Rational design of eukaryotic riboswitches that up-regulate IRES-mediated translation initiation with high switching efficiency through a kinetic trapping mechanism in vitro.

In general, riboswitches functioning through a cotranscriptional kinetic trapping mechanism (kt-riboswitches) show higher switching efficiencies in response to practical concentrations of their ligand molecules than eq-riboswitches, which function by an equilibrium mechanism. However, the former have been much more difficult to design due to their more complex mechanism. We here successfully developed a rational strategy for constructing eukaryotic kt-riboswitches that ligand-dependently enhance translation initiation mediated by an internal ribosome entry site (IRES). This was achieved both by utilizing some predicted structural features of a highly efficient bacterial kt-riboswitch identified through screening and by completely decoupling an aptamer domain from the IRES. Three kt-riboswitches optimized through this strategy, each responding to a different ligand, exhibited three- to sevenfold higher induction ratios (up to ∼90) than previously optimized eq-riboswitches regulating the same IRES-mediated translation in wheat germ extract. Because the IRES used functions well in various eukaryotic expression systems, these types of kt-riboswitches are expected to serve as major eukaryotic gene regulators based on RNA. In addition, the present strategy could be applied to the rational construction of other types of kt-riboswitches, including those functioning in bacterial expression systems.

Effects of Sichuan pepper (huajiao) powder on disease activity and caecal microbiota of dextran sodium sulphate-induced inflammatory bowel disease mouse model.

BACKGROUND: Sichuan pepper [Zanthoxylum bungeanum; huajiao (HJ)] is a widely used spice in China and has better antioxidative, anti-glycation, and bile acid-lowering properties than cumin and coriander seeds. HJ affects inflammation-related cytokines and caecal microbiota in mice fed a low-fibre and high-sucrose diet. METHODS AND RESULTS: To determine the ameliorative effect of HJ on inflammatory bowel disease, C57BL/6 mice were divided into three groups and fed distilled water (control) or 3% (w/v) dextran sodium sulphate (DSS) in drinking water with normal chow containing 0% or 5% (w/w) HJ powder for seven days. After 6 days of feeding, diarrhoea, decreased body weight, and blood in faeces were observed in the DSS group. DSS treatment increased the spleen weight and damaged the colon tissue. These inflammatory indices were inhibited by HJ treatment. Amplicon sequencing of the 16S rDNA (V4) gene of the caecal content revealed a decrease in the alpha diversity (Simpson index D) in the DSS treatment group compared to the control group. The abundance of caecal Desulfovibrio, an inflammation-related genus, was higher and the caecal Lachnospiraceae and Bacteroides levels were lower in the DSS-treated mice than those in the control mice. However, HJ suppressed the DSS-induced changes in the caecal microbiota. CONCLUSION: HJ intake contributes to the reduction in inflammation and maintenance of the gut microbiota. However, the strong antioxidant properties of phenolic compounds and fermentability of water-soluble dietary fibres in HJ and their relationship with other functional properties warrant further investigation.

Impact of salivary flow inhibition on masticatory behaviours in healthy individuals.

BACKGROUND: It remains unclear how the salivary flow and the fat content of food affect bolus formation during mastication. OBJECTIVES: We aimed to clarify: (1) how hyposalivation affects jaw-closing and hyoid-elevating muscle activities in bolus formation, and (2) if the effect of hyposalivation on muscle activity depends on the fat content of food. METHODS: Eighteen healthy male volunteers were instructed to freely ingest four test foods: Plain, Fat without seasoning, Fat with seasoning, and Soft rice crackers. Masseter and suprahyoid electromyographic activities were recorded before and 30 min after the administration of atropine sulfate, a muscarinic receptor antagonist that induces hyposalivation. RESULTS: Hyposalivation extended the masticatory duration significantly in all the test foods except Fat with seasoning. Masticatory cycle time was significantly longer with vs without hyposalivation for the Soft (p = .011). Suprahyoid activity/cycle was significantly greater with vs without hyposalivation (p = .013). Masticatory cycle time was significantly longer at the late stage with vs without hyposalivation for the Soft (p < .001). Suprahyoid activity/cycle was significantly greater at the middle (p = .045) and late stages (p = .002) with vs without hyposalivation for the Soft and greater at the late stage with vs without hyposalivation for the Plain (p = .043). Changes in masticatory cycle time and suprahyoid activity/cycle for these foods had significantly positive relationship (p < .001). CONCLUSION: Hyposalivation-induced changes in masticatory behaviours resulted from the middle and late stage suprahyoid activity. Fat content and seasoning compensate for salivary flow inhibition.

Detection of typical indigenous gut bacteria related to turmeric (Curcuma longa) powder in mouse caecum and human faecal cultures.

BACKGROUND: Turmeric (Curcuma longa; TM) is widely used as a spice and possesses anti-inflammatory, antioxidant, and antibacterial properties. The relationship between TM functions and gut microbiota is still unclear. METHODS AND RESULTS: To investigate the effect of TM on gut microbiota and to identify indigenous gut bacteria that are responsive to TM, we fed Institute of Cancer Research mice a diet containing either no fibre (NF, n = 6) or 5% (w/w) TM (n = 6) for 14 days. Moreover, we obtained human stool samples from four healthy volunteers and incubated the samples without (control) or with 2% (w/v) TM at 37 °C for 24 h. Subsequently, microbiota analysis in murine caecal samples and human faecal cultures was performed using 16S rRNA (V4) amplicon sequencing. Higher faecal weights (p < 0.01) and lower plasma triacylglycerol levels (p < 0.05) were measured in the TM-fed mice than in the NF-fed mice. Furthermore, TM feeding increased the abundance of butyrate-producing and other short-chain fatty acid (SCFA)-producing bacteria in mice as well as in human faecal cultures, and Roseburia bacteria were detected as TM-responsive indigenous gut bacteria (TM-RIB) both in mice and in human faecal cultures. Lastly, in the case of human faecal cultures, SCFA contents and antioxidant properties were higher in TM cultures than in control cultures (p < 0.05). CONCLUSION: TM appears to hold the potential to positively affect the host by altering the gut microbiota. Further studies are required to clarify the synergistic effects of TM and TM-RIB.

The effect of Sichuan pepper on gut microbiota in mice fed a high-sucrose and low-dietary fibre diet.

Sichuan pepper (Zanthoxylum bungeanum, HJ), a spice widely used in China, has antioxidative, anti-inflammatory, antibacterial, and anti-obesity properties. In this study, to confirm the value of HJ as a functional food, the in vitro antioxidant and bile acid-lowering capacities, as well as the effects on caecal microbiota, were compared with those of cumin (Cuminum cyminum, CM) and coriander (Coriandrum sativum, CR) seeds in Institute of Cancer Research (ICR) mice fed a high-sucrose and low-dietary fibre diet. The total phenolic content, superoxide anion radical-scavenging capacity, and Fe-reducing power of the HJ aqueous solution were higher than those of CM and CR (p < 0.05). The bile acid (taurocholic, glycocholic, and deoxycholic acids)-lowering capacity of the HJ suspension was also higher than those of CM and CR. Compared with mice fed a control diet (no fibre, NF), caecal Lactobacillus gasseri- and Muribaculum intestinale-like bacteria were higher in mice fed a diet containing 5% (w/w) of CM, CR, or HJ for 14 days. Bifidobacterium pseudolongum-, Lactobacillus murinus/animalis-, and Faecalibaculum rodentium-like bacteria were significantly increased, while Desulfovibrio-like bacteria were significantly decreased in the HJ group. In addition, CM and HJ may benefit specific metabolic functions of gut microbiota, such as starch, sucrose, and tyrosine metabolism. The tumour necrosis factor (TNF-α) concentration in the spleen tissue of ICR mice was decreased by the intake of spices. However, there were no changes in interleukin-2 (IL-2) and IL-10 levels in HJ fed mice. These results suggested that HJ has potential as a functional food related to gut microbiota. KEY POINTS: • Bididobacterium and Faecalibaculum in mice gut microbiota are increased by Sichuan pepper (HJ). • Desulfovibrionaceae, an inflammatory LPS producer, in mice gut microbiota is decreased by HJ. • HJ decreases pro-inflammatory TNF both in murine spleen tissue and in vitro macrophages.

Transition of microbial contamination on the surface of carcass during the cattle slaughter process.

In this study, we aimed to investigate how microbial contamination progresses on the carcass surface during the slaughter process. Cattle carcasses were tracked during a series of slaughter processes (five steps), and carcass surfaces (four parts) and equipment (nine types) were swabbed to investigate the bacterial contamination. Results showed that the outer surface (near the rear region of the flank [Top round] and [Top sirloin butt]) had significantly higher total viable counts (TVCs) than inner surface (p < 0.01) and that TVCs gradually decreased along the process. Enterobacteriaceae (EB) counts were high on the splitting saw and in top round region, and EB was detected on the inner surface of the carcasses. Furthermore, in some carcasses, Yersinia spp., Serratia spp., and Clostridium spp. present on top round and top sirloin butt immediately after skinning and remained on the carcass surface after the final process. These bacterial groups are detrimental to beef quality as they can grow in the package during cold distribution. Our results show that the skinning process is the most prone to microbial contamination, including psychrotolerant microorganisms. Moreover, this study provides information for understanding the dynamics of microbial contamination in the cattle slaughter process.

Eukaryotic artificial ON-riboswitches that respond efficiently to mid-sized short peptides.

We chose two types of mid-sized Arg-rich peptides (Rev-pep and Tat-pep) as ligands and used their aptamers to construct efficient eukaryotic ON-riboswitches (ligand-dependently upregulating riboswitches). Due to the aptamers' high affinities, the best Rev-pep-responsive and Tat-pep-responsive riboswitches obtained showed much higher switching efficiencies at low ligand concentrations than small ligand-responsive ON-riboswitches in the same mechanism. In addition, despite the high sequence similarity of Rev-pep and Tat-pep, the two best riboswitches were almost insensitive to each other's peptide ligand. Considering the high responsiveness and specificity along with the versatility of the expression platform used and the applicability of Arg-rich peptides, this orthogonal pair of riboswitches would be widely useful eukaryotic gene regulators or biosensors.

Detection of indigenous gut bacteria related to red chilli pepper (Capsicum annuum) in murine caecum and human faecal cultures.

BACKGROUND: Red chilli pepper (Capsicum annuum; RP) is a popular spice containing the active compound capsaicin. Indigenous gut bacteria and metabolism can affect host health. The functions of capsaicin, including the regulation of metabolic health and anti-oxidant properties, may be correlated with the gut microbiota. METHODS: To identify indigenous gut bacteria that are responsive to RP, Institute of Cancer Research mice fed a diet with no fibre or with 5% (w/w) RP for 14 days. Additionally, human stool samples collected from four healthy volunteers were incubated without (control) or with 2% (w/v) RP at 37 °C for 24 h. Microbiota in murine caecal samples and human faecal cultures were analysed using 16S rRNA (V4) amplicon sequencing. RESULTS: Compared with the microbiota in mice fed no-fibre diets, Lachnospiraceae spp.-, Muribaculaceae spp.-, and Phacaeicola vulgatus-like bacteria were defined as murine RP-responsive indigenous gut bacteria (RP-RIB). In the human faecal cultures, acetate and propionate levels were higher in RP cultures than in the control cultures. Subdoligranulum spp.-, Blautia spp.-, Faecalibacterium prausnitzii-, P. vulgatus-, and Prevotella copri-like bacteria were defined as human RP-RIB. Compared with control culture Fe-reducing power was increased in the culture with RP. CONCLUSION: RP increases the amount of short-chain fatty acid-producing bacteria and beneficial gut bacteria in mouse and human faecal cultures. Overall, RP could have a positive effect on the host by altering the gut microbiota.

Dynamics of microbiota in the imported beef primal cuts during storage at different chilled temperatures.

Primal cuts of Australian beef transported by sea were stored under different chilled temperatures (0, 2, and 4 °C) for 6 weeks in different packaging conditions (aerobic or anaerobic packaging). The number of microorganisms and the transition of the microbiota were investigated using culture methods and amplicon sequencing. After 6 weeks of storage, the beef tended to show a high total viable count under aerobic packaging conditions and a high lactic acid bacteria count under anaerobic packaging conditions. The result of amplicon sequencing analysis showed that different beef samples had different predominant bacterial groups. Moreover, at high storage temperatures, Serratia sp. having high putrefactive activity showed increased abundance, while at low storage temperatures, Lactobacillus sp. showed increased abundance. Thus, differences in the packaging conditions and distribution temperatures after import affect the number of bacteria and the type of microorganisms in the Australian beef primal cuts, which may affect their quality.

Coupled in vitro transcription/translation based on wheat germ extract for efficient expression from PCR-generated templates in short-time batch reactions.

We successfully constructed a coupled in vitro transcription/translation (cIVTT) system based on wheat germ extract (WGE) for efficient expression from PCR-generated DNA templates in short-time (∼3-h) batch reactions. The productivity of this system under optimized conditions was 85 µg (2.8 nmol) per 1 mL of reaction solution (corresponding to 425 µg per 1 mL of WGE), which was about 9-fold higher than that by the conventional batch method using mRNA as a template. The DNA template concentration required for efficient cIVTT was as low as 2.5 nM, which is much lower than those required for other eukaryotic cIVTT systems to maximize their productivity (30-50 nM). The productivity of the present system with a 2.5 nM template was 80-fold and 4-fold higher than that of a commercially available WGE-based cIVTT system with a 2.5 nM and a 40 nM template, respectively. In addition, the present system functioned well in a liposome (i.e., in an artificial cell) without a loss of productivity. Given that WGE-based systems have the advantage of being suitable for the expression of a broad range of proteins, the present cIVTT system is expected to be widely used in future cell-free synthetic biology.

Dynamics of microbiota in Japanese Black beef stored for a long time under chilled conditions.

In this study, changes in the microbiota of Japanese Black beef carcasses, which are expected to be transported for a long time in chilled temperatures, were investigated. Three Japanese Black beef samples (carcasses A, B, and C) immediately after slaughter were stored at 0 °C for 15 weeks under aerobic and vacuum conditions. The initial bacterial counts were 50 CFU/g for carcass A and less than the reliable quantitative detection limit for carcasses B and C. Under aerobic storage conditions, the bacterial count increased to 8.0 log CFU/g or higher, which is a measure of putrefaction, at 6-9 weeks. Under anaerobic storage conditions, the bacterial counts of carcasses A and C reached 3.5-6.5 log CFU/g, but carcass B showed no bacterial growth during the 15-week storage period. The predominant group was Pseudomonas spp. under aerobic conditions and Serratia spp. under anaerobic conditions. To the best of our knowledge, there is no previous study investigating the transition of microbiota when Japanese Black beef is stored at low temperatures for a long period of time, and the results of this study are considered very important findings for the expansion of international trade of Japanese Beef in the future.

In vitro antioxidant, anti-glycation and immunomodulation activities of fermented blue-green algae Aphanizomenon flos-aquae.

To clarify the antioxidant, anti-glycation and immunomodulatory capacities of fermented blue-green algae Aphanizomenon flos-aquae (AFA), hot aqueous extract suspensions made from 10% AFA were fermented by Lactobacillus plantarum AN7 and Lactococcus lactis subsp. lactis Kushiro-L2 strains isolated from a coastal region of Japan. The DPPH and O2- radical scavenging capacities and Fe-reducing power were increased in the fermented AFA. The increased DPPH radical scavenging capacity of the fermented AFA was fractionated to mainly < 3 kDa and 30-100 kDa. The increased O2- radical scavenging capacities were fractionated to mainly < 3 kDa. Anti-glycation activity in BSA-fructose model rather than BSA-methylglyoxal model was increased by the fermentation. The increased anti-glycation activity was fractionated to mainly 30-100 kDa. The NO concentration in the murine macrophage RAW264.7 culture media was high with the fermented AFA. The increased immunomodulation capacity was also fractionated to mainly 30-100 kDa. These results suggest that the fermented AFA is a more useful material for health foods and supplements.

Susceptibility of gut indigenous lactic acid bacteria in BALB/c mice to oral administered Lactobacillus plantarum.

Cells of Lactobacillus plantarum strains AN1 and Tennozu-SU2 exert anti-inflammatory responses in ICR mouse models of inflammatory bowel disease and protective effects against S. Typhimurium infection in BALB/c mice, respectively. To clarify the existence of L. plantarum-susceptible gut indigenous bacteria, AN1 and Tennozu-SU2 cells were administered to BALB/c mice via drinking water. Gene amplicon sequencing of 16S rRNA of caecal content revealed that the AN1 and Tennozu-SU2 cells affected the abundance of caecal indigenous lactobacilli, but the effect on the dominant Clostridiales and Bacteroidales was not clear. With Blood and Liver (BL) agar containing 5% v/v horse blood, six typical colonies from faecal samples were detected as the principal lactobacilli. Among them, two typical colonies were isolated and identified to be AN1 and Tennozu-SU2. Two and one typical colonies detected in all mice were identified to be L. reuteri and L. murinus, respectively. The other one was identified and estimated to be indigenous L. plantarum detected in the Tennozu-SU2 group.

[Development of the Genus and Species Determination Method for Histamine Producing Bacteria Isolated from Fishery Product with High-Resolution Melting Analysis].

Histamine in foods with a high histidine content may be produced by bacteria with histidine decarboxylase activity. Consumption of food enriched in histamine can produce symptoms of histamine poisoning that include flushing, headache, and urticaria. The number of histamine poisoning cases in Japan has decreased with developments in food hygiene management technology. However, approximately 10 cases are still reported each year. In addition, there have been cases where histamine was detected in the end products, prompting large product recalls. To prevent and identify causes of histamine toxicity, manufacturers must identify the bacteria causing the illness. A simple method of identification is needed, since sequence-based identification is complicated to perform and the analysis takes a long time. High-Resolution Melting Analysis (HRMA) is a method that detects differences in the base sequences of PCR products manifested as varied melting temperatures of double-stranded DNA. The present study was intended to develop a rapid identification method for major histamine-producing bacteria using HRMA. Species-specific HRMA primers were designed that specifically targeted the hdcA gene of 20 Gram-negative histamine-producing bacterial strains. The designed primers were used for HRM analysis of the 20 histamine-producing bacterial strains. The strains were divided into three groups (A, B, and C) based on differences in melting temperature values obtained by Tm Calling analysis program. Group A comprised terrestrial bacteria, such as Morganella, Enterobacter, and Raoultella, while Groups B and C comprised marine bacteria, such as those belonging to the genera Vibrio and Photobacterium. The melting profiles obtained in Group A by HRMA were used to identify the aforementioned terrestrial bacteria. The findings indicated that HRMA can easily identify the major gram-negative histamine-producing bacteria. A flow chart was created to identify histamine-producing bacterial species. This method enables the identification of histamine-producing bacterial species more quickly and easily than conventional sequence-based methods. Therefore, the method could be valuable for food companies to screen raw materials and products and track the source of contamination, which will in turn contribute to the prevention of histamine-food poisoning and investigation of its causes.

Canonical translation-modulating OFF-riboswitches with a single aptamer binding to a small molecule that function in a higher eukaryotic cell-free expression system.

We have found that OFF-riboswitches that ligand-dependently downregulate the canonical translation in a higher eukaryotic expression system (wheat germ extract) can be easily created by inserting a single aptamer into the 5' untranslated region (UTR) of mRNA, even if its ligand is as small as theophylline. The key is the position of the inserted aptamer: the 5' end (+0 position) is much better than other positions for inhibiting canonical translation with the aptamer-ligand complex. The data showed that ribosome loading is suppressed by a rigid structure in the 5' end, and this suppression is dependent on the structure's stability but not on its size. Although this preference of aptamer insertion point contradicts the results in a lower eukaryote, it accords with the fact that the 5'-end structural hindrance is more effective for blocking the ribosome in higher eukaryotes. Therefore, the present type of OFF-riboswitch would function in various higher eukaryotic expression systems.

Effects of fermented green-loofah and green-papaya on nitric oxide secretion from murine macrophage raw 264.7 cells.

To clarify the effect of lactic acid bacteria (LAB) fermentation on the immunomodulation capacity of green-loofah and green-papaya, aqueous suspensions prepared from the fresh and dry-powdered vegetables were fermented by Lactococcus lactis subsp. lactis Uruma-SU1 and Lactobacillus plantarum Uruma-SU4. Fermented and non-fermented suspensions were added to murine macrophage RAW264.7 culture with and without Escherichia coli O111 lipopolysaccharide (LPS). In the absence of LPS, nitric oxide (NO) secretion was elevated significantly in LAB fermented suspensions compared to that in non-fermented suspensions. NO production in fermented suspensions was observed even at low sample concentrations, but it was attenuated in the centrifuged supernatant. With LPS treatment, inhibition of NO secretion was shown with the high concentration of the non-fermented and also fermented samples. These results suggest that fermented green-loofah and green-papaya suspensions can play both immunostimulatory and anti-inflammatory roles at low and high doses, respectively.

Bacterial and fungal microbiota of spontaneously fermented Chinese products, Rubing milk cake and Yan-cai vegetable pickles.

The Rubing milk cake from Yunnan and the Yan-cai vegetable pickles from Guangdong are traditional spontaneously fermented foods in China. We evaluated the microbial properties of these products with the analysis of their bacterial and fungal microbiota using classical culture-dependent and culture-independent methods, including a 16S rDNA gene (V4) and an internal transcribed spacer (ITS) region pyrosequencing method with MiSeq system. The viable lactic acid bacteria (LAB) count was 8 and 6 log colony-forming units (CFU)/g in Rubing and Yan-cai samples, respectively. The yeast count was approximately 100-1000 times less than the LAB count in most samples, except one Yan-cai sample. In addition, the gram-negative rod count in half of the samples was similar to the LAB count. Pyrosequencing results revealed the high abundance (10%-20%) of gram-negative Pseudomonas spp. and Enterobacteriaceae in these samples. These results suggest that some of these traditional foods are undesirable as ready-to-eat (RTE) foods, even when these are typical lactic acid fermented foods.

Optical readout of hydrogen storage in films of Au and Pd.

For hydrogen sensor and storage applications, films of Au and Pd were (i) co-sputtered at different rates or (ii) deposited in a sequentially alternating fashion to create a layered structure on a cover glass. Peculiarities of hydrogen uptake and release were optically monitored using 1.3 µm wavelength light. Increase of optical transmission was observed for hydrogenated Pd-rich films of 10-30 nm thickness. Up to a three times slower hydrogen release took place as compared with the hydrogen uptake. Compositional ratio of Au:Pd and thermal treatment of films provided control over the optical extinction changes and hydrogen uptake/release time constants. Higher uptake and release rates were observed in the annealed Au:Pd films as compared to those deposited at room temperature and were faster for the Auricher films. Three main parameters relevant for sensors: sensitivity, selectivity, stability (reproducibility) are discussed together with the hydrogenation mechanism in Au:Pd alloys.

Effect of glucose on Listeria monocytogenes biofilm formation, and assessment of the biofilm's sanitation tolerance.

Listeria monocytogenes is an important cause of human foodborne infections and its ability to form biofilms is a serious concern to the food industry. To reveal the effect of glucose conditions on biofilm formation of L. monocytogenes, 20 strains were investigated under three glucose conditions (0.1, 1.0, and 2.0% w v(-1)) by quantifying the number of cells in the biofilm and observing the biofilm structure after incubation for 24, 72, and 168 h. In addition, the biofilms were examined for their sensitivity to sodium hypochlorite. It was found that high concentrations of glucose reduced the number of viable cells in the biofilms and increased extracellular polymeric substance production. Moreover, biofilms formed at a glucose concentration of 1.0 or 2.0% were more resistant to sodium hypochlorite than those formed at a glucose concentration of 0.1%. This knowledge can be used to help design the most appropriate sanitation strategy.

Adaptation of Campylobacter jejuni to biocides used in the food industry affects biofilm structure, adhesion strength, and cross-resistance to clinical antimicrobial compounds.

The emergence of biocide-adapted Campylobacter jejuni strains that developed into biofilms and their potential to develop clinical resistance to antimicrobial compounds was studied. C. jejuni was grown in sub-lethal concentrations of five biocides used in the food industry. C. jejuni exhibited adaptation to these biocides with increased minimum inhibitory concentrations. The 3-D structures of the biofilms produced by the biocide-adapted cells were investigated by atomic force microscopy (AFM). The results revealed marked variability in biofilm architecture, including ice-crystal-like structures. Adaptation to the biocides enhanced biofilm formation, with significant increases in biovolume, surface coverage, roughness, and the surface adhesion force of the biofilms. Adaptation to commercial biocides induced resistance to kanamycin and streptomycin. This study suggests that the inappropriate use of biocides may lead to cells being exposed to them at sub-lethal concentrations, which can result in adaptation of the pathogens to the biocides and a subsequent risk to public health.