In vitro gastrointestinal simulated digestion of three plant proteins: determination of digestion rate, free amino acids and peptide contents.

Cassava protein (CP), barley protein (BP) and yellow pea protein (YPP) are important nutrient and integral constituent of staple in pet foods. It is known that the digestion of proteins directly influences their absorption and utilisation. In the present work, we performed in vitro simulated gastrointestinal digestion of three plant proteins as a staple for dog and cat food. The digestion rate of CP, BP and YPP in dog food was 56.33 ± 0.90%, 48.53 ± 0.91%, and 66.96 ± 0.37%, respectively, whereas the digestion rate of CP, BP, and YPP in cat food was 66.25 ± 0.72%, 43.42 ± 0.83%, and 58.05 ± 0.85%, respectively. Using SDS-polyacrylamide gel electrophoresis to determine the molecular weight (MW) of each protein and the products of their digestion, it was revealed that MW of digestion samples decreased, and MW during the small intestine phase was lower than that during the gastric phase. Peptide sequences of digested products were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and it was found that the total number of peptides in the small intestine digestion samples was higher than that in the gastric phase samples. The MW of peptides obtained from CP was within the range of 1000-1500 Da, while MW of peptides derived from BP and YPP was within the range of 400-2000 Da. In addition, free amino acids were mainly produced in the small intestine phase. Furthermore, the percentage of essential amino acids in the small intestine phase (63 ~ 82%) was higher than that in the gastric phase (37 ~ 63%). Taken together, these findings contribute to the current understanding of the utilisation of plant proteins in dog and cat foods and provide important insights into the selection and application of plant proteins as a staple in dog and cat foods.

Efficient removal of Cr (VI) from coal gangue by indigenous bacteria-YZ1 bacteria: Adsorption mechanism and reduction characteristics of extracellular polymer.

The existence of heavy metals (especially Cr (VI)) in coal gangue has brought great safety risks to the environment. The indigenous bacteria (YZ1 bacteria) were separated and applied for removing Cr (VI) from the coal gangue, in which its tolerance to Cr (VI) was explored. The removal mechanism of Cr (VI) was investigated with pyrite in coal gangue, metabolite organic acids and extracellular polymer of YZ1 bacteria. The concentration of Cr (VI) could be stabilized around 0.012 mg/L by the treatment with YZ1 bacteria. The Cr (VI) tolerance of YZ1 bacteria reached 60 mg/L, and the removal efficiency of Cr (VI) was more than 95% by using YZ1 bacteria combined with pyrite. The organic acids had a certain reducing ability to Cr (VI) (removal efficiency of less than 10%). The extracellular polymers (EPS) were protective for the YZ1 bacteria resisting to Cr (VI). The polysaccharides and Humic-like substances in the soluble extracellular polymers (S-EPS) had strong adsorption and reduction effect on Cr (VI), in which the tryptophan and tyrosine proteins in the bound extracellular polymers (LB-EPS and TB-EPS) could effectively promote the reduction of Cr (VI). YZ1 bacteria could obviously reduce the damage of Cr (VI) from coal gangue to the environment.

Simvastatin restores pulmonary endothelial function in the setting of pulmonary over-circulation.

Statin therapy is a cornerstone in the treatment of systemic vascular diseases. However, statins have failed to translate as therapeutics for pulmonary vascular disease. Early pulmonary vascular disease in the setting of congenital heart disease (CHD) is characterized by endothelial dysfunction, which precedes the more advanced stages of vascular remodeling. These features make CHD an ideal cohort in which to re-evaluate the potential pulmonary vascular benefits of statins, with a focus on endothelial biology. However, it is critical that the full gamut of the pleiotropic effects of statins in the endothelium are uncovered. The purpose of this investigation was to evaluate the therapeutic potential of simvastatin for children with CHD and pulmonary over-circulation, and examine mechanisms of simvastatin action on the endothelium. Our data demonstrate that daily simvastatin treatment preserves endothelial function in our shunt lamb model of pulmonary over-circulation. Further, using pulmonary arterial endothelial cells (PAECs) isolated from Shunt and control lambs, we identified a new mechanism of statin action mediated by increased expression of the endogenous Akt1 inhibitor, C-terminal modifying protein (CTMP). Increases in CTMP were able to decrease the Akt1-mediated mitochondrial redistribution of endothelial nitric oxide synthase (eNOS) which correlated with increased enzymatic coupling, identified by increases in NO generation and decreases in NOS-derived superoxide. Together our data identify a new mechanism by which simvastatin enhances NO signaling in the pulmonary endothelium and identify CTMP as a potential therapeutic target to prevent the endothelial dysfunction that occurs in children born with CHD resulting in pulmonary over-circulation.

Correlations between flavor and fermentation days and changes in quality-related physiochemical characteristics of fermented Aurantii Fructus.

The effects of different fermentation times (0, 1, 2, 3, 4, and 5 days) on the physicochemical properties and flavor components of fermented Aurantii Fructus (FAF) were evaluated. Component analysis identified 66 compounds in positive ion mode and 32 compounds in negative ion mode. Flash GC e-nose results showed that propanal, (+)-limonene and n-nonanal may be the flavor characteristic components that distinguish FAF with different fermentation days. Furthermore, we found that the change of total flavonoid content was closely related to colony growth vitality. The total flavonoid content of FAF gradually decreased from 3rd day and then increased from 5th day (3rd day: 0.766 ± 0.123 mg/100 g; 4th day: 0.464 ± 0.001 mg/100 g; 5th day: 0.850 ± 0.192 mg/100 g). Finally, according to antioxidant activity correlation analysis, meranzin, (+)-limonene and total flavonoids were found to be the key substances affecting the fermentation days of FAF. Overall, the optimal fermentation time for FAF was 4 days.

Identification of Gut Microbiome and Metabolites Associated with Acute Diarrhea in Cats.

Changes in diet and environment can lead to acute diarrhea in companion animals, but the composition and interactions of the gut microbiome during acute diarrhea remain unclear. In this multicenter case-control study, we investigated the relationship between intestinal flora and acute diarrhea in two breeds of cats. Acutely diarrheic American Shorthair (MD, n = 12) and British Shorthair (BD, n = 12) and healthy American Shorthair (MH, n = 12) and British Shorthair (BH, n = 12) cats were recruited. Gut microbial 16S rRNA sequencing, metagenomic sequencing, and untargeted metabolomic analysis were performed. We observed significant differences in beta-diversity (Adonis, P < 0.05) across breeds and disease state cohorts. Profound differences in gut microbial structure and function were found between the two cat breeds. In comparison to healthy British Shorthair cats, Prevotella, Providencia, and Sutterella were enriched while Blautia, Peptoclostridium, and Tyzzerella were reduced in American Shorthair cats. In the case-control cohort, cats with acute diarrhea exhibited an increased abundance of Bacteroidota, Prevotella, and Prevotella copri and a decreased abundance of Bacilli, Erysipelotrichales, and Erysipelatoclostridiaceae (both MD and BD cats, P < 0.05). Metabolomic analysis identified significant changes in the BD intestine, affecting 45 metabolic pathways. Moreover, using a random forest classifier, we successfully predicted the occurrence of acute diarrhea with an area under the curve of 0.95. Our findings indicate a distinct gut microbiome profile that is associated with the presence of acute diarrhea in cats. However, further investigations using larger cohorts of cats with diverse conditions are required to validate and extend these findings. IMPORTANCE Acute diarrhea is common in cats, and our understanding of the gut microbiome variations across breeds and disease states remains unclear. We investigated the gut microbiome of two cat breeds (British Shorthair and American Shorthair) with acute diarrhea. Our study revealed significant effects of breeds and disease states on the structure and function of the gut microbiota in cats. These findings emphasize the need to consider breed-related factors in animal nutrition and research models. Additionally, we observed an altered gut metabolome in cats with acute diarrhea, closely linked to changes in bacterial genera. We identified a panel of microbial biomarkers with high diagnostic accuracy for feline acute diarrhea. These findings provide novel insights into the diagnosis, classification, and treatment of feline gastrointestinal diseases.

Fixating lead in coal gangue with phosphate using phosphate-dissolving bacteria: Phosphorus dissolving characteristics of bacteria and adsorption mechanism of extracellular polymer.

Controlling and preventing lead pollution is currently the focus of environmental remediation. Coal gangue contains large quantities of lead, and its environmental impact cannot be ignored. This study investigated the tolerance of Stenotrophomonas maltophilia (YZ-1 train) to lead ion and its fixation effect on lead in coal gangue. The fixation mechanism of lead ions by using the YZ-1 train was studied with CaHPO4 and Ca3(PO4)2. The tolerance mechanism and fixation characteristics of the three bacterial extracellular polymers and cell components to lead were analyzed. The results show that the YZ-1 train had a strong resistance to lead ions. The amount of lead released from coal gangue can be reduced by up to 91.1% upon treatment with the YZ-1 train, which can dissolve phosphate minerals to form stable hydroxyapatite (Pb5(PO4)3(OH)) and pyromorphite (Pb5(PO4)3Cl) with lead ions. Tryptophan and tyrosine from cellular components and extracellular polymers with loosely and tightly bound proteins are the main participants in the fixation of lead ions. The by-products of soluble microbes affect the fixation of lead ions in soluble extracellular polymers. The carboxylic acids and carboxylates secreted by bacteria are involved in the adsorption and fixation of lead ions.

Dose-related adaptive reconstruction of DMN in isoflurane administration: a study in the rat.

BACKGROUND: The anesthetic states are accompanied by functional alterations. However, the dose-related adaptive alterations in the higher-order network under anesthesia, e. g. default mode network (DMN), are poorly revealed. METHODS: We implanted electrodes in brain regions of the rat DMN to acquire local field potentials to investigate the perturbations produced by anesthesia. Relative power spectral density, static functional connectivity (FC), fuzzy entropy of dynamic FC, and topological features were computed from the data. RESULTS: The results showed that adaptive reconstruction was induced by isoflurane, exhibiting reduced static and stable long-range FC, and altered topological features. These reconstruction patterns were in a dose-related fashion. CONCLUSION: These results might impart insights into the neural network mechanisms underlying anesthesia and suggest the potential of monitoring the depth of anesthesia based on the parameters of DMN.

Reclamation of waste coal gangue activated by Stenotrophomonas maltophilia for mine soil improvement: Solubilizing behavior of bacteria on nutrient elements.

The coal gangue has occupied the farmland and caused severe pollution to the surrounding environment, which was discharged with vast amount as a by-product of coal mining and washing. A sustainable and ecological microorganism activation method was proposed to disposal coal gangue as mineral fertilizer. A Stenotrophomonas maltophilia YZ1 bacteria was separated and found to be useful in solubilizing nutrient elements in coal gangue. The contents of available P, available K and available Si in the treated coal gangue reached 278.4 mg/kg, 1305.3 mg/kg and 522.7 mg/kg, respectively. The YZ1 bacteria dissolved the minerals of monetite (CaHPO4), muscovite and annite by the organic acids, which were the metabolism product of YZ1 bacteria. The solubilizing mechanisms of phosphate minerals included the release of protic and the chelation of organic acid with calcium. The microbial activation method can provide nutrient elements for soil, which may realize the reclamation of coal gangue in a harmless way.

HIF-1α promotes cellular growth in lymphatic endothelial cells exposed to chronically elevated pulmonary lymph flow.

Normal growth and development of lymphatic structures depends on mechanical forces created by accumulating interstitial fluid. However, prolonged exposure to pathologic mechanical stimuli generated by chronically elevated lymph flow results in lymphatic dysfunction. The mechanisms that transduce these mechanical forces are not fully understood. Our objective was to investigate molecular mechanisms that alter the growth and metabolism of isolated lymphatic endothelial cells (LECs) exposed to prolonged pathologically elevated lymph flow in vivo within the anatomic and physiologic context of a large animal model of congenital heart disease with increased pulmonary blood flow using in vitro approaches. To this end, late gestation fetal lambs underwent in utero placement of an aortopulmonary graft (shunt). Four weeks after birth, LECs were isolated and cultured from control and shunt lambs. Redox status and proliferation were quantified, and transcriptional profiling and metabolomic analyses were performed. Shunt LECs exhibited hyperproliferative growth driven by increased levels of Hypoxia Inducible Factor 1α (HIF-1α), along with upregulated expression of known HIF-1α target genes in response to mechanical stimuli and shear stress. Compared to control LECs, shunt LECs exhibited abnormal metabolism including abnormalities of glycolysis, the TCA cycle and aerobic respiration. In conclusion, LECs from lambs exposed in vivo to chronically increased pulmonary lymph flow are hyperproliferative, have enhanced expression of HIF-1α and its target genes, and demonstrate altered central carbon metabolism in vitro. Importantly, these findings suggest provocative therapeutic targets for patients with lymphatic abnormalities.

DNMT1 deregulation of SOCS3 axis drives cardiac fibroblast activation in diabetic cardiac fibrosis.

Cardiac fibrosis is one of the main pathological manifestations of diabetic cardiomyopathy (DCM). Cardiac fibroblast activation is a key effector of cells resulting in diabetic cardiac fibrosis. However, the underlying mechanism of cardiac fibroblast activation and diabetic cardiac fibrosis remains unclear. Accumulating evidence suggests that DNA methylation alterations play a central role in cardiac fibroblast activation. In this study, we demonstrated that DNA methyltransferase 1 (DNMT1)-mediated suppression of cytokine signaling 3 (SOCS3) promoter hypermethylation leads to downregulation of SOCS3 expression in diabetic cardiac fibrosis. High glucose-induced expression of DNMT1 was increased in cardiac fibroblasts, while the expression of SOCS3 was decreased. Downregulation of SOCS3 facilitated activation of STAT3 to promote cardiac fibroblast activation and collagen deposition. Genetic or pharmacological inactivation of DNMT1 reversed the activated phenotype of cardiac fibroblasts. Clinically, we observed a significant inverse correlation between DNMT1 and SOCS3 expression levels, and loss of SOCS3 expression or increased expression of DNMT1. Taken together, these findings identify DNMT1 silencing of SOCS3 axis as a driver of cardiac fibroblast activation in diabetic cardiac fibrosis. These results provide a scientific and new explanation of the underlying mechanism of diabetic cardiac fibrosis.

Sad Music Modulates Pain Perception: An EEG Study.

BACKGROUND: Music has shown positive effects on pain management in previous studies. However, the relationship between musical emotional types and therapeutic effects remains unclear. To investigate this issue, this study tested three typical emotional types of music and discussed their neural mechanisms in relation to pain modulation. SUBJECTS AND METHODS: In this experiment, 40 participants were exposed to cold pain under four conditions: listening to happy music, listening to neutral music, listening to sad music and no sound. EEG and pain thresholds were recorded. The participants were divided into the remission group and the nonremission group for analysis. Differences among conditions were quantified by the duration of exposure to the pain-inducing stimulus in the remission group. EEG data were obtained using a fast Fourier transform (FFT) and then correlated with the behavioral data. RESULTS: We found that sad music had a significantly better effect on alleviating pain, as a result of brain oscillations in a higher beta band and the gamma band at the O2 and P4 electrodes. The comparison between the remission group and the nonremission group suggested that personality may affect music-induced analgesia, and dominance, liveliness and introvert and extrovert personality traits were associated with pain modulation by sad music. Additionally, in the network analysis, we compared brain networks under the three conditions and discussed the possible mechanisms underlying the better analgesic effect of sad music. CONCLUSION: Sad music may have a better effect on alleviating pain, and its neural mechanisms are also discussed. This work may help understand the effects of music on pain modulation, which also has potential value for clinical use.

CTBP1­AS2 inhibits proliferation and induces autophagy in ox­LDL­stimulated vascular smooth muscle cells by regulating miR­195­5p/ATG14.

Atherosclerosis (AS) is a chronic progressive disease caused by injury and functional changes in vascular smooth muscle cells (VSMCs). Long non­coding RNAs (lncRNAs) are pivotal regulators in AS development. The present study aimed to explore the roles and molecular mechanisms of lncRNA CTBP1­AS2 in AS progression. A dual­luciferase reporter assay confirmed that miR­195­5p is a downstream target miRNA of lncRNA CTBP1­AS2 and miR­195­5p was increased in AS. The expression levels of miR­195­5p and CTBP1­AS2 in the serums of patients with AS and human aorta vascular smooth muscle cells was increased or decreased, respectively, following treatment with oxidized low­density lipoprotein (ox­LDL). Functional experiments showed that the overexpression of lncRNA CTBP1­AS2 inhibited the proliferation of HA­VSMCs and promoted their autophagy following ox­LDL treatment. This effect could be reversed by treatment with ROC­325, the inhibitor of autophagy, or miR­195­5p mimics. Autophagy related 14 (ATG14) was identified to be a target of miR­195­5p, and lncRNA CTBP1­AS2 promoted ATG14 expression by serving as a competing endogenous RNA of miR­195­5p. The present study revealed that lncRNA CTBP1­AS2 may serve a role in AS by inhibiting the proliferation and promoting the autophagy of VSMCs through ATG14 modulation via miR­195­5p. These data may provide a novel therapeutic target for AS.

Mechanical forces alter endothelin-1 signaling: comparative ovine models of congenital heart disease.

The risk and progression of pulmonary vascular disease in patients with congenital heart disease is dependent on the hemodynamics associated with different lesions. However, the underlying mechanisms are not understood. Endothelin-1 is a potent vasoconstrictor that plays a key role in the pathology of pulmonary vascular disease. We utilized two ovine models of congenital heart disease: (1) fetal aortopulmonary graft placement (shunt), resulting in increased flow and pressure; and (2) fetal ligation of the left pulmonary artery resulting in increased flow and normal pressure to the right lung, to investigate the hypothesis that high pressure and flow, but not flow alone, upregulates endothelin-1 signaling. Lung tissue and pulmonary arterial endothelial cells were harvested from control, shunt, and the right lung of left pulmonary artery lambs at 3-7 weeks of age. We found that lung preproendothelin-1 mRNA and protein expression were increased in shunt lambs compared to controls. Preproendothelin-1 mRNA expression was modestly increased, and protein was unchanged in left pulmonary artery lambs. These changes resulted in increased lung endothelin-1 levels in shunt lambs, while left pulmonary artery levels were similar to controls. Pulmonary arterial endothelial cells exposed to increased shear stress decreased endothelin-1 levels by five-fold, while cyclic stretch increased levels by 1.5-fold. These data suggest that pressure or an additive effect of pressure and flow, rather than increased flow alone, is the principal driver of increased endothelin signaling in congenital heart disease. Defining the molecular drivers of the pathobiology of pulmonary vascular disease due to differing mechanical forces will allow for a more targeted therapeutic approach.

LncRNA ANRIL acts as a modular scaffold of WDR5 and HDAC3 complexes and promotes alteration of the vascular smooth muscle cell phenotype.

Many studies have shown that long-noncoding RNA (lncRNA) is associated with cardiovascular disease, but its molecular mechanism is still unclear. In this study, we explored the role of lncRNA ANRIL in ox-LDL-induced phenotypic transition of human aortic smooth muscle cells (HASMC). The results of quantitative fluorescence PCR showed that the expression of ANRIL in patients with coronary atherosclerotic heart disease (CAD) was significantly higher than that in normal subjects. RNA-FISH detection showed that the ANRIL expression increased in HASMC treated by ox-LDL. Ox-LDL could upregulate the expression of ANRIL and ROS and promote the phenotypic transition of HASMC. After downregulation of ANRIL by siRNA, ROS level decreased and HASMC phenotypic transition alleviated. ANRIL could act as a molecular scaffold to promote the binding of WDR5 and HDAC3 to form WDR5 and HDAC3 complexes, they regulated target genes such as NOX1 expression by histone modification, upregulated ROS level and promote HASMC phenotype transition. Therefore, we found a new epigenetic regulatory mechanism for phenotype transition of VSMC, ANRIL was a treatment target of occlusive vascular diseases.

LncRNA NRON alleviates atrial fibrosis through suppression of M1 macrophages activated by atrial myocytes.

The aim of the present study was to explore the role of long non-coding RNA (lncRNA) non-coding repressor of NFAT (NRON) in the atrial fibrosis and to explore whether its underlying mechanism was associated with macrophage polarization. Enzyme-linked immunosorbent assay (ELISA) analysis of pro-inflammatory cytokines revealed that NRON overexpression suppressed, whereas NRON silencing facilitated the angiotensin II (Ang II)-induced inflammatory response in primary cultured atrial myocytes. The chromatin immunoprecipitation (ChIP) results showed that nuclear factor of activated T cell 3 (NFATc3) was recruited to the promoter region of interleukin (IL) 12 (IL-12) in atrial myocytes. Further data showed that NRON overexpression suppressed, whereas NRON silencing further promoted the Ang II-induced NFATc3 nuclear transport and IL-12 expression in atrial myocytes. Moreover, RAW264.7 macrophages were incubated with the conditioned medium from the Ang II-treated atrial myocytes transfected with NRON and IL-12 overexpression vectors. IL-12 overexpression abrogated the NRON overexpression-mediated inhibition of RAW264.7 macrophage polarization to the M1-like phenotype. Additionally, mouse atrial fibroblasts were incubated with the culture medium from RAW264.7 macrophages treated as described above. IL-12 overexpression rescued the NRON overexpression-inhibited protein levels of fibrosis markers Collagen I/III in mouse atrial fibroblasts. Collectively, our data indicate that lncRNA NRON alleviates atrial fibrosis through suppression of M1 macrophages activated by atrial myocytes.

COL4A1 Mutations Cause Neuromuscular Disease with Tissue-Specific Mechanistic Heterogeneity.

Collagen type IV alpha 1 and alpha 2 chains form heterotrimers ([α1(IV)]2α2(IV)) that represent a fundamental basement membrane constituent. Dominant COL4A1 and COL4A2 mutations cause a multisystem disorder that is marked by clinical heterogeneity and variable expressivity and that is generally characterized by the presence of cerebrovascular disease with ocular, renal, and muscular involvement. Despite the fact that muscle pathology is reported in up to one-third of individuals with COL4A1 and COL4A2 mutations and in animal models with mutations in COL4A1 and COL4A2 orthologs, the pathophysiological mechanisms underlying COL4A1-related myopathy are unknown. In general, mutations are thought to impair [α1(IV)]2α2(IV) secretion. Whether pathogenesis results from intracellular retention, extracellular deficiency, or the presence of mutant proteins in basement membranes represents an important gap in knowledge and a major obstacle for developing targeted interventions. We report that Col4a1 mutant mice develop progressive neuromuscular pathology that models human disease. We demonstrate that independent muscular, neural, and vascular insults contribute to neuromyopathy and that there is mechanistic heterogeneity among tissues. Importantly, we provide evidence of a COL4A1 functional subdomain with disproportionate significance for tissue-specific pathology and demonstrate that a potential therapeutic strategy aimed at promoting [α1(IV)]2α2(IV) secretion can ameliorate or exacerbate myopathy in a mutation-dependent manner. These data have important translational implications for prediction of clinical outcomes based on genotype, development of mechanism-based interventions, and genetic stratification for clinical trials. Collectively, our data underscore the importance of the [α1(IV)]2α2(IV) network as a multifunctional signaling platform and show that allelic and tissue-specific mechanistic heterogeneities contribute to the variable expressivity of COL4A1 and COL4A2 mutations.

LncRNA NRON alleviates atrial fibrosis via promoting NFATc3 phosphorylation.

The aim of this study was to explore the role of NRON in the atrial fibrosis. The expression of NRON in atrial tissue was detected using qRT-PCR. The protein levels of collagen I, collagen III, NFATc3 and p-NFATc3 were determined by western blot. Immunohistochemistry assay were performed to observe expression and distribution of collagen I in atrial tissues. The atrial fibroblasts were authenticated by vimentin/troponin immunofluorescence staining. Fibroblast proliferation was detected by CCK-8 assay. The morphological changes of cardiac tissue were observed by HE staining. Myocardial fibrosis was detected by masson staining. NRON expression was significantly downregulated in atrial tissues of AF. NRON suppressed fibroblast proliferation; expression of collagen I and collagen III; activation of NFATc3 and nucleu import. NRON promoted p-NFATc3 expression and alleviated atrial fibrosis in vivo. Our data indicated that NRON alleviates atrial fibrosis via promoting NFATc3 phosphorylation.

Correction: Preservation of myocardial contractility during acute hypoxia with OMX-CV, a novel oxygen delivery biotherapeutic.

[This corrects the article DOI: 10.1371/journal.pbio.2005924.].