HPV16 E1 dysregulated cellular genes involved in cell proliferation and host DNA damage: A possible role in cervical carcinogenesis

HPV16 is the most prominent cause of cervical cancer. HPV16 E1, a helicase required for HPV replication exhibits increased expression in association with cervical cancer progression, suggesting that E1 has a similar effect on the host as the HPV16 E6 and E7 oncoproteins. This study aimed to determine whether expression of HPV16 E1 correlated with carcinogenesis by modulating cellular pathways involved in cervical cancer. HEK293T cells were transfected with pEGFP, pEGFPE1 or truncated forms of HPV16 E1. Cell proliferation, cell death, and the impact of HPV16 E1 on host gene expression was then evaluated. HPV16 E1 overexpression resulted in a significant reduction of cell viability and cellular proliferation (p-value<0.0001). Moreover, prolonged expression of HPV16 E1 significantly induced both apoptotic and necrotic cell death, which was partially inhibited by QVD-OPH, a broad-spectrum caspase inhibitor. Microarray, real time RT-PCR and kinetic host gene expression analyses revealed that HPV16 E1 overexpression resulted in the downregulation of genes involved in protein synthesis (RPL36A), metabolism (ALDOC), cellular proliferation (CREB5, HIF1A, JMJDIC, FOXO3, NFKB1, PIK3CA, TSC22D3), DNA damage (ATR, BRCA1 and CHEK1) and immune response (ISG20) pathways. How these genetic changes contribute to HPV16 E1-mediated cervical carcinogenesis warrants further studies.

Rumen Fermentation—Microbiota—Host Gene Expression Interactions to Reveal the Adaptability of Tibetan Sheep in Different Periods

As an important ruminant on the Qinghai-Tibet Plateau, Tibetan sheep can maintain their population reproduction rate in the harsh high-altitude environment of low temperature and low oxygen, which relies on their special plateau adaptations mechanism that they have formed for a long time. Microbiomes (known as “second genomes”) are closely related to the nutrient absorption, adaptability, and health of the host. In this study, rumen fermentation characteristics, the microbiota, and rumen epithelial gene expression of Tibetan sheep in various months were analyzed. The results show that the rumen fermentation characteristics of Tibetan sheep differed in different months. The total SCFAs (short-chain fatty acids), acetate, propionate, and butyrate concentrations were highest in October and lowest in June. The CL (cellulase) activity was highest in February, while the ACX (acid xylanase) activity was highest in April. In addition, the diversity and abundance of rumen microbes differed in different months. Bacteroidetes (53.4%) and Firmicutes (27.4%) were the dominant phyla. Prevotella_1 and Rikenellaceae_RC9_gut_group were the dominant genera. The abundance of Prevotella_1 was highest in June (27.8%) and lowest in December (17.8%). In addition, the expression of CLAUDIN4 (Claudin-4) and ZO1 (Zonula occludens 1) was significantly higher in April than in August and December, while the expression of SGLT1 (Sodium glucose linked transporter 1) was highest in August. Correlation analysis showed that there were interactions among rumen fermentation characteristics, the microbiota, and host gene expression, mainly by adjusting the amino acid metabolism pathway and energy metabolism pathway to improve energy utilization. At the same time, we adjusted the balance of the rumen “core microbiota” to promote the development of rumen and maintain the homeostasis of rumen environment, which makes Tibetan sheep better able to adapt to the harsh environment in different periods of the Qinghai-Tibet Plateau.

Differential Bumble Bee Gene Expression Associated With Pathogen Infection And Pollen Diet

Background: Diet and parasitism can have powerful effects on host gene expression. However, how specific dietary components affect host gene expression that could feed back to affect parasitism is relatively unexplored in many wild species. Recently, it was discovered that consumption of sunflower (Helianthus annuus) pollen reduced severity of gut protozoan pathogen Crithidia bombi infection in Bombus impatiens bumble bees. Despite the dramatic and consistent medicinal effect of sunflower pollen, very little is known about the mechanism(s) underlying this effect. However, sunflower pollen extract increases rather than suppresses C. bombi growth in vitro, suggesting that sunflower pollen reduces C. bombi infection indirectly via changes in the host. Here, we analyzed whole transcriptomes of B. impatiens workers to characterize the physiological response to sunflower pollen consumption and C. bombi infection to isolate the mechanisms underlying the medicinal effect. B. impatiens workers were inoculated with either C. bombi cells (infected) or a sham control (un-infected) and fed either sunflower or wildflower pollen ad libitum. Whole abdominal gene expression profiles were then sequenced with Illumina NextSeq 500 technology. Results: Among infected bees, sunflower pollen upregulated immune transcripts, including the anti-microbial peptide hymenoptaecin, Toll receptors and serine proteases. In both infected and un-infected bees, sunflower pollen upregulated putative detoxification transcripts and transcripts associated with the repair and maintenance of gut epithelial cells. Among wildflower-fed bees, infected bees downregulated immune transcripts associated with phagocytosis and the phenoloxidase cascade. Conclusions: Taken together, these results indicate dissimilar immune responses between sunflower- and wildflower-fed bumble bees infected with C. bombi, a response to physical damage to gut epithelial cells caused by sunflower pollen, and a strong detoxification response to sunflower pollen consumption. Identifying host responses that drive the medicinal effect of sunflower pollen in infected bumble bees may broaden our understanding of plant-pollinator interactions and provide opportunities for effective management of bee pathogens.

Comparing the Diagnostic Accuracy of Clinician Judgement to a Novel Host Response Diagnostic for Acute Respiratory Illness

Background Difficulty discriminating bacterial from viral infections drives antibacterial misuse. Host gene expression tests discriminate bacterial and viral etiologies, but their clinical utility has not been evaluated. Methods Host gene expression and procalcitonin levels were measured in 582 Emergency Department participants with suspected infection. We also recorded clinician diagnosis, and clinician-recommended treatment. These four diagnostic strategies were compared to clinical adjudication as the reference. To estimate the clinical impact of host gene expression, we calculated the change in overall net benefit (∆NB, the difference in net benefit comparing one diagnostic strategy to a reference) across a range of prevalence estimates while factoring in the clinical significance of false positive and negative errors. Results Gene expression correctly classified bacterial, viral, or non-infectious illness in 74.1% of subjects, similar to the other strategies. Clinical diagnosis and clinician-recommended treatment revealed a bias toward overdiagnosis of bacterial infection resulting in high sensitivity (92.6% and 94.5%, respectively), but poor specificity (67.2% and 58.8%, respectively) resulting in a 33.3% rate of inappropriate antibacterial use. Gene expression offered a more balanced sensitivity (79.0%) and specificity (80.7%), which corresponded to a statistically significant improvement in average weighted accuracy (79.9% vs. 71.5% for procalcitonin and 76.3% for clinician-recommended treatment, p&lt;0.0001 for both). Consequently, host gene expression had greater net benefit in diagnosing bacterial infection than clinician-recommended treatment (∆NB=6.4%) and procalcitonin (∆NB=17.4%). Conclusions Host gene expression-based tests to distinguish bacterial and viral infection can facilitate appropriate treatment, improving patient outcomes and mitigating the antibacterial resistance crisis.

1023. Host Gene Expression Biomarkers to Distinguish Between Causes of Acute Respiratory Symptoms in Lung Transplant Recipients

Background Long-term immunosuppression after lung transplantation increases susceptibility to a variety of respiratory infections that are often difficult to diagnose. Host gene expression patterns in circulating leukocytes may provide additional diagnostic information in these settings. Methods 107 lung transplant recipients (79% with cystic fibrosis) were enrolled at Duke University Medical Center over a 2-year period – 59% with acute respiratory symptoms, the remainder as healthy controls. Whole blood was collected by PAXGene for RNA sequencing. Prior to undergoing biomarker analysis, each case was adjudicated to the appropriate clinical phenotype: bacterial infection, viral infection, allograft rejection, and healthy. Logistic regression models were applied to gene expression data to identify classifiers capable of identifying each etiology. Results In lung transplant recipients, 117 genes were upregulated at least 2-fold in the presence of viral infection compared to healthy transplant controls. These genes clustered into expected antiviral pathways, including type I interferon signaling, interferon gamma mediated signaling, and defense response to virus, although the magnitude of gene expression was significantly less than that seen in non-transplant cohorts. Similar results were seen during bacterial infection (defense response to bacterium, antibacterial humoral response) and rejection (upregulation in defensins DEFA3 and DEFA4). Interestingly, despite the presence of immunosuppression, a previously published gene expression signature of respiratory infection (derived from non-immunosuppressed subjects) was able to differentiate between bacterial and viral infection with 100% accuracy. Conclusion Even in the presence of systemic immunosuppression and regardless of presence/absence of cystic fibrosis, core canonical components of the host response to infection and rejection are seen. Gene expression signatures based on these conserved components offer the potential for diagnostic capability in the setting of nonspecific respiratory illness in these vulnerable hosts. Disclosures Julie M. Steinbrink, MD, CareDx (Research Grant or Support) Alice Gray, MD, CareDx (Advisor or Review Panel member, Research Grant or Support, Speaker’s Bureau)Polarean (Advisor or Review Panel member)

Phenotypic Recovery of a Heterobasidion Isolate Infected by a Debilitation-Associated Virus Is Related to Altered Host Gene Expression and Reduced Virus Titer

The fungal genus Heterobasidion includes forest pathogenic species hosting a diverse group of partitiviruses. They include the host debilitating Heterobasidion partitivirus 13 strain an1 (HetPV13-an1), which was originally observed in a slowly growing H. annosum strain 94233. In this study, a relatively fast-growing sector strain 94233-RC3 was isolated from a highly debilitated mycelial culture of 94233, and its gene expression and virus transcript quantities as well as the genomic sequence of HetPV13-an1 were examined. The sequence of HetPV13-an1 genome in 94233-RC3 was identical to that in the original 94233, and thus not the reason for the partial phenotypic recovery. According to RNA-seq analysis, the HetPV13-an1 infected 94233-RC3 transcribed eight genes differently from the partitivirus-free 94233-32D. Three of these genes were downregulated and five upregulated. The number of differentially expressed genes was considerably lower and the changes in their expression were small compared to those of the highly debilitated original strain 94233 with the exception of the most highly upregulated ones, and therefore viral effects on the host transcriptome correlated with the degree of the virus-caused debilitation. The amounts of RdRp and CP transcripts of HetPV13-an1 were considerably lower in 94233-RC3 and also in 94233 strain infected by a closely related mildly debilitating virus HetPV13-an2, suggesting that the virus titer would have a role in determining the effect of HetPV13 viruses on their hosts.

Cross-Talk Between Intestinal Microbiota and Host Gene Expression in Gilthead Sea Bream (Sparus aurata) Juveniles: Insights in Fish Feeds for Increased Circularity and Resource Utilization

New types of fish feed based on processed animal proteins (PAPs), insect meal, yeast, and microbial biomasses have been used with success in gilthead sea bream. However, some drawback effects on feed conversion and inflammatory systemic markers were reported in different degrees with PAP- and non-PAP-based feed formulations. Here, we focused on the effects of control and two experimental diets on gut mucosal-adherent microbiota, and how it correlated with host transcriptomics at the local (intestine) and systemic (liver and head kidney) levels. The use of tissue-specific PCR-arrays of 93 genes in total rendered 13, 12, and 9 differentially expressed (DE) genes in the intestine, liver, and head kidney, respectively. Illumina sequencing of gut microbiota yielded a mean of 125,350 reads per sample, assigned to 1,281 operational taxonomic unit (OTUs). Bacterial richness and alpha diversity were lower in fish fed with the PAP diet, and discriminant analysis displayed 135 OTUs driving the separation between groups with 43 taxa correlating with 27 DE genes. The highest expression of intestinal pcna and alpi was achieved in PAP fish with intermediate values in non-PAP, being the pro-inflammatory action of alpi associated with the presence of Psychrobacter piscatorii. The intestinal muc13 gene was down-regulated in non-PAP fish, with this gene being negatively correlated with anaerobic (Chloroflexi and Anoxybacillus) and metal-reducing (Pelosinus and Psychrosinus) bacteria. Other inflammatory markers (igm, il8, tnfα) were up-regulated in PAP fish, positively correlating the intestinal igm gene with the inflammasome activator Escherichia/Shigella, whereas the systemic expression of il8 and tnfα was negatively correlated with the Bacilli class in PAP fish and positively correlated with Paracoccus yeei in non-PAP fish. Overall changes in the expression pattern of il10, galectins (lgals1, lgals8), and toll-like receptors (tlr2, tlr5, tlr9) reinforced the anti-inflammatory profile of fish fed with the non-PAP diet, with these gene markers being associated with a wide range of OTUs. A gut microbiota-liver axis was also established, linking the microbial generation of short chain fatty acids with the fueling of scd1- and elovl6-mediated lipogenesis. In summary, by correlating the microbiome with host gene expression, we offer new insights in the evaluation of fish diets promoting gut and metabolism homeostasis, and ultimately, the health of farmed fish.

Small Non-coding RNA Expression Following Respiratory Syncytial Virus or Measles Virus Infection of Neuronal Cells

Respiratory syncytial virus (RSV) or measles virus (MeV) infection modifies host responses through small non-coding RNA (sncRNA) expression. We show that RSV or MeV infection of neuronal cells induces sncRNAs including various microRNAs and transfer RNA fragments (tRFs). We show that these tRFs originate from select tRNAs (GCC and CAC for glycine, CTT and AAC for Valine, and CCC and TTT for Lysine). Some of the tRNAs are rarely used by RSV or MeV as indicated by relative synonymous codon usage indices suggesting selective cleavage of the tRNAs occurs in infected neuronal cells. The data implies that differentially expressed sncRNAs may regulate host gene expression via multiple mechanisms in neuronal cells.