The Non-Coding RNA Journal Club: Highlights on Recent Papers-12.

We are delighted to share with you our twelfth Journal Club and highlight some of the most interesting papers published recently [...].

Sustainable and Cost-Effective Gel Documentation.

A common laboratory method involves gel electrophoresis followed by photographic documentation of the results, a procedure which is performed worldwide by students and experienced scientists alike. Proprietary Gel Documentation Systems are convenient and useful for documentation of electrophoresis results, but the systems can be prohibitively expensive to purchase and repair, they contain features that are not necessary for everyday documentation, and some users may not find the systems intuitive to operate. We describe our gel documentation setup that meets the everyday needs for documentation in our lab. The setup is inexpensive, modular, user friendly, and increases sustainability through extending the working life of obsolete cell phones, iPads, or other electronic devices containing a camera. More importantly, the setup completely shields users from potentially damaging ultraviolet radiation.

Sustainable Methodologies for Efficient Gel Electrophoresis and Streamlined Screening of Difficult Plasmids.

We describe a workflow for efficient, environmentally attentive, and sustainable practices related to routine agarose gel electrophoresis. The methods reduce plastic waste and improve efficiency, especially for the exhaustive screening of difficult-to-obtain plasmids. Sustainability is increased when agarose is used ten times over by virtue of a thorough recycling regimen. The workflow optimizes workspaces and standardizes lab practices for handling potentially hazardous waste, minimizing environmental harm. Safety, efficiency, and sustainability improve laboratory productivity, help minimize environmental contamination, and increase cost-effectiveness.

Religiosity/Spirituality and Mental Health Outcomes in Mothers of Children with Autism Spectrum Disorder: The Mediating Role of Positive Thinking.

This study examines associations between dimensions of religiosity/spirituality (R/S) and anxiety symptoms in mothers of children with autism spectrum disorder (ASD). Mothers' positive ways of thinking and romantic relationship satisfaction were examined as mediators of associations. The extent to which child ASD symptom severity and maternal broad autism phenotype (BAP) behaviors predicted interactions with religious community members was also examined. Seventy-three Christian mothers of a child with ASD completed online surveys. Higher levels of spirituality were associated with viewing their child with ASD as providing a positive contribution to the family. These positive views were, in turn, associated with less anxiety symptoms. Mothers of children with more severe ASD symptoms also reported greater negative interactions with members of their congregation.

The Non-Coding RNA Journal Club: Highlights on Recent Papers-7.

We are delighted to share with you our seventh Journal Club and highlight some of the most interesting papers published recently [...].

Predictors of Daily Relationship Quality in Mothers of Children with Autism Spectrum Disorder.

Mothers of children with autism spectrum disorder (n = 70) completed online measures of global constructs (i.e., stable individual characteristics measured at time 1), which included resilience, depressive symptoms, and family functioning, followed by 14 daily questionnaires assessing relationship quality and affect on a given day. The global constructs were examined as predictors of daily relationship quality using multilevel modeling. Daily affect was examined in association with daily relationship factors (partner conflict, support from partner, and relationship happiness). Depressive symptoms and family flexibility predicted daily relationship quality. On a daily level, affect was associated with relationship quality. Results emphasize the potential of interventions to improve the quality of parents' relationships by addressing maternal mental health, family functioning, and daily affect.

The Relationship Between Optimism, Coping, and Depressive Symptoms in Hispanic Mothers and Fathers of Children with Autism Spectrum Disorder.

This study examined gender differences in the relationship between dispositional optimism, coping, and depressive symptoms of Hispanic mothers (n = 46) and fathers (n = 43) of children with autism spectrum disorder. Coping was hypothesized to mediate the relationship between optimism and depressive symptoms. The results revealed that mothers reported greater depressive symptoms and greater use of positive and support coping than fathers; however, both mothers and fathers reported similar levels of optimism and use of avoidant coping. In addition, positive and avoidant coping strategies mediated the association between optimism and depressive symptoms for both mothers and fathers. Clinical implications for this study include interventions for improving optimistic outlooks as well as interventions that improve parents' coping skills and therefore reduce negative outcomes.

The impact of maternal, child, and family characteristics on the daily well-being and parenting experiences of mothers of children with autism spectrum disorder.

This study utilized a daily diaries method to explore the global factors that impact daily general affect and daily parenting interactions of mothers of children with autism spectrum disorder. Eighty-three mothers of a child with autism spectrum disorder between the ages of 3 and 13 years completed global assessments of maternal depressive symptoms, child autism spectrum disorder symptom severity, and family functioning. Mothers then reported on their daily negative and positive affect as well as their daily positive and frustrating parenting interactions for 14 consecutive days. The results indicated that higher levels of maternal depressive symptoms were related to decreased daily positive affect, whereas greater child social motivation impairments were related to increased daily positive affect. Only maternal depressive symptoms were associated with increased daily negative affect. Furthermore, higher levels of family cohesion were related to increased daily positive parenting interactions. Finally, higher maternal depressive symptoms as well as family rigidity were related to increased daily frustrating parenting interactions. Implications for interventions focused on the family system are discussed.

The Power of Positivity: Predictors of Relationship Satisfaction for Parents of Children with Autism Spectrum Disorder.

The current study uses the actor-partner interdependence model to examine the predictors of relationship satisfaction for mothers and fathers of children with autism spectrum disorder. Sixty-seven couples completed measures of optimism, benefit finding, coping strategies, social support, and relationship satisfaction. Results indicated that parent's positive strengths predicted better personal relationship satisfaction. Moreover, parents' benefit finding, use of emotional support, and perceived social support from their partner also predicted their partner's relationship satisfaction. The results of this study highlight the importance of focusing on positive factors that can enhance relationship quality. Implications for the development of parent-focused interventions are discussed.

Systematic comparison of bacterial feeding strains for increased yield of Caenorhabditis elegans males by RNA interference-induced non-disjunction.

Rare Caenorhabditis elegans males arise when sex chromosome non-disjunction occurs during meiosis in self-fertilizing hermaphrodites. Non-disjunction is a relatively rare event, and males are typically observed at a frequency of less than one in five hundred wild-type animals. Males are required for genetic crosses and phenotypic analysis, yet current methods to generate large numbers of males can be cumbersome. Here, we identify RNAi reagents (dsRNA-expressing bacteria) with improved effectiveness for eliciting males. Specifically, we used RNAi to systematically reduce the expression of over two hundred genes with meiotic chromosome segregation functions, and we identified a set of RNAi reagents that robustly and reproducibly elicited male progeny.

The Receptor-Bound Guanylyl Cyclase DAF-11 Is the Mediator of Hydrogen Peroxide-Induced cGMP Increase in Caenorhabditis elegans [corrected]..

Adenosine 3', 5'-cyclic monophosphate (cAMP) and guanosine 3', 5'-cyclic monophosphate (cGMP) are well-studied second messengers that transmit extracellular signals into mammalian cells, with conserved functions in various other species such as Caenorhabditis elegans (C. elegans). cAMP is generated by adenylyl cyclases, and cGMP is generated by guanylyl cyclases, respectively. Studies using C. elegans have revealed additional roles for cGMP signaling in lifespan extension. For example, mutants lacking the function of a specific receptor-bound guanylyl cyclase, DAF-11, have an increased life expectancy. While the daf-11 phenotype has been attributed to reductions in intracellular cGMP concentrations, the actual content of cyclic nucleotides has not been biochemically determined in this system. Similar assumptions were made in studies using phosphodiesterase loss-of-function mutants or using adenylyl cyclase overexpressing mutants. In the present study, cyclic nucleotide regulation in C. elegans was studied by establishing a special nematode protocol for the simultaneous detection and quantitation of cyclic nucleotides. We also examined the influence of reactive oxygen species (ROS) on cyclic nucleotide metabolism and lifespan in C. elegans using highly specific HPLC-coupled tandem mass-spectrometry and behavioral assays. Here, we show that the relation between cGMP and survival is more complex than previously appreciated.

Natural and unanticipated modifiers of RNAi activity in Caenorhabditis elegans.

Organisms used as model genomics systems are maintained as isogenic strains, yet evidence of sequence differences between independently maintained wild-type stocks has been substantiated by whole-genome resequencing data and strain-specific phenotypes. Sequence differences may arise from replication errors, transposon mobilization, meiotic gene conversion, or environmental or chemical assault on the genome. Low frequency alleles or mutations with modest effects on phenotypes can contribute to natural variation, and it has proven possible for such sequences to become fixed by adapted evolutionary enrichment and identified by resequencing. Our objective was to identify and analyze single locus genetic defects leading to RNAi resistance in isogenic strains of Caenorhabditis elegans. In so doing, we uncovered a mutation that arose de novo in an existing strain, which initially frustrated our phenotypic analysis. We also report experimental, environmental, and genetic conditions that can complicate phenotypic analysis of RNAi pathway defects. These observations highlight the potential for unanticipated mutations, coupled with genetic and environmental phenomena, to enhance or suppress the effects of known mutations and cause variation between wild-type strains.

The Caenorhabditis elegans rsd-2 and rsd-6 genes are required for chromosome functions during exposure to unfavorable environments.

In Caenorhabditis elegans, exogenous dsRNA can elicit systemic RNAi, a process that requires the function of many genes. Considering that the activities of many of these genes are also required for normal development, it is surprising that exposure to high concentrations of dsRNA does not elicit adverse consequences to animals. Here, we report inducible phenotypes in attenuated C. elegans strains reared in environments that include nonspecific dsRNA and elevated temperature. Under these conditions, chromosome integrity is compromised in RNAi-defective strains harboring mutations in rsd-2 or rsd-6. Specifically, rsd-2 mutants display defects in transposon silencing, while meiotic chromosome disjunction is affected in rsd-6 mutants. RSD-2 proteins localize to multiple cellular compartments, including the nucleolus and cytoplasmic compartments that, in part, are congruent with calreticulin and HAF-6. We considered that the RNAi defects in rsd-2 mutants might have relevance to membrane-associated functions; however, endomembrane compartmentalization and endocytosis/exocytosis markers in rsd-2 and rsd-6 mutants appear normal. The mutants also possess environmentally sensitive defects in cell-autonomous RNAi elicited from transgene-delivered dsRNAs. Thus, the ultimate functions of rsd-2 and rsd-6 in systemic RNAi are remarkably complex and environmentally responsive.

Caenorhabditis elegans ABCRNAi transporters interact genetically with rde-2 and mut-7.

RNA interference (RNAi) mechanisms are conserved and consist of an interrelated network of activities that not only respond to exogenous dsRNA, but also perform endogenous functions required in the fine tuning of gene expression and in maintaining genome integrity. Not surprisingly, RNAi functions have widespread influences on cellular function and organismal development. Previously, we observed a reduced capacity to mount an RNAi response in nine Caenorhabditis elegans mutants that are defective in ABC transporter genes (ABC(RNAi) mutants). Here, we report an exhaustive study of mutants, collectively defective in 49 different ABC transporter genes, that allowed for the categorization of one additional transporter into the ABC(RNAi) gene class. Genetic complementation tests reveal functions for ABC(RNAi) transporters in the mut-7/rde-2 branch of the RNAi pathway. These second-site noncomplementation interactions suggest that ABC(RNAi) proteins and MUT-7/RDE-2 function together in parallel pathways and/or as multiprotein complexes. Like mut-7 and rde-2, some ABC(RNAi) mutants display transposon silencing defects. Finally, our analyses reveal a genetic interaction network of ABC(RNAi) gene function with respect to this part of the RNAi pathway. From our results, we speculate that the coordinated activities of ABC(RNAi) transporters, through their effects on endogenous RNAi-related mechanisms, ultimately affect chromosome function and integrity.

ABC transporters and RNAi in Caenorhabditis elegans.

RNAi is an evolutionarily conserved gene-silencing phenomenon that can be triggered by exogenous delivery of double stranded RNA to organisms. In Caenorhabditis elegans, the response to dsRNA is remarkably robust, and systemic RNAi responses are often observed. We have taken a genetic approach using this organism to better understand the mechanisms that facilitate RNAi. By analyzing strains of RNAi-defective mutants, we have uncovered an unexpected role for ABC transporters in RNAi and related silencing mechanisms. Ten of the sixty ABC transporter genes encoded in the C. elegans genome are required for robust RNAi. We will present data that highlights common features of these genes relative to their roles in RNAi, including genetic interactions with other components of the RNAi machinery. We will also describe unique roles for some transporter genes in endogenous RNAi-related processes.

Construction of plasmids for RNA interference and in vitro transcription of double-stranded RNA.

Double-stranded RNA (dsRNA)-induced gene silencing in Caenorhabditis elegans involves the manufacture and delivery of defined sequences of dsRNA to the organism, followed by a careful monitoring for loss-of-function phenocopies in treated animals. In this chapter, we describe how to generate DNAs that can be used as templates for transcription of dsRNA.

Delivery methods for RNA interference in C. elegans.

This chapter describes four methods for delivery of double-stranded RNA to Caenorhabditis elegans (injection, feeding, soaking, and in vivo delivery), and suggests schemes that should facilitate detection of specific gene silencing.

ATP-binding cassette transporters are required for efficient RNA interference in Caenorhabditis elegans.

RNA interference (RNAi) is a conserved gene-silencing phenomenon that can be triggered by delivery of double-stranded RNA (dsRNA) to cells and is a widely exploited technology in analyses of gene function. Although a number of proteins that facilitate RNAi have been identified, current descriptions of RNAi and interrelated mechanisms are far from complete. Here, we report that the Caenorhabditis elegans gene haf-6 is required for efficient RNAi. HAF-6 is a member of the ATP-binding cassette (ABC) transporter gene superfamily. ABC transporters use ATP to translocate small molecule substrates across the membranes in which they reside, often against a steep concentration gradient. Collectively, ABC transporters are involved in a variety of activities, including protective or barrier mechanisms that export drugs or toxins from cells, organellar biogenesis, and mechanisms that protect against viral infection. HAF-6 is expressed predominantly in the intestine and germline and is localized to intracellular reticular organelles. We further demonstrate that eight additional ABC genes from diverse subfamilies are each required for efficient RNAi in C. elegans. Thus, the ability to mount a robust RNAi response to dsRNA depends upon the deployment of two ancient systems that respond to environmental assaults: RNAi mechanisms and membrane transport systems that use ABC proteins.

Loss-of-function mutations in a glutathione S-transferase suppress the prune-Killer of prune lethal interaction.

The prune gene of Drosophila melanogaster is predicted to encode a phosphodiesterase. Null alleles of prune are viable but cause an eye-color phenotype. The abnormal wing discs gene encodes a nucleoside diphosphate kinase. Killer of prune is a missense mutation in the abnormal wing discs gene. Although it has no phenotype by itself even when homozygous, Killer of prune when heterozygous causes lethality in the absence of prune gene function. A screen for suppressors of transgenic Killer of prune led to the recovery of three mutations, all of which are in the same gene. As heterozygotes these mutations are dominant suppressors of the prune-Killer of prune lethal interaction; as homozygotes these mutations cause early larval lethality and the absence of imaginal discs. These alleles are loss-of-function mutations in CG10065, a gene that is predicted to encode a protein with several zinc finger domains and glutathione S-transferase activity.

Endogenous inhibitors of RNA interference in Caenorhabditis elegans.

In eukaryotes, double-stranded RNAs (dsRNAs) or short, interfering dsRNAs (siRNAs) can reduce the accumulation of a sequence-related mRNA, often resulting in a loss-of-function phenotype-a process termed RNA interference (RNAi). Unfortunately, some mRNAs are resistant to the effects of dsRNA. Experiments designed to unravel RNAi mechanisms in Caenorhabditis elegans have led to the identification of two worm proteins, RRF-31,2 and, now, ERI-1,3 that can inhibit RNAi responses. Animals defective in either protein can display enhanced RNAi phenotypes for mRNAs that were previously resistant to dsRNA. Since ERI-1 is a conserved protein, development of procedures to enhance RNAi effectiveness in other systems may be possible.