Thermostability of two cyanobacterial GrpE thermosensors

GrpE proteins act as co-chaperones for DnaK heat-shock proteins. The dimeric protein unfolds under heat stress conditions, which results in impaired interaction with a DnaK protein. Since interaction of GrpE with DnaK is crucial for the DnaK chaperone activity, GrpE proteins act as a thermosensor in bacteria. Here we have analyzed the thermostability and function of two GrpE homologs of the mesophilic cyanobacterium Synechocystis sp. PCC 6803 and of the thermophilic cyanobacterium Thermosynechococcus elongatus BP1. While in Synechocystis an N-terminal helix pair of the GrpE dimer appears to be the thermosensing domain and mainly mediates GrpE dimerization, the C-terminal four-helix bundle is involved in additional stabilization of the dimeric structure. The four-helix bundle domain has a key role in the thermophilic cyanobacterium, since dimerization of the Thermosynechococcus protein appears to be mediated by the four-helix bundle domain, and melting of this domain is linked to monomerization of the GrpE protein. Thus, in two related cyanobacteria the GrpE thermosensing function might be mediated by different protein domains.     

Two thermosensors in Drosophila have different behavioral functions

Insects inhabit extreme temperature environments and have evolved mechanisms to survive there. Small insects are especially susceptible to rapid changes in body temperature. Therefore, the rapid detection of environment and body temperature is important for their survival. Little, however, is known about the thermosensors that detect those temperatures. Using rapid thermosensitivity assays with temperature step gradients and a spatial learning paradigm (the heat-box) in which elevated temperature serves as the negative reinforcer, two thermosensors were identified and their behavioral functions assessed. A low-temperature thermosensor is located on the antenna, detects relatively low temperatures, and can detect spatial temperature gradients directly. Thus, the antennae can be used by Drosophila to quickly orient with respect to temperature cues. A high-temperature thermosensor of unknown location appears to have a roughly similar sensitivity to temperature differences as the low-temperature thermosensor (< or = 3 degrees C) and is both necessary and sufficient for memory formation in the heat-box spatial learning paradigm. Therefore, the high-temperature thermosensor is important for remembering spatial positions in which dangerously high temperatures were encountered.     

Generation of a synthetic lymphoid tissue-like organoid in mice

Stromal cells play an important role in the formation of the normal organized microarchitecture of secondary lymphoid organs. Here we demonstrate that a tissue-engineered, lymphoid tissue-like organoid, which was constructed by transplantation of stromal cells embedded in biocompatible scaffolds into the renal subcapsular space in mice, had an organized tissue structure similar to secondary lymphoid organs. This organoid contained compartmentalized B-cell and T-cell clusters, high endothelial venule-like vessels, germinal centers and follicular dendritic cell networks. Furthermore, the organoid was transplantable to naive normal or severe combined immunodeficiency (SCID) mice, and antigen-specific, IgG-isotype antibody formation could be induced soon after intravenous administration of the antigen. This simplified system of lymphoid tissue-like organoid construction will facilitate analyses of cell-cell interactions required for development of secondary lymphoid organs and efficient induction of adaptive immune responses, and may have possible applications in the treatment of immune deficiency.     

Evolutionary patterns of RNA-based duplication in non-mammalian chordates

The role of RNA-based duplication, or retroposition, in the evolution of new gene functions in mammals, plants, and Drosophila has been widely reported. However, little is known about RNA-based duplication in non-mammalian chordates. In this study, we screened ten non-mammalian chordate genomes for retrocopies and investigated their evolutionary patterns. We identified numerous retrocopies in these species. Examination of the age distribution of these retrocopies revealed no burst of young retrocopies in ancient chordate species. Upon comparing these non-mammalian chordate species to the mammalian species, we observed that a larger fraction of the non-mammalian retrocopies was under strong evolutionary constraints than mammalian retrocopies are, as evidenced by signals of purifying selection and expression profiles. For the Western clawed frog, Medaka, and Sea squirt, many retrogenes have evolved gonad and brain expression patterns, similar to what was observed in human. Testing of retrogene movement in the Medaka genome, where the nascent sex chrosomes have been well assembled, did not reveal any significant gene movement. Taken together, our analyses demonstrate that RNA-based duplication generates many functional genes and can make a significant contribution to the evolution of non-mammalian genomes.     

Miniature RT-PCR system for diagnosis of RNA-based viruses

This paper presents an innovative portable chip-based RT-PCR system for amplification of specific nucleic acid and detection of RNA-based viruses. The miniature RT-PCR chip is fabricated using MEMS (Micro-electro-mechanical-system) techniques, and comprises a micro temperature control module and a PDMS (polydimethylsiloxane)-based microfluidic control module. The heating and sensing elements of temperature control module are both made of platinum and are located within the reaction chambers in order to generate a rapid and uniform thermal cycling. The microfluidic control module is capable of automating testing process with minimum human intervention. In this paper, the proposed miniature RT-PCR system is used to amplify and detect two RNA-based viruses, namely dengue virus type-2 and enterovirus 71 (EV 71). The experimental data confirm the ability of the system to perform a two-step RT-PCR process. The developed miniature system provides a crucial tool for the diagnosis of RNA-based viruses.     

Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity

Combinatorial libraries of rearranged hypervariable V(H) and V(L) sequences from nonimmunized human donors contain antigen specificities, including anti-self reactivities, created by random pairing of V(H)s and V(L)s. Somatic hypermutation of immunoglobulin genes, however, is critical in the generation of high-affinity antibodies in vivo and occurs only after immunization. Thus, in combinatorial phage display libraries from nonimmunized donors, high-affinity antibodies are rarely found. Lengthy in vitro affinity maturation is often needed to improve antibodies from such libraries. We report the construction of human Fab libraries having a unique combination of immunoglobulin sequences captured from human donors and synthetic diversity in key antigen contact sites in heavy-chain complementarity-determining regions 1 and 2. The success of this strategy is demonstrated by identifying many monovalent Fabs against multiple therapeutic targets that show higher affinities than approved therapeutic antibodies. This very often circumvents the need for affinity maturation, accelerating discovery of antibody drug candidates.     

PETSTEP: Generation of synthetic PET lesions for fast evaluation of segmentation methods

PurposeThis work describes PETSTEP (PET Simulator of Tracers via Emission Projection): a faster and more accessible alternative to Monte Carlo (MC) simulation generating realistic PET images, for studies assessing image features and segmentation techniques.MethodsPETSTEP was implemented within Matlab as open source software. It allows generating three-dimensional PET images from PET/CT data or synthetic CT and PET maps, with user-drawn lesions and user-set acquisition and reconstruction parameters. PETSTEP was used to reproduce images of the NEMA body phantom acquired on a GE Discovery 690 PET/CT scanner, and simulated with MC for the GE Discovery LS scanner, and to generate realistic Head and Neck scans. Finally the sensitivity (S) and Positive Predictive Value (PPV) of three automatic segmentation methods were compared when applied to the scanner-acquired and PETSTEP-simulated NEMA images.ResultsPETSTEP produced 3D phantom and clinical images within 4 and 6 min respectively on a single core 2.7 GHz computer. PETSTEP images of the NEMA phantom had mean intensities within 2% of the scanner-acquired image for both background and largest insert, and 16% larger background Full Width at Half Maximum. Similar results were obtained when comparing PETSTEP images to MC simulated data. The S and PPV obtained with simulated phantom images were statistically significantly lower than for the original images, but led to the same conclusions with respect to the evaluated segmentation methods.ConclusionsPETSTEP allows fast simulation of synthetic images reproducing scanner-acquired PET data and shows great promise for the evaluation of PET segmentation methods.     

Generation of species-specific antihemoglobin antibodies by immunization with synthetic peptides of human hemoglobin

Four peptides (7–16 residues) representing nonconserved regions of human hemoglobin (Hb) were selected for synthesis by comparison of the amino acid sequence of human Hb with those of the most common domesticated animals. Mouse antisera resulting from immunization with the synthetic peptides were investigated for binding to a panel of animal Hbs using solid-phase radioimmunoassay (RIA). One of the peptides elicited antibodies which bound specifically to human Hb, but not to any Hb of the nonprimate animals tested. The results show that the peptide immunogen chosen on the basis of dissimilarity between regions of different species is useful for the generation of species-specific antibodies. Such antibodies could serve as valuable tools for clinical screening of fecal occult blood trait and for forensic identification of bloodstains of human origin.     

Generation of synthetic data and experimental designs in evaluating interactions for association studies

Complex diseases, by definition, involve multiple factors, including gene-gene interactions and gene-environment interactions. Researchers commonly rely on simulated data to evaluate their approaches for detecting high-order interactions in disease gene mapping. A publicly available simulation program to generate samples involving complex genetic and environmental interactions is of great interest to the community. We have developed a software package named gs1.0, which has been widely used since its publication. In this article, we present an upgraded version gs2.0, which not only inherits its capacity to generate realistic genotype data but also provides great functionality and flexibility to simulate various interaction models. In addition to a standalone version, a user-friendly web server (http://cbc.case.edu/gs) has been set up to help users to build complex interaction models. Furthermore, by utilizing three three-locus models as an example, we have shown how realistic model parameters can be chosen in generating simulated data.     

The emergence of ribozymes synthesizing membrane components in RNA-based protocells

A significant problem of the origin of life is the emergence of cellular self-replication. In the context of the “RNA world”, a crucial concern is how the RNA-based protocells could achieve the ability to produce their own membrane. Here we show, with the aid of a computer simulation, that for these protocells, there would be “immediately” a selection pressure for the emergence of a ribozyme synthesizing membrane components. The ribozyme would promote the enlargement of cellular space and favor the incoming (by permeation) of RNA's precursors, thus benefit the replication of inner RNA, including itself. Via growth and division, protocells containing the ribozyme would achieve superiority and spread in the system, and meanwhile the ribozyme would spread in the system. The present work is inspiring because it suggests that the transition from molecular self-replication to cellular self-replication might have occurred naturally (and necessarily) in the origin of life, leading to the emergence of Darwinian evolution at the cellular level.