High Content Screening as High Quality Assay for Biological Evaluation of Photosensitizers In Vitro

A novel single step assay approach to screen a library of photdynamic therapy (PDT) compounds was developed. Utilizing high content analysis (HCA) technologies several robust cellular parameters were identified, which can be used to determine the phototoxic effects of porphyrin compounds which have been developed as potential anticancer agents directed against esophageal carcinoma. To demonstrate the proof of principle of this approach a small detailed study on five porphyrin based compounds was performed utilizing two relevant esophageal cancer cell lines (OE21 and SKGT-4). The measurable outputs from these early studies were then evaluated by performing a pilot screen using a set of 22 compounds. These data were evaluated and validated by performing comparative studies using a traditional colorimetric assay (MTT). The studies demonstrated that the HCS assay offers significant advantages over and above the currently used methods (directly related to the intracellular presence of the compounds by analysis of their integrated intensity and area within the cells). A high correlation was found between the high content screening (HCS) and MTT data. However, the HCS approach provides additional information that allows a better understanding of the behavior of these compounds when interacting at the cellular level. This is the first step towards an automated high-throughput screening of photosensitizer drug candidates and the beginnings of an integrated and comprehensive quantitative structure action relationship (QSAR) study for photosensitizer libraries.     

Non-empirical study of the phosphorylation reaction catalyzed by 4-methyl-5-β-hydroxyethylthiazole kinase: relevance of the theory of intermolecular interactions

The subject of this study was an analysis of the role of active site residues in the phosphoryl transfer reaction catalyzed by 4-methyl-5-β-hydroxyethylthiazole kinase (ThiK). The ThiK-catalyzed reaction is of special interest due to the lack of a highly conserved aspartate residue serving as a catalytic base. ONIOM(B3LYP:PM3) models of stationary points along the reaction pathway consisted of reactants, two magnesium ions and several highly conserved ThiK active site residues. The results indicate that an S_N2-like mechanism of ThiK, with γ-phosphate acting as an alcohol-activating base is reasonable. Geometries of substrates, transition state and products were utilized in the non-empirical analysis of the physical nature of catalytic interactions taking place in the ThiK active site. The role of particular residues was investigated in terms of their ability to preferentially stabilize the transition state relative to substrates (differential transition state stabilization, DTSS) or products (differential product stabilization, DPS). It seems that Mg2, Glu126 and Cys198 play a major catalytic role, whereas Mg1 and the same Cys198 are responsible for product release. It is remarkable that no dominant role of an electrostatic term in the interactions involved in catalytic activity is observed for product release. Determination of catalytic fields expressing differential electrostatic potential of the transition state with respect to substrates revealed the optimal electrostatic features of an ideal catalyst for the studied reaction. The predicted catalytic environment is in agreement with experimental data showing increased catalytic activity of ThiK upon mutation of Cys198 to aspartate. Figure Catalytic fields for ThiK-catalyzed reaction juxtaposed with the positions of active site residues of a model system. Magnesium ions are considered part of the transition state/reactants. The surface of constant electronic density is colored according to differential electrostatic potential of transition state with respect to reactants. The sign of the differential potential reflects the electrostatic properties of a complementary molecular environment. Red (green) color denotes regions where a negative (positive) charge would be optimal for catalytic activity     

Comparative effect of different dietary inulin sources and probiotics on growth performance and blood characteristics in growing–finishing pigs

The study was focused on assessment of the effect of an extract of long-chain inulin (LCI) and dried tubers of Jerusalem artichoke (JA) and a multispecies probiotic preparation as well as a combination thereof on growth performance and blood parameters of fattening pigs. In total, 144 pigs (initial body weight 30.0 ± 0.5 kg) were used in a 98-d experiment. The six dietary treatments consisted of the control diet (Con), diet Con supplemented with probiotics (ConP) and four diets supplemented with LCI or JA alone or with probiotics (diets LCIP and JAP). Throughout the fattening period, there was a beneficial effect of the probiotic supplementation to the inulin-containing diets and the average daily gain (ADG) was increased by supplementation of probiotics in combination with inulin sources (p < 0.05). At the end of the fattening period, ADG and feed conversion ratio (FCR) were higher after supplementation of LCI only (p < 0.05). Compared with group ConP, in groups LCI and JA, the ADG and FCR were improved (p < 0.05). Only in the first fattening stage, the addition of the prebiotics and/or probiotics had an impact on the level of white blood cells and some biochemical indices in pigs. In younger animals, probiotic or LCI supplementation increased the IgG level (p < 0.05). There was also an interaction between the probiotics and JA resulting in increased IgG and IgA concentrations (p < 0.05). In the finishing period, LCI addition increased the IgM level (p < 0.05), whereas JA addition increased IgG and IgM levels as well (p < 0.05). In conclusion, both dietary sources of inulin and probiotic supplementation can improve the fattening performance and health status of growing pigs.     

Ubc9-induced inhibition of diadenosine triphosphate hydrolase activity of the putative tumor suppressor protein Fhit

Fhit protein is the product of the putative tumor suppressor fragile histidine triad (FHIT) gene. The way by which Fhit exerts its antitumor activity remains largely unknown, although the Fhit-Ap3A complex is believed to be the native signaling form of Fhit. Here, we have shown that Fhit protein interacts with hUbc9, a recombinant human SUMO-1 conjugating enzyme, in an adenosine(5')triphospho(5')nucleoside (Ap3N)-dependent manner. Our experiments showed that the dinucleoside polyphosphate hydrolase activity of Fhit is suppressed by interacting with hUbc9 protein. In the presence of equimolar hUbc9 the Vmax and Km activity of Fhit was decreased by 35%. Analysis of Fhit kinetics in the presence of different fixed concentrations of Ubc9 showed that Ubc9 is an uncompetitive inhibitor. Including SUMO-1 protein in the assay neither affected the Fhit activity nor modified the effect of Ubc9 on Fhit kinetics. Our data suggest that hUbc9-induced inhibition of Fhit may result in an elongation of the Fhit-Ap3A signaling complex lifetime leading to alteration of its antitumor activity.     

The utility of inflammation and platelet biomarkers in patients with acute coronary syndromes

Introduction

Thrombotic and inflammatory mechanisms are involved in the pathophysiology of acute coronary syndrome (ACS). The aim of the study was the evaluation of inflammation (white blood cells count/WBC, C-reactive protein/CRP, interleukin-6/IL-6) and platelet (platelet count/PLT, mean platelet volume/MPV, large platelet/LPLT, beta-thromboglobulin/β-TG) biomarkers in the groups of ACS patients depending on the severity of signs and symptoms and compared to controls without coronary artery disease.

Research on the ability of propionic acid and vitamin B12 biosynthesis by <Emphasis Type="Italic">Propionibacterium freudenreichii</Emphasis> strain T82

The purpose of this study was to determine the potential for biosynthesis of propionic acid and vitamin B12 by Propionibacterium freudenreichii T82 in a medium containing various sources of carbon (glucose, fructose, and saccharose). These sugars are present in apple pomaces, which are the waste from the production of apple juice. Using statistical analysis design of experiments (DoE), the results allowed us to determine which sugars (carbon sources) exert the most beneficial influence on the biosynthesis of propionic acid and cobalamin. The highest production of propionic acid by the tested bacterial strain was obtained in a medium in which glucose accounted for at least 50% of the available carbon sources. Depending on the culture medium, the concentration of this metabolite ranged from 23 to 40 g/L. P. freudenreichii T82 produced the smallest amount of acid in medium in which the dominant nutrient source was saccharose. The results obtained indicated an inverse relationship between the amount of acid produced by the bacteria and vitamin B12 biosynthesis. Because of the high efficiency of propionic acid biosynthesis by P. freudenreichii T82, the prospect of using this strain to obtain propionate with the simultaneous disposal of waste materials (such as apple pomaces) which contain glucose and/or fructose is very promising.     

Effects of head-down-tilt bed rest on cerebral hemodynamics during orthostatic stress

Zhang, Rong, Julie H. Zuckerman, James A. Pawelczyk, andBenjamin D. Levine. Effects of head-down-tilt bed rest on cerebralhemodynamics during orthostatic stress. J. Appl.Physiol. 83(6): 2139-2145, 1997.Our aim was todetermine whether the adaptation to simulated microgravity (µG)impairs regulation of cerebral blood flow (CBF) during orthostaticstress and contributes to orthostatic intolerance. Twelvehealthy subjects (aged 24 ± 5 yr) underwent 2 wk of 6°head-down-tilt (HDT) bed rest to simulate hemodynamic changes thatoccur when humans are exposed to µG. CBF velocity in the middlecerebral artery (transcranial Doppler), blood pressure, cardiac output(acetylene rebreathing), and forearm blood flow were measured at eachlevel of a ramped protocol of lower body negative pressure (LBNP;15, 30, and 40 mmHg × 5 min, 50 mmHg × 3 min, then 10 mmHg every 3 min to presyncope) beforeand after bed rest. Orthostatic tolerance was assessed by using thecumulative stress index (CSI; mmHg × minutes) for the LBNPprotocol. After bed rest, each individual's orthostatic tolerance wasreduced, with the group CSI decreased by 24% associated with greaterdecreases in cardiac output and greater increases in systemic vascularresistance at each level of LBNP. Before bed rest, mean CBF velocitydecreased by 14, 10, and 45% at 40 mmHg, 50 mmHg, andmaximal LBNP, respectively. After bed rest, mean velocity decreased by16% at 30 mmHg and by 21, 35, and 39% at 40 mmHg,50 mmHg, and maximal LBNP, respectively. Compared with pre-bedrest, post-bed-rest mean velocity was less by 11, 10, and 21% at30, 40, and 50 mmHg, respectively. However, therewas no significant difference at maximal LBNP. We conclude thatcerebral autoregulation during orthostatic stress is impaired byadaptation to simulated µG as evidenced by an earlier and greater fall in CBF velocity during LBNP. We speculate that impairment ofcerebral autoregulation may contribute to the reduced orthostatic tolerance after bed rest.

     

Perfusion-CT - Can We Predict Acute Pancreatitis Outcome within the First 24 Hours from the Onset of Symptoms?

Purpose

Severe acute pancreatitis (AP) is still a significant clinical problem which is associated with a highly mortality. The aim of this study was the evaluation of prognostic value of CT regional perfusion measurement performed on the first day of onset of symptoms of AP, in assessing the risk of developing severe form of acute pancreatitis.

Material and Methods

79 patients with clinical symptoms and biochemical criteria indicative of acute pancreatitis (acute upper abdominal pain, elevated levels of serum amylase and lipase) underwent perfusion CT within 24 hours after onset of symptoms. The follow-up examinations were performed after 4–6 days to detect progression of the disease. Perfusion parameters were compared in 41 people who developed severe form of AP (pancreatic and/or peripancreatic tissue necrosis) with parameters in 38 consecutive patients in whom course of AP was mild. Blood flow, blood volume, mean transit time and permeability surface area product were calculated in the three anatomic pancreatic subdivisions (head, body and tail). At the same time the patient''s clinical status was assessed by APACHE II score and laboratory parameters such as CRP, serum lipase and amylase, AST, ALT, GGT, ALP and bilirubin were compared.

Results

Statistical differences in the perfusion parameters between the group of patients with mild and severe AP were shown. Blood flow, blood volume and mean transit time were significantly lower and permeability surface area product was significantly higher in patients who develop severe acute pancreatitis and presence of pancreatic and/or peripancreatic necrosis due to pancreatic ischemia. There were no statistically significant differences between the two groups in terms of evaluated on admission severity of pancreatitis assessed using APACHE II score and laboratory tests.

Conclusions

CT perfusion is a very useful indicator for prediction and selection patients in early stages of acute pancreatitis who are at risk of developing pancreatic and/or peripancreatic necrosis already on the first day of the onset of symptoms and can be used for treatment planning and monitoring of therapy of acute pancreatitis. Early suspicion of possible pancreatic necrosis both on the basis of scores based on clinical status and laboratory tests have low predictive value.