Determination of plasmid copy number in yeast transformants by means of agarose plugs

The determination of plasmid copy number in Saccharomyces cerevisiaetransformants containing circular or linear plasmids is currently performed with total yeast DNA extracts obtained from cultures grown under selection. The determination is based essentially on quantitative Southern hybridization of an appropirate probe to a sequence present both on plasmid and chromosomal DNA in digested or undigested samples run out on conventional agarose gels. The DNA extraction procedure calls for treatment of cell lysates with organic solvents that could entail systemic losses of eithr plasmid or chromosomal DNA thus producing artifactual results. We propose here a method based on the assumption that quantitative analysis of plasmid and chromosomal DNA extracted from yeast cells embedded in agarose plugs will furnish more reliable results. With this procedure the cells are lysed in situ, thus avoiding possible losses of material, and the chromosomes and plasmid DNAs, trapped within the agarose matrix, can be separated by pulse field electrophoresis

Separating Reflection and Transmission Images in the Wild

The reflections caused by common semi-reflectors, such as glass windows, can impact the performance of computer vision algorithms. State-of-the-art methods can remove reflections on synthetic data and in controlled scenarios. However, they are based on strong assumptions and do not generalize well to real-world images. Contrary to a common misconception, real-world images are challenging even when polarization information is used. We present a deep learning approach to separate the reflected and the transmitted components of the recorded irradiance, which explicitly uses the polarization properties of light. To train it, we introduce an accurate synthetic data generation pipeline, which simulates realistic reflections, including those generated by curved and non-ideal surfaces, non-static scenes, and high-dynamic-range scenes.Comment: accepted at ECCV 201

Cardiovascular responses to postural change and aerobic capacity among middle-aged men and women before and after aerobic physical training

OBJETIVO: Comparar respostas cardiovasculares a Manobra Postural Passiva (Tilt Test) e capacidade cardiorrespiratória em homens e mulheres de meia-idade antes e após treinamento físico aeróbio. MATERIAIS E MÉTODOS: Sete homens - GH (44,6±2,1 anos) e sete mulheres - GM (51,7±4,8 anos), participaram de treinamento físico aeróbio por 12 semanas. Foi realizado protocolo de Tilt Test (cinco minutos supino, dez minutos inclinado 70º, cinco minutos supino), com monitoração da pressão arterial e freqüência cardíaca. Para mensuração da capacidade cardiorrespiratória foi realizado protocolo em cicloergômetro. RESULTADOS: Na condição sedentária, GH mostrou maior influência parassimpática no controle da freqüência cardíaca evidenciada por maior intervalo RR (iRR) durante Tilt Test. Na condição treinada, os valores de iRR de ambos os grupos se assemelham, tendo as mulheres iRR maior em supino, mas na inclinação os homens mantêm iRR mais elevado. Para pressão arterial, as mulheres permanecem com valores superiores após treino, mas a freqüência cardíaca tende a se assemelhar em ambos. Já na capacidade cardiorrespiratória, homens e mulheres têm um padrão de comportamento semelhante após treino. Com exceção dos valores absolutos da freqüência cardíaca, sem diferenças entre os grupos, para todas as outras variáveis os homens obtiveram valores superiores aos das mulheres. Observa-se ainda que, após o treinamento, houve redução significativa dos valores de pressão arterial no GM, mesmo continuando superiores aos dos homens. CONCLUSÃO: O treinamento parece ter reduzido os níveis pressóricos nas mulheres, além de serem observadas melhorias na capacidade cardiorrespiratória de ambos os grupos, permanecendo os homens com melhor desempenho do que as mulheres.OBJECTIVE: To compare the cardiovascular responses to passive postural maneuvers (tilt test) and the cardiorespiratory capacity in middle-aged men and women, before and after aerobic physical training. METHODS: Seven men (44.6±2.1 years old) and seven women (51.7±4.8 years old) participated in aerobic physical training for 12 weeks. The tilt test protocol (five minutes supine, ten minutes tilted at 70º and five minutes supine) was followed, with arterial blood pressure and heart rate monitoring. A cycle ergometer protocol was used to measure cardiorespiratory capacity. RESULTS: In the sedentary condition, men showed greater parasympathetic influence in heart rate control, as demonstrated by their higher RR interval (iRR) during the tilt test. After training, the iRR values became more similar in the two groups, although the women had higher iRR in the supine position and the men continued to present higher iRR under tilted conditions. The women's blood pressures continued to be higher after training, but heart rate tended to become similar in the two groups. The cardiorespiratory capacity patterns in the two groups were similar after training. Except for absolute heart rate values, for which there were no differences between the groups, the men's values were higher than those of the women for all other variables. It was also observed that, after the training, the women's blood pressures were significantly lower, even though their pressures remained higher than the men's. CONCLUSIONS: The training seemed to reduce the women's arterial blood pressure levels and improve both groups' cardiorespiratory capacity, but the men continued to present better performance than the women

Comparison of cardiorespiratory responses between constant and incremental load exercises below, above and at the ventilatory anaerobic threshold

OBJECTIVE: To apply different analytical methodologies to data from continuous ramp tests (CRT) and discontinuous step tests (DST), and compare responses from cardiorespiratory parameters. METHOD: Eight men performed spirometric tests on an electrically braked cycle ergometer: CRT increasing from 20 to 25 W.min-1 and DST in 15-min steps, each based on the ventilatory anaerobic threshold (VAT) for CRT. Step 1 was 70% VAT; step 2, 100% VAT; and step 3, 130% VAT. VAT was determined as loss of parallelism between O2 uptake (VO2) and CO2 output (VCO2). Heart rate (HR, bpm), VCO2, VO2 (ml.min-1), VO2 (ml.kg.min-1) and ventilation (VE L.min-1) values for CRT were obtained as moving averages of eight breath-to-breath respiratory cycles, using linear regression. For DST, means were applied from the third to fifteenth minute of the steps. Statistical comparisons utilized the Kolmogorov-Smirnov test, ANOVA, post-hoc Tukey-Kramer test and linear regression, with significance limit of p0.05), but VO2 was different between VAT and all steps (p0,05), porém VO2 relativo foi diferente (p<0,05) entre LAV e todos os degraus; a FC mostrou diferença (p<0,05) entre LAV e degrau 3, e na análise entre os três degraus houve diferença (p<0,05). CONCLUSÃO: Os resultados indicam que a regressão linear foi eficaz para estimar as variáveis cardiorrespiratórias. Em relação aos protocolos, verificou-se que para a obtenção no TDD de valores cardiorrespiratórios similares ao LAV do TCR foi necessário diminuir a potência em 30%.16316

{\phi}^4 Solitary Waves in a Parabolic Potential: Existence, Stability, and Collisional Dynamics

We explore a {\phi}^4 model with an added external parabolic potential term. This term dramatically alters the spectral properties of the system. We identify single and multiple kink solutions and examine their stability features; importantly, all of the stationary structures turn out to be unstable. We complement these with a dynamical study of the evolution of a single kink in the trap, as well as of the scattering of kink and anti-kink solutions of the model. We see that some of the key characteristics of kink-antikink collisions, such as the critical velocity and the multi-bounce windows, are sensitively dependent on the trap strength parameter, as well as the initial displacement of the kink and antikink

A Compensatory Mutation Provides Resistance to Disparate HIV Fusion Inhibitor Peptides and Enhances Membrane Fusion

Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide, are peptides designed to competitively inhibit the viral fusion protein gp41. With the emergence of drug resistance, there is an increased need for effective and unique alternatives within this class of antivirals. One such alternative is a class of cyclic, cationic, antimicrobial peptides known as θ-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the θ-defensin analog RC-101 is being developed as a microbicide due to its specific antiviral activity, lack of toxicity to cells and tissues, and safety in animals. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development. In previous studies, we identified a mutant, R5-tropic virus that had evolved partial resistance to RC-101 during in vitro selection. Here, we report that a secondary mutation in gp41 was found to restore replicative fitness, membrane fusion, and the rate of viral entry, which were compromised by an initial mutation providing partial RC-101 resistance. Interestingly, we show that RC-101 is effective against two enfuvirtide-resistant mutants, demonstrating the clinical importance of RC-101 as a unique fusion inhibitor. These findings both expand our understanding of HIV drug-resistance to diverse peptide fusion inhibitors and emphasize the significance of compensatory gp41 mutations. © 2013 Wood et al

Correction for Tarallo et al., “Altered Fecal Small RNA Profiles in Colorectal Cancer Reflect Gut Microbiome Composition in Stool Samples”

Dysbiotic configurations of the human gut microbiota have been linked to colorectal cancer (CRC). Human small noncoding RNAs are also implicated in CRC, and recent findings suggest that their release in the gut lumen contributes to shape the gut microbiota. Bacterial small RNAs (bsRNAs) may also play a role in carcinogenesis, but their role has been less extensively explored. Here, we performed small RNA and shotgun sequencing on 80 stool specimens from patients with CRC or with adenomas and from healthy subjects collected in a cross-sectional study to evaluate their combined use as a predictive tool for disease detection. We observed considerable overlap and a correlation between metagenomic and bsRNA quantitative taxonomic profiles obtained from the two approaches. We identified a combined predictive signature composed of 32 features from human and microbial small RNAs and DNA-based microbiome able to accurately classify CRC samples separately from healthy and adenoma samples (area under the curve [AUC] = 0.87). In the present study, we report evidence that host-microbiome dysbiosis in CRC can also be observed by examination of altered small RNA stool profiles. Integrated analyses of the microbiome and small RNAs in the human stool may provide insights for designing more-accurate tools for diagnostic purposes.IMPORTANCE The characteristics of microbial small RNA transcription are largely unknown, while it is of primary importance for a better identification of molecules with functional activities in the gut niche under both healthy and disease conditions. By performing combined analyses of metagenomic and small RNA sequencing (sRNA-Seq) data, we characterized both the human and microbial small RNA contents of stool samples from healthy individuals and from patients with colorectal carcinoma or adenoma. With the integrative analyses of metagenomic and sRNA-Seq data, we identified a human and microbial small RNA signature which can be used to improve diagnosis of the disease. Our analysis of human and gut microbiome small RNA expression is relevant to generation of the first hypotheses about the potential molecular interactions occurring in the gut of CRC patients, and it can be the basis for further mechanistic studies and clinical test

Altered Fecal Small RNA Profiles in Colorectal Cancer Reflect Gut Microbiome Composition in Stool Samples

Dysbiotic configurations of the human gut microbiota have been linked to colorectal cancer (CRC). Human small noncoding RNAs are also implicated in CRC, and recent findings suggest that their release in the gut lumen contributes to shape the gut microbiota. Bacterial small RNAs (bsRNAs) may also play a role in carcinogenesis, but their role has been less extensively explored. Here, we performed small RNA and shotgun sequencing on 80 stool specimens from patients with CRC or with adenomas and from healthy subjects collected in a cross-sectional study to evaluate their combined use as a predictive tool for disease detection. We observed considerable overlap and a correlation between metagenomic and bsRNA quantitative taxonomic profiles obtained from the two approaches. We identified a combined predictive signature composed of 32 features from human and microbial small RNAs and DNA-based microbiome able to accurately classify CRC samples separately from healthy and adenoma samples (area under the curve [AUC] = 0.87). In the present study, we report evidence that host-microbiome dysbiosis in CRC can also be observed by examination of altered small RNA stool profiles. Integrated analyses of the microbiome and small RNAs in the human stool may provide insights for designing more-accurate tools for diagnostic purposes.IMPORTANCE The characteristics of microbial small RNA transcription are largely unknown, while it is of primary importance for a better identification of molecules with functional activities in the gut niche under both healthy and disease conditions. By performing combined analyses of metagenomic and small RNA sequencing (sRNA-Seq) data, we characterized both the human and microbial small RNA contents of stool samples from healthy individuals and from patients with colorectal carcinoma or adenoma. With the integrative analyses of metagenomic and sRNA-Seq data, we identified a human and microbial small RNA signature which can be used to improve diagnosis of the disease. Our analysis of human and gut microbiome small RNA expression is relevant to generation of the first hypotheses about the potential molecular interactions occurring in the gut of CRC patients, and it can be the basis for further mechanistic studies and clinical test

BRain health and healthy AgeINg in retired rugby union players, the BRAIN Study: study protocol for an observational study in the UK.

INTRODUCTION: Relatively little is known about the long-term health of former elite rugby players, or former sportspeople more generally. As well as the potential benefits of being former elite sportspersons, there may be potential health risks from exposures occurring during an individual's playing career, as well as following retirement. Each contact sport has vastly different playing dynamics, therefore exposing its players to different types of potential traumas. Current evidence suggests that these are not necessarily comparable in terms of pathophysiology, and their potential long-term adverse effects might also differ. There is currently limited but increasing evidence that poorer age-related and neurological health exists among former professional sportsmen exposed to repetitive concussions; however the evidence is limited on rugby union players, specifically. METHODS AND ANALYSIS: We present the protocol for a cross-sectional study to assess the association between self-reported history of concussion during a playing career, and subsequent measures of healthy ageing and neurological and cognitive impairment. We are recruiting a sample of approximately 200 retired rugby players (former Oxford and Cambridge University rugby players and members of the England Rugby International Club) aged 50 years or more, and collecting a number of general and neurological health-related outcome measures though validated assessments. Biomarkers of neurodegeneration (neurofilaments and tau) will be also be measured. Although the study is focusing on rugby union players specifically, the general study design and the methods for assessing neurological health are likely to be relevant to other studies of former elite sportspersons. ETHICS AND DISSEMINATION: The study has been approved by the Ethical Committee of London School of Hygiene and Tropical Medicine (reference: 11634-2). It is intended that results of this study will be published in peer-reviewed medical journals, communicated to participants, the general public and all relevant stakeholders

Formation of regulatory modules by local sequence duplication

Turnover of regulatory sequence and function is an important part of molecular evolution. But what are the modes of sequence evolution leading to rapid formation and loss of regulatory sites? Here, we show that a large fraction of neighboring transcription factor binding sites in the fly genome have formed from a common sequence origin by local duplications. This mode of evolution is found to produce regulatory information: duplications can seed new sites in the neighborhood of existing sites. Duplicate seeds evolve subsequently by point mutations, often towards binding a different factor than their ancestral neighbor sites. These results are based on a statistical analysis of 346 cis-regulatory modules in the Drosophila melanogaster genome, and a comparison set of intergenic regulatory sequence in Saccharomyces cerevisiae. In fly regulatory modules, pairs of binding sites show significantly enhanced sequence similarity up to distances of about 50 bp. We analyze these data in terms of an evolutionary model with two distinct modes of site formation: (i) evolution from independent sequence origin and (ii) divergent evolution following duplication of a common ancestor sequence. Our results suggest that pervasive formation of binding sites by local sequence duplications distinguishes the complex regulatory architecture of higher eukaryotes from the simpler architecture of unicellular organisms