Collective Identity: Making the Case for a Stage Model Approach to Addressing Water Quality in the Chesapeake Bay Watershed.

The Chesapeake Bay watershed encompasses six states and the District of Columbia. Consequently, the people within it display great diversity in terms of values, allegiances, and experiences. That diversity may help to explain an apparent inability to coordinate actions aimed at redressing the dismal water quality throughout the watershed. In this paper, we bridge theory to an applied scenario to examine the importance of developing a collective identity within the watershed to bring about changes in individual behavior and policies. We present the current conditions of the Chesapeake Bay watershed, propose a stage model for the development of a collective watershed identity, outline theoretically grounded determinants of each stage, and discuss the challenges in developing a collective identity. We further suggest several guiding questions for future research.

Complete characterization of the human immune cell transcriptome using accurate full-length cDNA sequencing.

The human immune system relies on highly complex and diverse transcripts and the proteins they encode. These include transcripts encoding human leukocyte antigen (HLA) receptors as well as B cell and T cell receptors (BCR and TCR). Determining which alleles an individual possesses for each HLA gene (high-resolution HLA typing) is essential to establish donor-recipient compatibility in organ and bone marrow transplantations. In turn, the repertoires of millions of unique BCR and TCR transcripts in each individual carry a vast amount of health-relevant information. Both short-read RNA-seq-based HLA typing and BCR/TCR repertoire sequencing (AIRR-seq) currently rely on our incomplete knowledge of the genetic diversity at HLA and BCR/TCR loci. Here, we generated over 10,000,000 full-length cDNA sequences at a median accuracy of 97.9% using our nanopore sequencing-based Rolling Circle Amplification to Concatemeric Consensus (R2C2) protocol. We used this data set to (1) show that deep and accurate full-length cDNA sequencing can be used to provide isoform-level transcriptome analysis for more than 9000 loci, (2) generate accurate sequences of HLA alleles, and (3) extract detailed AIRR data for the analysis of the adaptive immune system. The HLA and AIRR analysis approaches we introduce here are untargeted and therefore do not require prior knowledge of the composition or genetic diversity of HLA and BCR/TCR loci.

Media Contributions to a Chesapeake Bay Watershed Collective Identity? A Tale of Three Cities.

Although collective action is needed to address many environmental challenges, it cannot proceed in the absence of collective identity, that is, evidence of group belongingness expressed in or via communicative behavior. This study looked for evidence of a collective identity in newspaper articles that referenced the Chesapeake Bay Watershed. The data were drawn from local papers published in municipalities located at the headwaters of the Susquehanna River, midway down the Susquehanna, and where the river meets the Bay. Computerized content analysis assessed the frequency with which the Chesapeake Bay and watershed were mentioned alongside a set of keywords thought to represent different facets of identity (e.g., agriculture, fishing, swimming). The results showed substantial variation in frequency across time and place but low absolute levels of coverage of the Bay and the watershed. Multidimensional scaling revealed different structures to collective identity as a function of place. These differences in content may be attributable to varying demographic and environmental characteristics along with proximity to the Bay. But, to the extent that media contribute to collective identity among residents of the watershed at all, they do so in a complex and heterogeneous manner.

A New Diploid Parthenogenetic Whiptail Lizard from Sonora, Mexico, Is the "Missing Link" in the Evolutionary Transition to Polyploidy.

AbstractTransitions between sexual and unisexual reproductive modes have significant consequences for the evolutionary trajectories of species. These transitions have occurred numerous times in vertebrates and are frequently mediated by hybridization events. Triploid unisexual vertebrates are thought to arise through hybridization between individuals of a diploid unisexual lineage and a sexual species, although additional evidence that confirms this mechanism is needed in numerous groups. North American whiptail lizards (Aspidoscelis) are notable for being one of the largest radiations of unisexual vertebrates, and the most diverse group of Aspidoscelis includes numerous triploid lineages that have no known diploid unisexual ancestors. This pattern of "missing" ancestors may result from the short evolutionary life span of unisexual lineages or the selective advantages of polyploidy, or it could suggest that alternative mechanisms of triploid formation are operating in nature. We leverage genomic, morphological, and karyotypic data to describe a new diploid unisexual whiptail and show that it is likely the unisexual progenitor of an extant triploid lineage, A. opatae. We also resolve patterns of polyploidization within the A. sexlineatus species group and test predictions about the phenotypic outcomes of hybridization.

Effectiveness of small group cognitive behavioural therapy for anxiety and depression in Ebola treatment centre staff in Sierra Leone.

Following the 2014 Ebola outbreak, South London and Maudsley NHS Foundation Trust (SLAM) were commissioned to provide a 'culturally appropriate, effective and sustainable' intervention to address the psychological needs of the Sierra Leonean Ebola Treatment Centre (ETC) staff. The study evaluated the effectiveness of group Cognitive Behavioural Therapy (CBT) developed to treat anxiety, depression and functional impairment amongst a sample of former ETC staff in Sierra Leone. Group comparisons explored whether the effect of the intervention differed dependent on the facilitators that delivered it, as well as whether it differed between former staff of high- and low-risk ETC roles. A sample of 253 former ETC staff attended the group CBT intervention comprised of eight sessions over six weeks. Outcome measures were administered upon entry and within two weeks after the intervention. At post-intervention, anxiety, depression and functional impairment significantly reduced. Reading ability (RA) was introduced as a covariate having impacted the outcomes. The intervention effect differed by facilitators delivering the sessions but not by ETC role risk. The implications of these results are discussed. Group CBT is a promising psychological intervention for treating the anxiety, depression and functional impairment of former ETC staff in Sierra Leone. Furthermore, as part of a stepped-care approach, it may provide a model for psychological support for staff that have worked on the frontline during future epidemics.

Improving nanopore read accuracy with the R2C2 method enables the sequencing of highly multiplexed full-length single-cell cDNA.

High-throughput short-read sequencing has revolutionized how transcriptomes are quantified and annotated. However, while Illumina short-read sequencers can be used to analyze entire transcriptomes down to the level of individual splicing events with great accuracy, they fall short of analyzing how these individual events are combined into complete RNA transcript isoforms. Because of this shortfall, long-distance information is required to complement short-read sequencing to analyze transcriptomes on the level of full-length RNA transcript isoforms. While long-read sequencing technology can provide this long-distance information, there are issues with both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) long-read sequencing technologies that prevent their widespread adoption. Briefly, PacBio sequencers produce low numbers of reads with high accuracy, while ONT sequencers produce higher numbers of reads with lower accuracy. Here, we introduce and validate a long-read ONT-based sequencing method. At the same cost, our Rolling Circle Amplification to Concatemeric Consensus (R2C2) method generates more accurate reads of full-length RNA transcript isoforms than any other available long-read sequencing method. These reads can then be used to generate isoform-level transcriptomes for both genome annotation and differential expression analysis in bulk or single-cell samples.

Tn5Prime, a Tn5 based 5' capture method for single cell RNA-seq.

RNA-sequencing (RNA-seq) is a powerful technique to investigate and quantify entire transcriptomes. Recent advances in the field have made it possible to explore the transcriptomes of single cells. However, most widely used RNA-seq protocols fail to provide crucial information regarding transcription start sites. Here we present a protocol, Tn5Prime, that takes advantage of the Tn5 transposase-based Smart-seq2 protocol to create RNA-seq libraries that capture the 5' end of transcripts. The Tn5Prime method dramatically streamlines the 5' capture process and is both cost effective and reliable. By applying Tn5Prime to bulk RNA and single cell samples, we were able to define transcription start sites as well as quantify transcriptomes at high accuracy and reproducibility. Additionally, similar to 3' end-based high-throughput methods like Drop-seq and 10× Genomics Chromium, the 5' capture Tn5Prime method allows the introduction of cellular identifiers during reverse transcription, simplifying the analysis of large numbers of single cells. In contrast to 3' end-based methods, Tn5Prime also enables the assembly of the variable 5' ends of the antibody sequences present in single B-cell data. Therefore, Tn5Prime presents a robust tool for both basic and applied research into the adaptive immune system and beyond.

A ligation-based single-stranded library preparation method to analyze cell-free DNA and synthetic oligos.

BACKGROUND: Cell-free DNA (cfDNA), present in circulating blood plasma, contains information about prenatal health, organ transplant reception, and cancer presence and progression. Originally developed for the genomic analysis of highly degraded ancient DNA, single-stranded DNA (ssDNA) library preparation methods are gaining popularity in the field of cfDNA analysis due to their efficiency and ability to convert short, fragmented DNA into sequencing libraries without altering DNA ends. However, current ssDNA methods are costly and time-consuming. RESULTS: Here we present an efficient ligation-based single-stranded library preparation method that is engineered to produce complex libraries in under 2.5 h from as little as 1 nanogram of input DNA without alteration to the native ends of template molecules. Our method, called Single Reaction Single-stranded LibrarY or SRSLY, ligates uniquely designed Next-Generation Sequencing (NGS) adapters in a one-step combined phosphorylation/ligation reaction that foregoes end-polishing. Using synthetic DNA oligos and cfDNA, we demonstrate the efficiency and utility of this approach and compare with existing double-stranded and single-stranded approaches for library generation. Finally, we demonstrate that cfDNA NGS data generated from SRSLY can be used to analyze DNA fragmentation patterns to deduce nucleosome positioning and transcription factor binding. CONCLUSIONS: SRSLY is a versatile tool for converting short and fragmented DNA molecules, like cfDNA fragments, into sequencing libraries while retaining native lengths and ends.

The Dbp5 cycle at the nuclear pore complex during mRNA export II: nucleotide cycling and mRNP remodeling by Dbp5 are controlled by Nup159 and Gle1.

Essential messenger RNA (mRNA) export factors execute critical steps to mediate directional transport through nuclear pore complexes (NPCs). At cytoplasmic NPC filaments, the ATPase activity of DEAD-box protein Dbp5 is activated by inositol hexakisphosphate (IP(6))-bound Gle1 to mediate remodeling of mRNA-protein (mRNP) complexes. Whether a single Dbp5 executes multiple remodeling events and how Dbp5 is recycled are unknown. Evidence suggests that Dbp5 binding to Nup159 is required for controlling interactions with Gle1 and the mRNP. Using in vitro reconstitution assays, we found here that Nup159 is specifically required for ADP release from Dbp5. Moreover, Gle1-IP(6) stimulates ATP binding, thus priming Dbp5 for RNA loading. In vivo, a dbp5-R256D/R259D mutant with reduced ADP binding bypasses the need for Nup159 interaction. However, NPC spatial control is important, as a dbp5-R256D/R259D nup42Δ double mutant is temperature-sensitive for mRNA export. Further analysis reveals that remodeling requires a conformational shift to the Dbp5-ADP form. ADP release factors for DEAD-box proteins have not been reported previously and reflect a new paradigm for regulation. We propose a model wherein Nup159 and Gle1-IP(6) regulate Dbp5 cycles by controlling its nucleotide-bound state, allowing multiple cycles of mRNP remodeling by a single Dbp5 at the NPC.

The Dbp5 cycle at the nuclear pore complex during mRNA export I: dbp5 mutants with defects in RNA binding and ATP hydrolysis define key steps for Nup159 and Gle1.

Nuclear export of messenger RNA (mRNA) occurs by translocation of mRNA/protein complexes (mRNPs) through nuclear pore complexes (NPCs). The DEAD-box protein Dbp5 mediates export by triggering removal of mRNP proteins in a spatially controlled manner. This requires Dbp5 interaction with Nup159 in NPC cytoplasmic filaments and activation of Dbp5's ATPase activity by Gle1 bound to inositol hexakisphosphate (IP(6)). However, the precise sequence of events within this mechanism has not been fully defined. Here we analyze dbp5 mutants that alter ATP binding, ATP hydrolysis, or RNA binding. We found that ATP binding and hydrolysis are required for efficient Dbp5 association with NPCs. Interestingly, mutants defective for RNA binding are dominant-negative (DN) for mRNA export in yeast and human cells. We show that the DN phenotype stems from competition with wild-type Dbp5 for Gle1 at NPCs. The Dbp5-Gle1 interaction is limiting for export and, importantly, can be independent of Nup159. Fluorescence recovery after photobleaching experiments in yeast show a very dynamic association between Dbp5 and NPCs, averaging <1 sec, similar to reported NPC translocation rates for mRNPs. This work reveals critical steps in the Gle1-IP(6)/Dbp5/Nup159 cycle, and suggests that the number of remodeling events mediated by a single Dbp5 is limited.

Molecular tools that block maturation of the nuclear lamin A and decelerate cancer cell migration.

Lamin A contributes to the structure of nuclei in all mammalian cells and plays an important role in cell division and migration. Mature lamin A is derived from a farnesylated precursor protein, known as prelamin A, which undergoes post-translational cleavage catalyzed by the zinc metalloprotease STE24 (ZPMSTE24). Accumulation of farnesylated prelamin A in the nuclear envelope compromises cell division, impairs mitosis and induces an increased expression of inflammatory gene products. ZMPSTE24 has been proposed as a potential therapeutic target in oncology. A library of peptidomimetic compounds were synthesized and screened for their ability to induce accumulation of prelamin A in cancer cells and block cell migration in pancreatic ductal adenocarcinoma cells. The results of this study suggest that inhibitors of lamin A maturation may interfere with cell migration, the biological process required for cancer metastasis.

Highly Accurate Sequencing of Full-Length Immune Repertoire Amplicons Using Tn5-Enabled and Molecular Identifier-Guided Amplicon Assembly.

Ab repertoire sequencing is a powerful tool to analyze the adaptive immune system. To sequence entire Ab repertoires, amplicons are created from Ab H chain (IgH) transcripts and sequenced on a high-throughput sequencer. The field of immune repertoire sequencing is growing rapidly and the protocols used are steadily improving; however, thus far, immune repertoire sequencing protocols have not been able to sequence full-length immune repertoires including the entire IgH V region and enough of the IgH C region to identify isotype subtypes. In this study, we present a method that combines Tn5 transposase and molecular identifiers for the highly accurate sequencing of amplicons >500 bp using Illumina short read paired-end sequencing. We then apply this method to Ab H chain amplicons to sequence the first, to our knowledge, highly accurate full-length immune repertoire.

Therapeutic leukocytapheresis in infants and children with leukemia and hyperleukocytosis: A single institution experience.

BACKGROUND: Hyperleukocytosis, defined as white blood cell (WBC) count above 100 × 109 /L, has high early morbidity and mortality from leukostasis-related complications, namely intracranial hemorrhage and pulmonary distress. Initiating chemotherapy without prior leukocytoreduction may lead to tumor lysis syndrome (TLS). Therapeutic leukocytapheresis (TL) is used as one leukocytoreductive intervention; however, its safety and efficacy in pediatric leukemia has not been established. The purpose of this study is to evaluate safety of TL in pediatric patients and assess the efficacy of TL in reducing WBC count in pediatric leukemia. METHODS: Retrospective chart review was conducted on 14 patients with acute lymphoblastic leukemia (ALL) and 5 with acute myeloid leukemia (AML) who underwent TL during the period 2000-2014 at a single institution. RESULTS: Mean WBC count of 19 patients who received TL was 483.2 × 109 /L (547.1 in ALL, 304.3 in AML); a portion of patients presented with central nervous system symptoms (15%), respiratory symptoms (10%), or both (10%). TL reduced WBC count (mean 50.7% reduction after a single TL procedure; additional 17.1% reduction after a second TL procedure in 6 patients). Short-term survival immediately following TL was 100% without any major procedural complication. Mean survival time in patients with AML was 1.5 years and with ALL was 6.5 years. CONCLUSIONS: TL significantly reduces WBC number in pediatric leukemia patients as young as 22 days old. In our retrospective study, TL was not associated with any significant complications and suggests that TL is a safe initial procedure in pediatric leukemia.

Congenital heart disease linked to maternal autoimmunity against cardiac myosin.

Structural congenital heart disease (CHD) has not previously been linked to autoimmunity. In our study, we developed an autoimmune model of structural CHD that resembles hypoplastic left heart syndrome (HLHS), a life-threatening CHD primarily affecting the left ventricle. Because cardiac myosin (CM) is a dominant autoantigen in autoimmune heart disease, we hypothesized that immunization with CM might lead to transplacental passage of maternal autoantibodies and a prenatal HLHS phenotype in exposed fetuses. Elevated anti-CM autoantibodies in maternal and fetal sera, as well as IgG reactivity in fetal myocardium, were correlated with structural CHD that included diminished left ventricular cavity dimensions in the affected progeny. Further, fetuses that developed a marked HLHS phenotype had elevated serum titers of anti-ß-adrenergic receptor Abs, as well as increased protein kinase A activity, suggesting a potential mechanism for the observed pathological changes. Our maternal-fetal model presents a new concept linking autoimmunity against CM and cardiomyocyte proliferation with cardinal features of HLHS. To our knowledge, this report shows the first evidence in support of a novel immune-mediated mechanism for pathogenesis of structural CHD that may have implications in its future diagnosis and treatment.

Yeast Integral Membrane Proteins Apq12, Brl1, and Brr6 Form a Complex Important for Regulation of Membrane Homeostasis and Nuclear Pore Complex Biogenesis.

Proper functioning of intracellular membranes is critical for many cellular processes. A key feature of membranes is their ability to adapt to changes in environmental conditions by adjusting their composition so as to maintain constant biophysical properties, including fluidity and flexibility. Similar changes in the biophysical properties of membranes likely occur when intracellular processes, such as vesicle formation and fusion, require dramatic changes in membrane curvature. Similar modifications must also be made when nuclear pore complexes (NPCs) are constructed within the existing nuclear membrane, as occurs during interphase in all eukaryotes. Here we report on the role of the essential nuclear envelope/endoplasmic reticulum (NE/ER) protein Brl1 in regulating the membrane composition of the NE/ER. We show that Brl1 and two other proteins characterized previously-Brr6, which is closely related to Brl1, and Apq12-function together and are required for lipid homeostasis. All three transmembrane proteins are localized to the NE and can be coprecipitated. As has been shown for mutations affecting Brr6 and Apq12, mutations in Brl1 lead to defects in lipid metabolism, increased sensitivity to drugs that inhibit enzymes involved in lipid synthesis, and strong genetic interactions with mutations affecting lipid metabolism. Mutations affecting Brl1 or Brr6 or the absence of Apq12 leads to hyperfluid membranes, because mutant cells are hypersensitive to agents that increase membrane fluidity. We suggest that the defects in nuclear pore complex biogenesis and mRNA export seen in these mutants are consequences of defects in maintaining the biophysical properties of the NE.

The myocardial and coronary histopathology and pathogenesis of hypoplastic left heart syndrome.

Hypoplastic left heart syndrome has the greatest mortality rate among all CHDs and without palliation is uniformly fatal. Despite noble efforts, the aetiology of this syndrome is unknown and a cure remains elusive. The genetic and anatomic heterogeneity of hypoplastic left heart syndrome supports a rethinking of old hypotheses and warrants further investigation into the histological and vascular variations recognised with this syndrome. In an effort to elucidate the pathogenesis of hypoplastic left heart syndrome, this review will focus on its unique myocardial and coronary pathology as well as evaluate the association of hypoplastic left heart syndrome with the endocardial fibroelastosis reaction.

Dissociation mediates the relationship between childhood maltreatment and subclinical psychosis.

More than a third of the population report childhood adversity, and these experiences are associated with an increased risk of clinical and subclinical psychosis. The reason why some people go on to develop mental health problems and others do not is a key question for study. It has been hypothesized that dissociative processes mediate the relationship between early adversity and psychosis. The current study assessed whether dissociation, and specifically depersonalization (one component of dissociation), plays a mediating role in the relationship between childhood maltreatment and both hallucination proneness and delusional ideation. The study used a cross-sectional design and recruited a student sample to assess these relationships in a nonclinical group. Dissociation mediated the relationship between early maltreatment and both hallucination proneness and delusional ideation. In terms of specific dissociative processes, depersonalization did not mediate hallucination proneness or delusional ideation. Absorption mediated hallucination proneness; dissociative amnesia (negatively) and absorption mediated delusional ideation. It is likely that dissociation interferes with the encoding of traumatic information in nonclinical as well as clinical groups and in certain ways. Absorption may be particularly relevant. For some people, traumatic memories may intrude into conscious awareness in adulthood as psychotic-type experience.

Non-synonymous variations in cancer and their effects on the human proteome: workflow for NGS data biocuration and proteome-wide analysis of TCGA data.

BACKGROUND: Next-generation sequencing (NGS) technologies have resulted in petabytes of scattered data, decentralized in archives, databases and sometimes in isolated hard-disks which are inaccessible for browsing and analysis. It is expected that curated secondary databases will help organize some of this Big Data thereby allowing users better navigate, search and compute on it. RESULTS: To address the above challenge, we have implemented a NGS biocuration workflow and are analyzing short read sequences and associated metadata from cancer patients to better understand the human variome. Curation of variation and other related information from control (normal tissue) and case (tumor) samples will provide comprehensive background information that can be used in genomic medicine research and application studies. Our approach includes a CloudBioLinux Virtual Machine which is used upstream of an integrated High-performance Integrated Virtual Environment (HIVE) that encapsulates Curated Short Read archive (CSR) and a proteome-wide variation effect analysis tool (SNVDis). As a proof-of-concept, we have curated and analyzed control and case breast cancer datasets from the NCI cancer genomics program - The Cancer Genome Atlas (TCGA). Our efforts include reviewing and recording in CSR available clinical information on patients, mapping of the reads to the reference followed by identification of non-synonymous Single Nucleotide Variations (nsSNVs) and integrating the data with tools that allow analysis of effect nsSNVs on the human proteome. Furthermore, we have also developed a novel phylogenetic analysis algorithm that uses SNV positions and can be used to classify the patient population. The workflow described here lays the foundation for analysis of short read sequence data to identify rare and novel SNVs that are not present in dbSNP and therefore provides a more comprehensive understanding of the human variome. Variation results for single genes as well as the entire study are available from the CSR website (http://hive.biochemistry.gwu.edu/dna.cgi?cmd=csr). CONCLUSIONS: Availability of thousands of sequenced samples from patients provides a rich repository of sequence information that can be utilized to identify individual level SNVs and their effect on the human proteome beyond what the dbSNP database provides.

The effectiveness and tolerability of trauma-focused psychotherapies for psychotic symptoms: A systematic review of trauma-focused psychotherapies.

INTRODUCTION: Psychological trauma is an established risk factor for psychosis. Trauma-focused psychotherapies (TFPT) have been suggested as a potential treatment for reducing psychotic symptoms in those who have experienced trauma. We therefore sought to investigate the effectiveness, tolerability, and acceptability of TFPT for psychotic symptoms. METHODS: We conducted a systematic review of studies of any form of TFPT that measured psychotic symptoms across a broad range of diagnoses. RESULTS: From 2584 papers initially identified, 17 studies (857 participants) met eligibility criteria. TFPT were found to be well tolerated, with very few adverse events. Acceptability was also high, with a mean dropout rate of 20%. CONCLUSIONS: Whilst the evidence of effectiveness for TFPT in reducing psychotic symptoms is weak, we found tentative evidence in favour of exposure-based interventions. Methodologically rigorous trials investigating the efficacy of TFPT for the treatment of psychotic symptoms are needed to assess this promising intervention.

Transport of messenger RNA from the nucleus to the cytoplasm.

All movement of molecules and macromolecules between the cytoplasm and the nucleus takes place through nuclear pore complexes (NPCs), very large macromolecular complexes that are the only channels connecting these compartments. mRNA export is mediated by multiple, highly conserved protein factors that couple steps of nuclear pre-mRNA biogenesis to mRNA transport. Mature messenger ribonucleoproteins (mRNPs) diffuse from sites of transcription to NPCs, although some active genes are positioned at the nuclear periphery where they interact physically with components of NPCs. As properly processed mRNPs translocate through the pore, certain mRNP proteins are removed, probably through the enzymatic action of the DEAD-box helicase Dbp5, which binds to Nup159 and Gle1, components of the cytoplasmic filaments of the NPC. Gle1 and the phosphoinositide IP6 activate Dbp5's ATPase activity in vitro and this could provide critical spatial regulation of Dbp5 activity in vivo.