Zika virus (ZIKV) has emerged as a cause of congenital brain anomalies and a range of placenta-related abnormalities, highlighting the need to unveil the modes of maternal-fetal transmission. The most likely route of vertical ZIKV transmission is via the placenta. The earliest events of ZIKV transmission in the maternal decidua, representing the maternal uterine aspect of the chimeric placenta, have remained unexplored. Here, we show that ZIKV replicates in first-trimester human maternal-decidual tissues grown ex vivo as three-dimensional (3D) organ cultures. An efficient viral spread in the decidual tissues was demonstrated by the rapid upsurge and continued increase of tissue-associated ZIKV load and titers of infectious cell-free virus progeny, released from the infected tissues. Notably, maternal decidual tissues obtained at midgestation remained similarly susceptible to ZIKV, whereas fetus-derived chorionic villi demonstrated reduced ZIKV replication with increasing gestational age. A genome-wide transcriptome analysis revealed that ZIKV substantially upregulated the decidual tissue innate immune responses. Further comparison of the innate tissue response patterns following parallel infections with ZIKV and human cytomegalovirus (HCMV) revealed that unlike HCMV, ZIKV did not induce immune cell activation or trafficking responses in the maternal-fetal interface but rather upregulated placental apoptosis and cell death molecular functions. The data identify the maternal uterine aspect of the human placenta as a likely site of ZIKV transmission to the fetus and further reveal distinct patterns of innate tissue responses to ZIKV. Our unique experimental model and findings could further serve to study the initial stages of congenital ZIKV transmission and pathogenesis and evaluate the effect of new therapeutic interventions. IMPORTANCE: In view of the rapid spread of the current ZIKV epidemic and the severe manifestations of congenital ZIKV infection, it is crucial to learn the fundamental mechanisms of viral transmission from the mother to the fetus. Our studies of ZIKV infection in the authentic tissues of the human maternal-fetal interface unveil a route of transmission whereby virus originating from the mother could reach the fetal compartment via efficient replication within the maternal decidual aspect of the placenta, coinhabited by maternal and fetal cells. The identified distinct placental tissue innate immune responses and damage pathways could provide a mechanistic basis for some of the placental developmental abnormalities associated with ZIKV infection. The findings in the unique model of the human decidua should pave the way to future studies examining the interaction of ZIKV with decidual immune cells and to evaluation of therapeutic interventions aimed at the earliest stages of transmission.

Although much has been done to understand how rearrangement of the Igkappa locus is regulated during B-cell development, little is known about the way the variable (V) segments themselves are selected. Here we show, using B6/Cast hybrid pre-B-cell clones, that a limited number of V segments on each allele is stochastically activated as characterized by the appearance of non-coding RNA and histone modifications. The activation states are clonally distinct, stable across cell division and developmentally important in directing the Ig repertoire upon differentiation. Using a new approach of allelic ATAC-seq, we demonstrate that the Igkappa V alleles have differential chromatin accessibility, which may serve as the underlying basis of clonal maintenance at this locus, as well as other instances of monoallelic expression throughout the genome. These findings highlight a new level of immune system regulation that optimizes gene diversity.

Several long noncoding RNAs (lncRNA) are abrogated in cancer but their precise contributions to oncogenesis are still emerging. Here we report that the lncRNA MALAT1 is upregulated in hepatocellular carcinoma and acts as a proto-oncogene through Wnt pathway activation and induction of the oncogenic splicing factor SRSF1. Induction of SRSF1 by MALAT1 modulates SRSF1 splicing targets, enhancing the production of antiapoptotic splicing isoforms and activating the mTOR pathway by modulating the alternative splicing of S6K1. Inhibition of SRSF1 expression or mTOR activity abolishes the oncogenic properties of MALAT1, suggesting that SRSF1 induction and mTOR activation are essential for MALAT1-induced transformation. Our results reveal a mechanism by which lncRNA MALAT1 acts as a proto-oncogene in hepatocellular carcinoma, modulating oncogenic alternative splicing through SRSF1 upregulation. Cancer Res; 77(5); 1155-67. ©2016 AACR.

Sex chromosomes pose an inherent genetic imbalance between genders. In mammals, one of the female’s X-chromosomes undergoes inactivation (Xi). Indirect measurements estimate that about 20% of Xi genes completely or partially escape inactivation. The identity of these escapee genes and their propensity to escape inactivation remain unsolved. A direct method for identifying escapees was applied by quantifying differential allelic expression from single cells. RNA-Seq fragments were assigned to informative SNPs which were labeled by the appropriate parental haplotype. This method was applied for measuring allelic specific expression from Chromosome-X (ChrX) and an autosomal chromosome as a control. We applied the protocol for measuring biallelic expression from ChrX to 104 primary fibroblasts. Out of 215 genes that were considered, only 13 genes (6%) were associated with biallelic expression. The sensitivity of escapees' identification was increased by combining SNP mapping for parental diploid genomes together with RNA-Seq from clonal single cells (25 lymphoblasts). Using complementary protocols, referred to as strict and relaxed, we confidently identified 25 and 31escapee genes, respectively. When pooled versions of 30 and 100 cells were used, <50% of these genes were revealed. We assessed the generality of our protocols in view of an escapee catalog compiled from indirect methods. The overlap between the escapee catalog and the genes’ list from this study is statistically significant (P-value of E-07). We conclude that single cells’ expression data are instrumental for studying X-inactivation with an improved sensitivity. Finally, our results support the emerging notion of the non-deterministic nature of genes that escape X-chromosome inactivation.

The 54 microRNAs (miRNAs) within the DLK-DIO3 genomic region on chromosome 14q32.31 (cluster-14-miRNAs) are organized into sub-clusters 14A and 14B. These miRNAs are downregulated in glioblastomas and might have a tumor suppressive role. Any association between the expression levels of cluster-14-miRNAs with overall survival (OS) is undetermined. We randomly selected miR-433, belonging to sub-cluster 14A and miR-323a-3p and miR-369-3p, belonging to sub-cluster 14B, and assessed their role in glioblastomas in vitro and in vivo. We also determined the expression level of cluster-14-miRNAs in 27 patients with newly diagnosed glioblastoma, and analyzed the association between their level of expression and OS. Overexpression of miR-323a-3p and miR-369-3p, but not miR-433, in glioblastoma cells inhibited their proliferation and migration in vitro. Mice implanted with glioblastoma cells overexpressing miR323a-3p and miR369-3p, but not miR433, exhibited prolonged survival compared to controls (P = .003). Bioinformatics analysis identified 13 putative target genes of cluster-14-miRNAs, and real-time RT-PCR validated these findings. Pathway analysis of the putative target genes identified neuregulin as the most enriched pathway. The expression level of cluster-14-miRNAs correlated with patients' OS. The median OS was 8.5 months for patients with low expression levels and 52.7 months for patients with high expression levels (HR 0.34; 95 % CI 0.12-0.59, P = .003). The expression level of cluster-14-miRNAs correlates directly with OS, suggesting a role for this cluster in promoting aggressive behavior of glioblastoma, possibly through ErBb/neuregulin signaling.

C/D box small nucleolar RNAs (SNORDs) are small noncoding RNAs, and their best-understood function is to target the methyltransferase fibrillarin to rRNA (for example, SNORD27 performs 2'-O-methylation of A27 in 18S rRNA). Unexpectedly, we found a subset of SNORDs, including SNORD27, in soluble nuclear extract made under native conditions, where fibrillarin was not detected, indicating that a fraction of the SNORD27 RNA likely forms a protein complex different from canonical snoRNAs found in the insoluble nuclear fraction. As part of this previously unidentified complex,SNORD27 regulates the alternative splicing of the transcription factor E2F7p re-mRNA through direct RNA-RNA interaction without methylating the RNA, likely by competing with U1 small nuclear ribonucleoprotein (snRNP). Furthermore, knockdown of SNORD27 activates previously "silent" exons in several other genes through base complementarity across the entire SNORD27 sequence, not just the antisense boxes. Thus, some SNORDs likely function in both rRNA and pre-mRNA processing, which increases the repertoire of splicing regulators and links both processes.

Aging is associated with progressive decline in cell function and with increased damage to macromolecular components. DNA damage, in the form of double-strand breaks (DSBs), increases with age and in turn, contributes to the aging process and age-related diseases. DNA strand breaks triggers a set of highly orchestrated signaling events known as the DNA damage response (DDR), which coordinates DNA repair. However, whether the accumulation of DNA damage with age is a result of decreased repair capacity, remains to be determined. In our study we showed that with age there is a decline in the resolution of foci containing γH2AX and pKAP-1 in diethylnitrosamine (DEN)-treated mouse livers, already evident at a remarkably early age of 6-months. Considerable age-dependent differences in global gene expression profiles in mice livers after exposure to DEN, further affirmed these age related differences in the response to DNA damage. Functional analysis identified p53 as the most overrepresented pathway that is specifically enhanced and prolonged in 6-month-old mice. Collectively, our results demonstrated an early decline in DNA damage repair that precedes 'old age', suggesting this may be a driving force contributing to the aging process rather than a phenotypic consequence of old age.

The molecular program underlying infrequent replication of pancreatic β-cells remains largely inaccessible. Using transgenic mice expressing green fluorescent protein in cycling cells, we sorted live, replicating β-cells and determined their transcriptome. Replicating β-cells upregulate hundreds of proliferation-related genes, along with many novel putative cell cycle components. Strikingly, genes involved in β-cell functions, namely, glucose sensing and insulin secretion, were repressed. Further studies using single-molecule RNA in situ hybridization revealed that in fact, replicating β-cells double the amount of RNA for most genes, but this upregulation excludes genes involved in β-cell function. These data suggest that the quiescence-proliferation transition involves global amplification of gene expression, except for a subset of tissue-specific genes, which are "left behind" and whose relative mRNA amount decreases. Our work provides a unique resource for the study of replicating β-cells in vivo.

Cellular senescence is thought to contribute to age-associated deterioration of tissue physiology. The senescence effector p16(Ink4a) is expressed in pancreatic beta cells during aging and limits their proliferative potential; however, its effects on beta cell function are poorly characterized. We found that beta cell-specific activation of p16(Ink4a) in transgenic mice enhances glucose-stimulated insulin secretion (GSIS). In mice with diabetes, this leads to improved glucose homeostasis, providing an unexpected functional benefit. Expression of p16(Ink4a) in beta cells induces hallmarks of senescence--including cell enlargement, and greater glucose uptake and mitochondrial activity--which promote increased insulin secretion. GSIS increases during the normal aging of mice and is driven by elevated p16(Ink4a) activity. We found that islets from human adults contain p16(Ink4a)-expressing senescent beta cells and that senescence induced by p16(Ink4a) in a human beta cell line increases insulin secretion in a manner dependent, in part, on the activity of the mechanistic target of rapamycin (mTOR) and the peroxisome proliferator-activated receptor (PPAR)-gamma proteins. Our findings reveal a novel role for p16(Ink4a) and cellular senescence in promoting insulin secretion by beta cells and in regulating normal functional tissue maturation with age.

Phage therapy has been proven to be more effective, in some cases, than conventional antibiotics, especially regarding multidrug-resistant biofilm infections. The objective here was to isolate an anti-Enterococcus faecalis bacteriophage and to evaluate its efficacy against planktonic and biofilm cultures. E. faecalis is an important pathogen found in many infections, including endocarditis and persistent infections associated with root canal treatment failure. The difficulty in E. faecalis treatment has been attributed to the lack of anti-infective strategies to eradicate its biofilm and to the frequent emergence of multidrug-resistant strains. To this end, an anti-E. faecalis and E. faecium phage, termed EFDG1, was isolated from sewage effluents. The phage was visualized by electron microscopy. EFDG1 coding sequences and phylogeny were determined by whole genome sequencing (GenBank accession number KP339049), revealing it belongs to the Spounavirinae subfamily of the Myoviridae phages, which includes promising candidates for therapy against Gram-positive pathogens. This analysis also showed that the EFDG1 genome does not contain apparent harmful genes. EFDG1 antibacterial efficacy was evaluated in vitro against planktonic and biofilm cultures, showing effective lytic activity against various E. faecalis and E. faecium isolates, regardless of their antibiotic resistance profile. In addition, EFDG1 efficiently prevented ex vivo E. faecalis root canal infection. These findings suggest that phage therapy using EFDG1 might be efficacious to prevent E. faecalis infection after root canal treatment.

The most critical stage in initiation of melanoma metastasis is the radial to vertical growth transition, yet the triggers of this transition remain elusive. We suggest that the microenvironment drives melanoma metastasis independently of mutation acquisition. Here we examined the changes in microenvironment that occur during melanoma radial growth. We show that direct contact of melanoma cells with the remote epidermal layer triggers vertical invasion via Notch signaling activation, the latter serving to inhibit MITF function. Briefly, within the native Notch ligand-free microenvironment, MITF, the melanocyte lineage master regulator, binds and represses miR-222/221 promoter in an RBPJK-dependent manner. However, when radial growth brings melanoma cells into contact with distal differentiated keratinocytes that express Notch ligands, the activated Notch intracellular domain impairs MITF binding to miR-222/221 promoter. This de-repression of miR-222/221 expression triggers initiation of invasion. Our findings may direct melanoma prevention opportunities via targeting specific microenvironments.

Multiple sclerosis (MS) is a demyelinating disorder predominantly affecting young people. Currently, interferon beta (IFNbeta) is a common treatment for MS. Despite a large effort in recent years, valid biomarkers with predictive value for clinical outcome and response to therapy are lacking. In order to identify predictive biomarkers of response to IFNbeta therapy in relapsing-remitting MS patients, we analyzed expression of 526 immune-related genes with the nCounter Analysis System (NanoString Technologies, Seattle, WA, USA) on total RNA extracted from peripheral blood mononuclear cells of 30 relapsing-remitting MS patients. We used a Wilcoxon signed-rank test to find an association between certain gene expression profiles and clinical responses to IFNbeta. We compared the expression profile of patients who responded to IFNbeta treatment (n=16) and non-responsive IFNbeta patients (n=14). The analysis revealed that the expression of eight genes could differentiate between responsive and non-responsive men (p0.005). This differentiation was not evident in women. We analyzed results from an additional cohort of 47 treated and untreated patients to validate the results and explore whether this eight gene cluster could also predict treatment response. Analysis of the validation cohort demonstrated that three out of the eight genes remained significant in only the treated men (p0.05). Our findings could be used as a basis for establishing a routine test for objective prediction of IFNbeta treatment response in male MS patients.

Induced pluripotent stem cells (iPSCs) undergo extensive nuclear reprogramming and are generally indistinguishable from embryonic stem cells (ESCs) in their functional capacity and transcriptome and DNA methylation profiles. However, direct conversion of cells from one lineage to another often yields incompletely reprogrammed, functionally compromised cells, raising the question of whether pluripotency is required to achieve a high degree of nuclear reprogramming. Here, we show that transient expression of Gata3, Eomes, and Tfap2c in mouse fibroblasts induces stable, transgene-independent trophoblast stem-like cells (iTSCs). iTSCs possess transcriptional profiles highly similar to blastocyst-derived TSCs, with comparable methylation and H3K27ac patterns and genome-wide H2A.X deposition. iTSCs generate trophoectodermal lineages upon differentiation, form hemorrhagic lesions, and contribute to developing placentas in chimera assays, indicating a high degree of nuclear reprogramming, with no evidence of passage through a transient pluripotent state. Together, these data demonstrate that extensive nuclear reprogramming can be achieved independently of pluripotency.