The rockefeller university

The multiprotein Mediator coactivator complex functions in large part by controlling the formation and function of the promoter-bound preinitiation complex (PIC), which consists of RNA polymerase II and general transcription factors. However, precisely how Mediator impacts the PIC, especially post-recruitment, has remained unclear. Here, we have studied Mediator effects on basal transcription in an in vitro transcription system reconstituted from purified components. Our results reveal a close functional interplay between Mediator and TFIIH in the early stages of PIC development. We find that under conditions when TFIIH is not normally required for transcription, Mediator actually represses transcription. TFIIH, whose recruitment to the PIC is known to be facilitated by the Mediator, then acts to relieve Mediator-induced repression to generate an active form of the PIC. Gel mobility shift analyses of PICs and characterization of TFIIH preparations carrying mutant XPB translocase subunit further indicate that this relief of repression is achieved through expending energy via ATP hydrolysis, suggesting that it is coupled to TFIIH's established promoter melting activity. Our interpretation of these results is that Mediator functions as an assembly factor that facilitates PIC maturation through its various stages. Whereas the overall effect of the Mediator is to stimulate basal transcription, its initial engagement with the PIC generates a transcriptionally inert PIC intermediate, which necessitates energy expenditure to complete the process. (C) 2016 Elsevier Ltd. All rights reserved.

Hepatocellular carcinoma (HCC) causes significant medical burdens worldwide. Diagnosis, especially in the early stages, is still challenging. Therapeutic options are limited and often ineffective. Although several risk factors have been known important for development of HCC, the molecular basis of the process is rather complex and has not been fully understood. We have found that a subpopulation of HCC cells which are resistant to oncolytic parvovirus H1 superinfection highly express serine protease inhibitor Kazal-type 6 (SPINK6). This protein is specifically reduced in all HCC cell lines and tissues we analyzed. When upregulated, SPINK6 could suppress the malignant phenotypes of the HCC cells in several in vitro models. The putative tumor suppression role of SPINK6 is, however, independent of its protease inhibitory activity. To suppress the malignancy of HCC cells, SPINK6 has to be secreted to trigger signals which regulate an intracellular signaling molecule, ERK1/2, as well as a series of downstream factors involved in cell cycle progression, apoptosis and migration. Our study supports that SPINK6 is an important tumor suppressor in liver, and further investigations may help develop more effective diagnostic and therapeutic approaches.

Caves are regarded as extreme habitats with appropriate conditions for the development of Actinobacteria. In comparison with other habitats, caves have not yet been the target of intensive screening for bioactive secondary metabolites produced by actinomycetes. As a primary screening strategy, we conducted a metagenomic analysis of the diversity and richness of a key gene required for non-ribosomal peptide (NRP) biosynthesis, focusing on cave-derived sediments from two Canadian caves (a lava tube and a limestone cave) to help us predict whether different types of caves may harbor drug-producing actinobacteria. Using degenerate PCR primers targeting adenylation domains (AD), a conserved domain in the core gene in NRP biosynthesis, a number of amplicons were obtained that mapped back to biomedically relevant NRP gene cluster families. This result guided our culture-dependent sampling strategy of actinomycete isolation from the volcanic caves of Canada (British Columbia) and Portugal (Azores) and subsequent characterization of their antibacterial and enzymatic activities. Multiple enzymatic and antimicrobial activities were identified from bacterial of the Arthrobacter and Streptomyces genera demonstrating that actinomycetes from volcanic caves are promising sources of antibacterial, antibiofilm compounds and industrially relevant enzymes.

Psoriasis vulgaris, affecting the skin, is one of the most common organ-specific autoimmune diseases in humans. Until recently, psoriasis was treated by agents or approaches discovered largely through serendipity. Many of the available drugs were inherently quite toxic when used as continuous treatment for many years in this chronic disease. However, an increasing understanding of disease-specific immune pathways has spurred development of pathway-targeted therapeutics during the past decade. Psoriasis is now the most effectively treated human autoimmune disease, with high-level clinical improvements possible in similar to 90% of patients using a new generation of drugs that selectively target the IL-23/Type 17 T cell axis. Thus, psoriasis is a model for the success of a translational-medicine approach based on cellular and molecular dissection of disease pathogenesis in humans.

Visualizing neural-circuit assembly in vivo requires tracking growth of optically resolvable neurites. The Caenorhabditis elegans embryonic nervous system, comprising 222 neurons and 56 glia, is attractive for comprehensive studies of development; however, embryonic reporters are broadly expressed, making single-neurite tracking/manipulation challenging. We present a method, using an infrared laser, for reproducible heat-dependent gene expression in small sublineages (one to four cells) without radiation damage. We go beyond proof-of-principle, and use our system to label and track single neurons during early nervous-system assembly. We uncover a retrograde extension mechanism for axon growth, and reveal the aetiology of axon-guidance defects in sax-3/Robo and vab-1/EphR mutants. We also perform cell-specific rescues, determining DAF-6/patched-related site of action during sensory-organ development. Simultaneous ablation and labelling of cells using our system reveals roles for glia in dendrite extension. Our method can be applied to other optically/IR-transparent organisms, and opens the door to high-resolution systematic analyses of C. elegans morphogenesis.

The nature of hematopoietic stem cells under normal hematopoiesis remained largely unknown due to the limited assays available to monitor their behavior in situ. Here, we develop a new mouse model to transfer genes specifically into the primitive hematopoietic stem cell compartment through the utilization of a modified Rcas/TVA system. We succeeded in transferring a GFP reporter gene into adult hematopoietic stem cells in vivo, which are predominantly quiescent, by generating pseudotyped-lentivirus. Furthermore, we demonstrate the utility of this system to study neonatal hematopoiesis, a developmental stage that has been difficult to analyze to date. Using the system developed in this study, we observed continuous multi-lineage hematopoietic cell supply in peripheral blood from Krt7-positive hematopoietic stem cells during unperturbed homeostatic condition. This powerful experimental system could provide a new standard tool to analyze hematopoiesis under physiological condition without transplantation.

AIDS is a preventable disease. Nevertheless, according to UNAIDS, 2.1 million individuals were infected with HIV-1 in 2015 worldwide. An effective vaccine is highly desirable. Most vaccines in clinical use today prevent infection because they elicit antibodies that block pathogen entry. Consistent with this general rule, studies in experimental animals have shown that broadly neutralizing antibodies to HIV-1 can prevent infection, suggesting that a vaccine that elicits such antibodies would be protective. However, despite significant efforts over the last 30 years, attempts to elicit broadly HIV-1 neutralizing antibodies by vaccination failed until recent experiments in genetically engineered mice were finally successful. Here, we review the key breakthroughs and remaining obstacles to the development of active and passive HIV-1 vaccines.

Some HIV-1-infected patients develop broad and potent HIV-1 neutralizing antibodies (bNAbs) that when passively transferred to mice or macaques can treat or prevent infection. However, bNAbs typically fail to neutralize coexisting autologous viruses due to antibody-mediated selection against sensitive viral strains. We describe an HIV-1 controller expressing HLA-B57*01 and HLA-B27*05 who maintained low viral loads for 30 years after infection and developed broad and potent serologic activity against HIV-1. Neutralization was attributed to three different bNAbs targeting nonoverlapping sites on the HIV-1 envelope trimer (Env). One of the three, BG18, an antibody directed against the glycan-V3 portion of Env, is the most potent member of this class reported to date and, as revealed by crystallography and electron microscopy, recognizes HIV-1 Env in a manner that is distinct from other bNAbs in this class. Single-genome sequencing of HIV-1 from serum samples obtained over a period of 9 years showed a diverse group of circulating viruses, 88.5% (31 of 35) of which remained sensitive to at least one of the temporally coincident autologous bNAbs and the individual's serum. Thus, bNAb-sensitive strains of HIV-1 coexist with potent neutralizing antibodies that target the virus and may contribute to control in this individual. When administered as a mix, the three bNAbs controlled viremia in HIV-1(YU2)-infected humanized mice. Our finding suggests that combinations of bNAbs may contribute to control of HIV-1 infection.

Purpose of reviewThe gastroenterology literature emphasizes factors that increase colorectal cancer (CRC) incidence but presents little about management after initial CRC treatments. The purpose of this review is to describe the remarkably increasing numbers of CRC survivors in whom surveillance guidelines are often not followed and patient care is fragmented. The gastroenterologist can play an important role in this care to improve prognosis and overall health.Recent findingsExisting surveillance recommendations by specialty societies for CRC survivors are fairly consistent but implementation occurs in less than half. The gastroenterologist can help to coordinate care to ensure appropriate surveillance and also can help to diagnose and treat chemotherapy and radiotherapy complications in survivors which can affect the quality of life long after the initial treatment. The gastroenterologist also can focus on host factors, including management of obesity, exercise programs, and the diet and can introduce potential chemopreventive agents such as nonsteroidal anti-inflammatory drugs when positive prospective studies are forthcoming. Interested gastroenterologists also have a role in participating in such prospective studies.SummaryThe gastroenterologist should enhance her/his role for coordinated management of CRC survivors to improve patient surveillance care, to treat posttherapy complications and encourage preventive measures to improve prognosis and quality of life.