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-sequencing.

BACKGROUND: We previously demonstrated that Shaziling and Yorkshire pigs differ in growth rate and meat quality. However, the molecular mechanisms responsible for such phenotypic differences remain unclear. In the present study, 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 indicated the two stages of 60-90 days and 150-210 days were critical for the muscle growth and development in Shaziling pigs. Genes such as FLNC, COL1A1, NRAP, SMYD1, TNNI3, CRYAB and PDLIM3 played vital roles in the muscle growth, and 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 the PPARß/δ signaling pathway. CONCLUSION: The present 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. © 2024 Society of Chemical Industry.

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.

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.

Mitochondrial Iron Metabolism: The Crucial Actors in Diseases.

Iron is a trace element necessary for cell growth, development, and cellular homeostasis, but insufficient or excessive level of iron is toxic. Intracellularly, sufficient amounts of iron are required for mitochondria (the center of iron utilization) to maintain their normal physiologic function. Iron deficiency impairs mitochondrial metabolism and respiratory activity, while mitochondrial iron overload promotes ROS production during mitochondrial electron transport, thus promoting potential disease development. This review provides an overview of iron homeostasis, mitochondrial iron metabolism, and how mitochondrial iron imbalances-induced mitochondrial dysfunction contribute to diseases.

Balanced branched-chain amino acids modulate meat quality by adjusting muscle fiber type conversion and intramuscular fat deposition in finishing pigs.

BACKGROUND: Pork is an important food for humans and improving the quality of pork is closely related to human health. This study was designed to investigate the effects of balanced branched-chain amino acid (BCAA)-supplemented protein-restricted diets on meat quality, muscle fiber types, and intramuscular fat (IMF) in finishing pigs. RESULTS: The results showed that, compared with the normal protein diet (160 g kg-1 crude protein), the reduced-protein diet (120 g kg-1 crude protein) supplemented with BCAAs to the ratio of 2:1:2 not only had higher average daily gain (P < 0.05) and carcass weight (P < 0.05) but also improved meat tenderness and juiciness by decreasing shear force (P < 0.05) and increasing water-holding capacity (P < 0.05). In particular, this treatment showed higher (P < 0.05) levels of phospho-acetyl-CoA carboxylase (P-ACC) and peroxisome proliferation-activated receptor-γ (PPARγ), and lower (P < 0.05) levels of P-adenosine 5'-monophosphate (AMP)-activated protein kinase (P-AMPK), increasing the composition of IMF and MyHC I (P < 0.05) in the longissimus dorsi muscle (LDM). In terms of health, this group increased eicosapentaenoic acid (EPA) (P < 0.01) and desirable hypocholesterolemic fatty acids (DHFA) (P < 0.05), and decreased atherogenicity (AI) (P < 0.01) and hypercholesterolemic saturated fatty acids (HSFA) (P < 0.05). CONCLUSION: Our findings suggest a novel role for a balanced BCAA-supplemented restricted protein (RP) diet in the epigenetic regulation of more tender and healthier pork by increasing IMF deposition and fiber type conversion, providing a cross-regulatory molecular basis for revealing the nutritional regulation network of meat quality. © 2021 Society of Chemical Industry.

Endogenous miRNA-Activated DNA Nanomachine for Intracellular miRNA Imaging and Gene Silencing.

The development of multifunctional nanoplatforms that integrate both diagnostic and therapeutic functions has always been extremely desirable and challenging in the cancer combat. Here, we report an endogenous miRNA-activated DNA nanomachine (EMDN) in living cells for concurrent sensitive miRNA imaging and activatable gene silencing. EMDN is constructed by interval hybridization of two functional DNA monomers (R/HP and F) to a DNA nanowire generated by hybridization chain reaction. After the target cell-specific transportation of EMDN, intracellular let-7a miRNA initiates the DNA nanomachine by DNA strand displacement cascades, resulting in an amplified fluorescence resonance energy-transfer signal and the release of many free HP sequences. The restoration of HP hairpin structures further activates the split-DNAzyme to identify and cleave the EGR-1 mRNA to realize gene silencing therapy. The proposed EMDN shows efficient cell internalization, good biological stability, rapid reaction kinetics, and the ability to avoid false-positive signals, thus ensuring reliable miRNA imaging in living cells. Meanwhile, the controlled activation of the split-DNAzyme activity regulated by the intracellular specific miRNA may be promising in the precise treatment of cancer. Collectively, this strategy provides a valuable nanoplatform for early clinical diagnosis and activatable gene therapy of tumors.

Identification of a New DNA Aptamer by Tissue-SELEX for Cancer Recognition and Imaging.

Cancer has become one of the most common diseases with high mortality in humans. Early and accurate diagnosis of cancer is of great significance to enhance the survival rate of patients. Therefore, effective molecular ligands capable of selectively recognizing cancer are urgently needed. In this work, we identified a new DNA aptamer named SW1 by tissue-based systematic evolution of ligands by exponential enrichment (tissue-SELEX), in which cancerous liver tissue sections were used as the positive control and adjacent normal liver tissue sections were used as the negative control. Taking immobilized liver cancer SMMC-7721 cells as the research object, aptamer SW1 exhibited excellent affinity with a Kd value of 123.62 ± 17.53 nM, and its binding target was preliminarily determined as a non-nucleic acid substance in the nucleus. Moreover, tissue imaging results showed that SW1 explicitly recognized cancerous liver tissues with a high detection rate of 72.7% but displayed a low detection rate to adjacent normal tissues. In addition to liver cancer cells and tissues, aptamer SW1 has been demonstrated to recognize various other types of cancer cells and tissues. Furthermore, SW1-A, an optimized aptamer of SW1, maintained its excellent affinity toward liver cancer cells and tissues. Collectively, these results indicate that SW1 possesses great potential for use as an effective molecular probe for clinical diagnosis of cancer.

Gut microbiota mediates the protective effects of dietary ß-hydroxy-ß-methylbutyrate (HMB) against obesity induced by high-fat diets.

Obesity increases the risk of developing insulin resistance and diabetes and is a major public health concern. Our previous study shows that dietary ß-hydroxy-ß-methylbutyrate (HMB) improves lipid metabolism in a pig model. However, it remains unclear whether HMB blocks obesity through gut microbiota. In this study, we found that HMB reduced body weight, alleviated the whitening of brown adipose tissue, and improved insulin resistance in mice fed a high-fat diet (HFD). High-throughput pyrosequencing of the 16S rRNA demonstrated that HMB administration significantly reversed the gut microbiota dysbiosis in HFD-fed mice, including the diversity of gut microbiota and relative abundances of Bacteroidetes and Firmicutes. Moreover, microbiota transplantation from HMB-treated mice attenuated HFD-induced lipid metabolic disorders. Furthermore, HFD-fed mice showed lower short-chain fatty acids, whereas administration of HMB increased the propionic acid production. Correlation analysis identified a significant correlation between propionic acid production and the relative Bacteroidetes abundance. Sodium propionate treatment also attenuated HFD-induced lipid metabolic disorders. Collectively, our results indicated that HMB might be used as a probiotic agent to reverse HFD-induced obesity, and the potential mechanism was associated with reprogramming gut microbiota and metabolism, especially Bacteroidetes-mediated propionic acid production. In future studies, more efforts should be made to confirm and expand the beneficial effects of HMB to human models.-Duan, Y., Zhong, Y., Xiao, H., Zheng, C., Song, B., Wang, W., Guo, Q., Li, Y., Han, H., Gao, J., Xu, K., Li, T., Yin, Y., Li, F., Yin, J., Kong, X. Gut microbiota mediates the protective effects of dietary ß-hydroxy-ß-methylbutyrate (HMB) against obesity induced by high-fat diets.

Colorimetric and fluorescent dual-mode detection of microRNA based on duplex-specific nuclease assisted gold nanoparticle amplification.

MicroRNAs (miRNAs) are attractive candidates for biomarkers for early cancer diagnosis, and play vital roles in physiological and pathological processes. In this work, we developed a colorimetric and fluorescent dual-mode sensor for miRNA detection based on the optical properties of gold nanoparticles (AuNPs) and the duplex-specific nuclease (DSN)-assisted signal amplification technique. In brief, FAM labelled hairpin probes (HPs) were immobilized on AuNPs, and fluorescence was efficiently quenched by the vicinity of the fluorophores to the AuNPs surface. In the presence of target miRNAs, the HPs could specifically hybridize with miRNAs and the DNA strand in the DNA/RNA heteroduplexes could be subsequently hydrolyzed by DSN. As a result, numbers of fluorophores were released into the solution, resulting in obvious fluorescence signal recovery. Meanwhile, the target miRNAs were able to participate in other hybridization reactions. With the DSN-assisted signal amplification technique, lots of gold nanoparticles were produced with short-chain DNA on their surface, which could aggregate in salt solution and result in a colorimetric detection. The proposed dual-mode strategy offers a sensitive, accurate and selective detection method for miRNAs. One reason is that the stem of the HPs was elaborately designed to avoid hydrolyzation by DSN under optimal conditions, which ensures a relatively low background and high sensitivity. The other is that the dual-mode strategy is more beneficial for enhancing the accuracy and reproducibility of the measurements. Moreover, the unique selective-cutting ability and single-base mismatch differentiation capability of the DSN also give rise to a satisfactory selectivity. This demonstrated that the developed method could quantitatively detect miR-21 down to 50 pM with a linear calibration range from 50 pM to 1 nM, and the analytical assay of target miRNAs in cell lysate samples revealed its great potential for application in biomedical research and clinical diagnostics.

Analysis of underwater wireless optical communication system performance.

Because the underwater channel environment is complicated, it is difficult to do an actual experiment in the ocean to analyze the performance of underwater wireless optical communication (UWOC) systems. In this study, the spot-expansion characteristics and time-domain broadening characteristics of underwater wireless optical signals are simulated and analyzed by a Monte Carlo statistical method. Thus, what we believe is an improved underwater channel transmission-attenuation model and time-domain broadening model based on UWOC are proposed, so the transmission distance characteristics of the UWOC system are obtained by combining the system parameters, and the transmission-rate characteristics can be analyzed by using the Shannon-Hartley theorem. The results show that the transmission distance is linear with the receiver sensitivity, and the transmission rate decays exponentially with the transmission distance and is limited by the receiver sensitivity in the UWOC system.

Enhanced Imaging of Specific Cell-Surface Glycosylation Based on Multi-FRET.

Cell-surface glycosylation contains abundant biological information that reflects cell physiological state, and it is of great value to image cell-surface glycosylation to elucidate its functions. Here we present a hybridization chain reaction (HCR)-based multifluorescence resonance energy transfer (multi-FRET) method for specific imaging of cell-surface glycosylation. By installing donors through metabolic glycan labeling and acceptors through aptamer-tethered nanoassemblies on the same glycoconjugate, intramolecular multi-FRET occurs due to near donor-acceptor distance. Benefiting from amplified effect and spatial flexibility of the HCR nanoassemblies, enhanced multi-FRET imaging of specific cell-surface glycosylation can be obtained. With this HCR-based multi-FRET method, we achieved obvious contrast in imaging of protein-specific GalNAcylation on 7211 cell surfaces. In addition, we demonstrated the general applicability of this method by visualizing the protein-specific sialylation on CEM cell surfaces. Furthermore, the expression changes of CEM cell-surface protein-specific sialylation under drug treatment was accurately monitored. This developed imaging method may provide a powerful tool in researching glycosylation functions, discovering biomarkers, and screening drugs.

A light-up fluorescence assay for tumor cell detection based on bifunctional split aptamers.

Light-up aptamers have attracted growing attention due to their advantages of being label-free and having low fluorescence background. In this work, we developed a light-up fluorescence assay for label-free detection of tumor cells based on a bifunctional split aptamer (BFSA) that contained two DNA strands (BFSA-a and BFSA-b). BFSA-a and BFSA-b were constructed by combining aptamers ZY11 and ThT.2-2, which could specifically bind to the tumor cell SMMC-7721 and activate the fluorescence of thioflavin T (ThT). A Helper strand was introduced to hybridize with BFSA-b, and then BFSA-a and BFSA-b were separated if the target cell was absent. Only when the target cell is present can BFSA-a approach and hybridize with BFSA-b due to the 'induced-fit effect', which made the Helper strand dissociate. Then ThT bound to BFSA and the fluorescence of ThT was activated. The results indicated that this fluorescence assay had a good linear response to the target cells in the range of 250-20 000 cells in 100 µL binding buffer; the lowest cell number actually detected was 125 cells in 100 µL buffer. This assay also displayed excellent selectivity and was successfully applied to detect target cells in 20% human serum samples. The design of bifunctional split aptamers realized no-washing, label-free, low-cost, one-step detection of tumor cells, which could generate detectable fluorescence signals just by mixing nucleic acid aptamers and fluorescent reporter molecules with target cells. Such a design of aptamer probes also has the potential to construct stimuli-responsive controlled drug delivery systems.

ß-hydroxy-ß-methyl butyrate promotes leucine metabolism and improves muscle fibre composition in growing pigs.

The aim of this study was to investigate the effects of excess leucine (Leu) vs. its metabolites α-ketoisocaproate (KIC) and ß-hydroxy-ß-methyl butyrate (HMB) on Leu metabolism, muscle fibre composition and muscle growth in growing pigs. Thirty-two pigs with a similar initial weight (9.55 ± 0.19 kg) were fed 1 of 4 diets for 45 days: basal diet, basal diet + 1.25% L-Leu, basal diet + 1.25% KIC-Ca, basal diet + 0.62% HMB-Ca. Results indicated that relative to the basal diet and HMB groups, Leu and KIC groups exhibited increased Leu concentrations and decreased concentrations of isoleucine, valine and EAAs in selected muscle (p < 0.05) and had lower mRNA levels of MyHC I and higher expression of MyHC IIx/IIb (p < 0.05), and there was no significant difference between the basal and HMB-supplemented groups. Moreover, the mRNA expression levels of AMPKα and UCP3 were higher but the myostatin mRNA levels were lower in the soleus muscle of the HMB group than those from other groups (p < 0.05). These findings demonstrated that doubling dietary Leu content exerted growth-depressing effects in growing pigs; dietary KIC supplementation induced muscular branched-chain amino acid imbalance and promoted muscle toward a more glycolytic phenotype; while dietary HMB supplementation promoted the generation of more oxidative muscle types and increased muscle growth specially in oxidative skeletal muscle, and these effects of HMB might be associated with the AMPKα-Sirt1-PGC-1α axis and mitochondrial biogenesis.

Design of a Modular DNA Triangular-Prism Sensor Enabling Ratiometric and Multiplexed Biomolecule Detection on a Single Microbead.

DNA nanostructures have emerged as powerful and versatile building blocks for the construction of programmable nanoscale structures and functional sensors for biomarker detection, disease diagnostics, and therapy. Here we integrated multiple sensing modules into a single DNA three-dimensional (3D) nanoarchitecture with a triangular-prism (TP) structure for ratiometric and multiplexed biomolecule detection on a single microbead. In our design, the complementary hybridization of three clip sequences formed TP nanoassemblies in which the six single-strand regions in the top and bottom faces act as binding sites for different sensing modules, including an anchor module, reference sequence module, and capture sequence module. The multifunctional modular TP nanostructures were thus exploited for ratiometric and multiplexed biomolecule detection on microbeads. Microbead imaging demonstrated that, after ratiometric self-calibration analysis, the imaging deviations resulting from uneven fluorescence intensity distribution and differing probe concentrations were greatly reduced. The rigid nanostructure also conferred the TP as a framework for geometric positioning of different capture sequences. The inclusion of multiple targets led to the formation of sandwich hybridization structures that gave a readily detectable optical response at different fluorescence channels and distinct fingerprint-like pattern arrays. This approach allowed us to discriminate multiplexed biomolecule targets in a simple and efficient fashion. In this module-designed strategy, the diversity of the controlled DNA assembly coupled with the geometrically well-defined rigid nanostructures of the TP assembly provides a flexible and reliable biosensing approach that shows great promise for biomedical applications.

High Signal-to-Background Ratio Detection of Cancer Cells with Activatable Strategy Based on Target-Induced Self-Assembly of Split Aptamers.

Highly sensitive detection of cancer cells with high signal-to-background ratio (SBR) is still urgently needed. Here, a self-assembling activatable probe (SAAP) based on split aptamers was developed to meet this purpose. The SAAP is formed with quenched fluorescence; only when target cells are present would the split aptamers self-assemble together and thus activate fluorescence by intramolecular and intermolecular fluorescence quenching strategies. As proof of concept, a split aptamer pair stemming from an intact aptamer, ZY11, developed by our lab was selected to construct SAAP. Owing to the design of self-assembly and activation strategy, the SBR of our approach could be raised to ∼40 and achieved a very low detection limit of seven target 7721 cells in 100 µL of binding buffer. Meanwhile, one-step detection of target cells was achieved within 15 min without any washing steps and pretreatment, which shows potential for point-of-care detection. Moreover, we succeeded in the specific recognition of target cells in 50% human serum and mixed cell samples, which indicated this strategy had great advantages in detection in complex biological samples. In addition, dual-signal detection was also successfully implemented, which may be helpful for accurate detection of target cells. Therefore, this rapid, facile, specific, and highly sensitive detection method for cancer cells may provide convenience in cancer research and medical diagnosis.

Effects of supplementation with branched-chain amino acids to low-protein diets on expression of genes related to lipid metabolism in skeletal muscle of growing pigs.

Branched-chain amino acids (BCAA), including leucine (Leu), isoleucine (Ile), and valine (Val), play critical roles in energy homeostasis and lipid metabolism in addition to their other functions, such as in protein metabolism. This study investigated the effects of different dietary BCAA ratios on the intramuscular fat (IMF) content and fatty acid composition in different location of skeletal muscles, including the longissimus dorsi (LD), biceps femoris (BF), and psoas major (PM) muscles of growing pigs, and also examined the mRNA expression levels of genes involved in lipid metabolism in these muscle tissues. The experiment was performed on 40 growing pigs (Large White × Landrace) with a similar initial weight (9.85 ± 0.35 kg). The pigs were randomly assigned to one of five diets: diet A was a positive control and contained 20 % crude protein (CP) with a Leu:Ile:Val ratio of 1:0.51:0.63 according to the recommendation of the National Research Council (NRC); for diets B to E, the CP level was reduced to 17 %, and the Leu:Ile:Val ratios were 1:1:1, 1:0.75:0.75, 1:0.51:0.63, and 1:0.25:0.25, respectively. No significant difference was observed in the average feed intake and feed efficiency of the pigs fed the low protein diet (17 % CP) with BCAA treatments relative to the positive control. However, there was a tendency for increased feed efficiency of the 1:0.75:0.75 group compared with the 1:1:1 group (P = 0.09). The BCAA ratio of 1:0.75:0.75 (17 % CP) increased the IMF content of BF muscle (P < 0.01). Moreover, varied dietary BCAA supplementation with a reduced protein level had different effects on the fatty acid composition of the LD, BF, and PM muscles. The BCAA ratio of 1:0.51:0.63-1:0.75:0.75 (17 % CP) significantly lowered the ratio of n-6 to n-3 polyunsaturated fatty acid in these muscles compared with the positive control group (20 % CP). This effect was associated with an increase in mRNA expression levels of acetyl-CoA carboxylase, lipoprotein lipase, fatty acid transport protein, and fatty acid binding protein 4 in the muscles (P < 0.05). The results indicated that the reduced protein diet (17 % CP) with the BCAA ratio within 1:0.25:0.25-1:0.75:0.75 could increase the IMF content in BF muscle and significantly improve the fatty acid composition in different skeletal muscles accompanied by changes in the expression of genes involved in lipid metabolism, compared with those in the pigs that received adequate dietary protein (20 %), which might result in improved eating quality and nutritional value of the meat.

Amplified fluorescence detection of adenosine via catalyzed hairpin assembly and host-guest interactions between ß-cyclodextrin polymer and pyrene.

Nowadays, enzyme-free nucleic acid-based signal amplification strategies are frequently utilized in the design of biosensors due to their excellent sensitivity. Developing more extended analytical methods is fundamental for basic studies in the biological and biomedical fields. Herein, we introduce an enzyme-free amplified detection strategy for the small molecule adenosine. The approach is based on adenosine-aptamer binding triggered catalyzed hairpin assembly and host-guest interactions between ß-cyclodextrin polymer (ß-CDP) and pyrene. Two hairpin probes (probe H1 and probe H2) and an aptamer-trigger/inhibitor duplex probe were employed in the system and the pyrene-labeled probe H1 was chosen as the signal unit. In the absence of adenosine, the aptamer-trigger was inhibited by the inhibitor strand. The hairpin probes were in the closed hairpin formation without activation of the trigger strand. Pyrene labeled at the 5'-termini of the single-stranded stem of probe H1 could be easily trapped in the hydrophobic cavity of ß-CDP because of weak steric hindrance, leading to a significant fluorescence enhancement. Once the hairpin assembly was catalyzed by the adenosine-aptamer binding event, a hybridized DNA duplex H1/H2 was created continuously. Pyrene labeled at the 5'-termini of the DNA duplex H1/H2 finds it difficult to enter the cavity of ß-CDP due to steric hindrance, leading to a weaker fluorescence signal. Thus, the target could be detected by this simple mix-and-detect amplification method without a need for expensive and perishable protein enzymes. As low as 42 nM of adenosine was detected by this assay, which is comparable to that of some reported colorimetric methods. Meanwhile, the proposed method was further successfully applied to detect adenosine in human serum samples, showing great potential for adenosine detection from complex fluids.

[Study on toxicity of 999 Ganmaoling grain and influence of diet on hepatic toxicity].

This paper was aimed to compare the acute toxicity of 999 Ganmaoling grain and its different ingredients, and investigate the influence of routine diet on the hepatic toxicity induced by Ganmaoling in mice, so as to provide experimental basis for the clinical safety evaluation. Mice were given a single dose of Ganmaoling grain or its different ingredients respectively by gavage, and then observed for 14 days. LD50 values of Ganmaoling grain or its chemical ingredient and the maximal tolerated dose of its herb ingredient were determined. Mice were divided into starvation and diet group, a single dose of Ganmaoling grain was administered by gavage. LD50 values were estimated after 14 day observation. Mice were divided into starvation and diet group. At the same time,control group was set up for each. A single dose of Ganmaoling grain was given. Serum biochemical indexes were detected, liver weight index was calculated and liver tissue morphological change was observed after 6 h. LD50 values were 4.42, 0.64 g•kg⁻¹ for Ganmaoling grain group and chemical ingredient group, respectively. The maximal tolerated dose of the herb ingredient group was close to 24.24 g•kg⁻¹. The toxic symptom was basically similar in the Ganmaoling grain and the chemical ingredient group. The body weight and food intake were decreased to a certain extent in both groups. There were pathological changes of liver and heart tissue in some of the surviving animals. The animals in the Ganmaoling grain group exhibited a lighter toxicity and recovered faster than that in the chemical ingredient group. LD50 values of Ganmaoling grain were 2.56, 6.93 g•kg⁻¹ for starvation and diet group respectively. TD50 values were 1.29, 6.31 g•kg⁻¹ for starvation and diet group respectively. The toxicity of 999 Ganmaoling was less, which may be related to the reduction of toxicity after the combination of herb and chemical ingredients. Compared with starvation group, the values of LD50 and TD50 of diet group was significantly increased, and toxicity was decreased. From the point of view of safety, it is safer to use Ganmaoling in the absence of hunger or after meal. The above tests provide experimental basis for the clinical safety use of Ganmaoling.

Cell-SELEX based selection and optimization of DNA aptamers for specific recognition of human cholangiocarcinoma QBC-939 cells.

Cholangiocarcinoma (CCA) is a very aggressive biliary tract malignancy with no efficient early diagnosis and therapeutics available, so there is a call for effective molecular probes. Herein, we performed cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX) to obtain aptamers for the specific recognition of human cholangiocarcinoma QBC-939 cells. By coordinating sequence homology analysis and secondary structure analysis, we successfully obtained two aptamers with dissociation constants (Kd) in the low nanomolar range. A 23 nt truncated sequence was identified after further analysis on the secondary structure. More importantly, because hepatocellular carcinoma SMMC-7721 cells were employed as the control in the counter selection, the obtained aptamers demonstrated excellent specificity to the target cells, and no binding to several other hepatocellular carcinoma cell lines was observed. Moreover, the aptamers were initially found to recognize membrane proteins, giving them great potential in the field of biomarker discovery. These newly generated aptamers may play a key role in the early diagnosis and clinical treatment of CCA.