An In Vivo Study of Self-Regulated Study Sequencing in Introductory Psychology Courses

Study sequence can have a profound influence on learning. In this study we investigated how students decide to sequence their study in a naturalistic context and whether their choices result in improved learning. In the study reported here, 2061 undergraduate students enrolled in an Introductory Psychology course completed an online homework tutorial on measures of central tendency, a topic relevant to an exam that counted towards their grades. One group of students was enabled to choose their own study sequence during the tutorial (Self-Regulated group), while the other group of students studied the same materials in sequences chosen by other students (Yoked group). Students who chose their sequence of study showed a clear tendency to block their study by concept, and this tendency was positively associated with subsequent exam performance. In the Yoked group, study sequence had no effect on exam performance. These results suggest that despite findings that blocked study is maladaptive when assigned by an experimenter, it may actually be adaptive when chosen by the learner in a naturalistic context.     

Novel escape mutants suggest an extensive TRIM5α binding site spanning the entire outer surface of the murine leukemia virus capsid protein

After entry into target cells, retroviruses encounter the host restriction factors such as Fv1 and TRIM5α. While it is clear that these factors target retrovirus capsid proteins (CA), recognition remains poorly defined in the absence of structural information. To better understand the binding interaction between TRIM5α and CA, we selected a panel of novel N-tropic murine leukaemia virus (N-MLV) escape mutants by a serial passage of replication competent N-MLV in rhesus macaque TRIM5α (rhTRIM5α)-positive cells using a small percentage of unrestricted cells to allow multiple rounds of virus replication. The newly identified mutations, many of which involve changes in charge, are distributed over the outer 'top' surface of N-MLV CA, including the N-terminal β-hairpin, and map up to 29 A(o) apart. Biological characterisation with a number of restriction factors revealed that only one of the new mutations affects restriction by human TRIM5α, indicating significant differences in the binding interaction between N-MLV and the two TRIM5αs, whereas three of the mutations result in dual sensitivity to Fv1(n) and Fv1(b). Structural studies of two mutants show that no major changes in the overall CA conformation are associated with escape from restriction. We conclude that interactions involving much, if not all, of the surface of CA are vital for TRIM5α binding.     

Identification,modeling and ligand affinity of early deuterostome CYP51s,and functional characterization of recombinant zebrafish sterol 14α-demethylase

Background

Sterol 14α-demethylase (cytochrome P450 51, CYP51, P450_14DM) is a microsomal enzyme that in eukaryotes catalyzes formation of sterols essential for cell membrane function and as precursors in biosynthesis of steroid hormones. Functional properties of CYP51s are unknown in non-mammalian deuterostomes.

Methods

PCR-cloning and sequencing and computational analyses (homology modeling and docking) addressed CYP51 in zebrafish Danio rerio, the reef fish sergeant major Abudefduf saxatilis, and the sea urchin Strongylocentrotus purpuratus. Following N-terminal amino acid modification, zebrafish CYP51 was expressed in Escherichia coli, and lanosterol 14α-demethylase activity and azole inhibition of CYP51 activity were characterized using GC-MS.

Results

Molecular phylogeny positioned S. purpuratus CYP51 at the base of the deuterostome clade. In zebrafish, CYP51 is expressed in all organs examined, most strongly in intestine. The recombinant protein bound lanosterol and catalyzed 14α-demethylase activity, at 3.2 nmol/min/nmol CYP51. The binding of azoles to zebrafish CYP51 gave K_S (dissociation constant) values of 0.26 μM for ketoconazole and 0.64 μM for propiconazole. Displacement of carbon monoxide also indicated zebrafish CYP51 has greater affinity for ketoconazole. Docking to homology models showed that lanosterol docks in fish and sea urchin CYP51s with an orientation essentially the same as in mammalian CYP51s. Docking of ketoconazole indicates it would inhibit fish and sea urchin CYP51s.

Conclusions

Biochemical and computational analyses are consistent with lanosterol being a substrate for early deuterostome CYP51s.

General significance

The results expand the phylogenetic view of animal CYP51, with evolutionary, environmental and therapeutic implications.     

A Revised Evolutionary History of the CYP1A Subfamily: Gene Duplication, Gene Conversion, and Positive Selection

Members of cytochrome P450 subfamily 1A (CYP1As) are involved in detoxification and bioactivation of common environmental pollutants. Understanding the functional evolution of these genes is essential to predicting and interpreting species differences in sensitivity to toxicity caused by such chemicals. The CYP1A gene subfamily comprises a single ancestral representative in most fish species and two paralogs in higher vertebrates, including birds and mammals. Phylogenetic analysis of complete coding sequences suggests that mammalian and bird paralog pairs (CYP1A1/2 and CYP1A4/5, respectively) are the result of independent gene duplication events. However, comparison of vertebrate genome sequences revealed that CYP1A genes lie within an extended region of conserved fine-scale synteny, suggesting that avian and mammalian CYP1A paralogs share a common genomic history. Algorithms designed to detect recombination between nucleotide sequences indicate that gene conversion has homogenized most of the length of the chicken CYP1A genes, as well as the 5′ end of mammalian CYP1As. Together, these data indicate that avian and mammalian CYP1A paralog pairs resulted from a single gene duplication event and that extensive gene conversion is responsible for the exceptionally high degree of sequence similarity between CYP1A4 and CYP1A5. Elevated nonsynonymous/synonymous substitution ratios within a putatively unconverted stretch of ∼250 bp suggests that positive selection may have reduced the effective rate of gene conversion in this region, which contains two substrate recognition sites. This work significantly alters our understanding of functional evolution in the CYP1A subfamily, suggesting that gene conversion and positive selection have been the dominant processes of sequence evolution. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Yves Van de Peer]     

The missing risk: MRI and MRS phenotyping of abdominal adiposity and ectopic fat

Individual compartments of abdominal adiposity and lipid content within the liver and muscle are differentially associated with metabolic risk factors, obesity and insulin resistance. Subjects with greater intra-abdominal adipose tissue (IAAT) and hepatic fat than predicted by clinical indices of obesity may be at increased risk of metabolic diseases despite their "normal" size. There is a need for accurate quantification of these potentially hazardous depots and identification of novel subphenotypes that recognize individuals at potentially increased metabolic risk. We aimed to calculate a reference range for total and regional adipose tissue (AT) as well as ectopic fat in liver and muscle in healthy subjects. We studied the relationship between age, body-mass, BMI, waist circumference (WC), and the distribution of AT, using whole-body magnetic resonance imaging (MRI), in 477 white volunteers (243 male, 234 female). Furthermore, we used proton magnetic resonance spectroscopy (MRS) to determine intrahepatocellular (IHCL) and intramyocellular (IMCL) lipid content. The anthropometric variable which provided the strongest individual correlation for adiposity and ectopic fat stores was WC in men and BMI in women. In addition, we reveal a large variation in IAAT, abdominal subcutaneous AT (ASAT), and IHCL depots not fully predicted by clinically obtained measurements of obesity and the emergence of a previously unidentified subphenotype. Here, we demonstrate gender- and age-specific patterns of regional adiposity in a large UK-based cohort and identify anthropometric variables that best predict individual adiposity and ectopic fat stores. From these data we propose the thin-on-the-outside fat-on-the-inside (TOFI) as a subphenotype for individuals at increased metabolic risk.     

Antibodies designed as effective cancer vaccines

Antigen/antibody complexes can efficiently target antigen presenting cells to allow stimulation of the cellular immune response. Due to the difficulty of manufacture and their inherent instability complexes have proved inefficient cancer vaccines. However, anti-idiotypic antibodies mimicking antigens have been shown to stimulate both antibody and T cell responses. The latter are due to T cell mimotopes expressed within the complementarity-determining regions (CDRs) of antibodies that are efficiently presented to dendritic cells in vivo. Based on this observation we have designed a DNA vaccine platform called ImmunoBody™, where cytotoxic T lymphocyte (CTL) and helper T cell epitopes replace CDR regions within the framework of a human IgG1 antibody. The ImmunoBody™ expression system has a number of design features which allow for rapid production of a wide range of vaccines. The CDR regions of the heavy and light chain have been engineered to contain unique restriction endonuclease sites, which can be easily opened, and oligonucleotides encoding the T cell epitopes inserted. The variable and constant regions of the ImmunoBody™ are also flanked by restriction sites, which permit easy exchange of other IgG subtypes. Here we show a range of T cell epitopes can be inserted into the ImmunoBody™ vector and upon immunization these T cell epitopes are efficiently processed and presented to stimulate high frequency helper and CTL responses capable of anti-tumor activity.Key words: DNA vaccines, cancer vaccines, melanoma, CTL, helper T cells     

A new Gateway vector and expression protocol for fast and efficient recombinant protein expression in Mycobacterium smegmatis

A major obstacle associated with recombinant protein over-expression in Escherichia coli is the production of insoluble inclusion bodies, a problem particularly pronounced with Mycobacterium tuberculosis proteins. One strategy to overcome the formation of inclusion bodies is to use an expression host that is more closely related to the organism from which the proteins are derived. Here we describe methods for efficiently identifying M. tuberculosis proteins that express in soluble form in Mycobacterium smegmatis. We have adapted the M. smegmatis expression vector pYUB1049 to the Gateway cloning system by the addition of att recombination recognition sequences. The resulting vector, designated pDESTsmg, is compatible with our in-house Gateway methods for E. coli expression. A target can be subcloned into pDESTsmg by a simple LR reaction using an entry clone generated for E. coli expression, removing the need to design new primers and re-clone target DNA. Proteins are expressed by culturing the M. smegmatis strain mc(2)4517 in autoinduction media supplemented with Tween 80. The media used are the same as those used for expression of proteins in E. coli, simplifying and reducing the cost of the switch to an alternative host. The methods have been applied to a set of M. tuberculosis proteins that form inclusion bodies when expressed in E. coli. We found that five of eight of these previously insoluble proteins become soluble when expressed in M. smegmatis, demonstrating that this is an efficient salvage strategy.     

Proteomic identification, cDNA cloning and enzymatic activity of glutathione S-transferases from the generalist marine gastropod, Cyphoma gibbosum

Glutathione S-transferases (GST) were characterized from the digestive gland of Cyphoma gibbosum (Mollusca; Gastropoda), to investigate the possible role of these detoxification enzymes in conferring resistance to allelochemicals present in its gorgonian coral diet. We identified the collection of expressed cytosolic Cyphoma GST classes using a proteomic approach involving affinity chromatography, HPLC and nano-spray liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two major GST subunits were identified as putative mu-class GSTs; while one minor GST subunit was identified as a putative theta-class GST, apparently the first theta-class GST identified from a mollusc. Two Cyphoma GST cDNAs (CgGSTM1 and CgGSTM2) were isolated by RT-PCR using primers derived from peptide sequences. Phylogenetic analyses established both cDNAs as mu-class GSTs and revealed a mollusc-specific subclass of the GST-mu clade. These results provide new insights into metazoan GST diversity and the biochemical mechanisms used by marine organisms to cope with their chemically defended prey.