Development and validation of an alternative parameter for quantification of signals emitted by fluorescently labelled bacteria in microscopic images

In this study, an alternative parameter for quantifying the signals of fluorescently labelled bacteria (e.g. propidium iodide, Cyanine 3, etc.) in microscopic images was investigated. Three common parameters (mean grey value (MGV), mean grey value which is corrected for the background (MGVcwB) and the signal to background ratio (SBR) per bacterial cell) are used as reference parameters. As an alternative, the coefficient of variation (CV) is defined as the ratio of the logarithm of the standard deviation and the logarithm of the mean grey value of a bacterial cell in a microscopic image. The actual fluorescence value was safeguarded by measuring commercially available fluorescence latex microspheres at regular time intervals within our study. The precision and the correlation of the respective values of MGV, MGVcwB, SBR and CV taken from identical images were measured and subsequently normalized in order to enhance the inter-parameter comparability. The average precision of CV was the highest (89% +/- 14) with decreasing numbers for MGVcwB, SBR, and MGV (78% +/- 25, 71% +/- 32, and, 52% +/- 22, respectively). Changes in operational parameters, e.g., microscope settings, protocol steps, etc., yielded good results for the CV but less precise results for MGV, MGVcwB, and SBR in the analyses of identical images. In conclusion, using the alternative parameter CV, changes in the composition of microbial ecosystems may thus be investigated at the highest precision level

Is methotrexate safe for men with an immune-mediated inflammatory disease and an active desire to become a father? Results of a prospective cohort study (iFAME-MTX)

Introduction Current scientific evidence guiding the decision whether men with an active desire to become a father should be treated with methotrexate (MTX) remains controversial. We aimed to prospectively evaluate the testicular toxicity profile of MTX focusing on several markers of male fertility, including semen parameters and sperm DNA fragmentation index (sDFI). As a secondary outcome, we aimed to evaluate whether MTX-polyglutamates can be detected in spermatozoa and seminal plasma and to evaluate the enzymatic activity in spermatozoa of folylpolyglutamate synthetase (FPGS). Methods In a prospective cohort study, men ≥18 years who started therapy with MTX were invited to participate (MTX-starters). Participants were instructed to produce two semen samples (a pre-exposure and a post-exposure sample after 13 weeks). Healthy men ≥18 years were invited to participate as controls. Conventional semen analyses, male reproductive endocrine axis and sDFI were compared between groups. FPGS enzymatic activity and MTX-PG1-5 concentrations were determined by mass spectrometry analytical methods. Results In total, 20 MTX-starters and 25 controls were included. The pre-exposure and postexposure semen parameters of MTX-starters were not statistically significant different. Compared with healthy controls, the conventional semen parameters and the sDFI of MTX-starters were not statistically significant different. These data were corroborated by the marginal accumulation of MTX-PGs in spermatozoa, consistent with the very low FPGS enzymatic activity associated with the expression of an alternative FPGS splice-variant. Discussion Treatment with MTX is not associated with testicular toxicity, consistent with the very low concentration of intracellular MTX-PG. Therefore, therapy with MTX can be safely started or continued in men and with a wish to become a father.</p

Genotype calling in tetraploid species from bi-allelic marker data using mixture models

<p>Abstract</p> <p>Background</p> <p>Automated genotype calling in tetraploid species was until recently not possible, which hampered genetic analysis. Modern genotyping assays often produce two signals, one for each allele of a bi-allelic marker. While ample software is available to obtain genotypes (homozygous for either allele, or heterozygous) for diploid species from these signals, such software is not available for tetraploid species which may be scored as five alternative genotypes (aaaa, baaa, bbaa, bbba and bbbb; nulliplex to quadruplex).</p> <p>Results</p> <p>We present a novel algorithm, implemented in the R package fitTetra, to assign genotypes for bi-allelic markers to tetraploid samples from genotyping assays that produce intensity signals for both alleles. The algorithm is based on the fitting of several mixture models with five components, one for each of the five possible genotypes. The models have different numbers of parameters specifying the relation between the five component means, and some of them impose a constraint on the mixing proportions to conform to Hardy-Weinberg equilibrium (HWE) ratios. The software rejects markers that do not allow a reliable genotyping for the majority of the samples, and it assigns a missing score to samples that cannot be scored into one of the five possible genotypes with sufficient confidence.</p> <p>Conclusions</p> <p>We have validated the software with data of a collection of 224 potato varieties assayed with an Illumina GoldenGate™ 384 SNP array and shown that all SNPs with informative ratio distributions are fitted. Almost all fitted models appear to be correct based on visual inspection and comparison with diploid samples. When the collection of potato varieties is analyzed as if it were a population, almost all markers seem to be in Hardy-Weinberg equilibrium. The R package fitTetra is freely available under the GNU Public License from <url>http://www.plantbreeding.wur.nl/UK/software_fitTetra.html</url> and as Additional files with this article.</p

Lactic acid production from lime-treated wheat straw by Bacillus coagulans: neutralization of acid by fed-batch addition of alkaline substrate

Conventional processes for lignocellulose-to-organic acid conversion requires pretreatment, enzymatic hydrolysis, and microbial fermentation. In this study, lime-treated wheat straw was hydrolyzed and fermented simultaneously to lactic acid by an enzyme preparation and Bacillus coagulans DSM 2314. Decrease in pH because of lactic acid formation was partially adjusted by automatic addition of the alkaline substrate. After 55 h of incubation, the polymeric glucan, xylan, and arabinan present in the lime-treated straw were hydrolyzed for 55%, 75%, and 80%, respectively. Lactic acid (40.7 g/l) indicated a fermentation efficiency of 81% and a chiral l(+)-lactic acid purity of 97.2%. In total, 711 g lactic acid was produced out of 2,706 g lime-treated straw, representing 43% of the overall theoretical maximum yield. Approximately half of the lactic acid produced was neutralized by fed-batch feeding of lime-treated straw, whereas the remaining half was neutralized during the batch phase with a Ca(OH)2 suspension. Of the lime added during the pretreatment of straw, 61% was used for the neutralization of lactic acid. This is the first demonstration of a process having a combined alkaline pretreatment of lignocellulosic biomass and pH control in fermentation resulting in a significant saving of lime consumption and avoiding the necessity to recycle lime

Spin Caloritronics

This is a brief overview of the state of the art of spin caloritronics, the science and technology of controlling heat currents by the electron spin degree of freedom (and vice versa).Comment: To be published in "Spin Current", edited by S. Maekawa, E. Saitoh, S. Valenzuela and Y. Kimura, Oxford University Pres

Standardizing Slimness: How Body Weight Quantified Beauty in the Netherlands, 1870–1940

This chapter investigates the history of one of the most powerful quantitative beauty standards: weight. The chapter argues that weight is neither a natural nor a neutral standard for the beauty ideals of slimness and fatness. It is shown first how, in late nineteenth-century Netherlands, weight had not yet become a standard of beauty but was rather a bodily curiosity, measured at fairgrounds. The chapter then analyses Dutch newspaper advertisements for slimming remedies to show that, by the 1930s, weight was strongly established as a standard of beauty, scales having ceased to be a fairground attraction. The chapter concludes with an exploration of the consequences of this new standard of beauty, which complicated its character by partially separating it from the visual

High susceptibility of c-KIT+CD34+ precursors to prolonged doxorubicin exposure interferes with Langerhans cell differentiation in a human cell line model

As neoadjuvant and adjuvant chemotherapy schedules often consist of multiple treatment cycles over relatively long periods of time, it is important to know what effects protracted drug administration can have on the immune system. Here, we studied the long-term effects of doxorubicin on the capacity of dendritic cell (DC) precursors to differentiate into a particular DC subset, the Langerhans cells (LC). In order to achieve high telomerase activity as detected in hematological stem cells, precursor cells from the acute-myeloid leukemia (AML)-derived cell line MUTZ3 were stably transduced with human telomerase reverse transcriptase (hTERT) to facilitate their growth potential, while preventing growth, and drug-induced senescence, and preserving their unique capacity for cytokine-dependent DC and LC differentiation. The hTERT-MUTZ3 cells were selected with increasing concentrations of the anthracyclin doxorubicin. After 1–2 months of selection with 30–90 nM doxorubicin, the cells completely lost their capacity to differentiate into LC. This inhibition turned out to be reversible, as the cells slowly regained their capacity to differentiate after a 3- to 4-month drug-free period and with this became capable again of priming allogeneic T cells. Of note, the loss and gain of this capacity to differentiate coincided with the loss and gain of a subpopulation within the CD34+ proliferative compartment with surface expression of the stem cell factor receptor (SCF-R/CD117/c-Kit). These data are in favor of cytostatic drug-free intervals before applying autologous DC-based vaccination protocols, as specific DC precursors may need time to recover from protracted chemotherapy treatment and re-emerge among the circulating CD34+ hematopoietic stem and precursor cells

Large-scale international validation of an indirect ELISA based on recombinant nucleocapsid protein of rift valley fever virus for the detection of IgG antibody in domestic ruminants

Diagnostic performance of an indirect enzyme-linked immunosorbent assay (I-ELISA) based on a recombinant nucleocapsid protein (rNP) of the Rift Valley fever virus (RVFV) was validated for the detection of the IgG antibody in sheep (n = 3367), goat (n = 2632), and cattle (n = 3819) sera. Validation data sets were dichotomized according to the results of a virus neutralization test in sera obtained from RVF-endemic (Burkina Faso, Democratic Republic of Congo, Mozambique, Senegal, Uganda, and Yemen) and RVF-free countries (France, Poland, and the USA). Cut-off values were defined using the two-graph receiver operating characteristic analysis. Estimates of the diagnostic specificity of the RVFV rNP I-ELISA in animals from RVF-endemic countries ranged from 98.6% (cattle) to 99.5% (sheep) while in those originating from RVF-free countries, they ranged from 97.7% (sheep) to 98.1% (goats). Estimates of the diagnostic sensitivity in ruminants from RVF-endemic countries ranged from 90.7% (cattle) to 100% (goats). The results of this large-scale international validation study demonstrate the high diagnostic accuracy of the RVFV rNP I-ELISA. Standard incubation and inactivation procedures evaluated did not have an adverse effect on the detectable levels of the anti-RVFV IgG in ruminant sera and thus, together with recombinant antigen-based I-ELISA, provide a simple, safe, and robust diagnostic platform that can be automated and carried out outside expensive bio-containment facilities. These advantages are particularly important for less-resourced countries where there is a need to accelerate and improve RVF surveillance and research on epidemiology as well as to advance disease control measures.The International Atomic Energy Agencyhttp://www.mdpi.com/journal/virusespm2021Medical Virolog

Methotrexate accumulation in target intestinal mucosa and white blood cells differs from non-target red blood cells of patients with Crohn's disease

BACKGROUND: Intracellular methotrexate polyglutamates (MTX-PGs) concentrations are measurable in red blood cells (RBCs) during MTX treatment. MTX-PG 3 concentrations correlate with efficacy in patients with Crohn's disease (CD). Since RBCs are not involved in pathogenesis of CD and lack extended MTX metabolism, we determined MTX-PGs accumulation in peripheral blood mononuclear cells (PBMCs: effector cells) and intestinal mucosa (target cells) and compared those with RBCs as a potential more precise biomarker. METHODS: In a multicentre prospective cohort study, blood samples of patients with CD were collected during the first year of MTX therapy. Mucosal biopsies were obtained from non-inflamed rectum and/or inflamed intestine. MTX-PGs concentrations in mucosa, PBMCs and RBCs were measured by liquid chromatography-tandem mass spectrometry. RESULTS: From 80 patients with CD, a total of 27 mucosal biopsies, 9 PBMC and 212 RBC samples were collected. From 12 weeks of MTX therapy onwards, MTX-PG 3 was the most predominant species (33%) in RBCs. In PBMCs, the distribution was skewed towards MTX-PG 1 (48%), which accounted for an 18 times higher concentration than in RBCs. Long-chain MTX-PGs were highly present in mucosa: 21% of MTX-PG total was MTX-PG 5. MTX-PG 6 was measurable in all biopsies. CONCLUSIONS: MTX-PG patterns differ between mucosa, PBMCs and RBCs of patients with CD

Ferric carboxymaltose infusion versus oral iron supplementation for preoperative iron deficiency anaemia in patients with colorectal cancer (FIT):a multicentre, open-label, randomised, controlled trial

Background: A third of patients with colorectal cancer who are eligible for surgery in high-income countries have concomitant anaemia associated with adverse outcomes. We aimed to compare the efficacy of preoperative intravenous and oral iron supplementation in patients with colorectal cancer and iron deficiency anaemia. Methods: In the FIT multicentre, open-label, randomised, controlled trial, adult patients (aged 18 years or older) with M0 stage colorectal cancer scheduled for elective curative resection and iron deficiency anaemia (defined as haemoglobin level of less than 7·5 mmol/L (12 g/dL) for women and less than 8 mmol/L (13 g/dL) for men, and a transferrin saturation of less than 20%) were randomly assigned to either 1–2 g of ferric carboxymaltose intravenously or three tablets of 200 mg of oral ferrous fumarate daily. The primary endpoint was the proportion of patients with normalised haemoglobin levels before surgery (≥12 g/dL for women and ≥13 g/dL for men). An intention-to-treat analysis was done for the primary analysis. Safety was analysed in all patients who received treatment. The trial was registered at ClincalTrials.gov, NCT02243735, and has completed recruitment. Findings: Between Oct 31, 2014, and Feb 23, 2021, 202 patients were included and assigned to intravenous (n=96) or oral (n=106) iron treatment. Treatment began a median of 14 days (IQR 11–22) before surgery for intravenous iron and 19 days (IQR 13–27) for oral iron. Normalisation of haemoglobin at day of admission was reached in 14 (17%) of 84 patients treated intravenously and 15 (16%) of 97 patients treated orally (relative risk [RR] 1·08 [95% CI 0·55–2·10]; p=0·83), but the proportion of patients with normalised haemoglobin significantly increased for the intravenous treatment group at later timepoints (49 [60%] of 82 vs 18 [21%] of 88 at 30 days; RR 2·92 [95% CI 1·87–4·58]; p&lt;0·0001). The most prevalent treatment-related adverse event was discoloured faeces (grade 1) after oral iron treatment (14 [13%] of 105), and no treatment-related serious adverse events or deaths were observed in either group. No differences in other safety outcomes were seen, and the most common serious adverse events were anastomotic leakage (11 [5%] of 202), aspiration pneumonia (5 [2%] of 202), and intra-abdominal abscess (5 [2%] 202). Interpretation: Normalisation of haemoglobin before surgery was infrequent with both treatment regimens, but significantly improved at all other timepoints following intravenous iron treatment. Restoration of iron stores was feasible only with intravenous iron. In selected patients, surgery might be delayed to augment the effect of intravenous iron on haemoglobin normalisation. Funding: Vifor Pharma.</p