Thermophile-fermented feed modulates the gut microbiota related to lactate metabolism in pigs.

AIMS: Extracts of fermented feed obtained via fermentation of marine animal resources with thermophilic Bacillaceae bacteria increase the fecundity of livestock. The intestinal bacterial profiles in response to long-term administration of this extract to pigs were investigated. METHODS AND RESULTS: Half of a swine farm was supplied with potable water containing an extract of fermented feed for more than 2 years, whereas the other half was supplied with potable water without the extract. Feces from 6-month-old pigs rearing in these two areas were collected. 16S rRNA gene sequencing and isolation of lactic acid bacteria revealed an increase in the D/L-lactate-producing bacterium, Lactobacillus amylovorus, and a decrease in several members of Clostridiales following administration of fermented feed. A lactate-utilizing bacterium, Megasphaera elsdenii, was more abundant in the feces of pigs in the fermented feed group. All representative isolates of M. elsdenii showed rapid utilization of D-lactate relative to L-lactate, and butyrate and valerate were the main products. CONCLUSION: The probiotic effect of fermented feed is associated with the modulation of lactate metabolism in the digestive organs of pigs.

Bleomycin-Induced Pulmonary Fibrosis in Transgenic Mice Carrying the Human MUC5B rs35705950 Variant.

Idiopathic pulmonary fibrosis (IPF) is a progressive, often fatal lung disease characterized by tissue scarring and declining lung function. The MUC5B promoter polymorphism rs35705950, a significant genetic predisposition for IPF, paradoxically associates with better survival and slower disease progression than other IPF genotypes. This study investigates the potential paradoxical protective effects of this MUC5B variant in lung fibrosis. For this purpose, we developed a transgenic mouse model overexpressing the human MUC5B rs35705950 variant in the proximal large airways. Lung fibrosis was induced through subcutaneous injection of bleomycin. Results demonstrated significantly reduced lung fibrosis severity in transgenic mice compared to wild-type mice, assessed by trichrome staining, Ashcroft scoring, and hydroxyproline levels. Additionally, transgenic mice showed significantly lower levels of inflammatory cells and cytokines (TNFα, IL-6, IFNγ) and growth factors (PDGF, CTGF, IL-13) in the bronchoalveolar lavage fluid and lung tissues. There was also a significant decrease in mRNA expressions of fibrosis-related markers (periostin, fibronectin, Col1a1). In summary, this study reveals that mucin overexpression related to the MUC5B rs35705950 variant in the large airways significantly attenuates lung fibrosis and inflammatory responses in transgenic mice. These findings suggest that the rs35705950 variant modulates inflammatory and fibrotic responses in the proximal airways, which may contribute to the slower disease progression observed in IPF patients carrying this variant. Our study offers a possible explanation for the paradoxical beneficial effects of the MUC5B variant despite its role as a significant predisposing factor for IPF.

Role of microbiota-derived corisin in coagulation activation during SARS-CoV-2 infection.

BACKGROUND: Coagulopathy is a major cause of morbidity and mortality in COVID-19 patients. Hypercoagulability in COVID-19 results in deep vein thrombosis, thromboembolic complications, and diffuse intravascular coagulation. Microbiome dysbiosis influences the clinical course of COVID-19. However, the role of dysbiosis in COVID-19-associated coagulopathy is not fully understood. OBJECTIVES: The present study tested the hypothesis that the microbiota-derived proapoptotic corisin is involved in the coagulation system activation during SARS-CoV-2 infection. METHODS: This cross-sectional study included 47 consecutive patients who consulted for symptoms of COVID-19. A mouse acute lung injury model was used to recapitulate the clinical findings. A549 alveolar epithelial, THP-1, and human umbilical vein endothelial cells were used to evaluate procoagulant and anticoagulant activity of corisin. RESULTS: COVID-19 patients showed significantly high circulating levels of corisin, thrombin-antithrombin complex, D-dimer, tumor necrosis factor-α, and monocyte-chemoattractant protein-1 with reduced levels of free protein S compared with healthy subjects. The levels of thrombin-antithrombin complex, D-dimer, and corisin were significantly correlated. A monoclonal anticorisin-neutralizing antibody significantly inhibited the inflammatory response and coagulation system activation in a SARS-CoV-2 spike protein-associated acute lung injury mouse model, and the levels of corisin and thrombin-antithrombin complex were significantly correlated. In an in vitro experiment, corisin increased the tissue factor activity and decreased the anticoagulant activity of thrombomodulin in epithelial, endothelial, and monocytic cells. CONCLUSION: The microbiota-derived corisin is significantly increased and correlated with activation of the coagulation system during SARS-CoV-2 infection, and corisin may directly increase the procoagulant activity in epithelial, endothelial, and monocytic cells.

Amelioration of Pulmonary Fibrosis by Matrix Metalloproteinase-2 Overexpression.

Idiopathic pulmonary fibrosis is a progressive and fatal disease with a poor prognosis. Matrix metalloproteinase-2 is involved in the pathogenesis of organ fibrosis. The role of matrix metalloproteinase-2 in lung fibrosis is unclear. This study evaluated whether overexpression of matrix metalloproteinase-2 affects the development of pulmonary fibrosis. Lung fibrosis was induced by bleomycin in wild-type mice and transgenic mice overexpressing human matrix metalloproteinase-2. Mice expressing human matrix metalloproteinase-2 showed significantly decreased infiltration of inflammatory cells and inflammatory and fibrotic cytokines in the lungs compared to wild-type mice after induction of lung injury and fibrosis with bleomycin. The computed tomography score, Ashcroft score of fibrosis, and lung collagen deposition were significantly reduced in human matrix metalloproteinase transgenic mice compared to wild-type mice. The expression of anti-apoptotic genes was significantly increased, while caspase-3 activity was significantly reduced in the lungs of matrix metalloproteinase-2 transgenic mice compared to wild-type mice. Active matrix metalloproteinase-2 significantly decreased bleomycin-induced apoptosis in alveolar epithelial cells. Matrix metalloproteinase-2 appears to protect against pulmonary fibrosis by inhibiting apoptosis of lung epithelial cells.

Noninvasive fecal metabolic profiling for the evaluation of characteristics of thermostable lactic acid bacteria, Weizmannia coagulans SANK70258, for broiler chickens.

Weizmannia coagulans SANK70258 is a spore-forming thermostable lactic acid bacterium and an effective probiotic for the growth of livestock animals, but its growth-promoting mechanism remains unclear. Here, the composition of fecal metabolites in broilers continuously administered with W. coagulans SANK70258 was assessed under a regular program with antibiotics, which was transiently given for 6 days after birth. Oral administration of W. coagulans to broiler chicks tended to increase the average daily gain of body weights thereafter. The composition of fecal metabolites in the early chick stage (day 10 after birth) was dramatically altered by the continuous exposure. The levels of short-chain fatty acids (SCFAs) propionate and butyrate markedly increased, while those of acetate, one of the SCFAs, and lactate were reduced. Simultaneously, arabitol, fructose, mannitol, and erythritol, which are carbohydrates as substrates for gut microbes to produce SCFAs, also increased along with altered correlation. Correlation network analyses classified the modularity clusters (|r| > 0.7) among carbohydrates, SCFAs, lactate, amino acids, and the other metabolites under the two conditions. The characteristic diversities by the exposure were visualized beyond the perspective associated with differences in metabolite concentrations. Further, enrichment pathway analyses showed that metabolic composition related to biosynthesis and/or metabolism for SCFAs, amino acids, and energy were activated. Thus, these observations suggest that W. coagulans SANK70258 dramatically modulates the gut metabolism of the broiler chicks, and the metabolomics profiles during the early chick stages may be associated with growth promotion.

The expression of alternative sigma-I7 factor induces the transcription of cellulosomal genes in the cellulolytic bacterium Clostridium thermocellum.

The composition of cellulosomal carbohydrate-active enzymes (CAZymes) secreted from a cellulolytic bacterium Clostridium thermocellum varies depending on the cellulosic substrate used during cultivation. C. thermocellum detects the polysaccharides in cellulosic material via anti-sigma factors and expresses the appropriate CAZyme gene via alternative sigma factors, SigIs. Previous studies on the regulation of CAZyme gene expression via SigIs in C. thermocellum have been conducted in vitro or in a heterologous host, because of the limited genetic tools available for C. thermocellum. To characterize the in vivo function of SigIs, in the present study, we established a sigI7 gene expression strain of C. thermocellum. Transcriptome analysis of this strain revealed that SigI7 induced the expression of cellulosomal CAZyme genes and cellulosomal scaffold genes. However, there was a decrease in the degradation ability of the exoproteome from the sigI7 expression strain; the product of the downregulated gene, Clo1313_1002, rescued the activity of the C. thermocellum exoproteome from the sigI7 expression strain. In this study, we demonstrate the in vivo function of SigI7 and discuss the CAZymes that are important for cellulosic biomass degradation by C. thermocellum.

Activation of fibroblast growth factor-inducible 14 in the early phase of childhood IgA nephropathy.

IgA nephropathy (IgAN) is the most common form of glomerulonephritis worldwide. Pediatric patients in Japan are diagnosed with IgAN at an early stage of the disease through annual urinary examinations. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible 14 (Fn14) have various roles, including proinflammatory effects, and modulation of several kidney diseases; however, no reports have described their roles in pediatric IgAN. In this study, we performed pathological and immunohistochemical analyses of samples from 14 pediatric IgAN patients. Additionally, gene expression arrays of glomeruli by laser-captured microdissection were performed in hemi-nephrectomized high serum IgA (HIGA) mice, a model of IgA nephropathy, to determine the role of Fn14. Glomeruli with intense Fn14 deposition were observed in 80% of mild IgAN cases; however, most severe cases showed glomeruli with little or no Fn14 deposition. Fn14 deposition was not observed in obvious mesangial proliferation or the crescent region of glomeruli, but was detected strongly in the glomerular tuft, with an intact appearance. In HIGA mice, Fn14 deposition was observed mildly beginning at 11 weeks of age, and stronger Fn14 deposition was detected at 14 weeks of age. Expression array analysis indicated that Fn14 expression was higher in HIGA mice at 6 weeks of age, increased slightly at 11 weeks, and then decreased at 26 weeks when compared with controls at equivalent ages. These findings suggest that Fn14 signaling affects early lesions but not advanced lesions in patients with IgAN. Further study of the TWEAK/Fn14 pathway will contribute to our understanding of the progression of IgAN.

Glucose production from cellulose through biological simultaneous enzyme production and saccharification using recombinant bacteria expressing the ß-glucosidase gene.

Efficient cellulosic biomass saccharification technologies are required to meet biorefinery standards. Biological simultaneous enzyme production and saccharification (BSES), which is glucose production from cellulosic biomass by Clostridium thermocellum, can be a reliable cellulose saccharification technology for biorefineries. However, the current BSES processes require purified ß-glucosidase supplementation. In this study, recombinant bacteria expressing the ß-glucosidase gene were developed and directly applied to BSES. The engineered Escherichia coli expressing the thermostable ß-glucosidase gene from Thermoanaerobacter brockii exhibited 0.5 U/ml of ß-glucosidase activities. The signal peptide sequence of lytF gene from Bacillus subtilis was the most appropriate for the ß-glucosidase secretion from Brevibacillus choshinensis, and the broth exhibited 0.74 U/ml of ß-glucosidase activities. The engineered E. coli and B. choshinensis expressing the thermostable ß-glucosidase gene produced 47.4 g/L glucose and 49.4 g/L glucose, respectively. Glucose was produced by the hydrolysis of 100 g/L Avicel cellulose for 10 days through BSES, and the product yield was similar to that obtained through BSES with purified ß-glucosidase supplementation. Our findings indicate that the direct supplementation of ß-glucosidase using bacterial cells expressing ß-glucosidase gene or their broth was applicable to BSES, suggesting the potential of this process as a cost-effective approach to cellulose saccharification.

Free space optical secret key agreement.

Free space optical (FSO) communications are enabling high-speed global wireless networks. Thanks to the highly directional nature of laser beam, they also yield a greater security advantage over radio frequency counterparts. When combined with a scheme of secret key agreement (SKA), FSO-SKA can establish at high speed a symmetric secret key which cannot be decrypted even by unbounded computer resources. Although there have been many theoretical studies on SKA, experimental investigations have been quite lacking, especially on quantifying eavesdropping risks and secret key rates in realistic environment. Here, we report the first full-field implementations of FSO-SKA in a 7.8-km terrestrial link with a probing station, enabling the estimation of eavesdropping risks. We attain the final key rates from 100 kbps to 7.77 Mbps under various atmospheric and beaming conditions even with total losses of 55dB or higher, in which known quantum key distribution schemes attain impractically low key rates.

Free-space optical wiretap channel and experimental secret key agreement in 7.8 km terrestrial link.

Secret key agreement using physical properties of a wireless channel is becoming a promising scheme to establish a secret key between two users, especially in short-distance radio frequency (RF) communications. In this scheme, the existence of codes or key distillation that can make the leaked information to an eavesdropper arbitrarily small can be derived in an information theoretical way, given a priori knowledge on the channel linking a sender (Alice), a legitimate receiver (Bob), and an eavesdropper (Eve), which is called the wiretap channel. In practice, however, it is often difficult for Alice and Bob to get sufficient knowledge on Eve. In this study, we implement a free-space optical wiretap channel in a 7.8 km-terrestrial link and study how to estimate Eve's tapping ability, demonstrating high speed secret key agreement in the optical domain under a certain restricted condition of line-of-sight.

Valerate production by Megasphaera elsdenii isolated from pig feces.

Megasphaera elsdenii is able to produce several short-chain fatty acids (SCFAs), such as acetate, propionate, butyrate, and valerate. These SCFAs serve as an energy source for host animals and play an important role in gut health. In this study, M. elsdenii was isolated from pig feces that had been collected from two farms located in distinct areas of Japan. These M. elsdenii isolates were genotyped, and 7 representative strains were selected. When these 7 strains and M. elsdenii JCM 1772T were cultured with lactate for 24 h, all 7 strains produced valerate as a predominant SCFA. Therefore, the valerate-producing M. elsdenii inhabits a wide area of Japan. In contrast, M. elsdenii JCM 1772T produced acetate, propionate, butyrate, and valerate at similar levels. When the Y2 strain, one of the 7 representative strains, was cultured without lactate, low levels of valerate accumulated. In contrast, in a time course of lactate fermentation by the Y2 strain, lactate was rapidly consumed, and acetate and propionate were produced after 6 h of incubation. Thereafter, acetate and propionate were consumed from 6 to 12 h after the start of the incubation, and valerate and butyrate were produced. In most of the previously described M. elsdenii strains, valerate was not a predominant SCFA. Therefore, the M. elsdenii Y2 strain showed an unique metabolism in which valerate was produced as a primary end product of lactate fermentation.

Preferential isolation of Megasphaera elsdenii from pig feces.

Lactic acid produced by intestinal bacteria is fermented by lactate-utilizing bacteria. In this study, we developed a selective culture medium (KMI medium) for Megasphaera elsdenii, a lactate-utilizing bacterium that is abundant in pig intestines. Supplementation of the medium with lactate and beef extract powder was necessary for the preferential growth of M. elsdenii. In addition, we designed a species-specific primer set to detect M. elsdenii. When pig fecal samples were plated on KMI agar medium, approximately 60-100% of the resulting colonies tested positive using the M. elsdenii-specific PCR primers. In fact, nearly all of the large, yellow-white colonies that grew on the KMI agar medium tested positive by PCR with this primer set. The 16S rRNA gene sequences of three representative PCR-positive strains showed strong similarities to that of M. elsdenii ATCC 25940T (98.9-99.2% identity). These three strains were approximately 1.5 µm sized cocci that were primarily arranged in pairs, as was observed for M. elsdenii JCM 1772T. The selective KMI medium and species-specific primer set developed in this study are useful for the isolation and detection of M. elsdenii and will be useful in research aimed at increasing our understanding of intestinal short-chain fatty acid metabolism in pigs.

Quantum photonic network and physical layer security.

Quantum communication and quantum cryptography are expected to enhance the transmission rate and the security (confidentiality of data transmission), respectively. We study a new scheme which can potentially bridge an intermediate region covered by these two schemes, which is referred to as quantum photonic network. The basic framework is information theoretically secure communications in a free space optical (FSO) wiretap channel, in which an eavesdropper has physically limited access to the main channel between the legitimate sender and receiver. We first review a theoretical framework to quantify the optimal balance of the transmission efficiency and the security level under power constraint and at finite code length. We then present experimental results on channel characterization based on 10 MHz on-off keying transmission in a 7.8 km terrestrial FSO wiretap channel.This article is part of the themed issue 'Quantum technology for the 21st century'.

Free-space optical channel estimation for physical layer security.

We present experimental data on message transmission in a free-space optical (FSO) link at an eye-safe wavelength, using a testbed consisting of one sender and two receiver terminals, where the latter two are a legitimate receiver and an eavesdropper. The testbed allows us to emulate a typical scenario of physical-layer (PHY) security such as satellite-to-ground laser communications. We estimate information-theoretic metrics including secrecy rate, secrecy outage probability, and expected code lengths for given secrecy criteria based on observed channel statistics. We then discuss operation principles of secure message transmission under realistic fading conditions, and provide a guideline on a multi-layer security architecture by combining PHY security and upper-layer (algorithmic) security.

Availability of Amino Acids Extends Chronological Lifespan by Suppressing Hyper-Acidification of the Environment in Saccharomyces cerevisiae.

The chronological lifespan of Saccharomyces cerevisiae represents the duration of cell survival in the postdiauxic and stationary phases. Using a prototrophic strain derived from the standard auxotrophic laboratory strain BY4742, we showed that supplementation of non-essential amino acids to a synthetic defined (SD) medium increases maximal cell growth and extends the chronological lifespan. The positive effects of amino acids can be reproduced by modulating the medium pH, indicating that amino acids contribute to chronological longevity in a cell-extrinsic manner by alleviating medium acidification. In addition, we showed that the amino acid-mediated effects on extension of chronological longevity are independent of those achieved through a reduction in the TORC1 pathway, which is mediated in a cell-intrinsic manner. Since previous studies showed that extracellular acidification causes mitochondrial dysfunction and leads to cell death, our results provide a path to premature chronological aging caused by differences in available nitrogen sources. Moreover, acidification of culture medium is generally associated with culture duration and cell density; thus, further studies are required on cell physiology of auxotrophic yeast strains during the stationary phase because an insufficient supply of essential amino acids may cause alterations in environmental conditions.

Thermophile-fermented compost extract as a possible feed additive to enhance fecundity in the laying hen and pig: Modulation of gut metabolism.

Recently, we reported that the oral administration of an extract of compost fermented with marine animal resources and thermophilic Bacillus species should confer health benefits in fish, pigs and rodents. Herein, the relations between fecundity and gut metabolites in laying hens and pigs on farms after oral exposure to compost were investigated. On the hen farms, the egg production of hens continuously administered the extract was maintained at significantly higher levels compared with the hens not administered the extract. On the swine farms, after the compost treatment, the shipping dates of fattening pigs were shortened, with an improvement in the death rate of the pigs. When the levels of fecal organic acids, such as short-chain fatty acids, lactate, and ammonium, as indicators of gut metabolism and energy sources for peripheral tissues, were examined, the levels of the acetate, propionate, and butyrate in the feces of the hens and pigs in the compost-treated group were not always different from those in the untreated control group. However, the levels of lactate were consistently low in the feces of both animals after the compost treatment. The fecal ammonium concentrations in old hens (age 597-672 days) and 2-month-old piglets from the compost-fed mother sows were low when compared with the untreated groups. The concentrations of free organic acids and their related compounds in the animal products (eggs and pig loins) were nearly equal to those in the untreated control products. Thus, the oral administration of the thermophile-fermented compost should improve the fecundity of hens and pigs by modifying their gut metabolism.

Transparent and Nonflammable Ionogel Photon Upconverters and Their Solute Transport Properties.

Photon upconversion based on triplet-triplet annihilation (TTA-UC) is a technology to convert presently wasted sub-bandgap photons to usable higher-energy photons. In this paper, ionogel TTA-UC samples are first developed by gelatinizing ionic liquids containing triplet-sensitizing and light-emitting molecules using an ionic gelator, resulting in transparent and nonflammable ionogel photon upconverters. The photophysical properties of the ionogel samples are then investigated, and the results suggest that the effect of gelation on the diffusion of the solutes is negligibly small. To further examine this suggestion and acquire fundamental insight into the solute transport properties of the samples, the diffusion of charge-neutral solute species over much longer distances than microscopic interpolymer distances is measured by electrochemical potential-step chronoamperometry. The results reveal that the diffusion of solute species is not affected by gelation within the tested gelator concentration range, supporting our interpretation of the initial results of the photophysical investigations. Overall, our results show that the advantage of nonfluidity can be imparted to ionic-liquid-based photon upconverters without sacrificing molecular diffusion, optical transparency, and nonflammability.

Thermophile-fermented compost as a fish feed additive modulates lipid peroxidation and free amino acid contents in the muscle of the carp, Cyprinus carpio.

Recently, a compost fermented with marine animals with thermophilic Bacillaceae in a clean and exclusive process at high temperature was reported as a possible feed additive to improve the healthy balance in sea fish and mammals (i.e., pigs and rodents). Here, the effects of the oral administration of the compost on the muscle and internal organs of carp (Cyprinus carpio) as a freshwater fish model were investigated. The fatty acid composition was different in the muscle of the carp fed with or without the compost extract, but there was little difference in the hepatopancreas. The accumulation of triacylglycerols, cholesterol, lipid peroxide and hydroxyl lipids decreased in the muscle after the oral administration of the compost extract in the carps over 12 weeks, but the accumulation did not always decrease in the hepatopancreas. In contrast, free-radical-scavenging activities and the concentrations of free amino acids in the muscle did not always increase and was dependent on the dose of the compost at 12 weeks. The scavenging activities and part of free amino acid levels in the muscle of the carp were improved at 24 weeks after a high dose of compost exposure, and then the survival rates of the carp were maintained. Thus, the oral administration of thermophile-fermented compost can prevent peroxidation and increase the content of free amino acids in the muscle of the freshwater fish, depending on the dose and term of the administration, and may be associated with the viability of the fish.

A spinach O-acetylserine(thiol)lyase homologue, SoCSaseLP, suppresses cysteine biosynthesis catalysed by other enzyme isoforms.

An enzyme, O-acetylserine(thiol)lyase (OASTL), also known as O-acetylserine sulfhydrylase or cysteine synthase (CSase), catalyses the incorporation of sulfide into O-acetylserine and produces cysteine. We previously identified a cDNA encoding an OASTL-like protein from Spinacia oleracea, (SoCSaseLP), but a recombinant SoCSaseLP produced in Escherichia coli did not show OASTL activity. The exon-intron structure of the SoCSaseLP gene shared conserved structures with other spinach OASTL genes. The SoCSaseLP and a Beta vulgaris homologue protein, KMT13462, comprise a unique clade in the phylogenetic tree of the OASTL family. Interestingly, when the SoCSaseLP gene was expressed in tobacco plants, total OASTL activity in tobacco leaves was reduced. This reduction in total OASTL activity was most likely caused by interference by SoCSaseLP with cytosolic OASTL. To investigate the possible interaction of SoCSaseLP with a spinach cytosolic OASTL isoform SoCSaseA, a pull-down assay was carried out. The recombinant glutathione S-transferase (GST)-SoCSaseLP fusion protein was expressed in E. coli together with the histidine-tagged SoCSaseA protein, and the protein extract was subjected to glutathione affinity chromatography. The histidine-tagged SoCSaseA was co-purified with the GST-SoCSaseLP fusion protein, indicating the binding of SoCSaseLP to SoCSaseA. Consistent with this interaction, the OASTL activity of the co-purified SoCSaseA was reduced compared with the activity of SoCSaseA that was purified on its own. These results strongly suggest that SoCSaseLP negatively regulates the activity of other cytosolic OASTL family members by direct interaction.