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Integrated small RNA and mRNA expression profiles reveal miRNAs and their target genes in response to Aspergillus flavus growth in peanut seeds
BMC Plant Biology volume 20, Article number: 215 (2020)
MicroRNAs are important gene expression regulators in plants immune system. Aspergillus flavus is the most common causal agents of aflatoxin contamination in peanuts, but information on the function of miRNA in peanut-A. flavus interaction is lacking. In this study, the resistant cultivar (GT-C20) and susceptible cultivar (Tifrunner) were used to investigate regulatory roles of miRNAs in response to A. flavus growth.
A total of 30 miRNAs, 447 genes and 21 potential miRNA/mRNA pairs were differentially expressed significantly when treated with A. flavus. A total of 62 miRNAs, 451 genes and 44 potential miRNA/mRNA pairs exhibited differential expression profiles between two peanut varieties. Gene Ontology (GO) analysis showed that metabolic-process related GO terms were enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses further supported the GO results, in which many enriched pathways were related with biosynthesis and metabolism, such as biosynthesis of secondary metabolites and metabolic pathways. Correlation analysis of small RNA, transcriptome and degradome indicated that miR156/SPL pairs might regulate the accumulation of flavonoids in resistant and susceptible genotypes. The miR482/2118 family might regulate NBS-LRR gene which had the higher expression level in resistant genotype. These results provided useful information for further understanding the roles of miR156/157/SPL and miR482/2118/NBS-LRR pairs.
Integration analysis of the transcriptome, miRNAome and degradome of resistant and susceptible peanut varieties were performed in this study. The knowledge gained will help to understand the roles of miRNAs of peanut in response to A. flavus.
Peanut (Arachis hypogaea. L), or groundnut, is one of the most important oil crops in the world. It is wildly planted in Asia, Africa, and North America. China, India and the United States are the world’s major producers of peanut. The world’s total volume of peanut production is about 29 million metric tons per year (http://www.peanutsusa.com), contributing to an estimated value of about $35 billion . However, peanut production is vulnerable to the threat of aflatoxin contamination, which is caused by the infection of Aspergillus flavus and Aspergillus parasiticus . The aflatoxin was first revealed in 1960, when the aflatoxin-contaminated peanut caused the death of a large number of turkeys in UK. Aflatoxins, can cause DNA damage and chromosome instability in host cells, are highly toxic and one of the most carcinogenic substances [3,4,5]. Peanut aflatoxin contamination is serious problem in China and limits the exportation of peanuts. Contamination can occur in the field, during harvest, or in storage and processing, and therefore, aflatoxin contamination is hard to prevent and control.
Although it is a widespread problem, the underlying molecular determinants and mechanisms of peanut-A. flavus interaction have remained elusive. The analysis of the molecular mechanism of peanut-fungus interactions, and mining peanut resistance information is the basis to develop resistance peanut varieties. To better understand the mechanisms, cDNA libraries derived from developing peanut seeds from a resistant cultivar (GT-C20) and a susceptible cultivar (Tifrunner) were constructed and a total of 21,777 EST sequences were generated . Soon afterwards, a peanut oligonucleotide microarray chip was used to identify resistance genes to A. flavus and A. parasiticus mixed infection, and found that 62 genes in resistant cultivar were up-regulated and the expression of 22 putative Aspergillus-resistance genes were higher in the resistant cultivar in comparison to the susceptible cultivar . In order to identify proteins related to the resistance to aflatoxin contamination, two-dimensional electrophoresis was employed with mass spectrometry as well as real time RT-PCR method and 12 potential different expressed proteins were identified . Another group also used the differential proteomic approach and identified an array of proteins responding to A. flavus infection . Recently, Wang et al. employed Next Generation Sequencing to survey the gene expression profiling of resistant and susceptible peanut genotypes in response to A. flavus growth [9, 10]. These studies provided valuable information for understanding the mechanism of peanut in response to aflatoxin production. However, due to the lack of peanut genomic sequences, it is hard for these studies to provide a comprehensive interpretation of the transcriptomic changes of peanut in response to A. flavus. Recently, the release of whole genome sequences of the diploid wild peanut species, A. duranensis and A. ipaensis [11, 12] and cultivars, Tifrunner and Shitouqi (https://peanutbase.org/, http://peanutgr.fafu.edu.cn/) provide new opportunity to understand the peanut resistance mechanism in response to A. flavus.
MicroRNAs (miRNAs) are a class of non-coding RNAs regulating gene expression and play roles in plant development and response to stresses [13,14,15]. Up to now, more than 5000 miRNAs have been identified from near 70 plant species (http://www.mirbase.org/). The functions of plant miRNAs in biotic stress responses have been extensively explored. For example, miR398 regulated plant productivity under oxidative stress conditions by targeting Cu/Zn superoxide dismutase [16, 17]. In tomato, the accumulation of miR169 was induced by drought stress. The expression of its target genes, nuclear factor Y subunit genes and multidrug resistance-associated protein gene, were significantly down-regulated by drought stress. Over-expression of miR169 enhanced drought tolerance of tomato [18, 19]. Many transcription factors, such as SPL [20, 21], MYB , NAC [23,24,25], ARF , HD-Zip  and AP2 [28, 29] were the targets of miRNAs, suggesting miRNAs located in the core position of the network in gene regulation.
Recently, the roles of miRNA in plant disease resistance were gradually discovered, and miRNAs were considered to play key roles in plant natural immune system [30, 31]. In Arabidopsis, it was proven that miR472/RDR6 silencing pathway modulated PAMP and effector triggered immunity (ETI) through the post-transcriptional control . In rice, multiple miRNAs are involved in resistance against the blast fungus Magnaporthe oryzae. A group of rice miRNAs were differentially expressed upon M. oryzae infection. Over-expression of miR160a and miR398b enhanced resistance to M. oryzae in the transgenic rice . Multiple miRNA target genes were related to disease resistance. For example, the expression and the variation of miR482 and miR2118 reflected the shift in the balance of plants NBS-LRR defense system . In our previous studies, we identified many known and novel miRNAs in peanut using high-throughput sequencing method . We found that the expression of some known and novel miRNAs was induced or inhibited upon Ralstonia solanacearum infection in cultivated and wild peanuts. Degradome sequencing results possibly indicated that some defense response genes were degraded by miRNA . These data suggested that miRNA might play important role in peanut disease resistance.
In this study, we aimed to identify potential A. flavus responsive miRNAs and their targets in peanut, and study their roles in peanut-A. flavus interaction. For this purpose, we employed high-throughput sequencing technology to simultaneously analyze the miRNA and mRNA expression profiles and their regulation in response to A. flavus growth using a resistant and a susceptible peanut variety. Our results provided valuable information for understanding the molecular mechanism of peanut resistance to A. flavus.
Different resistance of peanut varieties GT-C20 and Tifrunner to A. flavus
The resistance of peanut varieties GT-C20 and Tifrunner to A. flavus was validated. As a result, in two days after inoculation (DAI), hyphae were only observed in the seeds of Tifrunner, but not in GT-C20 (Fig. 1). From three DAI, the hyphae were very clear in both GT-C20 and Tifrunner. More hyphae were observed in the seeds of Tifrunner than that in GT-C20 (Fig. 1). Statistical analysis showed that GT-C20 was high resistant (Infection index of 4.7) and Tifrunner was high susceptible (Infection index of 95.3) to A. flavus growth according to the standard reported . Samples of two DAI were collected for miRNAome and transcriptome analysis.
High-throughput sequencing and identification of miRNAs
To study the role of peanut miRNAs in response to A. flavus growth, eight small RNA libraries were constructed and sequenced using Illumina HiSeq2000 platform. These small RNA libraries included two A. flavus-treated (GT1 and GT2) and two control (GC1 and GC2) libraries of resistant cultivar GT-C20, and two A. flavus-inoculated (TT1 and TT2) and two control (TC1 and TC2) libraries of susceptible cultivar Tifrunner. After sequencing, a total of 111,839,986 raw reads and 111,324,210 clean reads were obtained from eight libraries (Additional file: Table S1). The normalized clean reads were used for analysis of small RNA distribution. Most of the clean reads were 20–24 nucleotides (nt) in length, which accounted for 96.22, 96.67, 94.06 and 95.81% of all clean reads in TC, TT, GC and GT libraries, respectively (Fig. 2). The 24-nt small RNAs was the most abundant group, which was followed by the 21-nt small RNAs. These results were consistent with previous studies in peanut small RNA sequencing [35, 36, 38].
More than 85.5% unique small RNAs and 86% total small RNAs were mapped to peanut genome (A. duranensis and A. ipaensis, https://peanutbase.org/) using SOAP2 software (Additional file: Table S2). By blast search against the miRNA database (miRbase 21.0, http://www.mirbase.org/), we identified 113 known miRNAs, belongs to 30 miRNA families (Additional file: Table S3). In addition, clean small RNAs were also used to blast against with GenBank and Rfam database to remove rRNAs, tRNAs, snoRNAs and snRNAs (Additional file: Figure S1). The other small RNAs were used to explore novel miRNAs according the criteria described by the previous studies [35, 39, 40]. In total, we identified 67 novel miRNAs, ranging from 20 nt to 23 nt, and the negative folding free energies of their precursor’s hairpin structures ranged from 184.8 to 19.1 kcal/mol (Additional file: Table S4).
Expression of miRNAs in different peanut varieties response to A. flavus infection
To identify miRNAs in peanut that respond to A. flavus, the differential expressed miRNAs were identified at a significant level (adjusted p-value < 0.05 and fold change ≥1.5) by comparing the normalized miRNA expression level using TP10M (Tags per ten million). Among the 113 known miRNAs identified in peanut, eight and two were up-regulated, twelve and six were down-regulated in response to A. flavus in GT-C20 and Tifrunner, respectively (Fig. 3a, Additional file: Table S3). The total number of differentially expressed known miRNAs in GT-C20 (20) was more than that in Tifrunner (8). Among these differentially expressed known miRNAs, the expression of miR319 were both increased in GT-C20 and Tifrunner after A. flavus inoculation. While the expression of miR157a and miR157d were both decreased in GT-C20 and Tifrunner after treated with A. flavus. In addition, miR157a showed the highest expression abundance in all known miRNAs (Additional file: Table S3). There were also some miRNAs showing different expression trend in response to A. flavus in different peanut varieties. For example, the expression of miR156a was increased in GT-C20 in response to A. flavus, but unchanged in Tifrunner (Additional file: Table S3). Moreover, twelve differentially expressed novel miRNAs were identified in response to A. flavus, including six novel miRNAs from GT-C20 and six novel miRNAs from Tifrunner (Additional file: Table S4). In GT-C20, miRn21, miRn56 and miRn67 were induced or specifically expressed in response to A. flavus, and miRn3, miRn9, miRn18 were depressed in response to A. flavus. In Tifrunner, miRn12, miRn21 and miRn25 were induced or specifically expressed in response to A. flavus, and miRn1, miRn53 and miRn55 were depressed in response to A. flavus (Additional file: Table S4). To confirm the sequencing results, six miRNAs (miR156i, miR166g-3p, miR167a, miR167h, miR172a and miR396e-3p) were selected for stem-loop qRT-PCR analysis (Fig. 4). The expression profiles of these miRNAs were in accordance with the deep sequencing data (Additional file: Figure S2).
The miRNA abundance between two different varieties was compared. Before inoculation, the abundance of all 113 known miRNAs of GT-C20 was 245,769 (TP10M), which is lower than that in Tifrunner (615,962). After inoculation, the abundance of all 113 known miRNAs in GT-C20 (269,075.54 TP10M) was also lower than that in Tifrunner (442,962.68 TP10M) (Additional file: Table S3). Under the criterion of adjusted p value (padj) < 0.05 and fold change ≥1.5, a total of 23 and 39 differential expressed known miRNAs between two varieties were identified in control and A. flavus infected samples, respectively (Fig. 3a). Many known miRNAs showed higher expression level in Tifrunner than in GT-C20 in both control and A. flavus infected samples. For examples, in control samples, the expression level of miR157a was 52,711 TP10M in GT-C20 and 291,784 TP10M in Tifrunner. And in treatment samples, the expression level of miR157a-5p was 32,146 TP10M in GT-C20 and 154,326 TP10M in Tifrunner (Additional file: Table S3). In addition, the expression level of miR2118 and miR482a in GT-C20 was also lower than that in Tifrunner (Additional file: Table S3).
Targets identification of miRNAs by high throughout degradome analysis
To validate the targets of peanut miRNAs, a degradome pool was mixed with equal samples from TT, TC, GC and GT. Through high throughput sequencing, 14,338,349 raw reads representing 5,477,891 unique reads were obtained. All reads were used to align against the peanut genome and transcriptome using SOAP2 Program. As a result, 9,580,557 (66.82%) and 6,271,065 (43.73%) reads were successfully mapped in peanut reference genome and transcriptome, respectively (Additional file: Table S5). According to the signature number and abundance of putative cleaved position at each occupied transcript, these cleaved transcripts could be categorized into five classes according to the signature abundance at each occupied transcript position (0, 1, 2, 3 and 4) [41,42,43]. In our dataset, 249, 165, 554, 175 and 402 candidate targets were classified into categories 0, 1, 2, 3 and 4, respectively (Additional file: Table S6). In total, 93 and 176 candidate targets were identified for known and novel miRNAs, respectively (Additional file: Table S7-S8). We found many defense related genes were likely cleaved by miRNAs. For example, two PPR repeat-containing protein genes, Aradu.7H0DM.1 and Aradu.4L72B.1 were potentially cleaved by miR3514 and miRn10, respectively (Additional file: Table S8).
Global mRNA expression profiles in peanut in response to A. flavus growth
To study the target gene expression changes in response to A. flavus growth, eight transcriptome libraries were constructed using the same samples as used in miRNA study. Through transcriptome sequencing, a total of 97,021,101 raw reads were generated with an average of ∼12 million reads per libraries (Additional file: Table S9). Approximately 77.83% reads were successful mapped to the peanut reference genome, and about 8.18% reads were mapped to multiple regions (Additional file: Table S9). In peanut genome database, 40,636 genes from A. duranensis (A genome) and 46,984 genes from A. ipaensis (B genome) were deposited. In our RNA-seq data, 30,254 (74.45%) genes of A. duranensis and 34,172 (72.73%) genes of A. ipaensis were mapped, indicating that these genes were expressed in peanut seeds.
Differentially expressed genes in response to A. flavus
We further investigated the global gene expression profile of peanut in response to A. flavus. By applying a cut-off criterion of probability ≥0.8 and |log2(fold change) | ≥ 1, a total of 261 (125 up-regulated and 136 down-regulated) and 186 (69 up-regulated and 117 down-regulated) differentially expressed genes (DEGs) were identified in response to A. flavus from Tifrunner and GT-C20, respectively (Fig. 3b, Additional file Figure S3). Among these DEGs in response to A. flavus, 15 DEGs were up-regulated in both GT-C20 and Tifrunner, and 57 DEGs were down-regulated in both GT-C20 and Tifrunner (Fig. 5a). In addition, functional annotation results showed that 139 out of 447 (31.09%) DEGs were defense related genes, such as Chitinase, Heat shock protein, WRKY family transcription factor and temperature-induced lipocalin et al. (Table 1). Moreover, we found that an Indole-3-acetic acid inducible 2 gene (Araip.AN5V8.1) showed opposite expression trend in response to A. flavus. It was up-regulated in resistant variety GT-C20, but down-regulated in susceptible variety Tifrunner (Fig. 5a). The qRT-PCR analysis for several DEGs (Aradu.78 K64.1, Aradu.Q1D8Z.1, Aradu.412P9.1, Aradu.VK4DU.1, Aradu.39 V24.1, and Aradu.DAV01.1) confirmed the RNA-seq results (Fig. 4, Additional file Figure S2).
Differentially expressed genes between resistant and susceptible peanut varieties
We further analyzed the gene expression variation between two peanut varieties. By comparison with control samples (GC and TC), 451 DEGs (346 up-regulated and 105 down-regulated) were identified (Fig. 3b). For treatment samples (GT and TT), 703 DEGs (537 up-regulated and 166 down-regulated) were identified (Fig. 3b). Some disease related genes showed different expression level in two varieties. For example, one NBS-LRR disease resistance gene (Araip.WF303.1, log2 TC/GC = 9.47, log2 TT/GT = 4.66) was highly expressed in Tifrunner, but had a lower expression level in GT-C20. Meanwhile, another NBS-LRR encoding gene (Aradu.168 L7.1) showed the opposite expression trend, highly expressed in GT-C20, but had a lower expression level in Tifrunner (Fig. 3b).
GO and KEGG pathways of DEGs
GO analysis of the DEGs was performed and DEGs could be classified into three categories including molecular function, cellular component and biological process. In category of cellular component, “cell” and “cell part” represented the top terms. For the category of molecular function, the GO term of “catalytic activity” was enriched. For the category of biological processes, the most abundant terms were “metabolic process” and “cellular process” (Fig. 6a). The DEGs between control samples and treatments were also analyzed. Two peanut varieties have difference in many GO terms in both control samples and treatment samples, such as “metabolic process”, “catalytic activity” and “cellular process” (Fig. 6b).
To better understand the function and gene regulatory network, KEGG analysis was carried out. The DEGs in response to A. flavus were assigned into 53 and 41 KEGG pathways in Tifrunner and GT-C20, respectively (Fig. 7, Table S10–11). The top 16 enriched pathways possibly regulated by A. flavus were summarized (Fig. 7a, Table S10). Most enriched pathways were related to biosynthesis and metabolism, such as “biosynthesis of secondary metabolites”, “flavonoid biosynthesis”, “stilbenoid, diarylheptanoid”, “gingerol biosynthesis”, “metabolic pathways”, and “limonene and pinene degradation” etc. Interestingly, we observed that, in most of the enriched pathways including “biosynthesis of secondary metabolites” and “metabolic pathways”, the numbers of DEGs in Tifrunner was more than that in GT-C20, indicating the difference in metabolic active between two varieties. Interestingly, several pathways of biosynthesis and metabolism were also enriched, such as “biosynthesis of secondary metabolites”, “phenylpropanoid biosynthesis”, “phenylalanine metabolism”, “metabolic pathways”, “flavonoid biosynthesis” and “ascorbate and aldarate metabolism” etc. KEGG results also supported the difference in the metabolic activity between two peanut varieties (Fig. 7b, Table S11).
Correlation analysis of miRNAs expression profiles and their target genes
To specify the roles of miRNAs in response to A. flavus growth, the expression profiles of miRNAs and targets were analyzed. In response to A. flavus infection, twenty-two potential miRNAs/targets pairs were identified (Table S12). Degradome sequencing showed these target genes were cleaved by miRNAs. However, the expression profiles of miRNAs were not perfectly negative corresponding to these targets. For example, only five miRNAs/targets pairs showed one-to-one correspondence in Tifrunner, including five up-regulated miRNAs/down-regulated targets and two down-regulated miRNAs/up-regulated targets in response to A. flavus growth. In addition, five miRNAs/targets pairs showed opposite expression trend in Tifrunner after A. flavus infection. For other miRNAs/targets pairs in Tifrunner, only the miRNAs or targets showed differential expression in response to A. flavus, and the one-to-one correspondence expression was not observed.
Between two peanut varieties, forty-four differentially expressed miRNAs/targets pairs were identified (Table S13). We further compared the one-to-one expression correspondence of miRNAs/targets pairs between two peanut varieties. Between control samples and A. flavus infected samples (GC and TC), differentially expressed miRNAs and targets were simultaneously observed only in 16 miRNAs/targets pairs, and these results were not observed in other miRNAs/targets pairs. Among these 16 miRNAs/targets pairs, the expression of miRNA and targets were negatively correlated. For the rest five miRNAs/targets, the expression trend of miRNA and target was in the same trend, both up-regulated or down-regulated. For the flavus infected samples (GT and TT), differentially expressed miRNAs and targets were simultaneously observed in 14 miRNAs/targets pairs, ten of them showed negative correlation in expression of miRNAs and targets.
Strategies to reveal the disease resistant mechanism of peanut
Cultivated peanut (Arachis hypogaea. L) is an allotetraploid (2n = 4x = 40, AABB) organism, which emerged as a hybridization of two ancient diploid species, probably A. duranensis (A-genome) and A. ipaensis (B-genome), followed by spontaneous whole genome duplication [44,45,46]. Expressed Sequence Tag (EST) was first used to obtain gene sequence information and their response to biotic and abiotic stresses in peanut [47,48,49]. Based on EST sequencing and Microarray technology, dozens of different genes response to A. flavus infection were identified, which provide a useful information for understanding the molecular mechanism of peanut resistance to A. flavus [1, 6]. Recently, the whole genome sequencing of two diploid wild peanut species has been completed (http://peanutbase.org/). This achievement will greatly promote gene discovery and genetic improvement of this major crop. Here, we employed the high throughput mRNA and miRNA sequencing to investigate the gene expression and regulation of peanut upon A. flavus infection. Our data covered the expression information of more than 64,426 genes. It is the first report of genome-wide gene expression profiling study of peanut in response to A. flavus. Moreover, the integrated analysis of mRNAs and miRNAs might provide insight to understanding the miRNA regulatory in peanut in regulation of the resistance to A. flavus.
Differential expression of defense related genes in resistant and susceptible peanut lines
In the present study, a total of 447 differentially expressed genes (DEGs) were identified in GT-C20 and Tifrunner in response to A. flavus (Fig. 4). Functional annotation results showed that 139 of these genes were defense related genes (Table 1). The majority of defense related DEGs (132 out of 139) were differentially expressed in both peanut varieties, while only seven of the DEGs were differentially expressed only in one peanut variety in response to A. flavus.
Chitin is a key component of fungal cell wall and is considered as an essential factor for maintaining the pathogenicity of the fungus. Chitinases are hydrolytic enzymes that have the activity of degrading chitin. Accumulating evidence indicated that the activity of chitinase was important for plant resistance against pathogens infection [50,51,52], and some chitinases were pathogenesis related (PR) proteins [53,54,55]. We observed that the expression of a chitinase gene (Araip.8C3IU.1) had no change in Tifrunner but was induced in GT-C20. In addition, our data showed that two peroxidases (Aradu.BNR06.1 and Araip.595JK.1) and two copper amine oxidases (Aradu.NCJ0H.1 and Araip.26B5V.1 were up-regulated in GT-C20 but no significant expression difference in Tifrunner (Table 1). Interestingly, peroxidase and blue copper protein genes were also disease related genes [56, 57]. In plant, peroxidases were involved in plant disease resistance response through participating biosynthesis of bactericide and lignin. Copper amine oxidases contribute to catalyze the oxidative de-amination of polyamines and producing hydrogen peroxide (H2O2). In plants, H2O2 derived from polyamine oxidation mediated cell death and was correlated with stress response to pathogen invasion [58, 59]. Plant cell walls were considered as the first security system barrier of defense against bacterial and fungal pathogen. Pectin is an important component in plant cell wall. In plants, pectinesterase/pectinesterase inhibitor acts to modify the cell walls via degration of pectin. In our dataset, two pectinesterase/pectinesterase inhibitor genes (Aradu.S6DQM.1 and Araip.EQZ9W.1) were significantly depressed in Tifrunner but no significant expression difference in response to A. flavus (Table 1). Here, the difference expression trend of these genes in resistant and susceptible peanut varieties provided us a useful clue to reveal the molecular mechanism of peanut response to A. flavus.
Among these 139 defense related DEGs, 18 genes encode cytochrome P450 superfamily genes (Table 1). As a multi-functional gene, the function of cytochrome P450 in defense reaction were revealed in many plants [60, 61]. In rice, over-expressing of CYP71Z2 enhanced resistance to bacterial blight. Further studies confirmed that cytochrome P450 gene contributes to enhance rice disease resistance to pathogens both through mediating diterpenoid phytoalexin accumulation and via suppression of IAA signaling in rice .
NBS-LRR genes represent one of the largest disease resistance gene families in plants . So far, the majority of the disease-resistant genes identified by map-based cloning were NBS-LRR genes, such as resistance to rice blast , powdery mildew resistant genes Pm3/Pm8 in wheat , leaf rust resistance gene Lr10 in wheat . We found two NBS-NBS-LRR type disease resistance genes (Aradu.168 L7.1, Araip.VGW7F.1) were induced in both peanut varieties. However, the increased expression level of the two genes were more dramatic in resistant genotype than that of susceptible genotype peanut. For example, the expression level of Aradu.168 L7.1 was almost equal in both varieties in control samples (log2TC/GC = 0.11). In response to A. flavus, the expression of this gene was induced in both resistant (log2GT/GC = 3.28) and susceptible peanut (log2TT/TC = 0.68) genotype peanuts. After treatment with A. flavus, the expression of this Aradu.168 L7.1 in resistant genotype was significantly higher than that in susceptible genotype (log2GT/TT = 2.72). Araip.VGW7F.1 had the same expression patterns with that of Aradu.168 L7.1, which was induced in both peanut varieties but were more dramatically induced in resistant genotype (log2GT/TT = 2.84). Aradu.168 L7.1 and Araip.VGW7F.1 located in chromosome A05 (1880820–1,893,586) and B05 (1716985–1,720,472) of peanut, respectively, and were considered as orthologous genes. Interestingly, sequence alignment results showed that Aradu.168 L7.1 (Expect: 5e-146, Identities: 316/943(34%) and Positives: 506/943(53%)) and Araip.VGW7F.1 (Expect: 2e-119, Identities: 292/931(31%) and Positives: 490/931(52%)) were all homologous with the disease resistance locus, RPM1, of Arabidopsis. The RPM1 locus conferred resistance to the bacterial pathogen Pseudomonas syringae in Arabidopsis [67, 68] and was supposed to have functional conservation in bean, pea and other crop species .
Besides, other several defense related genes also showed differential expression profile between two different peanut genotypes including two cellulose synthase like E1 genes, two WRKY family transcription factor genes, two lipase/lipooxygenase (LOX) genes and one indole-3-acetic acid inducible 2 gene (Table 1). WRKY represents one of largest transcription factor gene families. Some WRKY gene members have been reported in regulating response to various biotic and abiotic stresses. Here, we found that two WRKY genes, Aradu.S7YD6 and Araip.RC4R7, located on chromosome A06 and B06, respectively, were homologous with WRKY33 genes of other plants. In Arabidopsis, WRKY33 was required for resistance to necrotrophic fungal pathogens. In our dataset, the above two WRKY genes were up-regulated in Tifrunner but remained unchanged in GT-C20 which seemed to be the opposite of what was expected in other plants. In addition, we found that two cellulose synthase like E1 genes (Aradu.BZH82, Araip.FL29A) were up-regulated in Tifrunner but did not showed significant differential expression in GT-C20 in response to A. flavus. Cell wall is an important barrier for plants against fungi which also seemed to be the opposite of what was expected in other plants. Previous studies have showed that lipoxygenase pathway was associated with the seed resistance against A. flavus in soybean and maize. For example, inactivation of the ZmLOX3 could increases susceptibility of maize to Aspergillus. Here, two other lipase/lipooxygenase (LOX) genes, Aradu.MAS03 and Araip.081EX were down regulated in the Tifrunner but unchanged in GT-C20. In addition, indole-3-acetic acid inducible genes were involved in response to auxin stimulus and lateral root morphogenesis in other plants. Here, we found that an indole-3-acetic acid inducible 2 gene (Araip.AN5V8.1) was upregulated in the resistant variety and downregulated in the susceptible.
Regulation of miRNAs in peanut response to A. flavus
Due to the sequence similarity, miR2118 was considered as member of miR482 superfamily [34, 70]. Emerging evidence indicated that miR482 and miR2118 both target the P-loop sequence motif of NBS-LRR defense genes in plants, and played a negative regulatory role in plant response to diseases . In soybean, constitutive expression of miR482 led to enhance degradation of targeted R genes, resulting in increased nodulation . In potato, the expression of miR482e was down-regulated in response to Verticillium dahliae infection which resulted in the up-regulation of its NBS-LRR targets. Overexpression of miR482e induced the decrease expression of its NBS-LRR targets, and the transgenic plantlets were highly susceptible to V. dahlia infection . In our datasets, one member of miR2118 and three members of miR482 were identified. We found the overall expression level of miR2118, miR482a, miR482b and miR482b were lower in resistant genotype than in susceptible genotype (Table S3). For example, in resistant genotype, the expression abundance of miR2118 was 74.10 and 91.53 TP10M in mock and treatment samples, respectively. While in susceptible genotype, the expression abundance of miR2118 was 170.22 and 261.62 TP10M in mock and treatment samples, respectively (Table S3). Interestingly, peanut miR482/2118 miRNA family also target NBS-LRR defense genes including Aradu.168 L7, which has the higher expression level in resistant genotype than that in susceptible genotype.
In this study, 66 potential miRNA/targets pairs were identified through a combined analysis of the datasets of small RNA, degradome sequences and mRNA expression profiling. Among them, many SPLs (SQUAMOSA promoter-binding protein-like) might be regulated by miR156/157 miRNA family (Table S12–13). For example, Aradu.0GH1S.1 encodes a SPL transcription factor, which is homologous with SPL12 of soybean (LOC100781289) and SPL2 of Arabidopsis (AT5G43270). Degradome sequencing showed that Aradu.0GH1S.1 was potentially cleaved by many miRNA members of miR156/157 family, such as miR156a, miR156e and miR156i, etc. (Table S7). Previous evidences showed that SPL negatively regulated the accumulation of anthocyanin. And further studies proved that miR156/SPL regulated the secondary metabolism and the content of anthocyanin and flavonol in plants . Both anthocyanin and flavonol were important flavonoids. Flavonoids have been shown to have antimicrobial properties against invading microorganisms including A. flavus . In this study, the expression level of Aradu.0GH1S.1 in susceptible genotype was significantly higher than that in resistant genotype, which might be caused by the regulation of miR156. In addition, GO and KEGG results showed that the GO term and pathway related with secondary metabolism of flavonoids was enriched between two peanut varieties. These results provided valuable information for understanding the roles of miR156/SPL in peanut.
Our study reported the integration analysis of the transcriptome, miRNAome and degradome of resistant and susceptible peanut varieties in response to A. flavus. A total of 30 differentially expressed miRNAs, 447 differentially expressed genes and 21 miRNA/targets pairs were identified in response to A. flavus. In addition, a total 62 differentially expressed miRNAs, 451 differentially expressed genes and 44 miRNA/targets pairs were identified between resistant and susceptible peanut varieties. Furthermore, the function of two miRNA/targets regulation pairs were discussed including miR482/2118/NBS-LRR and miR156/157/SPL (Fig. 8). Our study generated a comprehensive dataset for further understand the roles of miRNA in response to A. flavus in peanut.
Plant material and treatment condition
Two cultivated peanuts, including GT-C20 and Tifrunner, were obtained from Shandong Center of Crop Germplasm Resources, Jinan, China, and the voucher specimens were deposited in the Shandong Center of Crop Germplasm Resources. The resistance of GT-C20 and Tifrunner to A. flavus has ever been reported in previous studies . The A. flavus strain (AF-Wh2014) isolated from peanut was kindly provided by lab of Prof. Boshou Liao, Oil Crops Research Institute of Chinese Academy of Agricultural Sciences. The method for evaluating the resistance of the two peanut varieties in response to A. flavus referred to the previous studies and was improved in this study [9, 10, 37]. Briefly, A. flavus was cultured in fresh potato dextrose agar (PDA) medium at 30 °C for 7 d. The conidia were then collected and suspended into spores suspension (106 CFU/ml) with sterile distilled water. For inoculation with A. flavus, peanut seeds were surface sterilized with 70% alcohol for 1 min, and 3% sodium hypochlorite (NaClO) for 10 min. Then, the seed coat was wounded with an area of 6 mm × 5 mm. The wounded area was covered with the same size of a piece of cotton, and 20 μl of spores suspension was added on the cotton. For the control sample, 20 μl sterile water was added. To keep the humidity, the infected seeds were placed in the petri dish with two layers of filter paper wet with 5 ml sterilized water. Finally, seeds were placed into incubator at 30 °C in darkness. Evaluation of peanut resistance to A. flavus was performed as described in a previous study . Infection index was calculated based coverage of A. flavus mycelium in the testa of peanut seed. Peanut interaction with A. flavus was evaluated using the following criteria: high resistant (infection index < 5.0), moderate resistance (infection index ≥5.0 and < 10.0), moderate susceptible (infection index ≥10.0 and < 30.0), susceptible (infection index ≥30.0 and < 50.0).
Libraries construction and deep sequencing
Seeds of GT-C20 and Tifrunner were inoculated with A. flavus for 2 d for small RNA, transcriptome and degradome libraries construction. Un-inoculated seeds were used as control. For each sample, three individually biological replicates were prepared. Total RNA was isolated using Trizol Reagent (Invitrogen, USA) according to the manufacturer’s protocol. RNA quality and integrity was evaluated using electrophoresis on a 1% agarose gel and BioSpectrometer fluorescence (Eppendorf, GER). Then, the total RNAs were used for constructing small RNA, transcriptome and degradome libraries using the methods as described in previous studies, respectively [35, 36, 40, 77]. In brief, for constructing the transcriptome library, the total RNA was purified to obtain the mRNA using oligo (dT) magnetic beads. Then, the mRNA was fragmented into short fragments (about 200 bp) and further used for synthesizing the first strand of cDNA using random hexamer-primer. Next, the second-strand cDNA was synthesized using the buffer, dNTPs, RNase H and DNA polymerase I and then ligated to sequencing adapters. Following the cDNA fragments are enriched by PCR amplification. Finally, the library products are ready for sequencing. For constructing small RNA library, the total RNA was first used for purifying of small RNA molecules (18–30 nt) and then ligated of a pair of adaptors to 5′ and 3′ ends. Following, the sequencing library was prepared through reverse transcription and PCR amplification. For constructing degradome library, following steps were performed. First, mRNA was used as input RNA and annealing with Biotinylated Random Primers. Second, Strapavidin capture of RNA fragments through Biotinylated Random Primers. Third, 5′ adaptor ligation to only those RNAs containing 5-monophosphates. Finally, Reverse transcription and PCR amplification were performed. Before sequencing, the QC step was performed to qualify and quantify of the sample library using Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System. High-throughput sequencing of small RNA, transcriptome and degradome was performed in BGI (Shenzen, China) using Illumina HiSeq2000 platform.
MiRNA identification and bioinformatics analysis
Firstly, raw reads were cleaned up by removing low quality reads, 5′ primer contaminants, reads without 3′ primer, reads with poly A, and reads shorter than 18 nt. The clean reads were aligned by BLAST against Rfam database (version 11.0, http://rfam.janelia.org/) [78, 79] and GenBank noncoding RNA database to remove rRNA, scRNA, snoRNA, snRNA, and tRNA. Then, the rest of reads were aligned with mature miRNAs in miRbase (Version 21, http://www.mirbase.org/) [80, 81] to identify the known miRNAs. Novel miRNAs were identified according to the reported method . The clean reads were first mapped to whole genome sequences of A. duranensis and A. ipaensis (https://peanutbase.org/). Then, the hairpin structures of miRNA precursors were predicted using software Minreap (http://sourceforge.net/projects/mireap) according to the following parameters. The candidate sequences could form a proper secondary hairpin, where mature miRNAs mapped region was with a size of 18–25 nt, and the distance between miRNA and miRNA* is 16–300 nt. The maximal free energy allowed to form a hairpin structure is − 18 kcal/mol . The gene expression level of miRNAs from different sRNA libraries was normalized using TP10M (tags per ten million reads). Differentially expressed miRNAs between different samples were identified using R package DESeq (http://bioconductor.org/packages/DEGseq/) under the criteria of adjusted p value (padj) < 0.05 and fold change ≥1.5 .
Degradome sequencing and data analysis
To identify the potential targets regulated by miRNAs, equal amounts of RNAs from control (TC1, TC2, GC1 and GC2) and treatment samples (TT1, TT2, GT1 and GT2) of resistant and susceptible genotypes were mixed together for degradome library construction and deep sequencing. Through preprocessing, clean tags are generated. Then, clean tags were classified by alignment with Genbank, Rfam database, and miRNA database. Next, the reads were mapped to the transcriptome data of A. duranensis and A. ipaensis using SOAP2 program with allowing only two mismatches . The transcriptome data of A. duranensis and A. ipaensis were downloaded from peanutbase (https://peanutbase.org). The sense strand of peanut cDNA was used to predict miRNA cleavage sites using CLeaveLand pipeline . Based on the signature number and abundance of cleaved position at each occupied transcript, the cleaved transcripts could be categorized into five categories (0, 1, 2, 3 and 4) .
Transcriptome sequencing and data analysis
To analyze the expression profiles of the target genes, eight independent transcriptome libraries were sequenced. After sequencing, the adaptor sequences, low-quality reads, and empty reads were first removed. Then, the clean reads were aligned with the genome sequences of A. duranensis and A. ipaensis (https://peanutbase.org/) using SOAP2 program . The gene expression level was calculated using FPKM (expected number of fragments per kilobase of transcript sequence per millions base pairs sequenced) method. The relative gene expression level between different samples was calculated using log2 ratio method. Differential expression genes (DEGs) between two samples were identified with the criteria of probability ≥0.8 and |log2(fold change)| ≥ 1 using NOIseq method . To identify the putative biological functions and pathways of the target genes and DEGs, Gene Ontology (GO) annotation and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis were conducted as described previously [77, 86, 87].
Expression validation of miRNA and mRNA using qRT-PCR
To validate the high-throughput sequencing results of miRNAs and target genes, quantitative RT-PCR (qRT-PCR) was performed on ABI 7500 Real-Time PCR System (Applied Biosystems). Three biological replicates were prepared for each sample. For miRNA expression analysis, stem-loop RT-PCR method was used according to the method as described previously . For target genes, primers were designed using primer premier 5.0 software (www.premierbiosoft.com). All primers including the stem-loop miRNA primers, reference genes and targets genes were listed in Additional file: Table S14. The relative expression level between different samples was calculated using the 2-△△Ct method. Student’s T-test was performed to analyze the changes in gene expression after treated with A. flavus (*P < 0.05, **P < 0.01).
Availability of data and materials
The RNA-seq data in this study were available at NCBI BioProject: PRJNA438019 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA438019).
Kyoto Encyclopedia of Genes and Genomes
Nucleotide-binding site leucine-rich repeat
SQUAMOSA promoter binding protein-like
Differentially expressed genes
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The authors thank Prof. Boshou Liao and Prof. Yong Lei (Oil Crops Research Institute of Chinese Academy of Agricultural Sciences) for providing the A. flavus strain (AF-Wh2014).
This work was supported by the Natural Science Foundation of China ; Shandong Province Germplasm Innovation and Utilization Project [2016LZGC025], Shandong Province Natural Science Foundation [ZR2017MC005, ZR2015YL061, ZR2015CQ012 and ZR2018BC029], Shandong Provincial Key Research and Development Program (2019GNC106026 and 2019GSF107008) and Taishan Scholar Project of Shandong Province (ts20190964); Agricultural Scientific and Technological Innovation Project [CXGC2018E13, CXGC2016B02] and Young Talents Training Program of Shandong Academy of Agricultural Sciences. The founders did not play any roles in the design, analysis, and interpretation of this study or relevant data.
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Distinct RNA fragment categories in each library.
Comparison of sequencing and qRT-PCR results for the miRNAs and genes using heatmap. Heatmap was generated by online software Morpheus (https://software.broadinstitute.org/morpheus) according to the relative expression in comparison with the control sample TC.
Volcano plots showing the gene expression differences among different samples. The RPKM-normalized transcript count data sets were analysed by NOIseq: the x-axis shows the log-ratio (gene expression fold change after challenge) the y-axis the probability for each gene of being differentially expressed. The up-regulated and down-regulated genes were identified under the criterion of probability ≥0.8 and |log2(fold change)| ≥ 1.
Summary of small RNA reads from the individual libraries.
Statistical analysis of total sRNA mapped in peanut genome.
Known miRNA information in two peanut varieties.
Information of novel miRNAs identified from peanut miRNA libraries.
Classification analysis of reads from degradome library.
Categories of candidate cleaved sites.
Target genes of known miRNAs identification by degradome sequencing.
Target genes of novel miRNAs identification by degradome sequencing.
Summary of RNA-seq reads from the individual libraries.
Top sixteen KEGG pathways in Tifrunner and GT-C20 in response to AF infection.
Top sixteen enriched KEGG pathways between Tifrunner and GT-C20.
Correlation analysis of miRNAs expression profiles and their target genes in response to A. flavus.
Correlation analysis of miRNAs expression profiles and their target genes between peanut varieties.
Primers used in this study.
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Zhao, C., Li, T., Zhao, Y. et al. Integrated small RNA and mRNA expression profiles reveal miRNAs and their target genes in response to Aspergillus flavus growth in peanut seeds. BMC Plant Biol 20, 215 (2020). https://doi.org/10.1186/s12870-020-02426-z


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Ever since the release of Man of Steel back in 2013 many DC fans have been clamoring for a movie centralized around Lobo, a crude, loudmouthed intergalactic bounty hunter. It looks like they are going to get their wish! However, their director choice may stir up some controversy.
According to a story put out by The Wrap, Transformers director Michael Bay is being eyed to help the project. This would be Bay’s first superhero movie.
For the most part the internet has reacted negatively to Bay’s involvement once this news was released. The director has created good to great films in the past such as The Rock, Armageddon, and 13 Hours: The Secret Soldiers of Benghazi. However, the stench of the Transformers franchise is all fans can think about. Those films do make money, but are consistently critically panned, and have gradually become detested by audiences with each installment.
As of now Warner Brothers has not confirmed The Wrap’s story, but the film does fall within the director’s wheelhouse, so it’s probably true.
As of now the Lobo film does not yet have a release date.
Over the past several weeks there has been nothing but dreary news coming from Warner Bro. concerning their DC Universe. However, finally, this weekend has given way to some positivity!
A new trailer and poster for DC’s Wonder Woman has hit the web, showing off the Princess of the Amazon warriors, Diana, training on Themyscira. Check out the trailer and Poster below!
In previous trailers, we have seen Diana taking part in World War One, however this new trailer shows more of her training and honing her powers. In just this one trailer we see her as a young girl learning about her history, discovering her powers, and then her journey to London in order to save the world. This film is directed by Patty Jenkins, and stars Gal Gadot as Wonder Woman and Chris Pine as Steve Trevor who, in the comics, is Diana’s love interest.
This past Friday Jenkins unveiled the new poster for Wonder Woman for the film via her twitter account:
Wonder Woman is set to hit theaters this year on June 2nd, 2017.
While things lately have seen down for the DC Movie Universe, I have never given up hope that Patty Jenkins will give us a great Wonder Woman movie. And after seeing this trailer, I am very much looking forward to it.
Its still a few months before Harley Quinn’s big screen debut in Suicide Squad, however actress Margot Robbie seems to have impressed Warner Bros. enough to begin talking with her about a Harley Quinn movie.
According to a report that comes from The Hollywood Reporter, Margot Robbie is now attached to reprise her role as the character in the future as well as produce the currently untitled spinoff.
But don’t start screaming, “Harley Quinn solo film!!!” because that is not the case.
Although details are still being very closely guarded, the rumored project is not going to be a solo film, but rather a film that focuses on several of DC’s female heroes and villains. Maybe involving Batgirl or Birds of Prey?
I am all for this! The DC hero and villain lineup has a very strong female base. Characters like Harley Quinn, Batgirl, Poison Ivy, Katana, etc. I really hope this becomes a reality and that we get more of Robbie’s Harley Quinn because everything I have seen so far from the trailers show that she kills it as the psychologist turned psycho.

domain: entertainment


text: The development of strong social and emotional skills is central to personal wellbeing. Increasingly, these skills are being taught in schools through well researched curricula. Such social-emotional learning (SEL) curricula are most effective if reinforced by parents, thus transferring the skills into everyday contexts. Traditional SEL programs have however had limited success in engaging parents, and we argue that technology might be able to help bridge this school-home divide. Through interviews with SEL experts we identified central design considerations for technology and SEL content: The reliance on experiential learning and the need to scaffold the parents in scaffolding the interaction for their children. This informed the design of a technology probe comprising a magnet card and online SEL activities, deployed in a school and via Mturk. The results provide a nuanced understanding of how technology-based interventions could bridge the schoolhome gap in real-world settings and support at-home reinforcement of children's social-emotional skills.

domain: psychology


text:
I met her again after a couple of decades, yesterday. There in the open bay of her studio in Santa Fe on this gloriously warm and sunny morning in late October – the unseasonably lovely weather was providing an extended opportunity to enjoy being connected to the out-of-doors while working inside the cozy fire-pit. Yet, I imagine that there are many days when chilly outside that the crisp contrast is a welcome refresher to the intensity of the fire’s heat generated inside. But today was perfection.
Brilliant glass pumpkins of various colors of colorfully translucent glass were gathered in a welcoming autumnal display, catching the sunlight on the table out front. Piled beside them by surreal contrast were real, fresh-picked, enormously miss-shaped, world-class, organic deliciously edible pumpkins grown by a local champion.
Stepping through the large garage door opening revealed massive metal furnaces glowing and humming. The artist, her back to us was interacting with one of the units. As we watched, she thrust the rod into the intensely orange aperture twirling it gently with her fullest attention.
She then retracted the rod now displaying a wad of glowing goop and turned toward us to sit on her bench and continue to twirl the rod, grabbing the orb with clamps, pressing and twirling, to create before our very eyes, a perfect paperweight complete with inner swirls of orange and red. It’s like a being in a candy factory, molten glass like taffy is gooey and proves a most challenging medium.
Elodie Holmes has mastered the art of manipulating molten glass. After nearly 3 decades of owning her own studio, she has worked toward achieving perfection in her medium. Her work is sought by collectors as they wait anxiously for the completion of the firings that present her spectacular one-of-a-kind dimensional sculptures. As we spoke, I remarked that this reminded me of my first experience watching glass blowing in Williamsburg as a child. She looked up and said “me too!” And we discovered that we had both come from the Washington DC area and had transplanted to New Mexico’s Land of Enchantment decades ago pursuing art and an incredible interaction with the beauty of nature uniquely found here.
As an interior designer, seeing her work and watching the fascinating process, I appreciate the art of it all, the decorative sophistication of color and form and attention to detail required achieving the results – and the satisfaction of both the artist and her fans realizing those astonishing results!
I found Elodie in her studio today as a result of super sleuthing for a set of drinking glasses that I had enjoyed while visiting a friend. After a couple of phone calls, I traced her studio and spoke to her gallery manager, Marci. I stressed that I was looking for drinking vessels and she assured me that they offered some exquisite interpretations. Upon arrival, as I walked from the studio space into the small gallery/retail space, I saw what I was remembering…there they were – be speckled glasses tall and short, twisted and bulbous – their shapes are fun, comfortable to hold and colorful to delight.
Proudly, I bring these uniquely beautiful little glasses to our boutique gallery at PATRICIAN DESIGN to offer Elodie’s work among our other fine New Mexico artists. I love living with art. I love using art functionally. So always attracted to things that bring art to daily use, I am continually on the hunt for those special pieces of functional art – here now these beauties allow drinking by design.
See more at the studio tour: http://www.facebook.com/media/set/?set=a.10151069695516619.421546.232272436618&type=1


by Isaac Davis
If you've ever been to
the Vatican, which is actually a sovereign nation, you've seen the
unbelievable wealth owned by the Catholic Church, but how much is
the Vatican really worth?
The Rothschild family is believed to hold some five times more than the combined wealth of the world's 8 top private billionaires, but when it comes to the Vatican, it's much more difficult to assess its overall wealth.
In fact, it's actually practically impossible to pin down the Vatican's wealth, as the sum total depends greatly on how you add up their assets and affiliations with churches around the world, and also on who you ask.
For example, a 1987 investigation into the Vatican's finances by Forbes magazine, claims that the Vatican was nearly broke, spending almost twice as much annually as it brought in.
This is hard to fathom when you consider the sheer material extravagance held within the property itself which features thousands of priceless works of art and gold.
The Vatican is composed of two major entities:
As estimated by Forbes, Vatican wealth is nominal, merely in the hundreds of millions when factoring in the entity's investments and various funds.
The Holy See is,
Employing several thousand laborers tasked with maintaining and securing the premises, as well as members of the clergy, the Holy See serves as the central point for the Catholic Church around the world.
While labor costs are high, the Vatican maintains that its top members still work for 'peanuts.'
Fast forward to 2015, and a CNN Money analysis of the Vatican Bank suggests that the bank holds some $8 billion plus in assets, a number which includes the personal savings of many Vatican employees.
Looking at Vatican wealth on paper is also quite misleading.
Time Magazine ran this assessment of Vatican wealth in 1965, offering an estimate of total wealth, which at that time reached into the tens of billions of dollars.
In other words, estimates are all over the place, and contemporary reporting on this subject is rather slim.
Another dimension to this is the sheer value of all of the priceless works of art housed in Vatican City. Estimating the value of all of this in terms of dollars is impossible, but looking at just one well-known prize, the Sistine Chapel, offers a look at how difficult it is to appraise all of this.
Estimates range from $400 billion all the way up to $2 trillion for just this one masterpiece.
Regarding real estate values of properties held by the Vatican, a 2004 estimate claimed the number was just under a billion dollars.
In short, no one really knows how much wealth the Catholic Church controls, and the organization's secrecy and obfuscation of the facts surrounding its wealth continues to lead investigators on a wild goose chase.
The Vatican's cash-flow is in the hundreds of millions a year, individual holdings in the Vatican Bank total perhaps $15 billion, property held by the Vatican may be worth over a billion dollars, and the Church owns the largest store of the world's most priceless art...


Industrial videoscope VSC206
Always in the picture when it comes to indirect visual inspections
The industrial videoscope VSC206 unites wireless operation with razor-sharp videos and digital images in a mobile, user-friendly compact system, providing simple access to the test object.
The VSC206 features a robust, protector-equipped ABS housing providing equal protection from impact damage and the infiltration of dust or splashing water. Ideal prerequisites for the rough conditions of industrial applications.
Clear vision guaranteed – even in a tight corner
The probe of the VSC206, bendable to all sides, can be steplessly controlled via the joystick and with its titanium-coated camera head enables perfect panoramic views of the inspection area.
Its multi-ply construction of stainless steel wire mesh and PU on a flexible steel spiral ensures maximum range of motion with simultaneously high torsional strength of the push probe.
6 white LEDs with variable brightness control ensure sharp, detailed images that can be shown on the brilliant VGA display of the VSC206 and saved for documentation purposes either as video or individual image on an SD card.
All videoscopes in direct comparison:
To find the videoscope which exactly meets your requirements, please consult the concise overview of all videoscopes from Trotec which we're providing to you here so that you can compare them directly to each other.
Models which you do not wish to include in your comparison can be easily dismissed with only one click.
A practical benefit:
- Development, design, production: 100 % Trotec
- Hand-held, light and robust compact system
- Camera head infinitely bendable to all directions
- LED technology – less power consumption and heat production than with conventional lighting systems
- Recording photos and videos
- VGA LCD display for an outstanding image quality
- Robust, multi-walled probe for maximum flexibility with simultaneously high torsional strength
- Fast USB connection and analogue video output
- Easy handling
|Size||3.5 inch (640 x 480 pixels)|
|Internal (battery)||4 x 1.5 V, AA or 4 x 1.2 V Ni-MH battery|
|External (power adapter)|
|Via joystick control unit|
|Operation - min. temperature [°C]||-10|
|Operation - max. temperature [°C]||50|
|Operation – min. relative humidity [% r.F.]||0|
|Operation - max. relative humidity [% r.F.]||90|
|Type of protection probe with measuring head|
|SD card||Max. 32 GB|
|Internal flash [MB]|
|USB (type mini, 5-pin)|
|3.5 mm jack (video/audio in/out)|
|Video out (composite video)|
|Plastic - ABS|
|Length (packaging excluded) [mm]||265|
|Width (packaging excluded) [mm]||100|
|Height (packaging excluded) [mm]||125|
|(packaging excluded) [kg]||0.59|
|Standard scope of delivery|
|Standard scope of delivery|
|SD memory card (8 GB)|
|4 x 1.2 V Ni-MH battery|
|0.9 m probe / Ø 17 mm|
|1 m probe / Ø 17 mm|
|3.7 V Li-ion battery|
|Ascertainable measured values and functions|
|Functions and features|
|Multilingual on-screen menu|
|Fixed focus lens|
|Shooting and saving individual images and complete video sequences|
|Image rotation function 4 x 90°|
|Image rotation function 180°|


text:
I am an assistant professor of applied statistics and data science in the School of Mathematical and Statistical Sciences. My research interests include Bayesian statistical methodology, mixture models, clustering, hierarchical models, and applications in social sciences and human genetics. I am accepting Ph.D. students to work in these areas.
School of Mathematical and Statistical Sciences
Sianko, N. and Kunkel, D. “Longitudinal Patterns in Adolescent Intentions to Seek Help for Dating Violence: A Latent Transition Analysis,” Journal of Youth and Adolescence 51, 673-693 (2022). doi: 10.1007/s10964-022-01569-6.
Kunkel, D., Stuenkel, M., Sivaraj, L., Colenda,C., Pekarek, L., and Rennert, L. “Predic- tive value of clinical symptoms for COVID-19 diagnosis in young adults” (2022). Journal of American College Health. doi: 10.1080/07448481.2022.2068963.
Liu, Z., Zhang, Z., Kunkel, D., Singapogu, R. “Is Experience in Cannulation Re- lated to Expertise? A Metrics-based Investigation for Skills Assessment,” Annals of Biomedical Engineering, 49, 1688-1700 (2021). doi: 10.1007/s10439-020-02708-5.
Kunkel, D., Yan, Z., Peruggia, M., Craigmile, P.F, and Van Zandt, T. Hierarchical hidden Markov models for response time data. Computational Brain & Behavior (2020). Full text online. Supplemental material.
Dominic, J., Tubre, B., Houser, J., Ritter, C., Kunkel, D., Rodeghero, P. Program Comprehension in Virtual Reality. Proc. of the International Conference on Program Comprehension (ICPC'20 ERA), Seoul, Korea, May 23-24, 2020.
Sianko, N., Kunkel, D., Thompson, M.P, Small, M.A., McDonell, J.R. Trajectories of Dating Violence Victimization and Perpetration among Rural Adolescents. Journal of Youth and Adolescence, 1-17 (2019).
Byrne, K., Willis, H., Peters, C., Kunkel, D., Tibbett, T. Behind Closed Doors: The Role of Depressed Affect on Risky Choices Under Time Pressure. Clinical Psychological Science, 8(1), 198-207 (2019).
Kunkel, D. and Peruggia, M. Statistical inference with anchored Bayesian mixture of regressions models: A case study analysis of allometric data. arXiv, (2019).
Kunkel, D., Potter, K., Craigmile, P.F, Peruggia, M., and Van Zandt, T. A Bayesian race model for response times under cyclic stimulus discriminability. Annals of Applied Statistics, 13, 271-296 (2019).
Kunkel D. and Kaizar E. A comparison of existing methods for multiple imputation in individual participant data meta-analysis. Statistics in Medicine, 36, 3507-3532 (2017).
MATH 4000/6000, Theory of Probability, Summer 2022.
MATH 4030/6030, Introduction to Statistical Theory, Spring 2022.
STAT 8030, Regression and Least Squares Analysis, Fall 2021.
STAT 8020, Statistical Methods II, Summer 2021.
MATH 8820, Intro to Bayesian Statistics , Fall 2020, Fall 2022.
MATH 4820, Undergraduate Research , Fall 2020.
DSA 8010, Statistical Methods I , Fall 2020, Fall 2021, Fall 2022.
STAT 8010, Statistical Methods I, Fall 2018, Spring 2019, Fall 2019, Spring 2020.
STAT 8420, Intro to Statistical Methods, Summer 2019.
STAT 4020/6020, Statistical Computing , Fall 2018.

domain: literature


Woman wins appeal to stay in US hours after giving birth to 6th child
CHARLOTTE, NC (WBTV) - A Mt. Holly mother gave birth to her sixth child Thursday morning, then appeared at her deportation hearing just a few hours later.
Isaide Serrano, 39, won her cancellation of removal – that means she will be allowed to stay in the United States.
Tearful family and friends surrounded Serrano as she gingerly walked out of court.
Wiping away tears, her daughter Cinthya Serrano said "we're all really happy".
Serrano now has six children who are U.S. citizens, but she came to the country illegally as a 19-year-old.
The government now has 30 days to appeal the decision.
Serrano's lawyer says "the judge determined there was the required amount of hardship to grant the case".
Attorney Chris Greene says the hardship "was her children. One of her children has a medical condition that requires surgery. In the aggregate the Judge determined that that was exceptional, extremely unusual - which is required in these cases".
If the government does not appeal, Serrano will have permanent resident status and receive a green card in the mail.
Serrano gave birth to a boy at 5:08 a.m. and had to be medically cleared to appear at her hearing in U.S. Immigration Court just four hours later.
During an interview in her hospital room at Caromont Health Gaston Memorial Hospital - and through a hospital interpreter - Serrano said she was in a lot pain and could barely walk after delivering baby boy, Jayden.
She was due in court at 10a - but at 8:30a she couldn't change her clothes. Serrano says the nurses helped her get ready.
Serrano says while the post delivery pains were tough, nothing compared to the agony of leaving her newborn. "It was difficult because I couldn't have my baby with me", she said.
Serrano made it to Immigration Court in Charlotte on time. She says the last two years of waiting for a final decision about deportation were "very hard". And heading into court "she was thinking about what was going to happen".
Attorney Greene says the case was very close. "For this judge - he was right on the fence and I think what really tipped him over the edge was her and her presence here today in spite of giving birth hours ago. That was very impressive to him".
After her appearance before the Immigration Judge, Serrano had to go the Immigration office to present the judge's ruling. Then it was back to the hospital - where doctors and nurses are monitoring her and her newborn.
Serrano says "she feels very happy about the decision the judge made and because now she's back again with her son and other children".
Copyright 2012 WBTV. All rights reserved.


text:
It’s the twins’ 1st Christmas, and what better way to commemorate the event than with this delicately glittered keepsake featuring
two peas in a pod! Our talented personalizers will hand write the names, year and sentiment of your choice at no extra cost.
- Measures 4″
- Crafted from glossy resin
- Includes a satin ribbon for hanging

domain: design


text:
According to the latest research from Counterpoint’s Market Monitor service, the smartphone shipments in India grew a healthy 20% annually and 12% sequentially in Q3 2015 (July-Sept). More than one in three mobile phones shipped in India is now a smartphone.
The Indian mobile phone market demand is back on track after declining for two consecutive quarters fueled by cheaper smartphones as more and more feature phone users quickly upgrade to smartphones for the first time.
Market Summary for Q3 2015:
- Smartphones: More than One in Three mobile phones shipped were smartphones
- Online Sales: One in Three smartphones sold were through online channels such as Flipkart, Amazon, Snapdeal and so forth
- 4G LTE Smartphone: One in Three smartphones shipped were LTE smartphone
- Make in India: One in Four mobile phones sold during the quarter was “Made in India”
- Samsung continued to lead the overall mobile phone market and the smartphone segment during the quarter with market shares of 19.0% and 23.2% respectively
- Samsung benefitted from expanding its critical mid-tier portfolio with launch of J series during the quarter which drove smartphone volumes for the vendor
- However, the Korean vendor lost share sequentially with minor presence in sub $100 smartphone segment which is fastest-growing and dominated by Indian and Chinese brands
- Samsung’s A series brought in a welcome design change for Samsung with thinner and flatter designs in the mid- to high tier becoming the ‘halo devices’ taking focus away from S6 series. This design language is also trickling down to the new J series helping to strengthen its position in this segment. Additionally, strong offline distribution has also helped Samsung drive J series shipments during the quarter
- Though Samsung led the LTE smartphone segment as most of the mid-to high-tier Galaxy models are LTE ready.
- Micromax maintained the second position in both overall mobile phone market and the smartphone segment with a market share of 13.7% and 17.7% respectively.
- The brand’s share from online channel increased during the quarter. However, it is now facing pressure competition in $50-$100 price segment from Intex, Lava and others.
- Micromax’s Cyanogen based online only brand ‘Yu’ launched its cheapest LTE model ‘Yunique’ during the quarter and the brand has been growing steadily ever since its launch.
- Micromax Yu brand alone is now selling more smartphones than Xiaomi online, depicting it’s going to be challenging even for popular brands such as Xiaomi to scale in Indian market
- Continuing its momentum from Q2 2015, Intex cemented its position in top three brands in India third spot in both overall and smartphone segment.
- Intex’s smartphone market share jumped to double digit level for the first time ever capturing close to 12% of the smartphone market during the quarter.
- Demand for Intex products in the fastest growing sub-$100 smartphone price band remains strong which is the key reason for its growth. However, the vendor needs to improve its position in “affordable premium” segment.
- Intex also expanded its LTE portfolio during the quarter and remained aggressive in terms of promotional activities with “key feature” focused SKUs such as bigger batteries, bigger memory and so forth.
- Lava (excluding Xolo) surpassed Microsoft for the first time and thereby capturing fourth spot in the overall handset market.
- Lava managed to increase its smartphone market share to 7% level with healthy demand for Lava IRIS series, especially the ATOM 2 and X1 selfie smartphone were among the bestselling models
- Lenovo including Motorola raced to fourth position in smartphone segment, primarily due to strong sales of its K3 Note
- Lenovo’s K3 Note was the best-selling smartphone in India during Q3 2015 hitting the sweet spot in terms of specs vs price among all vendors.
- Lenovo (incl Motorola) crossed 2 million units in India for the first time ever continuing its momentum in India offsetting weakness in its home market China
- However, Lenovo will need to realign its offline vs online strategy going forward to position the two brands separately and overlapping of SKUs across brands affects the operational performance which has been weakened since Motorola’s acquisition.
- Xiaomi underwent its first sequential decline in India since its debut. The upstart Chinese vendor smartphone shipments declined by almost 46% due to fierce competition from Lenovo and Micromax’s Yu brands especially in the higher volume sub-$100 segment.
- Meanwhile, Apple’s finished its Fiscal year in India with record 1.7 Million units in India for the first time ever due to strong sales of its new iPhone 6 series
- However, Apple’s volume market share remains around 1% level but in terms of revenue share it is the third largest brand with almost 9% share which depicts the prowess of Apple’s brand equity and premiumness
- Microsoft also found its place among top five overall mobile phone brands during the quarter but Lumia shipments declined sequentially.
- Meanwhile, the other brands which grew significantly during the quarter were Panasonic, Vivo and Oppo
The comprehensive and in-depth Q3 2015 Market Monitor is available for subscribing clients. Please feel free to contact us at email@example.com for further questions regarding our in-depth latest research, insights or press enquiries.
The Market Monitor research is based on sell-in (shipments) estimates based on vendor’s IR results, vendor polling triangulated with sell-through (sales), supply chain checks and secondary research.

domain: economics


- For Photo, Video & Film Production
- Output: 183 Lux at 3.3' (5600K)
- 2700-7500K CCT; RGB Mode
- Onboard & App Control
Powered by an internal rechargeable battery, this tube light will give you approximately 65 minutes of runtime on a full charge at 100% power. It is ready for worldwide use via an included USB cable that can be connected to any USB-C source, including power banks, for added runtime. Compact and lightweight, the PavoTube II 6C mounts via 1/4"-20 receivers or simply attaches to metal surfaces with its included set of metal plates. It also comes with a wrist strap for convenience and a carrying bag for storage and transport.
YOUR RECENTLY VIEWED ITEMS


text:
I hadn’t thought too hard about a baby book for our little one. Up until now, this blog has acted as our main source of tracking baby’s progress. But, thinking back to my childhood I loved looking through my baby book and being able to see and touch pictures and my mom’s handwriting. So I […]
I haven’t embarked on my breastfeeding journey … but it’s coming. At the moment I’m a bit consumed by the science of it. But, these types of posts always catch my eye … read the “Nursing in Public War” and let me know what you think. I think she’s got a pretty valid point.
I have a plethora of reading to accomplish before baby arrives … good thing I learned how to skim and find the real important stuff in college! The Girlfriend’s Guide to Pregnancy by Vicki Iovine – in progress Gentle Birth Choices by Barbara Harper, R.N. – DONE! The Womanly Art of Breastfeeding by La Leche […]
My list of “to-read before baby” is quickly growing … I’ll be honest, the books on labor are being pushed to the bottom. I think I’ve gathered the overall message, “1,000 things could happen and every mom is different”. I’ve moved on to other topics like Healthy Meals for Babies and Toddlers. I found this […]
This is one of my most favorite children’s books … I can’t wait to snuggle up and read with my baby!
As of today I have identified 3 surprise monsters who are hiding away in the closet of life … the big unknowns of my pregnancy. If our baby will be a boy or girl – I do have some control over this one, but I’m sticking to my guns and going the old-fashioned route and […]
My co-worker, Kami, has the vibe. She gets what I mean when I get passionate about breastfeeding and knows why cloth diapers are so darn exciting :) Last week she gave me Gentle Birth, Gentle Mothering and it has skyrocketed to my top favorite baby books. It is written by Dr. Sarah Buckley, MD who […]
32 percent! That’s a 1 in 3 chance of a c-section (which is considered a major surgery). The highest rate ever reported. This baffles me on so many levels … so I’ll let the article speak for itself. The article is originally from the New York Times report summarized on FitPregnancy this week. DISCLAIMER – […]
The whole story is here.These are the snippets I appreciated … The lives of nearly 900 babies would be saved each year, along with billions of dollars, if 90 percent of U.S. women fed their babies breast milk only for the first six months of life, a cost analysis says. The magnitude of health benefits […]


Bill C-11 is in the hands of the government as Canadians await a decision on which Senate amendments it will accept, which might be rejected, and then how the Senate responds. A key question involves a fix to the regulation of user content provision, which provides that sound recordings are in, but user content is out. Senator Paula Simons, an independent Senator from Alberta nominated by Prime Minister Justin Trudeau to the Senate in 2018, co-crafted the compromise amendment and has been one of the most engaged and informed Senators throughout the Bill C-11 legislative process. She joins the Law Bytes podcast to discuss the hearings, her amendment, and what may lie ahead for both Bill C-11 and the upcoming Senate review of Bill C-18.
The podcast can be downloaded here, accessed on YouTube, and is embedded below. Subscribe to the podcast via Apple Podcast, Google Play, Spotify or the RSS feed. Updates on the podcast on Twitter at @Lawbytespod.


text:
Fletchall Family History & Genealogy
Fletchall Last Name History & Origin
Nationality & Ethnicity
These are the earliest records we have of the Fletchall family.
Fletchall Biographies & Family Trees
Fletchall Death Records & Life Expectancy
According to our database of 214 people with the last name Fletchall that have a birth and death date listed:
These are the longest-lived members of the Fletchall family on AncientFaces.
- Clida A Fletchall lived 99 years
- Maude M Fletchall lived 99 years
- Keith Fletchall lived 97 years
- Matilda E Fletchall lived 95 years
- Esta Fletchall lived 93 years
- Lucille Fletchall lived 94 years
- Adeline K Fletchall lived 92 years
- Dessie A Fletchall lived 92 years
- Faye L Fletchall lived 89 years
- Kathryn E Fletchall lived 89 years
Leave a message to start a discussion about the Fletchall family with other AncientFaces Members.

domain: history


JCSO says missing man in danger for his health & safety
Published 12:01 am Tuesday, February 11, 2020
NOME — The Jefferson County Sheriff’s Office is searching for Howard Lee Bosha, diagnosed with a cognitive impairment.
Police say he is 77 years old, 5’10” and 150 pounds.
He has black hair, brown eyes and was last seen wearing a maroon “Texas A&M” hat, blue t-shirt with “TX” and “Texas” printed on them and black pants.
The senior citizen was last seen at 10 a.m. Monday in the 1000 block of 2nd Street in Nome, driving a silver, 2019 Kia Optima with Texas license plate: MTV4963.
Law enforcement officials believe Bosha’s disappearance poses a credible threat to his health and safety.
If you have any information regarding this missing man, call the Jefferson County Sheriff’s Office at 409-835-8411.


text:
Analysis of Invesco Global manpower and management performance can provide insight into Invesco Global stock performance. Also an employee sentiment is becoming a valuable factor that investors can use to determine the amount of risk that may be associated with Invesco Global High future performance. Additionally see Investing Opportunities.
Invesco Global Workforce Comparison
Invesco Global High Income A AUD Inc Hdg is one of the top funds in number of employees among similar funds. The total workforce of High Yield Bond - Other Hedged category is currently estimated at about 2.0. Invesco Global totals roughly 1.0 in number of employees claiming about 50% of all funds under High Yield Bond - Other Hedged category.
Invesco Global Stakeholder
|Darren Hughes Fund Manager|
Create custom reports across your portfolios and generate quick suggestion pitch
|All Next||Launch Portfolio Reporting|
Invesco Global High Annual Yield
In accordance with recently published financial statements Invesco Global High Income A AUD Inc Hdg has Annual Yield of 0.0%. This is 100.0% lower than that of the Invesco Global Asset Management Limited family, and 100.0% lower than that of High Yield Bond - Other Hedged category, The Annual Yield for all funds is 100.0% higher than the company.
Additionally see Investing Opportunities. Please also try Headlines Timeline module to stay connected to all market stories and filter out noise. drill down to analyze hype elasticity.

domain: finance


We hear it grows on you
Growbot, a 2D sci-fi point & click adventure game, is now available on Switch. You can check out a new launch trailer for the game above.
In Growboth, you play as Nara, a growbot in training to become a captain, trying to save your home space station from a dark crystalline force. Set upon a beautiful biopunk space station bursting with fantastic plants and aliens, your station home is attacked by rapidly growing crystals.
The game is inspired by classic adventure games like Loom, modern adventure games like Machinarium, and aims to appeal to both seasoned and new gamers.