Circulating Tumor Reactive KIR+CD8+ T cells Suppress Anti-Tumor Immunity in Patients with Melanoma.

Effective anti-tumor immunity is largely driven by cytotoxic CD8+ T cells that can specifically recognize tumor antigens. However, the factors which ultimately dictate successful tumor rejection remain poorly understood. Here we identify a subpopulation of CD8+ T cells which are tumor antigen-specific in patients with melanoma but resemble KIR+CD8+ T cells with a regulatory function (Tregs). These tumor antigen-specific KIR+CD8+ T cells are detectable in both the tumor and the blood, and higher levels of this population are associated with worse overall survival. Our findings therefore suggest that KIR+CD8+ Tregs are tumor antigen-specific but uniquely suppress anti-tumor immunity in patients with melanoma.

The landscape of pioneer factor activity reveals the mechanisms of chromatin reprogramming and genome activation.

Upon fertilization, embryos undergo chromatin reprogramming and genome activation; however, the mechanisms that regulate these processes are poorly understood. Here, we generated a triple mutant for Nanog, Pou5f3, and Sox19b (NPS) in zebrafish and found that NPS pioneer chromatin opening at >50% of active enhancers. NPS regulate acetylation across core histones at enhancers and promoters, and their function in gene activation can be bypassed by recruiting histone acetyltransferase to individual genes. NPS pioneer chromatin opening individually, redundantly, or additively depending on sequence context, and we show that high nucleosome occupancy facilitates NPS pioneering activity. Nucleosome position varies based on the input of different transcription factors (TFs), providing a flexible platform to modulate pioneering activity. Altogether, our results illuminate the sequence of events during genome activation and offer a conceptual framework to understand how pioneer factors interpret the genome and integrate different TF inputs across cell types and developmental transitions.

Amplification of a broad transcriptional program by a common factor triggers the meiotic cell cycle in mice.

The germ line provides the cellular link between generations of multicellular organisms, its cells entering the meiotic cell cycle only once each generation. However, the mechanisms governing this initiation of meiosis remain poorly understood. Here, we examined cells undergoing meiotic initiation in mice, and we found that initiation involves the dramatic upregulation of a transcriptional network of thousands of genes whose expression is not limited to meiosis. This broad gene expression program is directly upregulated by STRA8, encoded by a germ cell-specific gene required for meiotic initiation. STRA8 binds its own promoter and those of thousands of other genes, including meiotic prophase genes, factors mediating DNA replication and the G1-S cell-cycle transition, and genes that promote the lengthy prophase unique to meiosis I. We conclude that, in mice, the robust amplification of this extraordinarily broad transcription program by a common factor triggers initiation of meiosis.

Isolating mitotic and meiotic germ cells from male mice by developmental synchronization, staging, and sorting.

Isolating discrete populations of germ cells from the mouse testis is challenging, because the adult testis contains germ cells at every step of spermatogenesis, in addition to somatic cells. We present a novel method for isolating precise, high-purity populations of male germ cells. We first synchronize germ cell development in vivo by manipulating retinoic acid metabolism, and perform histological staging to verify synchronization. We use fluorescence-activated cell sorting to separate the synchronized differentiating germ cells from contaminating somatic cells and undifferentiated spermatogonia. We achieve ~90% purity at each step of development from undifferentiated spermatogonia through late meiotic prophase. Utilizing this "3 S" method (synchronize, stage, and sort), we can separate germ cell types that were previously challenging or impossible to distinguish, with sufficient yield for epigenetic and biochemical studies. 3 S expands the toolkit of germ cell sorting methods, and should facilitate detailed characterization of molecular and biochemical changes that occur during the mitotic and meiotic phases of spermatogenesis.

Meioc maintains an extended meiotic prophase I in mice.

The meiosis-specific chromosomal events of homolog pairing, synapsis, and recombination occur over an extended meiotic prophase I that is many times longer than prophase of mitosis. Here we show that, in mice, maintenance of an extended meiotic prophase I requires the gene Meioc, a germ-cell specific factor conserved in most metazoans. In mice, Meioc is expressed in male and female germ cells upon initiation of and throughout meiotic prophase I. Mouse germ cells lacking Meioc initiate meiosis: they undergo pre-meiotic DNA replication, they express proteins involved in synapsis and recombination, and a subset of cells progress as far as the zygotene stage of prophase I. However, cells in early meiotic prophase-as early as the preleptotene stage-proceed to condense their chromosomes and assemble a spindle, as if having progressed to metaphase. Meioc-deficient spermatocytes that have initiated synapsis mis-express CYCLIN A2, which is normally expressed in mitotic spermatogonia, suggesting a failure to properly transition to a meiotic cell cycle program. MEIOC interacts with YTHDC2, and the two proteins pull-down an overlapping set of mitosis-associated transcripts. We conclude that when the meiotic chromosomal program is initiated, Meioc is simultaneously induced so as to extend meiotic prophase. Specifically, MEIOC, together with YTHDC2, promotes a meiotic (as opposed to mitotic) cell cycle program via post-transcriptional control of their target transcripts.

Application of a Rapid Knowledge Synthesis and Transfer Approach To Assess the Microbial Safety of Low-Moisture Foods.

Low-moisture foods (LMF) are increasingly implicated in outbreaks of foodborne illness, resulting in a significant public health burden. To inform the development of a new Codex Alimentarius code of hygienic practice for LMF, we applied a rapid knowledge synthesis and transfer approach to review global research on the burden of illness, prevalence, and interventions to control nine selected microbial hazards in eight categories of LMF. Knowledge synthesis methods included an integrated scoping review (search strategy, relevance screening and confirmation, and evidence mapping), systematic review (detailed data extraction), and meta-analysis of prevalence data. Knowledge transfer of the results was achieved through multiple reporting formats, including evidence summary cards. We identified 214 unique outbreaks and 204 prevalence and 126 intervention studies. Cereals and grains (n = 142) and Salmonella (n = 278) were the most commonly investigated LMF and microbial hazard categories, respectively. Salmonella was implicated in the most outbreaks (n = 96, 45%), several of which were large and widespread, resulting in the most hospitalizations (n = 895, 89%) and deaths (n = 14, 74%). Salmonella had a consistently low prevalence across all LMF categories (0 to 3%), but the prevalence of other hazards (e.g., Bacillus cereus) was highly variable. A variety of interventions were investigated in small challenge trials. Key knowledge gaps included underreporting of LMF outbreaks, limited reporting of microbial levels in prevalence studies, and a lack of intervention efficacy research under commercial conditions. Summary cards were a useful knowledge transfer format to inform complementary risk ranking activities. This review builds upon previous work in this area by synthesizing a broad range of evidence using a structured, transparent, and integrated approach to provide timely evidence informed inputs into international guidelines.

State of the art on the initiatives and activities relevant to risk assessment and risk management of nanotechnologies in the food and agriculture sectors.

The Food and Agriculture Organization of the United Nations (FAO) and World Health Organization (WHO) conducted an international expert meeting on the potential food safety implications of the application of nanotechnologies in the food and agriculture sectors in June 2009. The present report reviews national, regional and international activities on the risk assessment and risk management of nanomaterials in the food and agriculture sectors that have been carried out between 2009 and 2012. The full report of the work is presented in a FAO/WHO paper available at http://www.fao.org/food/food-safety-quality/a-z-index/nano. Information and data have been collected on national and international approaches that identify and implement strategies to address potential hazards associated with the use of nanotechnology-related products or techniques. Selected activities by international governmental and nongovernmental organizations were reviewed and the significant achievements are noted. Meta-analysis of scientific reviews addressing risk assessment of nanotechnologies in the food and agriculture sectors was conducted and key principles for the safety assessment of nanomaterials were identified. It was concluded that although the concepts of potential use of nanomaterials in food and the implied benefits for stakeholders including consumers have not changed significantly since 2009, there are new products being developed and claimed to enter the market and national and international interests in considering the needs for applying regulations on engineered nanomaterials are increasing. The number of published risk assessment of products used in foods that are nanomaterials or contain particles that fall within applicable definitions is growing slowly. Several data gaps with respect to interaction between nanomaterials and food matrices, behaviours of nanomaterials in the human body, methods to determine such interactions and behaviours, and the relevance of such data for risk assessment continue to exist. The international collaboration in the area of nanomaterials and nanotechnology in food and agriculture must be strengthened. International efforts on risk assessment and risk communication may benefit from the experience gained at the national and regional levels. Should a sufficient number of case studies of risk assessment of commercial products become available with time, a review of approaches applied and results obtained could support the development of risk assessment procedures acceptable at the international level.

A new system for comparative functional genomics of Saccharomyces yeasts.

Whole-genome sequencing, particularly in fungi, has progressed at a tremendous rate. More difficult, however, is experimental testing of the inferences about gene function that can be drawn from comparative sequence analysis alone. We present a genome-wide functional characterization of a sequenced but experimentally understudied budding yeast, Saccharomyces bayanus var. uvarum (henceforth referred to as S. bayanus), allowing us to map changes over the 20 million years that separate this organism from S. cerevisiae. We first created a suite of genetic tools to facilitate work in S. bayanus. Next, we measured the gene-expression response of S. bayanus to a diverse set of perturbations optimized using a computational approach to cover a diverse array of functionally relevant biological responses. The resulting data set reveals that gene-expression patterns are largely conserved, but significant changes may exist in regulatory networks such as carbohydrate utilization and meiosis. In addition to regulatory changes, our approach identified gene functions that have diverged. The functions of genes in core pathways are highly conserved, but we observed many changes in which genes are involved in osmotic stress, peroxisome biogenesis, and autophagy. A surprising number of genes specific to S. bayanus respond to oxidative stress, suggesting the organism may have evolved under different selection pressures than S. cerevisiae. This work expands the scope of genome-scale evolutionary studies from sequence-based analysis to rapid experimental characterization and could be adopted for functional mapping in any lineage of interest. Furthermore, our detailed characterization of S. bayanus provides a valuable resource for comparative functional genomics studies in yeast.

A microRNA network regulates proliferative timing and extracellular matrix synthesis during cellular quiescence in fibroblasts.

BACKGROUND: Although quiescence (reversible cell cycle arrest) is a key part in the life history and fate of many mammalian cell types, the mechanisms of gene regulation in quiescent cells are poorly understood. We sought to clarify the role of microRNAs as regulators of the cellular functions of quiescent human fibroblasts. RESULTS: Using microarrays, we discovered that the expression of the majority of profiled microRNAs differed between proliferating and quiescent fibroblasts. Fibroblasts induced into quiescence by contact inhibition or serum starvation had similar microRNA profiles, indicating common changes induced by distinct quiescence signals. By analyzing the gene expression patterns of microRNA target genes with quiescence, we discovered a strong regulatory function for miR-29, which is downregulated with quiescence. Using microarrays and immunoblotting, we confirmed that miR-29 targets genes encoding collagen and other extracellular matrix proteins and that those target genes are induced in quiescence. In addition, overexpression of miR-29 resulted in more rapid cell cycle re-entry from quiescence. We also found that let-7 and miR-125 were upregulated in quiescent cells. Overexpression of either one alone resulted in slower cell cycle re-entry from quiescence, while the combination of both together slowed cell cycle re-entry even further. CONCLUSIONS: microRNAs regulate key aspects of fibroblast quiescence including the proliferative state of the cells as well as their gene expression profiles, in particular, the induction of extracellular matrix proteins in quiescent fibroblasts.

Staying alive: metabolic adaptations to quiescence.

Quiescence is a state of reversible cell cycle arrest that can grant protection against many environmental insults. In some systems, cellular quiescence is associated with a low metabolic state characterized by a decrease in glucose uptake and glycolysis, reduced translation rates and activation of autophagy as a means to provide nutrients for survival. For cells in multiple different quiescence model systems, including Saccharomyces cerevisiae, mammalian lymphocytes and hematopoietic stem cells, the PI3Kinase/TOR signaling pathway helps to integrate information about nutrient availability with cell growth rates. Quiescence signals often inactivate the TOR kinase, resulting in reduced cell growth and biosynthesis. However, quiescence is not always associated with reduced metabolism; it is also possible to achieve a state of cellular quiescence in which glucose uptake, glycolysis and flux through central carbon metabolism are not reduced. In this review, we compare and contrast the metabolic changes that occur with quiescence in different model systems.