Mixing or regrouping of calves from different pens is a common animal management practice on the farm, which frequently occurs after weaning and has a negative effect on calve welfare.  Social integration before regrouping may relieve stresses, but more evidences are needed to verify this hypothesis.  The present study aimed to investigate acute physiological and behavioral variations of individually- or group-housed calves after being introduced into a mixed group.  A total of 132 postnatal calves were randomly divided into groups of 1, 3, 6 and 12 animals (S, G3, G6, and G12; 6 replicates in each group) until 59 days of age.  At 60 days of age, every two replicates from different groups (S, G3, G6 and G12) were introduced in a larger pen which containing 44 of the aboved experimental calves.  Before and after regrouping, physiological parameters of stress, including heart rate (HR), saliva cortisol (S-CORT), saliva secretory immunoglobulin A (SIgA), interleukin-2 (IL-2), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) levels, and behavioral responses were recorded.  After regrouping, HR and S-CORT increased immediately (P<0.05), and higher (P<0.05) levels of such molecules were found in S calves compared to those in group-housed calves.  Levels of SIgA and IL-2 were decreased (P<0.05), and the lowest (P<0.05) IL-2 values were found in S calves compared to those in group-housed calves.  In addition, the introduced calves displayed a distinct behavior, including altered active and rest time, which was associated with negative emotions triggered by the novel surroundings.  Allogrooming, play, exploration behaviors and lying time were increased significantly (P<0.05) in group-housed calves than those in S calves.  Conversely, self-grooming, aggressive behaviors, standing and walking time were increased (P<0.05) in S calves than those in group-housed calves.  These findings suggest that individually-housed calves may be more susceptible to stressors arising from regrouping than group-housed calves, which consequently negatively affected behavioral and neuroendocrine responses.  Furthermore, moving calves with previous social experience may help mitigate regrouping stress.

The application of fertilizer in agricultural production has become universally common for achieving high crop yields and economic benefits, but it has potential impacts on food safety, energy crisis and environmental pollution.  Optimal management of fertilization is thus necessary for maintaining sustainable agriculture.  Two-year (2013–2015) field experiment was conducted, in Yangling (108°24´E, 34°20´N, and 521 m a.s.l.), Shaanxi Province, China, to explore the effects of different nitrogen (N) applications on biomass accumulation, crop N uptake, nitrate N (NO₃^–-N) distribution, yield, and N use with a winter wheat/summer maize rotation system.  The N applications consisted of conventional urea (U) (at 80 (U80), 160 (U160), and 240 (U240) kg N ha–1; 40% applied as a basal fertilizer and 60% top-dressed at jointing stage) and controlled-release urea (CRU) (at 60 (C60), 120 (C120), 180 (C180), and 240 (C240) kg N ha^–1; all applied as a basal fertilizer) with no N application as a control (CK).  The continuous release of N from CRU matched well with the N demands of crop throughout entire growing stages.  Soil NO₃^–-N content varied less and peaked shallower in CRU than that in urea treatments.  The differences, however, were smaller in winter wheat than that in summer maize seasons.  The average yield of summer maize was the highest in C120 in CRU treatments and in U160 in urea treatments, and apparent N use efficiency (NUE) and N agronomic efficiency (NAE) were higher in C120 than in U160 by averages of 22.67 and 41.91%, respectively.  The average yield of winter wheat was the highest in C180 in CRU treatments and in U240 in urea treatments with C180 increasing NUE and NAE by averages of 14.89 and 35.62% over U240, respectively.  The annual yields under the two N fertilizers were the highest in C120 and U160.  The results suggested that CRU as a basal fertilizer once could be a promising alternative of urea as split application in semiarid areas. 

Granulosa cells (GCs) are somatic cells of ovary, the behaviors of GCs are important for ovarian function.  MicroRNAs (miRNAs) are a class of endogenous 18–24 nucleotide (nt) non-coding RNAs, some of which have been shown to be important regulators of GCs function.  miR-34c involved in the regulation of various biological processes and was identified to be a pro-apoptotic and anti-proliferative factor in many cell types.  However, the roles of miR-34c in GCs function remain unknown.  In this study, we used Annexin V-FITC and EdU assays to demonstrate that miR-34c exerted pro-apoptotic and anti-proliferative effects in porcine GCs.  Dual-luciferase reporter assays, quantitative real-time PCR (qRT-PCR) and Western blotting identified Forkhead box O3a (FoxO3a) as a direct target gene of miR-34c.  The overexpression of FoxO3a rescued the phenotypic change caused by miR-34c in porcine GCs.  In conclusion, miR-34c regulate the function of porcine GCs by targeting FoxO3a.

   MicroRNAs (miRNAs) are endogenous 18–24 nucleotide (nt) non-coding RNAs, some of which have been indicated to play key roles in granulosa cells (GCs) function. However, little is known about how the miRNA gene expression itself is regulated in the GCs. Our previous study showed that miR-34c, identified to be a pro-apoptotic and anti-proliferative factor in many cell types, exerted the same effects in porcine GCs. Here, the transcriptional regulation of miR-34c expression in GCs was further investigated. 5´ rapid amplification of cDNA ends (RACE) assay indicated that the pri-miR-34c transcription start site was located in 1 556 bp upstream of pre-miR-34c. With dual-luciferase reporter assay, we confirmed a 69 bp core promoter region (–1 799 bp/–1 730 bp) was indispensable for the transcription of miR-34c. Chromatin immunoprecipitation (ChIP) assay demonstrated that p53, p50, and p65 could bind to the transcription factor binding sites within the 69 bp core promoter region. In addition, deletion of transcripition factor binding sites resulted in obvious change of the miR-34c promoter activity. Finally, using overexpression and knockdown of p53, p50, and p65 strategies, we showed that p53 and p50 could positively regulated miR-34c expression, whereas p65 neletively regulated miR-34c expression in GCs. Our results provide new data about the transcription regulatory mechanism of miRNA genes in GCs.

Both bolting and flowering times influence taproot and seed production in radish. FLOWERING LOCUS C (FLC) plays a key role in plant flowering by functioning as a repressor. Two genomic DNA sequences, a 3 046-bp from an early- and a 2 959-bp from a late-bolting radish line were isolated and named as RsFLC1 and RsFLC2, respectively, for they share approximately 87.03% sequence identity to the FLC cDNA sequences. The genomic DNA sequences, 1 466-bp and 1 744-bp, flanking the 5´-regions of RsFLC1 and RsFLC2, respectively, were characterized. Since both of them harbor the basic promoter elements, the TATA box and CAAT box, they were designated as PRsFLC1 and PRsFLC2. The transcription start site (TSS) was identified at 424 and 336 bp upstream of the start codon in PRsFLC1 and PRsFLC2, respectively. cis-regulatory elements including CGTCA (MeJA-responsive) and ABRE (abscisic acid-responsive) motifs were found in both promoters, while some cis-regulatory elements including TCA element and GARE-motif were present only in PRsFLC1. These sequence differences lead to the diversity of promoter core elements, which could partially result in the difference of bolting and flowering time in radish line NauDY13 (early-bolting) and Naulu127 (late-bolting). Furthermore, to investigate the activity of these promoters, a series of 5´-deletion fragment-GUS fusions were constructed and transformed into tobacco. GUS activity was detected in PRsFLC1-(1 to 4)-GUS-PS1aG-3 and PRsFLC2-(1 to 4)-GUS-PS1aG-3 transgenic tobacco leaf discs, and this activity progressively decreased from PRsFLC-1-GUS-PS1aG-3 to PRsFLC-5-GUS-PS1aG-3. Deletion analysis indicated that the cis-regulatory elements located at –395 bp to +1 bp may be critical for specifying RsFLC gene transcription.

Myrosinase is a defense-related enzyme and is capable of hydrolyzing glucosinolates into a variety of compounds, some of which are toxic to pathogens and herbivores. Many studies revealed that a number of important vegetables or oil crops contain the myrosinase-glucosinolate system. However, the related promoter and genomic DNA sequences as well as expression profiles of myrosinase gene remain largely unexplored in radish (Raphanus sativus). In this study, the 2 798 bp genomic DNA sequence, designated as RsMyr2, was isolated and analyzed in radish. The RsMyr2 consisting of 12 exons and 11 introns reflected the common gene structure of myrosinases. Using the genomic DNA walking approach, the 5´-flanking region upstream of RsMyr2 with length of 1 711 bp was successfully isolated. PLACE and PlantCARE analyses revealed that this upstream region could be the promoter of RsMyr2, which contained several basic cis-regulatory elements including TATA-box, CAAT-box and regulatory motifs responsive to defense and stresses. Furthermore, recombinant pET-RsMyr2 protein separated by SDS-PAGE was identified as myrosinase with mass spectrometry. Real-time PCR analysis showed differential expression profiles of RsMyr2 in leaf, stem and root at different developmental stages (e.g., higher expression in leaf at cotyledon stage and lower in flesh root at mature stage). Additionally, the RsMyr2 gene exhibited up-regulated expression when treated with abscisic acid (ABA), methyl jasmonate (MeJA) and hydrogen peroxide (H2O2), whereas it was down-regulated by wounding (WO) treatment. The findings indicated that the expression of RsMyr2 gene was differentially regulated by these stress treatments. These results could provide new insight into elucidating the molecular characterization and biological function of myrosinase in radish.