Hydrolytic biotransformation of the bumetanide ester prodrug DIMAEB to bumetanide by esterases in neonatal human and rat serum and neonatal rat brain-A new treatment strategy for neonatal seizures?

OBJECTIVES: The loop diuretic bumetanide has been proposed previously as an adjunct treatment for neonatal seizures because bumetanide is thought to potentiate the action of γ-aminobutyric acid (GABA)ergic drugs such as phenobarbital by preventing abnormal intracellular accumulation of chloride and the subsequent "GABA shift." However, a clinical trial in neonates failed to demonstrate such a synergistic effect of bumetanide, most likely because this drug only poorly penetrates into the brain. This prompted us to develop lipophilic prodrugs of bumetanide, such as the N,N-dimethylaminoethyl ester of bumetanide (DIMAEB), which rapidly enter the brain where they are hydrolyzed by esterases to the parent compound, as demonstrated previously by us in adult rodents. However, it is not known whether esterase activity in neonates is sufficient to hydrolyze ester prodrugs such as DIMAEB. METHODS: In the present study, we examined whether esterases in neonatal serum of healthy term infants are capable of hydrolyzing DIMAEB to bumetanide and whether this activity is different from the serum of adults. Furthermore, to extrapolate the findings to brain tissue, we performed experiments with brain tissue and serum of neonatal and adult rats. RESULTS: Serum from 1- to 2-day-old infants was capable of hydrolyzing DIMAEB to bumetanide at a rate similar to that of serum from adult individuals. Similarly, serum and brain tissue of neonatal rats rapidly hydrolyzed DIMAEB to bumetanide. SIGNIFICANCE: These data provide a prerequisite for further evaluating the potential of bumetanide prodrugs as add-on therapy to phenobarbital and other antiseizure drugs as a new strategy for improving pharmacotherapy of neonatal seizures.

Erythrocytes Prevent Degradation of Carnosine by Human Serum Carnosinase.

The naturally occurring dipeptide carnosine (ß-alanyl-l-histidine) has beneficial effects in different diseases. It is also frequently used as a food supplement to improve exercise performance and because of its anti-aging effects. Nevertheless, after oral ingestion, the dipeptide is not detectable in human serum because of rapid degradation by serum carnosinase. At the same time, intact carnosine is excreted in urine up to five hours after intake. Therefore, an unknown compartment protecting the dipeptide from degradation has long been hypothesized. Considering that erythrocytes may constitute this compartment, we investigated the uptake and intracellular amounts of carnosine in human erythrocytes cultivated in the presence of the dipeptide and human serum using liquid chromatography-mass spectrometry. In addition, we studied carnosine's effect on ATP production in red blood cells and on their response to oxidative stress. Our experiments revealed uptake of carnosine into erythrocytes and protection from carnosinase degradation. In addition, no negative effect on ATP production or defense against oxidative stress was observed. In conclusion, our results for the first time demonstrate that erythrocytes can take up carnosine, and, most importantly, thereby prevent its degradation by human serum carnosinase.

Zinc Oxide Particles Catalytically Generate Nitric Oxide from Endogenous and Exogenous Prodrugs.

Nitric oxide (NO) is a potent biological molecule that contributes to a wide spectrum of physiological processes. However, the full potential of NO as a therapeutic agent is significantly complicated by its short half-life and limited diffusion distance in human tissues. Current strategies for NO delivery focus on encapsulation of NO donors into prefabricated scaffolds or an enzyme-prodrug therapy approach. The former is limited by the finite pool of NO donors available, while the latter is challenged by the inherent low stability of natural enzymes. Zinc oxide (ZnO) particles with innate glutathione peroxidase and glycosidase activities, a combination that allows to catalytically decompose both endogenous (S-nitrosoglutathione) and exogenous (ß-gal-NONOate) donors to generate NO at physiological conditions are reported. By tuning the concentration of ZnO particles and NO prodrugs, physiologically relevant NO levels are achieved. ZnO preserves its catalytic property for at least 6 months and the activity of ZnO in generating NO from prodrugs in human serum is demonstrated. The ZnO catalytic activity will be beneficial toward generating stable NO release for long-term biomedical applications.

Cholinesterase's activities of infected mice by Brucella ovis.

The role of cholinesterase in inflammatory reactions has been described in several infectious diseases. However, in Brucella spp. this has not yet been studied. Therefore, the objective of this study was to evaluate whether experimental infection by Brucella ovis alters the cholinergic activity in pro- or anti-inflammatory responses to the disease. For the study 48 mice were used, 24 infected by B. ovis and 24 non-infected. We collected samples of whole blood on days 7, 15, 30 and 60 post-infection (PI) by B. ovis. Acetylcholinesterase (AChE) activity in the blood increased on days 15 and 60 PI (P < 0.05). Butyrylcholinesterase (BChE) activity in serum increased on days 7 and 60 PI (P < 0.05). An increase in serum free radical levels occurred on days 7, 15 and 60 PI (P < 0.05), and consequently superoxide dismutase activity increased on day 15 PI (P < 0.05). A reduction in catalase activity occurred when the infection became chronic (60 PI). The increase in AChE and BChE characterized a pro-inflammatory response, since these enzymes regulate levels of acetylcholine (ACh) and butyrylcholine (BuSCh), molecules with anti-inflammatory properties. Therefore, with the increase of cholinesterase activity, there was an extracellular reduction of ACh, an inhibitor of several inflammatory mediators. This proinflammatory response of B. ovis infection leads to oxidative stress, and consequently to cellular damage.

Association of human serum paraoxonase-1 with some respiratory drugs.

In this study, we investigated the effects of certain respiratory drugs, which are mainly used on human serum paraoxonase-1 (hPON1; EC 3.1.8.1). hPON1 was purified from human serum, with 354.91 fold and 45% yield by using two simple step procedures including, first, ammonium sulfate precipitation, then, Sepharose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis showed a single protein band belonging to hPON1 with 43 kDa. All the pharmaceutical compounds inhibited the PON1 enzyme highly at the micromolar level. The obtained IC50 values for nine different pharmaceutics ranged from 0.219 µM (salbutamol sulfate) to 67.205 µM (montelukast sodium). So, all drugs could be considered as potent hPON1 inhibitors. Ki values and inhibition types were determined by Lineweaver-Burk graphs. While varenicline tartrate and moxifloxacin hydrochloride inhibited the enzyme in a noncompetitive manner, others inhibited it in a mixed manner.

An engineered cyclic peptide alleviates symptoms of inflammation in a murine model of inflammatory bowel disease.

Inflammatory bowel diseases (IBDs) are a set of complex and debilitating diseases for which there is no satisfactory treatment. Recent studies have shown that small peptides show promise for reducing inflammation in models of IBD. However, these small peptides are likely to be unstable and rapidly cleared from the circulation, and therefore, if not modified for better stability, represent non-viable drug leads. We hypothesized that improving the stability of these peptides by grafting them into a stable cyclic peptide scaffold may enhance their therapeutic potential. Using this approach, we have designed a novel cyclic peptide that comprises a small bioactive peptide from the annexin A1 protein grafted into a sunflower trypsin inhibitor cyclic scaffold. We used native chemical ligation to synthesize the grafted cyclic peptide. This engineered cyclic peptide maintained the overall fold of the naturally occurring cyclic peptide, was more effective at reducing inflammation in a mouse model of acute colitis than the bioactive peptide alone, and showed enhanced stability in human serum. Our findings suggest that the use of cyclic peptides as structural backbones offers a promising approach for the treatment of IBD and potentially other chronic inflammatory conditions.

Group IIA-Secreted Phospholipase A2 in Human Serum Kills Commensal but Not Clinical Enterococcus faecium Isolates.

Human innate immunity employs cellular and humoral mechanisms to facilitate rapid killing of invading bacteria. The direct killing of bacteria by human serum is attributed mainly to the activity of the complement system, which forms pores in Gram-negative bacteria. Although Gram-positive bacteria are considered resistant to killing by serum, we uncover here that normal human serum effectively kills Enterococcus faecium Comparison of a well-characterized collection of commensal and clinical E. faecium isolates revealed that human serum specifically kills commensal E. faecium strains isolated from normal gut microbiota but not clinical isolates. Inhibitor studies show that the human group IIA secreted phospholipase A2 (hGIIA), but not complement, is responsible for killing of commensal E. faecium strains in human normal serum. This is remarkable since the hGIIA concentration in "noninflamed" serum was considered too low to be bactericidal against Gram-positive bacteria. Mechanistic studies showed that serum hGIIA specifically causes permeabilization of commensal E. faecium membranes. Altogether, we find that a normal concentration of hGIIA in serum effectively kills commensal E. faecium and that resistance of clinical E. faecium to hGIIA could have contributed to the ability of these strains to become opportunistic pathogens in hospitalized patients.

Changes on the activity of cholinesterase's in an immunomodulatory response of cattle infected by Listeria monocytogenes.

The aim of this study was to evaluate whether Listeria monocytogenes infection alters the activity of cholinesterases in cattle to module their inflammatory response and neurotransmission. Thus, ten male bovines (Holstein) were divided into two groups of five animals each: uninfected (control) and L. monocytogenes infected. Blood samples were collected on days 0, 7 and 14 post-infection (PI) to evaluate the activity of acetylcholinesterase (AChE) in the blood, and seric butyrylcholinesterase (BChE) activity, as well as total protein, albumin, globulin and C-reactive protein (CPR) levels in serum. The AChE activity and acetylcholine (ACh) levels were measured in the central nervous system on day 14 PI, and histopathological analyses were also performed. The infected animals did not show apparent clinical signs of listeriosis, however histopathological alterations were seen in the intestines and spleens. On days 7 and 14 PI, AChE activity in the blood was lower in infected animals, as well the seric BChE activity on day 7 PI. In the cerebral cortex and cerebellum, AChE activity was lower in infected animals compared to the control group, while the ACh levels were higher in the cerebral cortex compared to uninfected animals. Moreover, seric levels of total protein, globulin and CRP were higher in infected animals on days 7 and 14 PI compared to the control group. Therefore, we concluded that acute infection by L. monocytogenes alters the cholinergic system through the reduction of cholinesterase enzymes in the blood, serum and cerebral tissues as an adaptive response to an anti-inflammatory effect in order to increase the ACh levels, an anti-inflammatory molecule with an important role in the host immunomodulation.

Evaluation of oxidative stress and antioxidant status, serum trace mineral levels and cholinesterases activity in cattle infected with Anaplasma marginale.

This study was undertaken to assess the influence of an Anaplasma marginale infection on oxidative stress and antioxidant status, trace elements and cholinesterase as markers of the inflammatory process and biomarkers of oxidative imbalance. An infected group comprised of 35 crossbred Holstein cattle, about 2-3 years old, naturally infected with Anaplasma marginale, were divided into 4 subgroups according to their parasitemia rates (<1%, 1-10%, 10-20%, >20%) and also 10 healthy cattle as control were selected. Blood samples were taken and hematological parameters, activities of antioxidant enzymes including erythrocyte glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), glucose-6-phosphate dehydrogenase (G6PD), total antioxidant capacity (TAC), median corpuscularfragility (MCF) as well as acetylcholinesterase (AChE), and serum concentrations of antioxidant trace minerals (copper, iron, zinc, manganese, and selenium) and butyrylcholinesterase (BChE) were determined. In addition, as an index of lipid peroxidation, the level of malondialdehyde (MDA) was measured. The results revealed a significant decrease (P < 0.05) in RBC count, packed cell volume (PCV) and Hb concentration as well as the activities of erythrocyte GSH-Px, SOD, CAT, G6PD, TAC, MCF and AChE and serum concentrations of Cu, Zn, Mn, Se and BchE in the infected cattle. In contrast, significantly increased (P < 0.05) levels of MDA and erythrocyte osmotic fragility as well as serum concentration of iron were recorded in the infected animals. The significant decrease in antioxidant enzyme activities and substantial elevated levels of lipid peroxidation and erythrocyte osmotic fragility associated with the notable increase in parasitemia indicate increased exposure of RBCs to oxidative damage. Furthermore, decrease of cholinesterase in infection by A. marginale can and directly or indirectly lead to increase acetylcholine levels potent anti-inflammatory molecules, thereby inhibiting inflammation.

Quantification of Human Kallikrein-Related Peptidases in Biological Fluids by Multiplatform Targeted Mass Spectrometry Assays.

Human kallikrein-related peptidases (KLKs) are a group of 15 secreted serine proteases encoded by the largest contiguous cluster of protease genes in the human genome. KLKs are involved in coordination of numerous physiological functions including regulation of blood pressure, neuronal plasticity, skin desquamation, and semen liquefaction, and thus represent promising diagnostic and therapeutic targets. Until now, quantification of KLKs in biological and clinical samples was accomplished by enzyme-linked immunosorbent assays (ELISA). Here, we developed multiplex targeted mass spectrometry assays for the simultaneous quantification of all 15 KLKs. Proteotypic peptides for each KLK were carefully selected based on experimental data and multiplexed in single assays. Performance of assays was evaluated using three different mass spectrometry platforms including triple quadrupole, quadrupole-ion trap, and quadrupole-orbitrap instruments. Heavy isotope-labeled synthetic peptides with a quantifying tag were used for absolute quantification of KLKs in sweat, cervico-vaginal fluid, seminal plasma, and blood serum, with limits of detection ranging from 5 to 500 ng/ml. Analytical performance of assays was evaluated by measuring endogenous KLKs in relevant biological fluids, and results were compared with selected ELISAs. The multiplex targeted proteomic assays were demonstrated to be accurate, reproducible, sensitive, and specific alternatives to antibody-based assays. Finally, KLK4, a highly prostate-specific protein and a speculated biomarker of prostate cancer, was unambiguously detected and quantified by immunoenrichment-SRM assay in seminal plasma and blood serum samples from individuals with confirmed prostate cancer and negative biopsy. Mass spectrometry revealed exclusively the presence of a secreted isoform and thus unequivocally resolved earlier disputes about KLK4 identity in seminal plasma. Measurements of KLK4 in either 41 seminal plasma or 58 blood serum samples revealed no statistically significant differences between patients with confirmed prostate cancer and negative biopsy. The presented multiplex targeted proteomic assays are an alternative analytical tool to study the biological and pathological roles of human KLKs.

Standardization of clinical enzyme analysis using frozen human serum pools with values assigned by the International Federation of Clinical Chemistry and Laboratory Medicine reference measurement procedures.

Variation in clinical enzyme analysis, particularly across different measuring systems and laboratories, represents a critical but long-lasting problem in diagnosis. Calibrators with traceability and commutability are imminently needed to harmonize analysis in laboratory medicine. Fresh frozen human serum pools were assigned values for alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), creatine kinase (CK) and lactate dehydrogenase (LDH) by six laboratories with established International Federation of Clinical Chemistry and Laboratory Medicine reference measurement procedures. These serum pools were then used across 76 laboratories as a calibrator in the analysis of five enzymes. Bias and imprecision in the measurement of the five enzymes tested were significantly reduced by using the value-assigned serum in analytical systems with open and single-point calibration. The median (interquartile range) of the relative biases of ALT, AST, GGT, CK and LDH were 2.0% (0.6-3.4%), 0.8% (-0.8-2.3%), 1.0% (-0.5-2.0%), 0.2% (-0.3-1.0%) and 0.2% (-0.9-1.1%), respectively. Before calibration, the interlaboratory coefficients of variation (CVs) in the analysis of patient serum samples were 8.0-8.2%, 7.3-8.5%, 8.1-8.7%, 5.1-5.9% and 5.8-6.4% for ALT, AST, GGT, CK and LDH, respectively; after calibration, the CVs decreased to 2.7-3.3%, 3.0-3.6%, 1.6-2.1%, 1.8-1.9% and 3.3-3.5%, respectively. The results suggest that the use of fresh frozen serum pools significantly improved the comparability of test results in analytical systems with open and single-point calibration.

Oxidative stress in rats experimentally infected by Sporothrix schenckii.

The aim of this study was to evaluate whether oxidative stress occurs in rats experimentally infected by Sporothrix schenckii, and its possible effect on disease pathogenesis. Thirty rats were divided into two groups: the group A (uninfected, n = 18) and the group B (infected by S. schenckii, n=21). Blood samples were collected on days 15, 30 and 40 post-infection (PI). At each sampling time, six rats of the group A, and seven of the group B were bled. TBARS (thiobarbituric acid reactive substances) levels in serum samples were measured to evaluate lipid peroxidation. In addition, catalase (CAT) and superoxide dismutase (SOD) activities, known as biomarkers of antioxidants levels, were verified in whole blood. Seric pro-inflammatory cytokine levels were measured (IFN-γ, TNF-α, and IL-6), which showed that these inflammatory mediators were at higher levels in the infected rats (P < 0.001). In comparison to uninfected animals, rats with sporotrichosis showed significantly higher (p < 0.01) levels of TBARS on day 40 PI; CAT activity was significantly increased (p < 0.01) on days 30 and 40 PI; and SOD activity was increased (p < 0.01) on day 40 PI. Infected rats showed larger testicles and granulomas in the testicular capsule, as well as hepatic granulomas and splenic follicular hyperplasia. All tissues (testicle, spleen, and liver) showed inflammation associated with numerous fungal structures. These results demonstrated that the intense inflammatory response (seric and tissue) in sporotrichosis is a likely mechanism for redox imbalance, and consequently cause the oxidative stress in experimentally infected rats.

[THE MODE OF EVALUATION OF FUNCTIONAL ACTIVITY OF C3-CONVERTASE OF CLASSIC PATH OF ACTIVATION OF COMPLEMENT].

The technique of detection of stabilization of C3-convertase classical way of activation of system of complement inhuman blood serum. The technique comprises two stages and is based on applying a reaction of lysis of erythrocytes of sheep sensitized by antibodies using 0.8% human blood serum. Preliminary an incubation of two samples (experimental and control) is applied during 10 min. and then reaction of activation of complement is stopped by adding a buffer containing 10 mM of EDTA. In control sample degree of lysis of erythrocytes is established and experimental sample is additionally incubated during 30 min at 37oC and then degree of lysis is determined. The activity of C3-convertase is calculated as a difference between degree of lysis and in experimental and control samples. The difference more than 10% is considered as a pathological state conditioned by stabilization of C3-convertase of classical way of activation of system of complement. The studies were carried out concerning stabilization of C3-convertase of classical way of activation of compliment in 31 patients with abdominal obesity. It is demonstrated that in 87% of patients with abdominal obesity stabilization of C3-convertase was established.

Levels of matrix metalloproteinases differ in plasma and serum - aspects regarding analysis of biological markers in cancer.

BACKGROUND: There are inconsistencies in the use of serum or plasma when analysing the matrix metalloproteinases (MMPs) as diagnostic or prognostic markers. The purpose of this study was to compare the concentration of MMP-1, -2, -7, -8, -9 and -13 in serum vs plasma samples. METHODS: Blood samples were obtained from sixty-five men and women. Samples were analysed for levels of MMPs in corresponding citrate plasma and serum. RESULTS: All MMPs expressed higher concentration in serum compared with plasma (P<0.01). There were no differences between genders. CONCLUSIONS: Present study demonstrated significant differences regarding concentrations of some MMPs using plasma vs serum. We conclude that future studies regarding MMPs as biological markers in cancer should consider the use of citrate plasma instead of serum.

Cholinergic enzymes and inflammatory markers in rats infected by Sporothrix schenckii.

The aim of this study was to evaluate the cholinesterase activity in serum, whole blood, and lymphocytes, as well as to verify its relation to immune response in rats experimentally infected by Sporothrix schenckii. For this study, 63 Wistar rats (Rattus norvegicus), male, adult were divided into three groups: the negative control group (GC: n = 21), the group infected subcutaneously (GSC: n = 21), and the group infected intraperitoneally (GIP: n = 21). The groups were divided into subgroups and the following variables were evaluated at 15, 30, and 40 days post-infection (PI): acetylcholinesterase (AChE) activity in lymphocytes and whole blood, butyrylcholinesterase (BChE) activity in serum, cytokines levels (IL-1, IL-6, TNFα, and INF-γ), immunoglobulins levels (IgA, IgG, IgM, and IgE), and protein profile by electrophoresis. Both infected groups showed increased levels of inflammatory parameters (P < 0.05) in tissue and inflammatory infiltrates. The activities of AChE in lymphocytes and BChE in serum increased (P < 0.05) significantly in animals from the GSC group on day 40 PI compared to the GC group. Regarding the GIP, there was a marked increase in the AChE activity in lymphocytes on days 30 and 40 PI, and in whole blood on days 15, 30, and 40 PI compared to GC. Furthermore, IL-10, an anti-inflammatory cytokine, was also present in high levels during chronic systemic S. schenckii infections in animals. Therefore, it is concluded that cholinesterase has an important modulatory role in the immune response during granulomatous infection by S. schenckii.

Involvement of cholinergic and purinergic systems during the inflammatory response caused by Aeromonas hydrophila in Rhamdia quelen.

The aim of this study was to evaluate the cholinergic (acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)) and purinergic (adenosine deaminase (ADA)) systems in head kidney, spleen, total blood and serum samples in experimentally infected fish with A. hydrophila, and the involvement of these systems during the inflammatory process. Silver catfish (Rhamdia quelen) juveniles were divided into two groups with seven fish each: uninfected (negative control) and infected (positive control). On day 2 post-infection, animals were euthanized and the head kidney, spleen, total blood and serum were collected. AChE and ADA activities in head kidney and spleen decreased in infected animals compared to uninfected animals, as well as AChE in total blood and seric ADA activities. BChE activity was not expressed in the evaluated tissues. Therefore, our results lead to the hypothesis that cholinergic and purinergic systems play an important role on the immune response against A. hydrophila with an anti-inflammatory effect. In summary, AChE and ADA activities reduced probably in order to protect against tissue inflammatory damage caused by infection.

Occurrence of a multimeric high-molecular-weight glyceraldehyde-3-phosphate dehydrogenase in human serum.

Cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a phylogenetically conserved, ubiquitous enzyme that plays an indispensable role in energy metabolism. Although a wealth of information is available on cellular GAPDH, there is a clear paucity of data on its extracellular counterpart (i.e., the secreted or extracellular GAPDH). Here, we show that the extracellular GAPDH in human serum is a multimeric, high-molecular-weight, yet glycolytically active enzyme. The high-molecular-weight multimers of serum GAPDH were identified by immunodetection on one- and two-dimensional gel electrophoresis using multiple antibodies specific for various epitopes of GAPDH. Partial purification of serum GAPDH by DEAE Affigel affinity/ion exchange chromatography further established the multimeric composition of serum GAPDH. In vitro data demonstrated that human cell lines secrete a multimeric, high-molecular-weight enzyme similar to that of serum GAPDH. Furthermore, LC-MS/MS analysis of extracellular GAPDH from human cell lines confirmed the presence of unique peptides of GAPDH in the high-molecular-weight subunits. Furthermore, data from pulse-chase experiments established the presence of high-molecular-weight subunits in the secreted, extracellular GAPDH. Taken together, our findings demonstrate the presence of a high-molecular-weight, enzymatically active secretory GAPDH in human serum that may have a hitherto unknown function in humans.

A Model to Predict the Severity of Acute Pancreatitis Based on Serum Level of Amylase and Body Mass Index.

BACKGROUND & AIMS: Most patients with acute pancreatitis (AP) develop mild disease, but up to 20% develop severe disease. Many clinicians monitor serum levels of amylase and lipase in an attempt to predict the disease course, but this strategy has not been recommended by practice guidelines. We performed a retrospective analysis to determine whether the percentage changes in amylase and lipase were associated with the severity of disease that developed in patients with AP. METHODS: We analyzed data collected from 182 consecutive patients with AP (21 with severe AP) admitted to the Cleveland Clinic from January 2008 through May 2010 (discover cohort). The association between 11 different factors and the severity of AP were assessed by univariable analysis; multivariable models were explored through stepwise selection regression. The percentage change in the serum level of amylase was calculated as follows: ([amylase day 1 - amylase day 2]/amylase day 1) × 100. The percentage change in amylase and body mass index (BMI) were combined to generate a z-score (z = -5.9 + [0.14 × BMI] + [0.01 × percentage change in amylase]), which was converted into a probability distribution called the change in amylase and BMI (CAB) score. The CAB score was validated using the AP database at the University of Pittsburgh Medical Center (140 patients, 35 with severe AP); we calculated p-scores for each patient and estimated the area under the receiver operating characteristics curve values. RESULTS: Univariable analysis identified the percentage change in the serum level of amylase and other factors to be associated significantly with the severity of AP (P = .017). The CAB score was best at identifying patients who developed severe AP, with an area under the receiver operating characteristics curve value of 0.79 in the discovery cohort (95% confidence interval, 0.71-0.87) and 0.731 in the validation cohort (95% confidence interval, 0.61-0.84). CONCLUSIONS: We developed a model to identify patients most likely to develop severe AP based on the percentage changes in serum level of amylase during the first 2 days after admission to the hospital and BMI.

Hepatic expression of miR-122, miR-126, miR-136 and miR-181a and their correlation to histopathological and clinical characteristics of patients with hepatitis C.

It has been shown for hepatitis C virus (HCV) infection that host miRNAs contribute to the replication of the viral RNA genome. However, the clinical impact of these and many other cellular miRNAs on HCV in humans is still largely unclear. We therefore analysed the expression of miR-122, miR-126, miR-181a and miR-136 in HCV-infected patients. The study included liver biopsies of 65 patients infected with HCV of different genotypes (gt 1, gt 1a, gt 1b, gt 3 and gt 4) and nine noninfected individuals. Expression analysis of miRNAs was performed by qPCR, and they were analysed for differences between patient gender and age, genotypes, stage of fibrosis, grade of inflammation, serum level of liver enzymes, serum viral load, the presence of steatosis and mode of transmission. Different target prediction algorithms were used to search for targets of analyzed miRNAs. Statistical analysis revealed significant up-regulation of miR-136 and down-regulation of miR-126 and miR-181a in patients infected with HCV of different genotypes compared with noninfected individuals. The same expression pattern was observed in different stages and grades of liver disease. miR-122 was up-regulated in women relative to men and associated to portal inflammation, miR-122 and miR-126 correlated with serum HCV load and miR-136 and miR-122 correlated with the presence of steatosis. miR-126 and miR-136 were differentially expressed between different modes of HCV transmission. There were approximately 2000 different targets predicted for all four miRNAs and each of the analyzed miRNAs could be involved in more than a 100 different biochemical pathways. miR-122, miR-126, miR-136 and miR-181a have been shown to be involved in HCV infection with different genotypes. Their expression has been associated with the gender, stage and grade of liver disease, mode of transmission, serum HCV load and the presence of steatosis. Numerous target genes and biochemical pathways are predicted for each of the analyzed miRNAs. All these results suggest their role in HCV-infected liver disease.

Single-step bioassays in serum and whole blood with a smartphone, quantum dots and paper-in-PDMS chips.

The development of nanoparticle-based bioassays is an active and promising area of research, where point-of-care (POC) diagnostics are one of many prospective applications. Unfortunately, the majority of nanoparticle-based assays that have been developed to date have failed to address two important considerations for POC applications: use of instrumentation amenable to POC settings, and measurement of analytes in biological sample matrices such as serum and whole blood. To address these considerations, we present design criteria and demonstrate proof-of-concept for a semiconductor quantum dot (QD)-based assay format that utilizes smartphone readout for the single-step, Förster resonance energy transfer (FRET)-based detection of hydrolase activity in serum and whole blood, using thrombin as a model analyte. Important design criteria for assay development included (i) the size and emission wavelength of the QDs, which had to balance brightness for smartphone imaging, optical transmission through blood samples, and FRET efficiency for signaling; (ii) the wavelength of a light-emitting diode (LED) excitation source, which had to balance transmission through blood and the efficiency of excitation of QDs; and (iii) the use of an array of paper-in-polydimethylsiloxane (PDMS)-on-glass sample chips to reproducibly limit the optical path length through blood to ca. 250 µm and permit multiplexing. Ultimately, CdSe/CdS/ZnS QDs with peak emission at 630 nm were conjugated with Alexa Fluor 647-labeled peptide substrates for thrombin and immobilized on paper test strips inside the sample cells. This FRET system was sensitive to thrombin activity, where the recovery of QD emission with hydrolytic loss of FRET permitted kinetic assays in buffer, serum and whole blood. Quantitative results were obtained in less than 30 min with a limit of detection 18 NIH units mL(-1) of activity in 12 µL of whole blood. Proof-of-concept for a competitive binding assay was also demonstrated with the same platform. Overall, this work demonstrates that the integration of QDs with smartphones and other consumer electronics can potentiate bioassays that are highly amenable to future point-of-care diagnostic applications.