SMN-assisted assembly of snRNP-specific Sm cores in trypanosomes.

Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring (U1, U5, spliced leader snRNPs), or variant Sm cores with snRNA-specific Sm subunits (U2, U4 snRNPs). Searching for specificity factors, we identified SMN and Gemin2 proteins that are highly divergent from known orthologs. SMN is splicing-essential in trypanosomes and nuclear-localized, suggesting that Sm core assembly in trypanosomes is nuclear. We demonstrate in vitro that SMN is sufficient to confer specificity of canonical Sm core assembly and to discriminate against binding to nonspecific RNA and to U2 and U4 snRNAs. SMN interacts transiently with the SmD3B subcomplex, contacting specifically SmB. SMN remains associated throughout the assembly of the Sm heteroheptamer and dissociates only when a functional Sm site is incorporated. These data establish a novel role of SMN, mediating snRNP specificity in Sm core assembly, and yield new biochemical insight into the mechanism of SMN activity

Strategies that reduce Stroop interference

A remarkable example of reducing Stroop interference is provided by the word blindness post-hypnotic suggestion (a suggestion to see words as meaningless during the Stroop task). This suggestion has been repeatedly demonstrated to halve Stroop interference when it is given to highly hypnotizable people. In order to explore how highly hypnotizable individuals manage to reduce Stroop interference when they respond to the word blindness suggestion, we tested four candidate strategies in two experiments outside of the hypnotic context. A strategy of looking away from the target words and a strategy of visual blurring demonstrated compelling evidence for substantially reducing Stroop interference in both experiments. However, the pattern of results produced by these strategies did not match those of the word blindness suggestion. Crucially, neither looking away nor visual blurring managed to speed up incongruent responses, suggesting that neither of these strategies is the likely underlying mechanism of the word blindness suggestion. Although the current results did not unravel the mystery of the word blindness suggestion, they showed that there are multiple voluntary ways through which participants can dramatically reduce Stroop interference

The structure of the complex of calmodulin with KAR-2: a novel mode of binding explains the unique pharmacology of the drug

3'-(beta- Chloroethyl)-2',4'-dioxo-3,5'-spiro-oxazolidino-4-deacetoxyvinblastine (KAR-2) is a potent anti-microtubular agent that arrests mitosis in cancer cells without significant toxic side effects. In this study we demonstrate that in addition to targeting microtubules, KAR-2 also binds calmodulin, thereby countering the antagonistic effects of trifluoperazine. To determine the basis of both properties of KAR-2, the three-dimensional structure of its complex with Ca2+-calmodulin has been characterized both in solution using NMR and when crystallized using x-ray diffraction. Heterocorrelation (H-1-N-15 heteronuclear single quantum coherence) spectra of N-15-labeled calmodulin indicate a global conformation change (closure) of the protein upon its binding to KAR-2. The crystal structure at 2.12-Angstrom resolution reveals a more complete picture; KAR-2 binds to a novel structure created by amino acid residues of both the N- and C- terminal domains of calmodulin. Although first detected by x-ray diffraction of the crystallized ternary complex, this conformational change is consistent with its solution structure as characterized by NMR spectroscopy. It is noteworthy that a similar tertiary complex forms when calmodulin binds KAR-2 as when it binds trifluoperazine, even though the two ligands contact (for the most part) different amino acid residues. These observations explain the specificity of KAR-2 as an anti-microtubular agent; the drug interacts with a novel drug binding domain on calmodulin. Consequently, KAR-2 does not prevent calmodulin from binding most of its physiological targets

PARP-Inhibitor Treatment Prevents Hypertension Induced Cardiac Remodeling by Favorable Modulation of Heat Shock Proteins, Akt-1/GSK-3beta and Several PKC Isoforms.

Spontaneously hypertensive rat (SHR) is a suitable model for studies of the complications of hypertension. It is known that activation of poly(ADP-ribose) polymerase enzyme (PARP) plays an important role in the development of postinfarction as well as long-term hypertension induced heart failure. In this study, we examined whether PARP-inhibitor (L-2286) treatment could prevent the development of hypertensive cardiopathy in SHRs. 6-week-old SHR animals were treated with L-2286 (SHR-L group) or placebo (SHR-C group) for 24 weeks. Wistar-Kyoto rats were used as aged-matched, normotensive controls (WKY group). Echocardiography was performed, brain-derived natriuretic peptide (BNP) activity and blood pressure were determined at the end of the study. We detected the extent of fibrotic areas. The amount of heat-shock proteins (Hsps) and the phosphorylation state of Akt-1Ser473, glycogen synthase kinase (GSK)-3betaSer9, forkhead transcription factor (FKHR)Ser256, mitogen activated protein kinases (MAPKs), and protein kinase C (PKC) isoenzymes were monitored. The elevated blood pressure in SHRs was not influenced by PARP-inhibitor treatment. Systolic left ventricular function and BNP activity did not differ among the three groups. L-2286 treatment decreased the marked left ventricular (LV) hypertrophy which was developed in SHRs. Interstitial collagen deposition was also decreased by L-2286 treatment. The phosphorylation of extracellular signal-regulated kinase (ERK)1/2Thr183-Tyr185, Akt-1Ser473, GSK-3betaSer9, FKHRSer256, and PKC epsilonSer729 and the level of Hsp90 were increased, while the activity of PKC alpha/betaIIThr638/641, zeta/lambda410/403 were mitigated by L-2286 administration. We could detect signs of LV hypertrophy without congestive heart failure in SHR groups. This alteration was prevented by PARP inhibition. Our results suggest that PARP-inhibitor treatment has protective effect already in the early stage of hypertensive myocardial remodeling

Can unconscious intentions be more effective than conscious intentions? Test of the role of metacognition in hypnotic response

While several theories assume that responses to hypnotic suggestions can be implemented without executive intentions, the metacognitive class of theories postulate that the behaviors produced by hypnotic suggestions are intended and the accompanying feeling of involuntariness is only a consequence of strategically not being aware of the intention. Cold control theory asserts that the only difference between a hypnotic and non-hypnotic response is this metacognitive one, that is, whether or not one is aware of one's intention to perform the relevant act. To test the theory, we compared the performance of highly suggestible participants in reducing the Stroop interference effect in a post-hypnotic suggestion condition (word blindness: that words will appear as a meaningless foreign script) and in a volitional condition (asking the participants to imagine the words as a meaningless foreign script). We found that participants had equivalent expectations that the posthypnotic suggestion and the volitional request would help control the conflicting information. Further, participants felt they had more control over experiencing the words as meaningless with the request rather than the suggestion; and they experienced the request largely as imagination and the suggestion largely as perception. That is, we set up the interventions we required for the experiment to constitute a test of cold control theory. Both the suggestion and the request reduced Stroop interference. Crucially, there was Bayesian evidence that the reduction in Stroop interference was the same between the suggestion and the volitional request. That is, the results support the claim that responding hypnotically does not grant a person greater first order abilities than they have non-hypnotically, consistent with cold control theory

Modeling and Rescue of RP2 Retinitis Pigmentosa Using iPSC-Derived Retinal Organoids

RP2 mutations cause a severe form of X-linked retinitis pigmentosa (XLRP). The mechanism of RP2-associated retinal degeneration in humans is unclear, and animal models of RP2 XLRP do not recapitulate this severe phenotype. Here, we developed gene-edited isogenic RP2 knockout (RP2 KO) induced pluripotent stem cells (iPSCs) and RP2 patient-derived iPSC to produce 3D retinal organoids as a human retinal disease model. Strikingly, the RP2 KO and RP2 patient-derived organoids showed a peak in rod photoreceptor cell death at day 150 (D150) with subsequent thinning of the organoid outer nuclear layer (ONL) by D180 of culture. Adeno-associated virus-mediated gene augmentation with human RP2 rescued the degeneration phenotype of the RP2 KO organoids, to prevent ONL thinning and restore rhodopsin expression. Notably, these data show that 3D retinal organoids can be used to model photoreceptor degeneration and test potential therapies to prevent photoreceptor cell death

Effective delivery of large genes to the retina by dual AAV vectors.

Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV's limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt's disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on "forced" packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns. Taking advantage of AAV's ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B. Thus, dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large gene

Phenomenological control as cold control

We first review recent work from our laboratory, which construes hypnotizability as an example of a more general trait of capacity for phenomenological control, which people can use to create subjective experiences in many nonhypnotic contexts where those experiences fulfill people’s goals. Second, we review recent work, which construes phenomenological control as a specifically metacognitive process, where intentional cognitive and motor action occurs without awareness of specific intentions (cold control theory). In terms of the reach of phenomenological control, we argue that various laboratory phenomena, namely vicarious pain, mirror-touch synesthesia, and the rubber hand illusion are to an unknown degree a construction of phenomenological control. The argument can of course be extended in principle to other findings. In terms of the reach of cold control, we present a new theory of intentional binding and show how intentional binding can measure the absence of conscious intentions in the hypnotic context. We obtain no evidence that cold control confers abilities beyond the changes in the metacognitive monitoring it postulates, and we explore the negative correlation between mindfulness and cold control viewed as a lack of mindfulness of intentions