Some Secrets of Fluorescent Proteins: Distinct Bleaching in Various Mounting Fluids and Photoactivation of cyan fluorescent proteins at YFP-Excitation

Background
The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins.

Methodology/Principal Findings
When we applied a commonly used FRET microscopy technique - the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10 - 15% after illumination at the YFP-excitation wavelength – a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor.

Conclusions/Significance
Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions

Twisting arms and sending messages: Terrorist tactics in civil war

We examine the strategic rationale for terrorist tactics in civil war. We identify conditions that favor terrorism as a tactic in armed civil conflicts as well as the specific targets as a function of rebel characteristics, goals, and government responses to political demands. Terrorist tactics can be helpful as an instrument to coerce the government in asymmetric conflicts, as rebels are typically weak relative to the government. But terrorism can also help communicate the goals and resolve of a group when there is widespread uncertainty. We consider the strategic importance and rationale for terrorism in terms of the frequency of attacks and specific targets, and analyze our propositions using new data linking actors from the Uppsala/PRIO Armed Conflict Data and the Global Terrorism Database. Consistent with our expectations, we find that terrorism is used more extensively in civil conflicts by weaker groups and when attacks can help the group convey its goals without undermining popular support. Groups with more inclusive audiences are more likely to focus on ‘hard’ or official targets, while groups with more sectarian audiences are more likely to attack ‘soft’ targets and civilians

Single-Atom Gating of Quantum State Superpositions

The ultimate miniaturization of electronic devices will likely require local and coherent control of single electronic wavefunctions. Wavefunctions exist within both physical real space and an abstract state space with a simple geometric interpretation: this state space--or Hilbert space--is spanned by mutually orthogonal state vectors corresponding to the quantized degrees of freedom of the real-space system. Measurement of superpositions is akin to accessing the direction of a vector in Hilbert space, determining an angle of rotation equivalent to quantum phase. Here we show that an individual atom inside a designed quantum corral can control this angle, producing arbitrary coherent superpositions of spatial quantum states. Using scanning tunnelling microscopy and nanostructures assembled atom-by-atom we demonstrate how single spins and quantum mirages can be harnessed to image the superposition of two electronic states. We also present a straightforward method to determine the atom path enacting phase rotations between any desired state vectors. A single atom thus becomes a real space handle for an abstract Hilbert space, providing a simple technique for coherent quantum state manipulation at the spatial limit of condensed matter.Comment: Published online 6 April 2008 in Nature Physics; 17 page manuscript (including 4 figures) + 3 page supplement (including 2 figures); supplementary movies available at http://mota.stanford.ed

Viral population estimation using pyrosequencing

The diversity of virus populations within single infected hosts presents a major difficulty for the natural immune response as well as for vaccine design and antiviral drug therapy. Recently developed pyrophosphate based sequencing technologies (pyrosequencing) can be used for quantifying this diversity by ultra-deep sequencing of virus samples. We present computational methods for the analysis of such sequence data and apply these techniques to pyrosequencing data obtained from HIV populations within patients harboring drug resistant virus strains. Our main result is the estimation of the population structure of the sample from the pyrosequencing reads. This inference is based on a statistical approach to error correction, followed by a combinatorial algorithm for constructing a minimal set of haplotypes that explain the data. Using this set of explaining haplotypes, we apply a statistical model to infer the frequencies of the haplotypes in the population via an EM algorithm. We demonstrate that pyrosequencing reads allow for effective population reconstruction by extensive simulations and by comparison to 165 sequences obtained directly from clonal sequencing of four independent, diverse HIV populations. Thus, pyrosequencing can be used for cost-effective estimation of the structure of virus populations, promising new insights into viral evolutionary dynamics and disease control strategies.Comment: 23 pages, 13 figure

Follow-up period of 13 years after endoscopic total extraperitoneal repair of inguinal hernias: a cohort study

Background: Endoscopic inguinal hernia repair was introduced in the Netherlands in the early 1990s. The authors' institution was among the first to adopt this technique. In this study, long-term hernia recurrence among patients treated by the total extraperitoneal (TEP) approach for an inguinal hernia is described. A cohort study was conducted. Methods: Between January 1993 and December 1997, 346 TEP hernia repairs were performed for 318 patients. After a mean follow-up period of 13-years, a senior resident examined each patient. An experienced surgeon subsequently examined the patients with a diagnosis of recurrent hernia. Data were collected on an intention-to-treat basis, meaning that conversions were included in the analysis. Univariant tests were used to analyze age older than 50 years, chronic obstructive pulmonary disease, body mass index, smoking habit, hernia type, history of open hernia repair, conversion, and surgeon as potential risk factors. Results: The analysis included 191 patients (62%) with 213 hernias. Of the original 318 patients, 59 patients died, and 68 were lost to follow-up evaluation. Perioperatively, 105 lateral, 55 medial, and 53 pantalon hernias were observed. Of the 213 hernias, 176 were primary and 37 were recurrent. The overall recurrence rate was 8.9% (8.5% for primary and 10.8% for recurrent hernias). Of the total study group, 48% of the patients experienced a bilateral inguinal hernia during their lifetime. No predicting factor for recurrent hernia could be identified. Conclusions: The current long-term results for TEP repair of primary and secondary inguinal hernia show an overall recurrence rate of 8.9%, which is slightly higher than in previous studies. The thorough examination at follow-up assessment, the learning curve effect, and the intention-to-treat-analysis may have influenced the observed recurrence rate. Also, the percentage of bilateral hernias was higher than known to date. Therefore, examination of the contralateral side should be standard procedure

Balancing Selection at the Tomato RCR3 Guardee Gene Family Maintains Variation in Strength of Pathogen Defense

Coevolution between hosts and pathogens is thought to occur between interacting molecules of both species. This results in the maintenance of genetic diversity at pathogen antigens (or so-called effectors) and host resistance genes such as the major histocompatibility complex (MHC) in mammals or resistance (R) genes in plants. In plant-pathogen interactions, the current paradigm posits that a specific defense response is activated upon recognition of pathogen effectors via interaction with their corresponding R proteins. According to the''Guard-Hypothesis,'' R proteins (the ``guards'') can sense modification of target molecules in the host (the ``guardees'') by pathogen effectors and subsequently trigger the defense response. Multiple studies have reported high genetic diversity at R genes maintained by balancing selection. In contrast, little is known about the evolutionary mechanisms shaping the guardee, which may be subject to contrasting evolutionary forces. Here we show that the evolution of the guardee RCR3 is characterized by gene duplication, frequent gene conversion, and balancing selection in the wild tomato species Solanum peruvianum. Investigating the functional characteristics of 54 natural variants through in vitro and in planta assays, we detected differences in recognition of the pathogen effector through interaction with the guardee, as well as substantial variation in the strength of the defense response. This variation is maintained by balancing selection at each copy of the RCR3 gene. Our analyses pinpoint three amino acid polymorphisms with key functional consequences for the coevolution between the guardee (RCR3) and its guard (Cf-2). We conclude that, in addition to coevolution at the ``guardee-effector'' interface for pathogen recognition, natural selection acts on the ``guard-guardee'' interface. Guardee evolution may be governed by a counterbalance between improved activation in the presence and prevention of auto-immune responses in the absence of the corresponding pathogen

Warming of Central European lakes and their response to the 1980s climate regime shift

Lake surface water temperatures (LSWTs) are sensitive to atmospheric warming and have previously been shown to respond to regional changes in the climate. Using a combination of in situ and simulated surface temperatures from 20 Central European lakes, with data spanning between 50 and ∼100 years, we investigate the long-term increase in annually averaged LSWT. We demonstrate that Central European lakes are warming most in spring and experience a seasonal variation in LSWT trends. We calculate significant LSWT warming during the past few decades and illustrate, using a sequential t test analysis of regime shifts, a substantial increase in annually averaged LSWT during the late 1980s, in response to an abrupt shift in the climate. Surface air temperature measurements from 122 meteorological stations situated throughout Central Europe demonstrate similar increases at this time. Climatic modification of LSWT has numerous consequences for water quality and lake ecosystems. Quantifying the response of LSWT increase to large-scale and abrupt climatic shifts is essential to understand how lakes will respond in the future

Do MRI findings identify patients with chronic low back pain and Modic changes who respond best to rest or exercise: A subgroup analysis of a randomised controlled trial

Background: No previous clinical trials have investigated MRI findings as effect modifiers for conservative treatment of low back pain. This hypothesis-setting study investigated if MRI findings modified response to rest compared with exercise in patients with chronic low back pain and Modic changes. Methods: This study is a secondary analysis of a randomised controlled trial comparing rest with exercise. Patients were recruited from a specialised outpatient spine clinic and included in a clinical trial if they had chronic low back pain and an MRI showing Modic changes. All patients received conservative treatment while participating in the trial. Five baseline MRI findings were investigated as effect modifiers: Modic changes Type 1 (any size), large Modic changes (any type), large Modic changes Type 1, severe disc degeneration and large disc herniation. The outcome measure was change in low back pain intensity measured on a 0-10 point numerical rating scale at 14-month follow-up (n = 96). An interaction = 1.0 point (0-10 scale) between treatment group and MRI findings in linear regression was considered clinically important. Results: The interactions for Modic Type 1, with large Modic changes or with large Modic changes Type 1 were all potentially important in size (-0.99 (95% CI -3.28 to 1.29), -1.49 (-3.73 to 0.75), -1.49 (-3.57 to 0.58), respectively) but the direction of the effect was the opposite to what we had hypothesized-that people with these findings would benefit more from rest than from exercise. The interactions for severe disc degeneration (0.74 (-1.40 to 2.88)) and large disc herniation (-0.92 (3.15 to 1.31)) were less than the 1.0-point threshold for clinical importance. As expected, because of the lack of statistical power, no interaction term for any of the MRI findings was statistically significant. Conclusions: Three of the five MRI predictors showed potentially important effect modification, although the direction of the effect was surprising and confidence intervals were wide so very cautious interpretation is required. Further studies with adequate power are warranted to study these and additional MRI findings as potential effect modifiers for common interventions