Cytoplasmic polyhedrosis virus-induced differential gene expression in two silkworm strains of different susceptibility

Digital gene expression (DGE) was performed to investigate the gene expression profiles of 4008 and p50 silkworm strains at 48 h after oral infection with BmCPV. 3,668,437 clean tags were identified in the BmCPV-infected p50 silkworms and 3,540,790 clean tags in the control p50. By contrast, 4,498,263 clean tags were identified in the BmCPV-infected 4008 silkworms and 4,164,250 clean tags in the control 4008. A total of 691 differentially expressed genes were detected in the infected 4008 DGE library and 185 were detected in the infected p50 DGE library, respectively. The expression profiles identified some important differentially expressed genes involved in signal transduction, enzyme activity and apoptotic changes, some of which were verified using quantitative real-time PCR (qRT-PCR). These results provide important clues on the molecular mechanism of BmCPV invasion and resistance mechanism of silkworms against BmCPV infection.     

Expression of genes in gastrointestinal and lymphatic tissues during parasite infection in sheep genetically resistant or susceptible to Trichostrongylus colubriformis and Haemonchus contortus

Resistance to an acute gastrointestinal nematode (GIN) infection is dependent on the ability of the host to recognise the parasite and mount a protective Th2 response. It is hypothesised that lambs which are genetically susceptible to GIN will differentially up-regulate Th1-type genes and therefore remain susceptible to chronic parasitism compared with genetically resistant lambs which will differentially up-regulate Th2-type genes and clear the parasite infection. Two selection flocks, in which lines of Merino sheep produced lambs genetically resistant or susceptible to GIN, were acutely challenged once or thrice with either Haemonchus contortus or Trichostrongylus colubriformis. Faecal-egg counts (FECs), and plasma and tissue anti-parasite (H. contortus or T. colubriformis) antibody isotype responses showed that resistant animals challenged three times with T. colubriformis established a protective Th2 response (negligible FEC, IgG1 and IgE) whereas susceptible animals required multiple challenges to establish a significant IgG1 response despite FECs remaining high. Trichostrongylus colubriformis elicited a more pronounced host response than H. contortus. RNA extracted from tissues at the site of each parasite infection and associated lymph nodes were interrogated by microarray and quantitative PCR analyses to correlate host gene expression to FECs and antibody responses. The IFN-γ inducible gene cxcl10 was up-regulated in the susceptible line of the Trichostrongylus selection flock sheep after a tertiary challenge with the parasites H. contortus and T. colubriformis. However, a uniform pattern of genes was not up-regulated in resistant animals from both selection flocks during both parasite infections, suggesting that the mode of host resistance to these parasites is different, although some similarities in host susceptibility were apparent.     

Gene Expression Profile of Bombyx mori Hemocyte under the Stress of Destruxin A

Destruxin A (DA) is a cyclo-peptidic mycotoxin from the entomopathogenic fungus Metarhizium anisopliae. To uncover potential genes associated with its molecular mechanisms, a digital gene expression (DGE) profiling analysis was used to compare differentially expressed genes in the hemocytes of silkworm larvae treated with DA. Ten DGE libraries were constructed, sequenced, and assembled, and the unigenes with least 2.0-fold difference were further analyzed. The numbers of up-regulated genes were 10, 20, 18, 74 and 8, as well as the numbers of down-regulated genes were 0, 1, 8, 13 and 3 at 1, 4, 8, 12 and 24 h post treatment, respectively. Totally, the expression of 132 genes were significantly changed, among them, 1, 3 and 12 genes were continually up-regulated at 4, 3 and 2 different time points, respectively, while 1 gene was either up or down-regulated continually at 2 different time points. Furthermore, 68 genes were assigned to one or multiple gene ontology (GO) terms and 89 genes were assigned to specific Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology. In-depth analysis identified that these genes putatively involved in insecticide resistance, cell apoptosis, and innate immune defense. Finally, twenty differentially expressed genes were randomly chosen and validated by quantitative real-time PCR (qRT-PCR). Our studies provide insights into the toxic effect of this microbial insecticide on silkworm''s hemocytes, and are helpful to better understanding of the molecular mechanisms of DA as a biological insecticide.     

Cisplatin Resistant Spheroids Model Clinically Relevant Survival Mechanisms in Ovarian Tumors

The majority of ovarian tumors eventually recur in a drug resistant form. Using cisplatin sensitive and resistant cell lines assembled into 3D spheroids we profiled gene expression and identified candidate mechanisms and biological pathways associated with cisplatin resistance. OVCAR-8 human ovarian carcinoma cells were exposed to sub-lethal concentrations of cisplatin to create a matched cisplatin-resistant cell line, OVCAR-8R. Genome-wide gene expression profiling of sensitive and resistant ovarian cancer spheroids identified 3,331 significantly differentially expressed probesets coding for 3,139 distinct protein-coding genes (Fc >2, FDR < 0.05) (S2 Table). Despite significant expression changes in some transporters including MDR1, cisplatin resistance was not associated with differences in intracellular cisplatin concentration. Cisplatin resistant cells were significantly enriched for a mesenchymal gene expression signature. OVCAR-8R resistance derived gene sets were significantly more biased to patients with shorter survival. From the most differentially expressed genes, we derived a 17-gene expression signature that identifies ovarian cancer patients with shorter overall survival in three independent datasets. We propose that the use of cisplatin resistant cell lines in 3D spheroid models is a viable approach to gain insight into resistance mechanisms relevant to ovarian tumors in patients. Our data support the emerging concept that ovarian cancers can acquire drug resistance through an epithelial-to-mesenchymal transition.     

Genetically determined resistance to lethal murine cytomegalovirus infection is mediated by interferon-dependent and -independent restriction of virus replication.

Susceptibility of 4-week-old mice of different strains to lethal murine cytomegalovirus (MCMV) infection was studied. Strains homozygous for H-2k and C57BL strains were resistant to greater than or equal to 10(5.5) PFU. B10.BR mice congenic for C57BL background genes and H-2k were about 10-fold more resistant than either C3H/HeN or C57BL strains. BALB/c mice (H-2d) were susceptible (50% lethal dose, 10(5.05) PFU). This susceptibility was dominant over resistance associated with H-2k but not that associated with C57BL background genes. The dominant susceptibility trait segregated in backcross mice as if carried by a single gene. Virus replication in spleen cells in vivo correlated with susceptibility to lethal infection. A similar trend was found in tests of salivary glands. Replication of MCMV in vitro in cultures of adherent spleen cells and primary mouse embryo cells correlated with replication in vivo. Neutralization of interferon (IFN) in cultures of adherent spleen cells reversed H-2k-linked restriction of viral replication but had minor effects on cells of other strains. Natural killer cell responses to infection were often higher in more resistant strains, but B10.BR mice developed minimal natural killer cell responses. Specific antibody and cytotoxic T cell responses in B10.BR mice were similar or lower than in other strains. Thus, resistance to lethal MCMV infection was not immunologically mediated, was dependent on and reflected by the capacity of cells from a given mouse strain to support replication in vivo and in vitro, and was IFN dependent and recessive if linked to H-2k but IFN independent when associated with C57BL background genes.     

An integrated transcriptomic and meta-analysis of hepatoma cells reveals factors that influence susceptibility to HCV infection

Hepatitis C virus (HCV) is a global problem. To better understand HCV infection researchers employ in vitro HCV cell-culture (HCVcc) systems that use Huh-7 derived hepatoma cells that are particularly permissive to HCV infection. A variety of hyper-permissive cells have been subcloned for this purpose. In addition, subclones of Huh-7 which have evolved resistance to HCV are available. However, the mechanisms of susceptibility or resistance to infection among these cells have not been fully determined. In order to elucidate mechanisms by which hepatoma cells are susceptible or resistant to HCV infection we performed genome-wide expression analyses of six Huh-7 derived cell cultures that have different levels of permissiveness to infection. A great number of genes, representing a wide spectrum of functions are differentially expressed between cells. To focus our investigation, we identify host proteins from HCV replicase complexes, perform gene expression analysis of three HCV infected cells and conduct a detailed analysis of differentially expressed host factors by integrating a variety of data sources. Our results demonstrate that changes relating to susceptibility to HCV infection in hepatoma cells are linked to the innate immune response, secreted signal peptides and host factors that have a role in virus entry and replication. This work identifies both known and novel host factors that may influence HCV infection. Our findings build upon current knowledge of the complex interplay between HCV and the host cell, which could aid development of new antiviral strategies.     

An initial comparative genomic autopsy of wasting disease in sea stars

Beginning in 2013, sea stars throughout the Eastern North Pacific were decimated by wasting disease, also known as “asteroid idiopathic wasting syndrome” (AIWS) due to its elusive aetiology. The geographic extent and taxonomic scale of AIWS meant events leading up to the outbreak were heterogeneous, multifaceted, and oftentimes unobserved; progression from morbidity to death was rapid, leaving few tell‐tale symptoms. Here, we take a forensic genomic approach to discover candidate genes that may help explain sea star wasting syndrome. We report the first genome and annotation for Pisaster ochraceus, along with differential gene expression (DGE) analyses in four size classes, three tissue types, and in symptomatic and asymptomatic individuals. We integrate nucleotide polymorphisms associated with survivors of the wasting disease outbreak, DGE associated with temperature treatments in P. ochraceus, and DGE associated with wasting in another asteroid Pycnopodia helianthoides. In P. ochraceus, we found DGE across all tissues, among size classes, and between asymptomatic and symptomatic individuals; the strongest wasting‐associated DGE signal was in pyloric caecum. We also found previously identified outlier loci co‐occur with differentially expressed genes. In cross‐species comparisons of symptomatic and asymptomatic individuals, consistent responses distinguish genes associated with invertebrate innate immunity and chemical defence, consistent with context‐dependent stress responses, defensive apoptosis, and tissue degradation. Our analyses thus highlight genomic constituents that may link suspected environmental drivers (elevated temperature) with intrinsic differences among individuals (age/size, alleles associated with susceptibility) that elicit organismal responses (e.g., coelomocyte proliferation) and manifest as sea star wasting mass mortality.     

Single Nucleotide Polymorphisms in IL8 and TLR4 Genes as Candidates for Digital Dermatitis Resistance/Susceptibility in Holstein Cattle

Relatedness between single nucleotide polymorphisms in IL8 and TLR4 genes and digital dermatitis resistance/susceptibility was investigated in seventy Holstein dairy cows. Animals were assigned into two groups, affected group (n?=?35) and resistant group (n?=?35) based on clinical signs and previous history of farm clinical records. Blood samples were collected for DNA extraction to ampliy fragments of 267-bp and 382-bp for IL8 and TLR4 genes, respectively. PCR-DNA sequencing revealed three SNPs in each of IL8 and TLR4 genes. The identified SNPs associated with digital dermatitis resistance were C94T, A220G, and T262A for IL8 and C118T for TLR4. However, the G349C and C355A SNPs in TLR4 gene were associated with digital dermatitis susceptibility. Chi-square analysis for comparison the distribution of all identified SNPs in both IL8 and TLR4 genes between resistant and affected animals showed no significant variation among the identified SNPs in IL8 gene. Meanwhile, there was a significant variation in case of TLR4 gene. As a pilot study, the present results revealed that identified SNPs in IL8 and TLR4 genes can be used as a genetic marker and predisposing factor for resistance/susceptibility to digital dermatitis in dairy cows. However, TLR4 gene may be a potential candidate for such disease.     

Genetic susceptibility to S. aureus mastitis in sheep: differential expression of mammary epithelial cells in response to live bacteria or supernatant

Staphylococcus aureus is a prevalent pathogen for mastitis in dairy ruminants and is responsible for both clinical and subclinical mastitis. Mammary epithelial cells (MEC) represent not only a physical barrier against bacterial invasion but are also active players of the innate immune response permitting infection clearance. To decipher their functions in general and in animals showing different levels of genetic predisposition to Staphylococcus in particular, MEC from ewes undergoing a divergent selection on milk somatic cell count were stimulated by S. aureus. MEC response was also studied according to the stimulation condition with live bacteria or culture supernatant. The early MEC response was studied during a 5 h time course by microarray to identify differentially expressed genes with regard to the host genetic background and as a function of the conditions of stimulation. In both conditions of stimulation, metabolic processes were altered, the apoptosis-associated pathways were considerably modified, and inflammatory and immune responses were enhanced with the upregulation of il1a, il1b, and tnfa and several chemokines known to enhance neutrophil (cxcl8) or mononuclear leukocyte (ccl20) recruitment. Genes associated with oxidative stress were increased after live bacteria stimulation, whereas immune response-related genes were higher after supernatant stimulation in the early phase. Only 20 genes were differentially expressed between Staphylococcus spp-mastitis resistant and susceptible animals without any clearly defined role on the control of infection. To conclude, this suggests that MEC may not represent the cell type at the origin of the difference of mastitis susceptibility, at least as demonstrated in our genetic model. Supernatant or heat-killed S. aureus produce biological effects that are essentially different from those induced by live bacteria.     

Avian Resistance to Campylobacter jejuni Colonization Is Associated with an Intestinal Immunogene Expression Signature Identified by mRNA Sequencing

Campylobacter jejuni is the most common cause of human bacterial gastroenteritis and is associated with several post-infectious manifestations, including onset of the autoimmune neuropathy Guillain-Barré syndrome, causing significant morbidity and mortality. Poorly-cooked chicken meat is the most frequent source of infection as C. jejuni colonizes the avian intestine in a commensal relationship. However, not all chickens are equally colonized and resistance seems to be genetically determined. We hypothesize that differences in immune response may contribute to variation in colonization levels between susceptible and resistant birds. Using high-throughput sequencing in an avian infection model, we investigate gene expression associated with resistance or susceptibility to colonization of the gastrointestinal tract with C. jejuni and find that gut related immune mechanisms are critical for regulating colonization. Amongst a single population of 300 4-week old chickens, there was clear segregation in levels of C. jejuni colonization 48 hours post-exposure. RNAseq analysis of caecal tissue from 14 C. jejuni-susceptible and 14 C. jejuni-resistant birds generated over 363 million short mRNA sequences which were investigated to identify 219 differentially expressed genes. Significantly higher expression of genes involved in the innate immune response, cytokine signaling, B cell and T cell activation and immunoglobulin production, as well as the renin-angiotensin system was observed in resistant birds, suggesting an early active immune response to C. jejuni. Lower expression of these genes in colonized birds suggests suppression or inhibition of a clearing immune response thus facilitating commensal colonization and generating vectors for zoonotic transmission. This study describes biological processes regulating C. jejuni colonization of the avian intestine and gives insight into the differential immune mechanisms incited in response to commensal bacteria in general within vertebrate populations. The results reported here illustrate how an exaggerated immune response may be elicited in a subset of the population, which alters host-microbe interactions and inhibits the commensal state, therefore having wider relevance with regard to inflammatory and autoimmune disease.     

Proteomic analysis of the abomasal mucosal response following infection by the nematode, Haemonchus contortus, in genetically resistant and susceptible sheep

Sheep have a variable ability to resist gastrointestinal nematode infection, but the key factors mediating this response are poorly defined. Here we report the first large-scale application of quantitative proteomic technologies to define proteins that are differentially abundant between sheep selectively bred to have an enhanced (resistant) or reduced (susceptible) ability to eliminate nematodes. Samples were collected from the abomasal mucosa three days after experimental challenge with the nematode, Haemonchus contortus. This timing reflects the initial interaction of host and parasite, and the tissue represents the immediate interface. We identified and quantified more than 4400 unique proteins, of which 158 proteins showed >1.5 fold difference between the resistant and susceptible sheep. Trefoil factor 2, a member of RAS oncogene family (RAP1A) and ring finger protein 126 were amongst the proteins found to be highly abundant in the abomasal surface of resistant sheep, whereas adenosine deaminase and the gastrokine-3 like precursor were found at higher levels in susceptible sheep. Construction of gut proteome interaction networks identified mitochondrial function and energetic partitioning as important components of an effective nematode eliminating response. The differentially abundant proteins may be useful targets for phenotypic tests that aim to identify sheep with an enhanced ability to resist nematode infection.     

Identification of Tuberculosis Susceptibility Genes with Human Macrophage Gene Expression Profiles

Although host genetics influences susceptibility to tuberculosis (TB), few genes determining disease outcome have been identified. We hypothesized that macrophages from individuals with different clinical manifestations of Mycobacterium tuberculosis (Mtb) infection would have distinct gene expression profiles and that polymorphisms in these genes may also be associated with susceptibility to TB. We measured gene expression levels of >38,500 genes from ex vivo Mtb-stimulated macrophages in 12 subjects with 3 clinical phenotypes: latent, pulmonary, and meningeal TB (n = 4 per group). After identifying differentially expressed genes, we confirmed these results in 34 additional subjects by real-time PCR. We also used a case-control study design to examine whether polymorphisms in differentially regulated genes were associated with susceptibility to these different clinical forms of TB. We compared gene expression profiles in Mtb-stimulated and unstimulated macrophages and identified 1,608 and 199 genes that were differentially expressed by >2- and >5-fold, respectively. In an independent sample set of 34 individuals and a subset of highly regulated genes, 90% of the microarray results were confirmed by RT-PCR, including expression levels of CCL1, which distinguished the 3 clinical groups. Furthermore, 6 single nucleotide polymorphisms (SNPs) in CCL1 were found to be associated with TB in a case-control genetic association study with 273 TB cases and 188 controls. To our knowledge, this is the first identification of CCL1 as a gene involved in host susceptibility to TB and the first study to combine microarray and DNA polymorphism studies to identify genes associated with TB susceptibility. These results suggest that genome-wide studies can provide an unbiased method to identify critical macrophage response genes that are associated with different clinical outcomes and that variation in innate immune response genes regulate susceptibility to TB.     

BoLA-DRB3 gene polymorphism and FMD resistance or susceptibility in Wanbei cattle

For the further characterization of foot-and-mouth disease virus (FMDV)-induced foot-and-mouth disease, we investigated the association between polymorphism of BoLA-DRB3 gene and FMD resistance/susceptibility of Wanbei cattle challenged with FMDV. One hundred cattle were challenged with FMDV and exon 2 of BoLA-DRB3 genes was amplified by hemi-nested polymerase chain reaction from asymptomatic animals and from animals with FMD. PCR products were characterized by the RFLP technique using restriction enzymes Hae III. The results revealed extensive polymorphisms, 6 RFLP patterns were identified. By analyzing alleles and genotypic frequencies between healthy and infection with FMD cattle, we found that allele Hae III A was associated with susceptibility to FMD in Wanbei cattle (P < 0.05), whereas Hae III C was associated with resistance to FMD (P < 0.01) and may have a strong protective effect against FMD. Hae IIICC and Hae III BC genotype were associated with resistance to FMD (P < 0.01). By contrast, Hae III AA genotype was associated with susceptibility to FMD (P < 0.01). Sequence analysis show that 89 amino acids were translated in exon 2 of BoLA-DRB3 and 13.70 % of nucleotide mutated, which resulted in 14.61 % of amino acid change. One PKC, one Tyr and one CAMP phosphorylation were increased; the hydrophobicity and secondary structure of proteins produced change after amino acid substitution. These results revealed that Wanbei cattle had the ability of resistance to disease by mutation which result changes of the protein structure to perform the regulation of the cell using different signaling pathways in the long process of choice evolution.