Participant recruitment challenges pervade the majority of publicly funded clinical trials. However, little is known about methods for enhancing participant accrual. The Epilepsy Phenome/Genome Project (EPGP), a multicenter study funded by the National Institute of Neurological Disorders and Stroke (NINDS), aimed to enroll a total of 5,250 participants to better understand the genetic causes and phenotypic manifestations of epilepsy. However, similar to other trials, EPGP encountered recruitment challenges, and by the end of its first year, net enrollment was only 48% of the target for that time. To address this, EPGP established a National Participant Recruitment Campaign and began implementing and tracking the enrollment outcomes of a variety of proven and relatively novel recruitment methods. At the conclusion of the project, EPGP had successfully enrolled a total of 5,445 participants, thus surpassing its enrollment target. Data pertaining to EPGP's National Participant Recruitment Campaign was analyzed retrospectively, and the results are reported here, so that other multicenter trials may consider these methods in their recruitment planning and potentially avoid the costly repercussions of participant accrual issues.

Most studies of HIV latency focus on the peripheral population of resting memory T cells, but the brain also contains a distinct reservoir of HIV-infected cells in microglia, perivascular macrophages, and astrocytes. Studying HIV in the brain has been challenging, since live cells are difficult to recover from autopsy samples and primate models of SIV infection utilize viruses that are more myeloid-tropic than HIV due to the expression of Vpx. Development of a realistic small animal model would greatly advance studies of this important reservoir and permit definitive studies of HIV latency. When radiation or busulfan-conditioned, immune-deficient NSG mice are transplanted with human hematopoietic stem cells, human cells from the bone marrow enter the brain and differentiate to express microglia-specific markers. After infection with replication competent HIV, virus was detected in these bone marrow-derived human microglia. Studies of HIV latency in this model would be greatly enhanced by the development of compounds that can selectively reverse HIV latency in microglial cells. Our studies have identified members of the CoREST repression complex as key regulators of HIV latency in microglia in both rat and human microglial cell lines. The monoamine oxidase (MAO) and potential CoREST inhibitor, phenelzine, which is brain penetrant, was able to stimulate HIV production in human microglial cell lines and human glial cells recovered from the brains of HIV-infected humanized mice. The humanized mice we have developed therefore show great promise as a model system for the development of strategies aimed at defining and reducing the CNS reservoir.

The bromodomain protein Brd4 promotes HIV-1 latency by competitively inhibiting P-TEFb-mediated transcription induced by the virus-encoded Tat protein. Brd4 is recruited to the HIV LTR by interactions with acetyl-histones3 (AcH3) and AcH4. However, the precise modification pattern that it reads and the writer for generating this pattern are unknown. By examining a pool of latently infected proviruses with diverse integration sites, we found that the LTR characteristically has low AcH3 but high AcH4 content. This unusual acetylation profile attracts Brd4 to suppress the interaction of Tat with the host super elongation complex (SEC) that is essential for productive HIV transcription and latency reversal. KAT5 (lysine acetyltransferase 5), but not its paralogs KAT7 and KAT8, is found to promote HIV latency through acetylating H4 on the provirus. Antagonizing KAT5 removes AcH4 and Brd4 from the LTR, enhances the SEC loading, and reverses as well as delays, the establishment of latency. The pro-latency effect of KAT5 is confirmed in a primary CD4+ T cell latency model as well as cells from ART-treated patients. Our data thus indicate the KAT5-AcH4-Brd4 axis as a key regulator of latency and a potential therapeutic target to reactivate latent HIV reservoirs for eradication.

The latent HIV reservoir is generated following HIV infection of activated effector CD4 T cells, which then transition to a memory phenotype. Here, we describe an ex vivo method, called QUECEL (quiescent effector cell latency), that mimics this process efficiently and allows production of large numbers of latently infected CD4⁺ T cells. Naïve CD4⁺ T cells were polarized into the four major T cell subsets (Th1, Th2, Th17, and Treg) and subsequently infected with a single-round reporter virus which expressed GFP/CD8a. The infected cells were purified and coerced into quiescence using a defined cocktail of cytokines, including tumor growth factor beta, interleukin-10 (IL-10), and IL-8, producing a homogeneous population of latently infected cells. Flow cytometry and transcriptome sequencing (RNA-Seq) demonstrated that the cells maintained the correct polarization phenotypes and had withdrawn from the cell cycle. Key pathways and gene sets enriched during transition from quiescence to reactivation include E2F targets, G₂M checkpoint, estrogen response late gene expression, and c-myc targets. Reactivation of HIV by latency-reversing agents (LRAs) closely mimics RNA induction profiles seen in cells from well-suppressed HIV patient samples using the envelope detection of in vitro transcription sequencing (EDITS) assay. Since homogeneous populations of latently infected cells can be recovered, the QUECEL model has an excellent signal-to-noise ratio and has been extremely consistent and reproducible in numerous experiments performed during the last 4 years. The ease, efficiency, and accuracy of the mimicking of physiological conditions make the QUECEL model a robust and reproducible tool to study the molecular mechanisms underlying HIV latency.IMPORTANCE Current primary cell models for HIV latency correlate poorly with the reactivation behavior of patient cells. We have developed a new model, called QUECEL, which generates a large and homogenous population of latently infected CD4⁺ memory cells. By purifying HIV-infected cells and inducing cell quiescence with a defined cocktail of cytokines, we have eliminated the largest problems with previous primary cell models of HIV latency: variable infection levels, ill-defined polarization states, and inefficient shutdown of cellular transcription. Latency reversal in the QUECEL model by a wide range of agents correlates strongly with RNA induction in patient samples. This scalable and highly reproducible model of HIV latency will permit detailed analysis of cellular mechanisms controlling HIV latency and reactivation.

Unbiased shRNA library screens revealed that the estrogen receptor-1 (ESR-1) is a key factor regulating HIV-1 latency. In both Jurkat T cells and a Th17 primary cell model for HIV-1 latency, selective estrogen receptor modulators (SERMs, i.e., fulvestrant, raloxifene, and tamoxifen) are weak proviral activators and sensitize cells to latency-reversing agents (LRAs) including low doses of TNF-α (an NF-κB inducer), the histone deacetylase inhibitor vorinostat (soruberoylanilide hydroxamic acid, SAHA), and IL-15. To probe the physiologic relevance of these observations, leukapheresis samples from a cohort of 12 well-matched reproductive-age women and men on fully suppressive antiretroviral therapy were evaluated by an assay measuring the production of spliced envelope (env) mRNA (the EDITS assay) by next-generation sequencing. The cells were activated by T cell receptor (TCR) stimulation, IL-15, or SAHA in the presence of either β-estradiol or an SERM. β-Estradiol potently inhibited TCR activation of HIV-1 transcription, while SERMs enhanced the activity of most LRAs. Although both sexes responded to SERMs and β-estradiol, females showed much higher levels of inhibition in response to the hormone and higher reactivity in response to ESR-1 modulators than males. Importantly, the total inducible RNA reservoir, as measured by the EDITS assay, was significantly smaller in the women than in the men. We conclude that concurrent exposure to estrogen is likely to limit the efficacy of viral emergence from latency and that ESR-1 is a pharmacologically attractive target that can be exploited in the design of therapeutic strategies for latency reversal.

Control of weeds in cultivated crops is a pivotal component in successful crop production allowing higher yield and higher quality. In rice-growing regions worldwide, weedy rice (Oryza sativa f. spontanea Rosh.) is a weed related to cultivated rice which infests rice fields. With populations across the globe evolving a suite of phenotypic traits characteristic of weeds and of cultivated rice, varying hypotheses exist on the origin of weedy rice. Here, we investigated the genetic diversity and possible origin of weedy rice in California using 98 simple sequence repeat (SSR) markers and an Rc gene-specific marker. By employing phylogenetic clustering analysis, we show that four to five genetically distinct biotypes of weedy rice exist in California. Analysis of population structure and genetic distance among individuals reveals diverse evolutionary origins of California weedy rice biotypes, with ancestry derived from indica, aus, and japonica cultivated rice as well as possible contributions from weedy rice from the southern United States and wild rice. Because this diverse parentage primarily consists of weedy, wild, and cultivated rice not found in California, most existing weedy rice biotypes likely originated outside California.

Two-dimensional materials constitute a promising platform for developing nanoscale devices and systems. Their physical properties can be very different from those of the corresponding three-dimensional materials because of extreme quantum confinement and dimensional reduction. Here we report a study of TiTe₂ from the single-layer to the bulk limit. Using angle-resolved photoemission spectroscopy and scanning tunneling microscopy and spectroscopy, we observed the emergence of a (2 × 2) charge density wave order in single-layer TiTe₂ with a transition temperature of 92 ± 3 K. Also observed was a pseudogap of about 28 meV at the Fermi level at 4.2 K. Surprisingly, no charge density wave transitions were observed in two-layer and multi-layer TiTe₂, despite the quasi-two-dimensional nature of the material in the bulk. The unique charge density wave phenomenon in the single layer raises intriguing questions that challenge the prevailing thinking about the mechanisms of charge density wave formation.Due to reduced dimensionality, the properties of 2D materials are often different from their 3D counterparts. Here, the authors identify the emergence of a unique charge density wave (CDW) order in monolayer TiTe₂ that challenges the current understanding of CDW formation.

Hermaphroditic (perfect) flowers were a key trait in grapevine domestication, enabling a drastic increase in yields due to the efficiency of self-pollination in the domesticated grapevine (Vitis vinifera L. ssp. vinifera). In contrast, all extant wild Vitis species are dioecious, each plant having only male or female flowers. In this study, we identified the male (M) and female (f) haplotypes of the sex-determining region (SDR) in the wild grapevine species V. cinerea and confirmed the boundaries of the SDR. We also demonstrated that the SDR and its boundaries are precisely conserved across the Vitis genus using shotgun resequencing data of 556 wild and domesticated accessions from North America, East Asia, and Europe. A high linkage disequilibrium was found at the SDR in all wild grape species, while different recombination signatures were observed along the hermaphrodite (H) haplotype of 363 cultivated accessions, revealing two distinct H haplotypes, named H1 and H2. To further examine the H2 haplotype, we sequenced the genome of two grapevine cultivars, 'Riesling' and 'Chardonnay'. By reconstructing the first two H2 haplotypes, we estimated the divergence time between H1 and H2 haplotypes at ∼6 million years ago, which predates the domestication of grapevine (∼8,000 y ago). Our findings emphasize the important role of recombination suppression in maintaining dioecy in wild grape species and lend additional support to the hypothesis that at least two independent recombination events led to the reversion to hermaphroditism in grapevine.