It has been more than 10 years since follicular dendritic cell (FDC) sarcoma was first reported to occur in extranodal sites, yet extranodal FDC sarcoma still appears underrecognized, and its clinical and pathological characteristics remain to be defined. This study analyzed the clinical and pathological findings of three such cases that the authors encountered recently and 43 previously reported cases identified in the literature. Assessment of all 46 cases showed a slight female predominance (1.2:1) with a median age of 41.5 years. One-third of the cases were misdiagnosed at initial evaluation mainly because the possibility of FDC sarcoma was not considered. When considered, this disease had distinct pathological characteristics that allowed an accurate diagnosis. Staining for FDC markers, CD21, CD35, and clusterin was particularly helpful. The pathogenesis of the disease appeared heterogeneous, and associated factors included Epstein-Barr virus infection (in hepatic cases) and inflammatory pseudotumor-like conditions. Treatment modality varied widely although surgical resection was often included. With a median follow-up of 18 months, 43% of the cases recurred and 7% died of disease. The 5-year recurrence-free survival was 27.4%. From data available at the current time, we were not able to identify prognostically significant pathologic factors.

Pyrosequencing of 16S rRNA (16S) variable tags has become the most popular method for assessing microbial diversity, but the method remains costly for the evaluation of large numbers of environmental samples with high sequencing depths. We developed a barcoded Illumina paired-end (PE) sequencing (BIPES) method that sequences each 16S V6 tag from both ends on the Illumina HiSeq 2000, and the PE reads are then overlapped to obtain the V6 tag. The average accuracy of Illumina single-end (SE) reads was only 97.9%, which decreased from B99.9% at the start of the read to less than 85% at the end of the read; nevertheless, overlapping of the PE reads significantly increased the sequencing accuracy to 99.65% by verifying the 3 0 end of each SE in which the sequencing quality was degraded. After the removal of tags with two or more mismatches within the medial 40-70 bases of the reads and of tags with any primer errors, the overall base sequencing accuracy of the BIPES reads was further increased to 99.93%. The BIPES reads reflected the amounts of the various tags in the initial template, but long tags and high GC tags were underestimated. The BIPES method yields 20-50 times more 16S V6 tags than does pyrosequencing in a single-flow cell run, and each of the BIPES reads costs less than 1/40 of a pyrosequencing read. As a laborsaving and cost-effective method, BIPES can be routinely used to analyze the microbial ecology of both environmental and human microbiomes.

Hypoxia-inducible factor 1␣ (HIF-1␣) has been frequently implicated in many cancers as well as viral pathogenesis. Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to several human malignancies. It can stabilize HIF-1␣ during latent infection and undergoes lytic replication in response to hypoxic stress. However, the mechanism by which KSHV controls its latent and lytic life cycle through the deregulation of HIF-1␣ is not fully understood. Our previous studies showed that the hypoxia-sensitive chromatin remodeler KAP1 was targeted by the KSHV-encoded latency-associated nuclear antigen (LANA) to repress expression of the major lytic replication and transcriptional activator (RTA). Here we further report that an RNA interferencebased knockdown of KAP1 in KSHV-infected primary effusion lymphoma (PEL) cells disrupted viral episome stability and abrogated sub-G 1 /G 1 arrest of the cell cycle while increasing the efficiency of KSHV lytic reactivation by hypoxia or using the chemical 12-O-tetradecanoylphorbol-13-acetate (TPA) or sodium butyrate (NaB). Moreover, KSHV genome-wide screening revealed that four hypoxia-responsive clusters have a high concurrence of both RBP-J and HIF-1␣ binding sites (RBS؉HRE) within the same gene promoter and are tightly associated with KAP1. Inhibition of KAP1 greatly enhanced the association of RBP-J with the HIF-1␣ complex for driving RTA expression not only in normoxia but also in hypoxia. These results suggest that both KAP1 and the concurrence of RBS؉HRE within the RTA promoter are essential for KSHV latency and hypoxia-induced lytic reactivation. IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV), a DNA tumor virus, is an etiological agent linked to several human malignancies, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). HIF-1␣, a key hypoxia-inducible factor, is frequently elevated in KSHV latently infected tumor cells and contributes to KSHV lytic replication in hypoxia. The molecular mechanisms of how KSHV controls the latent and lytic life cycle through deregulating HIF-1␣ remain unclear. In this study, we found that inhibition of hypoxia-sensitive chromatin remodeler KAP1 in KSHV-infected PEL cells leads to a loss of viral genome and increases its sensitivity to hypoxic stress, leading to KSHV lytic reactivation. Importantly, we also found that four hypoxiaresponsive clusters within the KSHV genome contain a high concurrence of RBP-J (a key cellular regulator involved in Notch signaling) and HIF-1␣ binding sites. These sites are also tightly associated with KAP1. This discovery implies that KAP1, RBP-J, and HIF-1␣ play an essential role in KSHV pathogenesis through subtle cross talk which is dependent on the oxygen levels in the infected cells.

TWIST1, a basic helix-loop-helix transcription factor is essential for the development of cranial mesoderm and cranial neural crest-derived craniofacial structures. We have previously shown that, in the absence of TWIST1, cells within the cranial mesoderm adopt an abnormal epithelial configuration via a process reminiscent of a mesenchymal to epithelial transition (MET). Here, we show by gene expression analysis that loss of TWIST1 in the cranial mesoderm is accompanied by a reduction in the expression of genes that are associated with cell-extracellular matrix interactions and the acquisition of mesenchymal characteristics. By comparing the transcriptional profiles of cranial mesoderm-specific Twist1 loss-of-function mutant and control mouse embryos, we identified a set of genes that are both TWIST1-dependent and predominantly expressed in the mesoderm. ChIP-seq was used to identify TWIST1-binding sites in an in vitro model of a TWIST1-dependent mesenchymal cell state, and the data were combined with the transcriptome data to identify potential target genes. Three direct transcriptional targets of TWIST1 (Ddr2, Pcolce and Tgfbi) were validated by ChIP-PCR using mouse embryonic tissues and by luciferase assays. Our findings reveal that the mesenchymal properties of the cranial mesoderm are likely to be regulated by a network of TWIST1 targets that influences the extracellular matrix and cell-matrix interactions, and collectively they are required for the morphogenesis of the craniofacial structures.

Chromatin immunoprecipitation (ChIP) coupled with high-throughput techniques (ChIP-X), such as next generation sequencing (ChIP-Seq) and microarray (ChIP–chip), has been successfully used to map active transcription factor binding sites (TFBS) of a transcription factor (TF). The targeted genes can be activated or suppressed by the TF, or are unresponsive to the TF. Microarray technology has been used to measure the actual expression changes of thousands of genes under the perturbation of a TF, but is unable to determine if the affected genes are direct or indirect targets of the TF. Furthermore, both ChIP-X and microarray methods produce a large number of false positives. Combining microarray expression profiling and ChIP-X data allows more effective TFBS analysis for studying the function of a TF. However, current web servers only provide tools to analyze either ChIP-X or expression data, but not both. Here, we present ChIP-Array, a web server that integrates ChIP-X and expression data from human, mouse, yeast, fruit fly and Arabidopsis. This server will assist biologists to detect direct and indirect target genes regulated by a TF of interest and to aid in the functional characterization of the TF. ChIP-Array is available at http://jjwanglab.hku.hk/ChIP-Array, with free access to academic users.

Pseudomonas aeruginosa possesses at least three well-defined quorum-sensing (QS) (las, rhl and pqs) systems that control a variety of important functions including virulence. RsaL is a QS repressor that reduces QS signal production and ensures homeostasis by functioning in opposition to LasR. However, its regulatory role in signal homeostasis remains elusive. Here, we conducted a ChIP-seq assay and revealed that RsaL bound to two new targets, the intergenic regions of PA2228/PA2229 and pqsH/cdpR, which are required for PQS synthesis. Deletion of rsaL reduced transcription of pqsH and cdpR, thus decreasing PQS signal production. The ΔrsaL strain exhibited increased pyocyanin production and reduced biofilm formation, which are dependent on CdpR or PqsH activity. In addition, we solved the structure of the RsaL–DNA complex at a 2.4 Å resolution. Although the overall sequence similarity is quite low, RsaL folds into a HTH-like structure, which is conserved among many transcriptional regulators. Complementation results of the rsaL knockout cells with different rsaL mutants further confirmed the critical role of the DNA-binding residues (including Arg20, Gln27, Gln38, Gly35, Ser37 and Ser42) that are essential for DNA binding. Our findings reveal new targets of RsaL and provide insight into the detailed characterization of the RsaL–DNA interaction.

No abstract

Intramuscular fat (IMF) content affects the tenderness, juiciness, and flavor of pork. An increasing number of studies are focusing on the functions of microRNAs (miRs) during porcine intramuscular preadipocyte development. Previous studies have proved that miR-425-5p was enriched in porcine skeletal muscles and played important roles in multiple physiological processes; however, its functions during intramuscular adipogenesis remain unclear. To explore the role of miR-425-5p in porcine intramuscular adipogenesis, miR-425-5p agomir and inhibitor were used to perform miR-425-5p overexpression and knockdown in intramuscular preadipocytes, respectively. Our results showed that the agomir of miR-425-5p dramatically inhibited intramuscular adipogenic differentiation and downregulated the expression levels of adipogenic marker genes PPARγ, FABP4, and FASN, whereas its inhibitor promoted adipogenesis. Interestingly, the agomir repressed proliferation of porcine intramuscular preadipocytes by downregulation of cyclin B and cyclin E. Furthermore, we demonstrated that miR-425-5p inhibited adipogenesis via targeting and repressing the translation of KLF13. Taken together, our findings identified that miR-425-5p is a novel inhibitor of porcine intramuscular adipogenesis possibly through targeting KLF13 and subsequently downregulating PPARγ.