Methods for a composite ecological suitability measure to inform cumulative restoration assessments in Gulf of Mexico estuaries.

Ecosystem management requires a systematic, holistic approach that considers ecological and social outcomes. Effective restoration practices promote a balance of ecological and social goals by addressing ecological integrity, efficiently maximizing benefits while minimizing investment, and encompassing collaborative stakeholder engagement. Socio-ecological assessments can inform adaptive management and be utilized to prioritize restoration activities and monitor restoration effectiveness. In estuarine systems, socio-ecological assessments should evaluate the ability of habitats to support both ecologically and locally important species. The composite measure presented utilizes a combination of ecological and social measures to characterize ecological suitability for individual and multiple Gulf of Mexico estuarine species. The ecological suitability value (ES) for a given spatial unit is based on a suite of biophysical measures of the quality and extent of suitable habitat for each species, the species' trophic importance in a food web context, and the importance of each species in relation to stakeholder values and benefits. ES values for individual spatial units can be aggregated to estimate the distribution of ecological suitability at the estuarine scale. The ES values are calculated using examples for each step in the process. The information provided by ecological suitability characterizations can support restoration prioritization decisions for Gulf of Mexico estuaries and can provide a baseline measure to gauge restoration effectiveness over time to inform cumulative restoration assessments.

Attention and Memory in Depersonalization-Spectrum Dissociative Disorders: Impact of Selective-Divided Attentional Condition, Stimulus Emotionality, and Stress.

We investigated cognition in depersonalization-spectrum dissociative disorders without comorbid posttraumatic stress disorder to explore evidence for emotionally avoidant information processing. Forty-eight participants with DSM-IV dissociative disorder (DD) (Depersonalization Disorder - 37, Dissociative Disorder NOS -11), 36 participants with Posttraumatic Stress Disorder (PTSD), and 56 healthy controls (HC) were administered the Weschler Adult Intelligence Scale-III (WAIS); the Weschler Memory Scale-III (WMS); and three Stroop tasks: the Standard Stroop, a selective-attention Emotional Stroop using neutral, dissociation, and trauma-related word categories, and a divided-attention Emotional Stroop using comparable words. Participants were also administered a paired-associates explicit and implicit memory test using emotionally neutral and negative words, before and after the Trier Social Stress Test. The DD and HC groups had comparable general intelligence and memory scores, though dissociation severity was inversely related to verbal comprehension and working memory. In the selective-attention condition, DD participants showed greater incidental recall across word categories with comparable interference. However in the divided-attention condition, DD participants significantly favored lesser attentional interference at the expense of remembering words. Across attentional conditions, DD participants had better recall for disorder-related than neutral words. Pre-stress, the DD group demonstrated better explicit memory for neutral versus negative words with reversal after stress, whereas the HC group demonstrated the opposite pattern; implicit memory did not differ. Cognition in the PTSD control group was generally dissimilar to the DD group. The findings in toto provide substantial evidence for emotionally avoidant information processing in DD, vulnerable to the impact of stress, at the level of both attention and memory.

A. thaliana Hybrids Develop Growth Abnormalities through Integration of Stress, Hormone and Growth Signaling.

Hybrids between Arabidopsis thaliana accessions are important in revealing the consequences of epistatic interactions in plants. F1 hybrids between the A. thaliana accessions displaying either defense or developmental phenotypes have been revealing the roles of the underlying epistatic genes. The interaction of two naturally occurring alleles of the OUTGROWTH-ASSOCIATED KINASE (OAK) gene in Sha and Lag2-2, previously shown to cause a similar phenotype in a different allelic combination in A. thaliana, was required for the hybrid phenotype. Outgrowth formation in the hybrids was associated with reduced levels of salicylic acid, jasmonic acid and abscisic acid in petioles and the application of these hormones mitigated the formation of the outgrowths. Moreover, different abiotic stresses were found to mitigate the outgrowth phenotype. The involvement of stress and hormone signaling in outgrowth formation was supported by a global transcriptome analysis, which additionally revealed that TCP1, a transcription factor known to regulate leaf growth and symmetry, was downregulated in the outgrowth tissue. These results demonstrate that a combination of natural alleles of OAK regulates growth and development through the integration of hormone and stress signals and highlight the importance of natural variation as a resource to discover the function of gene variants that are not present in the most studied accessions of A. thaliana.

Multiplexed Simian Immunodeficiency Virus-Specific Paired RNA-Guided Cas9 Nickases Inactivate Proviral DNA.

Human and simian immunodeficiency virus (HIV and SIV) infections establish lifelong reservoirs of cells harboring an integrated proviral genome. Genome editing CRISPR-associated Cas9 nucleases, combined with SIV-specific guiding RNA (gRNA) molecules, inactivate integrated provirus DNA in vitro and in animal models. We generated RNA-guided Cas9 nucleases (RGNu) and nickases (RGNi) targeting conserved SIV regions with no homology in the human or rhesus macaque genome. Assays in cells cotransfected with SIV provirus and plasmids coding for RGNus identified SIV long terminal repeat (LTR), trans-activation response (TAR) element, and ribosome slip site (RSS) regions as the most effective at virus suppression; RGNi targeting these regions inhibited virus production significantly. Multiplex plasmids that coexpressed these three RGNu (Nu3), or six (three pairs) RGNi (Ni6), were more efficient at virus suppression than any combination of individual RGNu and RGNi plasmids. Both Nu3 and Ni6 plasmids were tested in lymphoid cells chronically infected with SIVmac239, and whole-genome sequencing was used to determine on- and off-target mutations. Treatment with these all-in-one plasmids resulted in similar levels of mutations of viral sequences from the cellular genome; Nu3 induced indels at the 3 SIV-specific sites, whereas for Ni6 indels were present at the LTR and TAR sites. Levels of off-target effects detected by two different algorithms were indistinguishable from background mutations. In summary, we demonstrate that Cas9 nickase in association with gRNA pairs can specifically eliminate parts of the integrated provirus DNA; also, we show that careful design of an all-in-one plasmid coding for 3 gRNAs and Cas9 nuclease inhibits SIV production with undetectable off-target mutations, making these tools a desirable prospect for moving into animal studies. IMPORTANCE Our approach to HIV cure, utilizing the translatable SIV/rhesus macaque model system, aims at provirus inactivation and its removal with the least possible off-target side effects. We developed single molecules that delivered either three truncated SIV-specific gRNAs along with Cas9 nuclease or three pairs of SIV-specific gRNAs (six individual gRNAs) along with Cas9 nickase to enhance efficacy of on-target mutagenesis. Whole-genome sequencing demonstrated effective SIV sequence mutation and inactivation and the absence of demonstrable off-target mutations. These results open the possibility to employ Cas9 variants that introduce single-strand DNA breaks to eliminate integrated proviral DNA.

CrRLK1L receptor-like kinases HERK1 and ANJEA are female determinants of pollen tube reception.

Communication between the gametophytes is vital for angiosperm fertilisation. Multiple CrRLK1L-type receptor kinases prevent premature pollen tube burst, while another CrRLK1L protein, FERONIA (FER), is required for pollen tube reception in the female gametophyte. We report here the identification of two additional CrRLK1L homologues, HERCULES RECEPTOR KINASE 1 (HERK1) and ANJEA (ANJ), which act redundantly to promote pollen tube growth arrest at the synergid cells. HERK1 and ANJ localise to the filiform apparatus of the synergid cells in unfertilised ovules, and in herk1 anj mutants, a majority of ovules remain unfertilised due to pollen tube overgrowth, together indicating that HERK1 and ANJ act as female determinants for fertilisation. As in fer mutants, the synergid cell-specific, endomembrane protein NORTIA (NTA) is not relocalised after pollen tube reception; however, unlike fer mutants, reactive oxygen species levels are unaffected in herk1 anj double mutants. Both ANJ and HERK1 associate with FER and its proposed co-receptor LORELEI (LRE) in planta. Together, our data indicate that HERK1 and ANJ act with FER to mediate female-male gametophyte interactions during plant fertilisation.

A conceptual approach to characterizing ecological suitability: Informing socio-ecological measures for restoration effectiveness.

A combination of ecological and socio-economic outcome indicators is essential for understanding and assessing the effectiveness of the remediation and restoration of degraded ecosystems and revitalizing communities that could benefit from these ecosystem management activities. In this paper, we propose and develop a conceptual approach to characterize ecological suitability that incorporates ecological attributes that support ecosystem structural diversity and functionality, stakeholder values and perceptions, and the benefits derived from ecosystem goods and services. A structured literature review was used to identify existing restoration frameworks and indicators to inform the conceptual foundation for characterizing ecological suitability. The structure of the conceptual approach primarily builds from ecological and social attributes in the International Principles and Standards for the Practice of Ecological Restoration (Gann et al., 2019). We provide a conceptual example of the ecological suitability approach in estuaries. This example is based on habitat suitability and food web characterizations in combination with the provisioning of ecosystem services and desired social benefits to prioritize and evaluate restoration effectiveness. This foundational work sets the stage for developing a composite measure of ecological suitability. The holistic conceptual approach presented complements existing information regarding restoration effectiveness evaluations. Characterizing ecological suitability is a novel way to incorporate ecological and social information and communicate potential restoration outcomes to ecosystem managers and stakeholders.

Discovery of a Potent and Selective Covalent Inhibitor and Activity-Based Probe for the Deubiquitylating Enzyme UCHL1, with Antifibrotic Activity.

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a deubiquitylating enzyme that is proposed as a potential therapeutic target in neurodegeneration, cancer, and liver and lung fibrosis. Herein we report the discovery of the most potent and selective UCHL1 probe (IMP-1710) to date based on a covalent inhibitor scaffold and apply this probe to identify and quantify target proteins in intact human cells. IMP-1710 stereoselectively labels the catalytic cysteine of UCHL1 at low nanomolar concentration in cells. We further demonstrate that potent and selective UCHL1 inhibitors block pro-fibrotic responses in a cellular model of idiopathic pulmonary fibrosis, supporting the potential of UCHL1 as a potential therapeutic target in fibrotic diseases.

A malectin domain kinesin functions in pollen and seed development in Arabidopsis.

The kinesin family is greatly expanded in plants compared with animals and, with more than a third up-regulated in expression during cell division, it has been suggested that this expansion facilitated complex plant-specific cytoskeletal rearrangements. The cell cycle-regulated kinesins include two with an N-terminal malectin domain, a protein domain that has been shown to bind polysaccharides and peptides when found extracellularly in receptor-like kinases. Although malectin domain kinesins are evolutionarily deep rooted, their function in plants remains unclear. Here we show that loss of MALECTIN DOMAIN KINESIN 2 (MDKIN2) results in stochastic developmental defects in pollen, embryo, and endosperm. High rates of seed abnormalities and abortion occur in mdkin2 mutants through a partial maternal effect. No additive effect or additional developmental defects were noted in mdkin1 mdkin2 double mutants. MDKIN2 is expressed in regions of cell division throughout the plant. Subcellular localization of MDKIN2 indicates a role in cell division, with a possible secondary function in the nuclei. Our results reveal a non-essential but important role for a malectin domain kinesin during development in plants.

Silencing integrated SIV proviral DNA with TAR-specific CRISPR tools.

BACKGROUND: One approach for a functional HIV cure is to prevent transcription from integrated proviral DNA. A critical step in HIV transcription is the Tat protein interaction with the TAR element viral RNA. We tested the strategy of blocking this Tat-TAR interaction in the SIVmac model. METHODS: We designed five CRISPR short guiding RNAs (sgRNAs) targeting the SIVmac TAR element, along with inactive versions of Cas9 (dCas9). These sgRNA constructs were delivered as ribonucleoproteins or plasmid DNA, along with SIV DNA. The constructs were also tested in integrated viral DNA in a cell line chronically infected by SIV. RESULTS: The sgRNAs targeting the coding strand of the TAR element inhibited SIV RNA transcription in association with dCas9-KRAB, but not with dCas9. CONCLUSIONS: Induction of epigenetic modifications may be more effective in inactivating provirus than transcriptional interference and thus may be a better strategy to achieve a functional cure in vivo.

A neuroscience perspective of the gut theory of Parkinson's disease.

Parkinson's disease is caused by complex interactions between environmental factors and a genetic predisposition. Environmental factors include exposure to pesticides and toxins, heavy metals and accumulation of iron and/or manganese in the brain. However, accumulating evidence indicates that gut-brain health and function are impaired in Parkinson's disease, often a decade before motor symptoms are diagnosed. We present the gut-brain theory of Parkinson's disease and summarise the peripheral and central nervous system pathology, gastrointestinal symptoms experienced by many Parkinson's patients, the route by which gut-brain dysfunction may occur and changes in gut microbiota that are associated with disease expression. Finally, we consider future gut-based treatments to prevent or slow down the progression of Parkinson's disease and explore whether this knowledge may highlight biomarkers to be included in complex algorithms in the future to assess a person's risk of developing Parkinson's disease.

JMJ14, a JmjC domain protein, is required for RNA silencing and cell-to-cell movement of an RNA silencing signal in Arabidopsis.

JMJ14 is a histone H3 Lys4 (H3K4) trimethyl demethylase that affects mobile RNA silencing in an Arabidopsis transgene system. It also influences CHH DNA methylation, abundance of endogenous transposon transcripts, and flowering time. JMJ14 acts at a point in RNA silencing pathways that is downstream from RNA-dependent RNA polymerase 2 (RDR2) and Argonaute 4 (AGO4). Our results illustrate a link between RNA silencing and demethylation of histone H3 trimethylysine. We propose that JMJ14 acts downstream from the Argonaute effector complex to demethylate histone H3K4 at the target of RNA silencing.

The role of DNA (de)methylation in immune responsiveness of Arabidopsis.

DNA methylation is antagonistically controlled by DNA methyltransferases and DNA demethylases. The level of DNA methylation controls plant gene expression on a global level. We have examined impacts of global changes in DNA methylation on the Arabidopsis immune system. A range of hypo-methylated mutants displayed enhanced resistance to the biotrophic pathogen Hyaloperonospora arabidopsidis (Hpa), whereas two hyper-methylated mutants were more susceptible to this pathogen. Subsequent characterization of the hypo-methylated nrpe1 mutant, which is impaired in RNA-directed DNA methylation, and the hyper-methylated ros1 mutant, which is affected in DNA demethylation, revealed that their opposite resistance phenotypes are associated with changes in cell wall defence and salicylic acid (SA)-dependent gene expression. Against infection by the necrotrophic pathogen Plectosphaerella cucumerina, nrpe1 showed enhanced susceptibility, which was associated with repressed sensitivity of jasmonic acid (JA)-inducible gene expression. Conversely, ros1 displayed enhanced resistance to necrotrophic pathogens, which was not associated with increased responsiveness of JA-inducible gene expression. Although nrpe1 and ros1 were unaffected in systemic acquired resistance to Hpa, they failed to develop transgenerational acquired resistance against this pathogen. Global transcriptome analysis of nrpe1 and ros1 at multiple time-points after Hpa infection revealed that 49% of the pathogenesis-related transcriptome is influenced by NRPE1- and ROS1-controlled DNA methylation. Of the 166 defence-related genes displaying augmented induction in nrpe1 and repressed induction in ros1, only 25 genes were associated with a nearby transposable element and NRPE1- and/or ROS1-controlled DNA methylation. Accordingly, we propose that the majority of NRPE1- and ROS1-dependent defence genes are regulated in trans by DNA methylation.

N2-fixing tropical legume evolution: a contributor to enhanced weathering through the Cenozoic?

Fossil and phylogenetic evidence indicates legume-rich modern tropical forests replaced Late Cretaceous palm-dominated tropical forests across four continents during the early Cenozoic (58-42 Ma). Tropical legume trees can transform ecosystems via their ability to fix dinitrogen (N2) and higher leaf N compared with non-legumes (35-65%), but it is unclear how their evolutionary rise contributed to silicate weathering, the long-term sink for atmospheric carbon dioxide (CO2). Here we hypothesize that the increasing abundance of N2-fixing legumes in tropical forests amplified silicate weathering rates by increased input of fixed nitrogen (N) to terrestrial ecosystems via interrelated mechanisms including increasing microbial respiration and soil acidification, and stimulating forest net primary productivity. We suggest the high CO2 early Cenozoic atmosphere further amplified legume weathering. Evolution of legumes with high weathering rates was probably driven by their high demand for phosphorus and micronutrients required for N2-fixation and nodule formation.

Activation of the Arabidopsis thaliana immune system by combinations of common ACD6 alleles.

A fundamental question in biology is how multicellular organisms distinguish self and non-self. The ability to make this distinction allows animals and plants to detect and respond to pathogens without triggering immune reactions directed against their own cells. In plants, inappropriate self-recognition results in the autonomous activation of the immune system, causing affected individuals to grow less well. These plants also suffer from spontaneous cell death, but are at the same time more resistant to pathogens. Known causes for such autonomous activation of the immune system are hyperactive alleles of immune regulators, or epistatic interactions between immune regulators and unlinked genes. We have discovered a third class, in which the Arabidopsis thaliana immune system is activated by interactions between natural alleles at a single locus, ACCELERATED CELL DEATH 6 (ACD6). There are two main types of these interacting alleles, one of which has evolved recently by partial resurrection of a pseudogene, and each type includes multiple functional variants. Most previously studies hybrid necrosis cases involve rare alleles found in geographically unrelated populations. These two types of ACD6 alleles instead occur at low frequency throughout the range of the species, and have risen to high frequency in the Northeast of Spain, suggesting a role in local adaptation. In addition, such hybrids occur in these populations in the wild. The extensive functional variation among ACD6 alleles points to a central role of this locus in fine-tuning pathogen defenses in natural populations.

Rapid divergence and high diversity of miRNAs and miRNA targets in the Camelineae.

MicroRNAs (miRNAs) are short RNAs involved in gene regulation through translational inhibition and transcript cleavage. After processing from imperfect fold-back structures, miRNAs are incorporated into RNA-induced silencing complexes (RISCs) before targeting transcripts with varying degrees of complementarity. Some miRNAs are evolutionarily deep-rooted, and sequence complementarity with their targets is maintained through purifying selection. Both Arabidopsis and Capsella belong to the tribe Camelineae in the Brassicaceae, with Capsella rubella serving as an outgroup to the genus Arabidopsis. The genome sequence of C. rubella has recently been released, which allows characterization of its miRNA complement in comparison with Arabidopsis thaliana and Arabidopsis lyrata. Through next-generation sequencing, we identify high-confidence miRNA candidates specific to the C. rubella lineage. Only a few lineage-specific miRNAs have been studied for evolutionary constraints, and there have been no systematic studies of miRNA target diversity within or divergence between closely related plant species. Therefore we contrast sequence variation in miRNAs and their targets within A. thaliana, and between A. thaliana, A. lyrata and C. rubella. We document a surprising amount of small-scale variation in miRNA-target pairs, where many miRNAs are predicted to have species-specific targets in addition to ones that are shared between species. Our results emphasize that the transitive nature of many miRNA-target pairs can be observed even on a relatively short evolutionary time-scale, with non-random occurrences of differences in miRNAs and their complements in the miRNA precursors, the miRNA* sequences.

Regulation of pri-miRNA processing by the hnRNP-like protein AtGRP7 in Arabidopsis.

The hnRNP-like glycine-rich RNA-binding protein AtGRP7 regulates pre-mRNA splicing in Arabidopsis. Here we used small RNA-seq to show that AtGRP7 also affects the miRNA inventory. AtGRP7 overexpression caused a significant reduction in the level of 30 miRNAs and an increase for 14 miRNAs with a minimum log2 fold change of ± 0.5. Overaccumulation of several pri-miRNAs including pri-miR398b, pri-miR398c, pri-miR172b, pri-miR159a and pri-miR390 at the expense of the mature miRNAs suggested that AtGRP7 affects pri-miRNA processing. Indeed, RNA immunoprecipitation revealed that AtGRP7 interacts with these pri-miRNAs in vivo. Mutation of an arginine in the RNA recognition motif abrogated in vivo binding and the effect on miRNA and pri-miRNA levels, indicating that AtGRP7 inhibits processing of these pri-miRNAs by direct binding. In contrast, pri-miRNAs of selected miRNAs that were elevated or not changed in response to high AtGRP7 levels were not bound in vivo. Reduced accumulation of miR390, an initiator of trans-acting small interfering RNA (ta-siRNA) formation, also led to lower TAS3 ta-siRNA levels and increased mRNA expression of the target AUXIN RESPONSE FACTOR4. Furthermore, AtGRP7 affected splicing of pri-miR172b and pri-miR162a. Thus, AtGRP7 is an hnRNP-like protein with a role in processing of pri-miRNAs in addition to its role in pre-mRNA splicing.

Transposon variants and their effects on gene expression in Arabidopsis.

Transposable elements (TEs) make up the majority of many plant genomes. Their transcription and transposition is controlled through siRNAs and epigenetic marks including DNA methylation. To dissect the interplay of siRNA-mediated regulation and TE evolution, and to examine how TE differences affect nearby gene expression, we investigated genome-wide differences in TEs, siRNAs, and gene expression among three Arabidopsis thaliana accessions. Both TE sequence polymorphisms and presence of linked TEs are positively correlated with intraspecific variation in gene expression. The expression of genes within 2 kb of conserved TEs is more stable than that of genes next to variant TEs harboring sequence polymorphisms. Polymorphism levels of TEs and closely linked adjacent genes are positively correlated as well. We also investigated the distribution of 24-nt-long siRNAs, which mediate TE repression. TEs targeted by uniquely mapping siRNAs are on average farther from coding genes, apparently because they more strongly suppress expression of adjacent genes. Furthermore, siRNAs, and especially uniquely mapping siRNAs, are enriched in TE regions missing in other accessions. Thus, targeting by uniquely mapping siRNAs appears to promote sequence deletions in TEs. Overall, our work indicates that siRNA-targeting of TEs may influence removal of sequences from the genome and hence evolution of gene expression in plants.

Self-Reported Physical Tasks and Exercise Training in Special Weapons and Tactics (SWAT) Teams.

Davis, MR, Easter, RL, Carlock, JM, Weiss, LW, Longo, EA, Smith, LM, Dawes, JJ, and Schilling, BK. Self-reported physical tasks and exercise training in Special Weapons and Tactics (SWAT) teams. J Strength Cond Res 30(11): 3242-3248, 2016-Little research has been done examining the most physically demanding tasks a SWAT officer may perform in the line of duty. Our objective was to analyze the rankings of tasks by SWAT officers based on frequency, difficulty, and importance and assess if training is addressing traits needed for successful task completion. A survey was designed using Qualtrics (Qualtrics Labs Inc). The survey had a demographics section, performance section, and training section. Officers were contacted by phone or e-mail and asked about interest in participating. Officers who agreed were sent the survey. Our results found a strong correlation between frequency of task and importance (r = 0.69, p = 0.001), and a moderate correlation was found between task difficulty and importance (r = 0.37, p = 0.005). Task rankings were averaged across the 3 domains to assess "overall" importance, and the top 3 tasks were assessed for necessary traits for successful performance. Power and strength were determined to be the most important traits for successful performance. Officers ranked the top 2 focuses of their training program in the training section as stamina/muscular endurance and cardiovascular/respiratory endurance. Training programs for SWAT officers should be developed to improve performance of the tasks with the highest "overall" importance. Therefore, a training program should emphasize strength and power improvements while not neglecting other measures of fitness.