Development of an aptamer capable of multidrug resistance reversal for tumor combination chemotherapy.

Multidrug resistance (MDR) is a major factor in the failure of many forms of tumor chemotherapy. Development of a specific ligand for MDR-reversal would enhance the intracellular accumulation of therapeutic agents and effectively improve the tumor treatments. Here, an aptamer was screened against a doxorubicin (DOX)-resistant human hepatocellular carcinoma cell line (HepG2/DOX) via cell-based systematic evolution of ligands by exponential enrichment. A 50 nt truncated sequence termed d3 was obtained with high affinity and specificity for HepG2/DOX cells. Multidrug resistance protein 1 (MDR1) is determined to be a possible recognition target of the selected aptamer. Aptamer d3 binding was revealed to block the MDR of the tumor cells and increase the accumulation of intracellular anticancer drugs, including DOX, vincristine, and paclitaxel, which led to a boost to the cell killing of the anticancer drugs and lowering their survival of the tumor cells. The aptamer d3-mediated MDR-reversal for effective chemotherapy was further verified in an in vivo animal model, and combination of aptamer d3 with DOX significantly improved the suppression of tumor growth by treating a xenograft HepG2/DOX tumor in vivo. This work demonstrates the feasibility of a therapeutic DNA aptamer as a tumor MDR-reversal agent, and combination of the selected aptamer with chemotherapeutic drugs shows great potential for liver cancer treatments.

Dynamic transcriptome profiles of skeletal muscle growth and development in Shaziling and Yorkshire pigs using RNA-Seq.

BACKGROUD: We previously demonstrated that Shaziling and Yorkshire pigs differ in growth rate and meat quality. However, the molecular mechanisms responsible for such phenotypic differences remains unclear. Here, we performed a transcriptomic analysis of 36 longissimus dorsi (LM) and 36 soleus (SM) muscle samples from Shaziling and Yorkshire pigs at six postnatal stages (30, 60, 90, 150, 210, and 300 days) to explore the differences in postnatal skeletal muscle of Shaziling and Yorkshire pigs. RESULTS: Muscle morphological changes and the number of differentially expressed genes (DEGs) indicated the two stages of 60 to 90 d and 150 to 210 d were critical for the muscle growth and development in Shaziling pigs. The genes such as FLNC, COL1A1, NRAP, SMYD1, TNNI3, CRYAB, and PDLIM3 played vital roles in the muscle growth, and the genes such as CCDC71L, LPIN1, CPT1A, UCP3, NR4A3, and PDK4 played dominant roles in the lipid metabolism. Additionally, in contrast to the LM, the percentage of slow-twitch muscle fibers in the SM of both breeds consistently decreased from 30 to 150 days of age, but there was a significant rebound at 210 days of age. However, the percentage of slow-twitch muscle fibers in the SM of Shaziling pigs was higher than that in Yorkshire pigs, which may be associated with the calcium signaling pathway and PPARß/δ signaling pathway. CONCLUSION: This study detected two critical periods and many functional genes for the muscle growth and development of Shaziling pigs, and showed differences in muscle fiber characteristics between Shaziling and Yorkshire pigs. This article is protected by copyright. All rights reserved.

Effects of Dietary Eucommia ulmoides Leaf Extract Supplementation on Growth Performance, Meat Quality, Antioxidant Capacity, and Lipid Metabolism of Finishing Pigs.

This study aimed to investigate the effects of dietary Eucommia ulmoides leaf extract (ELE) on meat quality, antioxidant capacity, and lipid metabolism in finishing pigs. A total of 240 "Duroc × Landrace × Yorkshire" crossbred pigs with an initial weight of 74.70 ± 0.77 kg were randomly assigned to two groups: control group and 0.2% ELE group, with each group containing 10 replicates of 12 pigs per pen (half barrows and half gilts). The data showed dietary 0.2% ELE supplementation did not affect growth performance but tended to reduce the backfat thickness of the finishing pigs (p = 0.07). ELE diets increased pH value (p < 0.05) and meat color score (p = 0.01) and decreased 45 min L* value (p < 0.05), 24 h L* value (p = 0.01), pressurization loss (p = 0.01), and 24 h drip loss (p < 0.05) in longissimus dorsi (LD) muscle, accompanied by an increased (p < 0.05) proportion of monounsaturated fatty acids (MUFA) and decreased polyunsaturated fatty acids (PUFA) (p = 0.06) and n-6/n-3 PUFA ratio (p = 0.05) compared to controls. In addition, ELE supplementation increased inosine monophosphate (IMP) (p = 0.01), sweet amino acids (AAs) (p < 0.05), and total free AA content (p = 0.05) in LD. Meanwhile, increased activity of glutathione peroxidase (p < 0.05) and superoxide dismutase (p < 0.01) in both serum and LD muscle and decreased malondialdehyde content (p < 0.01) in LD muscle were detected with ELE treatment. Moreover, pigs fed ELE had a higher total protein (p < 0.01), albumin (p < 0.05), and high-density lipoprotein cholesterol (p < 0.05) and a lower total cholesterol (p < 0.01) and triacylglycerols (p = 0.06) in serum. Consistently, significant effects of dietary ELE were observed on the relative mRNA expression of lipid metabolism in the backfat and the LD muscle, respectively. ELE attenuated lipogenic processes in backfat, decreasing the relative expression of acetyl-CoA carboxylase and upregulating the relative expression of adipose triacyl glyceride lipase, carnitine palmitoyl transferase 1B, and fatty acid-binding protein 4 (p < 0.05). ELE also decreased the relative expression of CCAAT/enhancer-binding protein α (p < 0.05), fatty acid translocase (p < 0.05), carnitine palmitoyl transferase 1B (p < 0.01), and adipose triacyl glyceride lipase (p < 0.05) in LD muscle (p < 0.05). More specifically, lipogenesis appeared to be inhibited in both LD muscle and backfat, with the difference being that lipolysis was enhanced in backfat and inhibited in LD muscle. In conclusion, dietary ELE supplementation can potentially enhance carcass traits, sensory quality, and nutritional value of pork without negatively affecting intramuscular fat content. The underlying mechanism for these positive effects may be linked to the alterations in lipid metabolism and increased antioxidant capacity induced by ELE.

Leucine regulates lipid metabolism in adipose tissue through adipokine-mTOR-SIRT1 signaling pathway and bile acid-microbe axis in a finishing pig model.

This study was conducted to explore the regulatory mechanism of leucine (Leu) on lipid metabolism of finishing pigs. Twenty-four Duroc × Landrace × Large cross pigs with an average body weight of 68.33 ± 0.97 kg were randomly allocated into 3 treatment groups with 8 replicates per group (1 pig per replicate). The dietary treatments were as follows: control group (CON), 0.25% Leu group and 0.50% Leu group. The experimental period was 42 d. The results showed as follows. (1) Compared with the CON, 0.25% and 0.50% Leu increased (P < 0.01) the average daily gain (ADG), while the average backfat thickness (ABT) and the ratio of feed intake to body weight gain (F:G ratio) were decreased (P < 0.05). (2) In the 0.25% Leu group, the relative mRNA expression levels of sterol regulatory element binding protein-1c (SREBP1c), recombinant fatty acid transport protein 1 (FATP1), chemerin and peroxisome proliferator-activated receptor γ (PPARγ) were decreased but the level of fatty acid binding protein 4 (FABP4) and fatty acid translocase (FAT/CD36) were increased in backfat tissue. In the 0.25% Leu group, the protein levels of p-Rictor, p-Raptor, p-eIF4E-binding protein 1 (p-4EBP1), p-silent mating type information regulator 2 homolog 1 (p-SIRT1) and acetylation ribosome s6 protein kinase 1 (Ac-S6K1) were increased (P < 0.05). (3) Compared to the CON, the diversity of gut microbiota in the 0.25% Leu group was increased. Principal component analysis showed that the relative abundance of Bacteroidetes, Lactobacillus and Desulfovibrio was higher in the 0.25% Leu group than the CON, but the relative abundance of Firmicutes, Treponema and Shigella was lower than in the CON (P < 0.05). (4) Four different metabolites were screened out from the serum of finishing pigs including allolithocholic acid (alloLCA), isolithocholic acid (isoLCA), ursodeoxycholic acid (UDCA) and hyodeoxycholic acid (HDCA), which correlate to various degrees with the above microorganisms. In conclusion, Leu could promote adipose tissue lipolysis of finishing pigs through the mTOR-SIRT1 signaling pathway, and S6K1 is acetylated at the same time, and the interaction between gut microbiota and bile acid metabolism is also involved.

Dietary supplementation of Eucommia leaf extract to growing-finishing pigs alters muscle metabolism and improves meat quality.

OBJECTIVE: The objective of this study was to investigate the influence of dietary supplementation of Eucommia ulmoides leaf extract (ELE) on muscle metabolism and meat quality of pigs with and without pre-slaughter transportation. METHODS: In a 43-day feeding experiment, a total of 160 pigs with an initial body weight 60.00±2.00 kg were randomly assigned into four groups in a completely randomized design with 10 replicates. Pigs in groups A and C were fed a basal diet and pigs in groups B and D were fed a basal diet supplemented with 0.5% ELE. Pigs were slaughtered with (group B and D) or without (group A and C) pre-slaughter transport. Muscle chemical composition, postmortem glycolysis, meat quality and muscle metabolome were analyzed. RESULTS: Dietary ELE supplementation had no effect on the proximate composition of porcine muscle, but increased free phenylalanine, proline, citruline, norvaline, and the total free amino acids in muscle. In addition, dietary ELE increased decanoic acid and eicosapentaenoic acid, but decreased heptadecanoic acid, oleic acid, trans-oleic acid, and monounsaturated fatty acids in muscle. Meat quality measurement demonstrated that ELE improved meat water holding capacity and eliminated the negative effects of pre-slaughter transport on meat cooking yield and tenderness. Dietary ELE reduced muscle glycolytic potential, inhibited glycolysis and muscle pH decline in the postmortem conversion of muscle to meat and increased the activity of citrate synthase in muscle. Metabolomics analysis by liquid chromatographic tandem mass spectrometric showed that ELE enhanced muscle energy level, regulated AMP-activated protein kinase (AMPK) signaling, modulated glycogenolysis/glycolysis, and altered the metabolism of carbohydrate, fatty acids, ketone bodies, amino acids, purine, and pyrimidine. CONCLUSION: Dietary ELE improved meat quality and alleviated the negative effect of preslaughter transport on meat quality by enhancing muscle oxidative metabolism capacity and inhibiting glycolysis in postmortem muscle, which is probably involved its regulation of AMPK.

Development and Recovery of Liver Injury in Piglets by Incremental Injection of LPS.

This study aimed to explore the effects of the incremental injection of lipopolysaccharide (LPS) on liver histopathology, inflammation, oxidative status, and mitochondrial function in piglets. Forty healthy Duroc × Landrace × Yorkshire castrated boars (21 ± 2 days old, weight 6.84 ± 0.11 kg) were randomly assigned to five groups (n = 8) and then slaughtered on days 0 (group 0, without LPS injection), 1 (group 1), 5 (group 5), 9 (group 9), and 15 (group 15) of LPS injection, respectively. The results showed that, compared to the piglets without LPS injection, LPS injection caused liver injury in the early phase, as manifested by the increased activities of serum liver injury-related parameters (aspartate amino transferase, alanine aminotransferase, alkaline phosphatase, cholinesterase, and total bile acid) on day 1, and impaired liver morphology (disordered hepatic cell cord arrangement, dissolved and vacuolized hepatocytes, karyopycnosis, and inflammatory cell infiltration and congestion) on days 1 and 5. Meanwhile, LPS injection caused liver inflammation, oxidative stress, and mitochondrial dysfunction on days 1 and 5, as reflected by the upregulated mRNA expression of TNF-α, IL-6, IL-1ß, TLR4, MyD88, and NF-κB; increased MPO and MDA content; and impaired mitochondrial morphology. However, these parameters were ameliorated in the later phase (days 9~15). Taken together, our data indicate that the incremental injection of the LPS-induced liver injury of piglets could be self-repaired.

Organism type of infection is associated with prognosis in sepsis: an analysis from the MIMIC-IV database.

BACKGROUND: Sepsis has a high mortality rate, which is expensive to treat, and is a major drain on healthcare resources; it seriously impacts the quality of human life. The clinical features of positive or non-positive blood cultures have been reported, but the clinical features of sepsis with different microbial infections and how they contribute to clinical outcomes have not been adequately described. METHODS: We extracted clinical data of septic patients with a single pathogen from the online Medical Information Mart for Intensive Care(MIMIC)-IV database. Based on microbial cultures, patients were classified into Gram-negative, Gram-positive, and fungal groups. Then, we analyzed the clinical characteristics of sepsis patients with Gram-negative, Gram-positive, and fungal infections. The primary outcome was 28-day mortality. The secondary outcomes were in-hospital mortality, the length of hospital stay, the length of ICU stay, and the ventilation duration. In addition, Kaplan-Meier analysis was used for the 28-day cumulative survival rate of patients with sepsis. Finally, we performed further univariate and multivariate regression analyses for 28-day mortality and created a nomogram for predicting 28-day mortality. RESULTS: The analysis showed that bloodstream infections showed a statistically significant difference in survival between Gram-positive and fungal organisms; drug resistance only reached statistical significance for Gram-positive bacteria. Through univariate and multivariate analysis, it was found that both the Gram-negative bacteria and fungi were independent risk factors for the short-term prognosis of sepsis patients. The multivariate regression model showed good discrimination, with a C-index of 0.788. We developed and validated a nomogram for the individualized prediction of 28-day mortality in patients with sepsis. Application of the nomogram still gave good calibration. CONCLUSIONS: Organism type of infection is associated with mortality of sepsis, and early identification of the microbiological type of a patient with sepsis will provide an understanding of the patient's condition and guide treatment.

Engineered Bacillus subtilis alleviates intestinal oxidative injury through Nrf2-Keap1 pathway in enterotoxigenic Escherichia coli (ETEC) K88-infected piglet. / 工程枯草芽孢杆菌通过Nrf2-Keap1途径缓解ETEC K88æ„ŸæŸ“ä»”çŒªå¯¼è‡´çš„è‚ é“æ°§åŒ–æŸä¼¤.

Engineered probiotics can serve as therapeutics based on their ability of produce recombinant immune-stimulating properties. In this study, we built the recombinant Bacillus subtilis WB800 expressing antimicrobial peptide KR32 (WB800-KR32) using genetic engineering methods and investigated its protective effects of nuclear factor-E2-related factor 2 (Nrf2)|-Kelch-like ECH-associated protein 1 (Keap1) pathway activation in intestinal oxidative disturbance induced by enterotoxigenic Escherichia coli (ETEC) K88 in weaned piglets. Twenty-eight weaned piglets were randomly distributed into four treatment groups with seven replicates fed with a basal diet. The feed of the control group (CON) was infused with normal sterilized saline; meanwhile, the ETEC, ETEC+WB800, and ETEC+WB800-KR32 groups were orally administered normal sterilized saline, 5×1010 CFU (CFU: colony forming units) WB800, and 5×1010 CFU WB800-KR32, respectively, on Days 1|‒|14 and all infused with ETEC K88 1×1010 CFU on Days 15|‒|17. The results showed that pretreatment with WB800-KR32 attenuated ETEC-induced intestinal disturbance, improved the mucosal activity of antioxidant enzyme (catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx)) and decreased the content of malondialdehyde (MDA). More importantly, WB800-KR32 downregulated genes involved in antioxidant defense (GPx and SOD1). Interestingly, WB800-KR32 upregulated the protein expression of Nrf2 and downregulated the protein expression of Keap1 in the ileum. WB800-KR32 markedly changed the richness estimators (Ace and Chao) of gut microbiota and increased the abundance of Eubacterium_rectale_ATCC_33656 in the feces. The results suggested that WB800-KR32 may alleviate ETEC-induced intestinal oxidative injury through the Nrf2-Keap1 pathway, providing a new perspective for WB800-KR32 as potential therapeutics to regulate intestinal oxidative disturbance in ETEC K88 infection.

Profiles of muscular amino acids, fatty acids, and metabolites in Shaziling pigs of different ages and relation to meat quality.

Pork meat is closely related to physicochemical alterations during growth and development, resulting in differences in nutritional value and meat flavor. This study aimed to evaluate the composition of amino acids, fatty acids, and metabolic profiles in the longissimus thoracis muscle (LM) of Shaziling pigs aged 30, 90, 150, 210, and 300 days. The results showed that the predominant fatty acids identified in the LM of Shaziling pigs were C16:0, C16:1, C18:0, C18:1n9c, and C18:2n6c. An opposite correlation was observed for C18:2n6c and n6/n3 polyunsaturated fatty acids (P<0.05). Alanine, aspartate, glutamate, D-glutamine, and D-glutamate metabolism were the main metabolic pathways for the Shaziling pig meat flavor (P<0.05). Moreover, the correlation coefficients revealed that the contents of anserine, C16:0, C16:1, and C18:1n9c were positively correlated with intramuscular fat and/or pH24h and were negatively correlated with the values of L* (lightness) and b* (yellowness) (P<0.05). In conclusion, age greatly affected the meat quality of Shaziling pigs, and the contents of muscular anserine, C16:0, C16:1, and C18:1n9c might be promising indicators for better meat quality.

Sacubitril/valsartan alleviates sepsis-induced acute lung injury via inhibiting GSDMD-dependent macrophage pyroptosis in mice.

Sepsis-induced acute lung injury (ALI) is a life-threatening disorder with intricate pathogenesis. Macrophage pyroptosis reportedly plays a vital role in ALI. Although it has been established that angiotensin receptor blockers (ARBs) can reduce sepsis-induced organ injury, the efficacy of sacubitril/valsartan (SV) for sepsis has been largely understudied. Here, we aimed to investigate the role of SV in sepsis-induced ALI. Caecal ligation and puncture (CLP) were used to induce polymicrobial sepsis and related ALI. The therapeutic effects of SV in CLP mice were subsequently assessed. Gasdermin D (GSDMD)-/- mice were used to validate the signalling pathways affected by SV. In vitro, mouse bone marrow-derived macrophages (BMDMs) and Raw264.7 cells were treated with SV following exposure to lipopolysaccharide and adenosine triphosphate. Finally, the serum obtained from 42 septic patients was used for biochemical analysis. Compared to the other ARBs, SV yielded more pronounced anti-inflammatory effects on macrophages. In vivo, SV decreased mortality rates, significantly reduced lung damage and prevented the inflammatory response in CLP mice. In addition, SV suppressed GSDMD-mediated macrophage pyroptosis in mice. In BMDMs and Raw264.7 cells, the anti-inflammatory and anti-pyroptosis properties of SV were verified. SV treatment effectively inhibited NLRP3 inflammasome activation and prevented macrophage pyroptosis in a GSDMD-dependent manner. Furthermore, we found that septic individuals had considerably higher serum angiotensin II levels. Overall, we found that SV might prevent ALI in CLP mice by inhibiting GSDMD-mediated pyroptosis of macrophages. Thus, SV might be a viable drug for sepsis-induced ALI.

Dietary leucine and fish oil cooperatively regulate skeletal myofiber type transformation via the CaMKII signaling pathway of pigs.

The study investigated the effects of dietary leucine (Leu) and fish oil (FO) on skeletal myofiber type transformations in pigs and their potential interactions. The results showed that Leu increased the content of Leu, upregulated myocyte enhancer factor-2C (MEF2C) and activated the CaMKII-AMPK/SIRT1-PGC-1α pathway in the longissimus dorsi (LD) muscle. FO increased adiponectin and fatty acid beta-oxidation of LD muscle. Activation of the adiponectin signaling pathway by FO further enhanced the CaMKII pathway and upregulated the expression of MEF2C. Moreover, we found that Leu increased cyclic AMP and caffeine, and FO increased linoleic acid and glutamine in muscle metabolites, which may be the cause of myofiber conversion. In conclusion, this study demonstrated that dietary Leu and FO co-regulated the transformation from glycolytic to oxidative skeletal myofiber type. It is hypothesized that there is an interaction between amino acids and polyunsaturated fatty acids, possibly via the CaMKII signaling pathway to upregulate MEF2 and mitochondrial biogenesis.

Development of Intestinal Injury and Restoration of Weaned Piglets under Chronic Immune Stress.

This study aimed to investigate the effects of lipopolysaccharide (LPS)-induced chronic immune stress on intestinal morphology and function, immune system, oxidative status, and mitochondrial function in piglets. Fifty healthy Duroc × Landrace × Yorkshire piglets (21 ± 2 days old, barrow, 6.98 ± 0.14 kg body weight) were selected and randomly allotted to five groups, which were slaughtered at 0 (0 group), 1, 5, 9, and 15 d of LPS injection. The results showed that compared with the piglets without LPS injection, LPS injection significantly impaired the intestinal morphology and permeability at 1, 5, and 9 d, as manifested by the increased serum lactic acid and decreased ratio of villus height to crypt depth (p < 0.05). Moreover, intestinal inflammation and oxidative and mitochondrial injury were caused at 1 d, as manifested by upregulated IL-6 mRNA expression, increased malondialdehyde content, and impaired mitochondrial morphology (p < 0.05). However, these parameters were restored to levels identical to 0 group at 9~15 d, accompanied by significantly increased antioxidant capacity, enhanced protein expression of CD3+ and CD68+, and upregulated mRNA abundance of genes related to mitochondrial biogenesis and functions (p < 0.05). Collectively, these results suggest that the intestinal injury of piglets caused by chronic immune stress could be self-repaired.

Long-Term Protein Restriction Modulates Lipid Metabolism in White Adipose Tissues and Alters Colonic Microbiota of Shaziling Pigs.

Obesity is a matter of concern to the public. Abundant evidence has been accumulated that nutritional intervention is a promising strategy to address this health issue. The objective of this study is to investigate alterations in the lipid metabolism in white adipose tissues and the gut microbiota of Shaziling pigs challenged by long-term protein restriction. Results showed that compared with the control group, reducing the protein level by 20% (−20%) increased the mRNA abundance of FABP4 in white adipose tissues (p < 0.05). This occurred in conjunction with increases in PPARγ protein expression. Conversely, the protein expression of C/EBPα was reduced in the −20% group (p < 0.05). Moreover, the −20% group had increased/decreased phosphorylation of AMPKα/mTOR, respectively (p < 0.05). As for the colonic gut microbiota, a 20% reduction in the protein level led to increased Lachnospiraceae XPB1014 group abundance at the genus level (p < 0.01). Collectively, these results indicated that a 20% protein reduction could modulate lipid metabolism and alter the colonic microbiota of Shaziling pigs, an approach which might be translated into a treatment for obesity.

Role of brain-gut-muscle axis in human health and energy homeostasis.

The interrelationship between brain, gut and skeletal muscle plays a key role in energy homeostasis of the body, and is becoming a hot topic of research. Intestinal microbial metabolites, such as short-chain fatty acids (SCFAs), bile acids (BAs) and tryptophan metabolites, communicate with the central nervous system (CNS) by binding to their receptors. In fact, there is a cross-talk between the CNS and the gut. The CNS, under the stimulation of pressure, will also affect the stability of the intestinal system, including the local intestinal transport, secretion and permeability of the intestinal system. After the gastrointestinal tract collects information about food absorption, it sends signals to the central system through vagus nerve and other channels to stimulate the secretion of brain-gut peptide and produce feeding behavior, which is also an important part of maintaining energy homeostasis. Skeletal muscle has receptors for SCFAs and BAs. Therefore, intestinal microbiota can participate in skeletal muscle energy metabolism and muscle fiber conversion through their metabolites. Skeletal muscles can also communicate with the gut system during exercise. Under the stimulation of exercise, myokines secreted by skeletal muscle causes the secretion of intestinal hormones, and these hormones can act on the central system and affect food intake. The idea of the brain-gut-muscle axis is gradually being confirmed, and at present it is important for regulating energy homeostasis, which also seems to be relevant to human health. This article focuses on the interaction of intestinal microbiota, central nervous, skeletal muscle energy metabolism, and feeding behavior regulation, which will provide new insight into the diagnostic and treatment strategies for obesity, diabetes, and other metabolic diseases.

Nondestructive isolation of mesenchymal stem cells from bone marrow using DNA aptamers.

Mesenchymal stem cells (MSCs) mainly found in the bone marrow of adult mammals demonstrate unique capacities of differentiating into multiple cell lineages and undifferentiated MSCs are considered an ideal source of seed cells for cell therapy and tissue engineering. However, MSCs are heterogeneous and not abundant in bone marrow, and there are few specific markers for these cells currently. Therefore, new methods to isolate and characterize MSCs are urgently required. To address the problem, we successfully developed a high-specificity aptamer, called Apt-W2, to specifically recognize mouse bone marrow mesenchymal stem cells (mBMSCs). We synthesized Apt-W2 modified magnetic beads (Apt-W2-MBs) and used them as bait to fish out the MSCs from mouse bone marrow accurately by magnetic-activated cell sorting (MACS). Next, the sorted cells could break free from the Apt-W2-MBs by the competition of C-W2 (complementary strands of Apt-W2). As a result, the sorted cells were intact, and maintained the stem cell phenotype and good proliferative ability. Simultaneously, the sorted cells showed high pluripotency to differentiate into osteoblasts, chondrocytes, and adipocytes. More importantly, the Apt-W2-MB cocktail showed a fine capture performance for MSCs (∼88.33%). This new methodological approach can greatly facilitate MSC isolation efficiently and intactly, thereby enhancing the rate of in vitro differentiation of MSC-derived cells for the emerging field of tissue engineering and regenerative medicine. This new instrumental application of aptamers is an important innovation that achieved both high efficiency and nondestructive cell sorting, opening the door to novel cell sorting approaches.

Effects of Long-Term Protein Restriction on Meat Quality and Muscle Metabolites of Shaziling Pigs.

Background: It has been demonstrated that low-protein diets can improve the meat quality of pork. This study aimed to investigate the effects of long-term protein restriction from piglets to finishing pigs for 24 weeks on meat quality and muscle metabolites of Shaziling pigs. Results: Compared to the control group, reducing dietary protein levels by 20% reduced the L* value (p < 0.05), increased the a* value (p < 0.01), and tended to decrease pressing loss (p = 0.06) of longissimus thoracis muscle (LTM). Furthermore, compared to the control group, the −20% group had significantly lower levels of muscular danazol, N,N-dimethyl-Safingol, and cer(d18:0/14:0) (p < 0.05), all of which were positively associated with the L* value and negatively associated with the a* value (p < 0.05). Therefore, danazol, N,N-dimethyl-Safingol, and cer(d18:0/14:0) might be potential biomarkers for meat color. Conclusions: These results indicated that reducing dietary crude protein by 20% for 24 weeks could improve meat quality and alter muscular metabolites of Shaziling pigs, and the improvement in meat quality might be ascribable to decreased danazol, N,N-dimethyl-Safingol and cer(d18:0/14:0).

Effectiveness and safety evaluation of graded levels of N-carbamylglutamate in growing-finishing pigs.

The aim of this study was as follows: 1) to investigate the effects of graded levels of N-carbamylglutamate (NCG) on performance, blood biochemical indexes, carcass traits and related indicators in growing-finishing pigs, and 2) to determine the optimal supplemental level. The toxicity of high-dose (much higher than recommended levels) NCG was assessed by routine blood tests and blood biochemical and histopathologic examinations of the heart, liver, spleen, lung, kidney and stomach. One hundred and forty-four growing-finishing pigs (Duroc × Large White × Landrace, 32.24 ± 1.03 kg) were used in a 74-d experiment and each treatment was replicated 6 times with 4 pigs (2 barrows and 2 gilts) per replicate. The dietary treatments were a corn-soybean meal basal diet supplemented with 0% (control), 0.05%, 0.1%, 0.15%, 0.2% or 1% NCG. The first 5 groups were used to explore the optimal supplemental level of NCG, while the control, 0.1% and 1% NCG groups were used to explore the safety of high-dose NCG. Compared with the normal control group, the final body weight and average daily gain tended to be higher in the 0.1% group (P = 0.08), the lean percentage tended to be higher in the 0.05% group (P = 0.07), the levels of free amino acids in the blood significantly increased in the 0.1% group (P < 0.05), both 0.1% and 0.15% NCG supplementation increased the levels of nitric oxide (NO) in serum (P = 0.07) and muscle growth- and lipid metabolism-related gene expression (P < 0.05) and NCG supplementation improved C18:1N9C monounsaturated fatty acids (MUFA) in a dose-dependent manner (P = 0.08). In addition, routine blood tests, blood biochemical indexes and histopathological examination revealed no abnormalities. Overall, increasing the levels of NCG did not linearly improve the above indicators; the 0.1% dose showed the best effect, and a high dose (1%) did not pose a toxicity risk.

Binaprofen induces zebrafish liver injury via the mitochondrial pathway.

Binaprofen (C18H23NO5) is a drug not commercially available that causes liver injury; however, the underlying mechanism is unknown. The aim of the present study was to determine the mechanism underlying binaprofen­induced liver injury at the genetic level. Zebrafish were treated with binaprofen. Serum biomarkers [alanine transaminase (ALT), aspartate transaminase (AST) and lactate dehydrogenase (LDH)], malondialdehyde (MDA) and glutathione (GSH) content analysis, liver cell morphology examination, DAPI staining, electron microscopy, microarray analysis and reverse transcription­quantitative (RT­q)PCR were performed 12, 24 and 48 h post­treatment to analyze the mechanism underlying binaprofen­induced liver injury. Following exposure to binaprofen, zebrafish serum levels of ALT, AST and LDH increased; MDA content of liver tissue increased and GSH content decreased. Liver cells exhibited mild to moderate vacuolization and mitochondria exhibited vacuolization and disrupted cristae. Liver cell apoptosis rate increased. There were 190 common differentially expressed genes at 12, 24 and 48 h. Gene Ontology analysis showed that the function of downregulated genes was primarily associated with 'DNA replication', 'DNA metabolic process', 'cell cycle', 'cell redox homeostasis', 'mitochondrion' and 'lipid transport'. The function of upregulated genes was primarily associated with 'peroxisome proliferator', 'oxidation activity', 'peroxisome' and 'apoptosis'. Pathway analysis showed that downregulated genes were those pertaining to 'cell cycle', 'DNA replication', 'ribosome', 'spliceosome', 'pyrimidine metabolism', 'purine metabolism', upregulated genes were those pertaining to 'PPAR signaling pathway', 'p53 signaling pathway'; RT­qPCR assay supported the microarray results. The mechanism underlying binaprofen­induced liver injury was associated with lipid peroxidation and apoptosis. Binaprofen downregulated genes associated with lipid transport and anti­apoptosis genes, upregulated pro­apoptosis genes and induces liver cell injury via the mitochondrial signaling pathway.

The Effect of Dietary Leucine Supplementation on Antioxidant Capacity and Meat Quality of Finishing Pigs under Heat Stress.

This study examined the effects of dietary leucine supplements on antioxidant capacity and meat quality in growing-finishing pigs. A total of 24 crossbred (Duroc × Landrace × Yorkshire) pigs with an average initial weight of 68.33 ± 0.97 kg were randomly allotted to three treatment groups. All pigs were exposed to constant heat stress. Each group of pigs was fed a basal diet, or a diet supplemented with increasing levels of leucine (0.25% or 0.50%). The results showed that leucine intake could improve average daily gain and reduce feed/gain of finishing pigs under heat stress (p < 0.05). The supplementation of leucine could improve the carcass slant length (p = 0.09), and dramatically increased loin-eye area of the finishing pigs (p < 0.05) but had no significant effect on other carcass traits. Compared with the control group, 0.50% leucine markedly reduced drip loss and shear force of longissimus dorsi muscle, and increased pH value at 24 h after slaughter (p < 0.05). Dietary supplementation of 0.25% leucine increased the contents of inosine monophosphate and intramuscular fat in biceps femoris muscle (p < 0.05). Supplementation of 0.25% or 0.50% leucine significantly stimulated the activities of antioxidant enzymes while reduced the level of MDA in serum, liver and longissimus dorsi muscle (p < 0.05). Compared with the control group, 0.50% leucine supplementation markedly modulated the relative mRNA expression levels of genes related to muscle fiber type and mitochondrial function in longissimus dorsi muscle and the gene relative antioxidant in the liver (p < 0.05). In conclusion, dietary leucine supplementation could improve the growth performance and meat quality of the finishing pigs under heat stress, and the pathway of Keap1-NRF2 and PGC-1α-TFAM might be involved.

Taurine Attenuates Oxidized Fish Oil-Induced Oxidative Stress and Lipid Metabolism Disorder in Mice.

The objective of this study was to determine the effect of dietary taurine on lipid metabolism and liver injury in mice fed a diet high in oxidized fish oil. The ICR mice (six weeks old) were randomly assigned to six groups and fed different diets for 10 weeks: control (CON), normal plus 15% fresh fish oil diet (FFO), normal plus 15% oxidized fish oil diet (OFO), or OFO plus 0.6% (TAU1), 0.9% (TAU2) or 1.2% (TAU3) taurine. Compared to the CON group, OFO mice showed increased liver index, aspartate aminotransferase (AST) and malondialdehyde (MDA) levels in serum (p < 0.05). In addition, OFO mice had increased cholesterol (CHOL)/high-density lipoprotein cholesterol (HDL-C) and decreased HDL-C/low-density lipoprotein cholesterol (LDL-C) and n-6/n-3 polyunsaturated fatty acid (PUFA) ratio in serum (p < 0.05) compared with CON mice. Notably, dietary taurine ameliorated the liver index and AST and MDA levels in serum and liver in a more dose-dependent manner than OFO mice. In addition, compared to OFO mice, decreased levels of CHOL and ratio of CHOL/HDL-C and n-6 PUFA/n-3 PUFA in serum were found in TAU3-fed mice. Supplementation with TAU2 and TAU3 increased the relative mRNA expression levels of peroxisome proliferator-activated receptor α, adipose triglyceride lipase, lipoprotein lipase, hormone-sensitive lipase and carnitine palmitoyl transferase 1 in liver compared with the OFO group (p < 0.05). Moreover, impaired autophagy flux was detected in mice fed with the OFO diet, and this was prevented by taurine. These findings suggested that dietary taurine might provide a potential therapeutic choice against oxidative stress and lipid metabolism disorder.