectrometry (GC/MS), which is the conventional technique used for AAS detection. Furthermore, the accuracy and precision obtained with MALDI are very good, since an internal mass calibration is performed with the matrix ions. For the inorganic matrices, laser fluences higher than those used with organic matrices are required to obtain good MALDI signals. When inorganic matrices were used in combination with glycerol as a dispersing agent, an important reduction of the background noise was observed. Urine samples spiked with the study compounds were processed by solid-phase extraction (SPE) and the screening was consistently positive., (Copyright (c) 2009 John Wiley & Sons, Ltd.)