Microbial Butyrate Synthesis Indicates Therapeutic Efficacy of Azathioprine in IBD Patients.

BACKGROUND AND AIMS: The microbial ecosystem seems to be an important player for therapeutic intervenption in inflammatory bowel disease [IBD]. We assessed longitudinal microbiome changes in IBD patients undergoing therapy with either azathioprine [AZA] or anti-tumour necrosis factor [anti-TNF] antibodies. We predicted the metabolic microbial community exchange and linked it to clinical outcome. METHODS: Faecal and blood samples were collected from 65 IBD patients at baseline and after 12 and 30 weeks on therapy. Clinical remission was defined as Crohn's Disease Activity Index [CDAI] < 150 in Crohn´s disease [CD], partial Mayo score <2 in ulcerative colitis [UC], and faecal calprotectin values <150 µg/g and C-reactive protein <5 mg/dl. 16S rRNA amplicon sequencing was performed. To predict microbial community metabolic processes, we constructed multispecies genome-scale metabolic network models. RESULTS: Paired Bray-Curtis distance between baseline and follow-up time points was significantly different for UC patients treated with anti-TNF antibodies. Longitudinal changes in taxa composition at phylum level showed a significant decrease of Proteobacteria and an increase of Bacteroidetes in CD patients responding to both therapies. At family level, Lactobacilli were associated with persistent disease and Bacteroides abundance with remission in CD. In-silico simulations of microbial metabolite exchange predicted a 1.7-fold higher butyrate production capacity of patients in remission compared with patients without remission [p = 0.041]. In this model, the difference in butyrate production between patients in remission and patients without remission was most pronounced in the CD group treated with AZA [p = 0.008]. CONCLUSIONS: In-silico simulation identifies microbial butyrate synthesis predictive of therapeutic efficacy in IBD.

Prospective multicentre clinical study on inter- and intrapatient genetic variability for antimicrobial resistance of Helicobacter pylori.

OBJECTIVES: We report on a large prospective, multicentre clinical investigation on inter- and intrapatient genetic variability for antimicrobial resistance of Helicobacter pylori. METHODS: Therapy-naive patients (n = 2004) who had undergone routine diagnostic gastroscopy were prospectively included from all geographic regions of Austria. Gastric biopsy samples were collected separately from antrum and corpus. Samples were analysed by histopathology and real-time PCR for genotypic resistance to clarithromycin and quinolones. Clinical and demographic information was analysed in relation to resistance patterns. RESULTS: H. pylori infection was detected in 514 (26%) of 2004 patients by histopathology and confirmed in 465 (90%) of 514 patients by real-time PCR. PCR results were discordant for antrum and corpus in 27 (5%) of 514 patients, indicating inhomogeneous infections. Clarithromycin resistance rates were 17% (77/448) and 19% (84/455), and quinolone resistance rates were 12% (37/310) and 10% (32/334) in antrum and corpus samples, respectively. Combination of test results per patient yielded resistance rates of 21% (98/465) and 13% (50/383) for clarithromycin and quinolones, respectively. Overall, infection with both sensitive and resistant H. pylori was detected in 65 (14%) of 465 patients. CONCLUSIONS: Anatomically inhomogeneous infection with different, multiple H. pylori strains is common. Prospective clinical study design, collection of samples from multiple sites and microbiologic methods that allow the detection of coinfections are mandatory for collection of reliable data on antimicrobial resistance patterns in representative patient populations. (ClinicalTrials.gov identifier: NCT02925091).

Universal Linear Fit Identification: A Method Independent of Data, Outliers and Noise Distribution Model and Free of Missing or Removed Data Imputation.

Data processing requires a robust linear fit identification method. In this paper, we introduce a non-parametric robust linear fit identification method for time series. The method uses an indicator 2/n to identify linear fit, where n is number of terms in a series. The ratio Rmax of amax - amin and Sn - amin*n and that of Rmin of amax - amin and amax*n - Sn are always equal to 2/n, where amax is the maximum element, amin is the minimum element and Sn is the sum of all elements. If any series expected to follow y = c consists of data that do not agree with y = c form, Rmax > 2/n and Rmin > 2/n imply that the maximum and minimum elements, respectively, do not agree with linear fit. We define threshold values for outliers and noise detection as 2/n * (1 + k1) and 2/n * (1 + k2), respectively, where k1 > k2 and 0 ≤ k1 ≤ n/2 - 1. Given this relation and transformation technique, which transforms data into the form y = c, we show that removing all data that do not agree with linear fit is possible. Furthermore, the method is independent of the number of data points, missing data, removed data points and nature of distribution (Gaussian or non-Gaussian) of outliers, noise and clean data. These are major advantages over the existing linear fit methods. Since having a perfect linear relation between two variables in the real world is impossible, we used artificial data sets with extreme conditions to verify the method. The method detects the correct linear fit when the percentage of data agreeing with linear fit is less than 50%, and the deviation of data that do not agree with linear fit is very small, of the order of ±10-4%. The method results in incorrect detections only when numerical accuracy is insufficient in the calculation process.

Outlier detection method in linear regression based on sum of arithmetic progression.

We introduce a new nonparametric outlier detection method for linear series, which requires no missing or removed data imputation. For an arithmetic progression (a series without outliers) with n elements, the ratio (R) of the sum of the minimum and the maximum elements and the sum of all elements is always 2/n : (0,1]. R ≠ 2/n always implies the existence of outliers. Usually, R < 2/n implies that the minimum is an outlier, and R > 2/n implies that the maximum is an outlier. Based upon this, we derived a new method for identifying significant and nonsignificant outliers, separately. Two different techniques were used to manage missing data and removed outliers: (1) recalculate the terms after (or before) the removed or missing element while maintaining the initial angle in relation to a certain point or (2) transform data into a constant value, which is not affected by missing or removed elements. With a reference element, which was not an outlier, the method detected all outliers from data sets with 6 to 1000 elements containing 50% outliers which deviated by a factor of ±1.0e - 2 to ±1.0e + 2 from the correct value.

The feed-forward loop between YB-1 and MYC is essential for multiple myeloma cell survival.

Y-box binding protein 1 (YB-1) functions as a translational regulator and has been suggested to elevate MYC mRNA translation via an internal ribosome entry segment (IRES) point mutation in multiple myeloma (MM). We show that YB-1-mediated translation of MYC mRNA occurs independently of the reported IRES mutation, as 87 MM patients (n=88) and all tested human MM cell lines (HMCLs) were negative for the mutation. We show for the first time that positive MYC staining predicts YB-1 co-expression in malignant plasma cells and YB-1/MYC co-expression increases from 30% in medullary to 70% in extramedullary MM. YB-1 knockdown in HMCLs reduced both MYC protein levels and MYC mRNA in the polysomal fraction, providing a mechanism by which YB-1 controls MYC translation. MYC transcription of YB-1 is demonstrated in HMCLs as MYC knockdown resulted in reduced YB-1 protein and mRNA levels. Furthermore, MYC activation in non-malignant mouse embryonic fibroblasts (MEFs) increased YB-1 mRNA, clearly indicating that MYC drives YB-1 transcription. Importantly, perturbation of the MYC/YB-1 oncogenic circuit leads to apoptosis in HMCLs. Here, we demonstrate that these two proteins co-regulate each other via combined transcriptional/translational activity establishing their pivotal role in MM cell survival. We therefore suggest that targeting the YB-1/mRNA interaction provides a new strategy for MM drug development.

Effects of six second generation antipsychotics on body weight and metabolism - risk assessment and results from a prospective study.

BACKGROUND: Due to the association of second generation antipsychotics (SGAs) with weight gain and alterations of glucose and lipid homeostasis, we aimed to group six commonly prescribed SGAs into classes of differing risks. METHODS: Twenty-eight patients meeting the criteria for a diagnosis of schizophrenic disorder according to ICD-10 were assigned to monotherapy with olanzapine, clozapine, quetiapine, amisulpride, ziprasidone or risperidone. The levels of glucose and lipid metabolism were assessed before and after 28 days of treatment. RESULTS: Based on cluster analysis, olanzapine and clozapine were found to constitute a high-risk group for metabolic dysregulation while amisulpride, quetiapine, risperidone and ziprasidone could be assigned to a non-high-risk group. Subjects from the high-risk group displayed significant weight gain with concomitant increases of HOMA-IR, levels of insulin, total cholesterol, TG, LDL-C and leptin. No significant changes were observed in the non-high-risk group. CONCLUSION: The results of this study support the conclusion of the Consensus Development Conference on Antipsychotic Drugs and Obesity and Diabetes that certain SGAs are associated with a higher risk for weight gain, insulin resistance and dyslipidemia.

Quantification of Cryptosporidium parvum in anaerobic digesters treating manure by (reverse-transcription) quantitative real-time PCR, infectivity and excystation tests.

The survival of Cryptosporidium parvum oocysts in anaerobic digesters treating manure was investigated for mesophilic, thermophilic, and a combined treatment (mesophilic-thermophilic-mesophilic) under different retention times of oocysts in the reactors. C. parvum DNA was extracted with an optimised protocol, and its amount determined by quantitative real-time PCR (qPCR). Results indicated noteworthy differences in DNA content after the different treatments. DNA was not degraded during the process. However, excystation and infectivity tests showed a reduction of viable oocyst numbers of > or = 2 and > or = 5 log units after the thermophilic treatment in two different experiments. Thus qPCR-targeting DNA can overestimate the number of oocysts that survive and remain viable after anaerobic digestion. However, targeting DNA is suitable to indicate the presence or absence of oocysts. Reverse transcription qPCR (RT-qPCR) targeting C. parvum hsp70 mRNA successfully indicated the presence of viable fraction of oocysts.

Mesophilic-thermophilic-mesophilic anaerobic digestion of liquid dairy cattle manure.

The potential of a mesophilic-thermophilic-mesophilic anaerobic digestion system was investigated with respect to improvement of both digestion and sanitation efficiencies during treatment of liquid cattle manure. The pilot plant produced a high methane yield from liquid dairy cattle manure of 0.24 m3 (kg VSfed)(-1) Considering the low system loading rate of 1.4-1.5 kg VS (m3 d)(-1), digestion efficiency compared to conventional processes did not appear improved. The minimum guaranteed retention time in the tubular thermophilic reactor was increased compared to a continuously stirred tank reactor. Levels of intestinal enterococci in raw liquid manure as determined with cultivation methods were reduced by 2.5 -3 log units to a level of around 10(2) cfu/mL. This sanitizing effect was achieved both during mesophilic-thermophilic-mesophilic and thermophilic-mesophilic treatment, provided the thermophilic digester was operated at 53-55 degrees C. A change in feeding interval from 1 h to 4 h did not significantly alter methane yield and sanitation efficiency. It was proposed that a two-stage, thermophilic-mesophilic anaerobic digestion system would be able to achieve the same sanitizing effect and equal or better digestion efficiency at lower costs.

Hygienization by anaerobic digestion: comparison between evaluation by cultivation and quantitative real-time PCR.

In order to assess hygienization by anaerobic digestion, a comparison between evaluation by cultivation and quantitative real-time PCR (qPCR) including optimized DNA extraction and quantification was carried out for samples from a full-scale fermenter cascade (F1, mesophilic; F2, thermophilic; F3, mesophilic). The system was highly effective in inactivating (pathogenic) viable microorganisms, except for spore-formers. Conventionally performed cultivation underestimated viable organisms particularly in F2 and F3 by a factor of at least 10 as shown by data from extended incubation times, probably due to the rise of sublethally injured (active but not cultivable) cells. Incubation should hence be extended adequately in incubation-based hygiene monitoring of stressed samples, in order to minimize contamination risks. Although results from qPCR and cultivation agreed for the equilibrated compartments, considerably higher qPCR values were obtained for the fermenters. The difference probably corresponded to DNA copies from decayed cells that had not yet been degraded by the residual microbial activity. An extrapolation from qPCR determination to the quantity of viable organisms is hence not justified for samples that had been exposed to lethal stress.

Evaluating real-time PCR for the quantification of distinct pathogens and indicator organisms in environmental samples.

We evaluated quantitative real-time PCR (qPCR) and RTqPCR (for RNA species) for their ability to quantify microorganisms and viruses in problematic environmental samples such as cattle manure, digester material, wastewater and soil. Important developments included a standard spiking approach which compensated for methodological bias and allowed sample-to-sample comparison and reliable quantification. Programme CeTe was developed to calculate endogenous concentrations of target organisms (nucleic acid copies) for each sample separately from the generated standard curves. The approach also permitted assessment of the detection limit of the complete method, including extraction. It varied from sample to sample, due to different extraction efficiencies and variable co-extraction of PCR inhibitors. False negative results were thereby avoided. By using this approach we were able to optimise a DNA extraction protocol from the different tested sample types. Protocols for the extraction of RNA species from environmental samples were also optimised. DNA was (almost) not degraded after lethal shock (autoclaving) in the sterile environment. In contrast, the parallel selective cultivation and qPCR results for various microbial parameters from an anaerobic digester chain suggested that DNA from decaying organisms was readily recycled in metabolically active environments. It may, therefore, be used to determine viable organisms in samples exhibiting substantial metabolic turnover. It is proposed that our standard spiking approach, including data evaluation by the program CeTe, should be considered in future standardisation and norms for the quantification of nucleic acid containing organisms in environmental and product samples.

Using quantitative real-time PCR to determine the hygienic status of cattle manure.

We developed a suitable system of DNA extraction and real-time quantitative polymerase chain reaction (qPCR) for the specific and sensitive quantification of pathogens and other relevant (indicator) organisms in recalcitrant material such as cattle manure. PCR inhibition by coextraction of humic compounds was minimized in this system, resulting in detection sensitivity of one target DNA copy per reaction well. Data from qPCR analysis for Escherichia coli agreed with cultivation based results, but orders of magnitude more fecal enterococci, Enterobacteriaceae and Campylobacter jejuni, were determined by qPCR than by cultivation. These bacteria may have been in a potentially hazardous active but non-cultivable state. The qPCR system is much less time consuming than conventional cultivation, highly specific, can detect non-cultivable organisms, provides high measurement throughput, and is cost attractive. It should be considered as an alternative in various application areas for (prescribed routine) cultivation based assays, e.g. for biosafety and hygiene monitoring.

Mapping of cholinoceptive(nicotinoceptive)neurons in the lower brainstem: with special reference to the ventral surface of the medulla.

The distribution of cholinoceptive neurons in the lower brainstem of the rat was investigated by means of a histochemical method for specific acetylcholinesterase. Nicotinoceptive neurons were characterized using an alpha-bungarotoxin-horseradish peroxidase conjugate for the detection of nicotinic acetylcholine receptors. For the first time a nearly complete mapping of the location of cholinoceptive (nicotinoceptive) neurons of the lower brainstem was achieved. Special attention was focused on the organization of the cholinoceptive neuronal matrix of the ventral surface of the medulla, where regulative centers for vasomotor and respiratory control are located.