Real-time RT-PCR expression analysis of chitinase and endoglucanase genes in the three-way interaction between the biocontrol strain Clonostachys rosea IK726, Botrytis cinerea and strawberry

Clonostachys rosea is a well-known biocontrol agent against Botrytis cinerea, the causal agent of gray mold in strawberry. The activity of cell wall-degrading enzymes might play a significant role for successful biocontrol by C. rosea. The expression pattern of four chitinases, and two endoglucanase genes from C. rosea strain IK726 was analyzed using real-time RT-PCR in vitro and in strawberry leaves during interaction with B. cinerea. Specific primers were designed for beta-tubulin genes from C. rosea and B. cinerea, respectively, and a gene encoding a DNA-binding protein (DBP) from strawberry, allowing in situ activity assessment of each fungus in vitro and during their interaction on strawberry leaves. Growth of B. cinerea was inhibited in all pathogen-antagonist interactions while the activity of IK726 was slightly increased. In all in vitro interactions, four of the six genes were upregulated while no change in expression of two endochitinases was measured. In strawberry leaves, the chitinase genes were upregulated 2-12-fold, except one of the endochitinases, whereas no change in expression of the two endoglucanases was measured. The results suggest that three out of four chitinase genes of IK726 are involved in biocontrol on leaves. This is the first example of monitoring of expression of chitinolytic genes in interactions between biocontrol agents and pathogens in plant material.     

Microbial transformation of hydrocortisone by Acremonium strictum PTCC 5282

The ability of a genus of cephalosporium-like fungus isolated from soil, Acremonium strictum PTCC 5282, for hydrocortisone biotransformation has been investigated. This potential had not been previously examined. The fermentation yielded 11beta,17beta-dihydroxyandrost-4-en-3-one, 11beta,17alpha,20beta,21-tetrahydroxypregn-4-en-3-one and 21-acetoxy-11beta,17alpha,20-trihydroxypregn-4-en-3-one. Each microbial metabolite was purified and characterized using spectroscopic methods.     

Electrochemical studies of DNA immobilization onto the azide-terminated monolayers and its interaction with taxol

A surface modification procedure for the creation of self-assembled monolayers (SAMs) that can be used as a scaffold for double-stranded DNA (dsDNA) incorporation onto the gold surfaces is described. The SAMs of an azidohexane thiol derivative were prepared on the Au electrode and then used for the immobilization of dsDNA. The electrochemical characteristics of dsDNA onto the SAM-modified gold electrode were investigated by cyclic voltammetry and electrochemical impedance spectroscopy, and the surface concentration of dsDNA onto the SAMs surface was estimated. The interaction of dsDNA with the anticancer drug, taxol (paclitaxel), was also studied on the surface of DNA/SAM/Au electrode. The observed decrease in the guanine oxidation peak current was used to monitor the interaction of taxol with DNA. The resulting Langmuir isotherm for taxol binding to DNA at the modified electrode was used to evaluate the binding constant of taxol-DNA. The results obtained supported the groove binding interaction of taxol with DNA. The modified electrode was used as a sensitive sensor for quantification of taxol in human serum sample.     

Establishment and in vitro differentiation of a new embryonic stem cell line from human blastocyst

Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. These cells have, therefore, potential for in vitro differentiation studies, gene function, and so on. The aim of this study was to produce a human embryonic stem cell line. An inner cell mass of a human blastocyst was separated and cultured on mouse embryonic fibroblasts in embryonic stem cell medium with related additives. The established line was evaluated by morphology; passaging; freezing and thawing; alkaline phosphatase; Oct-4 expression; anti-surface markers including Tra-1-60 and Tra-1-81; and karyotype and spontaneous differentiation. Differentiated cardiomyocytes and neurons were evaluated by transmission electron microscopy and immunocytochemistry. Here, we report the derivation of a new embryonic stem cell line (Royan H1) from a human blastocyst that remains undifferentiated in morphology during continuous passaging for more than 30 passages, maintains a normal XX karyotype, is viable after freezing and thawing, and expresses alkaline phosphatase, Oct-4, Tra-1-60, and Tra-1-81. These cells remain undifferentiated when grown on mouse embryonic fibroblast feeder layers in the presence or absence of recombinant human leukemia inhibitory factor. Royan H1 cells can differentiate in vitro in the absence of feeder cells and can produce embryoid bodies that can further differentiate into beating cardiomyocytes as well as neurons. These results define Royan H1 cells as a new human embryonic stem cell line.     

Temporal expression profile of CXC chemokines in serum of patients with spinal cord injury

Chemokines, a subclass of cytokine superfamily have both pro-inflammatory and migratory role and serve as chemoattractant of immune cells during the inflammatory responses ensuing spinal cord injury (SCI). The chemokines, especially CXCL-1, CXCL-9, CXCL-10 and CXCL-12 contribute significant part in the inflammatory secondary damage of SCI. Inhibiting chemokine’s activity and thereby the secondary damage cascades has been suggested as a chemokine-targeted therapeutic approach to SCI. To optimize the inhibition of secondary injury through targeted chemokine therapy, accurate knowledge about the temporal profile of these cytokines following SCI is required. Hence, the present study was planned to determine the serum levels of CXCL-1, CXCL-9, CXCL-10 and CXCL-12 at 3–6 h, 7 and 28 days and 3 m after SCI in male and female SCI patients (n = 78) and compare with age- and sex-matched patients with non-spinal cord injuries (NSCI, n = 70) and healthy volunteers (n = 100). ANOVA with Tukey post hoc analysis was used to determine the differences between the groups. The data from the present study show that the serum level of CXCL-1, CXCL-9 and CXCL-10 peaked on day 7 post-SCI and then declined to the control level. In contrast, significantly elevated level of CXCL-12 persisted for 28 days post SCI. In addition, post-SCI expression of CXCL-12 was found to be sex-dependent. Male SCI patients expressed significantly higher CXCL-12 when compared to control and SCI female. We did not observe any change in chemokines level of NSCI. Further, the age of the patients did not influence chemokines expression after SCI. These observations along with SCI-induced CSF-chemokine level should contribute to the identification of selective and temporal chemokine targeted therapy after SCI.     

A selective 19F nuclear magnetic resonance spectroscopic method for the assay of the neuroleptic drug cis(Z)-flupentixol in human serum

This investigation was carried out to evaluate 19F nuclear magnetic resonance as an analytical tool for the measurement of the cis(Z) and trans(E) stereoisomers of the antipsychotic drug flupentixol in human serum. The method is based on the integration of appropriate signals of both analytes and an internal standard. The proposed method was applied to the analysis of real samples without any interference, manipulation of large samples, and lengthy instrument time. Experimental parameters were selected to optimize accuracy, precision, and analysis time. The calibration curves in human serum matrix were linear for cis(Z)- and trans(E)-flupentixol over the ranges 4.0-50.0 and 2.6-25.0 microg/mL, respectively, with respective minimum detectable limits (S/N=3) of 1.67 and 1.72 microg/mL. The method was validated through spike and recovery for the two isomers of flupentixol from a human serum matrix.     

Simulation of Major Histocompatibility Complex (MHC) structure and peptide loading into an MHC binding pocket with teachers’hands

Molecular understanding of three-dimensional (3D) peptide: MHC models require both basic knowledge of computational modeling and skilled visual perception, which are not possessed by all students. The present model aims to simulate MHC molecular structure with the hands and make a profound impression on the students.Key Words: MHC molecules, Simulation, Hand, Binding pocket, Physical model using handsCurrently, lecturers can use many instructional models to enhance teaching of molecular structures to undergraduate students. However, we sometimes find it necessary to present an immunological concept with a touchable model to create a more understandable and profound image than can be found in books. The realization of paratope-epitope interaction with both hands and an apple is one example of this simulation in immunology teaching (1). MHC class I and II structures and MHC peptide-loading compartments are considered to be fundamental concepts in immunology. Molecular understanding of 3D peptide: MHC models require both basic knowledge of computational modeling and skilled visual perception, which are not possessed by all audiences. However, no models are more accessible, efficient, and affordable than teachers’ hands. This simulation aims to simplify perception of MHC molecular structures and provide an enduring image of MHC-peptide interactions. Simulation of MHC class I structure and its peptide loading compartment To imitate MHC class I structure with the hands, bend the index, middle, and ring fingers toward the palm and then touch the thumb with the little finger as shown in Fig. 1. The three bent fingers and the connected thumb and little finger simulate the pair of parallel α-helices that form the peptide binding groove. One helix belongs to the α1 domain and the other to the α2 domain. The palm represents the beta-pleated sheet structure, which forms the base of the groove. The narrow groove between the bent and connected fingers represents the peptide-loading compartment. A flexible rod inserted into this groove can be imagined as an antigenic peptide located in the closed groove of the MHC class I molecule. The rod must bend in the middle to simulate the loaded peptide. Introduce your left hand to the students as an α-chain of MHC class I structure, which is encoded on chromosome 6 in humans. Then, introduce your watch as a β2-microglobulin chain that is non-covalently associated with the α-chain and encoded on chromosome 15 in humans. It should be emphasized that the polypeptide chains are encoded by genes on different chromosomes. The MHC class I α-chain is inserted into the cell membrane, indicated by the shirt sleeve in our simulation (2, 3). Open in a separate windowFig. 1Simulation of MHC class I structure and its peptide loading compartment (A). In B this simulation has been superimposed with two α-helices and a beta-pleated sheet of the α-chain from a 3D view of human class I MHC HLA-A2 (3PWJ). Simulation of MHC class II structure and its peptide loading compartment To demonstrate MHC class II structure, bend all the fingers of both hands toward the palms and then close both hands from the forearms as shown in Fig. 2. Try to hide your watch, which represented β2-microglobulin. The bent fingers and palm of the left hand represent the α-helix and β-pleated sheet of the α-chain, respectively (Fig. 2), and the same status could be imagined for the right hand as a β-chain of MHC class II. The open space between the bent fingers of both hands represents the peptide anchoring site, which is an open-ended groove in MHC class II. A flexible rod with overhanging portions projecting from both sides of groove inserted in this space simulates an antigenic peptide that has been located in an open-ended groove of the MHC class II molecule. MHC class II molecules bind peptides of 13-25 amino acids, which is considerably longer than MHC class I-binding peptides of 8-11 amino acids. The two chains of MHC class II molecules are inserted into the cell membrane, represented by the shirt sleeves in our simulation. In this simulation, the two chains of the MHC class II molecules are represented by the two hands, while the MHC class I molecule was modeled by a hand and a watch. This is a good time to remind the students that both chains of MHC class II molecules, in contrast to class I molecules, are encoded by the MHC gene cluster on chromosome 6 in humans, and that the two chains associate non-covalently (2, 3).Open in a separate windowFig. 2Simulation of MHC class II structure and its peptide loading compartment (A). This simulation has been superimposed with two α-helices and two beta-pleated sheets of α-chain and β-chain from the crystal structure of HLA-DR1 (3PDO) (B).Certainly, some MHC structural features are not completely duplicated with this model, but this visualization of a molecular structure makes a profound impression on the students.     

The Vital Role of Blood Flow-Induced Proliferation and Migration in Capillary Network Formation in a Multiscale Model of Angiogenesis

Sprouting angiogenesis and capillary network formation are tissue scale phenomena. There are also sub-scale phenomena involved in angiogenesis including at the cellular and intracellular (molecular) scales. In this work, a multiscale model of angiogenesis spanning intracellular, cellular, and tissue scales is developed in detail. The key events that are considered at the tissue scale are formation of closed flow path (that is called loop in this article) and blood flow initiation in the loop. At the cellular scale, growth, migration, and anastomosis of endothelial cells (ECs) are important. At the intracellular scale, cell phenotype determination as well as alteration due to blood flow is included, having pivotal roles in the model. The main feature of the model is to obtain the physical behavior of a closed loop at the tissue scale, relying on the events at the cellular and intracellular scales, and not by imposing physical behavior upon it. Results show that, when blood flow is considered in the loop, the anastomosed sprouts stabilize and elongate. By contrast, when the loop is modeled without consideration of blood flow, the loop collapses. The results obtained in this work show that proper determination of EC phenotype is the key for its survival.     

Trends in Cardiovascular Disease Risk Factors in People with and without Diabetes Mellitus: A Middle Eastern Cohort Study

Aims/Hypothesis

To investigate secular trends in cardiovascular disease (CVD) risk factors during a decade of follow-up in a Middle Eastern cohort, and to compare observed trends between diabetic and non-diabetic populations.

Methods

In a population of 6181 participants (2622 males and 3559 females), diabetes status and CVD risk factors were evaluated in 4 study phases from 1999–2011. 1045 subjects had type 2 diabetes mellitus at baseline and 5136 participants were diabetes-free. To examine the trends of CVD risk factors, generalized estimation equation models were constructed. The interaction between the diabetes status and each phase of the study was checked in a separate model.

Results

During the follow-up period diabetic females significantly gained better control of their blood pressure, serum low density lipoprotein cholesterol and general and central obesity measures compared to non-diabetic counterparts, although 60% of them had high BP and 64% had high serum LDL-C levels till the end of the study. Diabetic males however, experienced significantly better control on their serum LDL-C and general and central obesity measures compared to their non-diabetic controls; but 24% of them were still smoker, 63% had high BP and 60% had high serum LDL-C levels at the end of the follow-up (all Ps _interaction <0.05). Use of lipid-lowering and antihypertensive medications increased consistently in both diabetic and non-diabetic populations.

Conclusions/Interpretation

Although CVD risk factors have been controlled to some extent among diabetic population in Iran, still high numbers of people with diabetes have uncontrolled CVD risk factors that prompt more attention.