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Background: Pre-diagnostic stages of psychotic illnesses, including 'clinical high risk' (CHR), are marked by sleep disturbances. These sleep disturbances appear to represent a key aspect in the etiology and maintenance of psychotic disorders. We aimed to examine the relationship between self-reported sleep dysfunction and attenuated psychotic symptoms (APS) on a day-to-day basis. Methods: Seventy-six CHR young people completed the Experience Sampling Methodology (ESM) component of the European Union Gene-Environment Interaction Study, collected through PsyMate® devices, prompting sleep and symptom questionnaires 10 times daily for 6 days. Bayesian multilevel mixed linear regression analyses were performed on time-variant ESM data using the brms package in R. We investigated the day-to-day associations between sleep and psychotic experiences bidirectionally on an item level. Sleep items included sleep onset latency, fragmentation, and quality. Psychosis items assessed a range of perceptual, cognitive, and bizarre thought content common in the CHR population. Results: Two of the seven psychosis variables were unidirectionally predicted by previous night's number of awakenings: every unit increase in number of nightly awakenings predicted a 0.27 and 0.28 unit increase in feeling unreal or paranoid the next day, respectively. No other sleep variables credibly predicted next-day psychotic symptoms or vice-versa. Conclusion: In this study, the relationship between sleep disturbance and APS appears specific to the item in question. However, some APS, including perceptual disturbances, had low levels of endorsement amongst this sample. Nonetheless, these results provide evidence for a unidirectional relationship between sleep and some APS in this population. [ABSTRACT FROM AUTHOR]

The C[O.sub.2]-concentrating mechanism (CCM) of Chlamydomonas reinhardtii and other microalgal species is essential for photosynthetic growth in most natural settings. A great deal has been learned regarding the CCM in cyanobacteria, including identification of inorganic carbon (Ci; C[O.sub.2] and HC[O.sup.-.sub.3]) transporters; however, specific knowledge of analogous transporters has remained elusive in eukaryotic microalgae such as C. reinhardtii. Here we investigated whether the limiting-C[O.sub.2]--inducible, putative ABC-type transporter HLA3 might function as a HC[O.sup.-.sub.3] transporter by evaluating the effect of pH on growth, photosynthetic Ci affinity, and [[sup.14]C]-Ci uptake in very low C[O.sub.2] conditions following RNA interference (RNAi) knockdown of HLA3 mRNA levels in wild-type and mutant cells. Although knockdown of HLA3 mRNA alone resulted in only modest but high-pH--dependent decreases in photosynthetic Ci affinity and Ci uptake, the combination of nearly complete knockdown of HLA3 mRNA with mutations in LCIB (which encodes limiting-Ci--inducible plastid-localized protein required for normal Ci uptake or accumulation in low-C[O.sub.2] conditions) and/or simultaneous, apparently off-target knockdown of LCIA mRNA (which encodes limiting-Ci--inducible plastid envelope protein reported to transport HC[O.sup.-.sub.3]) resulted in dramatic decreases in growth, Ci uptake, and photosynthetic Ci affinity, especially at pH 9, at which HC[O.sup.-.sub.3] is the predominant form of available Ci. Collectively, the data presented here provide compelling evidence that HLA3 is directly or indirectly involved in HC[O.sup.-.sub.3] transport, along with additional evidence supporting a role for LCIA in chloroplast envelope HC[O.sup.-.sub.3] transport and a role for LCIB in chloroplast Ci accumulation. ABC transporter | C[O.sub.2]-concentrating mechanism | microalgae | RNAi

The availability of the complete DNA sequence of the Chlamydomonas reinhardtii genome and advanced computational biology tools has allowed elucidation and study of the small ubiquitin-like modifier (SUMO) system in this unicellular photosynthetic alga and model eukaryotic cell system. SUMO is a member of a ubiquitin-like protein superfamily that is covalently attached to target proteins as a post-translational modification to alter the localization, stability, and/or function of the target protein in response to changes in the cellular environment. Three SUMO homologs (CrSUMO96, CrSUMO97, and CrSUMO148) and three novel SUMO-related proteins (CrSUMO-like89A, CrSUMO-like89B, and CrSUMO-like90) were found by diverse gene predictions, hidden Markov models, and database search tools inferring from Homo sapiens, Saccharomyces cerevisiae, and Arabidopsis thaliana SUMOs. Among them, CrSUMO96, which can be recognized by the A. thaliana anti-SUMO1 antibody, was studied in detail. Free CrSUMO96 was purified by immunoprecipitation and identified by mass spectrometry analysis. A SUMO-conjugating enzyme (SCE) (E2, Ubc9) in C. reinhardtii was shown to be functional in an Escherichia coli-based in vivo chimeric SUMOylation system. Antibodies to CrSUMO96 recognized free and conjugated forms of CrSUMO96 in Western blot analysis of whole-cell extracts and nuclear localized SUMOylated proteins with in situ immunoflnorescence. Western blot analysis showed a marked increase in SUMO conjugated proteins when the cells were subjected to environmental stresses, such as heat shock and osmotic stress. Related analyses revealed multiple potential ubiquitin genes along with two Rub1 genes and one Ufm1 gene in the C. reinhardtii genome.

Glucose control and high cell density fermentation produces heterologous, cyanobacterial recombinant hexadecameric (L8S8) protein, and ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) in Escherichia coli. Optimization of culture and plasmid stability conditions produces about 1 gram of soluble, active recombinant RubisCO per litre. Lactose-induced high cell density fermentation of E. coli K-12 also gives recombinant RubisCO.

The inducible CO 2 concentrating mechanism (CCM) of microalgae is essential for their acclimation to highly variable aquatic inorganic carbon levels. The key transcriptional regulator, CIA5, affects expression of thousands of genes in Chlamydomonas reinhardtii , but the molecular characteristics of this important protein are poorly understood. This study identifies a functional activation domain in CIA5 and demonstrates the functionality of a mini-CIA5 including only the zinc-binding domain and the identified activation domain. A highly conserved 130aa region from CIA5 exhibits auto-activation in yeast and appears responsible for the markedly slow migration of CIA5 when analyzed by SDS-PAGE. This 130aa region or either half of this region also effectively replaced the activation domain of a modified designer Transcription Activator-like Element (dTALE) in targeted activation of an endogenous Chlamydomonas gene. Additionally, a mini-CIA5 combining the conserved zinc-binding domain with the 130aa putative activation domain complemented the growth phenotype of the cia5 mutant and triggered CO 2 -regulated gene expression patterns similar to wild-type cells or cia5 complemented with the full-length CIA5 . Although the mini-CIA5 complementation did not fully restore wild-type growth rates or full gene induction/repression amplitudes, especially in very low CO 2 , this newly identified activation domain combined with the previously described zinc-binding domain are demonstrated to be the key essential components of CIA5 that permit rapid CIA5-mediated responses to changes in CO 2 concentrations. [ABSTRACT FROM AUTHOR]

BackgroundEvidence has accumulated that implicates childhood trauma in the aetiology of psychosis, but our understanding of the putative psychological processes and mechanisms through which childhood trauma impacts on individuals and contributes to the development of psychosis remains limited. We aimed to investigate whether stress sensitivity and threat anticipation underlie the association between childhood abuse and psychosis.MethodWe used the Experience Sampling Method to measure stress, threat anticipation, negative affect, and psychotic experiences in 50 first-episode psychosis (FEP) patients, 44 At-Risk Mental State (ARMS) participants, and 52 controls. Childhood abuse was assessed using the Childhood Trauma Questionnaire.ResultsAssociations of minor socio-environmental stress in daily life with negative affect and psychotic experiences were modified by sexual abuse and group (all pFWE < 0.05). While there was strong evidence that these associations were greater in FEP exposed to high levels of sexual abuse, and some evidence of greater associations in ARMS exposed to high levels of sexual abuse, controls exposed to high levels of sexual abuse were more resilient and reported less intense negative emotional reactions to socio-environmental stress. A similar pattern was evident for threat anticipation.ConclusionsElevated sensitivity and lack of resilience to socio-environmental stress and enhanced threat anticipation in daily life may be important psychological processes underlying the association between childhood sexual abuse and psychosis. [ABSTRACT FROM AUTHOR]

In multiple system atrophy (MSA) and progressive supranuclear palsy (PSP), the absence of surrogate endpoints makes clinical trials long and expensive. We aim to determine annualized whole-brain atrophy rates (a-WBAR) in idiopathic Parkinson’s disease (IPD), MSA, and PSP. Ten healthy controls, 20 IPD, 12 PSP, and 8 MSA patients were studied using a volumetric MRI technique (SIENA). In controls, the a-WBAR was 0.37%±0.28 (CI 95% 0.17–0.57), while in IPD a-WBAR was 0.54%±0.38 (CI 95% 0.32–0.68). The IPD patients did not differ from the controls. In PSP, the a-WBAR was 1.26%±0.51 (CI 95%: 0.95–1.58). In MSA, a-WBAR was 1.65%±1.12 (CI 95%: 0.71–2.59). MSA did not differ from PSP. The a-WBAR in PSP and MSA were significantly higher than in the IPD group (p=0.004 and p<0.001, resp.). In PSP, the use of a-WBAR required one-half of the patients needed for clinical scales to detect a 50% reduction in their progression. In MSA, one-quarter of the patients would be needed to detect the same effect. a-WBAR is a reasonable candidate to consider as a surrogate endpoint in short clinical trials using smaller sample sizes. The confidence intervals for a-WBAR may add a potential retrospective application for a-WBAR to improve the diagnostic accuracy of MSA and PSP versus IPD. [ABSTRACT FROM AUTHOR]

A significantly improved viral 2A peptide system for dependable high-level expression of dicistronic genes in Chlamydomonas reinhardtii has been developed. Data are presented demonstrating that use of an especially proficient 'extended FMDV 2A' coding region allows production of two independent protein products from a dicistronic gene with almost complete efficiency. Importantly, results are also presented that demonstrate the utility of this 2A system for efficient high-level expression of foreign genes in C. reinhardtii, which has not previously been reliably achievable in this algal model system. To expand the versatility of the 2A expression system, a number of commonly used selectable marker proteins were assessed for their compatibility with the extended FMDV 2A peptide. Additional experiments demonstrate the feasibility and utility of 2A-containing dicistronic systems that rely on a strong conditional promoter for transcriptional control and a low-expression marker gene for selection. This strategy allows easy and efficient delivery of genes of interest whose expression levels require regulation either to mitigate potential toxicity or allow differential expression under controlled experimental conditions. Finally, as an additional practical demonstration of the utility of the extended FMDV 2A system, confocal fluorescence microscopy is used to demonstrate that native and foreign proteins of interest bearing post-translational remnants of the extended FMDV 2A peptide localize correctly to various cellular compartments, including a striking demonstration of the almost exclusive localization of the Rubisco small subunit protein to the pyrenoid of the C. reinhardtii chloroplast in cells maintained under ambient CO2 concentrations. [ABSTRACT FROM AUTHOR]

The CO2-concentrating mechanism (CCM) of Chlamydomonas reinhardtii and other microalgal species is essential for photosynthetic growth in most natural settings. A great deal has been learned regarding the CCM in cyanobacteria, including identification of inorganic carbon (Ci; CO2 and HCO3-) transporters; however, specific knowledge of analogous transporters has remained elusive in eukaryotic microalgae such as C. reinhardtii. Here we investigated whether the limiting-CO2-inducible, putative ABC-type transporter HLA3 might function as a HCO3- transporter by evaluating the effect of pH on growth, photosynthetic Ci affinity, and [14C]-Ci uptake in very low CO2 conditions following RNA interference (RNAi) knockdown of HLA3 mRNA levels in wild-type and mutant cells. Although knockdown of HLA3 mRNA alone resulted in only modest but high-pH-dependent decreases in photosynthetic Ci affinity and Ci uptake, the combination of nearly complete knockdown of HLA3 mRNA with mutations in LCIB (which encodes limiting-Ci-inducible plastid-localized protein required for normal Ci uptake or accumulation in low-CO2 conditions) and/or simultaneous, apparently off-target knockdown of LCIA mRNA (which encodes limiting-Ci-inducible plastid envelope protein reported to transport HCO3-) resulted in dramatic decreases in growth, Ci uptake, and photosynthetic Ci affinity, especially at pH 9, at which HCO3- is the predominant form of available Ci. Collectively, the data presented here provide compelling evidence that HLA3 is directly or indirectly involved in HCO3- transport, along with additional evidence supporting a role for LCIA in chloroplast envelope HCO3- transport and a role for LCIB in chloroplast Ci accumulation. [ABSTRACT FROM AUTHOR]

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Characterizes the `green' form I ribulose 1,5-biphosphate carboxylase/oxygenase (RubisCO) from the nonsulfur purple bacterium Rhodobacter capsulatus. RubisCO gene expression and enzyme purification; Enzymatic characterization of R. capsulatus form I RubisCO; Expression of wild-type, hybrid, and chimeric RubisCO in Escherichia coli.

Objective Conversion disorder is the presence of neurological symptoms that are not due to neurological disease and are thought to be psychological in origin. It is assumed that patients have normal brain anatomy; structural brain abnormalities of potential aetiological relevance generally preclude the diagnosis. However, it remains possible there are subtle neuroanatomical differences that are only discernable at the group, rather than at the patient level. We aimed to test this hypothesis by comparing high-resolution MRI scans of patients with motor conversion disorder with healthy controls using a Region Of Interest (ROI) approach. Method 15 patients with ICD-10 diagnoses of motor conversion disorder with symptom onset within 2 years and 31 age- and sexmatched healthy controls had high resolution Spoiled Gradient Recalled sequence scanning on a 3 T MRI scanner. ROIs, chosen on the basis of previous reports or on theoretical grounds, were placed on the bilateral amygdala, thalamus, caudate and lentiform nuclei. Freesurfer V.5.0 software was used to identify, label and measure the anatomical structure volumes. Results There were uncorrected reductions in bilateral thalamic volume in patients compared to controls (left thalamus p=0.001, right thalamus p=0.002). Intracranial volume was also reduced in patients, however (p=0.012), and when corrected for this the thalamic reductions remained significant (left thalamus p=0.02, right thalamus p=0.05) and there was a significant (p=0.05) reduction in the volume of the left, but not the right, lentiform nucleus. There were no other ROI differences. Conclusion We have found evidence for reduced volume of the thalamus in conversion disorder. These changes could be secondary to chronic limb immobility as there is some evidence of similar thalamic volume losses after limb amputation. Longitudinal studies of conversion disorder patients, particularly re-scanning post recovery, would be needed to address this question. If thalamic volume changes are found to correlate to level of limb disuse such changes would be a strong indicator of the severity of this disorder in terms of the amount of actual, as well as reported, immobility. However, a functional imaging study (Vuilleumier et al, 2001) has found evidence of reduced activation of the thalamus (and basal ganglia) in symptomatic conversion disorder patients and it therefore remains possible these changes could be of aetiological or mechanistic significance. [ABSTRACT FROM AUTHOR]

The availability of the complete DNA sequence of the Chlamydomonas reinhardtii genome and advanced computational biology tools has allowed elucidation and study of the small ubiquitin-like modifier (SUMO) system in this unicellular photosynthetic alga and model eukaryotic cell system. SUMO is a member of a ubiquitin-like protein superfamily that is covalently attached to target proteins as a post-translational modification to alter the localization, stability, and/or function of the target protein in response to changes in the cellular environment. Three SUMO homologs (CrSUMO96, CrSUMO97, and CrSUMO148) and three novel SUMO-related proteins (CrSUMO-like89A, CrSUMO-like89B, and CrSUMO-like90) were found by diverse gene predictions, hidden Markov models, and database search tools inferring from Homo sapiens, Saccharomyces cerevisiae, and Arabidopsis thaliana SUMOs. Among them, CrSUMO96, which can be recognized by the A. thaliana anti-SUMO1 antibody, was studied in detail. Free CrSUMO96 was purified by immunoprecipitation and identified by mass spectrometry analysis. A SUMO-conjugating enzyme (SCE) (E2, Ubc9) in C. reinhardtii was shown to be functional in an Escherichia coli-based in vivo chimeric SUMOylation system. Antibodies to CrSUMO96 recognized free and conjugated forms of CrSUMO96 in Western blot analysis of whole-cell extracts and nuclear localized SUMOylated proteins with in situ immunofluorescence. Western blot analysis showed a marked increase in SUMO conjugated proteins when the cells were subjected to environmental stresses, such as heat shock and osmotic stress. Related analyses revealed multiple potential ubiquitin genes along with two Rub1 genes and one Ufm1 gene in the C. reinhardtii genome. [ABSTRACT FROM AUTHOR]

The eriophyid mite transmitted Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) shares a common genome organization with aphid transmitted species of the genus Potyvirus. Although both tritimoviruses and potyviruses encode helper component-proteinase (HC-Pro) homologues (required for nonpersistent aphid transmission of potyviruses), sequence conservation is low (amino acid identity, ∼16%), and a role for HC-Pro in semipersistent transmission of WSMV by the wheat curl mite (Aceria tosichella [Keifer]) has not been investigated. Wheat curl mite transmissibility was abolished by replacement of WSMV HC-Pro with homologues of an aphid transmitted potyvirus (Turnip mosaic virus), a rymovirus (Agropyron mosaic virus) vectored by a different eriophyid mite, or a closely related tritimovirus (Oat necrotic mottle virus; ONMV) with no known vector. In contrast, both WSMV-Sidney 81 and a chimeric WSMV genome bearing HC-Pro of a divergent strain (WSMV-El Batán 3; 86% amino acid sequence identity) were efficiently transmitted by A. tosichella. Replacing portions of WSMV-Sidney 81 HC-Pro with the corresponding regions from ONMV showed that determinants of wheat curl mite transmission map to the 5′-proximal half of HC-Pro. WSMV genomes bearing HC-Pro of heterologous species retained the ability to form virions, indicating that loss of vector transmissibility was not a result of failure to encapsidate. Although titer in systemically infected leaves was reduced for all chimeric genomes relative to WSMV-Sidney 81, titer was not correlated with loss of vector transmissibility. Collectively, these results demonstrate for the first time that HC-Pro is required for virus transmission by a vector other than aphids. [ABSTRACT FROM AUTHOR]