SILAC-based proteomic quantification of chemoattractant-induced cytoskeleton dynamics on a second to minute timescale

Cytoskeletal dynamics during cell behaviours ranging from endocytosis and exocytosis to cell division and movement is controlled by a complex network of signalling pathways, the full details of which are as yet unresolved. Here we show that SILAC-based proteomic methods can be used to characterize the rapid chemoattractant-induced dynamic changes in the actin–myosin cytoskeleton and regulatory elements on a proteome-wide scale with a second to minute timescale resolution. This approach provides novel insights in the ensemble kinetics of key cytoskeletal constituents and association of known and novel identified binding proteins. We validate the proteomic data by detailed microscopy-based analysis of in vivo translocation dynamics for key signalling factors. This rapid large-scale proteomic approach may be applied to other situations where highly dynamic changes in complex cellular compartments are expected to play a key role

Nanopore-based kinetics analysis of individual antibody-channel and antibody-antigen interactions

<p>Abstract</p> <p>Background</p> <p>The UNO/RIC Nanopore Detector provides a new way to study the binding and conformational changes of individual antibodies. Many critical questions regarding antibody function are still unresolved, questions that can be approached in a new way with the nanopore detector.</p> <p>Results</p> <p>We present evidence that different forms of channel blockade can be associated with the same antibody, we associate these different blockades with different orientations of "capture" of an antibody in the detector's nanometer-scale channel. We directly detect the presence of antibodies via reductions in channel current. Changes to blockade patterns upon addition of antigen suggest indirect detection of antibody/antigen binding. Similarly, DNA-hairpin anchored antibodies have been studied, where the DNA linkage is to the carboxy-terminus at the base of the antibody's Fc region, with significantly fewer types of (lengthy) capture blockades than was observed for free (un-bound) IgG antibody. The introduction of chaotropic agents and its effects on protein-protein interactions have also been observed.</p> <p>Conclusion</p> <p>Nanopore-based approaches may eventually provide a direct analysis of the complex conformational "negotiations" that occur upon binding between proteins.</p

A novel, fast, HMM-with-Duration implementation – for application with a new, pattern recognition informed, nanopore detector

<p>Abstract</p> <p>Background</p> <p>Hidden Markov Models (HMMs) provide an excellent means for structure identification and feature extraction on stochastic sequential data. An HMM-with-Duration (HMMwD) is an HMM that can also exactly model the hidden-label length (recurrence) distributions – while the regular HMM will impose a best-fit geometric distribution in its modeling/representation.</p> <p>Results</p> <p>A Novel, Fast, HMM-with-Duration (HMMwD) Implementation is presented, and experimental results are shown that demonstrate its performance on two-state synthetic data designed to model Nanopore Detector Data. The HMMwD experimental results are compared to (i) the ideal model and to (ii) the conventional HMM. Its accuracy is clearly an improvement over the standard HMM, and matches that of the ideal solution in many cases where the standard HMM does not. Computationally, the new HMMwD has all the speed advantages of the conventional (simpler) HMM implementation. In preliminary work shown here, HMM feature extraction is then used to establish the first pattern recognition-informed (PRI) sampling control of a Nanopore Detector Device (on a "live" data-stream).</p> <p>Conclusion</p> <p>The improved accuracy of the new HMMwD implementation, at the same order of computational cost as the standard HMM, is an important augmentation for applications in gene structure identification and channel current analysis, especially PRI sampling control, for example, where speed is essential. The PRI experiment was designed to inherit the high accuracy of the well characterized and distinctive blockades of the DNA hairpin molecules used as controls (or blockade "test-probes"). For this test set, the accuracy inherited is 99.9%.</p

The Surgical Infection Society revised guidelines on the management of intra-abdominal infection

Background: Previous evidence-based guidelines on the management of intra-abdominal infection (IAI) were published by the Surgical Infection Society (SIS) in 1992, 2002, and 2010. At the time the most recent guideline was released, the plan was to update the guideline every five years to ensure the timeliness and appropriateness of the recommendations. Methods: Based on the previous guidelines, the task force outlined a number of topics related to the treatment of patients with IAI and then developed key questions on these various topics. All questions were approached using general and specific literature searches, focusing on articles and other information published since 2008. These publications and additional materials published before 2008 were reviewed by the task force as a whole or by individual subgroups as to relevance to individual questions. Recommendations were developed by a process of iterative consensus, with all task force members voting to accept or reject each recommendation. Grading was based on the GRADE (Grades of Recommendation Assessment, Development, and Evaluation) system; the quality of the evidence was graded as high, moderate, or weak, and the strength of the recommendation was graded as strong or weak. Review of the document was performed by members of the SIS who were not on the task force. After responses were made to all critiques, the document was approved as an official guideline of the SIS by the Executive Council. Results: This guideline summarizes the current recommendations developed by the task force on the treatment of patients who have IAI. Evidence-based recommendations have been made regarding risk assessment in individual patients; source control; the timing, selection, and duration of antimicrobial therapy; and suggested approaches to patients who fail initial therapy. Additional recommendations related to the treatment of pediatric patients with IAI have been included. Summary: The current recommendations of the SIS regarding the treatment of patients with IAI are provided in this guideline

Massive migration from the steppe is a source for Indo-European languages in Europe

We generated genome-wide data from 69 Europeans who lived between 8,000-3,000 years ago by enriching ancient DNA libraries for a target set of almost four hundred thousand polymorphisms. Enrichment of these positions decreases the sequencing required for genome-wide ancient DNA analysis by a median of around 250-fold, allowing us to study an order of magnitude more individuals than previous studies and to obtain new insights about the past. We show that the populations of western and far eastern Europe followed opposite trajectories between 8,000-5,000 years ago. At the beginning of the Neolithic period in Europe, ~8,000-7,000 years ago, closely related groups of early farmers appeared in Germany, Hungary, and Spain, different from indigenous hunter-gatherers, whereas Russia was inhabited by a distinctive population of hunter-gatherers with high affinity to a ~24,000 year old Siberian6 . By ~6,000-5,000 years ago, a resurgence of hunter-gatherer ancestry had occurred throughout much of Europe, but in Russia, the Yamnaya steppe herders of this time were descended not only from the preceding eastern European hunter-gatherers, but from a population of Near Eastern ancestry. Western and Eastern Europe came into contact ~4,500 years ago, as the Late Neolithic Corded Ware people from Germany traced ~3/4 of their ancestry to the Yamnaya, documenting a massive migration into the heartland of Europe from its eastern periphery. This steppe ancestry persisted in all sampled central Europeans until at least ~3,000 years ago, and is ubiquitous in present-day Europeans. These results provide support for the theory of a steppe origin of at least some of the Indo-European languages of Europe

Effects of Fusion between Tactile and Proprioceptive Inputs on Tactile Perception

Tactile perception is typically considered the result of cortical interpretation of afferent signals from a network of mechanical sensors underneath the skin. Yet, tactile illusion studies suggest that tactile perception can be elicited without afferent signals from mechanoceptors. Therefore, the extent that tactile perception arises from isomorphic mapping of tactile afferents onto the somatosensory cortex remains controversial. We tested whether isomorphic mapping of tactile afferent fibers onto the cortex leads directly to tactile perception by examining whether it is independent from proprioceptive input by evaluating the impact of different hand postures on the perception of a tactile illusion across fingertips. Using the Cutaneous Rabbit Effect, a well studied illusion evoking the perception that a stimulus occurs at a location where none has been delivered, we found that hand posture has a significant effect on the perception of the illusion across the fingertips. This finding emphasizes that tactile perception arises from integration of perceived mechanical and proprioceptive input and not purely from tactile interaction with the external environment

Reduced Mature MicroRNA Levels in Association with Dicer Loss in Human Temporal Lobe Epilepsy with Hippocampal Sclerosis

Hippocampal sclerosis (HS) is a common pathological finding in patients with temporal lobe epilepsy (TLE) and is associated with altered expression of genes controlling neuronal excitability, glial function, neuroinflammation and cell death. MicroRNAs (miRNAs), a class of small non-coding RNAs, function as post-transcriptional regulators of gene expression and are critical for normal brain development and function. Production of mature miRNAs requires Dicer, an RNAase III, loss of which has been shown to cause neuronal and glial dysfunction, seizures, and neurodegeneration. Here we investigated miRNA biogenesis in hippocampal and neocortical resection specimens from pharmacoresistant TLE patients and autopsy controls. Western blot analysis revealed protein levels of Dicer were significantly lower in certain TLE patients with HS. Dicer levels were also reduced in the hippocampus of mice subject to experimentally-induced epilepsy. To determine if Dicer loss was associated with altered miRNA processing, we profiled levels of 380 mature miRNAs in control and TLE-HS samples. Expression of nearly 200 miRNAs was detected in control human hippocampus. In TLE-HS samples there was a large-scale reduction of miRNA expression, with 51% expressed at lower levels and a further 24% not detectable. Primary transcript (pri-miRNAs) expression levels for several tested miRNAs were not different between control and TLE-HS samples. These findings suggest loss of Dicer and failure of mature miRNA expression may be a feature of the pathophysiology of HS in patients with TLE