A multi-enzyme model for Pyrosequencing

Pyrosequencing is a DNA sequencing technique based on sequencing-by-synthesis enabling rapid real-time sequence determination. This technique employs four enzymatic reactions in a single tube to monitor DNA synthesis. Nucleotides are added iteratively to the reaction and in case of incorporation, pyrophosphate (PPi) is released. PPi triggers a series of reactions resulting in production of light, which is proportional to the amount of DNA and number of incorporated nucleotides. Generated light is detected and recorded by a detector system in the form of a peak signal, which reflects the activity of all four enzymes in the reaction. We have developed simulations to model the kinetics of the enzymes. These simulations provide a full model for the Pyrosequencing four-enzyme system, based on which the peak height and shape can be predicted depending on the concentrations of enzymes and substrates. Simulation results are shown to be compatible with experimental data. Based on these simulations, the rate-limiting steps in the chain can be determined, and K_M and k_cat of all four enzymes in Pyrosequencing can be calculated.     

The Effect of Lactobacillus casei Consumption in Improvement of Obsessive–Compulsive Disorder: an Animal Study

Obsessive–compulsive disorder (OCD) is an important neuropsychiatric disorder worldwide. Common treatments of OCD include serotonergic antidepressants, which can cause potentially serious side effects. We assessed the effects of Lactobacillus casei (L. casei) Shirota consumption in an animal model of OCD. OCD-like symptoms were induced in rats by the chronic injection of the D2/D3 dopamine agonist quinpirole hydrochloride. Rats were classified into five groups of 6 rats. Four groups were injected chronically with quinpirole (0.5 mg/kg, twice weekly for 5 weeks). They were fed with L. casei Shirota (10⁹ CF/g, daily for 4 weeks) (group 1), fluoxetine (10 mg/kg, daily for 4 weeks) (group 2), combination of L. casei Shirota and fluoxetine (group 3), and normal saline (positive control group). The last group did not receive dopamine agonist and was only injected with saline (negative control group). Expression levels of brain-derived neurotrophic factor (Bdnf), solute carrier family 6 member 4 (Slc6a4), and 5-hydroxytryptamine receptor type 2A (Htr2a) were assessed in orbitofrontal cortex tissues of all rats. Behavioral tests showed improvement of OCD signs in rats treated with L. casei Shirota, fluoxetine, and a combination of drugs. Quantitative PCR analysis showed a remarkable decrease in the expression of Bdnf and an increase in the expression of Htr2a in quinpirole-treated rats. After treatment with L. casei Shirota and fluoxetine, the expression level of Bdnf was increased remarkably, whereas Htr2a expression was decreased. The current study showed the effectiveness of L. casei Shirota in the treatment of OCD in a rat model. The beneficial effects of this probiotic are possibly exerted through the modulation of serotonin-related genes expression.

     

Study of β-lactam-based drug interaction with albumin protein using optical,sensing, and docking methods

Journal of Biological Physics - The quality and strength of drug and albumin interaction affecting the drug-free concentration and physiological activity are important issues in pharmacokinetic...     

Effect of amrinone on diaphragm blood flow

The purpose of the present study was to examine the effect of amrinone, a drug known to augment cardiac output and dilate peripheral vascular beds, on diaphragm blood flow. Studies were performed on 12 anesthetized mechanically ventilated dogs in which strips of left costal diaphragm were developed in situ. Strip blood flow was assessed with a drop counter attached to a catheter tied into the phrenic veins' draining strips. Strip tension was measured with an isometric force transducer. Amrinone was administered as an intravenous bolus of 2 mg/kg followed by a continuous infusion of 25 micrograms.kg-1.min-1. Amrinone increased cardiac output and resting diaphragm blood flow [from 1.8 +/- 0.1 to 3.2 +/- 3 (SE) l/min and from 13 +/- 2 to 29 +/- 6 (SE) ml.100 g-1.min-1, respectively, P less than 0.001 for both comparisons]. Amrinone also increased blood flow during periods of rhythmic contraction (tension time indexes of 0.1-0.4, P less than 0.05 for comparisons of flow with and without amrinone at each tension time index) and increased the magnitude of the postcontraction hyperemia (P less than 0.02 for comparisons of hyperemic flow with and without amrinone at tension time indexes of 0.3 and 0.4). Graded occlusion of the inferior vena cava produced reductions in arterial pressure, cardiac output, and diaphragm blood flow both before and after amrinone. Both cardiac output and diaphragm blood flow were greater after amrinone, however, at all levels of blood pressure examined. These findings indicate that amrinone can override diaphragm vasoregulatory systems and augment diaphragm blood flow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Auswirkungen von abgestufter pflanzenschutzmittel‐intensität und unkrautbesatz auf laufkäferzönosen (coleoptera; carabidae) während einer fruchtfolgerotation unter den spezifischen Bedingungen des mitteldeutschen trockengebietes*

Die Untersuchungen fanden von 1991 bis 1995 auf einem 43 ha großen Schlag in Obhausen (Querfurter Platte) statt. Zielstellung der Untersuchungen war es, Auswirkungen abgestufter Pflanzenschutzintensitäten auf Laufkäfer zu erfassen. Dazu wurden auf dem Feld 6 Parzellen von 72 × 200 m Größe angelegt, von denen zwei als pflanzenschutzmittelfreie Kontrollflächen dienten. Auf zwei weiteren Teilstücken erfolgten intensive chemische Pflanzenschutzmaßnahmen. In den anderen beiden Varianten wurden im Sinne eines integrierten Pflanzenschutzes unter Beachtung von Schadschwellenwerten (Unkräuter, pilzliche und tierische Schaderreger) gezielte Pflanzenschutzmaßnahmen praktiziert. Zur Erfassung der Laufkäfer dienten Bodenfallen. Die Fruchtfolge begann 1991 mit Wintergerste, die aber wegen Trockenheit umgebrochen werden mußte. Die Intensitätsabstufungen des Pflanzenschutzmitteleinsatzes erfolgten ab 1992 im Mais und 1993 sowie 1994 in Winterweizen. Letztes Fruchtfolgeglied war 1995 Winterroggen, bei dem alle Parzellen wieder konventionell bewirtschaftet wurden. Im Mais kam es in den unbehandelten Kontrollen zu einer starken Verunkrautung. In diesen Parzellen erreichte die Fangsumme der Laufkäfer den 2,5 fachen Wert gegenüber den anderen Prüfgliedern. In den darauffolgenden Jahren, in den Winterweizenbeständen, lagen die höchsten Fangsummen wiederum in den Kontrollen vor, doch fielen die Differenzen zu den anderen Prüfgliedern nicht so groß aus. Die wenigsten Laufkäfer wurden in den Intensiv‐Varianten erfaßt. Die Bewirtschaftung nach dem Schadschwellenprinzip erbrachte wesentlich höhere Fangsummen. Während der Intensitätsabstufung besiedelten die Arten Calosoma auropunc‐tatum, Harpalus affinis, Pseudoophonus rufipes, Harpalus distinguendus, Poecilus punctulatus, Harpalus tardus sowie Harpalus zabroides überwiegend die Kontrollparzellen. Bei den abschließenden Untersuchungen im Winterroggen 1995 traf dies nur noch für Harpalus affinis zu. Ob für das Überwiegen dieser Arten in den Kontrollen der starke Unkrautbesatz im Jahre 1992 bzw. nachfolgend der Vorrat an Diasporen oder der völlige Verzicht auf Pflanzenschutzmittel ausschlaggebend war, konnte nicht eindeutig geklärt werden. Entgegen allen Erwartungen lag im Winterroggen die höchste Fangsumme der Laufkäfer in den ehemals intensiv behandelten Parzellen vor.

Ein Verzicht auf Pflanzenschutzmittel geht oft mit nicht mehr tolerierbaren Ertragsverlusten einher, wie z.B. im Mais 1992. Bei einem Einsatz von Pflanzenschutzmitteln nach Schadschwellenwerten entsprechend den Prinzipien eines integrierten Pflanzenschutzes sind langfristig keine negativen Effekte auf den Naturhaushalt zu erwarten.     

Trichomonas vaginalis: Investigation of a novel diagnostic method in urine samples using cysteine proteinase 4 gene and PCR technique

Trichomonas vaginalis is the agent of a highly prevalent sexually transmitted disease that leads to vaginitis, urethritis, ectocervicitis and has been associated with human immunodeficiency virus (HIV). Detection of T. vaginalis based on wet-mount microscopy and culture methods is insensitive and time consuming, respectively. Thus the quest for reliable PCR techniques of T. vaginalis in vaginal discharge and urine sample is more importance. In this study, 500 urine and vaginal-discharge samples were collected from women referred to Sexual Transmitted Disease Clinic of Mirzakuchakkhan Hospital in Tehran, Iran between May 2008 and March 2009. Wet-mount and culture methods were done on the vaginal discharges, and PCR assay targeting cysteine proteinase 4 (CP4) was performed on the urine samples. The present study demonstrated 16 (3.2%) of patients were infected with T. vaginalis using culture and wet-mount, whereas PCR assay using CP4 could detect 12 (2.4%) positivity. Sensitivity and specificity of urine PCR assay compared to culture were 80% (95% CI, 54-96) and 99.6% (95% CI, 98.96-100), respectively. These results indicate that using urine-based detection method for T. vaginalis may not be appropriate in women.     

The Importance of Bank Vole Density and Rainy Winters in Predicting Nephropathia Epidemica Incidence in Northern Sweden

Pathogenic hantaviruses (family Bunyaviridae, genus Hantavirus) are rodent-borne viruses causing hemorrhagic fever with renal syndrome (HFRS) in Eurasia. In Europe, there are more than 10,000 yearly cases of nephropathia epidemica (NE), a mild form of HFRS caused by Puumala virus (PUUV). The common and widely distributed bank vole (Myodes glareolus) is the host of PUUV. In this study, we aim to explain and predict NE incidence in boreal Sweden using bank vole densities. We tested whether the number of rainy days in winter contributed to variation in NE incidence. We forecast NE incidence in July 2013–June 2014 using projected autumn vole density, and then considering two climatic scenarios: 1) rain-free winter and 2) winter with many rainy days. Autumn vole density was a strong explanatory variable of NE incidence in boreal Sweden in 1990–2012 (R² = 79%, p<0.001). Adding the number of rainy winter days improved the model (R² = 84%, p<0.05). We report for the first time that risk of NE is higher in winters with many rainy days. Rain on snow and ground icing may block vole access to subnivean space. Seeking refuge from adverse conditions and shelter from predators, voles may infest buildings, increasing infection risk. In a rainy winter scenario, we predicted 812 NE cases in boreal Sweden, triple the number of cases predicted in a rain-free winter in 2013/2014. Our model enables identification of high risk years when preparedness in the public health sector is crucial, as a rainy winter would accentuate risk.     

Fourier transform infrared study of the effect of different cations on bacteriorhodopsin protein thermal stability

The effect of divalent ion binding to deionized bacteriorhodopsin (dI-bR) on the thermal transitions of the protein secondary structure have been studied by using temperature-dependent Fourier transform infrared (FT-IR) spectroscopy. The native metal ions in bR, Ca(2+), and Mg(2+), which we studied previously, are compared with Mn(2+), Hg(2+), and a large, synthesized divalent organic cation, ((Et)(3)N)(2)Bu(2+). It was found that in all cases of ion regeneration, there is a pre-melting, reversible conformational transition in which the amide frequency shifts from 1665 to 1652 cm(-1). This always occurs at approximately 80 degrees C, independent of which cation is used for the regeneration. The irreversible thermal transition (melting), monitored by the appearance of the band at 1623 cm(-1), is found to occur at a lower temperature than that for the native bR but higher than that for acid blue bR in all cases. However, the temperature for this transition is dependent on the identity of the cation. Furthermore, it is shown that the mechanism of melting of the organic cation regenerated bR is different than for the metal cations, suggesting a difference in the type of binding to the protein (either to different sites or different binding to the same site). These results are used to propose specific direct binding mechanisms of the ions to the protein of deionized bR.