Exploring the gastrointestinal role of hydrogen sulfide (H2S) is difficult because of its volatility and the absence of a precisely controllable model system for manipulating the gut environment. ...Hayes et al. address this issue by engineering Escherichia coli to titrate H2S levels in a gas-impermeable gut-on-chip device.
Exploring the gastrointestinal role of hydrogen sulfide (H2S) is difficult because of its volatility and the absence of a precisely controllable model system for manipulating the gut environment. Hayes et al. address this issue by engineering Escherichia coli to titrate H2S levels in a gas-impermeable gut-on-chip device.
The probiotic yeast Saccharomyces boulardii (Sb) is a promising chassis to deliver therapeutic proteins to the gut due to Sb's innate therapeutic properties, resistance to phage and antibiotics, and ...high protein secretion capacity. To maintain therapeutic efficacy in the context of challenges such as washout, low rates of diffusion, weak target binding, and/or high rates of proteolysis, it is desirable to engineer Sb strains with enhanced levels of protein secretion. In this work, we explored genetic modifications in both cis- (i.e. to the expression cassette of the secreted protein) and trans- (i.e. to the Sb genome) that enhance Sb's ability to secrete proteins, taking a Clostridioides difficile Toxin A neutralizing peptide (NPA) as our model therapeutic. First, by modulating the copy number of the NPA expression cassette, we found NPA concentrations in the supernatant could be varied by sixfold (76-458 mg/L) in microbioreactor fermentations. In the context of high NPA copy number, we found a previously-developed collection of native and synthetic secretion signals could further tune NPA secretion between 121 and 463 mg/L. Then, guided by prior knowledge of S. cerevisiae's secretion mechanisms, we generated a library of homozygous single gene deletion strains, the most productive of which achieved 2297 mg/L secretory production of NPA. We then expanded on this library by performing combinatorial gene deletions, supplemented by proteomics experiments. We ultimately constructed a quadruple protease-deficient Sb strain that produces 5045 mg/L secretory NPA, an improvement of > tenfold over wild-type Sb. Overall, this work systematically explores a broad collection of engineering strategies to improve protein secretion in Sb and highlights the ability of proteomics to highlight under-explored mediators of this process. In doing so, we created a set of probiotic strains that are capable of delivering a wide range of protein titers and therefore furthers the ability of Sb to deliver therapeutics to the gut and other settings to which it is adapted.
As is known, alfalfa saponins can be used as a feed additive in a pig's diet and the addition of alfalfa saponins to a pig's diet could improve its antioxidant capacity. However, the mechanism by ...which alfalfa saponins exert their antioxidant effects has not been studied. To address this issue, H
2
O
2
-induced rat intestinal epithelial cells were used to establish an oxidative stress model to explore the protective mechanism of alfalfa saponins in this study. The results demonstrated that alfalfa saponins could rescue the cell proliferation activity, elevate the amount of antioxidant enzymes and downregulate the release of MDA and LDH in H
2
O
2
-induced cells. The antioxidant activity of alfalfa saponins was achieved by restoring GSH homeostasis. Further results demonstrated that alfalfa saponins could inhibit cell apoptosis through activating the MAPK signaling pathway. These results elucidated the mechanism by which alfalfa saponins exert their antioxidant effects and provided a potential strategy for alleviating oxidative stress in monogastric animals.
Alfalfa saponins defend against oxidative stress by enhancing the antioxidant system and further inhibit cell apoptosis by activating the MAPK signaling pathway.
Water molecules and cations with mono, binary, and triple valences have been intercalated into V2O5 to significantly improve its electrochemical properties as a cathode material of zinc-ion ...batteries. Sn as a tetravalent element is supposed to interact aggressively with the V2O5 layer and have a significant impact on the electrochemical performance of V2O5. However, it has been rarely investigated as a pre-intercalated ion in previous works. Hence, it is intriguing and beneficial to develop water molecules and Sn co-doped V2O5 for zinc-ion batteries. Herein, Sn-doped hydrated V2O5 nanosheets were prepared by a one-step hydrothermal synthesis, and they demonstrated that they had a high specific capacity of 374 mAh/g at 100 mA/g. Meanwhile, they also showed an exceptional rate capability with 301 mAh/g even at a large current density of 10 A/g, while it was only 40 mAh/g for the pristine hydrated V2O5, and an excellent cycling life (87.2% after 2500 cycles at 5 A/g), which was far more than the 25% of the pure hydrated V2O5. The dramatic improvement of the rate and cycling performance is mainly attributed to the faster charge transfer kinetics and the enhanced crystalline framework. The remarkable electrochemical performance makes the Sn-doped hydrate V2O5 a potential cathode material for zinc-ion batteries.
In pregnant and lactating sows, metabolism and immunity undergo drastic changes, which can lead to constipation, abortion, and intrauterine growth restriction (IUGR) and reduce production ...performance. Dietary fiber can regulate animal gut microbiota, alleviate inflammatory responses, and improve performance. Here, 48 sows (Large × Landrace) were randomly allocated to groups including, control, and with alfalfa meal (AM), beet pulp, and soybean skin dietary supplementation for 60 days of gestation. The AM diet decreased IUGR, increased food intake during lactation, and promoted the reproductive performance and physical condition of sows. Further, the AM diet significantly reduced markers of intestinal permeability (reactive oxygen species and endotoxin) in sow serum, and of systemic inflammation (interleukin-6 IL-6 and tumor necrosis factor alpha) in sow feces and serum, as well as piglet serum, while it increased the anti-inflammatory marker, IL-10, in sow serum and feces. The AM diet also significantly affected gut microbiota by increasing the relative abundance of proinflammatory bacteria, while decreasing anti-inflammatory bacteria. Moreover, the total short-chain fatty acid (SCFA) content was higher in feces from sows fed an AM diet, with butyric acid content significantly higher during lactation, than in controls. Sow performance was correlated with intestinal permeability, inflammation, and gut microbiota, which were also vertically transmitted to piglets. Our results are significant for guiding feed management in the pig breeding industry. Further, the "sows to piglets" model provides a reference for the effect of dietary fiber on the gastrointestinal function of human mothers and infants.
Although the direct effects of dietary fiber on gut microbiota composition have been studied extensively, systematic evaluation of different fiber sources on gut health and inflammatory responses of sows and their offspring has rarely been conducted. Excessive reactive oxygen species produced by overactive metabolic processes during late pregnancy and lactation of sows leads to increased endotoxin levels, disordered gut microbiota, decreased SCFA production, and secretion of proinflammatory factors, which in turn causes local inflammation of the gut, potential damage of the gut microbial barrier, increased gut permeability, increased blood endotoxin levels (resulting in systemic inflammation), and ultimately decreased sow and piglet performance. Our results showed that supplementation of the diet with alfalfa meal in mid and late pregnancy can reverse this process. Our findings lay a foundation for improving the gut health of sows and piglets and provide insights into the study of the gastrointestinal tract function in human mothers and infants.
Roughage quality is a crucial factor influencing the growth performance and feeding cost of ruminants; however, a systematic investigation of the mechanisms underlying this is still lacking. In this ...study, we examined the growth performance, meat quality, ruminal fermentation parameters, rumen microbiome, and tissue methylomes of post-weaned Hu sheep fed low- or high-quality forage-based diets. Our results showed that sheep in the alfalfa hay (AG) and peanut vine (PG) groups exhibited better growth performance, slaughter performance, and meat quality than sheep in the wheat straw group (WG). The sheep in the AG possessed relatively higher contents of serum immunoglobins (IgA, IgG, and IgM) and lower contents of serum inflammation factors (TNF-α, IL-1β, IL-6, and IL-8) than those in the WG and the PG did. In addition, the levels of blood T lymphocytes (CD4
+
and CD8
+
) and the CD4-to-CD8 ratio were significantly higher in the AG sheep than in the WG sheep and PG sheep. The concentration of ruminal NH
3
-N was highest in WG sheep, whereas the concentrations of individual and total short-chain fatty acids (SCFAs) were highest in the PG sheep. The length, width, and surface area of ruminal papillae were markedly different among the three groups, with the sheep in the PG being the most morphologically developed. The main ruminal microbes at the genus level include
Prevotella 1
,
Rikenellaceae RC9 gut group, norank f F082
,
Ruminococcus 1
, and
Ruminococcus 2
. The relative abundances of certain species are positively or negatively associated with fermentation parameters and growth index. For example, the fibrolytic bacteria
Ruminococcaceae UGG-001
showed positive relationships with the concentration of SCFAs, except propionate. In addition, the relative abundances of fibrolytic bacteria (e.g.,
Ruminoccus 1
) showed a negative relationship with starch-degrading bacteria (e.g.,
Prevotellaceae
). The genome-wide DNA methylation analysis revealed that rumen tissues in the PG sheep and WG sheep occupied different global DNA methylomes. The genes with differentially methylated promoters were involved in known pathways (e.g., the FoxO signaling pathway) and the Gene Ontology (GO) terms (e.g., anatomical structure morphogenesis) pertaining to rumen development. Two candidate genes (
ACADL
and
ENSOARG00020014533
) with hyper- and hypo-methylated promoters were screened as potential regulators of rumen development. In conclusion, roughage quality determines sheep growth performance via directly influencing rumen fermentation and microbiome composition, and indirectly affecting rumen development at the epigenetic level.
Andrographis paniculata (A. paniculata, Chuanxinlian), a medicinal herb with an extremely bitter taste that is native to China and other parts of Southeast Asia, possesses immense therapeutic value; ...however, its therapeutic properties have rarely been applied in the field of skin care. In this study, we investigated the effect of an A. paniculata extract (APE) on human epidermal stem cells (EpSCs), and confirmed its anti-aging effect through in vitro, ex vivo, and in vivo study. An MTT assay was used to determine cell proliferation. A flow cytometric analysis, with propidium iodide, was used to evaluate the cell cycle. The expression of integrin β1 (CD29), the stem cell marker, was detected with antibodies, using flow cytometry in vitro, and immunohistochemical assays in ex vivo. Type 1 collagen and VEGF (vascular endothelial growth factor) were measured using an enzyme-linked immunosorbent assay (ELISA). During the clinical study, skin hydration, elasticity, wrinkling, sagging, and dermal density were evaluated before treatment and at four and eight weeks after the treatment with the test product (containing the APE) on the face. The proliferation of the EpSCs, treated with the APE, increased significantly. In the cell cycle analysis, the APE increased the G2/M and S stages in a dose-dependent manner. The expression of integrin β1, which is related to epidermal progenitor cell expansion, was up-regulated in the APE-treated EpSCs and skin explants. In addition, the production of VEGF in the EpSCs increased significantly in response to the APE treatment. Consistent with these results, the VEGF and APE-treated EpSCs conditioned medium enhanced the Type 1 collagen production in normal human fibroblasts (NHFs). In the clinical study, the APE improved skin hydration, dermal density, wrinkling, and sagging significantly. Our findings revealed that the APE promotes a proliferation of EpSCs, through the up-regulation of the integrin β1 and VEGF expression. The VEGF might affect the collagen synthesis of NHF as a paracrine factor. Clinical studies further suggested that treatment with formulations containing APE confers anti-aging benefits. Based on these results, we suggest that APE may be introduced as a possible anti-aging agent.
Directed evolution is a powerful technique for identifying beneficial mutations in defined DNA sequences with the goal of improving desired phenotypes. Recent methodological advances have made the ...evolution of short DNA sequences quick and easy. However, the evolution of DNA sequences >5kb in length, notably gene clusters, is still a challenge for most existing methods. Since many important microbial phenotypes are encoded by multigene pathways, they are usually improved via adaptive laboratory evolution (ALE), which while straightforward to implement can suffer from off-target and hitchhiker mutations that can adversely affect the fitness of the evolved strain. We have therefore developed a new directed evolution method (Inducible Directed Evolution, IDE) that combines the specificity and throughput of recent continuous directed evolution methods with the ease of ALE. Here, we present detailed methods for operating Inducible Directed Evolution (IDE), which enables long (up to 85kb) DNA sequences to be mutated in a high throughput manner via a simple series of incubation steps. In IDE, an intracellular mutagenesis plasmid (MP) tunably mutagenizes the pathway of interest, located on the phagemid (PM). MP contains a mutagenic operon (
, and
) that can be expressed via the addition of a chemical inducer. Expression of the mutagenic operon during a cell cycle represses DNA repair mechanisms such as proofreading, translesion synthesis, mismatch repair, and base excision and selection, which leads to a higher mutation rate. Induction of the P1 lytic cycle results in packaging of the mutagenized phagemid, and the pathway-bearing phage particles infect naïve cells, generating a mutant library that can be screened or selected for improved variants. Successive rounds of IDE enable optimization of complex phenotypes encoded by large pathways (as of this writing up to 36 kb), without requiring inefficient transformation steps. Additionally, IDE avoids off-target genomic mutations and enables decoupling of mutagenesis and screening steps, establishing it as a powerful tool for optimizing complex phenotypes in
.
Sustainably enhancing crop production is a global necessity to meet the escalating demand for staple crops while sustainably managing their associated carbon/nitrogen inputs. Leveraging ...plant-associated microbiomes is a promising avenue for addressing this demand. However, studying these communities and engineering them for sustainable enhancement of crop production have remained a challenge due to limited genetic tools and methods. In this work, we detail the development of the Maize Root Microbiome ToolKit (MRMTK), a rapid Modular Cloning (MoClo) toolkit that only takes 2.5 h to generate desired constructs (5400 potential plasmids) that replicate and express heterologous genes in Enterobacter ludwigii strain AA4 (Elu), Pseudomonas putida strain AA7 (Ppu), Herbaspirillum robiniae strain AA6 (Hro), Stenotrophomonas maltophilia strain AA1 (Sma), and Brucella pituitosa strain AA2 (Bpi), which comprise a model maize root synthetic community (SynCom). In addition to these genetic tools, we describe a highly efficient transformation protocol (107–109 transformants/μg of DNA) 1 for each of these strains. Utilizing this highly efficient transformation protocol, we identified endogenous Expression Sequences (ES; promoter and ribosomal binding sites) for each strain via genomic promoter trapping. Overall, MRMTK is a scalable and adaptable platform that expands the genetic engineering toolbox while providing a standardized, high-efficiency transformation method across a diverse group of root commensals. These results unlock the ability to elucidate and engineer plant–microbe interactions promoting plant growth for each of the 5 bacterial strains in this study.
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