New synchronization method for <i>Plasmodium falciparum</i>

&lt;b&gt;Background&lt;/b&gt;: Plasmodium falciparum is usually asynchronous during in vitro culture. Although various synchronization methods are available, they are not able to narrow the range of ages of parasites. A newly developed method is described that allows synchronization of parasites to produce cultures with an age range as low as 30 minutes. &lt;b&gt;Methods&lt;/b&gt;: Trophozoites and schizonts are enriched using Plasmion. The enriched late stage parasites are immobilized as a monolayer onto plastic Petri dishes using concanavalin A. Uninfected erythrocytes are placed onto the monolayer for a limited time period, during which time schizonts on the monolayer rupture and the released merozoites invade the fresh erythrocytes. The overlay is then taken off into a culture flask, resulting in a highly synchronized population of parasites. &lt;b&gt;Results&lt;/b&gt;: Plasmion treatment results in a 10- to 13-fold enrichment of late stage parasites. The monolayer method results in highly synchronized cultures of parasites where invasion has occurred within a very limited time window, which can be as low as 30 minutes. The method is simple, requiring no specialized equipment and relatively cheap reagents. &lt;b&gt;Conclusions&lt;/b&gt;: The new method for parasite synchronization results in highly synchronized populations of parasites, which will be useful for studies of the parasite asexual cell cycle

Gut microbial activity as influenced by fiber digestion: dynamic metabolomics in an in vitro colon simulator

Understanding the interaction between the gut microbial activity and the host is essential, and in vitro models are being used to test and develop hypotheses regarding the impact of food components/drugs on the human gut ecosystem. However, while in vitro models provide excellent possibilities for dynamic investigations, studies have commonly been restricted to analyses of few, targeted metabolites. In the present study, we employed NMR-based metabolomics combined with multilevel data analysis as a tool to characterize the impact of polydextrose (PDX) fiber on the in vitro derived fecal metabolome. This approach enabled us to identify and quantify the fiber-induced response on several fecal metabolites; we observed higher levels of butyrate, acetate, propionate, succinate, N-acetyl compound and a lower level of amino acids (leucine, valine, isoleucine, phenylalanine, and lysine), valerate, formate, isovalerate and trimethylamine among the PDX-treated sample compared to the control samples. In addition, by the application of multilevel data analysis we were able to examine the specific inter-individual variations, and caprylic acid was identified to be the main marker of distinct microbial compositions among the subjects. Our work is expected to provide a useful approach to understand the metabolic impact of potential prebiotic compounds and get deeper insight into the molecular regulation of gut-microbe activities in the complex gut system

Inexperienced clinicians can extract pathoanatomic information from MRI narrative reports with high reproducibility for use in research/quality assurance

<p>Abstract</p> <p>Background</p> <p>Although reproducibility in reading MRI images amongst radiologists and clinicians has been studied previously, no studies have examined the reproducibility of inexperienced clinicians in extracting pathoanatomic information from magnetic resonance imaging (MRI) narrative reports and transforming that information into quantitative data. However, this process is frequently required in research and quality assurance contexts. The purpose of this study was to examine inter-rater reproducibility (agreement and reliability) among an inexperienced group of clinicians in extracting spinal pathoanatomic information from radiologist-generated MRI narrative reports.</p> <p>Methods</p> <p>Twenty MRI narrative reports were randomly extracted from an institutional database. A group of three physiotherapy students independently reviewed the reports and coded the presence of 14 common pathoanatomic findings using a categorical electronic coding matrix. Decision rules were developed after initial coding in an effort to resolve ambiguities in narrative reports. This process was repeated a further three times using separate samples of 20 MRI reports until no further ambiguities were identified (total n = 80). Reproducibility between trainee clinicians and two highly trained raters was examined in an arbitrary coding round, with agreement measured using percentage agreement and reliability measured using unweighted Kappa (<it>k</it>). Reproducibility was then examined in another group of three trainee clinicians who had not participated in the production of the decision rules, using another sample of 20 MRI reports.</p> <p>Results</p> <p>The mean percentage agreement for paired comparisons between the initial trainee clinicians improved over the four coding rounds (97.9-99.4%), although the greatest improvement was observed after the first introduction of coding rules. High inter-rater reproducibility was observed between trainee clinicians across 14 pathoanatomic categories over the four coding rounds (agreement range: 80.8-100%; reliability range <it>k </it>= 0.63-1.00). Concurrent validity was high in paired comparisons between trainee clinicians and highly trained raters (agreement 97.8-98.1%, reliability <it>k </it>= 0.83-0.91). Reproducibility was also high in the second sample of trainee clinicians (inter-rater agreement 96.7-100.0% and reliability <it>k </it>= 0.76-1.00; intra-rater agreement 94.3-100.0% and reliability <it>k </it>= 0.61-1.00).</p> <p>Conclusions</p> <p>A high level of radiological training is not required in order to transform MRI-derived pathoanatomic information from a narrative format to a quantitative format with high reproducibility for research or quality assurance purposes.</p

Experience with onabotulinumtoxinA (BOTOX) in chronic refractory migraine: focus on severe attacks

The objective of this study is to analyse our experience in the treatment of refractory chronic migraine (CM) with onabotulinumtoxinA (BTA) and specifically in its effects over disabling attacks. Patients with CM and inadequate response or intolerance to oral preventatives were treated with pericranial injections of 100 U of TBA every 3 months. The dose was increased up to 200 U in case of no response. The patients kept a headache diary. In addition, we specifically asked on the effect of BTA on the frequency of disabling attacks, consumption of triptans and visits to Emergency for the treatment of severe attacks. This series comprises a total of 35 patients (3 males), aged 24–68 years. All except three met IHS criteria for analgesic overuse. The number of sessions with BTA ranged from 2 to 15 (median 4) and nine (26%) responded (reduction of >50% in headache days). However, the frequency of severe attacks was reduced to an average of 46%. Oral triptan consumption (29 patients) was reduced by 50% (from an average of 22 to 11 tablets/month). Those six patients who used subcutaneous sumatriptan reduced its consumption to a mean of 69% (from 4.5 to 1.5 injections per month). Emergency visits went from an average of 3 to 0.4 per trimester (−83%). Six patients complained of mild adverse events, transient local cervical pain being the most common. Although our data must be taken with caution as this is an open trial, in clinical practice treatment of refractory CM with BTA reduces the frequency of disabling attacks, the consumption of triptans and the need of visits to Emergency, which makes this treatment a profitable option both clinically and pharmacoeconomically

Smallest detectable change in volume differs between mass flow sensor and pneumotachograph

<p>Abstract</p> <p>Background</p> <p>To assess a pulmonary function change over time the mass flow sensor and the pneumotachograph are widely used in commercially available instruments. However, the smallest detectable change for both devices has never been compared. Therefore, the aim of this study is to determine the smallest detectable change in vital capacity (VC) and single-breath diffusion parameters measured by mass flow sensor and or pneumotachograph.</p> <p>Method</p> <p>In 28 healthy pulmonary function technicians VC, transfer factor for carbon monoxide (DLCO) and alveolar volume (VA) was repeatedly (10×) measured. The smallest detectable change was calculated by 1.96 x Standard Error of Measurement ×√2.</p> <p>Findings</p> <p>The mean (range) of the smallest detectable change measured by mass flow sensor and pneumotachograph respectively, were for VC (in Liter): 0.53 (0.46-0.65); 0.25 (0.17-0.36) (<it>p </it>= 0.04), DLCO (in mmol*kPa^-1*min^-1): 1.53 (1.26-1.7); 1.18 (0.84-1.39) (<it>p </it>= 0.07), VA (in Liter): 0.66. (0.53-0.82); 0.43 (0.34-0.53) (<it>p </it>= 0.04) and DLCO/VA (in mmol*kPa^-1*min^-1*L^-1): 0.22 (0.19-0.28); 0.19 (0.14-0.22) (<it>p </it>= 0.79).</p> <p>Conclusions</p> <p>Smallest detectable significant change in VC and VA as measured by pneumotachograph are smaller than by mass flow sensor. Therefore, the pneumotachograph is the preferred instrument to estimate lung volume change over time in individual patients.</p

Bias in MRI Measurements of Apparent Diffusion Coefficient and Kurtosis: Implications for Choice of Maximum Diffusion Encoding

Tissue water diffusion is non-Gaussian and the expressions used to calculate diffusion parameters are approximations which introduce systematic errors dependent on the maximum diffusion encoding, diffusion time, etc. This study aimed at characterizing biases in estimates of both apparent diffusion coefficient and kurtosis, and determines their dependence on these parameters. Similar to the approach of several previous studies, Taylor expansion of the diffusion signal was used to calculate biases. Predicted errors were compared with data from one volunteer. Predicted errors agreed well with the measured errors and also the published diffusion tensor imaging measurements. The equations derived predict biases in measured diffusion parameters and explain much of the discrepancy between measurements obtained with different acquisition protocols. The equations may also be used to choose appropriate diffusion encoding for diffusion weighted, tensor, and kurtosis imaging

Genome-Wide Characterization of Menin-Dependent H3K4me3 Reveals a Specific Role for Menin in the Regulation of Genes Implicated in MEN1-Like Tumors

Inactivating mutations in the MEN1 gene predisposing to the multiple endocrine neoplasia type 1 (MEN1) syndrome can also cause sporadic pancreatic endocrine tumors. MEN1 encodes menin, a subunit of MLL1/MLL2-containing histone methyltransferase complexes that trimethylate histone H3 at lysine 4 (H3K4me3). The importance of menin-dependent H3K4me3 in normal and transformed pancreatic endocrine cells is unclear. To study the role of menin-dependent H3K4me3, we performed in vitro differentiation of wild-type as well as menin-null mouse embryonic stem cells (mESCs) into pancreatic islet-like endocrine cells (PILECs). Gene expression analysis and genome-wide H3K4me3 ChIP-Seq profiling in wild-type and menin-null mESCs and PILECs revealed menin-dependent H3K4me3 at the imprinted Dlk1-Meg3 locus in mESCs, and all four Hox loci in differentiated PILECs. Specific and significant loss of H3K4me3 and gene expression was observed for genes within the imprinted Dlk1-Meg3 locus in menin-null mESCs and the Hox loci in menin-null PILECs. Given that the reduced expression of genes within the DLK1-MEG3 locus and the HOX loci is associated with MEN1-like sporadic tumors, our data suggests a possible role for menin-dependent H3K4me3 at these genes in the initiation and progression of sporadic pancreatic endocrine tumors. Furthermore, our investigation also demonstrates that menin-null mESCs can be differentiated in vitro into islet-like endocrine cells, underscoring the utility of menin-null mESC-derived specialized cell types for genome-wide high-throughput studies

The Transcription Factor PU.1 Regulates γδ T Cell Homeostasis

T cell development results in the generation of both mature αβ and γδ T cells. While αβ T cells predominate in secondary lymphoid organs, γδ T cells are more abundant in mucosal tissues. PU.1, an Ets family transcription factor, also identified as the spleen focus forming virus proviral integration site-1 (Sfpi1) is essential for early stages of T cell development, but is down regulated during the DN T-cell stage.In this study, we show that in mice specifically lacking PU.1 in T cells using an lck-Cre transgene with a conditional Sfpi1 allele (Sfpi1(lck-/-)) there are increased numbers of γδ T cells in spleen, thymus and in the intestine when compared to wild-type mice. The increase in γδ T cell numbers in PU.1-deficient mice is consistent in γδ T cell subsets identified by TCR variable regions. PU.1-deficient γδ T cells demonstrate greater proliferation in vivo and in vitro.The increase of γδ T cell numbers in Lck-Cre deleter strains, where deletion occurs after PU.1 expression is diminished, as well as the observation that PU.1-deficient γδ T cells have greater proliferative responses than wild type cells, suggests that PU.1 effects are not developmental but rather at the level of homeostasis. Thus, our data shows that PU.1 has a negative influence on γδ T cell expansion

The translation, validity and reliability of the German version of the Fremantle Back Awareness Questionnaire

Background: The Fremantle Back Awareness Questionnaire (FreBAQ) claims to assess disrupted self-perception of the back. The aim of this study was to develop a German version of the Fre-BAQ (FreBAQ-G) and assess its test-retest reliability, its known-groups validity and its convergent validity with another purported measure of back perception. Methods: The FreBaQ-G was translated following international guidelines for the transcultural adaptation of questionnaires. Thirty-five patients with non-specific CLBP and 48 healthy participants were recruited. Assessor one administered the FreBAQ-G to each patient with CLBP on two separate days to quantify intra-observer reliability. Assessor two administered the FreBaQ-G to each patient on day 1. The scores were compared to those obtained by assessor one on day 1 to assess inter-observer reliability. Known-groups validity was quantified by comparing the FreBAQ-G score between patients and healthy controls. To assess convergent validity, patient\u27s FreBAQ-G scores were correlated to their two-point discrimination (TPD) scores. Results: Intra- and Inter-observer reliability were both moderate with ICC3.1 = 0.88 (95%CI: 0.77 to 0.94) and 0.89 (95%CI: 0.79 to 0.94), respectively. Intra- and inter-observer limits of agreement (LoA) were 6.2 (95%CI: 5.0±8.1) and 6.0 (4.8±7.8), respectively. The adjusted mean difference between patients and controls was 5.4 (95%CI: 3.0 to 7.8, p\u3c0.01). Patient\u27s FreBAQ-G scores were not associated with TPD thresholds (Pearson\u27s r = -0.05, p = 0.79). Conclusions: The FreBAQ-G demonstrated a degree of reliability and known-groups validity. Interpretation of patient level data should be performed with caution because the LoA were substantial. It did not demonstrate convergent validity against TPD. Floor effects of some items of the FreBAQ-G may have influenced the validity and reliability results. The clinimetric properties of the FreBAQ-G require further investigation as a simple measure of disrupted self-perception of the back before firm recommendations on its use can be made

Extracellular Matrix Aggregates from Differentiating Embryoid Bodies as a Scaffold to Support ESC Proliferation and Differentiation

Embryonic stem cells (ESCs) have emerged as potential cell sources for tissue engineering and regeneration owing to its virtually unlimited replicative capacity and the potential to differentiate into a variety of cell types. Current differentiation strategies primarily involve various growth factor/inducer/repressor concoctions with less emphasis on the substrate. Developing biomaterials to promote stem cell proliferation and differentiation could aid in the realization of this goal. Extracellular matrix (ECM) components are important physiological regulators, and can provide cues to direct ESC expansion and differentiation. ECM undergoes constant remodeling with surrounding cells to accommodate specific developmental event. In this study, using ESC derived aggregates called embryoid bodies (EB) as a model, we characterized the biological nature of ECM in EB after exposure to different treatments: spontaneously differentiated and retinoic acid treated (denoted as SPT and RA, respectively). Next, we extracted this treatment-specific ECM by detergent decellularization methods (Triton X-100, DOC and SDS are compared). The resulting EB ECM scaffolds were seeded with undifferentiated ESCs using a novel cell seeding strategy, and the behavior of ESCs was studied. Our results showed that the optimized protocol efficiently removes cells while retaining crucial ECM and biochemical components. Decellularized ECM from SPT EB gave rise to a more favorable microenvironment for promoting ESC attachment, proliferation, and early differentiation, compared to native EB and decellularized ECM from RA EB. These findings suggest that various treatment conditions allow the formulation of unique ESC-ECM derived scaffolds to enhance ESC bioactivities, including proliferation and differentiation for tissue regeneration applications. © 2013 Goh et al