Íleo meconial y falsos negativos del resultado de tripsina inmunorreactiva en una niña portadora de fibrosis quística / Meconium Ileus and false negative immunoreactive trypsine results in a cystic fibrosis carrier infant / Íleo meconial e resultados falso-negativos de tripsina imunorreativa em uma menina com fibrose cística

Introducción: la fibrosis quística (FQ) es una enfermedad genética, multisistémica, caracterizada por la disfunción de las glándulas de secreción exocrina secundaria a la mutación de una proteína transmembrana que actúa como canal de cloro. La inclusión de su búsqueda en el Sistema Nacional de Pesquisa Neonatal (SNPN) determinó un gran impacto sobre la morbimortalidad de esta enfermedad, permitiendo un diagnóstico y tratamiento precoz. El íleo meconial (IM) es la manifestación más precoz de la enfermedad, ocurriendo en un 10% a 18% de los pacientes. Objetivo: describir una forma de presentación precoz de FQ con resultado de pesquisa neonatal normal. Caso clínico: 18 meses. Sexo femenino; 33 semanas de edad gestacional. Retraso en la expulsión del meconio. Al tercer día de vida íleo meconial con oclusión intestinal, vólvulo de intestino medio y necrosis intestinal. Se realiza resección quirúrgica. Tripsina inmunorreactiva (TIR) normal a los 7 y 23 días de vida. No se solicita proteína asociada a la pancreatitis (PAP). Desnutrición crónica, bronquiolitis grave a los 3 meses, neumonías virales a los 7 y 11 meses. Ingreso con diagnóstico de neumonía aguda comunitaria con insuficiencia respiratoria. Test del sudor alterado en dos oportunidades. Elastasa pancreática insuficiente. Se aísla Staphylococcus aureus y Pseudomonas aeruginosa en exudado nasal y de secreciones respiratorias. Estudio genético: mutación Delta F508. Con diagnóstico de FQ se inició abordaje multidisciplinario, tratamiento y derivación al centro especializado. Conclusiones: la existencia de IM puede dar falsos negativos en el valor de la TIR, siendo necesaria la dosificación de la PAP. El pronóstico de estos pacientes va a depender de un diagnóstico precoz y el manejo terapéutico oportuno en centros especializados.

Introduction: cystic fibrosis (CF) is a genetic, multisystemic disease, characterized by dysfunction of the exocrine secretion glands secondary to the mutation of a transmembrane protein that acts as a chloride channel. Including its research by the National Neonatal Screening System (SNPN) caused a great impact on the morbidity and mortality of this disease, enabling early diagnosis and treatment. Meconium ileus (MI) is the earliest manifestation of the disease, occurring in 10% to 18% of patients. Objective: to describe a form of early presentation of CF with normal neonatal screening results. Clinical case: 18 months. Female, 33 weeks gestational age. Delay in the expulsion of meconium. On the third day of life, meconium ileus with intestinal obstruction, midgut volvulus and intestinal necrosis. Surgical resection is performed. Trypsin immunoreactive (TIR) normal at 7 and 23 days of age. Pancreatitis-associated protein (PAP) is not requested. Chronic malnutrition, severe bronchiolitis at 3 months, viral pneumonia at 7 and 11 months. Admission with a diagnosis of acute community pneumonia with respiratory failure. Sweat test altered on two occasions. Insufficient pancreatic elastase. Staphylococcus aureus and Pseudomonas aeruginosa were isolated in nasal exudate and respiratory secretions. Genetic study: Delta F508 mutation. With the CF diagnosis, a multidisciplinary approach, treatment and referral to a specialized center began. Conclusions: the existence of MI can provide false negatives in the IRR value, making PAP dosage necessary. The prognosis of these patients will depend on early diagnosis and timely therapeutic management at specialized centers.

Introdução: a fibrose cística (FC) é uma doença genética, multissistêmica, caracterizada pela disfunção das glândulas secretoras exócrinas secundária à mutação de uma proteína transmembrana que age como canal de cloreto. A inclusão da sua pesquisa no Sistema Nacional de Triagem Neonatal (SNPN) determinou grande impacto na morbimortalidade desta doença, permitindo diagnóstico e tratamento precoces. O íleo meconial (IM) é a manifestação mais precoce da doença, ocorrendo em 10 a 18% dos pacientes. Objetivo: descrever uma forma de apresentação precoce da FC com resultados normais de triagem neonatal. Caso clínico: 18 meses. Sexo feminino. 33 semanas de idade gestacional. Atraso na expulsão do mecônio. No terceiro dia de vida, íleo meconial com obstrução intestinal, vólvulo de intestino médio e necrose intestinal. A ressecção cirúrgica é realizada. Imunorreativo à tripsina (TIR) normal aos 7 e 23 dias de vida. A proteína associada à pancreatite (PAP) não é solicitada. Desnutrição crônica, bronquiolite grave aos 3 meses, pneumonia viral aos 7 e11 meses. Internação com diagnóstico de pneumonia comunitária aguda com insuficiência respiratória. Teste do suor alterado em duas ocasiões. Elastase pancreática insuficiente. Staphylococcus aureus e Pseudomonas aeruginosa foram isolados em exsudato nasal e secreções respiratórias. Estudo genético: mutação Delta F508. Com o diagnóstico de FC iniciou-se abordagem multidisciplinar, tratamento e encaminhamento para centro especializado. Conclusões: a existência de IM pode dar falsos negativos no valor da TIR, tornando necessária a dosagem de PAP. O prognóstico destes pacientes dependerá do diagnóstico precoce e do manejo terapêutico oportuno em centros especializados.

Trypsin Detection Strategies: A Review.

Trypsin, a pancreatic serine protease, due to its narrow specificity and selectivity, has been tremendously used in food technology, proteome analysis, modulating soy protein allergenicity, antihypertensive peptide production, as well as, a biomarker in diseases such as pancreatitis, cystic fibrosis etc. Therefore, simple, sensitive and selective biosensors, for trypsin evaluation, are strongly recommended. This review provides, for the first time, an overview of various sensing systems, developed for the detection of trypsin. We have categorized various sensors, developed in the last ten years, according to their signal output as optical (fluorescence, colorimeter, surface plasmon resonance, liquid crystals), electrochemical (photo-electrochemical, nanopore sensors), piezoelectric and enzyme linked immunosorbant assay based sensors. Under each section, further subsections explain, construction of biosensors on the basis of various natural proteins acting as substrate unit and nucleic acids, nanoparticles, inorganic dyes, polymers etc. as participant interacting units. Their working principles and strategies along with salient features, such as, limit of detection, linearity range, time etc. have been critically analyzed to highlight their comparative merits and demerits. The most sensitive biosensors for trypsin detection is a photo-electrochemical anti-trypsin based immunosensor, with a lowest limit of detection 0.02 ng mL-1; linearity 0.10-100 ng mL-1.

Bcl-10, trypsin and synaptophysin helps recognize acinar cell and mixed acinar neuroendocrine cell carcinoma of the pancreas on both preoperative cytological samples and needle biopsy specimens.

OBJECTIVE: Acinar cell carcinoma (ACC) of the pancreas are known to be rare and difficult to be recognize because they mimic other unrelated tumors (neuroendocrine, solid pseudopapillary) with different clinical behavior. Especially in the setting of inoperable patients, fine needle aspiration cytology (FNAC), core needle biopsy (FNAB) and immunocyto/histochemistry (ICC/IHC) play a crucial role in the differential diagnosis. The biological material available for ICC tests obtained by minimal invasive procedures is usually limited. Aim of the current study was to evaluate diagnostic panel based on a limited number of ICC markers for typing preoperatively ACC of the pancreas. METHODS: Of 1820 needle sampling procedures performed and related to pancreatic lesions, 21 cases were extracted with a confirmed diagnosis of ACC on histology. Of them,12 were pure ACC and 9 mixed acinar-neuroendocrine carcinoma (MANEC). Smears of ACC, MANEC and a control group composed of 34neuroendocrine, 7solid pseudopapillary, 50ductal and 4 adenosquamous carcinoma were assessed with an ICC panel made up of BCL10, trypsin, synaptophysin, chromograninA, ß-catenin. RESULTS: On cytology, BCL10 sensitivity and specificity for ACC was 100%. Trypsin correctly recognized 90% of the cases. Synaptophysin was helpful to correctly identify all the cases with a mixed neuroendocrine component. No significant cross-reaction was observed between BCL10 and trypsin in any of the control group case. CONCLUSIONS: BCL10 is a determinant marker for the diagnosis of acinar cell carcinoma and mixed acinar neuroendocrine cell carcinoma of the pancreas in a pre-operative citologic/histologic setting.

Modulation of Gold Nanorod Growth via the Proteolysis of Dithiol Peptides for Enzymatic Biomarker Detection.

Gold nanorods possess optical properties that are tunable and highly sensitive to variations in their aspect ratio (length/width). Therefore, the development of a sensing platform where the gold nanorod morphology (i.e., aspect ratio) is modulated in response to an analyte holds promise in achieving ultralow detection limits. Here, we use a dithiol peptide as an enzyme substrate during nanorod growth. The sensing mechanism is enabled by the substrate design, where the dithiol peptide contains an enzyme cleavage site in-between cysteine amino acids. When cleaved, the peptide dramatically impacts gold nanorod growth and the resulting optical properties. We demonstrate that the optical response can be correlated with enzyme concentration and achieve a 45 pM limit of detection. Furthermore, we extend this sensing platform to colorimetrically detect tumor-associated inhibitors in a biologically relevant medium. Overall, these results present a subnanomolar method to detect proteases that are critical biomarkers found in cancers, infectious diseases, and inflammatory disorders.

Ultrasensitive detection of trypsin in serum via nanochannel device.

A highly sensitive trypsin sensing system in serum was developed by using an anodic alumina oxide (AAO)-based, trypsin substrate-decorated hybrid ion permeation membrane. Owing to the trypsin-triggered peptide hydrolyzation reaction, the surface electrical feature of the peptide-decorated hybrid ion membrane changed. The electric double layer effect reduces the effective ion current diameter in the AAO nano unit, so that the ion current rectification ratio will be enhanced, realizing the quantitative detection of trypsin. The lowest detection concentration can be achieved as low as 0.1 pM. This method is no need for sample pre-preparation, easy to operate, highly sensitive, and also applicable to other enzyme evaluation systems by changing corresponding substrates. This study provides a new idea for selective measurements of proteases in complex biological samples.

Quantitative detection of ricin in beverages using trypsin/Glu-C tandem digestion coupled with ultra-high-pressure liquid chromatography-tandem mass spectrometry.

The toxic protein of ricin has drawn wide attention in recent years as a potential bioterrorism agent due to its high toxicity and wide availability. For the verification of the potential anti-terrorism activities, it is urgent for the quantification of ricin in food-related matrices. Here, a novel strategy of trypsin/Glu-C tandem digestion was introduced for quantitative detection of ricin marker peptides in several beverage matrices using isotope-labeled internal standard (IS)-mass spectrometry. The ricin in beverages was captured and enriched by biotinylated anti-ricin polyclonal antibodies conjugated to streptavidin magnetic beads. The purified ricin was cleaved using the developed trypsin/Glu-C tandem digestion method and then quantitatively detected by ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with isotope-labeled T7A and TG11B selected as IS. The use of trypsin/Glu-C digestion allows shorter peptides, which are more suitable for MS detection, to be obtained than the use of single trypsin digestion. Under the optimized tandem digestion condition, except for T7A in the A-chain, two resulting specific peptides of TG13A, TG28A from the A-chain and two of TG11B, TG33B from the B-chain were chosen as novel marker peptides with high MS response. The uniqueness of the selected marker peptides allows for unambiguous identification of ricin among its homologous proteins in a single run. The MS response of the four novel marker peptides is increased by more than 10 times compared with that of individual corresponding tryptic peptides. Both the marker peptides of A-chain T7A and B-chain TG11B were selected as quantitative peptides based on the highest MS response among the marker peptides from their individual chains. The limit of detection (LOD) of ricin is 0.1 ng/mL in PBS and 0.5 ng/mL in either milk or orange juice. The linear range of calibration curves for ricin were 0.5-300 ng/mL in PBS, 1.0-400 ng/mL in milk, and 1.0-250 ng/mL in orange juice. The method accuracy ranged between 82.6 and 101.8% for PBS, 88.9-105.2% for milk, and 95.3-118.7% for orange juice. The intra-day and inter-day precision had relative standard deviations (%RSD) of 0.3-9.4%, 0.7-8.9%, and 0.2-6.9% in the three matrices respectively. Furthermore, whether T7A or TG11B is used as a quantitative peptide, the quantitative results of ricin are consistent. This study provides not only a practical method for the absolute quantification of ricin in beverage matrices but also a new strategy for the investigation of illegal use of ricin in chemical weapon verification tasks such as OPCW biotoxin sample analysis exercises.

Label-free analytical performances of a peptide-based QCM biosensor for trypsin.

A label-free biosensor was fabricated for the detection of trypsin by using a peptide-functionalized quartz crystal microbalance gold electrode. The synthetized peptide chains were immobilized tightly on the QCM electrode via a self-assembly method, which formed a thin and approximate rigid layer of peptides. The detection signal was achieved by calculating the mass changes on the QCM electrode because the peptide chains could be specifically cleaved in the carboxyl terminuses of arginine and lysine by trypsin. When gold nanoparticles were coupled to the peptide chains, the sensing signal would be amplified 10.9 times. Furthermore, the sensor interface shows a lower resonance resistance change when the peptide chain is immobilized horizontally. Independent detections in parallel on different electrodes have a wide linear range. Under the optimum conditions, the signal-amplified biosensor allowed the measurement of trypsin over the range of 0-750 ng mL-1 with a detection limit of 8.6 ng mL-1. Moreover, for screening the inhibitor of trypsin, the IC50 values were obtained to be 1.85 µg mL-1 for benzamidine hydrochloride and 20.5 ng mL-1 for the inhibitor from soybean.

Miniaturisation of a peptide-based electrochemical protease activity sensor using platinum microelectrodes.

Proteases are ideal target biomarkers as they have been implicated in many disease states, including steps associated with cancer progression. Electrochemical peptide-based biosensors have attracted much interest in recent years. However, the significantly large size of the electrodes typically used in most of these platforms has led to performance limitations. These could be addressed by the enhancements offered by microelectrodes, such as rapid response times, improved mass transport, higher signal-to-noise and sensitivity, as well as more localised and less invasive measurements. We present the production and characterisation of a miniaturised electrochemical biosensor for the detection of trypsin, based on 25 µm diameter Pt microelectrodes (rather than the ubiquitous Au electrodes), benchmarked by establishing the equivalent Pt macroelectrode response in terms of quantitative response to the protease, the kinetics of cleavage and the effects of non-specific protein binding and temperature. Interestingly, although there was little difference between Au and Pt macroelectrode response, significant differences were observed between the responses of the Pt macroelectrode and microelectrode systems indicative of increased reproducibility in the microelectrode SAM structure and sensor performance between the electrodes, increased storage stability and a decrease in the cleavage rate at functionalised microelectrodes, which is mitigated by measurement at normal body temperature. Together, these results demonstrate the robustness and sensitivity of the miniaturised sensing platform and its ability to operate within the clinically-relevant concentration ranges of proteases in normal and disease states. These are critical features for its translation into implantable devices.

Low-concentration trypsin detection from a metal-enhanced fluorescence (MEF) platform: Towards the development of ultra-sensitive and rapid detection of proteolytic enzymes.

Proteolytic enzymes, which serve to degrade proteins to their amino acid building blocks, provide a distinct challenge for both diagnostics and biological research fields. Due to their ubiquitous presence in a wide variety of organisms and their involvement in disease, proteases have been identified as biomarkers for various conditions. Additionally, low-levels of proteases may interfere with biological investigation, as contamination with these enzymes can physically alter the protein of interest to researchers, resulting in protein concentration loss or subtler polypeptide clipping that leads to a loss of functionality. Low levels of proteolytic degradation also reduce the shelf-life of commercially important proteins. Many detection platforms have been developed to achieve low-concentration or low-activity detection of proteases, yet many suffer from limitations in analysis time, label stability, and ultimately sensitivity. Herein we demonstrate the potential utility of fluorescein derivatives as fluorescent labels in a new, turn-off enzymatic assay based on the principles of metal-enhanced fluorescence (MEF). For fluorescein sodium salt alone on nano-slivered 96-well plates, or Quanta Plates™, we report up to 11,000x enhancement for fluorophores within the effective coupling or enhancement volume region, defined as ~100 nm from the silver surface. We also report a 9% coefficient of variation, and detection on the picomolar concentration scale. Further, we demonstrate the use of fluorescein isothiocyanate-labeled YebF protein as a coating layer for a MEF-based, Quanta Plate™ enzymatic activity assay using trypsin as the model enzyme. From this MEF assay we achieve a detection limit of ~1.89 ng of enzyme (2.8 mBAEE activity units) which corresponds to a minimum fluorescence signal decrease of 10%. The relative success of this MEF assay sets the foundation for further development and the tuning of MEF platforms for proteolytic enzyme sensing not just for trypsin, but other proteases as well. In addition, we discuss the future development of ultra-fast detection of proteases via microwave-accelerated MEF (MAMEF) detection technologies.

Peptide-induced aggregation of glutathione-capped gold nanoclusters: A new strategy for designing aggregation-induced enhanced emission probes.

A series of polymers and metal ions have been observed to be useful in triggering aggregation-induced emission (AIE) and AIE enhancement (AIEE) of thiolated gold nanoclusters (AuNCs). However, peptide-induced AIEE of thiolated AuNCs and their applications in biosensors have rarely been investigated. In this study, we showed that positively charged peptides induced efficient AIEE of negatively charged glutathione-capped AuNCs (GSH-AuNCs) through electrostatic attraction. In contrast to GSH-AuNCs, polyarginine (polyArg), a cationic peptide, stimulated the AIEE of the GSH-AuNCs, resulting in a 3.5-fold luminescence enhancement, 10-fold enhancement in quantum yield, 8-nm blueshift in the luminescence maximum, and a 2.1-fold increase in the mean luminescence lifetime. Four different AIEE-based biosensors with excellent selectivity and acceptable sensitivity were fabricated using cationic peptides as an AIEE-active trigger and as a biorecognition element. A heparin biosensor with a limit of detection (LOD) of 3 nM was constructed by combining AG73 peptide-mediated AIEE of the GSH-AuNCs and the specific interaction of AG73 peptides with heparin macromolecules. The concentration of human trypsin was selectively detected at a concentration as low as 1 nM using an arginine-glycine repeat peptide as an enzymatic substrate and as an AIEE-active trigger. Alkaline phosphatase (ALP)-catalyzed dephosphorylation of phosphopeptides paired with the corresponding product-mediated AIEE of the GSH-AuNCs was used for ALP sensing with an LOD of 0.3 U L-1. A peptide consisting of a cyclic RGD unit and an AIEE-active unit was designed to synthesize RGD-modified GSH-AuNC aggregates that can target αvß3 integrin receptors. These AIEE-based sensors were practically applied for the quantitative determination of heparin in human plasma, trypsin in human urine, and ALP in human plasma as well as for luminescent imaging of αvß3 integrin-overexpressing HeLa cells.

A pyrene linked peptide probe for quantitative analysis of protease activity via MALDI-TOF-MS.

We report herein a rationally designed pyrene linked substrate for quantitative protease activity assay via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In this proof-of-concept study, a trypsin-specific peptide with the sequence of GGGGRG was selected to conjugate with pyrene forming a pyrene linked peptide probe, Py-GGGGRG. In the presence of trypsin, the Py-GGGGRG probe can be specifically hydrolyzed into Py-GGGGR. The introduction of pyrene greatly increased ionization efficiency of Py-peptides, and Py-peptides could be selectively captured from complex mixtures by a facially fabricated polystyrene coated MALDI plate through hydrophobic and π-π stacking interactions. As a result, trypsin activity can be directly quantified by relative intensity ratio of product and substrate via MALDI-TOF-MS without the use of external internal standard. A linear range of 0.1-10 µg/mL and a relatively low detection limit of 29 ng/mL were obtained. This method has also been successfully used for quantification of trypsin activity in urine and screening the inhibitors of trypsin. Besides, the proposed strategy was also validated for another protease, chymotrypsin, by using the probe Py-GGGGGGYG. Therefore, owing to simplicity, high-throughput capacity and quantificational accuracy, the proposed method shows great potential for activity assay of various proteases and screening their inhibitors via application of specific peptide sequences.

Aprotinin Encapsulated Gold Nanoclusters: A Fluorescent Bioprobe with Dynamic Nuclear Targeting and Selective Detection of Trypsin and Heavy Metal.

Fluorescence imaging has currently emerged as one of the most frequently used noninvasive imaging technologies to selectively monitor biological processes in living systems. In past decades, gold nanoclusters (Au NCs) has received increasing attraction because of their intrinsic fluorescence and their inherent biocompatibility. As a stabilizing and reducing agent, an abundant, sustainable, and widely used polypeptide derived drug molecule, aprotinin (Ap), is selected for the synthesis of Au nanoclusters (Ap-Au NCs) due to characteristic bioactivity, excellent biocompatibility, biodegradability, and non-allergenic character. Herein, Ap encapsulated Au NCs with desirable red fluorescence was facilely produced for the first time, which were subsequently used for cell imaging and detection of various analytes. Much interestingly, dynamically subcellular targeting  from the cytoplasm to the nucleus in HeLa cells was observed. Besides, it has shown that, the selective and quantitative detection of trypsin has been established by using Ap-Au NCs. Finally, Ap-Au NCs were readily used for quantitative detection of mercury and copper. The photoluminescence of the Ap-Au NCs was quenched with the addition of the aforementioned analytes. This study not only  discusses a multifunctional nanomaterial  for cell imaging, dynamically nuclear targeting and biosensing, but also opens crucial insights on the integration of funtional biomolecule with metal nanoclusters intended for extensively biomedical applications.

A highly sensitive peptide-based biosensor using NiCo2O4 nanosheets and g-C3N4 nanocomposite to construct amplified strategy for trypsin detection.

Here, a simple electrochemical biosensor was proposed based on the specific recognition between trypsin and peptide. Initially, NiCo2O4-PAMAM nanocomposite was casted on the bare electrode to achieve the electrochemical signal amplification in 0.1 mM [Ru(NH3)6]3+ solution owing to the great electronic conductivity and high electrochemical activity induced by the special structure of NiCo2O4 nanosheets (Ni3+ cations in octachedral sites of the Co3O4). Subsequently, a declined electrochemical signal was obtained when g-C3N4 labeled peptide composites were anchored on the electrode. However, after trypsin was added into solution and incubated with the biosensor, the electrochemical signal was re-promoted. Therefore, the as-synthesized biosensor could realize the sensitive detection of trypsin by virtue of the specific recognition between trypsin and peptide. As a result, the developed peptide-based exhibited a linear range from 10-10 to 10-4 mg mL-1 with an ultralow detection limit of 10-10 mg mL-1, providing sensitive analytical performance and acceptable application potential in clinical test and disease diagnosis due to its high stability, excellent selectivity, acceptable reproducibility and accurate signal output.

Infrared laser ablation and capture of enzymes with conserved activity.

Infrared (IR) laser ablation at 3 µm wavelength was used to extract enzymes from tissue and quantitatively determine their activity. Experiments were conducted with trypsin, which was ablated, captured and then used to digest bovine serum albumin (BSA). BSA digests were evaluated using matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) and sequence coverage of 59% was achieved. Quantification was performed using trypsin and catalase standards and rat brain tissue by fluorescence spectroscopy. Both enzymes were reproducibly transferred with an efficiency of 75 ±â€¯8% at laser fluences between 10 and 30 kJ/m2. Trypsin retained 37 ±â€¯2% of its activity and catalase retained 50 ±â€¯7%. The activity of catalase from tissue was tested using three consecutive 50 µm thick rat brain sections. Two 4 mm2 regions were ablated and captured from the cortex and cerebellum regions. The absolute catalase concentration in the two regions was consistent with previously published data, demonstrating transfer of intact enzymes from tissue.

Dual emissive bispyrene peptide probes for highly sensitive measurements of trypsin activity and evaluation of trypsin inhibitors.

Peptide substrates were double labeled with pyrenes to prepare fluorescent probes for highly sensitive detection of protease activity and evaluation of protease inhibitors using pyrene monomer/excimer signals. Two proximate pyrene moieties formed excited state dimers in the probes, and these pyrene excimer formations were dissociated by tryptic digestion. The specificity constant of the optimum bispyrene peptide probe was 2.7 times higher than that of the conventional peptide-4-methylcoumarin amide. Moreover, our probe had high sensitivity with an estimated detection limit for trypsin of 4.11 pM. The half maximal inhibitory concentration and dissociation constant of the Bowman-Birk inhibitor were successfully estimated.

PRSS3 is a prognostic marker in invasive ductal carcinoma of the breast.

OBJECTIVE: Serine protease 3 (PRSS3) is an isoform of trypsinogen, and plays an important role in the development of many malignancies. The objective of this study was to determine PRSS3 mRNA and protein expression levels in invasive ductal carcinoma of the breast and normal surrounding tissue samples. RESULTS: Both PRSS3 mRNA and protein levels were significantly higher in invasive ductal carcinoma of the breast tissues than in normal or benign tissues (all P < 0.05). High PRSS3 protein levels were associated with patients' age, histological grade, Her-2 expression level, ki-67 expression, and the 5.0-year survival rate. These high protein levels are independent prognostic markers in invasive ductal carcinoma of the breast. MATERIALS AND METHODS: We used real-time quantitative polymerase chain reactions (N = 40) and tissue microarray immunohistochemistry analysis (N = 286) to determine PRSS3 mRNA and protein expression, respectively. PRSS3 protein levels in invasive ductal carcinoma of the breast tissues were correlated with the clinical characteristics of patients with invasive ductal carcinoma of the breast and their 5.0-year survival rate. CONCLUSIONS: PRSS3 acts as an oncogene in invasive ductal carcinoma of the breast development and progression. This finding implies that detection of PRSS3 expression can be a useful prognosis marker and the targeting of PRSS3 can potentially represent a new strategy for invasive ductal carcinoma of the breast treatment.

Photoelectrochemical biosensor constructed using TiO2 mesocrystals based multipurpose matrix for trypsin detection.

Herein, a delicate photoelectrochemical biosensor for quantitative detection of trypsin was successfully established by virtue of polyethylenimine-sensitized TiO2 mesocrystal as the photoactive matrix integrated with Boron-doped carbon quantum dots labeled peptide as the signal amplification tags. Specifically, polyethylenimine with fine photo-stability was introduced here as the electron transporting layer to reduce the energy barrier of TiO2 mesocrystal, thereby facilitating the carriers transfer and improving the photocurrent response. Moreover, the Boron-doped carbon dots-peptide bioconjugates could noticeably decrease the photocurrent due to the competitively light harvesting by Boron-doped carbon dots and the steric hindrance of peptide chains, leading to less light energy arriving at the TiO2 mesocrystal and hindering the electrons transfer between the electrolyte and electrode. The anchored conjugates synergistically promoted the decline of photocurrent signal, evidently enhancing the sensitivity of this detection protocol. When trypsin was incubated, the photoelectric signal was obviously re-promoting because arginine-containing peptide chains could be specifically cleaved by trypsin and the Boron-doped carbon quantum dots was affranchised from the electrode, making the most of the previous suppression effects released. Therefore, the intensity of photocurrent signal was proportional to the trypsin concentration in a wide linger range from 1×10-7mg/mL to 1.0mg/mL. This practical and elegant "on-off-on" biosensor with high sensitivity offered a promising scheme to monitor various proteases and the inhibitors screening for early diagnoses of different diseases.

Effects of Hull Scratching, Soaking, and Boiling on Antinutrients in Japanese Red Sword Bean (Canavalia gladiata).

The effects of hull processing, soaking, and boiling on the content or activity of antinutrients in the red sword bean (RSB; Canavalia gladiata) were investigated. RSB seeds were compared with kidney bean (KB; Phaseolus vulgaris) seeds that are starch based and often used as processed products in Japan. RSB seeds had higher weight, thicker hull, and higher protein content, but lower moisture content compared with KB seeds. Because of the strong and thick hull, the relative water absorption of untreated RSB seeds was very low after soaking. Seeds were soaked after dehulling, scratching, and roasting. The results showed that hull scratching was the optimal method for increasing water absorption during soaking compared with dehulling and roasting. After soaking, the water used for soaking was discarded, since it had a high content of polyphenols and bitter taste, and RSB seeds were boiled in fresh water for 20, 40, and 60 min. The results showed that polyphenol and tannin contents, antioxidant activity, and hemagglutinating activity, as well as maltase, sucrase, and trypsin inhibitor activities in scratched RSB seeds decreased significantly after boiling compared with those in raw seeds, whereas amylase inhibitor activity showed no significant change. Overall, it was concluded that the combination of hull scratching, soaking, and boiling in fresh water can reduce thermal-stable or sensitive antinutrients in RSB and thus, significantly improve its nutritional value.

Proteomic studies with a novel nano-magnetic chelating system to capture metalloproteins and its application in the preliminary study of monocyte and macrophage sub-secretome.

A new chelating chromatography method was developed based in the use of magnetic iron oxide nanoparticles functionalized with EDTA-TMS ((N-(trimethoxysilylpropyl)ethylenediaminetriacetate trisodium salt). These particles combine a high surface area, biocompatibility and magnetic removal from solution, with the chelating affinity towards metal ions. The particles were used to selectively capture metallo-dependant proteins in secretome obtained from human monocytes and mouse macrophages. Secreted metallo-dependant proteins are highly important sources of information since they are involved in several pathological processes. The identification of secreted proteins involved in these processes is highly important for diagnosis or monitoring the progression of a disease. In this multiple-approach study it was possible to not only selectively capture several secreted metallo-dependant proteins, but also to significantly avoid masking proteins such as the highly abundant albumin form the fetal bovine serum used to supplement the cell culture medium. Overall, the magnetic nanoparticle-based chelating chromatography method developed here has proved to be a sensitive, low cost, and a quick tool for sample treatment in order to selectively enrich metalloproteins while overcoming the contamination of highly abundant proteins.

Detection of Cancer-Specific Proteases Using Magnetic Relaxation of Peptide-Conjugated Nanoparticles in Biological Environment.

Protease expression is closely linked to malignant phenotypes of different solid tumors; as such, their detection is promising for diagnosis and treatment of cancers, Alzheimer's, and vascular diseases. Here, we describe a new method for detecting proteases by sensitively monitoring the magnetic relaxation of monodisperse iron oxide nanoparticles (IONPs) using magnetic particle spectrometer (MPS). In this assay, tailored peptides functioning as activatable nanosensors link magnetic nanoparticles and possess selective sites that are recognizeable and cleaveable by specific proteases. When these linker peptides, labeled with biotin at N- and C-terminals, are added to the neutravidin functionalized IONPs, nanoparticles aggregate, resulting in well-defined changes in the MPS signal. However, as designed, in the presence of proteases these peptides are cleaved at predetermined sites, redispersing IONPs, and returning the MPS signal(s) close to its preaggregation state. These changes observed in all aspects of the MPS signal (peak intensity, its position as a function of field amplitude, and full width at half-maximum-when combined, these three also eliminate false positives), help to detect specific proteases, relying only on the magnetic relaxation characteristics of the functionalized nanoparticles. We demonstrate the general utility of this assay by detecting one each from the two general classes of proteases: trypsin (digestive serine protease, involved in various cancers, promoting proliferation, invasion, and metastasis) and matrix metalloproteinase (MMP-2, observed through metastasis and tumor angiogenesis). This MPS based protease-assay is rapid, reproducible, and highly sensitive and can form the basis of a feasible, high-throughput method for detection of various other proteases.