The Effect of Freeze-Thaw Cycles on Gene Expression Levels in Lymphoblastoid Cell Lines

Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs) are a widely used renewable resource for functional genomic studies in humans. The ability to accumulate multidimensional data pertaining to the same individual cell lines, from complete genomic sequences to detailed gene regulatory profiles, further enhances the utility of LCLs as a model system. However, the extent to which LCLs are a faithful model system is relatively unknown. We have previously shown that gene expression profiles of newly established LCLs maintain a strong individual component. Here, we extend our study to investigate the effect of freeze-thaw cycles on gene expression patterns in mature LCLs, especially in the context of inter-individual variation in gene expression. We report a profound difference in the gene expression profiles of newly established and mature LCLs. Once newly established LCLs undergo a freeze-thaw cycle, the individual specific gene expression signatures become much less pronounced as the gene expression levels in LCLs from different individuals converge to a more uniform profile, which reflects a mature transformed B cell phenotype. We found that previously identified eQTLs are enriched among the relatively few genes whose regulations in mature LCLs maintain marked individual signatures. We thus conclude that while insight drawn from gene regulatory studies in mature LCLs may generally not be affected by the artificial nature of the LCL model system, many aspects of primary B cell biology cannot be observed and studied in mature LCL cultures.     

Biohybrid Polymer-Antimicrobial Peptide Medium against Enterococcus faecalis

Antimicrobial peptides (AMPs) are conserved evolutionary components of the innate immune system that are being tested as alternatives to antibiotics. Slow release of AMPs using biodegradable polymers can be advantageous in maintaining high peptide levels for topical treatment, especially in the oral environment in which dosage retention is challenged by drug dilution with saliva flow and by drug inactivation by salivary enzymatic activity. Enterococcus faecalis is a multidrug resistant nosocomial pathogen and a persistent pathogen in root canal infections. In this study, four ultra-short lipopeptides (C16-KGGK, C16-KLLK, C16-KAAK and C16-KKK) and an amphipathic α-helical antimicrobial peptide (Amp-1D) were tested against E. faecalis. The antibacterial effect was determined against planktonic bacteria and bacteria grown in biofilm. Of the five tested AMPs, C16-KGGK was the most effective. Next C16-KGGK was formulated with one of two polymers poly (lactic acid co castor oil) (DLLA) or ricinoleic acid-based poly (ester-anhydride) P(SA-RA). Peptide-synthetic polymer conjugates, also referred to as biohybrid mediums were tested for antibacterial activity against E. faecalis grown in suspension and in biofilms. The new formulations exhibited strong and improved anti- E. faecalis activity.     

Separase biosensor reveals that cohesin cleavage timing depends on phosphatase PP2A(Cdc55) regulation

In anaphase, sister chromatids separate abruptly and are then segregated by the mitotic spindle. The protease separase triggers sister separation by cleaving the Scc1/Mcd1 subunit of the cohesin ring that holds sisters together. Polo-kinase phosphorylation of Scc1 promotes its cleavage, but the underlying regulatory circuits are unclear. We developed a separase biosensor in Saccharomyces cerevisiae that provides a quantitative indicator of cohesin cleavage in single cells. Separase is abruptly activated and cleaves most cohesin within 1?min, after which anaphase begins. Cohesin near centromeres and telomeres is cleaved at the same rate and time. Protein phosphatase PP2A(Cdc55) inhibits cohesin cleavage by counteracting polo-kinase phosphorylation of Scc1. In early anaphase, the previously described separase inhibition of PP2A(Cdc55) promotes cohesin cleavage. Thus, separase acts directly on Scc1 and also indirectly, through inhibition of PP2A(Cdc55), to stimulate cohesin cleavage, providing a feedforward loop that may contribute to a robust and timely anaphase.     

Standardization of the Teratoma Assay for Analysis of Pluripotency of Human ES Cells and Biosafety of Their Differentiated Progeny

Teratoma tumor formation is an essential criterion in determining the pluripotency of human pluripotent stem cells. However, currently there is no consistent protocol for assessment of teratoma forming ability. Here we present detailed characterization of a teratoma assay that is based on subcutaneous co-transplantation of defined numbers of undifferentiated human embryonic stem cells (hESCs) with mitotically inactivated feeder cells and Matrigel into immunodeficient mice. The assay was highly reproducible and 100% efficient when 100,000 hESCs were transplanted. It was sensitive, promoting teratoma formation after transplantation of 100 hESCs, though larger numbers of animals and longer follow-up were required. The assay could detect residual teratoma forming cells within differentiated hESC populations however its sensitivity was decreased in the presence of differentiated cells. Our data lay the foundation, for standardization of a teratoma assay for pluripotency analysis. The assay can also be used for bio-safety analysis of pluripotent stem cell-derived differentiated progeny.     

White Blood Cell Count and the Risk for Coronary Artery Disease in Young Adults

Background

The association between white blood cell (WBC) count and coronary artery disease (CAD) is unknown in young adults. Our objective was to assess the association between WBC count and its changes over time with CAD incidence in the Metabolic, Life-style and Nutrition Assessment in Young adults (MELANY) study, a cohort of Israeli army personnel.

Methods and Findings

29,120 apparently healthy young men (mean age; 31.2±5.5 years) with a normal baseline WBC count (3,000–12,000 cells/mm³) were followed during a mean follow up of 7.5±3.8 years for incidence of CAD. Participants were screened every 3–5 years using a stress test, and CAD was confirmed by coronary angiography. In a multivariate model adjusted for age, body mass index (BMI), LDL- and HDL-cholesterol, blood pressure, family history of CAD, physical activity, diabetes, triglycerides and smoking status, WBC levels (divided to quintiles) above 6,900 cells/mm³ (quintile 4) were associated with a 2.17-fold increase (95%CI = 1.18–3.97) in the risk for CAD as compared with men in quintile 1 (WBC≤5,400 cells/mm³). When modeled as a continuous variable, a WBC increment of 1000 cells/mm³ was associated with a 17.4% increase in CAD risk (HR 1.174; 95%CI = 1.067–1.290, p = 0.001). A decrease in the WBC level (within the normal range) during the follow-up period was associated with increased physical activity and decreased triglyceride levels as well as with reduced incidence of CAD.

Conclusions

WBC count is an independent risk factor for CAD in young adults at values well within the normal range. WBC count may assist in detecting subgroups of young men at either low or high risk for progression to CAD.     

A cortical attractor network with Martinotti cells driven by facilitating synapses

The population of pyramidal cells significantly outnumbers the inhibitory interneurons in the neocortex, while at the same time the diversity of interneuron types is much more pronounced. One acknowledged key role of inhibition is to control the rate and patterning of pyramidal cell firing via negative feedback, but most likely the diversity of inhibitory pathways is matched by a corresponding diversity of functional roles. An important distinguishing feature of cortical interneurons is the variability of the short-term plasticity properties of synapses received from pyramidal cells. The Martinotti cell type has recently come under scrutiny due to the distinctly facilitating nature of the synapses they receive from pyramidal cells. This distinguishes these neurons from basket cells and other inhibitory interneurons typically targeted by depressing synapses. A key aspect of the work reported here has been to pinpoint the role of this variability. We first set out to reproduce quantitatively based on in vitro data the di-synaptic inhibitory microcircuit connecting two pyramidal cells via one or a few Martinotti cells. In a second step, we embedded this microcircuit in a previously developed attractor memory network model of neocortical layers 2/3. This model network demonstrated that basket cells with their characteristic depressing synapses are the first to discharge when the network enters an attractor state and that Martinotti cells respond with a delay, thereby shifting the excitation-inhibition balance and acting to terminate the attractor state. A parameter sensitivity analysis suggested that Martinotti cells might, in fact, play a dominant role in setting the attractor dwell time and thus cortical speed of processing, with cellular adaptation and synaptic depression having a less prominent role than previously thought.     

Enhancing protein crystallization through precipitant synergy

Suitable conditions for protein crystallization are commonly identified by screening combinations of independent factors that affect crystal formation. Because precipitating agents are prime determinants of crystallization, we investigated whether a systematic exploration of combinations of mechanistically distinct precipitants would enhance crystallization. A crystallization screen containing 64 precipitant mixtures was devised. Tests with ten HIV envelope-related proteins demonstrated that use of precipitant mixtures significantly enhanced both the probability of crystallization as well as the quality of optimized crystals. Tests with hen egg white lysozyme generated a novel C2 crystal from a salt/organic solvent mixture; structure solution at 2 A resolution revealed a lattice held together by both hydrophobic and electrostatic dyad interactions. The results indicate that mechanistically distinct precipitants can synergize, with precipitant combinations adding unique dimensions to protein crystallization.     

Concerted release of substrate domains from GroEL by ATP is demonstrated with FRET

The chaperonin GroEL assists protein folding by undergoing ATP-induced conformational changes that are concerted within each of its two back-to-back stacked rings. Here we examined whether concerted allosteric switching gives rise to all-or-none release and folding of domains in a chimeric fluorescent protein substrate, CyPet-YPet. Using this substrate, it was possible to determine the folding yield of each domain from its intrinsic fluorescence and that of the entire chimera by measuring Förster resonance energy transfer between the two domains. Hence, it was possible to determine whether release of one domain is accompanied by release of the other domain (concerted mechanism), or whether their release is not coupled. Our results show that the chimera's release tends to be concerted when folding is assisted by a wild-type GroEL variant, but not when assisted by the F44W/D155A mutant that undergoes a sequential allosteric switch. A connection between the allosteric mechanism of this molecular machine and its biological function in assisting folding is thus established.