Protein disulfide-isomerase interacts with a substrate protein at all stages along its folding pathway

In contrast to molecular chaperones that couple protein folding to ATP hydrolysis, protein disulfide-isomerase (PDI) catalyzes protein folding coupled to formation of disulfide bonds (oxidative folding). However, we do not know how PDI distinguishes folded, partly-folded and unfolded protein substrates. As a model intermediate in an oxidative folding pathway, we prepared a two-disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) and showed by NMR that it is partly-folded and highly dynamic. NMR studies show that it binds to PDI at the same site that binds peptide ligands, with rapid binding and dissociation kinetics; surface plasmon resonance shows its interaction with PDI has a Kd of ca. 10−5 M. For comparison, we characterized the interactions of PDI with native BPTI and fully-unfolded BPTI. Interestingly, PDI does bind native BPTI, but binding is quantitatively weaker than with partly-folded and unfolded BPTI. Hence PDI recognizes and binds substrates via permanently or transiently unfolded regions. This is the first study of PDI's interaction with a partly-folded protein, and the first to analyze this folding catalyst's changing interactions with substrates along an oxidative folding pathway. We have identified key features that make PDI an effective catalyst of oxidative protein folding – differential affinity, rapid ligand exchange and conformational flexibility

Transcriptomics reveal an integrative role for maternal thyroid hormones during zebrafish embryogenesis

Thyroid hormones (THs) are essential for embryonic brain development but the genetic mechanisms involved in the action of maternal THs (MTHs) are still largely unknown. As the basis for understanding the underlying genetic mechanisms of MTHs regulation we used an established zebrafish monocarboxylic acid transporter 8 (MCT8) knock-down model and characterised the transcriptome in 25hpf zebrafish embryos. Subsequent mapping of differentially expressed genes using Reactome pathway analysis together with in situ expression analysis and immunohistochemistry revealed the genetic networks and cells under MTHs regulation during zebrafish embryogenesis. We found 4,343 differentially expressed genes and the Reactome pathway analysis revealed that TH is involved in 1681 of these pathways. MTHs regulated the expression of core developmental pathways, such as NOTCH and WNT in a cell specific context. The cellular distribution of neural MTH-target genes demonstrated their cell specific action on neural stem cells and differentiated neuron classes. Taken together our data show that MTHs have a role in zebrafish neurogenesis and suggest they may be involved in cross talk between key pathways in neural development. Given that the observed MCT8 zebrafish knockdown phenotype resembles the symptoms in human patients with Allan-Herndon-Dudley syndrome our data open a window into understanding the genetics of this human congenital condition.Portuguese Fundacao para Ciencia e Tecnologia (FCT) [PTDC/EXPL/MARBIO/0430/2013]; CCMAR FCT Plurianual financing [UID/Multi/04326/2013]; FCT [SFRH/BD/111226/2015, SFRH/BD/108842/2015, SFRH/BPD/89889/2012]; FCT-IF Starting Grant [IF/01274/2014]info:eu-repo/semantics/publishedVersio

Simultaneous Activation of Complement and Coagulation by MBL-Associated Serine Protease 2

The complement system is an important immune mechanism mediating both recognition and elimination of foreign bodies. The lectin pathway is one pathway of three by which the complement system is activated. The characteristic protease of this pathway is Mannan-binding lectin (MBL)-associated serine protease 2 (MASP2), which cleaves complement proteins C2 and C4. We present a novel and alternative role of MASP2 in the innate immune system. We have shown that MASP2 is capable of promoting fibrinogen turnover by cleavage of prothrombin, generating thrombin. By using a truncated active form of MASP2 as well as full-length MASP2 in complex with MBL, we have shown that the thrombin generated is active and can cleave both factor XIII and fibrinogen, forming cross-linked fibrin. To explore the biological significance of these findings we showed that fibrin was covalently bound on a bacterial surface to which MBL/MASP2 complexes were bound. These findings suggest that, as has been proposed for invertebrates, limited clotting may contribute to the innate immune response

A robotic wheelchair trainer: design overview and a feasibility study

<p>Abstract</p> <p>Background</p> <p>Experiencing independent mobility is important for children with a severe movement disability, but learning to drive a powered wheelchair can be labor intensive, requiring hand-over-hand assistance from a skilled therapist.</p> <p>Methods</p> <p>To improve accessibility to training, we developed a robotic wheelchair trainer that steers itself along a course marked by a line on the floor using computer vision, haptically guiding the driver's hand in appropriate steering motions using a force feedback joystick, as the driver tries to catch a mobile robot in a game of "robot tag". This paper provides a detailed design description of the computer vision and control system. In addition, we present data from a pilot study in which we used the chair to teach children without motor impairment aged 4-9 (n = 22) to drive the wheelchair in a single training session, in order to verify that the wheelchair could enable learning by the non-impaired motor system, and to establish normative values of learning rates.</p> <p>Results and Discussion</p> <p>Training with haptic guidance from the robotic wheelchair trainer improved the steering ability of children without motor impairment significantly more than training without guidance. We also report the results of a case study with one 8-year-old child with a severe motor impairment due to cerebral palsy, who replicated the single-session training protocol that the non-disabled children participated in. This child also improved steering ability after training with guidance from the joystick by an amount even greater than the children without motor impairment.</p> <p>Conclusions</p> <p>The system not only provided a safe, fun context for automating driver's training, but also enhanced motor learning by the non-impaired motor system, presumably by demonstrating through intuitive movement and force of the joystick itself exemplary control to follow the course. The case study indicates that a child with a motor system impaired by CP can also gain a short-term benefit from driver's training with haptic guidance.</p

Rare mutations predisposing to familial adenomatous polyposis in Greek FAP patients

BACKGROUND: Familial Adenomatous Polyposis (FAP) is caused by germline mutations in the APC (Adenomatous Polyposis Coli) gene. The vast majority of APC mutations are point mutations or small insertions / deletions which lead to truncated protein products. Splicing mutations or gross genomic rearrangements are less common inactivating events of the APC gene. METHODS: In the current study genomic DNA or RNA from ten unrelated FAP suspected patients was examined for germline mutations in the APC gene. Family history and phenotype were used in order to select the patients. Methods used for testing were dHPLC (denaturing High Performance Liquid Chromatography), sequencing, MLPA (Multiplex Ligation – dependent Probe Amplification), Karyotyping, FISH (Fluorescence In Situ Hybridization) and RT-PCR (Reverse Transcription – Polymerase Chain Reaction). RESULTS: A 250 Kbp deletion in the APC gene starting from intron 5 and extending beyond exon 15 was identified in one patient. A substitution of the +5 conserved nucleotide at the splice donor site of intron 9 in the APC gene was shown to produce frameshift and inefficient exon skipping in a second patient. Four frameshift mutations (1577insT, 1973delAG, 3180delAAAA, 3212delA) and a nonsense mutation (C1690T) were identified in the rest of the patients. CONCLUSION: Screening for APC mutations in FAP patients should include testing for splicing defects and gross genomic alterations

Childhood adversity, mental ill-health and aggressive behavior in an African orphanage: Changes in response to trauma-focused therapy and the implementation of a new instructional system

<p>Abstract</p> <p>Background</p> <p>The number of orphans in Sub-Saharan Africa is constantly rising. While it is known that family or community care is preferable over institutional care of African orphans, little is known about the quality of care in orphanages and possibilities of improvement.</p> <p>Study 1</p> <p>Methods</p> <p>Exposure to traumatic stress, experiences of violence in the home, school and orphanage, as well as mental ill-health and aggression of 38 children (mean age of <it>M </it>= 8.64 years) living in an orphanage in rural Tanzania were assessed at two time points. The severity of post-traumatic stress disorder symptoms (PTSD), depressive symptoms, and internalizing and externalizing problems were used as indicators of mental ill-health.</p> <p>Results</p> <p>Violence experienced in the orphanage correlated more strongly with all indicators of mental ill-health than violence in the former home, school or neighborhood at time point 1. Additionally, violence experienced in the orphanage had a positive relationship with the aggressive behavior of the children at time point 2.</p> <p>Study 2</p> <p>Methods</p> <p>With the help of the pre-post assessment of Study 1, the implementation of a new instructional system and psychotherapeutic treatment (KIDNET) for trauma-related illness were evaluated.</p> <p>Results</p> <p>In response to both, a change in the instructional system and psychotherapeutic treatment of PTSD, a massive decline in experienced violence and in the severity of PTSD-symptoms was found, whereas depressive symptoms and internalizing and externalizing problems exhibited little change.</p> <p>Conclusions</p> <p>These studies show that violence, especially in the orphanage, can severely contribute to mental ill-health in orphans and that mental health can be improved by implementing a new instructional system and psychotherapeutic treatment in an orphanage. Moreover, the results indicate that the experience of violence in an orphanage also plays a crucial role in aggressive behavior of the orphans.</p

Manipulation of Plant Defense Responses by the Tomato Psyllid (Bactericerca cockerelli) and Its Associated Endosymbiont Candidatus Liberibacter Psyllaurous

Some plant pathogens form obligate relationships with their insect vector and are vertically transmitted via eggs analogous to insect endosymbionts. Whether insect endosymbionts manipulate plant defenses to benefit their insect host remains unclear. The tomato psyllid, Bactericerca cockerelli (Sulc), vectors the endosymbiont “Candidatus Liberibacter psyllaurous” (Lps) during feeding on tomato (Solanum lycopersicum L.). Lps titer in psyllids varied relative to the psyllid developmental stage with younger psyllids harboring smaller Lps populations compared to older psyllids. In the present study, feeding by different life stages of B. cockerelli infected with Lps, resulted in distinct tomato transcript profiles. Feeding by young psyllid nymphs, with lower Lps levels, induced tomato genes regulated by jasmonic acid (JA) and salicylic acid (SA) (Allene oxide synthase, Proteinase inhibitor 2, Phenylalanine ammonia-lyase 5, Pathogenesis-related protein 1) compared to feeding by older nymphs and adults, where higher Lps titers were found. In addition, inoculation of Lps without insect hosts suppressed accumulation of these defense transcripts. Collectively, these data suggest that the endosymbiont-like pathogen Lps manipulates plant signaling and defensive responses to benefit themselves and the success of their obligate insect vector on their host plant

Cost-effectiveness of nurse-led self-help for recurrent depression in the primary care setting: design of a pragmatic randomized trial

<p>Abstract</p> <p>Background</p> <p>Major Depressive Disorder is a leading cause of disability, tends to run a recurrent course and is associated with substantial economic costs due to increased healthcare utilization and productivity losses. Interventions aimed at the prevention of recurrences may reduce patients' suffering and costs. Besides antidepressants, several psychological treatments such as preventive cognitive therapy (PCT) are effective in the prevention of recurrences of depression. Yet, many patients find long-term use of antidepressants unattractive, do not want to engage in therapy sessions and in the primary care setting psychologists are often not available. Therefore, it is important to study whether PCT can be used in a nurse-led self-help format in primary care. This study sets out to test the hypothesis that usual care plus nurse-led self-help for recurrent depression in primary care is feasible, acceptable and cost-effective compared to usual care only.</p> <p>Design</p> <p>Patients are randomly assigned to ‘nurse-led self-help treatment plus usual care’ (134 participants) or ‘usual care’ (134 participants). Randomisation is stratified according to the number of previous episodes (2 or 3 previous episodes versus 4 or more). The primary clinical outcome is the cumulative recurrence rate of depression meeting DSM-IV criteria as assessed by the Structured-Clinical-Interview-for-DSM-IV- disorders at one year after completion of the intervention. Secondary clinical outcomes are quality of life, severity of depressive symptoms, co-morbid psychopathology and self-efficacy. As putative effect-moderators, demographic characteristics, number of previous episodes, type of treatment during previous episodes, age of onset, self-efficacy and symptoms of pain and fatigue are assessed. Cumulative recurrence rate ratios are obtained under a Poisson regression model. Number-needed-to-be-treated is calculated as the inverse of the risk-difference. The economic evaluation is conducted from a societal perspective, both as a cost-effectiveness analysis (costs per depression free survival year) and as a cost-utility analysis (costs per quality adjusted life-year).</p> <p>Discussion</p> <p>The purpose of this paper is to outline the rationale and design of a nurse-led, cognitive therapy based self-help aimed at preventing recurrence of depression in a primary care setting. Only few studies have focused on psychological self-help interventions aimed at the prevention of recurrences in primary care patients.</p> <p>Trial registration</p> <p>NTR3001 (<url>http://www.trialregister.nl</url>)</p