Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca²⁺) often termed a ''Ca²⁺ wave''. While the speed and number of oscillations of the Ca²⁺ wave varies between species, the change in intracellular Ca²⁺ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca²⁺-dependent events. Here we present a protocol for imaging the Ca²⁺ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca²⁺ wave and the downstream changes associated with it.     

Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system

Type II restriction-modification (R-M) systems encode a restriction endonuclease that cleaves DNA at specific sites, and a methyltransferase that modifies same sites protecting them from restriction endonuclease cleavage. Type II R-M systems benefit bacteria by protecting them from bacteriophages. Many type II R-M systems are plasmid-based and thus capable of horizontal transfer. Upon the entry of such plasmids into a naïve host with unmodified genomic recognition sites, methyltransferase should be synthesized first and given sufficient time to methylate recognition sites in the bacterial genome before the toxic restriction endonuclease activity appears. Here, we directly demonstrate a delay in restriction endonuclease synthesis after transformation of Escherichia coli cells with a plasmid carrying the Esp1396I type II R-M system, using single-cell microscopy. We further demonstrate that before the appearance of the Esp1396I restriction endonuclease the intracellular concentration of Esp1396I methyltransferase undergoes a sharp peak, which should allow rapid methylation of host genome recognition sites. A mathematical model that satisfactorily describes the observed dynamics of both Esp1396I enzymes is presented. The results reported here were obtained using a functional Esp1396I type II R-M system encoding both enzymes fused to fluorescent proteins. Similar approaches should be applicable to the studies of other R-M systems at single-cell level.     

Oil for Fish: An Energy Return on Investment Analysis of Selected European Union Fishing Fleets

World food production has increased substantially in the past century, thanks mostly to the increase in the use of oil as input in the production processes. This growing use of fossil fuels has negative effects, both on the environment and the production costs. Fishing is a fuel consuming food production activity, and its energy efficiency performance has worsened over time. World‐wide fisheries are also suffering from overexploitation, which contributes to the poor efficiency performance, adding more pressure and criticism on this economic activity. In this paper we analyzed the energy efficiency performance of more than 20,000 European Union (EU) fishing vessels for the period 2002–2008, using the edible energy return on investment (EROI) indicator. The vessels analyzed, grouped in 49 different fleets, represented 25% of the vessels and 33% of the landings of the EU fishing sector. These EU fishing fleets’ average EROI for 2008 was 0.11, which translates to an energy content of the fuel burned that is 9 times greater than the edible energy content of the catch. Hence, the significance of this study arises from the use of time‐series data on a relevant part of the EU fleet that showed stable or even slight improvements on the EROI over time. Moreover, results showed that the energy efficiency of the different fleets varied significantly (from 0.02 to 1.12), mainly depending on the fishing gear and the vessel length. The performance of the most efficient fleets, such as large pelagic trawlers and seiners, was comparable to many agricultural production activities. The plausible drivers behind these trends are further considered.     

Coupling spatial segregation with synthetic circuits to control bacterial survival

Engineered bacteria have great potential for medical and environmental applications. Fulfilling this potential requires controllability over engineered behaviors and scalability of the engineered systems. Here, we present a platform technology, microbial swarmbot, which employs spatial arrangement to control the growth dynamics of engineered bacteria. As a proof of principle, we demonstrated a safeguard strategy to prevent unintended bacterial proliferation. In particular, we adopted several synthetic gene circuits to program collective survival in Escherichia coli: the engineered bacteria could only survive when present at sufficiently high population densities. When encapsulated by permeable membranes, these bacteria can sense the local environment and respond accordingly. The cells inside the microbial swarmbot capsules will survive due to their high densities. Those escaping from a capsule, however, will be killed due to a decrease in their densities. We demonstrate that this design concept is modular and readily generalizable. Our work lays the foundation for engineering integrated and programmable control of hybrid biological–material systems for diverse applications.     

A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2): HINTS FOR AN INTRACELLULAR FUNCTION AT THE SYNAPSE*

Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (N_cyt/C_exo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders.     

A comparison and user-based evaluation of models of textual information structure in the context of cancer risk assessment

Background  

Many practical tasks in biomedicine require accessing specific types of information in scientific literature; e.g. information about the results or conclusions of the study in question. Several schemes have been developed to characterize such information in scientific journal articles. For example, a simple section-based scheme assigns individual sentences in abstracts under sections such as Objective, Methods, Results and Conclusions. Some schemes of textual information structure have proved useful for biomedical text mining (BIO-TM) tasks (e.g. automatic summarization). However, user-centered evaluation in the context of real-life tasks has been lacking.     

Risk of all-cause mortality in HIV infected patients is associated with clinical, immunologic predictors and the CCR5 Δ32 deletion

Objective

Investigation of the interplay between the CCR5 Δ32/wt genotype and demographic, epidemiological, clinical and immunological factors associated with mortality in the cART era.

Design

Longitudinal data from 507 HIV-infected patients following the Δ32 allele detection were analyzed.

Methods

Cumulative 15 years mortality was calculated using Kaplan-Meyer methodology. Hazard ratios were estimated using univariate Cox models. Basing on Akakie information criteria and statistical significance multivariate Cox model was constructed and effect plots presenting adjusted hazard ratio time-dependency were drawn. Analysis of the association of all-cause mortality and CCR5 Δ32/wt genotype prior to the antiretroviral treatment (cART) initiation (n = 507) and on the therapy (n = 422) was also performed.

Results

A mortality rate of 2.66 (CI 2.57–3.19) per 100 person-years was observed. Univariate analysis factors modifying the risk of death included the CCR5 genotype, gender, history of cART, AIDS diagnosis and also CD4 lymphocyte nadir, zenith, the latest CD4 count and stable levels >500 cells/µl. For multivariate analysis the following predictors were selected: CCR5 genotype (HR for wt/wt 2.53, CI 1.16–5.53, p = 0.02), gender (HR for males 1.91, 95%CI 1.1–3.36, p = 0.023), introduction of combined antiretroviral treatment (HR 4.85, CI 3.0–7.89, if untreated or treated <1 month, p<0.0001) CD4 count of 500 cells/µl for six months or more (HR 4.16, CI 1.95–8.88 if not achieved, p = 0.028), the latest CD4 count (HR 5.44, CI 3.39–8.74 for <100 cells/µl, p<0.0001) and history of AIDS (HR 1.69, CI 1.03–2.79, p = 0.039). Among untreated individuals the Δ32/wt genotype was associated with notably better survival (p = 0.026), while among cART treated individuals the Δ32 mutation did not correlate significantly with higher survival rates (p = 0.23).

Conclusions

The Δ32 CCR5 allele is associated with a reduction of the risk of all-cause mortality in HIV (+) patients alongside clinical and immunologic predictors such as AIDS, history of cART, lymphocyte CD4 cell count and gender.     

Global architecture of human poly(A)-specific ribonuclease by atomic force microscopy in liquid and dynamic light scattering

Deadenylation is the initial and often rate-limiting step in the main pathways of eukaryotic mRNA decay. Poly(A)-specific ribonuclease (PARN) is a eukaryotic enzyme that efficiently degrades mRNA poly(A) tails. Structural and functional studies have shown that human PARN is composed of at least three functional domains, i.e. the catalytic nuclease domain and two RNA binding domains, the R3H and the RNA recognition motif (RRM), respectively. However, the complete structure of the full length protein is still unknown. We have investigated the global architecture of human PARN by atomic force microscopy (AFM) imaging in buffered milieu and report for the first time the dimensions of the full length protein at subnanometer resolution. The AFM images of single PARN molecules reveal compact ellipsoidal dimers (10.9 × 7.6 × 4.6nm). The dimeric form of PARN was confirmed by dynamic light scattering (DLS) measurements that rendered a molecular weight of 161 kDa, in accordance with previous crystal structures of PARN fragments showing a dimeric composition. We discuss a putative internal arrangement of three functional domains within the full length PARN dimer.