microRNA as a Maternal Marker for Prenatal Stress-Associated ASD, Evidence from a Murine Model.

Autism Spectrum Disorder (ASD) has been associated with a complex interplay between genetic and environmental factors. Prenatal stress exposure has been identified as a possible risk factor, although most stress-exposed pregnancies do not result in ASD. The serotonin transporter (SERT) gene has been linked to stress reactivity, and the presence of the SERT short (S)-allele has been shown to mediate the association between maternal stress exposure and ASD. In a mouse model, we investigated the effects of prenatal stress exposure and maternal SERT genotype on offspring behavior and explored its association with maternal microRNA (miRNA) expression during pregnancy. Pregnant female mice were divided into four groups based on genotype (wildtype or SERT heterozygous knockout (Sert-het)) and the presence or absence of chronic variable stress (CVS) during pregnancy. Offspring behavior was assessed at 60 days old (PD60) using the three-chamber test, open field test, elevated plus-maze test, and marble-burying test. We found that the social preference index (SPI) of SERT-het/stress offspring was significantly lower than that of wildtype control offspring, indicating a reduced preference for social interaction on social approach, specifically for males. SERT-het/stress offspring also showed significantly more frequent grooming behavior compared to wildtype controls, specifically for males, suggesting elevated repetitive behavior. We profiled miRNA expression in maternal blood samples collected at embryonic day 21 (E21) and identified three miRNAs (mmu-miR-7684-3p, mmu-miR-5622-3p, mmu-miR-6900-3p) that were differentially expressed in the SERT-het/stress group compared to all other groups. These findings suggest that maternal SERT genotype and prenatal stress exposure interact to influence offspring behavior, and that maternal miRNA expression late in pregnancy may serve as a potential marker of a particular subtype of ASD pathogenesis.

Transcriptome analysis reveals differences in cell cycle, growth and migration related genes that distinguish fibroblasts derived from pre-invasive and invasive breast cancer.

Background/Introduction: As the most common form of pre-invasive breast cancer, ductal carcinoma in situ (DCIS) affects over 50,000 women in the US annually. Despite standardized treatment involving lumpectomy and radiation therapy, up to 25% of patients with DCIS experience disease recurrence often with invasive ductal carcinoma (IDC), indicating that a subset of patients may be under-treated. As most DCIS cases will not progress to invasion, many patients may experience over-treatment. By understanding the underlying processes associated with DCIS to IDC progression, we can identify new biomarkers to determine which DCIS cases may become invasive and improve treatment for patients. Accumulation of fibroblasts in IDC is associated with disease progression and reduced survival. While fibroblasts have been detected in DCIS, little is understood about their role in DCIS progression. Goals: We sought to determine 1) whether DCIS fibroblasts were similar or distinct from normal and IDC fibroblasts at the transcriptome level, and 2) the contributions of DCIS fibroblasts to breast cancer progression. Methods: Fibroblasts underwent transcriptome profiling and pathway analysis. Significant DCIS fibroblast-associated genes were further analyzed in existing breast cancer mRNA databases and through tissue array immunostaining. Using the sub-renal capsule graft model, fibroblasts from normal breast, DCIS and IDC tissues were co-transplanted with DCIS.com breast cancer cells. Results: Through transcriptome profiling, we found that DCIS fibroblasts were characterized by unique alterations in cell cycle and motility related genes such as PKMYT1, TGF-α, SFRP1 and SFRP2, which predicted increased cell growth and invasion by Ingenuity Pathway Analysis. Immunostaining analysis revealed corresponding increases in expression of stromal derived PKMYT1, TGF-α and corresponding decreases in expression of SFRP1 and SFRP2 in DCIS and IDC tissues. Grafting studies in mice revealed that DCIS fibroblasts enhanced breast cancer growth and invasion associated with arginase-1+ cell recruitment. Conclusion: DCIS fibroblasts are phenotypically distinct from normal breast and IDC fibroblasts, and play an important role in breast cancer growth, invasion, and recruitment of myeloid cells. These studies provide novel insight into the role of DCIS fibroblasts in breast cancer progression and identify some key biomarkers associated with DCIS progression to IDC, with important clinical implications.

Transcriptomic data indicating differential expressed genes between HIV-1 Associated Nephropathy (HIVAN) mouse model (Tg26) and wildtype mice.

Tg26 mice are robust models of human immunodeficiency virus 1 associated nephropathy (HIVAN). These mice are useful for HIVAN pathology analysis, and recent studies suggest that the Tg26 mouse model is an excellent model of other chronic kidney diseases. We performed RNA seq analysis of differential gene expression in the kidneys of Tg26 mice. Kidneys were collected from Tg26 mice and wildtype (WT) littermates at 3 months of age. The raw data were analyzed for differential gene expression using a negative binomial generalized linear model in the DeSeq2 software package. We used P-Value ≤0.05 and an absolute fold change of 1.5 to identify top 50 upregulated and top 50 downregulated differentially expressed genes between the WT and Tg26 mice. As expected inflammatory genes were among the top differentially regulated genes. Our data provides yet another level of information to help gain a more comprehensive understanding of disease progression and identify potential drug targets for HIVAN and chronic kidney diseases.

Robust gene expression and mutation analyses of RNA-sequencing of formalin-fixed diagnostic tumor samples.

Current genomic studies are limited by the availability of fresh tissue samples. Here, we show that Illumina RNA sequencing of formalin-fixed diagnostic tumor samples produces gene expression that is strongly correlated with matched frozen tumor samples (r > 0.89). In addition, sequence variations identified from FFPE RNA show 99.67% concordance with that from exome sequencing of matched frozen tumor samples. Because FFPE is a routine diagnostic sample preparation, the feasibility results reported here will facilitate the setup of large-scale research and clinical studies in medical genomics that are currently limited by the availability of fresh frozen samples.

Concurrent expression of latent and a limited number of lytic genes with immune modulation and antiapoptotic function by Kaposi's sarcoma-associated herpesvirus early during infection of primary endothelial and fibroblast cells and subsequent decline of lytic gene expression.

Kaposi's sarcoma-associated herpesvirus (KSHV) infection of in vitro target cells is characterized by the expression of the latency-associated open reading frame (ORF) 73 gene (LANA-1) and the absence of progeny virus production. This default latent infection can be switched into lytic cycle by phorbol ester and by the lytic cycle ORF 50 (RTA) protein. In this study, the kinetics of latent and lytic gene expression immediately following KSHV infection of primary human dermal microvascular endothelial (HMVEC-d) and foreskin fibroblast (HFF) cells were examined by real-time reverse transcriptase PCR and whole-genome array. Within 2 h postinfection (p.i.), high levels of ORF 50 transcripts were detected in both cell types, which declined sharply by 24 h p.i. In contrast, comparatively low levels of ORF 73 expression were detected within 2 h p.i., increased subsequently, were maintained at a steady state, and declined slowly by 120 h p.i. The RTA and LANA-1 proteins were detected in the majority of infected cells by immunoperoxidase assays. In genome array, only 29 of 94 (31%) KSHV genes were expressed, which included 11 immediate-early/early, 8 early, and 5 late lytic genes and 4 latency-associated genes. While the expression of latent ORF 72, 73, and K13 genes continued, nearly all of the lytic genes declined or were undetectable by 8 and 24 h p.i. in HMVEC-d and HFF cells, respectively. Only a limited number of RTA-activated KSHV genes were expressed briefly, and the majority of KSHV genes involved in viral DNA synthesis and structural proteins were not expressed. However, early during infection, the lytic K2, K4, K5, K6, and vIRF2 genes with immune modulation functions and the K7 gene with antiapoptotic function were expressed. Expression of K5 was detected for up to 5 days of observation, and vIRF2 was expressed up to 24 h p.i. The full complement of lytic cycle genes were expressed when 12-O-tetradecanoylphorbol-13-acetate was added to the HMVEC-d cells after 48 h p.i. These data suggest that in contrast to alpha- and betaherpesviruses and some members of gammaherpesviruses, gamma-2 KSHV in vitro infection is characterized by the concurrent expression of latent and a limited number of lytic genes immediately following infection and a subsequent decline and/or absence of lytic gene expression with the persistence of latent genes. Expression of its limited lytic cycle genes could be a "strategy" that evolved in KSHV allowing it to evade the immune system and to provide the necessary factors and time to establish and/or maintain latency during the initial phases of infection. These are unique observations among in vitro herpesvirus infections and may have important implications in KSHV biology and pathogenesis.

Host gene induction and transcriptional reprogramming in Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8)-infected endothelial, fibroblast, and B cells: insights into modulation events early during infection.

Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) is etiologically linked to the endothelial tumor Kaposi's sarcoma and with two lymphoproliferatve disorders, primary effusion lymphoma and multicentric Castleman's disease. HHV-8 infects a variety of target cells both in vivo and in vitro, binds to the in vitro target cells via cell surface heparan sulfate, and uses the alpha(3)beta(1) integrin as one of the entry receptors. Within minutes of infection, HHV-8 induced the integrin-mediated signaling pathways and morphological changes in the target cells (S. M. Akula et al., Cell, 108: 407-419, 2002; P. P. Naranatt et al., J. Virol., 77: 1524-1539, 2003). As an initial step toward understanding the role of host genes in HHV-8 infection and pathogenesis, modulation of host cell gene expression immediately after infection was examined. To reflect HHV-8's broad cellular tropism, mRNAs collected at 2 and 4 h after infection of primary human endothelial [human adult dermal microvascular endothelial cells (HMVECd)] and foreskin fibroblast [human foreskin fibroblast (HFF)] cells and human B cell line (BJAB) were analyzed by oligonucleotide array with approximately 22,000 human transcripts. With a criteria of >2-fold gene induction as significant, approximately 1.72% of the genes were differentially expressed, of which, 154 genes were shared by at least two cells and 33 genes shared by all three cells. HHV-8-induced transcriptional profiles in the endothelial and fibroblast cells were closely similar, with substantial differences in the B cells. In contrast to the antiapoptotic regulators induced in HMVECd and HFF cells, proapoptotic regulators were induced in the B cells. A robust increase in the expression of IFN-induced genes suggestive of innate immune response induction was observed in HMVECd and HFF cells, whereas there was a total lack of immunity related protein inductions in B cells. These striking cell type-specific behaviors suggest that HHV-8-induced host cell gene modulation events in B cells may be different compared with the adherent endothelial and fibroblast target cells. Functional clustering of modulated genes identified several host molecules hitherto unknown to HHV-8 infection. These results indicate that early during infection, HHV-8 reprograms the host transcriptional machinery regulating a variety of cellular processes including apoptosis, transcription, cell cycle regulation, signaling, inflammatory response, and angiogenesis, all of which may play important roles in the biology and pathogenesis of HHV-8.