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Abstract Stool consistency is evaluated mainly in reference to indirect indicators such as water content or the appearance of stool forms using Bristol Stool Form Scale (BSFS). Methods of measurement are limited. We thus aimed to develop a simple protocol for direct measurement of stool consistency using the TA.XTExpress Texture Analyser (Stable Micro Systems Ltd.). We developed a protocol which enables mechanical quantification of the gram-force against a cylindrical probe (ø 6 mm) pushed into the stool surface at 2.0 mm/s to 5 mm depth. The consistency of 252 stools collected from 40 healthy Belgians was evaluated by the direct method and by the indirect indicators (water content and BSFS) for comparison. The log-transformed stool consistency values measured by the texture analyzer had a negative linear correlation with the stool water contents (r rm = − 0.781) with homoscedastic variance, suggesting the appropriateness of the new protocol. They showed a similar correlation with the BSFS, but with a large variance in the consistency values of normal stool forms. This correlation was much smaller for BSFS scored by subjects (r rm = − 0.587) than by experts (r rm = − 0.789), collectively indicating BSFS as a rough indicator of stool consistency susceptible to subjective bias despite its effectiveness in clinical use. The optimized direct method using the texture analyzer enables the accurate quantification of stool consistency, which facilitates understanding of the intestinal environment and function and thus may enhance the value of the stool as a predictor of human health.

Since the viability throughout the human gastro-intestinal (GI) tract is a key metric to select new probiotics, we aimed to develop an evolutive and robust semi-dynamic model, named Gastro-Intestinal Digestive Simulator (GIDS), mimicking pH variations of the upper GI tract and based on the latest Infogest in vitro digestion protocol recommendations. We first tested the GIDS and its reproducibility by measuring, during in vitro digestion, the cultivability of bacterial strain contained in two marketed fermented milk products. Then, we used this model to assess inter- and intra-species variability among 16 bacterial strains from Bifidobacterium animalis, Bifidobacterium breve, and Lactobacillus paracasei species. Finally, we further explored the applicability of propidium monoazide-quantitative PCR and flow cytometry to GIDS samples. To summarize, our study showed that i) the GIDS model is reproducible and ii) the complementary methods to plating tested here are compatible with GI environments; providing insights that will guide future developments.

Abstract Background The importance of the gut microbiota at the early stage of life and their longitudinal effect on host health have recently been well investigated. In particular, Bifidobacterium longum subsp. longum, a common component of infant gut microbiota, appears in the gut shortly after birth and can be detected there throughout an individual’s lifespan. However, it remains unclear whether this species colonizes in the gut over the long term from early infancy. Here, we investigated the long-term colonization of B. longum subsp. longum by comparing the genotypes of isolates obtained at different time points from individual subjects. Strains were isolated over time from the feces of 12 subjects followed from early infancy (the first six months of life) up to childhood (approximately six years of age). We also considered whether the strains were transmitted from their mothers’ perinatal samples (prenatal feces and postnatal breast milk). Results Intra-species diversity of B. longum subsp. longum was observed in some subjects’ fecal samples collected in early infancy and childhood, as well as in the prenatal fecal samples of their mothers. Among the highlighted strains, several were confirmed to colonize and persist in single individuals from as early as 90 days of age for more than six years; these were classified as long-term colonizers. One of the long-term colonizers was also detected from the corresponding mother’s postnatal breast milk. Quantitative polymerase chain reaction data suggested that these long-term colonizers persisted in the subjects’ gut despite the existence of the other predominant species of Bifidobacterium. Conclusions Our results showed that several strains belonging to B. longum subsp. longum colonized in the human gut from early infancy through more than six years, confirming the existence of long-term colonizers from this period. Moreover, the results suggested that these strains persisted in the subjects’ gut while co-existing with the other predominant bifidobacterial species. Our findings also suggested the importance of microbial-strain colonization in early infancy relative to their succession and showed the possibility that probiotics targeting infants might have longitudinal effects. Trial Registration TRN: ISRCTN25216339. Date of registration: 11/03/2016. Prospectively registered.

The majority of mutations studied in animal models are designated as recessive based on the absence of visible phenotypes in germline heterozygotes. Accordingly, genetic studies primarily rely on homozygous loss-of-function to determine gene requirements, and a conceptually-related ‘two-hit model’ remains the central paradigm in cancer genetics. Here we investigate pathogenesis due to somatic mutation in epithelial tissues, a process that predominantly generates heterozygous cell clones. To study somatic mutation in Drosophila, we generated inducible alleles that mimic human Juvenile polyposis-associated BMPR1A mutations. Unexpectedly, four of these mutations had no phenotype in heterozygous carriers but exhibited clear tissue-level effects when present in somatic clones of heterozygous cells. We conclude that these alleles are indeed recessive when present in the germline, but nevertheless deleterious when present in heterozygous clones. This unforeseen effect, deleterious heteromosaicism, suggests a ‘one-hit’ mechanism for disease initiation that may explain some instances of pathogenesis associated with spontaneous mutation.

It has been suggested that the Klason method overestimates the lignin content of non-wood tissues of plants. To evaluate the effect of pre-extraction treatments on lignin determination in leaves, nine kinds of pre-extraction treatment were applied to ginkgo leaves and zelkova leaves. The apparent lignin contents (lignin determination by the Klason method) of ginkgo and zelkova leaves without pre-extraction treatments were 30.7% and 42.6%, respectively. After the various pre-extraction treatments, the apparent lignin contents were still high. On the other hand, the yield of nitrobenzene oxidation products (NPs) per apparent lignin content was maintained at a very low level before and after pre-extraction treatments (maximum value was 6% for ginkgo leaf and 11% for zelkova leaf after extraction treatment) compared with the value from wood (25 to 60%). These results suggested that the Klason method overestimates the lignin content of leaves even after the pre-extraction treatments examined in this study. In addition, a considerable part of the sample from which NPs or neutral sugars originate was lost during these pre-extraction stages. These results implied that some parts of the cell wall components were also removed by these pre-extraction treatments.

The degradation kinetics of monosaccharides during sulfurous acid treatment was compared to hydrochloric acid and to sulfuric acid treatments. Reaction conditions corresponded to the range found in previous research to allow for the production of hemicelluloses-derived monosaccharides through hydrolysis of wood. Degradation behavior of monosaccharides during treatment with each acid was expressed by a second-order reaction rate constant with respect to substrate and acid concentrations, and the activation energy and frequency factor were calculated using the Arrhenius equation. Results demonstrated that the second-order reaction rate of a monosaccharide was dependent on the type of acid, indicating that monosaccharides degrade at different rates under different acids, even when the molar concentration of the acid is the same. The degradation of monosaccharides in sulfurous acid was much slower than that in hydrochloric acid and in sulfuric acid. A comparison of two sequential treatments with sulfuric acid, with and without the bisulfite ion, showed that sulfurous acid has a protective effect on the degradation of monosaccharides.

Endoplasmic reticulum (ER) stress is caused by accumulation of unfolded and misfolded proteins in the ER, thereby compromising its vital cellular functions in protein production and secretion. Genome wide association studies in humans as well as experimental animal models linked ER stress in intestinal epithelial cells (IECs) with intestinal disorders including inflammatory bowel diseases. However, the mechanisms linking the outcomes of ER stress in IECs to intestinal disease have not been clarified. In this study, we investigated the impact of ER stress on intestinal epithelial barrier function using human colon carcinoma-derived Caco-2 monolayers. Tunicamycin-induced ER stress decreased the trans-epithelial electrical resistance of Caco-2 monolayers, concomitant with loss of cellular plasma membrane integrity. Epithelial barrier disruption in Caco-2 cells after ER stress was not caused by caspase- or RIPK1-dependent cell death but was accompanied by lysosomal rupture and up-regulation of the ER stress markers Grp78, sXBP1 and Chop. Interestingly, several bifidobacteria species inhibited tunicamycin-induced ER stress and thereby diminished barrier disruption in Caco-2 monolayers. Together, these results showed that ER stress compromises the epithelial barrier function of Caco-2 monolayers and demonstrate beneficial impacts of bifidobacteria on ER stress in IECs. Our results identify epithelial barrier loss as a potential link between ER stress and intestinal disease development, and suggest that bifidobacteria could exert beneficial effects on this phenomenon.

The transmission performance for a downlink mobile WiMAX system with multiuser multiple-input multiple-output (MU-MIMO) systems in a computer simulation and field experiment is described. In computer simulation, a MU-MIMO transmission system can be realized by using the block diagonalization (BD) algorithm, and each user can receive signals without any signal interference from other users. The bit error rate (BER) performance and channel capacity in accordance with modulation schemes and the number of streams were simulated in a spatially correlated multipath fading environment. Furthermore, we propose a method for evaluating the transmission performance for this downlink mobile WiMAX system in this environment by using the computer simulation. In the field experiment, the received power and downlink throughput in the UDP layer were measured on an experimental mobile WiMAX system developed in Azumino City in Japan. In comparison with the simulated and experimented results, the measured maximum throughput performance in the downlink had almost the same performance as the simulated throughput. It was confirmed that the experimental mobile WiMAX system for MU-MIMO transmission successfully increased the total channel capacity of the system.

Clostridium difficile is the main cause of nosocomial diarrhea, but is also found in asymptomatic subjects that are potentially involved in transmission of C. difficile infection. A sensitive and accurate detection method of C. difficile, especially toxigenic strains is indispensable for the epidemiological investigation.TaqMan-based quantitative-PCR (qPCR) method for targeting 16S rRNA, tcdB, and tcdA genes of C. difficile was developed. The detection limit and accuracy of qPCR were evaluated by analyzing stool samples spiked with known amounts of C. difficile. A total of 235 stool specimens collected from 82 elderly nursing home residents were examined by qPCR, and the validity was evaluated by comparing the detection result with that by C. difficile selective culture (CDSC).The analysis of C. difficile-spiked stools confirmed that qPCR quantified whole C. difficile (TcdA+TcdB+, TcdA-TcdB+, and TcdA-TcdB- types), TcdB-producing strains (TcdA+TcdB+ and TcdA-TcdB+ types), and TcdA-producing strains (TcdA+TcdB+ type), respectively, with a lower detection limit of 103 cells/g of stool. Of the 235 specimens examined, 12 specimens (5.1%) were C. difficile-positive by qPCR: TcdA+TcdB+ strain in six specimens and TcdA-TcdB- strain in the other six. CDSC detected C. difficile in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method. Moreover, the qPCR examination revealed that the carriage rate of whole C. difficile and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively. An average qPCR count of C. difficile detected was 104.5 cells/g of stool, suggesting that C. difficile constituted a very small fraction of intestinal microbiota.Our qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of C. difficile.

Heparan sulfate (HS) and chondroitin sulfate (CS) are evolutionarily conserved glycosaminoglycans that are found in most animal species, including the genetically tractable model organism Drosophila. In contrast to extensive in vivo studies elucidating co-receptor functions of Drosophila HS proteoglycans (PGs), only a limited number of studies have been conducted for those of CSPGs. To investigate the global function of CS in development, we generated mutants for Chondroitin sulfate synthase (Chsy), which encodes the Drosophila homolog of mammalian chondroitin synthase 1, a crucial CS biosynthetic enzyme. Our characterizations of the Chsy mutants indicated that a fraction survive to adult stage, which allowed us to analyze the morphology of the adult organs. In the ovary, Chsy mutants exhibited altered stiffness of the basement membrane and muscle dysfunction, leading to a gradual degradation of the gross organ structure as mutant animals aged. Our observations show that normal CS function is required for the maintenance of the structural integrity of the ECM and gross organ architecture. [ABSTRACT FROM AUTHOR]

Sjögren syndrome (SS) is one of several collagen vascular diseases that occasionally involve the central nervous system. We report two cases of SS involving young patients who initially presented with aseptic meningitis. A male with recurrent AM was found to have anti-Ro/SSA and La/SSB antibodies in a screening test for autoimmune process. A minor salivary gland biopsy revealed lymphocytic infiltrations compatible with SS, although the patient did not exhibit sicca symptoms. A female presenting with AM and polyarthritis also reported xerophthalmia. Anti-Ro/SSA antibody testing and a positive result in a minor salivary gland biopsy led to the diagnosis of SS. In the literature review, we found that AM or aseptic meningoencephalitis (AME) preceded or had a concomitant onset with SS in approximately 70% of cases. Screening for anti-Ro/SSA antibody, as well as systemic assessment for rheumatic symptoms, may be useful for diagnosing AM/AME of unknown etiology. [ABSTRACT FROM AUTHOR]

Lignin analyses were performed on the reaction woods of five tropical wood species. The reaction woods of these five species and that of Gnetum gnemon L. (previously reported) were categorized into three types based on eccentric thickening growth and p-hydroxyphenyl/guaiacyl/syringyl (H/G/S) ratios: compression wood (CW) containing GH-lignin observed in gymnosperms (GH-lignin-CW), tension wood (TW) containing GS-lignin observed in angiosperms (GS-lignin-TW), and reaction wood that resembles CW and contains GS-lignin (GSlignin-CW). GS-lignin-CW is an unusual type that was found in the angiosperm Eusideroxylon zwageri and in the gymnosperm G. gnemon. The erythro/threo ratio of the β-O-4 structures and the S/G ratio were higher on the upper side (usW) of the leaning wood stem or branch, and both ratios decreased along the periphery of the stem toward the lower side (lsW). Except for a difference in thickening growth, these distribution patterns were similar to the GSlignin-TW patterns for Melia azedarach L. and Avicennia sp. Reaction wood of Paraserianthes falcataria (L.) Nielsen was also classified as a GS-lignin-TW, but this was lacking a clear distribution pattern. In contrast, the GH-lignin-CW of the usW of Pinus merkusii Jungh. et de Vriese had a low erythro/threo ratio, which increased toward the lsW along with increasing lignin contents and H/G ratios. [ABSTRACT FROM AUTHOR]

A 4-O-5-tetramer lignin model compound carrying β-O-4 linkages on each of the side-chain moieties was synthesized, as well as 4-O-5-coupled dehydrodiconiferyl alcohol. By comparison with their NMR data, two cross-signals in the HSQC spectrum of pine milled wood lignin recorded in DMSO-d6 were assigned to H2/C2 and H6/C6 correlations on the aromatic rings of 4-O-5-linked units. Although the H2/C2 correlation peak appeared in the same region as syringyl units, nitrobenzene oxidation of the pine lignin did not yield any syringyl-type product, but did release a 4-O-5-type product. [ABSTRACT FROM AUTHOR]

The article discusses the contribution of an operon consisted of LacS symporter, integral membrane protein, and β-galactosidase to bifidobacterial galactooligosaccharides (GOS), a prebiotics, consumption. Topics discussed include analysis of GOS consumption by 14 bifidobacterial strains; transcription analysis of GOS consumer; and the growth of bifidobacteria on GOS.

The potential of sulfurous acid prehydrolysis followed by chemical pulping was examined at the laboratory scale using radiata pine. The residue obtained by sulfurous acid prehydrolysis with base addition was sufficiently delignified in a subsequent soda pulping stage, while the delignification of the residue was completely insufficient in subsequent kraft pulping. The residues prepared by prehydrolyses of hydrochloric and sulfuric acids were not delignified sufficiently regardless of the pulping method. The yield of hemicellulose-derived monosaccharides was about 90% on a theoretical basis in the optimal sulfurous acid prehydrolysis. In subsequent soda pulping under various conditions, pulps with kappa number 21 to 34, pulp yield 37 to 41%, and high cellulose content could be prepared from the residue obtained by sulfurous acid prehydrolysis with base addition. It was suggested from these results that sulfurous acid prehydrolysis with base addition in combination with subsequent soda pulping is an effective method for the utilization of wood hemicelluloses. [ABSTRACT FROM AUTHOR]

AbstractIn the structural analysis of lignins by 13C-NMR, signal overlap limits definitive assignment and accurate intensity measurement. Selective labeling by 13C-enrichment of a specific carbon in lignin enhances its signal intensity in the spectrum. Further enhancement of the specifically labeled carbons can be realized via difference spectra created from the enriched and unenriched samples. Difference 2D 13C-1H correlation (HSQC) NMR spectra, derived from the spectra of specifically 13C-enriched lignin model polymers (so-called dehydrogenation polymers) and their unenriched counterparts, take advantage of the enhanced dispersion afforded by both 13C and 1H chemical shifts, diminishing the difficulties arising from the signal-overlap problem and aiding in definitive signal assignments. In this research, protolignin in xylem cell walls was specifically 13C-enriched at all of the individual phenylpropanoid side-chain carbons by feeding 13C-enriched coniferins to growing stems of Ginkgo biloba. The whole xylem fractions containing 13C-enriched and unenriched protolignins were dissolved in a mixture of N-methylimidazole and DMSO, and then acetylated. Solution state 2D-NMR (HSQC) spectra of the acetylated whole cell wall were acquired. Difference spectra between the walls containing 13C-enriched and unenriched lignins afforded simplified 2D spectra in which well-separated signals were assigned exclusively to the specifically enriched carbons. This novel NMR technique provides a useful tool for elucidation of entire protolignin in the cell wall of ginkgo xylem. [ABSTRACT FROM AUTHOR]

The proportion of erythro - and threo -forms of O-4-structures in lignin was elucidated by ozonation analysis of 21 wood species, and the relationship to the syringyl and guaiacyl composition was investigated. For all hardwood species, the erythro form of O-4-structures predominated, although the extent varied widely, depending on wood species. In contrast, the proportion and amount of erythro - and threo -forms were very similar in all softwood species. The proportion of the erythro form was greater in species with a higher methoxyl content in the lignin (correlation coefficient, R 2 =0.83). The S/V ratio (molar ratio of syringaldehyde and syringic acid to that of vanillin and vanillic acid) obtained by nitrobenzene oxidation was also strongly correlated with the proportion of the erythro form (R 2 =0.99). Accordingly, the syringyl/guaiacyl ratio is closely related to the erythro / threo ratio. This stereochemical characteristic of O-4-structures is discussed in relation to the process of lignin formation. [ABSTRACT FROM AUTHOR]

Heparan sulfate proteoglycans (HSPGs) are composed of a core protein and glycosaminoglycan (GAG) chains and serve as coreceptors for many growth factors and morphogens. To understand the molecular mechanisms by which HSPGs regulate morphogen gradient formation and signaling, it is important to determine the relative contributions of the carbohydrate and protein moieties to the proteoglycan function. To address this question, we generated ΔGAG alleles for dally and dally-like protein (dlp), two Drosophila HSPGs of the glypican family, in which all GAG-attachment serine residues are substituted to alanine residues using CRISPR/Cas9 mutagenesis. In these alleles, the glypican core proteins are expressed from the endogenous loci with no GAG modification. Analyses of the dallyΔGAG allele defined Dally functions that do not require heparan sulfate (HS) chains and that need both core protein and HS chains. We found a new, dallyΔGAGspecific phenotype, the formation of a posterior ectopic vein, which we have never seen in the null mutants. Unlike dallyΔGAG, dlpΔGAG mutants do not show most of the dlp null mutant phenotypes, suggesting that HS chains are dispensable for these dlp functions. As an exception, HS is essentially required for Dlp's activity at the neuromuscular junction. Thus, Drosophila glypicans show strikingly different levels of HS dependency. The ΔGAG mutant alleles of the glypicans serve as new molecular genetic toolsets highly useful to address important biological questions, such as molecular mechanisms of morphogen gradient formation. [ABSTRACT FROM AUTHOR]

Asymptomatic infant carriers of toxigenic Clostridium difficile are suggested to play a role in the transmission of C. difficile infection (CDI) in adults. However, the mode of C. difficile carriage in infants remains to be fully elucidated. We investigated longitudinal changes in carriage rates, counts, and strain types of toxigenic C. difficile in infants. Stools collected from 111 healthy infants in Belgium periodically from birth until the age of 6 months were examined by quantitative PCR targeting 16S rRNA and toxin genes. Toxigenic C. difficile was detected in 18 of 111 infants (16%) in the period up to the age of 6 months. The carriage rate of toxigenic C. difficile remained below 5% until the age of 3 months. The carriage rate increased to 13% 1 week after weaning (average age, 143 days) and reached 16% at the age of 6 months. Counts of toxigenic C. difficile bacteria ranged from 104 to 108 cells/g of stool. Notably, two infants retained>108 cells/g of stool for at least several weeks. Average counts in the 18 infants hovered around 107 cells/g of stool from the age of 3 days until the age of 6 months, showing no age-related trend. Genotyping of toxigenic C. difficile isolates from the 18 infants revealed that 11 infants each retained a particular monophyletic strain for at least a month. The genotype most frequently identified was the same as that frequently identified in symptomatic adult CDI patients. Thus, toxigenic C. difficile strains--potential causes of CDI in adults--colonized the infants' intestines. [ABSTRACT FROM AUTHOR]