Neuron-derived neurotrophic factor promotes the differentiation of intramuscular and subcutaneous adipocytes in goat.

Adipocyte play an important role in human health and meat quality by influencing the tenderness, flavor, and juiciness of mutton It has been shown that neuron-derived neurotrophic factor (NENF) is closely related to energy metabolism and adipocyte differentiation in bovine. However, the role of NENF in the goats remains unclear. The aim of this study was to detect the expression of NENF in goat subcutaneous and intramuscular adipocytes, temporal expression profiles of the NENF, and overexpressed NENF on the differentiation of different adipocytes. In this study, PCR amplification successfully cloned the goat NENF gene with a fragment length of 521 bp. In addition, the time point of highest expression of NENF differed between these two adipocytes differentiation processes. Overexpression of NENF in intramuscular and subcutaneous adipocytes promoted the expression levels of differentiation markers CEBPß and SREBP, which in turn promoted the differentiation of intramuscular and subcutaneous adipocytes. This study will provide basic data for further study of the role of goats in goat adipocyte differentiation and for the final elucidation of its molecular mechanisms in regulating goat adipocyte deposition.

Integrated pharmacokinetic-pharmacodynamic modeling and metabolomic research on polyphenol-rich fraction of Thymus quinquecostatus Celak. Alleviating cerebral ischemia-reperfusion injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Thymus quinquecostatus Celak., a member of thymus genus in Lamiaceae family, has been used as a folk medicine for relieving exterior syndrome and alleviating pain in China. The polyphenol-rich fraction (PRF) derived from Thymus quinquecostatus Celak. had been validated that it can protect cerebral ischemia-reperfusion injury (CIRI) by activating Keap1/Nrf2/HO-1 signaling pathway. AIM OF THIS STUDY: To explore effective components and their pharmacokinetic and pharmacodynamic characteristics as well as possible mechanisms of PRF in treating CIRI. MATERIALS AND METHODS: Normal treated group (NTG) and tMCAO model treated group (MTG) rats were administrated PRF intragastrically. The prototype components and metabolites of PRF in plasma and brain were analyzed by the UPLC-Q-Exactive Orbitrap MSn method. Subsequently, the pharmacokinetics properties of indicative components were performed based on HPLC-QQQ-MS/MS. SOD and LDH activities were determined to study the pharmacodynamic (PD) properties of PRF. The PK-PD relationship of PRF was constructed. In addition, the effect of PRF on endogenous metabolites in plasma and brain was investigated using metabolomic method. RESULTS: Salvianic acid A, caffeic acid, rosmarinic acid, scutellarin, and apigenin-7-O-glucuronide were selected as indicative components based on metabolic analysis. The non-compartmental parameters were calculated for indicative components in plasma and brain of NTG and MTG rats. Furthermore, single-component and multi-component PK-PD modeling involved Emax, Imax PD models for effect indexes were fitted as well as ANN models were established, which indicated that these components can work together to regulate SOD and LDH activities in plasma and SOD activity in brain tissue to improve CIRI. Additionally, PRF may ameliorate CIRI by regulating the disorder of endogenous metabolites in lipid metabolism, amino acid metabolism, and purine metabolism pathways in vivo, among which lipid metabolism and purine metabolism are closely related to oxidative stress. CONCLUSION: The PK-PD properties of effect substances and mechanisms of PRF anti-CIRI were further elaborated. The findings provide a convincing foundation for the application of T. quinquecostatus Celak. in the maintenance of human health disorders.

Left ventricular strain changes at high altitude in rats: a cardiac magnetic resonance tissue tracking imaging study.

BACKGROUND: Long-term exposure to a high altitude environment with low pressure and low oxygen could cause abnormalities in the structure and function of the heart. Myocardial strain is a sensitive indicator for assessing myocardial dysfunction, monitoring myocardial strain is of great significance for the early diagnosis and treatment of high altitude heart-related diseases. This study applies cardiac magnetic resonance tissue tracking technology (CMR-TT) to evaluate the changes in left ventricular myocardial function and structure in rats in high altitude environment. METHODS: 6-week-old male rats were randomized into plateau hypoxia rats (plateau group, n = 21) as the experimental group and plain rats (plain group, n = 10) as the control group. plateau group rats were transported from Chengdu (altitude: 360 m), a city in a plateau located in southwestern China, to the Qinghai-Tibet Plateau (altitude: 3850 m), Yushu, China, and then fed for 12 weeks there, while plain group rats were fed in Chengdu(altitude: 360 m), China. Using 7.0 T cardiac magnetic resonance (CMR) to evaluate the left ventricular ejection fraction (EF), end-diastolic volume (EDV), end-systolic volume (ESV) and stroke volume (SV), as well as myocardial strain parameters including the peak global longitudinal (GLS), radial (GRS), and circumferential strain (GCS). The rats were euthanized and a myocardial biopsy was obtained after the magnetic resonance imaging scan. RESULTS: The plateau rats showed more lower left ventricular GLS and GRS (P < 0.05) than the plain rats. However, there was no statistically significant difference in left ventricular EDV, ESV, SV, EF and GCS compared to the plain rats (P > 0.05). CONCLUSIONS: After 12 weeks of exposure to high altitude low-pressure hypoxia environment, the left ventricular global strain was partially decreased and myocardium is damaged, while the whole heart ejection fraction was still preserved, the myocardial strain was more sensitive than the ejection fraction in monitoring cardiac function.

Aggravated visual toxicity in zebrafish larvae upon co-exposure to titanium dioxide nanoparticles and bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate.

The bioavailability and toxicity of organic pollutants in aquatic organisms can be largely affected by the co-existed nanoparticles. However, the impacts of such combined exposure on the visual system remain largely unknown. Here, we systematically investigated the visual toxicity in zebrafish larvae after single or joint exposure to titanium dioxide nanoparticles (n-TiO2) and bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate (TBPH) at environmentally relevant levels. Molecular dynamics simulations revealed the enhanced transmembrane capability of the complex than the individual, which accounted for the increased bioavailability of both TBPH and n-TiO2 when combined exposure to zebrafish. Transcriptome analysis showed that co-exposure to n-TiO2 and TBPH interfered with molecular pathways related to eye lens structure and sensory perception of zebrafish. Particularly, n-TiO2 or TBPH significantly suppressed the expression of ßB1-crystallin and rhodopsin in zebrafish retina and lens, which was further enhanced after co-exposure. Moreover, we detected disorganized retinal histology, stunted lens development and significant visual behavioral changes of zebrafish under co-exposure condition. The overall results suggest that combined exposure to water borne n-TiO2 and TBPH increased their bioavailability, resulted in severer damage to optic nerve development and ultimately abnormal visual behavior patterns, highlighting the higher potential health risks of co-exposure to aquatic vertebrates.

CORRELATIONS BETWEEN SUSPENDED HYPERREFLECTIVE MATERIAL WITH THE MACULAR MICROSTRUCTURE AND SPONTANEOUS CLOSURE OF FULL-THICKNESS MACULAR HOLES.

PURPOSE: To investigate the characteristics of spontaneously closed full-thickness macular holes (FTMHs) and to seek potential predictors for the spontaneous closure of FTMHs. METHODS: In this retrospective cohort study, the clinical data and optical coherence tomography images were reviewed from 19 eyes with spontaneously closed FTMHs (spontaneous closure group) and 37 control eyes with FTMHs that were delayed for nonmedical reasons, but ultimately required surgery (control group). The term, suspended hyperreflective material, was defined as hyperreflective material suspended within the FTMHs observed via optical coherence tomography; the presence of suspended hyperreflective material was evaluated in these eyes. RESULTS: The median time from diagnosis to spontaneous closure of the FTMHs was 13.7 (range, 2.4-32.4) weeks in the spontaneous closure group. The mean diameter of FTMHs in the spontaneous closure group was significantly smaller than that in the control group (191.68 ± 70.57 vs. 401.68 ± 162.19 µ m, P < 0.0001). The incidence of vitreomacular traction was higher in the spontaneous closure group compared with the control group (9/19 vs. 5/37, P = 0.009, odds ratio [95% confidence interval], 5.76 [1.56-21.21]); in seven of the nine eyes with vitreomacular traction from the spontaneous closure group, spontaneous vitreomacular traction separation and subsequent FTMH closure was observed. Suspended hyperreflective material was observed in nine eyes (47%) from the spontaneous closure group and three eyes (8%) from the control group ( P = 0.001, odds ratio [95% confidence interval], 10.20 [2.31‒45.02]). CONCLUSION: Smaller diameters, vitreomacular traction, and presence of suspended hyperreflective material may be suggestive of the potential for spontaneous closure of FTMHs.

Integrated Studies on Male Reproductive Toxicity of Bis(2-ethylhexyl)-tetrabromophthalate: in Silico, in Vitro, ex Vivo, and in Vivo.

Bis(2-ethylhexyl)tetrabromophthalate (TBPH) has been widely detected in the environment and organisms; thus, its toxic effects on male reproduction were systematically studied. First, we found that TBPH can stably bind to the androgen receptor (AR) based on in silico molecular docking results and observed an antagonistic activity, but not agonistic activity, on the AR signaling pathway using a constructed AR-GRIP1 yeast assay. Subsequently, we validated the adverse effects on male germ cells by observing inhibited androgen production and proliferation in Leydig cells upon in vitro exposure and affected general motility and motive tracks of zebrafish sperm upon ex vivo exposure. Finally, the in vivo reproductive toxicity was demonstrated in male zebrafish by reduced mating behavior in F0 generation when paired with unexposed females and abnormal development of their offspring. In addition, reduced sperm motility and impaired germ cells in male zebrafish were also observed, which may be related to the disturbed homeostasis of sex hormones. Notably, the specifically suppressed AR in the brain provides further evidence for the antagonistic effects as above-mentioned. These results confirmed that TBPH affected male reproduction through a classical nuclear receptor-mediated pathway, which would be helpful for assessing the ecological and health risks of TBPH.

Effect of DHCR7 on adipocyte differentiation in goats.

Cholesterol is regarded as a signaling molecule in regulating the metabolism and function of fat cells, in which 7-Dehydrocholesterol reductase (DHCR7) is a key enzyme that catalyzes the conversion of 7-dehydrocholesterol to cholesterol, however, the exact function of DHCR7 in goat adipocytes remains unknown. Here, the effect of DHCR7 on the formation of subcutaneous and intramuscular fat in goats was investigated in vitro, and the result indicated that the mRNA level of DHCR7 showed a gradual downward trend in subcutaneous adipogenesis, but an opposite trend in intramuscular adipogenesis. In the process of subcutaneous preadipocytes differentiation, overexpression of DHCR7 inhibited the expression of adipocytes differentiation marker genes (CEBP/α, CEBP/ß, SREBP1 and AP2), lipid metabolism-related genes (AGPAT6, FASN, SCD1 and LPL), and the lipid accumulation. However, in intramuscular preadipocyte differentiation, DHCR7 overexpression showed a promoting effect on adipocyte differentiation marker genes (CEBP/α, CEBP/ß, PPARγ and SREBP1) and lipid metabolism-related genes (GPAM, AGPAT6, DGAT1 and SCD1) expression, and on lipid accumulation. In summary, our work demonstrated that DHCR7 played an important role in regulating adipogenic differentiation and lipid metabolism in preadipocytes in goats, which is of great significance for uncovering the underlying molecular mechanism of adipocyte differentiation and improving goat meat quality.

FOS Inhibits the Differentiation of Intramuscular Adipocytes in Goats.

Goat intramuscular fat (IMF) deposition is precisely regulated by many key genes as well as transcription factors. Nevertheless, the potential of the regulators of goat IMF deposition remains undefined. In this work, we reported that the transcription factor FOS is expressed at a low level at the early differentiation stage and at a high level in late differentiation. The overexpression of FOS inhibited intramuscular adipocyte lipid accumulation and significantly downregulated the expressions of PPARγ, C/EBPß, C/EBPα, AP2, SREBP1, FASN, ACC, HSL, and ATGL. Consistently, the knockdown of FOS, facilitated by two distinct siRNAs, significantly promoted intramuscular adipocyte lipid accumulation. Moreover, our analysis revealed multiple potential binding sites for FOS on the promoters of PPARγ, C/EBPß, and C/EBPα. The expression changes in PPARγ, C/EBPß, and C/EBPα during intramuscular adipogenesis were opposite to that of FOS. In summary, FOS inhibits intramuscular lipogenesis in goats and potentially negatively regulates the expressions of PPARγ, C/EBPß, and C/EBPα genes. Our research will provide valuable data for the underlying molecular mechanism of the FOS regulation network of intramuscular lipogenesis.

Rapid detection of Cryptosporidium spp. in diarrheic cattle feces by isothermal recombinase polymerase amplification assays.

As a zoonotic parasite, Cryptosporidium spp. could cause severe diarrhea mainly in calves and children globally. Monitoring and prevention of Cryptosporidium spp.'s prevalence is of great significance in both economy and public health aspects. In this study, specific primers and probes were designed within the conserved region of 18S rRNA gene for Cryptosporidium spp. and recombinase polymerase amplification assays based on the fluorescence monitoring (real-time RPA) as well as combined with a lateral flow strip (LFS RPA) were developed. Both of the two RPA assays allowed the exponential amplification of the target fragment within 20 min. After incubation on a metal bath at 42 °C, the LFS RPA results were displayed on the lateral flow strip within 5 min while real-time RPA allowed the real-time observation of the results in Genie III at 39 °C. The RPA assays showed high specificity for Cryptosporidium spp. without any cross-reaction with other tested pathogens causing diarrhea in cattle. With the recombinant plasmid DNA containing the 18S rRNA gene of Cryptosporidium spp. serving as a template, the limit of detection for real-time RPA and LFS RPA assays were 14.6 and 12.7 copies/reaction, respectively. Moreover, the RPA assays were validated by testing diarrheic cattle fecal samples and compared with a real-time PCR. The positive ratio of Cryptosporidium spp. was 24.04 % (44/183) and 26.23 % (48/183) in both RPA assays and real-time PCR assay, respectively, and the kappa coefficient value was 0.942. The diagnostic specificity and diagnostic sensitivity of both RPA assays were 100 % and 91.67 %, respectively. Forty-one of 48 positive samples were successfully sequenced and four Cryptosporidium species were detected, including C. parvum (n = 20), C. andersoni (n = 17), C. bovis (n = 3) and C. ryanae (n = 1). The developed RPA assays are easy to operate and faster to obtain the detection results, and they are suiting for the point-of-care detection and facilitating the prevention and control of Cryptosporidium spp. infections.

Cataract, glaucoma, and diabetic retinopathy are independent risk factors affecting falls in the older adult with eye diseases.

OBJECTIVES: Falls are the leading cause of injury-related hospitalization in older adult, presenting a significant public health concern. To examine the specific eye diseases for risk factors of falls in the older adult. METHODS: A total of 775 older adults admitted to tertiary care hospitals were divided into a fall or non-fall group based on a questionnaire. Logistic regression analysis was used to identify factors associated with falls. RESULTS: With 208 falls, 775 participants were recruited. The major associated factors of falls were older age (Odds ratios [OR]: 1.05), female (OR: 1.91), cardiovascular diseases (OR: 1.65), more outdoor activities (OR: 2.81), cataract (OR: 1.65), glaucoma (OR: 1.63), diabetic retinopathy (OR: 2.72). CONCLUSIONS: Our study demonstrates that cataract, glaucoma, and diabetic retinopathy in the older adult with eye diseases are independent risk factors of falls, which may shed light on the prevention of falls in the older adult with eye diseases.

Discovery of hepatoprotective activity components from Thymus quinquecostatus celak. by molecular networking, biological evaluation and molecular dynamics studies.

Thymus quinquecostatus Celak. is an edible herb that widely cultivated in Asia and possesses hepatoprotective activity, but the underlying non-volatile components of this protective activity are not well studied. In this study, combining molecular networking visualization and bioassay-guided fractionation strategies, a pair of novel skeleton diterpenoid enantiomers, (+)- and (-)-thymutatusone A [(+)- and (-)-1], along with one new and one known biogenetically related compounds (2-3) and 16 other known compounds (4-19), were identified from T. quinquecostatus. Their structures were exhaustively characterized by comprehensive spectroscopic data, X-ray diffraction analysis, and ECD calculations. Compounds (±)-1, (-)-1, and (+)-1, with a rare tricyclo [7.3.1.02,7] tridecane skeleton, exhibited potent hepatoprotective activity in HepG2 cells injured by acetaminophen, with EC50 values of 11.5 ± 2.8, 8.4 ± 1.9, and 12.2 ± 0.3 µM respectively. They were more potent than positive drug bifendate (EC50 15.2 ± 1.3). Further, the underlying mechanism for the hepatoprotective activity of compound (-)-1 related to activating the Nrf 2 signaling pathway. What's more, molecular docking and molecular dynamics simulation analysis showed that compound (-)-1 could dock with the active site of Nrf 2 protein and form a stable system through hydrogen bonding. These results suggest that T. quinquecostatus can be used as a valuable source of hepatoprotective activity compounds.

Development of the isothermal recombinase polymerase amplification assays for rapid detection of the genus Capripoxvirus.

Sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV) belong to the genus Capripoxvirus (CaPV), and are important pathogens of sheep, goat and cattle, respectively. Rapid and reliable detection of CaPV is critical to prevent its spread and promote its eradication. This study aimed to develop the recombinase polymerase amplification (RPA) assays combined with real-time fluorescence (real-time RPA) and naked-eye visible lateral flow strip (LFS RPA) for rapid detection of CaPV. Both developed RPA assays worked well at 39 °C within 20 min. They were highly specific for the detection of GTPV, SPPV and LSDV, while no cross-reactivity was observed for other non-targeted pathogens and genomic DNA of goat, sheep and cattle. The limit of detection for real-time RPA and LFS RPA were 1.0 × 102 and 1.0 × 101 copies per reaction, respectively. In the artificially contaminated samples with GTPV, the detection results of RPA assays were consistent with those of real-time PCR. For 15 clinical samples, LSDV was detected by real-time RPA, LFS RPA and real-time PCR in 13, 15 and 15 samples, respectively. The developed RPA assays were specific, sensitive, and user-friendly for the rapid detection of CaPV, and could be a better alternative method applied in low-resources settings.

Mitochondrial Dysfunction Was Involved in Decabromodiphenyl Ethane-Induced Glucolipid Metabolism Disorders and Neurotoxicity in Zebrafish Larvae.

Decabromodiphenyl ethane (DBDPE), a novel brominated flame retardant, is becoming increasingly prevalent in environmental and biota samples. While DBDPE has been shown to cause various biological adverse effects, the molecular mechanism behind these effects is still unclear. In this research, zebrafish embryos were exposed to DBDPE (50-400 µg/L) until 120 h post fertilization (hpf). The results confirmed the neurotoxicity by increased average swimming speed, interfered neurotransmitter contents, and transcription of neurodevelopment-related genes in zebrafish larvae. Metabolomics analysis revealed changes of metabolites primarily involved in glycolipid metabolism, oxidative phosphorylation, and oxidative stress, which were validated through the alterations of multiple biomarkers at various levels. We further evaluated the mitochondrial performance upon DBDPE exposure and found inhibited mitochondrial oxidative respiration accompanied by decreased mitochondrial respiratory chain complex activities, mitochondrial membrane potential, and ATP contents. However, addition of nicotinamide riboside could effectively restore DBDPE-induced mitochondrial impairments and resultant neurotoxicity, oxidative stress as well as glycolipid metabolism in zebrafish larvae. Taken together, our data suggest that mitochondrial dysfunction was involved in DBDPE-induced toxicity, providing novel insight into the toxic mechanisms of DBDPE as well as other emerging pollutants.

Ultrasensitive Detection of Multidrug-Resistant Bacteria Based on Boric Acid-Functionalized Fluorescent MOF@COF.

The widespread use of antibiotics has made multidrug-resistant bacteria (MDRB) one of the greatest threats toward global health. Current conventional microbial detection methods are usually time-consuming, labor-intensive, expensive, and unable to detect low concentrations of bacteria, which cause great difficulties in clinical diagnosis and treatment. Herein, we constructed a versatile biosensing platform on the basis of boric acid-functionalized porous framework composites (MOF@COF-BA), which were able to realize highly efficient and sensitive label-free MDRB detection via fluorescence. In this design, MDRB were captured using aptamer-coated nanoparticles and the fluorescent probe MOF@COF-BA was tightly anchored onto the surface of MDRB due to interactions between boric acid groups and glycolipids on bacteria cells. Benefitting from the remarkable fluorescence performance of MOF@COF-BA, rapid and specific detection of MDRB, such as methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii (AB), was realized with a detection range of 20-108 CFU/mL (for both) and limits of detection of 7 CFU/mL (MRSA) and 5 CFU/mL (AB). The feasibility of using the developed platform to selectively detect MRSA and AB from complex urine, human serum, and cerebrospinal fluid samples was also demonstrated. This work provides a promising strategy for accurate MDRB diagnosis, avoiding serious infection using rational antibiotic therapy.

Knockdown of KLF7 inhibits the differentiation of both intramuscular and subcutaneous preadipocytes in goat.

KLF7 belongs to the Krüppel-like factors (KLFs) family, which function as transcriptional regulators controlling a number of basic cellular processes, involving proliferation, differentiation, and migration. Here, we reveal insights into the differentiated expression of KLF7 in different goat tissues and different stages of growth, and the inhibition role of KLF7 knockdown to differentiation by using goat intramuscular and subcutaneous preadipocytes. We demonstrate that KLF7 expression is obviously changed during the differentiation of preadipocytes into mature adipocytes. Knockdown of KLF7 inhibited lipid droplet accumulation, reduced the expression of adipogenic markers both in intramuscular and subcutaneous preadipocytes in goats, suggesting that KLF7 is a novel regulator of adipogenesis. KLF7 expression changed also up or down-regulation the other KLF family members, but there were differences between these two types of cells. Investigation into the mechanism that KLF7 regulates preadipocyte differentiation revealed that KLF family members KLF1, KLF5, KLF6, KLF8, KLF11, KLF12, KLF16, KLF17 and adipogenic markers C/EBPα and SREBP1 promoter region present KLF7 transcriptional binding sites. Altogether, the data here identify KLF7 as a novel regulator of adipogenesis.

FGF1 promotes the differentiation of goat intramuscular and subcutaneous preadipocytes.

Fibroblast growth factor 1(FGF1) has been proved to bind to specific signal molecules and activate intracellular signal transduction, leading to proliferation or differentiation of cells. However, the role of FGF1 in goat adipocytes is still unclear. Here, we investigated its role in lipogenesis of goats, which depends on the activation of FGFRs. In goat intramuscular and subcutaneous adipocytes, we observed that adipocytes accumulation was inhibited by interfering of FGF1, the expression of C/EBPα, C/EBPß, LPL, Pref-1, PPARγ, AP2, KLF4, KLF6, KLF10 and KLF17 were significantly down-regulated (p < 0.05). When the FGF1 was up-regulated, the opposite result was found, while the expression of C/EBPß, LPL, PPARγ, SREBP1, AP2, KLF4, KLF7, KLF15, KLF16 and KLF17 were increased significantly (p < 0.05) in goat intramuscular and subcutaneous adipocytes. The expression level of FGFR1 was significantly and decreased with the interference of FGF1, and increased with the overexpression of FGF1. But in goat subcutaneous adipocytes, only the expression of FGFR2 was consistent with the expression of FGF1. Interference methods confirmed that FGFR1 or FGFR2 and FGF1 have the similarly promoting function in adipocytes differentiation. With the co-transfection technology, we confirmed that FGF1 promoted the differentiation of intramuscular and subcutaneous adipocytes might via FGFR1 or FGFR2, respectively.

[Effects of seed grading on population regularity degree and yield of summer maize]. / 种子分级对夏玉米群体整齐度和产量的影响.

Grading seeds based on grain size is an effective measure to improve population regularity degree and increase the yield of summer maize. Taking Denghai 605 as the experimental material, we set up a field experiment with treatments based on grain size: large seeds (L), medium-round seeds (MR), medium-flat seeds (MF), medium-round and medium-flat mixed seeds (MRF), and small seeds (S), with no-grading seeds as control (CK). We investigated seedling emergence rate, population regularity degree (including height, ear height and stem diameter), seedling sturdiness index, photosynthetic characteristics, dry matter accumulation and distribution characteristics, and yield. The results showed that the emergence rate followed an order of L>MR>MRF>MF>CK>S, with that of L treatment differed little from MR, MF and MRF treatments, but being significantly higher than S and CK treatments. Plant height and stem diameter population regularity degree of MRF treatment before seven-leaf stage was not different from those of L, MR, MF and S treatments, but significantly higher than those of CK. At the tasseling stage, all treatments had higher population regularity degree of plant height than other stages. Ear height population regularity degree of L, MR, MF, MRF, and S increased by 11.1%, 10.3%, 9.5%, 7.1%, and 6.4% compared with CK, respectively. The seedling sturdiness index of MRF treatment increased by 36.7% compared with S treatment, but was not significantly different from L treatment. The leaf area index of the L and MRF treatments was significantly higher than that of CK, and both had higher population photosynthetic properties. The population dry matter accumulation showed a pattern as L>MR>MRF>MF>CK>S. There was no significant difference among L, MR, and MRF treatments, but that in L being obviously higher than MF, CK, and S treatments. After seed grading, the number of harvested ears of the L and MRF treatments increased significantly, and the yield were shown as L>MR>MRF>MF>CK>S. There was no difference between the yield of MRF, MR and MF treatments. In conclusion, the performance of L treatment was improved but the number was small. Considering the grading cost and yield, the MRF treatment can save the seed amounts of sowing, realize mechanized sowing and precision sowing.

Overexpression of goat STEAP4 promotes the differentiation of subcutaneous adipocytes.

Objective: The focus of this study was the six-transmembrane epithelial antigen of the prostate 4 (STEAP4) gene, on the basis of the cloned goat STEAP4 gene sequence. Its molecular and expression characteristics were analyzed, and its influence on the differentiation of goat subcutaneous adipocytes was explored through overexpression. Method: Reverse-transcription PCR (RT-PCR) was used to clone the goat STEAP4 sequence, and online tools were used to analyze the molecular characteristic. Real-time quantitative PCR (qPCR) was used to detect the expression level of STEAP4 in goat tissues and subcutaneous adipocyte differentiation. Liposome transfection, BODIPY, Oil Red O staining, and qPCR were used to explore the effect of overexpression of STEAP4 on adipocyte differentiation. Results: The cloned goat STEAP4 gene sequence was 1388 bp, and the complete coding sequence (CDS) region was 1197 bp, which encoded a total of 398 amino acids. Compared with the predicted sequence (XM_005679300.3), there were three base mutations in the CDS region of goat STEAP4, A188G, T281C, and A507G. Among them, A507G changed the amino acid at position 170 from Ile to Val. Analysis of the physical and chemical properties of the protein showed that STEAP4 was a stable hydrophilic basic protein. STEAP4 gene expression level was highest in goat liver tissue ( P < 0.01 ), followed by lung and back subcutaneous adipose tissue. STEAP4 showed different expression levels in goat subcutaneous adipocytes at different times during the induction of differentiation. The expression in the late stage of differentiation was higher than that before differentiation and lowest at 12 h ( P < 0.01 ). Overexpression of STEAP4 promoted the accumulation of intracellular lipid droplets; C/EBP ß (CCAAT enhancer binding protein) was extremely significantly up-regulated ( P < 0.01 ), and aP2 (fatty acid binding protein) was significantly up-regulated ( P < 0.05 ). Conclusion: Overexpression of STEAP4 could promote the differentiation of goat subcutaneous preadipocytes. This study lays the foundation for an in-depth study of the role of STEAP4 in goat lipid deposition.

Rapid detection of bovine rotavirus a by isothermal reverse transcription recombinase polymerase amplification assays.

BACKGROUND: Bovine rotavirus A (BRVA) is considered to be the most common pathogen of severe diarrhea in cattle worldwide, which could lead to the death of newborn calves and cause the significant economic losses to the cattle industry. As a novel isothermal nucleic acid amplification technique, recombinase polymerase amplification (RPA) has been applied widely for the rapid detection of different important pathogens in human and animals. RESULTS: An RT-RPA assay based on the real time fluorescence monitoring (real-time RT-RPA) and an RT-RPA assay combined with a lateral flow strip (LFS RT-RPA) were successfully developed by targeting the VP6 gene of BRVA. The RT-RPA assays allowed the exponential amplification of the target fragment in 20 min. After incubation of the LFS RT-RPA on a metal bath at 40 °C, the results were displayed on the lateral flow strip within 5 min, while real-time RT-RPA allowed the real-time observation of the results in Genie III at 42 °C. Both of the two assays showed high specificity for BRVA without any cross-reaction with the other tested pathogens causing diarrhea in cattle. With the standard RNA of BRVA serving as a template, the limit of detection for real-time RT-RPA and LFS RT-RPA were 1.4 × 102 copies per reaction and 1.4 × 101 copies per reaction, respectively. In the 134 fecal samples collected from cattle with diarrhea, the BRVA positive rate were 45.52% (61/134) and 46.27% (62/134) in real-time RT-RPA and LFS RT-RPA, respectively. Compared to a previously published real-time PCR, the real-time RT-RPA and LFS RT-RPA showed a diagnostic specificity of 100%, diagnostic sensitivity of 98.39% and 100%, and a kappa coefficient of 0.985 and 1.0, respectively. CONCLUSIONS: In this study, BRVA was successfully detected in cattle fecal samples by the developed real-time RT-RPA and LFS RT-RPA assays. The developed RT-RPA assays had great potential for the rapid detection of BRVA in under-equipped diagnostic laboratory and the point-of-need diagnosis at quarantine stations and farms, which is of great importance to control BRVA-associated diarrhea in cattle herds.

Development of a new TaqMan-based real-time RT-PCR assay for the specific detection of bovine kobuvirus.

Bovine kobuvirus (BKV) is a novel kobuvirus considered to be closely related to calf diarrhea and has become a worldwide epidemic. Currently, the BKV lacks an efficient and convenient detection method to assist the research on BKV prevalence. In this study, a new and specific TaqMan-based real-time RT-PCR for the detection of BKV was developed using the conserved region of the 3D gene. The assay was highly specific for BKV, without cross-amplification with other non-targeted pathogens. The limit of detection of this assay was 102 copies. Standard curves showed a strong linear correlation from 102 to 106 copies of BKV standard RNA per reaction, and the parameters revealed as a slope of -3.54, efficiency of 91.64%, and regression coefficients (R2) of 0.998. The assay was also reproducible, with the intra-assay and inter-assay coefficient of variation <1.0%. The newly developed real-time RT-PCR was validated using 243 fecal samples collected from diarrheic or non-diarrheic cattle from nine regions in Hebei province and revealed the positive detection of BKV at a ratio of 19.34% (47/243). Sequencing of partial 3D genes from 13 positive samples and the following phylogenetic analysis demonstrated the reliability of the assay. In conclusion, the newly developed TaqMan-based real-time RT-PCR could be used for the screening and epidemic monitoring of BKV.