Clinical Characteristics and Progression of Pachychoroid and Conventional Geographic Atrophy.

Purpose: To elucidate the clinical characteristics and progression rates of pachychoroid and conventional geographic atrophy (GA). Design: Retrospective, multicenter, observational study. Participants: A total of 173 eyes from 173 patients (38 eyes with pachychoroid GA and 135 with conventional GA) from 6 university hospitals in Japan were included. All patients were Japanese, aged ≥50 years and with fundus autofluorescence images having analyzable image quality. A total of 101 eyes (22 with pachychoroid GA and 79 with conventional GA) were included in the follow-up group. Methods: The studied eyes were classified as having pachychoroid or conventional GA; the former was diagnosed if the eye had features of pachychoroid and no drusen. The GA area was semiautomatically measured on fundus autofluorescence images, and the GA progression rate was calculated for the follow-up group. Multivariable linear regression analysis was used to determine whether the rate of GA progression was associated with GA subtype. Main Outcome Measures: Clinical characteristics and progression rates of pachychoroid and conventional GA. Results: The pachychoroid GA group was significantly younger (70.3 vs. 78.7 years; P < 0.001), more male-dominant (89.5 vs. 55.6%; P < 0.001), and had better best-corrected visual acuity (0.15 vs. 0.40 in logarithm of the minimum angle of resolution; P = 0.002), thicker choroid (312.4 vs. 161.6 µm; P < 0.001), higher rate of unifocal GA type (94.7 vs. 49.6%; P < 0.001), and smaller GA area (0.59 vs. 3.76 mm2;P < 0.001) than the conventional GA group. In the follow-up group, the mean GA progression rate (square-root transformation) was significantly lower in the pachychoroid GA group than in the conventional GA group (0.11 vs. 0.27 mm/year; P < 0.001). Conclusions: Demographic and ocular characteristics differed between GA subtypes. The progression rate of pachychoroid GA, adjusted for age and baseline GA area, was significantly lower than that of conventional GA. Japanese patients with conventional GA showed characteristics and progression rates similar to those in White populations. Some characteristics of GA in Japanese population differ from those in Waucasian populations, which may be due to the inclusion of pachychoroid GA. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

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.

Cell-Free Biosensors Based on Modular Eukaryotic Riboswitches That Function in One Pot at Ambient Temperature.

Artificial riboswitches responsive to user-defined analytes can be constructed by successfully inserting in vitro selected aptamers, which bind to the analytes, into untranslated regions of mRNA. Among them, eukaryotic riboswitches are more promising as biosensors than bacterial ones because they function well at ambient temperature. In addition, cell-free expression systems allow the broader use of these riboswitches as cell-free biosensors in an environmentally friendly manner without cellular limitations. The current best cell-free eukaryotic riboswitch regulates eukaryotic canonical translation initiation through self-cleavage mediated by an implanted analyte-responsive ribozyme (i.e., an aptazyme, an aptamer-ribozyme fusion). However, it has critical flaws as a sensor: due to the less-active ribozyme used, self-cleavage and translation reactions must be conducted separately and sequentially, and a different aptazyme has to be selected to change the analyte specificity, even if an aptamer for the next analyte is available. We here stepwise engineered novel types of cell-free eukaryotic riboswitches that harness highly active self-cleavage and thus require no reaction partitioning. Despite the single-step and one-pot reaction, these riboswitches showed higher analyte dose dependency and sensitivities than the current best cell-free eukaryotic riboswitch requiring multistep reactions. In addition, the analyte specificity can be changed in an extremely facile way, simply by aptamer substitution (and the subsequent simple fine-tuning for giant aptamers). Given that cell-free systems can be lyophilized for storage and transport, the present one-pot and thus easy-to-handle cell-free biosensors utilizing eukaryotic riboswitches are expected to be widely used for on-the-spot sensing of analytes at ambient temperature.

Characterization of rice endosperm-derived antidepressant-like peptide (REAP): An orally active novel tridecapeptide derived from rice protein.

It is ideal to ingest bioactive substances from daily foods to stay healthy. Rice is the staple food for almost half of the human population. We found that an orally administered enzymatic digest of rice endosperm protein exhibits antidepressant-like effects in the tail suspension test (TST) using mice. A comprehensive peptide analysis of the digest using liquid chromatography-tandem mass spectrometry was performed, and a tridecapeptide QQFLPEGQSQSQK, detected in the digest, was chemosynthesized. Oral administration of the tridecapeptide exhibited antidepressant-like effects at a low dose comparable to classical antidepressant in the TST. This also exhibited anti-depressant-like effect in the forced swim test. We named it rice endosperm-derived antidepressant-like peptide (REAP). Intriguingly, intraperitoneal administration had no effect. Orally administered REAP(8-13) but not REAP(1-7) exhibited antidepressant-like activity, suggesting that the C-terminal structure is important for the antidepressant-like effect. We confirmed the presence of REAP, corresponding to rice glutelin type B4(130-142) and B5(130-142), in the digest. The effects of REAP were blocked by both dopamine D1 and D2 antagonists. These results suggest that it exerts its antidepressant-like activity through activation of the dopamine system. Taken together, oral administration of a novel tridecapeptide exhibited antidepressant-like effects via the dopamine system. This is the first report of a rice-derived peptide that exhibits antidepressant-like effects.

Detection of typical indigenous gut bacteria related to kanpyo Lagenaria siceraria var. hispida powder in murine caecum and human faecal cultures.

Kanpyo (KP) is an edible dried product produced by peeling the fruit of the gourd Lagenaria siceraria var. hispida; it is used in the traditional Japanese cuisine. The health functionality of KP due to its rich dietary fibre is expected to include a possible combined effect of KP-responsive indigenous gut bacteria (KP-RIB). However, its effect on the gut microbiota is unclear. To determine the effects of the KP on the gut microbiota and their host, Institute of Cancer Research mice were fed a high-sucrose diet containing no fibre (NF) or 5% (w/w) KP for 14 days, and their caecal microbiota was analysed by 16S rRNA (V4) amplicon sequencing. Higher faecal frequency and weight and lower spleen weight and spleen tumour necrosis factor-α levels were observed in KP-fed mice than in NF-fed mice (p < 0.05). KP increased and decreased the abundance of short-chain fatty acid producer Lachnospiraceae and obesity-inflammation related Allobaculum species, respectively. In the case of human faecal cultures, stool samples from five healthy volunteers were inoculated and incubated at 37 °C for 24 h anaerobically; 3.2% (w/v) KP suppressed putrefactive compounds (indole, phenol, and ammonia). KP increased butyrate-producer Faecalibacterium, acetate/lactate-producer Bifidobacterium, and Lachnospira. Furthermore, KP cultures showed high antioxidant and RAW264.7 macrophage cell activation capacities. These results suggest that KP-RIB and KP intake may synergistically affect host health. However, further studies are required to clarify the synergistic effects of KP and KP-RIB.

Differences in collateral vessel formation after experimental retinal vein occlusion in spontaneously hypertensive rats and wild-type rats.

Objective: Retinal vein occlusion (RVO) can lead to visual impairment, but the development of collateral vessels can sometimes mitigate significant damage. This study aimed to investigate the relationship between collateral vessels and hypertension, the most common underlying condition associated with RVO, by comparing spontaneously hypertensive rats (SHRs) and wild-type Wister rats (WWRs). We also examined the differences between WWRs and SHRs in terms of sphingosine 1-phosphate receptor 1 (S1PR1) expression and its product nitric oxide synthase 3 (NOS3) expression, which are involved in the formation of collateral vessels after vascular occlusion. Methods: Laser photocoagulation (PC) was used to occlude one randomly selected retinal vein in WWRs and SHRs, and the area surrounding the occluded vessel was examined using optical coherence tomography angiography. If reperfusion of the occluded vessel occurred within 2 weeks, the vessel was re-occluded repeatedly by PC. The number of eyes with successfully occluded vessels accompanied by collateral vessels was recorded. Then, WWRs and SHRs were divided into the following four groups: 1) control (no treatment), 2) vehicle (20% DMSO), 3) S1PR1 agonist (2 mg/mL SEW2871), and 4) S1PR1 antagonist (0.25 mg/mL VPC 23019) groups. The drugs were administered intravitreally in all groups except the control. The number of laser shots required for successful RVO was recorded. Histological evaluation and quantitative real-time PCR of S1PR1 and NOS3 were performed to elucidate the mechanisms underlying collateral vessel formation. Results: The proportion of eyes achieving successful vein occlusion was lower in SHRs (4/12 eyes, 33.3%) than in WWRs (8/10 eyes, 80%, p = 0.043). NOS3 expression at 6 h after PC was significantly higher in WWRs than in SHRs (p = 0.021). In WWRs treated with SEW2871, vein occlusion failed in 7 of 10 eyes (70%). The expression of NOS3 was significantly higher in the SEW2871 treatment group than in the untreated group (p < 0.001). Furthermore, NOS3 expression was significantly higher after SEW2871 treatment in WWRs than in SHRs (p = 0.011). Conclusion: In hypertensive environments, collateral vessels are less likely to develop, and S1PR1 may be involved in this phenomenon.

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.

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.

The Effect of the Rice Endosperm Protein Hydrolysate on the Subjective Negative Mood Status in Healthy Humans: A Randomized, Double-Blind, and Placebo-Controlled Clinical Trial.

The rice endosperm protein (REP) hydrolysate containing the following rice endosperm protein derived oligopeptides QQFLPEGQSQSQK, LPEGQSQSQK, and pEQFLPEGQSQSQK (a N-terminal pyroglutamate residue-modified peptide) reportedly showed an antidepressant-like effect in an animal model. We investigated the effect of the REP hydrolysate on healthy humans who self-reported mental fatigue with subjectively low vigor. Seventy-six participants (age: 20-64 years) were randomly allocated to two groups. The influence of the REP hydrolysate on the mood state was evaluated in two studies: single intake (Study 1) and repeated intake over 4 weeks (Study 2). A salivary stress marker, Chromogranin A (CgA), was measured in Study 1. The single intake of the REP hydrolysate significantly improved the Profile of Mood Status 2nd edition for adults (POMS 2) subscale of Tension-Anxiety. Additionally, the salivary CgA concentrations were remarkably reduced after the single intake of the REP hydrolysate. Though a single intake of the REP hydrolysate did not significantly influence the other subscales and the TMD of the POMS 2 and the Euthymia Scale, both the subjective and objective results supported the possible effect of the REP hydrolysate on reducing anxiety and nervousness. No significant positive effects on the subjective mood state (Euthymia Scale and POMS 2) and sleep quality (Insomnia Severity Index) were observed in the trial setting employed for Study 2. In conclusion, a single intake of REP hydrolysate might help relax the subjective feelings of tension and anxiety. The effectiveness of repeated REP hydrolysate intake needs to be tested in a different clinical setting.

Lacticaseibacillus paracasei K71 Alleviates UVB-Induced Skin Barrier Dysfunction by Attenuating Inflammation via Increased IL-10 Production in Mice.

SCOPE: Ultraviolet B (UVB) radiation causes skin barrier dysfunction, leading to decreased water-holding capacity, impaired epidermal barrier function, and increased skin thickness. This study investigates the protective effects of oral administration of Lacticaseibacillus paracasei K71 against skin barrier dysfunction in UVB-irradiated mice. METHODS AND RESULTS: Mice are fed diets with or without K71 and irradiated with UVB three times a week for 12 weeks. Oral administration of K71 suppresses UVB-induced decrease in stratum corneum water content, mitigates the increase of transepidermal water loss, and decreases epidermal thickness of the dorsal skin. Treatment with K71 reverses the upregulation of inflammatory cytokines and the activation of nuclear factor-κB induced by UVB irradiation and upregulates the expression of anti-inflammatory IL-10 in the dorsal skin. Notable upregulation of IL-10 is observed in the spleens of K71-treated mice. K71 treatment enhances IL-10 production in J774.1 macrophages; however, this enhancement is diminished by inhibiting K71 phagocytosis and TLR3. Furthermore, transfection using K71 RNAs significantly increases IL-10 production. CONCLUSION: These results indicate that K71 may alleviate UVB-induced skin barrier dysfunction by attenuating inflammation via increasing IL-10 production and that K71 RNAs may induce IL-10 production in macrophages. Therefore, K71 may be beneficial for preventing skin barrier dysfunction.

Clinical Characteristics and Progression of Geographic Atrophy in a Japanese Population.

PURPOSE: To elucidate the clinical characteristics and progression rate of geographic atrophy (GA) associated with age-related macular degeneration (AMD) in a Japanese population. DESIGN: Retrospective, multicenter, observational study. PARTICIPANTS: A total of 173 eyes from 173 patients from 6 university hospitals in Japan were included. Of 173 study eyes, 101 eyes from 101 patients were included in the follow-up group. All patients were Japanese, aged ≥ 50 years and had definite GA associated with AMD in at least 1 eye. METHODS: The GA area was measured semiautomatically using fundus autofluorescence (FAF) images. In the follow-up group followed for > 6 months with FAF images, the GA progression rate was calculated by 2 methods: mm2 per year and mm per year using the square-root transformation (SQRT) strategy. Simple and multiple linear regression analyses were used to identify the baseline factors associated with the GA progression rate. MAIN OUTCOME MEASURES: Clinical characteristics of GA and the GA progression rate. RESULTS: The mean age was 76.8 ± 8.8 years, and 109 (63.0%) were males. Sixty-two (35.8%) patients had bilateral GA. The mean GA area was 3.06 ± 4.00 mm2 (1.44 ± 1.00 mm [SQRT]). Thirty-eight eyes (22.0%) were classified as having pachychoroid GA. Drusen and reticular pseudodrusen were detected in 115 (66.5%) and 73 (42.2%) eyes, respectively. The mean subfoveal choroidal thickness was 194.7 ± 105.5 µm. In the follow-up group (follow-up period: 46.2 ± 28.9 months), the mean GA progression rate was 1.01 ± 1.09 mm2 per year (0.23 ± 0.18 mm/year [SQRT]). In the multivariable analysis, the baseline GA area (SQRT; P = 0.002) and the presence of reticular pseudodrusen (P < 0.001) were significantly associated with a greater GA progression rate (SQRT). CONCLUSIONS: Certain clinical characteristics of GA in Asian populations may differ from those in White populations. Asian patients with GA showed male dominance and relatively thicker choroid than White patients. There was a group with GA without drusen but with features of pachychoroid. The GA progression rate in this Asian population was relatively lower than that in White populations. Large GA and reticular pseudodrusen were associated with a greater GA progression rate. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

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.

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.

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.

Rice Endoplasmic Protein-Derived Peptides, Rice-Ghretropins A and B, Stimulate Ghrelin Release in MGN3-1 Cells and Increase Plasma Acylated Ghrelin and Food Intake in Mice.

In this study, we demonstrated that novel rice-derived bioactive peptides promote the secretion of ghrelin, an endogenous orexigenic hormone secreted from the stomach. The enzymatic digest of rice endosperm protein with subtilisin, a microorganism-derived enzyme, stimulated acylated ghrelin secretion in the ghrelin-releasing cell line MGN3-1 and increased food intake after oral administration in mice. By performing a comprehensive analysis based on structure-activity relationships, we selected candidate peptides from over 30,000 peptides in the rice digest. Among them, we found that QAFEPIRSV and TNPWHSPRQGSF, corresponding to the amino acid sequence of the rice endoplasmic proteins glutelin A1 or A2(52-60) and B1 or B2(31-42), respectively, stimulated acylated ghrelin release in MGN3-1 cells. We named them rice-ghretropins A and B. Pyroglutamate formation of rice-ghretropin A, [pyr1]-rice-ghretropin A, also promoted ghrelin secretion. Furthermore, oral administration of rice-ghretropins increased food intake, plasma ghrelin concentration, and small intestinal transit in mice. In addition, the subtilisin digest of the rice protein significantly increased food intake for 4 h in 9 month-old (control: 0.61 ± 0.049 g; digest: 0.83 ± 0.059 g) and 24 month-old mice (control: 0.52 ± 0.067 g; digest: 1.01 ± 0.064 g). In summary, we found that novel bioactive peptides, namely, rice-ghretropins, from the enzymatic digest of rice endosperm stimulated acylated ghrelin secretion and increased food intake. This is the first report of rice-derived exogenous bioactive peptides that increase acylated ghrelin secretion.

Facile Expansion of the Variety of Orthogonal Ligand/Aptamer Pairs for Artificial Riboswitches.

An RNA aptamer that induces suitable conformational changes upon binding to a user-defined ligand allows us to artificially construct a riboswitch, a ligand-dependent and cis-acting gene regulatory RNA. Although such an aptamer can be obtained through in vitro selection, it is still challenging to rationally expand the variety of orthogonal ligand/aptamer (ligand/riboswitch) pairs. To achieve this in a facile, selection-free way, we herein focused on a specific type of ligand, 6-nt nanosized DNA (nDNA) and its aptamer that was previously selected to construct a eukaryotic artificial riboswitch. Specifically, we merely mutated one or more possible Watson-Crick base pairs in the nDNA/aptamer (nDNA/riboswitch) interactions into another base pair or pairs. Using two sets that each had 16 comprehensive mutations, we obtained three groups of several orthogonal nDNA/riboswitch pairs. These pairs could be used to create complex gene circuits, including multiple simultaneous and/or multistep cascading regulations in synthetic biology.

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.

Distribution of psychrophilic microorganisms in a beef slaughterhouse in Japan after cleaning.

The purpose of this study was to investigate the abundance and distribution of psychrophilic microorganisms associated with spoilage in beef slaughterhouse environments after cleaning. The processing lines and equipment used in slaughtering and boning were swabbed, and the microbial count was determined using a TSA and MRS medium and Chromocult® Coliform agar incubated at 15ºC and 37ºC, respectively. As a result, the brisket saw (handle side) and trolley hook were the most heavily contaminated with microorganisms, with each having a microbial adhesion rate of 66.7%. The microbial adhesion rates of the apron and milling cutter (edge side) were 50%, respectively, and those of the foot cutter (edge and handle side), splitting saw (edge side), and knife (handle side) were 33.3%, respectively. Next, four colonies were randomly isolated from the petri dish used for the bacterial count measurement to identify the predominant microbial species of the microorganisms attached to each equipment. As a result of Sanger sequencing analysis, yeasts such as Candida zeylanoides and Rhodotorula sp. and bacteria including Pseudomonas sp. and Rhodococcus sp. were identified from the equipment used in the slaughtering line, and it was assumed that these microorganisms were of environmental origin. In contrast, only Pseudomonas sp. and Candida zeylanoides were isolated from the boning line. Despite the use of cleaning operations, this study identified some equipment was contaminated with microorganisms. Since this equipment frequently comes into direct contact with the carcass, it is critical to thoroughly remove the microorganisms through accurate cleaning to prevent the spread of microbial contamination on the carcasses.

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.

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.