Library preparation and MiSeq sequencing for the genotyping-by-sequencing of the Huntington disease HTT exon one trinucleotide repeat and the quantification of somatic mosaicism [Protocol]

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG repeat in the first exon of the HTT gene. Affected individuals inherit more than 40 repeats and the CAG repeat is genetically unstable in both the germline and soma. Molecular diagnosis and genotyping of the CAG repeat is traditionally performed by estimation of PCR fragment size. However, this approach is complicated by the presence of an adjacent polymorphic CCG repeat and provides no information on the presence of variant repeats, flanking sequence variants or on the degree of somatic mosaicism. To overcome these limitations, we have developed an amplicon-sequencing protocol that allows the sequencing of hundreds of samples in a single MiSeq run. The composition of the HTT exon one trinucleotide repeat locus can be determined from the MiSeq sequencing reads generated. With sufficient sequencing depth, such MiSeq data can also be used to quantify the degree of somatic mosaicism of the HTT CAG repeat in the tissue analysed

Algal genomes reveal evolutionary mosaicism and the fate of nucleomorphs

Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote–eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have <21, 000 protein genes and are intron rich, and B. natans exhibits unprecedented alternative splicing for a single-celled organism. Phylogenomic analyses and subcellular targeting predictions reveal extensive genetic and biochemical mosaicism, with both host- and endosymbiont-derived genes servicing the mitochondrion, the host cell cytosol, the plastid and the remnant endosymbiont cytosol of both algae. Mitochondrion-to-nucleus gene transfer still occurs in both organisms but plastid-to-nucleus and nucleomorph-to-nucleus transfers do not, which explains why a small residue of essential genes remains locked in each nucleomorph. © 2012 Macmillan Publishers Limited. All rights reserved

A review of psychosocial interventions targeting families of children with cancer

Objective Psychosocial interventions in families of children with cancer are considered an effective way of empowering family members to tackle the complex hurdles they face. The ability of parents to develop adaptive coping strategies during the child&apos;s treatment is not only important to their own mental and physical health, but also to their child&apos;s well-being and long-term adjustment with the disease. Methods The aim of this review was to evaluate the existing literature for the period from 2009 to 2017 on psychosocial interventions targeting families of children with cancer. We searched the PubMed database using the following combination of keywords: cancer AND children AND (intervention OR training) AND (mothers OR primary caregivers OR parents OR fathers OR siblings). Results After careful evaluation of 995 papers, 17 full-text papers were found to match our criteria (12 randomized controlled trials and 5 quasi-experimental studies). The quality of the studies was assessed using the Delphi score questionnaire, and the score of the reviewed studies ranged from 3 to 5. The findings suggest that most interventions reduced distress and improved coping strategies among participants. Interventions, mainly cognitive behavioral therapy and problem-solving skills training targeting maternal distress, were associated with improved adjustment outcomes in mothers of children with cancer. Significance of results Psychosocial interventions are helpful, and efforts should be made to promote them in a larger scale. Protocols should be implemented to ensure that all parents benefit. Computer-assisted methods may provide additional benefit by improving cancer-related knowledge and cancer-related communication. Copyright © The Author(s), 2020. Published by Cambridge University Press

Factors Affecting Opioid Treatment in Cancer Patients

Purpose: Pain is prevalent in cancer patients, appearing to be moderate to severe in more than one third of them. Despite the fact that fentanyl is widely used with effective analgesic results, some patients do not correspond to treatment, resulting in opioid change. Methods: This is a cohort study, performed in Greek patients with cancer. Its scope was to identify potential reasons responsible for opioid change, due to transdermal-fentanyl intolerance, resulting from inadequate analgesia (pain relief% in 1week) and/or unacceptable adverse-events (grade≥3 at Common Terminology Criteria-v4.0). The final sample included 289 participants. To investigate responsible reasons for transdermal-fentanyl intolerance we studied its relation with patients’ history, haematology, biochemistry, body-mass-index, demographic and disease related characteristics. The Eastern Cooperative Oncology Group performance status scale, the Mini Mental State Examination questionnaire, the M.D.Anderson Symptom Inventory and the Greek Brief Pain Inventory were also used to measure performance status and quality-of-life for the same reason. Results: Almost one third of the patients had to change to an alternative opioid oral-morphine in order to achieve adequate analgesia or/and avoid adverse-events. The most common adverse-events observed were nausea/vomiting and sleepiness. Statistical analysis demonstrated that younger age (OR=0.976) and obesity (OR=0.29 against underweight, OR=0.39 against normal, OR=0.48 against pre-obese) had a higher possibility to contribute to modification of the analgesic treatment. Furthermore, a higher impact of symptoms in patient’s life (OR=1.184) and chemotherapy (OR=2.109) could also contribute to the need of change of the opioid analgesic medication. Conclusion: This study found significant variables for transdermal-fentanyl intolerance. This knowledge may help person-center care in moderate to severe cancer pain. © 2019 Journal of Pharmacy and Pharmaceutical Sciences. All rights reserved

Evaluation of instrumental activities of daily living in Greek patients with advanced cancer

Translation of the instrumental activities of daily living (IADL) was carried out and its psychometric properties were assessed in a Greek sample of patients with advanced cancer. The scale was translated with the forward-backward procedure into the Greek language. It was initially administered to 136 advanced cancer patients. To assess reliability, it was administered to 45 patients 3 days later. To assess the effect of treatment, 75 patients were studied. The patients also completed the Eastern Cooperative Oncology Group (ECOG) performance status and the linear analogue scale assessment (LASA) quality of life (QoL) scale. Confirmatory factor analysis of the IADL was carried out. Reliability was assessed in terms of internal consistency (Cronbach&apos;s α) and test-retest correlation (Pearson and ICC) of the IADL scale. Construct validity was assessed through correlation of IADL with ECOG and LASA QoL scores. Confirmatory factor analysis yielded a single-factor model. The homogeneity of the instrument proved to be satisfactory (α was 0.88 for men and 0.83 for women). Test-retest reliability was also satisfactory (P&lt;0.0005). High correlation with ECOG (men, r=-0.87; women, r=-0.85) and LASA QoL (men, r=0.55; women, r=0.53) was observed. The Greek version of the IADL in cancer patients treated in a palliative care unit is a reliable and valid clinical instrument. © 2013 Wolters Kluwer Health | Lippincott Williams &amp; Wilkins

The chromatin landscape of the moss <em>Physcomitrella patens</em> and its dynamics during development and drought stress

International audienceThe moss Physcomitrella patens is an important model organism for evo-devo studies. Here, we determined the genome-wide chromatin landscape of five important histone three (H3) modifications (H3K4me3, H3K27me3, H3K27Ac, H3K9Ac and H3K9me2) and describe the changes to these histone marks in two contrasted situations, developmental transition and abiotic (drought) stress. Integrative analysis of these histone H3 modifications revealed their preferential association into 15 chromatin states (CS) in genic regions of the P. patens genome. Synergistic relationships that influence expression levels were revealed for the three activating marks H3K4me3, H3K27Ac and H3K9Ac, while an antagonistic relationship was found between CS containing the H3K27me3 and H3K27Ac marks, suggesting that H3K27 is a key indexing residue regarding transcriptional output. Concerning the alteration of histone marks in response to developmental transition (juvenile to adult) and drought stress, the three activating marks H3K4me3, H3K27Ac and H3K9Ac show significant changes in both situations. However, changes to H3K27me3 are central only for genes differentially expressed during development. Interestingly, genes induced during drought stress show significant histone mark toggling during developmental transition. This situation suggests that drought induced adult (gametophore expressed) genes are primed to respond to this stress during the juvenile to adult transition

The BIR2/BIR3-Associated Phospholipase Dγ1 Negatively Regulates Plant Immunity

Plants have evolved effective strategies to defend themselves against pathogen invasion. Starting from the plasma membrane with the recognition of microbe-associated molecular patterns (MAMPs) via pattern recognition receptors, internal cellular signaling pathways are induced to ultimately fend off the attack. Phospholipase D (PLD) hydrolyzes membrane phospholipids to produce phosphatidic acid (PA), which has been proposed to play a second messenger role in immunity. The Arabidopsis (Arabidopsis thaliana) PLD family consists of 12 members, and for some of these, a specific function in resistance toward a subset of pathogens has been shown. We demonstrate here that Arabidopsis PLDγ1, but not its close homologs PLDγ2 and PLDγ3, is specifically involved in plant immunity. Genetic inactivation of PLDγ1 resulted in increased resistance toward the virulent bacterium Pseudomonas syringae pv. tomato DC3000 and the necrotrophic fungus Botrytis cinerea As pldγ1 mutant plants responded with elevated levels of reactive oxygen species to MAMP treatment, a negative regulatory function for this PLD isoform is proposed. Importantly, PA levels in pldγ1 mutants were not affected compared to stressed wild-type plants, suggesting that alterations in PA levels are not likely the cause for the enhanced immunity in the pldγ1 line. Instead, the plasma-membrane-attached PLDγ1 protein colocalized and associated with the BAK1-INTERACTING RECEPTOR-LIKE KINASES BIR2 and BIR3, which are known negative regulators of pattern-triggered immunity. Moreover, complex formation of PLDγ1 and BIR2 was further promoted upon MAMP treatment. Hence, we propose that PLDγ1 acts as a negative regulator of plant immune responses in complex with immunity-related proteins BIR2 and BIR3

The BIR2/BIR3-interacting Phospholipase D gamma 1 negatively regulates immunity in Arabidopsis

Plants have evolved effective strategies to defend themselves against pathogen invasion. Starting from the plasma membrane with the recognition of microbe-associated molecular patterns (MAMPs) via pattern recognition receptors, internal cellular signaling pathways are induced to ultimately fend off the attack. Phospholipase D (PLD) hydrolyzes membrane phospholipids to produce phosphatidic acid (PA), which has been proposed to play a second messenger role in immunity. The Arabidopsis PLD family consists of 12 members and for some a specific function in resistance towards a subset of pathogens has been shown. We demonstrate here that Arabidopsis PLDγ1, but not its close homologs PLDγ2 and PLDγ3, is specifically involved in plant immunity. Genetic inactivation of PLDγ1 resulted in increased resistance towards the virulent bacterium Pseudomonas syringae pv. tomato DC3000 and the necrotrophic fungus Botrytis cinerea. As pldγ1 mutant plants responded with elevated levels of reactive oxygen species to MAMP-treatment, a negative regulatory function for this PLD isoform is proposed. Importantly, PA levels in pldγ1 mutants were not affected compared to stressed wild-type plants, suggesting that alterations in PA levels are unlikely the cause for the enhanced immunity in the pldγ1 line. Instead, the plasma-membrane-attached PLDγ1 protein colocalized and associated with the receptor-like kinases BIR2 and BIR3, which are known negative regulators of pattern-triggered immunity. Moreover, complex formation of PLDγ1 and BIR2 was further promoted upon MAMP-treatment. Hence, we propose that PLDγ1 acts as a negative regulator of plant immune responses in complex with immunity-related proteins BIR2 and BIR3

GO annotation of R40 transcripts

GO annotation of R40 transcript

Combination of the endogenous lhcsr1 promoter and codon usage optimization boosts protein expression in the Moss Physcomitrella patens

The moss Physcomitrella patens is used both as an evo-devo model and biotechnological production system for metabolites and pharmaceuticals. Strong in vivo expression of genes of interest is important for production of recombinant proteins, e.g. selectable markers, fluorescent proteins or enzymes. In this regard, the choice of the promoter sequence as well as codon usage optimization are two important inside factors to consider in order to obtain optimum protein accumulation level. To reliably quantify fluorescence we transfected protoplasts with promoter:GFP fusion constructs and measured fluorescence intensity of living protoplasts in a plate reader system. We used the red fluorescent protein mCherry under 2x 35S promoter control as second reporter to normalize for different transfection efficiencies. We derived a novel endogenous promoter and compared deletion variants with exogenous promoters. We used different codon-adapted green fluorescent protein (GFP) genes to evaluate the influence of promoter choice and codon optimization on protein accumulation in P. patens, and show that the promoter of the gene of P. patens chlorophyll a/b binding protein LHCSR1 drives expression of GFP in protoplasts significantly (more than 2-fold) better than the commonly used 2x 35S promoter or the rice actin1 promoter. We identified a shortened 677 bp version of the lhcsr1 promoter that retains full activity in protoplasts. The codon optimized GFP yields significantly (more than 2-fold) stronger fluorescence signals and thus demonstrates that adjusting codon usage in P. patens can increase expression strength. In combination, new promotor and codon optimized GFP conveyed 6-fold increases fluorescence signal