Advancing towards practice: A novel LC-MS/MS method for detecting retinol in dried blood spots.

BACKGROUND: To date, clinical laboratories face challenges in quantifying retinol from DBS samples. Disputes arise throughout the whole detection process, encompassing the storage condition, the release strategy as well as the selection of internal standards. METHODS: We incubated DBS with ascorbic acid solution. Then, retinol-d4 in acetonitrile was introduced to incorporate isotopic internal standard and promote protein precipitation. Afterward, sodium carbonate solution was added to ionize cytochromes (such as bilirubin), which amplified the difference of their hydrophobicity to retinol. Subsequently, cold-induced phase separation could be facilitated to separate retinol from the impurities. In the end, the upper layer was injected for LC-MS/MS analysis. RESULTS: By comparing the detected retinol content in whole blood and DBS samples prepared from the same volume, we confirmed the established pretreatment was capable to extract most of retinol from DBS (recovery >90 %). Thereafter, we verified that within DBS, retinol possessed satisfying stability without antioxidation. Indoor-light exposure and storage duration would not cause obvious degradation (<10 %). Following systematic validation, the established method well met the criteria outlined in the relevant guidelines. After comparing with detected DBS results to the paired plasma samples, 54 out of 60 met the acceptance limit for cross-validation of ±20 %. CONCLUSIONS: We realized precise quantification of retinol from one 3.2 mm DBS disc. By circumventing conventional antioxidation, liquid-liquid/solid-phase extraction and organic solvent evaporation, the pretreatment could be completed within 15 min consuming only minimal amounts of low-toxicity chemicals (ascorbic acid, acetonitrile, and sodium carbonate). We expect this contribution holds the potential to significantly facilitate the evaluation of patients' vitamin A status by using DBS samples in the future.

Associations Between Vitamin K and Suicide Attempts in Patients with Depression: A Case-Control Study.

Background: Hypovitaminosis K has been linked to depression and suicide, but epidemiological research is scarce. This study aimed to explore the association among vitamin K with depression and suicidal attempts. Methods: This was a retrospective cross-sectional study involving 146 cases with a history of suicidal attempts and 149 subjects without a lifetime history of suicidal attempts. The levels of thyroid hormones, lipid profile, inflammatory cytokines, and vitamins were measured. Results: Subjects who had suicidal attempts presented with a significant decrease in FT4, TC, vitamin D, and vitamin K but increased CRP levels. In these variables, vitamin K has a better diagnostic value for suicidal attempts in depressed patients, with a sensitivity of 0.842 and a specificity of 0.715. Correlation analysis suggested that vitamin K was significantly and positively related to FT4, TC, LDL, and sdLDL. Multivariate analysis showed that serum vitamin K level predicts suicidal attempts in depressive patients (OR = 0.614, P = 0.004, 95% CI 0.153-0.904). Moreover, a negative correlation between vitamin K and suicidal attempts was also noted for partial FT4, CRP, and vitamin D strata analysis. Conclusion: Our study suggests that low vitamin K levels were correlated with suicidal attempts in patients with depression, indicating that vitamin K deficiency might be a biological risk factor for depression.

Effect of melatonin on ATG2B-mediated autophagy regulation in sheep granulosa cells with different FecB genotypes.

Melatonin (MLT) protects cells by reducing reactive oxygen species (ROS) levels, which are key for inducing cellular autophagy. The aim of this study was to investigate the molecular mechanisms underlying MLT regulation of autophagy in granulosa cells (GCs) with BMPR-1B homozygous (FecB BB) and wild type (FecB ++) mutations. GCs collected from small-tailed Han sheep with different FecB genotypes were typed using a TaqMan probe assay, and autophagy levels were found to be significantly higher in GCs with FecB BB than the levels in those with FecB ++. Autophagy-related 2 homolog B (ATG2B) was associated with cell autophagy and was highly expressed in GCs with the FecB BB genotype in small-tailed Han sheep. Overexpression of ATG2B in the GCs of sheep with both FecB genotypes promoted GC autophagy, and the contrary was observed after the inhibition of ATG2B expression. Subsequently, treatment of GCs with different genotypes of FecB and MLT revealed a significant decrease in cellular autophagy and an increase in ATG2B expression. Addition of MLT to GCs with inhibited ATG2B expression revealed that MLT could protect GCs by decreasing ROS levels, especially in GCs with FecB ++ genotype. In conclusion, this study determined that autophagy levels were significantly higher in sheep GCs with FecB BB genotype than the levels in those with FecB ++ genotype, which may have contributed to the difference in lambing numbers between the two FecB genotypes. Autophagy was regulated by ATG2B and was able to protect GCs by reducing the high levels of ROS produced following inhibition of ATG2B through the addition of MLT in vitro.

Progress on the effect of FecB mutation on BMPR1B activity and BMP/SMAD pathway in sheep.

BMPR1B is the first major gene of litter size identified in sheep. However, the molecular mechanism of the FecB mutation that increases the ovulation rate in sheep is still unclear. In recent years, it has been demonstrated that BMPR1B activity is regulated by the small molecule repressor protein FKBP1A, which acts as a key activity switch of the BMPR1B in the BMP/SMAD pathway. The FecB mutation is located close to the binding site of FKBP1A and BMPR1B. In this review, we summarize the structure of BMPR1B and FKBP1A proteins, and clarify the spatial interactive domains of the two proteins with respect to the location of the FecB mutation. Then the relationship between the FecB mutation and the degree of affinity of the two proteins are predicted. Finally, the hypothesis that FecB mutation causes change of activity in BMP/SMAD pathway by affecting the intensity of the interactions between BMPR1B and FKBP1A is proposed. This hypothesis provides a new clue to investigate the molecular mechanism of FecB mutation affecting ovulation rate and litter size in sheep.

Clinical significance of serum miR-129-5p in patients with diabetes mellitus presenting macrovascular complications.

BACKGROUND: Diabetic macrovascular complications (DMCs) are the most common complications encountered during the course of diabetes mellitus (DM) with extremely high mortality rates. Therefore, there is an urgent need to identify specific and sensitive biomarkers for the early diagnosis of DMCs. AIM: To investigate the expression and significance of serum miR-129-5p in patients with DM and macrovascular complications. METHODS: Serum samples were collected from 36 healthy controls, 58 patients with DM presenting no macrovascular complications, and 62 patients with DMCs. The expression of miR-129-5p was detected using quantitative real-time polymerase chain reaction. Pearson's correlation assay was performed to analyze the correlation between serum miR-129-5p levels and clinical indicators. Receiver operator characteristic (ROC) analysis was conducted to analyze the diagnostic value of serum miR-129-5p in patients with DM or DMCs. RESULTS: There was a 4.378-fold and 7.369-fold increase in serum miR-129-5p expression in the DM (5.346 ± 0.405) and DMCs (8.998 ± 0.631) groups, respectively (P < 0.001), compared with the control group (1.221±0.090). In addition, the expression of serum miR-129-5p in patients with DMCs was higher than that in patients with DM, revealing a 1.683-fold increase (P < 0.001). Additionally, serum miR-129-5p expression significantly correlated with smoking history, disease duration, and glycated hemoglobin (HbA1c) in patients with DMCs (P < 0.001). The area under the ROC curve (AUC) of miR-129-5p as a serum marker was 0.964 (95% confidence interval [CI]: 0.930-0.997, P < 0.001) in distinguishing between patients with DM and healthy controls, whereas the AUC of miR-129-5p as a serum marker was 0.979 (95%CI: 0.959-0.999, P < 0.001) in distinguishing between patients with DMCs and healthy controls. CONCLUSION: Elevated serum miR-129-5p expression levels correlate with the development of DMCs and can be utilized as a novel early diagnostic biomarker for DM combined with macrovascular complications.

Role of exosomal long non-coding RNAs in colorectal cancer.

Exosomes are a class of small extracellular vesicles, 30-150 nm in diameter, that transfer biological information (e.g., DNA, RNA, and protein) via cell-to-cell communication. Exosomes play critical roles in the occurrence and development of human cancers, including colorectal cancer (CRC). Recent studies have shown that long non-coding RNAs (lncRNAs) can be encapsulated in exosomes, which transfer lncRNAs from secretory cells into recipient cells. This process affects the progression of CRC, since exosomal lncRNAs display special and extensive functions in CRC tumorigenesis, including malignant proliferation, metastasis, chemoresistance, and inflammatory response. Moreover, due to their specificity and sensitivity, exosomal lncRNAs are released into body fluids (e.g., urine, sputum, and plasma), which have the potential to be biomarkers of CRC tumorigenesis within screening efforts and medical and epidemiologic research. In this review, we aim to clarify the function and mechanism of exosomal lncRNAs in CRC tumorigenesis and provide a strategy for early diagnosis and medical treatment of this malignancy.

Polymorphism, expression and structure analysis of key genes in the ovarian steroidogenesis pathway in sheep (Ovis aries).

BACKGROUND: Litter size is an important factor that significantly affects the development of the sheep industry. Our previous TMT proteomics analysis found that three key proteins in the ovarian steroidogenesis pathway, STAR, HSD3B1, and CYP11A1, may affect the litter size trait of Small Tail Han sheep. OBJECTIVE: The purpose of this study was to better understand the relationship between polymorphisms of these three genes and litter size. MATERIAL AND METHOD: Sequenom MassARRAY detected genetic variance of the three genes in 768 sheep. Real-time qPCR of the three genes was used to compare their expression in monotocous and polytocous sheep in relevant tissues. Finally, bioinformatics analysis predicted the protein sequences of the different SNP variants. RESULT: Association analysis showed that there was a significant difference in litter size among the genotypes at two loci of the CYP11A1 gene (p < 0.05), but no significant difference was observed in litter size among all genotypes at all loci of the STAR and HSD3B1 genes (p > 0.05). However, STAR expression was significantly different in polytocous and monotocous sheep in the pituitary (p < 0.01). Tissue-specific expression in the ovary was observed for HSD3B1 (p < 0.05), but its expression was not different between polytocous and monotocous sheep. Bioinformatics analysis showed that the g.33217408C > T mutation of CYP11A1 resulted in major changes to the secondary and tertiary structures. In contrast, gene polymorphisms in STAR and HSD3B1 had minimal impacts on their protein structures. DISCUSSION: This may explain why the CYP11A1 variant impacted litter size while the others did not. The single nucleotide polymorphism of the CYP11A1 gene would serve as a good molecular marker when breeding to increase litter size in sheep. Our study provides a basis for further revealing the function of the ovarian steroidogenesis pathway in sheep reproduction and sheep breeding.

Expression analysis of DIO2, EYA3, KISS1 and GPR54 genes in year-round estrous and seasonally estrous rams.

The expression characteristics of the hypothalamic-pituitary-gonadal (HPG) axis-related candidate genes, DIO2, EYA3, KISS1 and GPR54, were analyzed in year-round estrous rams (small-tail Han sheep, STH) and seasonally estrous rams (Sunite sheep, SNT) using qPCR. The results were as follows: DIO2 was mainly expressed in pituitary, and KISS1 was specifically expressed in hypothalamus in the two groups. However, EYA3 and GPR54 were widely expressed in the cerebrum, cerebellum, hypothalamus, pituitary, testis, epididymis, vas deferens and adrenal gland tissues in both breeds, with significant differences in the cerebellum, hypothalamus, pituitary, testis and vas deferens tissues. We speculated that DIO2 and KISS1 may have positive roles in different regions in ram year-round estrus. Moreover, the expression patterns of EYA3 and GPR54 suggested that they may regulate the estrous mode of ram via testis and vas deferens. This is the first study to systematically analyze the expression patterns of HPG axis-related genes in rams.

[The molecular mechanism of sheep seasonal breeding and artificial regulatory techniques for estrus and mating in anestrus].

Seasonal breeding is an important factor limiting sheep production efficiency. Detailed analysis on the molecular mechanisms of seasonal breeding is the premise for improving estrus and mating rate of sheep during anestrus. Recent research showed that under long-photoperiod and short-photoperiod conditions, a series of changes in signaling molecules and cell morphology could be observed in ovine seasonal reproduction pathway. Based on the molecular mechanisms of seasonal reproduction, several technologies or methods for inducing estrus and mating of ewes in anestrus have been developed. In this review, photoperiod-induced changes in signaling molecules and cell morphology in pituitary and hypothalamic tissue are first summarized in terms of the molecular mechanisms and characteristics of seasonal reproduction. The application effect, advantages and disadvantages for applying these technologies for inducing estrus and mating of ewes in anestrus are then discussed, thereby providing the critical insights in identifying a new technology, which is environmentally friendly and efficient, to improve breeding rate in anestrus.

Atorvastatin prevents glomerular extracellular matrix formation by interfering with the PKC signaling pathway.

Platelet-activating factor (PAF) promotes glomerular extracellular matrix (ECM) deposition, primarily through activation of the protein kinase C (PKC) pathway. The present study was designed to investigate whether atorvastatin, which mediates a protective effect against glomerular ECM deposition and diabetic neuropathy, may interfere with the PKC­transforming growth factor­ß1 (TGF­ß1) pathway in a model of human mesangial cells (HMCs) exposed to a high glucose (HG) and lysophosphatidylcholine (LPC) environment. HMCs were divided into three treatment groups: Control, high glucose and lysophosphatidylcholine (HG+LPC), and HG+LPC+atorvastatin. Cells were cultured for 24 h. The levels of the ECM­associated molecules collagen IV (Col IV) and fibronectin (Fn) in the supernatant were detected using an ELISA kit. PKC­ß1, TGF­ß1 and PAF­receptor gene expression was detected by reverse transcription­quantitative polymerase chain reaction. PKC­ß1 and TGF­ß1 protein expression was detected by western blotting, and the subcellular localization of PKC­ß1 was assessed using immunofluorescence. The results indicated that atorvastatin may reduce the secretion of ECM components (Fn and Col IV) in HMCs in a HG and LPC environment, by inhibiting the increase in PAF secretion and the activation of the PKC­TGF­ß1 signaling pathway.

High glucose/lysophosphatidylcholine levels stimulate extracellular matrix deposition in diabetic nephropathy via platelet­activating factor receptor.

Platelet-activating factor (PAF), protein kinase C (PKC)ßI, transforming growth factor (TGF)­ß1 and aberrant extracellular matrix (ECM) deposition have been associated with diabetic nephropathy (DN). However, the mechanistic basis underlying this association remains to be elucidated. The present study investigated the association among the aforementioned factors in a DN model consisting of human mesangial cells (HMCs) exposed to high glucose (HG) and lysophosphatidylcholine (LPC) treatments. HMCs were divided into the following treatment groups: Control; PAF; PAF+PKCßI inhibitor LY333531; HG + LPC; PAF + HG + LPC; and PAF + HG + LPC + LY333531. Cells were cultured for 24 h, and PKCßI and TGF­ß1 expression was determined using the reverse transcription­quantitative polymerase chain reaction and western blotting. The expression levels of the ECM­associated molecules collagen IV and fibronectin in the supernatant were detected using ELISA analysis. Subcellular localization of PKCßI was assessed using immunocytochemistry. PKCßI and TGF­ß1 expression was increased in the PAF + HG + LPC group compared with the other groups (P<0.05); however, this effect was abolished in the presence of LY333531 (P<0.05). Supernatant fibronectin and collagen IV levels were increased in the PAF + HG + LPC group compared with the others (P<0.05); this was reversed by treatment with LY333531 (P<0.05). In cells treated with PAF, HG and LPC, PKCßI was translocated from the cytosol to the nucleus, an effect which was blocked when PKCßI expression was inhibited (P<0.05). The findings of the present study demonstrated that PAF stimulated ECM deposition in HMCs via activation of the PKC­TGF­ß1 axis in a DN model.

Subchronic feeding study of high-free-lysine transgenic rice in Sprague-Dawley rats.

Lysine is considered to be the first essential amino acid in rice. An elite High-Free-Lysine transgenic line HFL1 was previously produced by metabolic engineering to regulate lysine metabolism. In this study, a 90-day toxicology experiment was undertaken to investigate the potential health effect of feeding different doses of HFL1 rice to Sprague-Dawley rats. During the trial, body weight gain, food consumption and food efficiency were recorded, and no adverse effect was observed in rats fed transgenic (T) rice diets compared with non-transgenic (N) or control diets. At both midterm and final assessments, hematological parameters and serum chemistry were measured, and organ weights and histopathology were examined at the end of the trial. There was no diet-related difference in most hematological or serum chemistry parameters or organ weights between rats fed the T diets and those fed the N or control diets. Some parameters were found to differ between T groups and their corresponding N and/or control groups, but no adverse histological effect was observed. Taken together, the data from the current trial demonstrates that high lysine transgenic rice led to no adverse effect in Sprague-Dawley rats given a diet containing up to 70% HFL1 rice in 90 days.

Selection signature in domesticated animals.

Domesticated animals play an important role in the life of humanity. All these domesticated animals undergo same process, first domesticated from wild animals, then after long time natural and artificial selection, formed various breeds that adapted to the local environment and human needs. In this process, domestication, natural and artificial selection will leave the selection signal in the genome. The research on these selection signals can find functional genes directly, is one of the most important strategies in screening functional genes. The current studies of selection signal have been performed in pigs, chickens, cattle, sheep, goats, dogs and other domestic animals, and found a great deal of functional genes. This paper provided an overview of the types and the detected methods of selection signal, and outlined researches of selection signal in domestic animals, and discussed the key issues in selection signal analysis and its prospects.

The effect of genetically modified Lactobacillus plantarum 590 on the gut health of Sprague-Dawley rats.

Lp was a generally recognized as safe microorganism. Lactobacillus plantarum 590 was obtained by inserting nisI gene into Lp genome to help it tolerate higher concentration nisin. As the unintended effects of the genetically modified microorganism (GMM) are the most important barriers to the progress of GMM, we have performed a useful exploration to establish a new in vivo evaluation model for GMM from the point of view of intestinal health. In this study, Sprague-Dawley rats were orally administered with Lp 590 and Lp for 4 weeks. Fecal samples were collected to determine the number of beneficial bacteria Bifidobacterium and harmful bacteria Clostridium perfringens. Denaturing gradient gel electrophoresis was used to detect the bacterial profiles of every group. Fecal enzyme activities and short-chain fatty acids as main metabolites were also examined. Real time PCR (RT-PCR) and immunohistochemistry were used to analyze two proteins (ZO-1 and occludin) and secretory immunoglobulin A to detect intestinal permeability and mucosal immunity, gut permeability and gut mucosal immunity were analyzed to see whether GM Lp 590 can induce changes of the gut health when compared with non-GM Lp group, andeventually we concluded that there is no significant difference between GM Lp 590-fed group and non-GM Lp-fed group. The conclusion of gut health test was comparable withthat from traditional subchronic test. Evaluation of intestinal health will be a new approach of assessing the safety of GMM.

A 90-day toxicology study of transgenic lysine-rich maize grain (Y642) in Sprague-Dawley rats.

The gene for a lysine-rich protein (sb401) obtained from potatoes (Solanum berthaultii) was inserted into maize seed to produce Y642 transgenic maize. Compositional analysis of Y642 grain demonstrated that the concentrations of lysine and total protein were higher than those observed in maize grain from a near-isogenic non-genetically modified (non-GM) commercially available control quality protein maize (Nongda 108). The safety of Y642 maize grain was assessed by comparison of toxicology response variables in Sprague-Dawley (SD) rats consuming diets containing Y642 maize grain with those containing Nongda 108 maize grain. Maize grains from Y642 or Nongda 108 were incorporated into rodent diets at low (30%) or high concentrations (76%) and administered to SD rats (n=10/sex/group) for 90 days. An additional group of negative control group of rats (n=10/sex/group) were fed AIN93G diets. No adverse diet-related differences in body weights, feed consumption/utilization, clinical chemistry, hematology, absolute and relative organ weights were observed. Further, no differences in gross or microscopic pathology were observed between rats consuming diets with Y642 maize grain compared with rats consuming diets containing Nongda 108 maize grain. These results demonstrated that Y642 lysine-rich maize is as safe and nutritious as conventional quality protein maize.