Transfection,Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.     

Impaired Chloroplast Biogenesis in Immutans,an Arabidopsis Variegation Mutant,Modifies Developmental Programming,Cell Wall Composition and Resistance to Pseudomonas syringae

The immutans (im) variegation mutation of Arabidopsis has green- and white- sectored leaves due to action of a nuclear recessive gene. IM codes for PTOX, a plastoquinol oxidase in plastid membranes. Previous studies have revealed that the green and white sectors develop into sources (green tissues) and sinks (white tissues) early in leaf development. In this report we focus on white sectors, and show that their transformation into effective sinks involves a sharp reduction in plastid number and size. Despite these reductions, cells in the white sectors have near-normal amounts of plastid RNA and protein, and surprisingly, a marked amplification of chloroplast DNA. The maintenance of protein synthesis capacity in the white sectors might poise plastids for their development into other plastid types. The green and white im sectors have different cell wall compositions: whereas cell walls in the green sectors resemble those in wild type, cell walls in the white sectors have reduced lignin and cellulose microfibrils, as well as alterations in galactomannans and the decoration of xyloglucan. These changes promote susceptibility to the pathogen Pseudomonas syringae. Enhanced susceptibility can also be explained by repressed expression of some, but not all, defense genes. We suggest that differences in morphology, physiology and biochemistry between the green and white sectors is caused by a reprogramming of leaf development that is coordinated, in part, by mechanisms of retrograde (plastid-to-nucleus) signaling, perhaps mediated by ROS. We conclude that variegation mutants offer a novel system to study leaf developmental programming, cell wall metabolism and host-pathogen interactions.     

Targeting Medication Non-Adherence Behavior in Selected Autoimmune Diseases: A Systematic Approach to Digital Health Program Development

Background

29 autoimmune diseases, including Rheumatoid Arthritis, gout, Crohn’s Disease, and Systematic Lupus Erythematosus affect 7.6-9.4% of the population. While effective therapy is available, many patients do not follow treatment or use medications as directed. Digital health and Web 2.0 interventions have demonstrated much promise in increasing medication and treatment adherence, but to date many Internet tools have proven disappointing. In fact, most digital interventions continue to suffer from high attrition in patient populations, are burdensome for healthcare professionals, and have relatively short life spans.

Objective

Digital health tools have traditionally centered on the transformation of existing interventions (such as diaries, trackers, stage-based or cognitive behavioral therapy programs, coupons, or symptom checklists) to electronic format. Advanced digital interventions have also incorporated attributes of Web 2.0 such as social networking, text messaging, and the use of video. Despite these efforts, there has not been little measurable impact in non-adherence for illnesses that require medical interventions, and research must look to other strategies or development methodologies. As a first step in investigating the feasibility of developing such a tool, the objective of the current study is to systematically rate factors of non-adherence that have been reported in past research studies.

Methods

Grounded Theory, recognized as a rigorous method that facilitates the emergence of new themes through systematic analysis, data collection and coding, was used to analyze quantitative, qualitative and mixed method studies addressing the following autoimmune diseases: Rheumatoid Arthritis, gout, Crohn’s Disease, Systematic Lupus Erythematosus, and inflammatory bowel disease. Studies were only included if they contained primary data addressing the relationship with non-adherence.

Results

Out of the 27 studies, four non-modifiable and 11 modifiable risk factors were discovered. Over one third of articles identified the following risk factors as common contributors to medication non-adherence (percent of studies reporting): patients not understanding treatment (44%), side effects (41%), age (37%), dose regimen (33%), and perceived medication ineffectiveness (33%). An unanticipated finding that emerged was the need for risk stratification tools (81%) with patient-centric approaches (67%).

Conclusions

This study systematically identifies and categorizes medication non-adherence risk factors in select autoimmune diseases. Findings indicate that patients understanding of their disease and the role of medication are paramount. An unexpected finding was that the majority of research articles called for the creation of tailored, patient-centric interventions that dispel personal misconceptions about disease, pharmacotherapy, and how the body responds to treatment. To our knowledge, these interventions do not yet exist in digital format. Rather than adopting a systems level approach, digital health programs should focus on cohorts with heterogeneous needs, and develop tailored interventions based on individual non-adherence patterns.     

Heat Shock Inhibition of CDK5 Increases NOXA Levels through miR-23a Repression

Hyperthermia is a proteotoxic stress that is lethal when exposure is extreme but also cytoprotective in that sublethal exposure leads to the synthesis of heat shock proteins, including HSP70, which are able to inhibit stress-induced apoptosis. CDK5 is an atypical cyclin-dependent kinase family member that regulates many cellular functions including motility and survival. Here we show that exposure of a human lymphoid cell line to hyperthermia causes CDK5 insolubilization and loss of tyrosine-15 phosphorylation, both of which were prevented in cells overexpressing HSP70. Inhibition of CDK5 activity with roscovitine-sensitized cells to heat induced apoptosis indicating a protective role for CDK5 in inhibiting heat-induced apoptosis. Both roscovitine and heat shock treatment caused increased accumulation of NOXA a pro-apoptotic BH3-only member of the BCL2 family. The increased abundance of NOXA by CDK5 inhibition was not a result of changes in NOXA protein turnover. Instead, CDK5 inhibition increased NOXA mRNA and protein levels by decreasing the expression of miR-23a, whereas overexpressing the CDK5 activator p35 attenuated both of these effects on NOXA and miR-23a expression. Lastly, overexpression of miR-23a prevented apoptosis under conditions in which CDK5 activity was inhibited. These results demonstrate that CDK5 activity provides resistance to heat-induced apoptosis through the expression of miR-23a and subsequent suppression of NOXA synthesis. Additionally, they indicate that hyperthermia induces apoptosis through the insolubilization and inhibition of CDK5 activity.     

Infection of Anastrepha ludens following soil applications of Heterorhabditis bacteriophora in a mango orchard

To determine the efficacy of Heterorhabditis bacteriophora Poinar (Nematoda: Heterorhabditidae) for control of Anastrepha ludens (Loew) (Diptera: Tephritidae), field experiments were performed in a mango orchard with soil temperatures of 24–29 °C. The density of third‐instar A. ludens (50–500 larvae per plot) released into 0.25 m² wood‐framed experimental plots containing soil (16% wt/wt moisture) previously treated with 125 infective juveniles per square centimetre soil surface did not significantly influence the prevalence of infection by H. bacteriophora. In subsequent experiments, the percentages of infection of fly pupae were positively correlated with the concentration of infective stages applied to soil plots. The highest average percentage of infection (74% at 250 infective juveniles per square centimetre) was observed when fly larvae were released simultaneously onto soil, compared to larvae that emerged from laboratory‐infested mangoes over a period of 8 days (52% infection at 500 infective juveniles per square centimetre). Double applications of infective juveniles at an interval of 4 days did not greatly improve the prevalence of infection (～10% higher) compared to single applications. Between 9 and 15% of larvae that remained within infested mangoes became infected by nematodes, irrespective of the concentration of nematodes applied to each experimental plot. We conclude that effective control of A. ludens requires very high densities of H. bacteriophora. The successful use of this nematode for biocontrol of A. ludens will depend on identifying ways of overcoming the fly's ability to avoid infection.     

Responses of elbow flexors to two strenuous eccentric exercise bouts separated by three days

This study investigated whether the second eccentric exercise performed 3 days after the initial bout would exacerbate muscle damage and retard the recovery. Fifty-one athletes performed 30 eccentric actions of the elbow flexors using a dumbbell weighted 100% of the maximal isometric force (MIF) at the elbow joint angle of 90 degrees (ECC1). Three days after ECC1, all subjects except those in the control group (n = 12) performed the second bout (ECC2) with the same (100%) intensity (n = 12), 90% (n = 13), or 80% (n = 14) of the ECC1. Some subjects, especially in the 100% group, required spotting for ECC2 but made maximal effort to complete the exercise. MIF, range of motion, upper-arm circumference, muscle soreness, muscle proteins in the blood, and ultrasound images were used to assess muscle damage. Changes in these measures for 9 days following ECC1 were compared among groups by 2-way analysis of variance (ANOVA) with repeated measures. All criterion measures changed significantly after ECC1; however, no significant differences between the groups were evident for any of the changes in the measures. These results suggest that it is possible for athletes to complete the second bout if the intensity is reduced 10-20% from the initial bout. No significant differences between the control group and other groups indicate that the second eccentric exercise performed 3 days after the initial bout does not exacerbate muscle damage and retard the recovery regardless of the intensity of the second bout. It is concluded that the elbow flexors can perform high-intensity eccentric exercise in the early stage of recovery from the initial bout and are not damaged further by performing a subsequent bout 3 days after the first.     

Virulence of Serratia Strains against Costelytra zealandica

Strains of Serratia spp. showed a high level of virulence when injected into the hemocoel of larvae Costelytra zealandica, with Serratia entomophila, S. plymuthica, and S. marcescens showing significantly higher virulence than S. proteamaculans. Toxicity was independent of the amber disease-causing plasmid pADAP, suggesting a generalized Serratia toxin.     

Carbon isotope variations in a plantation of Sitka spruce,and the effect of acid mist

Intra- and inter-tree variations in ¹³C/¹²C ratios were studied within a single clone plantation of 20-year-old Sitka spruce, some of which were treated with mist simulating acidic cloud water. For groups of trees of similar height and the same treatment, sampled at the same whorl height, ¹³C values for current year needles showed variations (1 SD) of between 0.2 and 0.7. The variations reflect the seasonally averaged influences, on intercellular CO₂ concentrations, of slight variations in the microhabitat within a group. For a typical intra-group variation of 0.4 one may be able to distinguish between groups whose mean intercellular CO₂ concentrations differ by only 8 ppm. Acid misting resulted in a lowering of ¹³C values by c. 0.7 (significant at the P0.05 level). This reflects higher intercellular CO₂ concentrations for acid misted trees, which can be interpreted in terms of their having assimilation rates c. 10% lower than those of control trees, and might explain the observed reduction in stem growth for acid-misted trees. Without careful attention to sampling strategy, however, these small inter-tree ¹³C variations can be easily masked by the much larger intra-tree variations with height. Large gradients of increasing needle ¹³C with height, of c. 0.5 m^-1, were observed in two untreated trees of different total height. The gradient was similar for both trees so, though ¹³C values of both trees were identical close to their leaders (–27), the taller tree displayed much lower values close to the ground (–31). The gradients are believed to reflect lower light levels close to the ground, rather than the accumulation of respired CO₂ in the atmosphere. The different height response of stems versus needles, reflected by an increase in ¹³C_stems–¹³C_needles with height (for cellulose), is discussed in terms of stem photosynthetic recapture of internally respired CO₂.     

OPA1 deficiency promotes secretion of FGF21 from muscle that prevents obesity and insulin resistance

Mitochondrial dynamics is a conserved process by which mitochondria undergo repeated cycles of fusion and fission, leading to exchange of mitochondrial genetic content, ions, metabolites, and proteins. Here, we examine the role of the mitochondrial fusion protein optic atrophy 1 (OPA1) in differentiated skeletal muscle by reducing OPA1 gene expression in an inducible manner. OPA1 deficiency in young mice results in non‐lethal progressive mitochondrial dysfunction and loss of muscle mass. Mutant mice are resistant to age‐ and diet‐induced weight gain and insulin resistance, by mechanisms that involve activation of ER stress and secretion of fibroblast growth factor 21 (FGF21) from skeletal muscle, resulting in increased metabolic rates and improved whole‐body insulin sensitivity. OPA1‐elicited mitochondrial dysfunction activates an integrated stress response that locally induces muscle atrophy, but via secretion of FGF21 acts distally to modulate whole‐body metabolism.     

Visual Cues of Object Properties Differentially Affect Anticipatory Planning of Digit Forces and Placement

Studies on anticipatory planning of object manipulation showed initial task failure (i.e., object roll) when visual object shape cues are incongruent with other visual cues, such as weight distribution/density (e.g., symmetrically shaped object with an asymmetrical density). This suggests that shape cues override density cues. However, these studies typically only measured forces, with digit placement constrained. Recent evidence suggests that when digit placement is unconstrained, subjects modulate digit forces and placement. Thus, unconstrained digit placement might be modulated on initial trials (since it is an explicit process), but not forces (since it is an implicit process). We tested whether shape and density cues would differentially influence anticipatory planning of digit placement and forces during initial trials of a two-digit object manipulation task. Furthermore, we tested whether shape cues would override density cues when cues are incongruent. Subjects grasped and lifted an object with the aim of preventing roll. In Experiment 1, the object was symmetrically shaped, but with asymmetrical density (incongruent cues). In Experiment 2, the object was asymmetrical in shape and density (congruent cues). In Experiment 3, the object was asymmetrically shaped, but with symmetrical density (incongruent cues). Results showed differential modulation of digit placement and forces (modulation of load force but not placement), but only when shape and density cues were congruent. When shape and density cues were incongruent, we found collinear digit placement and symmetrical force sharing. This suggests that congruent and incongruent shape and density cues differentially influence anticipatory planning of digit forces and placement. Furthermore, shape cues do not always override density cues. A continuum of visual cues, such as those alluding to shape and density, need to be integrated.