Prebiotic effect of galacto-N-biose on the intestinal lactic acid bacteria as enhancer of acetate production and hypothetical colonization.

Galacto-N-biose (GNB) is an important core structure of glycan of mucin glycoproteins in the gastrointestinal (GI) mucosa. Because certain beneficial bacteria inhabiting the GI tract, such as bifidobacteria and lactic acid bacteria, harbor highly specialized GNB metabolic capabilities, GNB is considered a promising prebiotic for nourishing and manipulating beneficial bacteria in the GI tract. However, the precise interactions between GNB and beneficial bacteria and their accompanying health-promoting effects remain elusive. First, we evaluated the proliferative tendency of beneficial bacteria and their production of beneficial metabolites using gut bacterial strains. By comparing the use of GNB, glucose, and inulin as carbon sources, we found that GNB enhanced acetate production in Lacticaseibacillus casei, Lacticaseibacillus rhamnosus, Lactobacillus gasseri, and Lactobacillus johnsonii. The ability of GNB to promote acetate production was also confirmed by RNA-seq analysis, which indicated the upregulation of gene clusters that catalyze the deacetylation of N-acetylgalactosamine-6P and biosynthesize acetyl-CoA from pyruvate, both of which result in acetate production. To explore the in vivo effect of GNB in promoting acetate production, antibiotic-treated BALB/cA mice were administered with GNB with L. rhamnosus, resulting in a fecal acetate content that was 2.7-fold higher than that in mice administered with only L. rhamnosus. Moreover, 2 days after the last administration, a 3.7-fold higher amount of L. rhamnosus was detected in feces administered with GNB with L. rhamnosus than in feces administered with only L. rhamnosus. These findings strongly suggest the prebiotic potential of GNB in enhancing L. rhamnosus colonization and converting L. rhamnosus into higher acetate producers in the GI tract. IMPORTANCE: Specific members of lactic acid bacteria, which are commonly used as probiotics, possess therapeutic properties that are vital for human health enhancement by producing immunomodulatory metabolites such as exopolysaccharides, short-chain fatty acids, and bacteriocins. The long residence time of probiotic lactic acid bacteria in the GI tract prolongs their beneficial health effects. Moreover, the colonization property is also desirable for the application of probiotics in mucosal vaccination to provoke a local immune response. In this study, we found that GNB could enhance the beneficial properties of intestinal lactic acid bacteria that inhabit the human GI tract, stimulating acetate production and promoting intestinal colonization. Our findings provide a rationale for the addition of GNB to lactic acid bacteria-based functional foods. This has also led to the development of therapeutics supported by more rational prebiotic and probiotic selection, leading to an improved healthy lifestyle for humans.

Mucin glycans and their degradation by gut microbiota.

The human intestinal tract is inhabited by a tremendous number of microorganisms, which are collectively termed "the gut microbiota". The intestinal epithelium is covered with a dense layer of mucus that prevents penetration of the gut microbiota into underlying tissues of the host. Recent studies have shown that the maturation and function of the mucus layer are strongly influenced by the gut microbiota, and alteration in the structure and function of the gut microbiota is implicated in several diseases. Because the intestinal mucus layer is at a crucial interface between microbes and their host, its breakdown leads to gut bacterial invasion that can eventually cause inflammation and infection. The mucus is composed of mucin, which is rich in glycans, and the various structures of the complex carbohydrates of mucins can select for distinct mucosa-associated bacteria that are able to bind mucin glycans, and sometimes degrade them as a nutrient source. Mucin glycans are diverse molecules, and thus mucin glycan degradation is a complex process that requires a broad range of glycan-degrading enzymes. Because of the increased recognition of the role of mucus-associated microbes in human health, how commensal bacteria degrade and use host mucin glycans has become of increased interest. This review provides an overview of the relationships between the mucin glycan of the host and gut commensal bacteria, with a focus on mucin degradation.

Exploring role of sleep aids in sleep problems in preschool children.

It is well known that children use sleep aids, such as blankets or soft toys, at bedtime. However, there is a lack of understanding regarding the factors associated with their use and role in addressing sleep problems. This study investigated 96 Japanese children aged 40 to 47 months to examine these associations. We measured children's stress (through a questionnaire and salivary cortisol [cortisol awakening response]), anxiety symptoms, behavioral problems, and temperament, and created a model to predict the status of sleep aid use. Furthermore, we explored the association between sleep aid use and children's sleep problems as evaluated by their caregivers. We found that children who used sleep aids were more likely to experience anxiety symptoms. Moreover, most children used sleep aids even when they co-slept with their caregivers and/or siblings. Their use was not uniquely associated with sleep problems. These findings suggest that sleep aid serves as a defense against anxiety, including that caused by the absence of a caregiver, rather than as a substitute for a caregiver. Our study sheds light on their role and emphasizes the importance of viewing development within the complex interactive processes of humans and objects.

Did Children Interact With Their Personified Objects During the COVID-19 Pandemic?

Although previous studies revealed the characteristics of children with imaginary companions, the characteristics of children alone could not explain why some children create and interact with imaginary companions. The current cross-sectional study examined the impact of the situational factors, decreased opportunities to meet and play with real playmate due to the COVID-19 pandemic, on the prevalence of imaginary companions. Five hundred sixty caregivers of children aged 2-9 years (half of them were girls) were asked whether their children currently had imaginary companions (personified objects and invisible friends) before (September 2019) and during the pandemic (April 2020). The logistic regression model showed that only the prevalence of personified objects increased during the pandemic, OR = 2.01, 95%CI [1.34, 3.00], even when potential variables were controlled. The results suggest that children more frequently played with their personified objects during the pandemic compared to before the pandemic.

Process for the Purification of cis-p-Coumaric Acid by Cellulose Column Chromatography after the Treatment of the trans Isomer with Ultraviolet Irradiation.

A methanolic solution of trans-p-coumaric acid was exposed to ultraviolet radiation and a mixture solution of the trans and cis isomers was subjected to cellulose column chromatography, eluting with an aqueous 0.1% trifluoroacetic acid solution containing methanol (90:10, v/v). Separation of the trans and cis isomers was achieved. The identity of the cis isomer was confirmed by TLC, HPLC, and NMR. Since both the support and eluent are inexpensive, the cis isomers can be obtained economically on both the laboratory and industrial scales.

Identification and characterization of a sulfoglycosidase from Bifidobacterium bifidum implicated in mucin glycan utilization.

Human gut symbiont bifidobacteria possess carbohydrate-degrading enzymes that act on the O-linked glycans of intestinal mucins to utilize those carbohydrates as carbon sources. However, our knowledge about mucin type O-glycan degradation by bifidobacteria remains fragmentary, especially regarding how they decompose sulfated glycans, which are abundantly found in mucin sugar-chains. Here, we examined the abilities of several Bifidobacterium strains to degrade a sulfated glycan substrate and identified a 6-sulfo-ß-d-N-acetylglucosaminidase, also termed sulfoglycosidase, encoded by bbhII from Bifidobacterium bifidum JCM 7004. A recombinant BbhII protein showed a substrate preference toward 6-sulfated and 3,4-disulfated N-acetylglucosamines over non-sulfated and 3-sulfated N-acetylglucosamines. The purified BbhII directly released 6-sulfated N-acetylglucosamine from porcine gastric mucin and the expression of bbhII was moderately induced in the presence of mucin. This de-capping activity may promote utilization of sulfated glycans of mucin by other bacteria including bifidobacteria, thereby establishing the symbiotic relationship between human and gut microbes.

Sulfonylcarbamate as a versatile and unique hydroxy-protecting group: a protecting group stable under severe conditions and labile under mild conditions.

The sulfonylcarbamate group is a unique hydroxyl protecting group. In contrast to typical acyl protecting groups, the sulfonylcarbamate group is stable under harsh basic conditions, while showing labile behavior under mild basic conditions. Its compatibility with other hydroxyl protecting groups and application to carbohydrate chemistry is demonstrated.

Lacto-N-biosidase encoded by a novel gene of Bifidobacterium longum subspecies longum shows unique substrate specificity and requires a designated chaperone for its active expression.

Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N-tetraose (Galß1-3GlcNAcß1-3Galß1-4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Galß1-3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase(+) strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAcß1-3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fucα1-2Galß1-3GlcNAcß1-3Galß1-4Glc), and sialyllacto-N-tetraose a (Neu5Acα2-3Galß1-3GlcNAcß1-3Galß1-4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-ß-lacto-N-bioside I (Galß1-3GlcNAcß-pNP) and GalNAcß1-3GlcNAcß-pNP. GalNAcß1-3GlcNAcß linkage has been found in O-mannosyl glycans of α-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca(2+) and Mg(2+)) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches.

Surveillance for an outbreak of Encephalitozoon cuniculi infection in rabbits housed at a zoo and biosecurity countermeasures.

An outbreak of encephalitozoonosis occurred in a rabbit colony at a zoo in Japan. Throughout the two years after the onset, all 42 rabbits were investigated clinically, pathologically and serologically for prevention and control of the disease. Eleven rabbits (11/42, 26.2%) showed clinical symptoms. Of 38 rabbits examined to detect specific antibodies against Encephalitozoon cuniculi, 71.1% (n=27) were found seropositive; 20 out of 30 clinically healthy rabbits (except for 8 clinical cases) were seropositive. The infection rate was 76.2% (32/42), including 5 pathologically diagnosed cases. The results of serological survey revealed that asymptomatic infection was widespread, even among clinically healthy rabbits. However, encephalitozoonosis was not found by pathological examination in any other species of animals kept in the same area within the zoo. Isolation and elimination of the rabbits with suspected infection based on the results of serological examination were carried out immediately; however, encephalitozoonosis continued to occur sporadically. Therefore, all the remaining rabbits were finally slaughtered. Then, the facility was closed, and all the equipment was disinfected. After a two-month interval, founder rabbits were introduced from encephalitozoonosis-free rabbitries for new colony formation. Since then, encephalitozoonosis has not been seen in any animals at the zoo. In this study, biosecurity countermeasures including staff education, epidemiological surveillance and application of an "all-out and all-in" system for rabbit colony establishment based on serological examination were successfully accomplished with regard to animal hygiene and public health for the eradication of E. cuniculi.

Mechanism for the hydrolysis of hyaluronan oligosaccharides by bovine testicular hyaluronidase.

Synthetic hyaluronan oligosaccharides with defined structures and their pyridylaminated derivatives were used to investigate the mechanism of hydrolysis of hyaluronan by bovine testicular hyaluronidase. The products of the hydrolysis were analyzed by HPLC and ion-spray mass spectroscopy (MS). It was confirmed that the minimum substrate for bovine testicular hyaluronidase is the hyaluronan hexasaccharide, even though it is a poor substrate that is barely cleaved, even on prolonged incubation. When hyaluronan octasaccharide was the substrate, increasing amounts of tetrasaccharide and hexasaccharide were produced with increasing time of incubation. Whereas disaccharide was not detectable in the reaction mixture by HPLC, MS analysis revealed trace amounts. The data suggest that the enzyme generates a disaccharide intermediate from hyaluronan oligosaccharide, the majority of which is transferred to the nonreducing ends of other oligosaccharides, only traces being released as free disaccharide. When hyaluronan octasaccharide, with an unsaturated glucuronic acid at the nonreducing end, was used as a substrate, only a tetrasaccharide was detected by HPLC. However, MS showed that the product was a mixture of equal amounts of two tetrasaccharides, one with and the other without the unsaturated glucuronic acid. This suggests that, in the case of substrates with a double bond at the nonreducing end, a tetrasaccharide is cleaved off instead of a disaccharide. The results of the experiments with pyridylaminated oligosaccharides were entirely consistent with these conclusions, and in addition showed the importance of the reducing end of the substrate for the enzyme to recognize the length of the saccharide.

Penta-aqua-tri-µ(3)-hydroxido-tris-(imino-diacetato)-µ(3)-oxido-tetra-hedro-calcium(II)tricobalt(III) 2.54-hydrate.

In the title compound, [CaCo(3)(C(4)H(5)NO(4))(3)(OH)(3)O(H(2)O)(5)]·2.54H(2)O, the Co atom is octa-hedrally coordinated by one imino-diacetate (ida) dianion as a facial O,N,O'-tridentate ligand, two µ(3)-OH groups and one µ(3)-O ligand, forming an partial Co(3)O(4) cubane cluster. This unit coordinates to a Ca(II) cation in an O,O',O''-tridentate fashion, generating a distorted CaCo(3)O(4) cubane-type cluster. The Ca-µ(3)-O distances [2.429 (5)-2.572 (6) Å] are much longer than the Co-µ(3)-O bonds [1.895 (5)-1.941 (5) Å]. The Ca(II) cation is also coord-inated by five water mol-ecules with Ca-O distances in the range 2.355 (6)-2.543 (6) Å. There are three additional uncoordinated water mol-ecules in the asymmetric unit, the occupancy of which refined to 0.54 (3). In H(2)O (or D(2)O), the title complex hydrolyses to Ca(2+) (aq) cations and [Co(3)(ida)(3)(µ(2)-OH)(3)(µ(3)-O)](2-) anions.

Novel proteoglycan glycotechnology: chemoenzymatic synthesis of chondroitin sulfate-containing molecules and its application.

Proteoglycans consist of a protein core, with one or more glycosaminoglycan chains (i.e., chondroitin sulfate, dermatan sulfate and heparin sulfate) bound covalently to it. The glycosaminoglycan chains account for many of the functions and properties of proteoglycans. The development of proteoglycan glycotechnology to exploit the functionality of glycosaminoglycan chains is an extremely important aspect of glycobiology. Here we describe an efficient and widely applicable method for chemoenzymatic synthesis of conjugate compounds comprising intact long chondroitin sulfate (ChS) chains. An alkyne containing ChS was prepared by an enzymatic transfer reaction and linked with a chemically synthesized core compound containing an azido group using click chemistry. This method enabled highly efficient introduction of ChS into target materials. Furthermore, the ChS-introduced compounds had marked stability against proteolysis, and the chemically linked ChS chain contributed to the stability of these core compounds. We believe the present method will contribute to the development of proteoglycan glycobiology and technology.

[Prevalence of intestinal canine and feline parasites in Saitama Prefecture, Japan].

We studied the prevalence of intestinal parasites in animal companions in Saitama Prefecture, Japan, where no detailed data is currently available. Between May 1999 and December 2007, fecal samples were collected from 906 dogs and 1,079 cats in public animal shelters and examined by microscopy. Overall, prevalence of intestinal parasites in dogs was 38.6% and cats 43.1%. Trichuris vulpis was the most prevalent canine parasite species (22.3%), followed by Toxocara canis (12.5%), Ancylostoma caninum (10.4%), Isospora ohioensis (2.1%), Spirometra erinaceieuropaei (1.0%), Crypstosporidium sp. (0.9%), Giardia intestinalis (0.9%), I. canis (0.6%), Taeniidae (0.3%), Dipylidium caninum (0.2%), Echinostoma sp. (0.1%), and Pentatrichomonas hominis (0.1%). T. cati was the most prevalent feline parasite species (21.8%), followed by A. tubaeforme (13.2%), S. erinaceieuropaei (8.3%), I. felis (4.5%), Cryptosporidium sp. (2.8%), I. rivolta (2.2%), Pharyngostomum cordatum (1.6%), D. caninum (1.4%), Eimeria sp. (0.3%), Taeniidae (0.2%), Trichuris sp. (0.2%), Capillaria sp. (0.1%), Diphyllobothrium nihonkaiense (0.1%), and Metagonimus yokogawai (0.1%). Further molecular analysis to identify canine Taeniidae species and canine and feline Cryptosporidium species identified one canine taeniid positive species as Echinococcus multilocularis. Cryptosporidium species were identified as C. canis and C. felis. Parasites E. multilocularis and Cryptosporidium spp. in animal hosts were the first to be recorded in this prefecture. Compared to previous surveys conducted in the same area, the endemicity of some parasites appeared to have decreased, but some others remain. Given that most of these parasites have zoonotic potential, indicates the importance of having current data on parasite dissemination among animal companions. Government public health agencies should be responsible for educating pet owners about the control and prevention of zoonotic risk from such parasites.

Novel products in hyaluronan digested by bovine testicular hyaluronidase.

When the products of hyaluronan (HA) digested by bovine testicular hyaluronidase (BTH) were analyzed by high-performance liquid chromatography (HPLC), minor peaks were detected just before the main even-numbered oligosaccharide peaks. The amount of each minor peak was dependent on the reaction conditions for transglycosylation, rather than hydrolysis, by the BTH. Mainly based on HPLC and MS analysis, each minor peak was found to correspond to its oligosaccharide with one N-acetyl group removed from the reducing terminal N-acetylglucosamine. Enzymatic studies showed that the N-deacetylation activity was closely related to reaction temperature, pH, and the concentration of NaCl contained in the buffer, and glycosaminoglycan types and chain lengths of substrates. These findings strongly suggest that the N-deacetylation reaction in minor peaks was due to a novel enzyme contaminant in the BTH, N-deacetylase, that carries out N-deacetylation at the reducing terminal N-acetylglucosamine of oligosaccharides and is dependent on HA hydrolysis by BTH.

Bifidobacterium bifidum lacto-N-biosidase, a critical enzyme for the degradation of human milk oligosaccharides with a type 1 structure.

Breast-fed infants often have intestinal microbiota dominated by bifidobacteria in contrast to formula-fed infants. We found that several bifidobacterial strains produce a lacto-N-biosidase that liberates lacto-N-biose I (Galbeta1,3GlcNAc; type 1 chain) from lacto-N-tetraose (Galbeta1,3GlcNAcbeta1,3Galbeta1,4Glc), which is a major component of human milk oligosaccharides, and subsequently isolated the gene from Bifidobacterium bifidum JCM1254. The gene, designated lnbB, was predicted to encode a protein of 1,112 amino acid residues containing a signal peptide and a membrane anchor at the N and C termini, respectively, and to possess the domain of glycoside hydrolase family 20, carbohydrate binding module 32, and bacterial immunoglobulin-like domain 2, in that order, from the N terminus. The recombinant enzyme showed substrate preference for the unmodified beta-linked lacto-N-biose I structure. Lacto-N-biosidase activity was found in several bifidobacterial strains, but not in the other enteric bacteria, such as clostridia, bacteroides, and lactobacilli, under the tested conditions. These results, together with our recent finding of a novel metabolic pathway specific for lacto-N-biose I in bifidobacterial cells, suggest that some of the bifidobacterial strains are highly adapted for utilizing human milk oligosaccharides with a type 1 chain.

Total synthesis of 6-O-sulfo-sialylparagloboside: a widely useful glycoprobe for biochemical research.

The total synthesis of 6-O-sulfo-sialylparagloboside is described. A suitably protected beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-D-GlcpOSE derivative was glycosylated with an alpha-D-Neup5Ac-(2-->3)-D-Galp derived imidate to give the corresponding protected alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-d-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-D-GlcpOSE pentasaccharide derivative. Proper manipulation of the protecting groups of the pentasaccharide afforded the corresponding glycosyl imidate, which was coupled with (2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol. Selective reduction of the azido group, N-acylation with octadecanoic acid, 6-O-sulfation of the GlcpNAc residue, and complete removal of the protecting groups gave the desired 6-O-sulfo-sialylparagloboside.

myo-Inositol catabolism in Bacillus subtilis.

The iolABCDEFGHIJ operon of Bacillus subtilis is responsible for myo-inositol catabolism involving multiple and stepwise reactions. Previous studies demonstrated that IolG and IolE are the enzymes for the first and second reactions, namely dehydrogenation of myo-inositol to give 2-keto-myo-inositol and the subsequent dehydration to 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione. In the present studies the third reaction was shown to be the hydrolysis of 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione catalyzed by IolD to yield 5-deoxy-d-glucuronic acid. The fourth reaction was the isomerization of 5-deoxy-D-glucuronic acid by IolB to produce 2-deoxy-5-keto-D-gluconic acid. Next, in the fifth reaction 2-deoxy-5-keto-D-gluconic acid was phosphorylated by IolC kinase to yield 2-deoxy-5-keto-D-gluconic acid 6-phosphate. IolR is known as the repressor controlling transcription of the iol operon. In this reaction 2-deoxy-5-keto-D-gluconic acid 6-phosphate appeared to be the intermediate acting as inducer by antagonizing DNA binding of IolR. Finally, IolJ turned out to be the specific aldolase for the sixth reaction, the cleavage of 2-deoxy-5-keto-D-gluconic acid 6-phosphate into dihydroxyacetone phosphate and malonic semialdehyde. The former is a known glycolytic intermediate, and the latter was previously shown to be converted to acetyl-CoA and CO(2) by a reaction catalyzed by IolA. The net result of the inositol catabolic pathway in B. subtilis is, thus, the conversion of myo-inositol to an equimolar mixture of dihydroxyacetone phosphate, acetyl-CoA, and CO(2).

Up-regulation of hyaluronan synthase genes in cultured human epidermal keratinocytes by UVB irradiation.

Hyaluronan controls keratinocyte proliferation and regeneration. We examined effect of UV on the expression of hyaluronan synthases (HASs) and hyaluronidases in cultured normal human newborn foreskin epidermal keratinocytes, NHEK(F). HAS3 mRNA was expressed predominantly and HAS2 mRNA expressed in lesser amounts and both were up-regulated after a single irradiation with moderate UVB but hyaluronidases was unchanged. Increased accumulation of hyaluronan in the culture medium mirrored the UVB-induced increase in the mRNA levels of HAS3 and HAS2. Unexpectedly, hyaluronan derived from UVB-irradiated and non-irradiated cells had identical size distribution. Increased expression of KGF and IL-1beta was detected just prior to the increase of HAS3 and HAS2 mRNAs after UVB irradiation. Antibody-neutralization study revealed that KGF and/or IL-1beta were at least involved in the up-regulation of HAS3 and HAS2 expressions. UVB-irradiated cells may enhance hyaluronan production to maintain homeostasis through up-regulation of HAS3 and HAS2 genes via cytokine response mechanism.