Engineering Escherichia coli for high-yielding 2,5-Dimethylpyrazine synthesis from L-Threonine by reconstructing metabolic pathways and enhancing cofactors regeneration.

2,5-Dimethylpyrazine (2,5-DMP) is important pharmaceutical raw material and food flavoring agent. Recently, engineering microbes to produce 2,5-DMP has become an attractive alternative to chemical synthesis approach. In this study, metabolic engineering strategies were used to optimize the modified Escherichia coli BL21 (DE3) strain for efficient synthesis of 2,5-DMP using L-threonine dehydrogenase (EcTDH) from Escherichia coli BL21, NADH oxidase (EhNOX) from Enterococcus hirae, aminoacetone oxidase (ScAAO) from Streptococcus cristatus and L-threonine transporter protein (EcSstT) from Escherichia coli BL21, respectively. We further optimized the reaction conditions for synthesizing 2,5-DMP. In optimized conditions, the modified strain can convert L-threonine to obtain 2,5-DMP with a yield of 2897.30 mg/L. Therefore, the strategies used in this study contribute to the development of high-level cell factories for 2,5-DMP.

The Pea Oligosaccharides Could Stimulate the In Vitro Proliferation of Beneficial Bacteria and Enhance Anti-Inflammatory Effects via the NF-κB Pathway.

The oligosaccharides extracted from the seeds of peas, specifically consisting of raffinose, stachyose, and verbascose, fall under the category of raffinose family oligosaccharides (RFOs). The effect of RFOs on intestinal microflora and the anti-inflammatory mechanism were investigated by in vitro fermentation and cell experiments. Firstly, mouse feces were fermented in vitro and different doses of RFOs (0~2%) were added to determine the changes in the representative bacterial community, PH, and short-chain fatty acids in the fermentation solution during the fermentation period. The probiotic index was used to evaluate the probiotic proliferation effect of RFOs and the optimal group was selected for 16S rRNA assay with blank group. Then, the effects of RFOs on the inflammatory response of macrophage RAW264.7 induced by LPS were studied. The activity of cells, the levels of NO, ROS, inflammatory factors, and the expression of NF-κB, p65, and iNOS proteins in related pathways were measured. The results demonstrated that RFOs exerted a stimulatory effect on the proliferation of beneficial bacteria while concurrently inhibiting the growth of harmful bacteria. Moreover, RFOs significantly enhanced the diversity of intestinal flora and reduced the ratio of Firmicutes-to-Bacteroides (F/B). Importantly, it was observed that RFOs effectively suppressed NO and ROS levels, as well as inflammatory cytokine release and expression of NF-κB, p65, and iNOS proteins. These findings highlight the potential of RFOs in promoting intestinal health and ameliorating intestinal inflammation.

A NILM load identification method based on structured V-I mapping.

With the increasing number and types of global power loads and the development and popularization of smart grid technology, a large number of researches on load-level non-intrusive load monitoring technology have emerged. However, the unique power characteristics of the load make NILM face the difficult problem of low robustness of feature extraction and low accuracy of classification and identification in the recognition stage. This paper proposes a structured V-I mapping method to address the inherent limitations of traditional V-I trajectory mapping methods from a new perspective. In addition, for the verification of the V-I trajectory mapping method proposed in this paper, the complexity of load characteristics is comprehensively considered, and a lightweight convolutional neural network is designed based on AlexNet. The experimental results on the NILM dataset show that the proposed method significantly improves recognition accuracy compared to existing VI trajectory mapping methods.

Production of monoclonal antibody against tylosin and tilmicosin with homogeneous cross-reactivity and its application in lateral flow immunoassay.

To avoid false negative results due to the low cross-reactivity rate (CR) in rapid immunoassay, a group-specific antibody with homogeneous CR toward target compounds is needed for accuracy. In this study, tylosin (TYL) and tilmicosin (TM) were selected as model molecules. Firstly, two-dimensional similarity, electrostatic potential energy, spatial conformation and charge distribution of the haptens TYL-CMO, TYL-6-ACA, TYL-4-APA, TYL-CHO and DES-CMO and target compounds of TYL and TM were obtained using Gaussian 09W and Discovery Studio. The optimal hapten was DES-CMO because it is the most similar to TYL and TM. Subsequently, the mAb 14D5 cell line was obtained with IC50 values of 1.59 and 1.72 ng/mL for TYL and TM, respectively, and a CR of 92.44%. Finally, amorphous carbon nanoparticles (ACNPs) were conjugated with mAb 14D5 to develop an accurate lateral flow immunoassay (LFA) for detection of TYL and TM by the reflectance value under natural light. The recoveries of TYL and TM ranged from 77.18 to 112.04% with coefficient of variation < 13.43%. The cut-off value in milk samples was 8 ng/mL, and the limits of detection were 11.44, 15.96, 22.29 and 25.53 µg/kg for chicken muscle, bovine muscle, porcine muscle and porcine liver samples, respectively, and the results being consistent with HPLC-UV. The results suggest that the developed LFA is accurate and potentially useful for on-site screening of TYL and TM in milk and animal tissue samples.

The Impact of High Temperature on Microbial Communities in Pork and Duck Skin.

Pork skin and duck skin are highly favored by consumers in China, and high-temperature processing methods are widely employed in cooking and food preparation. However, the influence of high-temperature treatment on the microbial communities within pork skin and duck skin remains unclear. In this study, a high-temperature treatment method simulating the cooking process was utilized to treat samples of pork skin and duck skin at temperatures ranging from 60 °C to 120 °C. The findings revealed that high-temperature treatment significantly altered the microbial communities in both pork skin and duck skin. Heat exposure resulted in a decrease in microbial diversity and induced changes in the relative abundance of specific microbial groups. In pork skin, high-temperature treatment led to a reduction in bacterial diversity and a decline in the relative abundance of specific bacterial taxa. Similarly, the relative abundance of microbial communities in duck skin also decreased. Furthermore, potential pathogenic bacteria, including Gram-positive and Gram-negative bacteria, as well as aerobic, anaerobic, and facultative anaerobic bacteria, exhibited different responses to high-temperature treatment in pork skin and duck skin. These findings highlighted the substantial impact of high-temperature processing on the composition and structure of microbial communities in pork skin and duck skin, potentially influencing food safety and quality. This study contributed to an enhanced understanding of the microbial mechanisms underlying the alterations in microbial communities during high-temperature processing of pork skin and duck skin, with significant implications for ensuring food safety and developing effective cooking techniques.

Development of a monoclonal antibody-based lateral flow immunoassay for the detection of benzoic acid in liquid food.

To monitor benzoic acid (BA) residues in liquid food samples, a monoclonal antibody (mAb)-based lateral flow immunoassay (LFA) was developed in this study. First, 2-aminobenzoic acid (2-AA), 3-aminobenzoic acid (3-AA), and 4-aminobenzoic acid (4-AA) were conjugated to BSA and used as immunogens. After cell fusion, mAb 6D8 from 4-AA-BSA performed best with an IC50 value of 0.21 mg L-1 using 3-AA-OVA as a heterogeneous antigen, which represented a 3.4-fold improvement compared with the homogeneous antigen 4-AA-BSA. Subsequently, eight kinds of CGNPs with sizes varying from 20.94 nm to 90.00 nm were synthesized for screening the suitable size to develop a sensitive LFA. Finally, a sensitive LFA based on colloidal gold (23.27 nm) nanoparticles was developed for screening BA with a cut-off value of 4 mg L-1, which could meet the requirement of BA detection in milk, Fanta, Sprite, Coca-Cola, and Smart samples.

Effect of 2'-Fucosyllactose on Beige Adipocyte Formation in 3T3-L1 Adipocytes and C3H10T1/2 Cells.

2'-Fucosyllactose (2'-FL), the functional oligosaccharide naturally present in milk, has been shown to exert health benefits. This study was aimed to investigate the effect of 2'-fucosyllactose (2'-FL) on the browning of white adipose tissue in 3T3-L1 adipocytes and C3H10T1/2 cells. The results revealed that 2'-FL decreased lipid accumulations with reduced intracellular triglyceride contents in vitro. 2'-FL intervention increased the mitochondria density and the proportion of UCP1-positive cells. The mRNA expressions of the mitochondrial biogenesis-related and browning markers (Cox7a, Cyto C, Tfam, Ucp1, Pgc1α, Prdm16, Cidea, Elovl3, Pparα, CD137, and Tmem26) were increased after 2'-FL intervention to some extent. Similarly, the protein expression of the browning markers, including UCP1, PGC1α, and PRDM16, was up-regulated in the 2'-FL group. Additionally, an adenosine monophosphate-activated protein kinase (AMPK) inhibitor, compound C (1 µM), significantly decreased the induction of thermogenic proteins expressions mediated by 2'-FL, indicating that the 2'-FL-enhanced beige cell formation was partially dependent on the AMPK pathway. In conclusion, 2'-FL effectively promoted the browning of white adipose in vitro.

Improving the quality of soluble dietary fiber from Poria cocos peel residue following steam explosion.

Poria cocos peel residue (PCPR) still contains much soluble dietary fiber (SDF), steam explosion (SE) treatment was applied to PCPR to create a superior SDF. Steam pressure of 1.2 MPa, residence period of 120 s, and moisture content of 13% were the optimized parameters for SE treatment of PCPR. Under optimized circumstances, SE treatment of PCPR enhanced its SDF yield from 5.24% to 23.86%. Compared to the original SDF, the SE-treated SDF displayed improved enzyme inhibition, including the inhibition of α-amylase and pancreatic lipase, also enhanced water holding, oil holding, water swelling, nutrient adsorption including cholesterol, nitrite ions, and glucose and antioxidant abilities. Additionally, it had a decreased molecular weight, improved thermal stability, and a rough surface with many pores of different sizes. Given that SDF had been improved physiochemical and functional characteristics thanks to SE treatment, it might be the excellent functional ingredient for the food business.

Merge Loss Calculation Method for Highly Imbalanced Data Multiclass Classification.

In real classification scenarios, the number distribution of modeling samples is usually out of proportion. Most of the existing classification methods still face challenges in comprehensive model performance for imbalanced data. In this article, a novel theoretical framework is proposed that establishes a proportion coefficient independent of the number distribution of modeling samples and a general merge loss calculation method independent of class distribution. The loss calculation method of the imbalanced problem focuses on both the global and batch sample levels. Specifically, the loss function calculation introduces the true-positive rate (TPR) and the false-positive rate (FPR) to ensure the independence and balance of loss calculation for each class. Based on this, global and local loss weight coefficients are generated from the entire dataset and batch dataset for the multiclass classification problem, and a merge weight loss function is calculated after unifying the weight coefficient scale. Furthermore, the designed loss function is applied to different neural network models and datasets. The method shows better performance on imbalanced datasets than state-of-the-art methods.

GABA Application Enhances Drought Stress Tolerance in Wheat Seedlings (Triticum aestivum L.).

In this study, the effects of γ-aminobutyric acid (GABA) on physio-biochemical metabolism, phenolic acid accumulation, and antioxidant system enhancement in germinated wheat under drought stress was investigated. The results showed that exogenous GABA reduced the oxidative damage in wheat seedlings caused by drought stress and enhanced the content of phenolics, with 1.0 mM being the most effective concentration. Six phenolic acids were detected in bound form, including p-hydroxybenzoic acid, vanillic acid, syringic acid, p-coumaric acid, ferulic acid, and sinapic acid. However, only syringic acid and p-coumaric acid were found in free form. A total of 1.0 mM of GABA enhanced the content of total phenolic acids by 28% and 22%, respectively, compared with that of drought stress, on day four and day six of germination. The activities of phenylalanine ammonia lyase (PAL), cinnamic acid 4-hydroxylase (C4H) and 4-coumarate coenzyme A ligase (4CL) were activated by drought stress plus GABA treatment. Antioxidant enzyme activities were also induced. These results indicate that GABA treatment may be an effective way to relieve drought stress as it activates the antioxidant system of plants by inducing the accumulation of phenolics and the increase in antioxidant enzyme activity.

A self-assembled DNA double-crossover-based fluorescent aptasensor for highly sensitivity and selectivity in the simultaneous detection of aflatoxin M1 and aflatoxin B1.

Realizing the simultaneous speedy detection of multiple mycotoxins in contaminated food and feed is of great practical importance in the domain of food manufacturing and security. Herein, a fluorescent aptamer sensor based on self-assembled DNA double-crossover was developed and used for effective simultaneous quantitative detection of aflatoxins M1 and B1 by fluorescence resonance energy transfer (FRET). Fluorescent dye-modified aflatoxin M1 and B1 aptamers are selected as recognition elements and signal probes, and DNA double crosses are consistently locked by the aflatoxin aptamers, which results in a "turn-off" of the fluorescent signal. In the presence of AFM1 and AFB1, the aptamer sequences are more inclined to form Apt-AFM1 and Apt-AFB1 complexes, and the fluorescent probes are released from the DNA double-crossing platform, leading to an enhanced fluorescent signal (Cy3: 568 nm; Cy5: 660 nm). Under the optimal conditions, the signal response of the constructed fluorescent aptamer sensor showed good linearity with the logarithm of AFM1 and AFB1 concentrations, with detection limits of 6.24 pg/mL and 9.0 pg/mL, and a wide linear range of 0.01-200 ng/mL and 0.01-150 ng/mL, respectively. In addition, the effect of potential interfering substances in real samples was analyzed, and the aptasensor presented a good interference immunity. Moreover, by modifying and designing aptamer probes, the sensor can be applied to high-throughput simultaneous screening of other analytes, providing a new approach for the development of fluorescent aptamer sensors.

Development of a Lateral Flow Immunoassay Based on a Highly Specific Monoclonal Antibody To Detect 4-Methylaminoantipyrine.

To avoid false-positive results in immunoassays due to cross-reactivity of antibodies with structural analogues, especially metabolites of target compounds, the preparation of highly specific antibodies is crucial. Preserving the characteristic structure of a target compound when designing a hapten is important when preparing highly specific antibodies. Here, we designed a novel hapten, 4-(((1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4yl)amino)methyl)benzoic acid, named AA-BA, to improve the specificity of antibodies for detection of 4-methylaminoantipyrine (MAA), a residual marker of dipyrone, an important antipyretic-analgesic and anti-inflammatory drug. The structural features of the hapten remained almost the same as those of MAA. After experimental validation, monoclonal antibody 6A4 (mAb 6A4) was prepared with the half maximal inhibitory concentration (IC50) value of 4.03 ng/mL and negligible cross-reactivity with dipyrone metabolites and other antibiotics. In addition, a specific lateral flow immunoassay (LFA) strip based on colloidal gold was developed for screening MAA with a cutoff value of 25 ng/mL in milk. The developed LFA is a useful tool for rapid and accurate detection of MAA.

Differential expression of SLC30A10 and RAGE in mouse pups by early life lead exposure.

BACKGROUND: SLC30A10 and RAGE are widely recognized as pivotal regulators of Aß plaque transport and accumulation. Prior investigations have established a link between early lead exposure and cerebral harm in offspring, attributable to Aß buildup and amyloid plaque deposition. However, the impact of lead on the protein expression of SLC30A10 and RAGE has yet to be elucidated. This study seeks to confirm the influence of maternal lead exposure during pregnancy, specifically through lead-containing drinking water, on the protein expression of SLC30A10 and RAGE in mice offspring. Furthermore, this research aims to provide further evidence of lead-induced neurotoxicity. METHODS: Four cohorts of mice were subjected to lead exposure at concentrations of 0 mM, 0.25 mM, 0.5 mM, and 1 mM over a period of 42 uninterrupted days, spanning from pregnancy to the weaning phase. On postnatal day 21, the offspring mice underwent assessments. The levels of lead in the blood, hippocampus, and cerebral cortex were scrutinized, while the mice's cognitive abilities pertaining to learning and memory were probed through the utilization of the Morris water maze. Furthermore, Western blotting and immunofluorescence techniques were employed to analyze the expression levels of SLC30A10 and RAGE in the hippocampus and cerebral cortex. RESULTS: The findings revealed a significant elevation in lead concentration within the brains and bloodstreams of mice, mirroring the increased lead exposure experienced by their mothers during the designated period (P < 0.05). Notably, in the Morris water maze assessment, the lead-exposed group exhibited noticeably diminished spatial memory compared to the control group (P < 0.05). Both immunofluorescence and Western blot analyses effectively demonstrated the concomitant impact of varying lead exposure levels on the hippocampal and cerebral cortex regions of the offspring. The expression levels of SLC30A10 displayed a negative correlation with lead doses (P < 0.05). Surprisingly, under identical circumstances, the expression of RAGE in the hippocampus and cortex of the offspring exhibited a positive correlation with lead doses (P < 0.05). CONCLUSION: SLC30A10 potentially exerts distinct influence on exacerbated Aß accumulation and transportation in contrast to RAGE. Disparities in brain expression of RAGE and SLC30A10 may contribute to the neurotoxic effects induced by lead.

Tea Polyphenols Inhibit the Activity and Toxicity of Staphylococcus aureus by Destroying Cell Membranes and Accumulating Reactive Oxygen Species.

Staphylococcus aureus can cause bacterial food intoxication and seriously affect human health. Tea polyphenols (TP) are a kind of natural, safe, and broad-spectrum bacteriostatic substances, with a wide range of bacteriostatic effects. In the study, we explored the possible bacteriostatic mode of TP. The minimum inhibitory concentration of TP against S. aureus was 64 µg/mL. Protein, DNA, and K+ leak experiments, fluorescence microscopy, and transmission electron microscopy suggested that TP disrupt cell membranes, leading to intracellular component loss. By studying the effect of TP on the toxicity of S. aureus, it was found that the expression levels of two toxin genes, coa and spa, were downregulated by 2.37 and 32.6, respectively. Furthermore, after treatment with TP, a large number of reactive oxygen species (ROS) were propagated and released, leading to oxidative stress in cells. We speculated that the bacteriostatic mechanism of TP may be through the destruction of the cell membrane and ROS-mediated oxidative stress. Meanwhile, the hemolysis activity proved the safety of TP. Our results suggested that TP may be a potential antimicrobial agent for food.

(-)-Epigallocatechin-3-Gallate Attenuates the Adverse Reactions Triggered by Selenium Nanoparticles without Compromising Their Suppressing Effect on Peritoneal Carcinomatosis in Mice Bearing Hepatocarcinoma 22 Cells.

Increasing evidence shows that selenium and polyphenols are two types of the most reported compounds in tumor chemoprevention due to their remarkable antitumor activity and high safety profile. The cross-talk between polyphenols and selenium is a hot research topic, and the combination of polyphenols and selenium is a valuable strategy for fighting cancer. The current work investigated the combination anti-peritoneal carcinomatosis (PC) effect of selenium nanoparticles (SeNPs) and green tea (Camellia sinensis) polyphenol (-)-epigallocatechin-3-gallate (EGCG) in mice bearing murine hepatocarcinoma 22 (H22) cells. Results showed that SeNPs alone significantly inhibited cancer cell proliferation and extended the survival time of mice bearing H22 cells. Still, the potential therapeutic efficacy is accompanied by an approximately eighty percent diarrhea rate. When EGCG was combined with SeNPs, EGCG did not affect the tumor proliferation inhibition effect but eliminated diarrhea triggered by SeNPs. In addition, both the intracellular selectively accumulated EGCG without killing effect on cancer cells and the enhanced antioxidant enzyme levels in ascites after EGCG was delivered alone by intraperitoneal injection indicated that H22 cells were insensitive to EGCG. Moreover, EGCG could prevent SeNP-caused systemic oxidative damage by enhancing serum superoxide dismutase, glutathione, and glutathione peroxidase levels in healthy mice. Overall, we found that H22 cells are insensitive to EGCG, but combining EGCG with SeNPs could protect against SeNP-triggered diarrhea without compromising the suppressing efficacy of SeNPs on PC in mice bearing H22 cells and attenuate SeNP-caused systemic toxicity in healthy mice. These results suggest that EGCG could be employed as a promising candidate for preventing the adverse reactions of chemotherapy including chemotherapy-induced diarrhea and systemic toxicity in cancer individuals.

Antioxidant, antihyperglycemic, and antihyperlipidemic properties of Chimonanthus salicifolius S. Y. Hu leaves in experimental animals: modulation of thioredoxin and glutathione systems, renal water reabsorption, and gut microbiota.

Introduction: Excessive calorie intake and physical inactivity have dramatically increased nutrient overload-associated disease, becoming a global public health issue. Chimonanthus salicifolius S. Y. Hu (CHI) is a homology plant of food and medicine in China and shows several health benefits. Methods: This work investigated the antioxidant activity, the alleviating effects, and the mechanism of action on diabetes and hyperlipidemia of CHI leaves. Results and discussion: Results showed that CHI leaves infusion displayed in vitro antioxidant activity measured by ABTS and ferric reducing antioxidant power methods. In wild-type Kunming mice, CHI leaves infusion consumption activated the hepatic antioxidant enzymes, including glutathione reductase, glutathione S-transferase, glutathione peroxidase and thioredoxin reductase as well as thioredoxin reductase 1. In alloxan-induced type 1 diabetic mice, CHI leaves infusion ameliorated diabetic symptoms, including polyuria, polydipsia, polyphagia and hyperglycemia, in a dose-dependent and time-course manners. The mechanism involved CHI leaves up-regulating renal water reabsorption associated protein - urine transporter A1-and promoting the trafficking of urine transporter A1 and aquaporin 2 to the apical plasma membrane. Despite this, in high-fat diet-induced hyperlipidemic golden hamsters, CHI leaves powder did not significantly effect on hyperlipidemia and body weight gain. This might be attributed to CHI leaves powder increasing the calorie intake. Interestingly, we found that CHI leaves extract containing a lower dose of total flavonoid than CHI leaves powder pronouncedly reduced the levels of total cholesterol, triglyceride, and low-density lipoprotein cholesterol in serum in golden hamsters fed a high-fat diet. Furthermore, CHI leaves extract elevated the diversity of gut microbiota and the abundance of Bifidobacterium and Ruminococcaceae_UCG-014. It also decreased the abundance of Lactobacillus at the genus level in golden hamsters fed a high-fat diet. Overall, CHI leaves benefit oxidative stress prevention and metabolic syndrome amelioration in vivo.

A fluorescent lateral flow immunoassay based on CdSe/CdS/ZnS quantum dots for sensitive detection of olaquindox in feedstuff.

A portable fluorescence immunosensor based on the CdSe/CdS/ZnS quantum dots (QDs) with multiple-shell structure was fabricated for the precise quantification of olaquindox (OLA). The QDs labeled anti-OLA antibody used as bioprobe played an important role in the design and preparation of a lateral flow test strip. Due to the strong fluorescent intensity of QDs, the sensitivity is greatly improved. The quantitative results were obtained using a fluorescent strip scan reader within 8 min, and the calculated limit of detection for OLA at 0.12 µg/kg, which was 2.7 times more sensitive than that of the conventional colloidal gold-based strips method. Acceptable recovery of 85.0%-95.5% was obtained by the spiked samples. This newly established QDs-based strip immunoassay method is suitable for the on-site detection and rapid initial screening of OLA in swine feedstuff, and is potentially applied for the detection of other veterinary drugs to ensure food safety.

A highly sensitive lateral flow immunoassay based on a group-specific monoclonal antibody and amorphous carbon nanoparticles for detection of sulfonamides in milk.

To meet high-throughput screening of the residues of sulfonamides (SAs) with high sensitivity toward sulfamethazine (SM2) in milk samples, a new highly sensitive lateral flow immunoassay (LFA) based on amorphous carbon nanoparticles (ACNs) was developed. First, a group-specific monoclonal antibody 10H7 (mAb 10H7) that could recognize 25 SAs with high sensitivity toward SM2 (IC50 value of 0.18 ng/mL) was prepared based on H1 as an immune hapten and H4 as a heterologous coating hapten. Then, mAb 10H7 was conjugated to ACNs as an immune probe for LFA development. Under the optimized conditions, the LFA could detect 25 SAs with the cut-off value toward SM2 of 2 ng/mL, which could meet the requirement for detection of SAs. In addition, the LFA developed was also used for screening SAs' residues in real milk samples, with results being consistent with HPLC-MS/MS. Thus, this LFA can be used as a high-throughput screening tool for detection of SAs.

Melatonin improved glucose homeostasis is associated with the reprogrammed gut microbiota and reduced fecal levels of short-chain fatty acids in db/db mice.

Accumulated evidence shows that melatonin possesses the potential to improve lipid metabolism by modifying gut microbiota and glucose metabolism via regulating the melatonin receptor signaling pathway. However, the contribution of melatonin consumption on glucose homeostasis by affecting gut microbiota has not been investigated in diabetes. In the current work, we investigated the effect of melatonin administration on gut microbiota and glucose homeostasis in db/db mice, a type 2 diabetes model with leptin receptor deficiency. Administration of melatonin through drinking water (at 0.25% and 0.50%) for 12 weeks decreased diabetic polydipsia and polyuria, increased insulin sensitivity and impeded glycemia. The accumulated fecal levels of total short-chain fatty acids (SCFAs) and acetic acid are positively correlated with diabetes-related parameters-homeostasis model assessment of insulin resistance (HOMA-IR) index and fasting blood glucose (FBG) level. The reprogramming of gut microbiota structure and abundance and the reduction of fecal levels of SCFAs, including acetic acid, butyric acid, isovaleric acid, caproic acid, and isobutyric acid, by melatonin may be beneficial for enhancing insulin sensitivity and lowering FBG, which were verified by the results of correlation analysis between acetic acid or total SCFAs and HOMA-IR and FBG. In addition, the melatonin downregulated hepatic genes, including fructose-1,6-bisphosphatase 1, forkhead box O1 alpha, thioredoxin-interacting protein, phosphoenolpyruvate carboxy-kinase (PEPCK), PEPCK1 and a glucose-6-phosphatase catalytic subunit, that responsible for gluconeogenesis support the result that melatonin improved glucose metabolism. Overall, results showed that the melatonin supplementation reduced fecal SCFAs level via reprogramming of gut microbiota, and the reduction of fecal SCFAs level is associated with improved glucose homeostasis in db/db mice.

Production of high-affinity monoclonal antibody and development of immunoassay for 3-methyl-quinoxaline-2-carboxylic acid detection in swine muscle and liver.

Production of high-affinity and specific antibodies to small molecules with molecular weight (MW) lower than 200 Da is challenging. Here, we designed a novel hapten, named hapten H6, for the detection of 3-methyl-quinoxaline-2-carboxylic acid (MQCA, MW of 189 Da), a residual marker of olaquindox, one of important veterinary antibiotics. The hapten H6 maintained all structural features of MQCA, especially in mulliken atomic charge distribution. Then, a monoclonal antibody (mAb) named 8C9 was obtained with an IC50 value of 0.2 µg/L, yielding a 15.5- to 88.5-fold improvement compared to previously prepared specific antibodies against MQCA. In addition, mAb 8C9 exhibited ignorable cross-reactivity with other structural analogs. Finally, a highly sensitive and specific indirect competitive ELISA based on mAb 8C9 was developed for the detection of MQCA in swine muscle and liver samples with limit of detection values of 0.04 µg/kg and 0.09 µg/kg, respectively.