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The Delta variant of SARS-CoV-2 has now become the predominant strain in the global COVID-19 pandemic. Strain coverage of some detection assays developed during the early pandemic stages has declined due to periodic mutations in the viral genome. We have developed a real-time RT-PCR (RT-qPCR) for SARS-CoV-2 detection that provides nearly 100% strain coverage, and differentiation of highly transmissible Delta variant strains. All full or nearly full (≥28 kb) SARS-CoV-2 genomes (n = 403,812), including 6422 Delta and 280 Omicron variant strains, were collected from public databases at the time of analysis and used for assay design. The two amino acid deletions in the spike gene (S-gene, Δ156-157) that is characteristic of the Delta variant were targeted during the assay design. Although strain coverage for the Delta variant was very high (99.7%), detection coverage for non-Delta wild-type strains was 93.9%, mainly due to the confined region of design. To increase strain coverage of the assay, the design for CDC N1 target was added to the assay. In silico analysis of 403,812 genomes indicated a 95.4% strain coverage for the CDC N1 target, however, in combination with our new non-Delta S-gene target, total coverage for non-Delta wild-type strains increased to 99.8%. A human 18S rRNA gene was also analyzed and used as an internal control. The final four-plex RT-qPCR assay generated PCR amplification efficiencies between 95.4% and 102.0% with correlation coefficients (R

Electricity load forecasting plays an indispensable role in the electric power systems. However, its characteristics of uncertainty and complexity are hard to handle. This paper proposes a probabilistic load forecasting approach named QRCNN-E. Specifically, the deep convolutional neural network is applied to model the non-linear relationship with the electricity load and its influencing factors. By replacing the traditional loss function with pinball loss, the network can eventually forecast loads in quantiles. Then, kernel density estimation takes quantile forecasts as inputs and produces deterministic and probabilistic results. Case studies on GEFCom2014 show that the proposed method presents better performance than other cutting-edge models.

With the advancement of power market reforms, electricity price prediction has attracted increasing attention. This paper proposes a novel probabilistic forecasting approach based on deep neural network for electricity prices. Firstly, reasonable price distributions are constructed from historical data based on the nearest neighbors. Then, a deep convolutional neural network(DCNN) is employed to extract high-level features. Meanwhile, these features are fed to label distribution learning forests (LDLFs) to generate probabilistic forecasts. The proposed framework, dubbed DCNN–LDLFs, can jointly learn the price distributions. The DCNN–LDLFs provides three types of forecasts, including deterministic forecasts, prediction intervals (PIs), and probability density functions. Unlike most popular models, the DCNN–LDLFs can be trained in an end-to-end manner, which has the opportunity to obtain a globally optimal solution. The case study on Singapore shows that the proposed method provides superior forecasts over the existing approaches.

A novel respiratory-associated Mycoplasma species (M. sp. nov.) of unknown clinical significance was recently identified that causes false positive results with multiple published PCR methods reported to specifically detect Mycoplasma ovipneumonaie, a well-known respiratory pathogen in small ruminants. This necessitates our objective to develop a real-time PCR (qPCR) assay for improved specificity and sensitivity, and more rapid detection and differentiation of M. ovipneumoniae and the M. sp. nov. in domestic sheep (DS) and domestic goat (DG) samples, as compared to a conventional PCR and sequencing (cPCR-seq) assay. Primers and probes were designed based on available M. ovipneumoniae 16S rRNA gene sequences in the GenBank database, and partial 16S rRNA gene sequences provided by the United States Department of Agriculture, Agricultural Research Service (USDA-ARS) for M. ovipneumoniae and M. sp. nov. USDA-ARS provided DS (n = 153) and DG (n = 194) nasal swab nucleic acid that previously tested positive for either M. ovipneumoniae (n = 117) or M. sp. nov. (n = 138), or negative for both targets (n = 92) by cPCR-seq. A host 18S rRNA gene was included as an internal control to monitor for the failure of nucleic acid extraction and possible PCR inhibition. For samples positive by cPCR-seq, qPCR agreement was 88.0% (103/117; κ = 0.81) and 89.9% (124/138; κ = 0.84) for M. ovipneumoniae and M. sp. nov., respectively; 12 of 255 (4.7%) cPCR-seq positive samples were qPCR positive for both targets. Of samples negative by cPCR for both mycoplasmas, qPCR detected M. ovipneumoniae and M. sp. nov. in 6.5% (6/92) and 4.3% (4/92), respectively. Samples with discordant results between the cPCR and sequencing assay and the new qPCR were analyzed by target sequencing; successfully sequenced samples had identity matches that confirmed the qPCR result. The increased target specificity of this qPCR is predicted to increase testing accuracy as compared to other published assays.

The SARS-CoV-2 virus is the causative agent of COVID-19 and has undergone continuous mutations throughout the pandemic. The more transmissible Omicron variant has quickly spread and is replacing the Delta variant as the most prevalent strain globally, including in the United States. A new molecular assay that can detect and differentiate both the Delta and Omicron variants was developed. A collection of 660,035 SARS-CoV-2 full- or near-full genomes, including 169,454 Delta variant and 24,202 Omicron variant strains, were used for primer and probe designs. In silico data analysis predicted an assay coverage of 99% of all strains, including 99% of the Delta and 99% of Omicron strains. The Omicron variant differential test was designed based on the Δ31-33 aa deletion in the N-gene, which is present in the original B.1.1.529 main genotype, BA.1, as well as in BA.2 and BA.3 subtypes. Therefore, the assay should detect the majority of all Omicron variant strains. Standard curves generated with human clinical samples indicated that the PCR amplification efficiencies were 104%, 90.7% and 90.4% for the Omicron, Delta, and non-Delta/non-Omicron wild-type genotypes, respectively. Correlation coefficients of the standard curves were all 0.99. The detection limit of the assay was 14.3, 32.0, and 21.5 copies per PCR reaction for Omicron, Delta, and wild-type genotypes, respectively. The assay was designed to specifically detect SAR-CoV-2 strains. Selected samples with Omicron, Delta and wild-type genotypes identified by the RT-qPCR assay were also confirmed by sequencing. The assay did not detect any animal coronavirus-positive samples that were tested. Human nasal swab samples that previously tested positive (n = 182) or negative (n = 42) for SARS-CoV-2 by the ThermoFisher TaqPath COVID-19 Combo Kit, produced the same result with the new assay. Among positive samples, 55.5% (101/182), 23.1% (42/182), and 21.4% (39/182) were identified as Omicron, Delta, and non-Omicron/non-Delta wild-type genotypes, respectively.

Influenza is a common and contagious respiratory disease caused by influenza A, B, C, and D viruses (IAV, IBV, ICV, and IDV). A multiplex real-time RT-PCR assay was developed for simultaneous detection of IAV, IBV, ICV, and IDV. The assay was designed to target unique sequences in the matrix gene of IBV and ICV, the RNA polymerase subunit PB1 of IDV, and combined with USDA and CDC IAV assays, both target the matrix gene. The host 18S rRNA gene was included as an internal control. In silico analyses indicated high strain coverages: 97.9% for IBV, 99.5% for ICV, and 100% for IDV. Transcribed RNA, viral isolates and clinical samples were used for validation. The assay specifically detected target viruses without cross-reactivity, nor detection of other common pathogens. The limit of detection was approximately 30 copies for each viral RNA template, which was equivalent to a threshold cycle value of ~37.

Infectious bovine keratoconjunctivitis (IBK), also known as pinkeye, is one of the most common eye diseases in cattle. Several pathogens have been associated with IBK cases, however, Moraxella bovis, Moraxella bovoculi, Mycoplasma bovis, Mycoplasma bovoculi and bovine herpesvirus type 1 (BHV-1) are most frequently observed. A multiplex real-time PCR assay using two reactions was developed for the detection and differentiation of these five pathogens. Detection sensitivities of the multiplex assays were compared to singleplex reactions testing for the same targets. Correlation coefficients (R2) of >0.99, and PCR efficiencies between 92 and 106% were demonstrated in all singleplex and multiplex real-time PCR reactions. The limits of detection (LOD) of multiplex assays for Moraxella bovis, Moraxella bovoculi, Mycoplasma bovis, Mycoplasma bovoculi and BHV-1 were 19, 23, 25, 24 and 26 copies per reaction, respectively. No cross amplification was observed for specificity testing of 179 IBK positive clinical samples and 55 non-target clinical samples. Percentage of clinical samples positive for Mycoplasma bovoculi, Moraxella bovoculi, Moraxella bovis, BHV-1 and Mycoplasma bovis were 88.8% (159/179), 75.9% (136/179), 60.3% (108/179), 11.7% (21/179) and 10.0% (18/179), respectively. Moraxella bovis, Moraxella bovoculi and Mycoplasma bovoculi were more prevalent than Mycoplasma bovis and BHV-1 in IBK samples collected from animals in this study population. Our data indicates that the multiplex real-time PCR panel assay is highly sensitive and highly specific for the detection and differentiation of the five major pathogens associated with bovine pinkeye., Highlights • A multiplex real-time PCR is developed for the detection of five major IBK pathogens. • Correlation coefficients of all standard curves were >0.99. • PCR amplification efficiencies for the five pathogens were between 92% and 106%. • Limits of detection were between 19 and 26 copies per reaction for the five pathogens. • Moraxella bovoculi, Mycoplasma bovoculi, and Moraxella bovis were more prevalent IBK pathogens.

Background The lower Dipteran fungus fly, Sciara coprophila, has many unique biological features that challenge the rule of genome DNA constancy. For example, Sciara undergoes paternal chromosome elimination and maternal X chromosome nondisjunction during spermatogenesis, paternal X elimination during embryogenesis, intrachromosomal DNA amplification of DNA puff loci during larval development, and germline-limited chromosome elimination from all somatic cells. Paternal chromosome elimination in Sciara was the first observation of imprinting, though the mechanism remains a mystery. Here, we present the first draft genome sequence for Sciara coprophila to take a large step forward in addressing these features. Results We assembled the Sciara genome using PacBio, Nanopore, and Illumina sequencing. To find an optimal assembly using these datasets, we generated 44 short-read and 50 long-read assemblies. We ranked assemblies using 27 metrics assessing contiguity, gene content, and dataset concordance. The highest-ranking assemblies were scaffolded using BioNano optical maps. RNA-seq datasets from multiple life stages and both sexes facilitated genome annotation. A set of 66 metrics was used to select the first draft assembly for Sciara. Nearly half of the Sciara genome sequence was anchored into chromosomes, and all scaffolds were classified as X-linked or autosomal by coverage. Conclusions We determined that X-linked genes in Sciara males undergo dosage compensation. An entire bacterial genome from the Rickettsia genus, a group known to be endosymbionts in insects, was co-assembled with the Sciara genome, opening the possibility that Rickettsia may function in sex determination in Sciara. Finally, the signal level of the PacBio and Nanopore data support the presence of cytosine and adenine modifications in the Sciara genome, consistent with a possible role in imprinting.

Rotavirus C (RVC) is associated with acute diarrhoea in both children and young animals. Because of its frequent occurrence, additional sequences have recently been generated. In this study, we sequenced 21 complete genomes from porcine diarrhoea samples and analysed them together with all available reference sequences collected from the GenBank database [National Center for Biotechnology Information (NCBI)]. Based on phylogenetic analysis and genetic distance calculation, the number of each segment was identified as 31G, 26P, 13I, 5R, 5C, 5M, 12A, 10 N, 9T, 8E and 4 H for genotypes encoding VP7, VP4, VP6, VP1, VP2, VP3 and NSP1, NSP2, NSP3, NSP4 and NSP5, respectively. From the analysis, genotypes G19–G31, P[22]–P[26], R5, A9–A12, N9–N10, T7–T9 and E6–E8 were defined as newly identified genotypes, and genotype C6 was combined with C5, and M6 was combined with M1, due to their closely related nature. Estimated with the identity frequency ratio between the intergenotype and intragenotype, the nucleotide identity cutoff values for different genotypes were determined as 85, 85, 86, 84, 83, 84, 82, 87, 84, 81 and 79 % for VP7, VP4, VP6, VP1, VP2, VP3, NSP1, NSP2, NSP3, NSP4 and NSP5, respectively. Genotyping of the 49 US strains indicated possible segment reassortment in 9 of the 11 segments, with the exceptions being VP1 and NSP5, and the most prevalent genotypes for each segment genes in the USA were G6/G5/G21/G9-P5/P4-I6/I5-R1-C5-M1-A8-N1/N10-T1-E1-H1. Our study updated the genotypes of RVC strains and provided more evidence of RVC strain diversity that may be relevant to better understand genetic diversity, and the distribution and evolution of RVC strains.

Infectious respiratory disease is one of the most common diseases in dogs worldwide. Several bacterial and viral pathogens can serve as causative agents of canine infectious respiratory disease (CIRD), including Mycoplasma cynos, Mycoplasma canis, Bordetella bronchiseptica, canine adenovirus type 2 (CAdV-2), canine herpesvirus 1 (CHV-1), canine parainfluenza virus (CPIV), canine distemper virus (CDV), canine influenza virus (CIA) and canine respiratory coronavirus (CRCoV). Since these organisms cause similar clinical symptoms, disease diagnosis based on symptoms alone can be difficult. Therefore, a quick and accurate test is necessary to rapidly identify the presence and relative concentrations of causative CIRD agents. In this study, a multiplex real-time PCR panel assay was developed and composed of three subpanels for detection of the aforementioned pathogens. Correlation coefficients (R

Porcine reproductive and respiratory syndrome (PRRS) remains one of the most economically devastating diseases in swine population in the United States of America. Due to high mutation rate of the PRRS virus (PRRSV) genome, it is difficult to develop an accurate diagnostic assay with high strain coverage. Differentiation of field strains from the four vaccines that have been used in the USA, namely Ingelvac PRRS MLV, Ingelvac ATP, Fostera PRRS and Prime Pac PRRS, adds an additional challenge. It is difficult to use current real-time PCR systems to detect and differentiate the field strains from the vaccine strains. Luminex xTAG technology allows us to detect more molecular targets in a single reaction with a cost similar to a single real-time PCR reaction. By analyzing all available 678 type 2 PRRSV (PRRSV-2) complete genome sequences, including the 4 vaccine strains, two pairs of detection primers were designed targeting the conserved regions of ORF4-ORF7, with strain coverage of 98.8% (670/678) based on in silico analysis. The virus strains sharing ≥98% identity of the complete genomes with the vaccine strains were considered vaccine or vaccine-like strains. One pair of primers for each vaccine strain were designed targeting the nsp2 region. In silico analysis showed the assay matched 96.5% (55/57) of Ingelvac PRRS® MLV (MLV) strain and the MLV-like strains, and 100% of the other three vaccine strains. Analytical sensitivity of the Luminex assay was one to two logs lower than that of the reverse transcription real-time PCR assay. Evaluated with 472 PRRSV-2 positive clinical samples, 93.8% samples were detected by the Luminex assay. Compared to ORF5 sequencing results, the Luminex assay detected 92.4% (73/79) of MLV strains, 78.3% (18/23) of Fostera strains and 50% (2/4) of ATP strains. None of the 472 samples were the Prime Pac strain tested by either ORF5 sequencing or the Luminex assay.

We identified influenza C virus (ICV) in samples from US cattle with bovine respiratory disease through real-time PCR testing and sequencing. Bovine ICV isolates had high nucleotide identities (≈98%) with each other and were closely related to human ICV strains (≈95%). Further research is needed to determine bovine ICV's zoonotic potential.

The lower Dipteran fungus fly,Sciara coprophila, has many unique biological features. For example,Sciaraundergoes paternal chromosome elimination and maternal X chromosome nondisjunction during spermatogenesis, paternal X elimination during embryogenesis, intrachromosomal DNA amplification of DNA puff loci during larval development, and germline-limited chromosome elimination from all somatic cells. Paternal chromosome elimination inSciarawas the first observation of imprinting, though the mechanism remains a mystery. Here, we present the first draft genome sequence forSciara coprophilato take a large step forward in aiding these studies. We approached assembling theSciaragenome using multiple sequencing technologies: PacBio, Oxford Nanopore MinION, and Illumina. To find an optimal assembly using these datasets, we generated 44 Illumina assemblies using 7 short-read assemblers and 50 long-read assemblies of PacBio and MinION sequence data using 6 long-read assemblers. We ranked assemblies using a battery of reference-free metrics, and scaffolded a subset of the highest-ranking assemblies using BioNano Genomics optical maps. RNA-seq datasets from multiple life stages and both sexes facilitated genome annotation. Moreover, we anchored nearly half of theSciaragenome sequence into chromosomes. Finally, we used the signal level of both the PacBio and Oxford Nanopore data to explore the presence or absence of DNA modifications in theSciaragenome since DNA modifications may play a role in imprinting inSciara, as they do in mammals. These data serve as the foundation for future research by the growing community studying the unique features of this emerging model system.

Streptococcus equi subsp. equi is a Gram positive bacterial pathogen commonly associated with strangles in horses, a respiratory disease characterized by abscessation of submandibular and retropharyngeal lymph nodes which can lead to obstruction of the airway. Several real-time PCR (qPCR) assays have been developed for detection of S. equi from horses with many targeting conserved regions of the S. equi cell wall-associated M-protein (SeM), a major virulence factor and immunogen of S. equi. Our objective was to develop a nested PCR (nPCR) targeting SeM and an 18S rRNA internal control gene for detection of S. equi from horses with potential improvement in detection sensitivity compared to a qPCR. Primers and probes from the Kansas State Veterinary Diagnostic Laboratory (KSVDL) S. equi clinical testing assay were utilized for all qPCR testing. Primers flanking the SeM qPCR target region were selected for an initial end-point PCR step of the nested assay; PCR product from the end-point reaction then served as template for the qPCR reaction step of the nested assay. Sample nucleic acid was also tested directly with qPCR to allow for assay comparison. Nucleic acid from clinical specimens (n = 188) submitted to KSVDL were tested in parallel with each assay. The nPCR and qPCR assays identified 22.9% (43/188) and 13.3% (25/188) of samples positive for S. equi, respectively. None of the samples positive by qPCR were negative by nPCR. The PCR products from all positive samples were submitted for DNA sequencing. Each of the 25 samples positive by both assays had a high nucleotide identity match (>96%) to the SeM gene. Among the samples positive by nPCR but negative by qPCR, 17 of 18 were sequence confirmed for SeM at greater than 96% nucleotide identity. Based on the nPCR Ct (37.8) of the one sequence un-confirmed case, it is likely that the S. equi bacterial load in this sample was below the necessary concentration for successful sequencing. Limit of detection (LOD) for the nPCR was established at a Ct of 37, and based both on the LOD of the qPCR assay (Ct of 37), as determined by standard curve data, and on the highest nPCR Cts (~37) of clinical samples able to result in SeM sequence-confirmation. As demonstrated by sequencing confirmation, the nPCR assay targeting the SeM gene is highly specific to S. equi. The increased sensitivity of the nPCR, compared to the qPCR, may reduce the number of false negative sample results in clinical testing and provide a superior detection method during low bacterial shedding periods.

Escherichia coli serogroups O157, O26, O45, O103, O111, O121, and O145, when carrying major virulence genes, the Shiga toxin genes stx(1) and stx(2) and the intimin gene eae, are important foodborne pathogens. They are referred to as the “top 7” Shiga toxin-producing E. coli (STEC) serogroups and were declared by the USDA as adulterants to human health. Since top 7 serogroup-positive cattle feces and ground beef can also contain nonadulterant E. coli strains, regular PCR cannot confirm whether the virulence genes are carried by adulterant or nonadulterant E. coli serogroups. Thus, traditional gold-standard STEC detection requires bacterial isolation and characterization, which are not compatible with high-throughput settings and often take a week to obtain a definitive result. In this study, we demonstrated that the partition-based multichannel digital PCR (dPCR) system can be used to detect and associate the E. coli serogroup-specific gene with major virulence genes and developed a single-cell-based dPCR approach for rapid (within 1 day) and accurate detection and confirmation of major STEC serogroups in high-throughput settings. Major virulence genes carried by each of the top 7 STEC serogroups were detected by dPCR with appropriately diluted intact bacterial cells from pure cultures, culture-spiked cattle feces, and culture-spiked ground beef. Furthermore, from 100 randomly collected, naturally shed cattle fecal samples, 3 O103 strains carrying eae and 2 O45 strains carrying stx(1) were identified by this dPCR assay and verified by the traditional isolation method. This novel and rapid dPCR assay is a culture-independent, high-throughput, accurate, and sensitive method for STEC detection and confirmation.

In recent years, reports indicated that PCV3 may be involved in porcine dermatitis and nephropathy syndrome (PDNS)-like disease similar to that linked to PCV2. A total of 2,125 porcine samples from 910 cases were collected during 2016-2018 and tested for presence of PCV3 and PCV2 by real-time PCR assays. Results showed high prevalence of PCV3 and PCV2: 28.4% samples from 41.2% cases were PCV3 positive and 16.4% samples from 16.7% cases were PCV2 positive. The overall coinfection rate was 5.4% and 8.4% at the sample and case level, respectively. Temporal analysis indicated that PCV3 positive case rate increased from 31.6% in 2016, 40.9% in 2017, to 55.6% in 2018. Although its prevalence was lower, PCV2-positive case rate in 2018 (28.8%) doubled that in 2017 (14.4%). The coinfection case rate also increased from 3.4% in 2016, 8.0% in 2017 to 16.1% in 2018. The high positive rate of PCV3 (56.9%) and PCV2 (33.8%) in oral fluids, PCV3 in foetuses (57.1%) and PCV2 in tonsils (54.8%) implied viral transmission route and tissue tropism. In phylogenetic analysis, two small PCV3 clusters (1 and 2) were separated but others were clustered with low bootstrapping values indicating overall low genetic diversity. Genotypes, PCV2a-h, were confirmed by analysing 2,944 strains, with a new genotype proposed as PCV2i. In this study, 61 PCV3 unique whole genomes were sequenced; 12 belonged to a separate cluster that were characterized by five consistent amino acid changes in the capsid protein (24V, 27K, 56D, 98R and 168K) and may be associated with potential differences in immunogenicity. Among the 43 unique PCV2 whole genomes sequenced, 31 belonged to PCV2d, 7 to PCV2a and 5 to PCV2b. Thus, our study demonstrates that PCV2d is the predominant genotype and PCV3 is widely circulating in the Midwest of the USA.

Viruses belonging to the genus Bocaparvovirus (BoV) are a genetically diverse group of DNA viruses known to cause respiratory, enteric, and neurological diseases in animals, including humans. An intestinal sample from an alpaca (Vicugna pacos) herd with reoccurring diarrhea and respiratory disease was submitted for next-generation sequencing, revealing the presence of a BoV strain. The alpaca BoV strain (AlBoV) had a 58.58% whole genome nucleotide percent identity to a camel BoV from Dubai, belonging to a tentative ungulate BoV 8 species (UBoV8). Recombination events were lacking with other UBoV strains. The AlBoV genome was comprised of the NS1, NP1, and VP1 proteins. The NS1 protein had the highest amino acid percent identity range (57.89–67.85%) to the members of UBoV8, which was below the 85% cut-off set by the International Committee on Taxonomy of Viruses. The low NS1 amino acid identity suggests that AlBoV is a tentative new species. The whole genome, NS1, NP1, and VP1 phylogenetic trees illustrated distinct branching of AlBoV, sharing a common ancestor with UBoV8. Walker loop and Phospholipase A2 (PLA2) motifs that are vital for virus infectivity were identified in NS1 and VP1 proteins, respectively. Our study reports a novel BoV strain in an alpaca intestinal sample and highlights the need for additional BoV research.

Zearalenone (ZEA) is a non-steroidal estrogen mycotoxin produced by several Gibberella and Fusarium species. Accumulating evidence has indicated that ZEA strongly stimulates cell proliferation. However the detailed molecular and cellular mechanisms of ZEA-mediated induction of cell proliferation have not yet been completely explained. The aim of this study was to detect the role of miRNAs in ZEA-mediated induction of cell proliferation. The effects of ZEA on cell proliferation were assessed using a cell counting kit assay and xCELLigence system. Micro-RNA sequencing was performed after treatment of TM3 cells with ZEA (0.01 &mu, mol/L) for different time periods (0, 2, 6 and 18 h). Cell function and pathway analysis of the miRNA target genes were performed by Ingenuity Pathway Analysis (IPA). We found that ZEA promotes TM3 cell proliferation at low concentrations. miRNA sequenceing revealed 66 differentially expressed miRNAs in ZEA-treated cells in comparison to the untreated control ( p &lt, 0.05). The miRNA sequencing indicated that compared to control group, there were 66 miRNAs significant change (p &lt, 0.05) in ZEA-treated groups. IPA analysis showed that the predicated miRNAs target gene involved in cell Bio-functions including cell cycle, growth and proliferation, and in signaling pathways including MAPK and RAS-RAF-MEK-ERK pathways. Results from flow cytometry and Western Blot analysis validated the predictions that ZEA can affect cell cycle, and the MAPK signaling pathway. Taking these together, the cell proliferation induced ZEA is regulated by miRNAs. The results shed light on the molecular and cellular mechanisms for the mediation of ZEA to induce proliferation.

Rice (Oryza sativa L.) is not susceptible to rust fungi, including the wheat leaf rust fungus Puccinia triticina. Upon inoculation with P. triticina spores, infection hyphae and appressoria were observed on the leaf surfaces of the rice cultivar Nipponbare. The cultivar responded to the inoculation with brown discoloration of the local tissue and fragmentation of rust infection hyphae and appressoria. A microarray gene-expression analysis of the host transcriptional response was performed 24 h after inoculation, revealing rice genes that were up- or down-regulated following the interaction. In particular, the loci represented by five probe sets (Os.55776.1. S1_x_at, Os.55647.1. A1_at, Os.55776.1. S1_at, OsAffx.10944.1. S1_x_at, and OsAffx.10944.1. S1_at) displayed the highest increase in gene expression compared to the control inoculation. The probe sets included members of the receptor-like kinase family (RLK) that occurs within a cluster of RLK genes on chromosome 1. Other RLK genes, within the RLK gene cluster and at another location, also showed increases in gene expression after P. triticina inoculation. The RLK genes varied in response to challenges with different rust strains or when challenged with several non-rust wheat pathogens that are also non-pathogenic to rice. The results indicate that rice has an active transcriptional and possible defense priming reaction in response to P. triticina and other non-host fungal pathogens.

Zearalenone (ZEA) is a non-steroidal estrogen mycotoxin produced by several Gibberella and Fusarium species. Accumulating evidence has indicated that ZEA strongly stimulates cell proliferation. However the detailed molecular and cellular mechanisms of ZEA-mediated induction of cell proliferation have not yet been completely explained. The aim of this study was to detect the role of miRNAs in ZEA-mediated induction of cell proliferation. The effects of ZEA on cell proliferation were assessed using a cell counting kit assay and xCELLigence system. Micro-RNA sequencing was performed after treatment of TM3 cells with ZEA (0.01 μmol/L) for different time periods (0, 2, 6 and 18 h). Cell function and pathway analysis of the miRNA target genes were performed by Ingenuity Pathway Analysis (IPA). We found that ZEA promotes TM3 cell proliferation at low concentrations. miRNA sequenceing revealed 66 differentially expressed miRNAs in ZEA-treated cells in comparison to the untreated control (p < 0.05). The miRNA sequencing indicated that compared to control group, there were 66 miRNAs significant change (p < 0.05) in ZEA-treated groups. IPA analysis showed that the predicated miRNAs target gene involved in cell Bio-functions including cell cycle, growth and proliferation, and in signaling pathways including MAPK and RAS-RAF-MEK-ERK pathways. Results from flow cytometry and Western Blot analysis validated the predictions that ZEA can affect cell cycle, and the MAPK signaling pathway. Taking these together, the cell proliferation induced ZEA is regulated by miRNAs. The results shed light on the molecular and cellular mechanisms for the mediation of ZEA to induce proliferation.

Zearalenone (ZEA), a non-steroidal estrogen mycotoxin produced by several species of Fusarium fungi, can be metabolized into many other derivatives by microorganisms, plants, animals and humans. It can affect mammalian reproductive capability by impacting the synthesis and secretion of sex hormones, including testosterone, estradiol and progesterone. This review summarizes the mechanisms in which ZEA and its derivatives disturb the synthesis and secretion of sex steroid hormones. Because of its structural analogy to estrogen, ZEA and its derivatives can exert a variety of estrogen-like effects and engage in estrogen negative feedback regulation, which can result in mediating the production of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the pituitary gland. ZEA and its derivatives can ultimately reduce the number of Leydig cells and granulosa cells by inducing oxidative stress, endoplasmic reticulum (ER) stress, cell cycle arrest, cell apoptosis, and cell regeneration delay. Additionally, they can disrupt the mitochondrial structure and influence mitochondrial functions through overproduction of reactive oxygen species (ROS) and aberrant autophagy signaling ways. Finally, ZEA and its derivatives can disturb the expressions and activities of the related steroidogenic enzymes through cross talking between membrane and nuclear estrogen receptors.

Influenza C virus (ICV) has been identified for the first time from bovine respiratory disease complex (BRDC) samples in the United States. Here, we report the complete genome sequence of the strain C/bovine/Montana/12/2016, identified from a nasal swab sample collected from a sick calf with clinical signs of respiratory disease in Montana.

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease resulting from abnormal interactions between T and B cells. The acquisition of SLE is linked to genetic susceptibility, and diverse environmental agents can trigger disease onset in genetically susceptible individuals. However, the strongest risk factor for developing SLE is being female (9:1 female to male ratio). The female sex steroid, estradiol, working through its receptors, contributes to the gender bias in SLE although the mechanisms remain enigmatic. In a small clinical trial, monthly administration of the estrogen receptor (ERα) antagonist, ICI182,780 (fulvestrant), significantly reduced disease indicators in SLE patients. In order to identify changes that could account for improved disease status, the present study utilized fulvestrant (Faslodex) to block ERα action in cultured SLE T cells that were purified from blood samples collected from SLE patients (n = 18, median age 42 years) and healthy control females (n = 25, median age 46 years). The effects of ERα antagonism on estradiol-dependent gene expression and canonical signaling pathways were analyzed. Pathways that were significantly altered by addition of Faslodex included T helper (Th) cell differentiation, steroid receptor signaling [glucocorticoid receptor (GR), ESR1 (ERα)], ubiquitination, and sumoylation pathways. ERα protein expression was significantly lower (p 0.05) between SLE patients and controls. A previously undetected interaction between GR and ERα signaling pathways suggests posttranslational modification of steroid receptors in SLE T cells may alter ERα/GR actions and contribute to the strong gender bias of this autoimmune disorder.

We developed a high-resolution expression microarray based on 2456 unique transcripts from a cDNA library of the aquatic midge (Chironomus tentans). By using the microarray, we detected that 146, 434 and 243 genes were differentially expressed after C. tentans larvae were exposed to chlorpyrifos (organophosphate insecticide) at 0.1 and 0.5 μg/L, and atrazine (triazine herbicide) at 1000 μg/L, respectively, for 48 h. The number of differentially expressed genes in the larvae exposed to chlorpyrifos at 0.5 μg/L was three times of that in the larvae exposed to chlorpyrifos at 0.1 μg/L. Among the differentially expressed genes in response to chlorpyrifos exposures, 76 genes showed significant Blast hits, and among them 42 were in common between the chlorpyrifos and atrazine exposures. In 19 differentially expressed xenobiotic detoxification genes, 16 were significantly up-regulated in the larvae exposed to chlorpyrifos and/or atrazine. Two cytochrome P450 genes (CtCYP6EV1 and CtCYP4DG2) were specifically up-regulated by chlorpyrifos, whereas three cytochrome P450 genes (CtCYP4DG1, CtCYP6EX3 and CtCYP6EV3) were specifically up-regulated by atrazine. Our results showed that chlorpyrifos exposures even at low concentrations can lead to significant changes in gene expression. The significant transcriptional responses are likely attributed to larval intoxication by the insecticide. These results not only support our previous studies in which candidate gene approaches were used, but also can potentially help develop specific molecular markers for monitoring pesticide exposures in non-target organisms in aquatic systems.

Differentiation of Brucella canis from other Brucella species are mainly performed through PCR-based methods and multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) procedures. Both PCR-based and MLVA methods are limited in discriminating B. canis strains. A new MLVA-13Bc method for B. canis genotyping was established by combining eight newly-developed VNTRs with five published ones. During 2010 and 2016, 377 B. canis PCR-positives were identified from 6,844 canine blood samples from 22 U.S. states, resulting in 229 B. canis isolates. The MLVA-13Bc method was able to differentiate each of these 229 isolates. The Hunter-Gaston Discriminatory Index of the individual VNTR loci ranged from 0.516 to 0.934 and the combined discriminatory index reached 1.000. Three major clusters (A, B and C) and 10 genotype groups were identified from the 229 B. canis isolates. Cluster A mainly contains genotype groups 1 and 2, and a few group 3 isolates; nearly all Cluster B isolates were from group 6; other genotype groups were classified into Cluster C. Our newly developed MLVA-13Bc assay is a highly discriminatory assay for B. canis genotyping, and can serve as a useful molecular epidemiological tool, especially for tracing the source of contamination in an event of a B. canis outbreak.

We developed a microarray based on 2895 unique transcripts assembled from 15,000 cDNA sequences from the European corn borer (Ostrinia nubilalis) larval gut. This microarray was used to monitor gene expression in early third-instar larvae of Bacillus thuringiensis (Bt)-susceptible O. nubilalis after 6 h feeding on diet, with or without the Bt Cry1Ab protoxin. We identified 174 transcripts, for which the expression was changed more than two-fold in the gut of the larvae fed Cry1Ab protoxin (p &lt, 0.05), representing 80 down-regulated and 94 up-regulated transcripts. Among 174 differentially expressed transcripts, 13 transcripts putatively encode proteins that are potentially involved in Bt toxicity, and these transcripts include eight serine proteases, three aminopeptidases, one alkaline phosphatase, and one cadherin. The expressions of trypsin-like protease and three aminopeptidase transcripts were variable, but two potential Bt-binding proteins, alkaline phosphatase and cadherin were consistently up-regulated in larvae fed Cry1Ab protoxin. The significantly up and down-regulated transcripts may be involved in Cry1Ab toxicity by activation, degradation, toxin binding, and other related cellular responses. This study is a preliminary survey of Cry1Ab protoxin-induced transcriptional responses in O. nubilalis gut and our results are expected to help with further studies on Bt toxin-insect interactions at the molecular level.

Viruses belonging to the genus Bocaparvovirus (BoV) are a genetically diverse group of DNA viruses known to cause respiratory, enteric, and neurological diseases in animals, including humans. An intestinal sample from an alpaca (Vicugna pacos) herd with reoccurring diarrhea and respiratory disease was submitted for next-generation sequencing, revealing the presence of a BoV strain. The alpaca BoV strain (AlBoV) had a 58.58% whole genome nucleotide percent identity to a camel BoV from Dubai, belonging to a tentative ungulate BoV 8 species (UBoV8). Recombination events were lacking with other UBoV strains. The AlBoV genome was comprised of the NS1, NP1, and VP1 proteins. The NS1 protein had the highest amino acid percent identity range (57.89−67.85%) to the members of UBoV8, which was below the 85% cut-off set by the International Committee on Taxonomy of Viruses. The low NS1 amino acid identity suggests that AlBoV is a tentative new species. The whole genome, NS1, NP1, and VP1 phylogenetic trees illustrated distinct branching of AlBoV, sharing a common ancestor with UBoV8. Walker loop and Phospholipase A2 (PLA2) motifs that are vital for virus infectivity were identified in NS1 and VP1 proteins, respectively. Our study reports a novel BoV strain in an alpaca intestinal sample and highlights the need for additional BoV research.

A microarray developed on the basis of 2895 unique transcripts from larval gut was used to compare gut gene expression profiles between a laboratory-selected Cry1Ab-resistant (R) strain and its isoline susceptible (S) strain of the European corn borer (Ostrinia nubilalis) after the larvae were fed the leaves of transgenic corn (MON810) expressing Cry1Ab or its non-transgenic isoline for 6 h. We revealed 398 gut genes differentially expressed (i.e., either up- or down-regulated genes with expression ratio ≥2.0) in S-strain, but only 264 gut genes differentially expressed in R-strain after being fed transgenic corn leaves. Although the percentages of down-regulated genes among the total number of differentially expressed genes (50% in S-strain and 45% in R-strain) were similar between the R- and S-strains, the expression ratios of down-regulated genes were much higher in S-strain than in R-strain. We revealed that 17 and 9 significantly up- or down-regulated gut genes from S and R-strain, respectively, including serine proteases and aminopeptidases. These genes may be associated with Cry1Ab toxicity by degradation, binding, and cellular defense. Overall, our study suggests enhanced adaptation of Cry1Ab-resistant larvae on transgenic Cry1Ab corn as revealed by lower number and lower ratios of differentially expressed genes in R-strain than in S-strain of O. nubilalis.

We differentiated mouse bone marrow cells in the presence of recombinant macrophage colony stimulating (rM-CSF) factor for 14 days during the flight of space shuttle Space Transportation System (STS)-126. We tested the hypothesis that the receptor expression for M-CSF, c-Fms was reduced. We used flow cytometry to assess molecules on cells that were preserved during flight to define the differentiation state of the developing bone marrow macrophages; including CD11b, CD31, CD44, Ly6C, Ly6G, F4/80, Mac2, c-Fos as well as c-Fms. In addition, RNA was preserved during the flight and was used to perform a gene microarray. We found that there were significant differences in the number of macrophages that developed in space compared to controls maintained on Earth. We found that there were significant changes in the distribution of cells that expressed CD11b, CD31, F4/80, Mac2, Ly6C and c-Fos. However, there were no changes in c-Fms expression and no consistent pattern of advanced or retarded differentiation during space flight. We also found a pattern of transcript levels that would be consistent with a relatively normal differentiation outcome but increased proliferation by the bone marrow macrophages that were assayed after 14 days of space flight. There also was a surprising pattern of space flight influence on genes of the coagulation pathway. These data confirm that a space flight can have an impact on the in vitro development of macrophages from mouse bone marrow cells.

The insect cuticle is a composite biomaterial made up primarily of chitin and proteins. The physical properties of the cuticle can vary greatly from hard and rigid to soft and flexible. Understanding how different cuticle types are assembled can aid in the development of novel biomimetic materials for use in medicine and technology. Toward this goal, we have taken a combined proteomics and transcriptomics approach with the red flour beetle, Tribolium castaneum, to examine the protein and gene expression profiles of the elytra and hindwings, appendages that contain rigid and soft cuticles, respectively. Two-dimensional gel electrophoresis analysis revealed distinct differences in the protein profiles between elytra and hindwings, with four highly abundant proteins dominating the elytral cuticle extract. MALDI/TOF mass spectrometry identified 19 proteins homologous to known or hypothesized cuticular proteins (CPs), including a novel low complexity protein enriched in charged residues. Microarray analysis identified 372 genes with a 10-fold or greater difference in transcript levels between elytra and hindwings. CP genes with higher expression in the elytra belonged to the Rebers and Riddiford family (CPR) type 2, or cuticular proteins of low complexity (CPLC) enriched in glycine or proline. In contrast, a majority of the CP genes with higher expression in hindwings were classified as CPR type 1, cuticular proteins analogous to peritrophins (CPAP), or members of the Tweedle family. This research shows that the elyra and hindwings, representatives of rigid and soft cuticles, have different protein and gene expression profiles for structural proteins that may influence the mechanical properties of these cuticles.

Background Genome assembly remains an unsolved problem. Assembly projects face a range of hurdles that confound assembly. Thus a variety of tools and approaches are needed to improve draft genomes. Results We used a custom assembly workflow to optimize consensus genome map assembly, resulting in an assembly equal to the estimated length of the Tribolium castaneum genome and with an N50 of more than 1 Mb. We used this map for super scaffolding the T. castaneum sequence assembly, more than tripling its N50 with the program Stitch. Conclusions In this article we present software that leverages consensus genome maps assembled from extremely long single molecule maps to increase the contiguity of sequence assemblies. We report the results of applying these tools to validate and improve a 7x Sanger draft of the T. castaneum genome. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1911-8) contains supplementary material, which is available to authorized users.

ChLGs before and after super scaffolding. Alignments of Tcas5.0 and Tcas5.2 in silico maps to consensus genome maps for all ChLGs. Consensus genome maps (blue with molecule coverage shown in dark blue) aligned to the in silico maps (green with contigs overlaid as translucent colored squares). Alignment to both Tcas5.2 super scaffolds (top alignment) and Tcas5.0 scaffolds (bottom alignment) are shown. (PDF 408 kb)

Exercise has been linked to a reduced cancer risk in animal models. However, the underlying mechanisms are unclear. This study assessed the effect of exercise with dietary consideration on the phospholipid profile in 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced mouse skin tissues. CD-1 mice were randomly assigned to one of the three groups: ad libitum–fed sedentary control; ad libitum–fed treadmill exercise at 13.4 m/min for 60 min/d, 5 d/wk (Ex+AL); and treadmill-exercised but pair-fed with the same amount as the control (Ex+PF). After 14 weeks, Ex+PF but not Ex+AL mice showed ∼25% decrease in both body weight and body fat when compared with the controls. Of the total 338 phospholipids determined by electrospray ionization–tandem mass spectrometry, 57 were significantly changed, and 25 species could distinguish effects of exercise and diet treatments in a stepwise discriminant analysis. A 36% to 75% decrease of phosphatidylinositol (PI) levels in Ex+PF mice occurred along with a significant reduction of PI 3-kinase in TPA-induced skin epidermis, as measured by both Western blotting and immunohistochemistry. In addition, ∼2-fold increase of the long-chain polyunsaturated fatty acids, docosahexaenoic and docosapentaenoic acids, in phosphatidylcholines, phosphatidylethanolamines, and lysophosphatidylethanolamines was observed in the Ex+PF group. Microarray analysis indicated that the expression of fatty acid elongase-1 increased. Taken together, these data indicate that exercise with controlled dietary intake, but not exercise alone, significantly reduced body weight and body fat as well as modified the phospholipid profile, which may contribute to cancer prevention by reducing TPA-induced PI 3-kinase and by enhancing ω-3 fatty acid elongation. Cancer Prev Res; 3(4); 466–77

Assembly and super scaffolding with multiple genera. We examined experiments from 16 different genera to determine if the results seen for the Tribolium castaneum genome are typical for other genomes as well. (PDF 876 kb)

ChLGs before and after super scaffolding. Alignments of Tcas5.0 and Tcas5.2 in silico maps to consensus genome maps for all ChLGs. Consensus genome maps (blue with molecule coverage shown in dark blue) aligned to the in silico maps (green with contigs overlaid as translucent colored squares). Alignment to both Tcas5.2 super scaffolds (top alignment) and Tcas5.0 scaffolds (bottom alignment) are shown. (PDF 408 kb)

Assembly and super scaffolding with multiple genera. We examined experiments from 16 different genera to determine if the results seen for the Tribolium castaneum genome are typical for other genomes as well. (PDF 876 kb)