Quasi-cellular Systems: Stochastic Simulation Analysis at Nanoscale Range

I complessi sistemi di reazioni biochimiche all’interno della cellula sono altamente compartimentalizzati, conseguenza di un importante fenomeno di macromolecolar crowding (sovraffollamento molecolare). E’ dunque importante determinare il comportamento e le proprietà di un sistema di reazioni in piccoli volumi. Sono stati riprodotti con successo diversi sistemi di semplici reazioni all’interno di vescicole lipidiche (liposomi) nell’ordine del micro/nanometro di diametro, osservando in molti casi una risposta cinetica diversa dalle reazioni in esame rispetto al comportamento in sistemi di grandi volumi. Questo fenomeno di divergenza tra piccoli e grandi volumi è in gran parte dipendente da fenomeni non completamente chiariti, quali l’incapsulamento delle specie e il crowding molecolare, aspetti sempre più importanti man mano che l’attenzione si sposta verso i piccoli volumi. Recenti dati sperimentali dimostrano che il fenomeno dell'intrappolamento sembra non seguire un andamento casuale squisitamente probabilistico, ma un comportamento di tipo power-law (a legge di potenza), in cui solo pochissime vescicole intrappolano tante specie, mentre la maggior parte resta completamente vuota. A tal proposito è stato intrapreso uno studio sui meccanismi generativi delle distribuzioni a legge di potenza calate nel contesto dell’incapsulamento (entrapment) delle specie all'interno di vescicole lipidiche. Utilizzando un sistema cell-free di trascrizione/traduzione (PURESYSTEM™), volto alla produzione di EGFP all’interno di liposomi di POPC, è possibile monitorare la produzione di proteina fluorescente in liposomi di differente grandezza. Tuttavia, è molto difficile osservare la produzione di molecole fluorescenti in singole vescicole di 100 nm di diametro; diventa così importante poter studiare in silico la di produzione di proteina in singole vescicole virtuali, utilizzando un modello formalmente valido del complesso sistema di reazioni del PURESYSTEM™. QDC (Quick Direct-Method Controlled), è un software di simulazione stocastico precedentemente sviluppato in laboratorio, basato sull’algoritmo di simulazione SSA Direct-Method di Gillespie, tra i più usati in biologia computazionale/systems biology. L’argomento della tesi riguarda l’uso di questo software nello studio delle oltre 100 reazioni biochimiche del PURESYSTEM™, comparando i risultati ottenuti in diverse condizioni (volume totale di reazione, concentrazioni delle specie, costanti cinetiche delle singole reazioni). Dopo aver affinato il modello in silico di Trascrizione/traduzione coupled (accoppiato), sono state effettuate delle simulazioni variando alcune variabili macroscopiche (concentrazioni delle specie e costanti cinetiche), mostrando un'importante dipendenza della traduzione dalla trascrizione, soprattutto considerando il grande limite energetico di un sistema che non produce al suo interno nucleotidi trifosfato

Reaction Prediction: The Case of Tweets from Luxury Fashion Brands

Social media platforms represent an essential tool for both consumers and marketers. Meanwhile, luxury fashion brands play a key role in fashion, one of the most important industries of the world economy. Despite assumptions to the contrary, social media platforms and luxury fashion brands do mix, especially in the recent time. Consequently, it is worth asking whether it is possible to predict the reaction a post will generate in the audience of luxury fashion brands. This new question is the one this thesis intends to answer. To do so, the concept of reaction is defined through a novel composite index that is created and named Tweet reaction overall score (TROS), which is one of the solid and relevant contributions this thesis makes. Then, several predictive models are implemented, based on a wide range of different learning algorithms. The results show that it is indeed possible to predict the TROS that a post on Twitter will obtain in the audience of luxury fashion brands the day it is posted

Clinical Applications of Neuromonitoring Following Acute Brain Injury

Various invasive and non-invasive cranial monitoring techniques can be applied clinically to describe the extent to which cerebral hemodynamics and subsequently, patient outcome, have been impacted following acute brain injury (ABI). This Ph.D. thesis examines both prospective and retrospective patient data in both neurocritical and general intensive care patients. Thirty neurotrauma patients and forty general intensive care patients with neurological complications were prospectively monitored after ABI. Retrospective patient data was harvested from a database of 1,023 traumatic brain injury (TBI) patients with invasive intracranial pressure (ICP), arterial blood pressure (ABP), and transcranial Doppler ultrasonography (TCD) recordings. Data analysis focused on ICP microsensor accuracy, compensatory reserve, the pulsatility of brain signals (ICP and TCD), and cerebral arterial blood volume (CaBV) based on TCD. The main results are summarized below: I. Intracranial hypertension has a profound negative influence on cerebrovascular parameters and patient outcome. II. ICP microsensor accuracy is limited, with an average error of approximately ± 6.0 mm Hg. III. ICP weighted with the compensatory reserve better predicts outcome than mean ICP alone. IV. ICP and TCD pulsatility are functions of mean ICP and cerebral perfusion pressure (CPP). V. Continuous blood flow forward (CFF) and pulsatile blood flow forward (PFF) models can approximate CaBV with derived TCD signals; CFF best models TCD pulsatility. VI. The pressure reactivity index (PRx) and the pulse amplitude index (PAx) can be estimated non-invasively using slow waves of TCD estimated by CaBV with similar outcome-predictive power. VII. Multi-parametric TCD-based monitoring of general intensive care patients is clinically feasible; the joint estimation of autoregulation, dysautonomia, non-invasive ICP, and critical closing pressure is possible. The culmination of these projects should have an impact on current monitoring practices in ABI patients, emphasizing the continued validation and refinement of TCD methodology in clinical neurosciences

Ribo-seQC: comprehensive analysis of cytoplasmic and organellar ribosome profiling data

Summary: Ribosome profiling enables genome-wide analysis of translation with unprecedented resolution. We present Ribo-seQC, a versatile tool for the comprehensive analysis of Ribo-seq data, providing in-depth insights on data quality and translational profiles for cytoplasmic and organelle ribosomes. Ribo-seQC automatically generates platform-independent HTML reports, offering a detailed and easy-to-share basis for collaborative Ribo-seq projects. Availability: Ribo-seQC is available at https://github.com/ohlerlab/RiboseQC and submitted to Bioconductor. Contact: uwe.ohler{at}mdc-berlin.d

Quasi-cellular systems: Stochastic simulation analysis at nanoscale range

Background: The wet-lab synthesis of the simplest forms of life (minimal cells) is a challenging aspect in modern synthetic biology. Quasi-cellular systems able to produce proteins directly from DNA can be obtained by encapsulating the cell-free transcription/translation system PURESYSTEM™(PS) in liposomes. It is possible to detect the intra-vesicle protein production using DNA encoding for GFP and monitoring the fluorescence emission over time. The entrapment of solutes in small-volume liposomes is a fundamental open problem. Stochastic simulation is a valuable tool in the study of biochemical reaction at nanoscale range. QDC (Quick Direct-Method Controlled), a stochastic simulation software based on the well-known Gillespie's SSA algorithm, was used. A suitable model formally describing the PS reactions network was developed, to predict, from inner species concentrations (very difficult to measure in small-volumes), the resulting fluorescence signal (experimentally observable).Results: Thanks to suitable features specific of QDC, we successfully formalized the dynamical coupling between the transcription and translation processes that occurs in the real PS, thus bypassing the concurrent-only environment of Gillespie's algorithm. Simulations were firstly performed for large liposomes (2.67μm of diameter) entrapping the PS to synthetize GFP. By varying the initial concentrations of the three main classes of molecules involved in the PS (DNA, enzymes, consumables), we were able to stochastically simulate the time-course of GFP-production. The sigmoid fit of the GFP-production curves allowed us to extract three quantitative parameters which are significantly dependent on the various initial states. Then we extended this study for small-volume liposomes (575 nm of diameter), where it is more complex to infer the intra-vesicle composition, due to the expected anomalous entrapment phenomena. We identified almost two extreme states that are forecasted to give rise to significantly different experimental observables.Conclusions: The present work is the first one describing in the detail the stochastic behavior of the PS. Thanks to our results, an experimental approach is now possible, aimed at recording the GFP production kinetics in very small micro-emulsion droplets or liposomes, and inferring, by using the simulation as a reverse-engineering procedure, the internal solutes distribution, and shed light on the still unknown forces driving the entrapment phenomenon. © 2013 Calviello et al.; licensee BioMed Central Ltd

Evaluation of reactivity of horses in the presence of unknown stimulus

The study aimed to evaluate reactivity of horses during usual brushing management against the repeated presence of an unknown sonorous stimulus. Twenty Mangalarga Marchador horses, distributed in different categories (mares and foals), were evaluated. The animals were allocated into the control treatment (N = 10) and the treatment with unknown sonorous stimulus (N = 10) from a rattle and a tambourine. Four consecutive evaluations were carried out first (day 0, 1, 2, 3). Two consecutive assessments were carried out after 30 days of the first collection (day 30 and 31), and two consecutive assessments were carried out 15 days after the second evaluation (day 45 and 46). The behavioral observations were made by assigning a score to behaviors of movement, position of ears and eyes, breathing, and vocalization during brushing management. A response variable called reactivity was attributed to each animal, ranging from score 1 (not reactive or calm animal) to reactivity score 4 (very reactive or aggressive animal). For statistical analysis, the results were adjusted to a logistic regression model using the categories, day, and treatment as covariates. The animals of the unknown stimuli showed greater reactivity. The days of the experimental period influenced the reactivity of animals between 6 and 7 months old, with a decrease in the possibilities of the animals to have a higher reactivity. The maturity of the foal with repeated exposure to the unknown sound stimulus may decrease the possibility of the animal being reactive

Int J Tuberc Lung Dis

BackgroundTherapeutic effects of antiretroviral therapy (ART) in patients with multidrug resistant tuberculosis (MDR-TB) and HIV infection have not been established.ObjectiveThe objective of this study was to assess therapeutic outcomes of ART integration with MDR-TB treatment.DesignA subgroup of MDR-TB patients from the SAPiT study, a randomized controlled trial, conducted in an out-patient clinic in Durban, South Africa from 2008\u20132012MethodsClinical outcomes at 18 months were compared in patients randomized to receive ART within 12 weeks of standard first-line tuberculosis treatment initiation with those who commenced ART after completing tuberculosis treatment.ResultsMycobacterium tuberculosis drug susceptibility was available in 489 (76%) of 642 SAPiT patients; 23 had MDR-TB, 14 in the integrated treatment arm and 9 in the sequential treatment arm. At 18 months, the mortality rate was 11.9/100 person-years (95% confidence interval (CI): 1.4\u201342.8) in the combined integrated treatment arm and 56.0/100 person-years (95%CI: 18.2\u2013130.8) in the sequential treatment arm, (Hazard Ratio adjusted for baseline CD4 count and whether MDR-TB treatment was initiated: 0.14; 95% CI: 0.02\u20130.94; P=0.04).ConclusionDespite the small sample size, the 86% reduction in mortality due to early initiation of ART in MDR-TB patients was statistically significant.D43TW00231/TW/FIC NIH HHS/United StatesD43 TW000231/TW/FIC NIH HHS/United States5U26PS001350/PS/NCHHSTP CDC HHS/United StatesU19 AI051794/AI/NIAID NIH HHS/United StatesU2G PS001350/PS/NCHHSTP CDC HHS/United StatesPEPFAR/United States2016-02-29T00:00:00Z24429305PMC47700138583vault:1580

n–3 PUFA dietary supplementation inhibits proliferation and store-operated calcium influx in thymoma cells growing in Balb/c mice

The antitumor effect of daily individual administration of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (2 g/kg body weight) in Balb/c mice bearing a transplantable thymoma was investigated. Mice received oleic acid (control group), EPA and DHA ethyl esters starting 10 days before tumor inoculation. Analysis of phospholipid composition of neoplastic cell revealed that EPA and DHA levels were significantly increased (63 and 22% increase) after EPA and DHA treatments, respectively. Conversely, decreased levels of arachidonic acid were found in both cases (19 and 24% decrease in EPA and DHA groups, respectively). EPA and DHA delayed the appearance of macroscopic ascites (100% of animal, from 7 to 28 days), prolonged animal survival (100% of animal, from 22 to 32 and 33 days, respectively) and reduced the percentage of proliferating tumor cells detected by immunostaining of proliferation cell nuclear antigen (PCNA) (80 and 85% decrease, respectively). Moreover, the regulatory effects of these dietary n–3 fatty acids on the influx of Ca2+, activated by depletion of intracellular stores with thapsigargin (Tg), were investigated. By using a Ca2+-free/Ca2+-reintroduction protocol and Fura-2 as fluorescent indicator of intracellular free Ca2+([Ca2+]i), we observed that EPA and DHA treatments markedly decreased Tg-induced rise in [Ca2+]i (49 and 37% decrease, respectively). This effect was related to the inhibition of the store-operated Ca2+ influx, as confirmed also by Mn2+ influx experiments. The inhibitory action of EPA and DHA on the store-operated Ca2+ influx could explain, at least in part, their antitumoral activity, as this Ca2+ mobilization pathway appears to be involved in the cell signaling occurring in non-excitable cells to evoke many cellular processes, including cell proliferation. —Calviello, G., P. Palozza, F. Di Nicuolo, N. Maggiano, and G. M. Bartoli. N–3 PUFA dietary supplementation inhibits proliferation and store-operated calcium influx in thymoma cells growing in Balb/c mice