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Saccharomyces cerevisiae is an important unicellular yeast species within the biotechnological and the food and beverage industries. A significant application of this species is the production of ethanol, where concentrations are limited by cellular toxicity, often at the level of the cell membrane. Here, we characterize 61 S. cerevisiae strains for ethanol tolerance and further analyze five representatives with various ethanol tolerances. The most tolerant strain, AJ4, was dominant in coculture at 0 and 10% ethanol. Unexpectedly, although it does not have the highest noninhibitory concentration or MIC, MY29 was the dominant strain in coculture at 6% ethanol, which may be linked to differences in its basal lipidome. Although relatively few lipidomic differences were observed between strains, a significantly higher phosphatidylethanolamine concentration was observed in the least tolerant strain, MY26, at 0 and 6% ethanol compared to the other strains that became more similar at 10%, indicating potential involvement of this lipid with ethanol sensitivity. Our findings reveal that AJ4 is best able to adapt its membrane to become more fluid in the presence of ethanol and that lipid extracts from AJ4 also form the most permeable membranes. Furthermore, MY26 is least able to modulate fluidity in response to ethanol, and membranes formed from extracted lipids are least leaky at physiological ethanol concentrations. Overall, these results reveal a potential mechanism of ethanol tolerance and suggest a limited set of membrane compositions that diverse yeast species use to achieve this. [ABSTRACT FROM AUTHOR]

Our previous studies on the human β globin gene cluster revealed the presence of intergenic transcripts throughout the locus, and demonstrated that transcription of the locus control region (LCR) initiates within an ERV9 endogenous retroviral long-terminal repeat (LTR) upstream of DNase I hypersensitive site 5. We show, using a combination of assays, that there are additional sites of transcription initiation within the LCR at hypersensitive sites 2 and 3. We have defined sites of transcription initiation, which occurs at discrete positions in a direction towards the globin genes. In addition, we show that mutation of specific transcription factor binding sites within HS2 leads to a reduction in transcription levels from within this site. We propose that these initiation events within the LCR can account for the observed orientation dependence of LCR function, and contribute to the open chromatin configuration of the β globin locus. In addition, transcription from within the LCR hypersensitive sites could compensate for the absence of the ERV9 LTR in many transgenic mice lines, which nevertheless regulate their globin clusters correctly. [Copyright &y& Elsevier]

Abstract Background Pichia pastoris is a widely-used host for recombinant protein production. Initial screening for both suitable clones and optimum culture conditions is typically carried out in multi-well plates. This is followed by up-scaling either to shake-flasks or continuously stirred tank bioreactors. A particular problem in these formats is foaming, which is commonly prevented by the addition of chemical antifoaming agents. Intriguingly, antifoams are often added without prior consideration of their effect on the yeast cells, the protein product or the influence on downstream processes such as protein purification. In this study we characterised, for the first time, the effects of five commonly-used antifoaming agents on the total amount of recombinant green fluorescent protein (GFP) secreted from shake-flask cultures of this industrially-relevant yeast. Results Addition of defined concentrations of Antifoam A (Sigma), Antifoam C (Sigma), J673A (Struktol), P2000 (Fluka) or SB2121 (Struktol) to shake-flask cultures of P. pastoris increased the total amount of recombinant GFP in the culture medium (the total yield) and in the case of P2000, SB2121 and J673A almost doubled it. When normalized to the culture density, the GFP specific yield (μg OD595-1) was only increased for Antifoam A, Antifoam C and J673A. Whilst none of the antifoams affected the growth rate of the cells, addition of P2000 or SB2121 was found to increase culture density. There was no correlation between total yield, specific yield or specific growth rate and the volumetric oxygen mass transfer coefficient (kLa) in the presence of antifoam. Moreover, the antifoams did not affect the dissolved oxygen concentration of the cultures. A comparison of the amount of GFP retained in the cell by flow cytometry with that in the culture medium by fluorimetry suggested that addition of Antifoam A, Antifoam C or J673A increased the specific yield of GFP by increasing the proportion secreted into the medium. Conclusions We show that addition of a range of antifoaming agents to shake flask cultures of P. pastoris increases the total yield of the recombinant protein being produced. This is not only a simple method to increase the amount of protein in the culture, but our study also provides insight into how antifoams interact with microbial cell factories. Two mechanisms are apparent: one group of antifoams (Antifoam A, Antifoam C and J673A) increases the specific yield of GFP by increasing the total amount of protein produced and secreted per cell, whilst the second (P2000 or SB2121) increases the total yield by increasing the density of the culture.

The first intracellular loop (ICL1) of G protein-coupled receptors (GPCRs) has received little attention, although there is evidence that, with the 8 th helix (H8), it is involved in early conformational changes following receptor activation as well as contacting the G protein β subunit. In class B1 GPCRs, the distal part of ICL1 contains a conserved R 12.48 KLRCxR 2.46b motif that extends into the base of the second transmembrane helix; this is weakly conserved as a [R/H] 12.48 KL[R/H] motif in class A GPCRs. In the current study, the role of ICL1 and H8 in signaling through cAMP, i Ca 2+ and ERK1/2 has been examined in two class B1 GPCRs, using mutagenesis and molecular dynamics. Mutations throughout ICL1 can either enhance or disrupt cAMP production by CGRP at the CGRP receptor. Alanine mutagenesis identified subtle differences with regard elevation of i Ca 2+ , with the distal end of the loop being particularly sensitive. ERK1/2 activation displayed little sensitivity to ICL1 mutation. A broadly similar pattern was observed with the glucagon receptor, although there were differences in significance of individual residues. Extending the study revealed that at the CRF1 receptor, an insertion in ICL1 switched signaling bias between i Ca 2+ and cAMP. Molecular dynamics suggested that changes in ICL1 altered the conformation of ICL2 and the H8/TM7 junction (ICL4). For H8, alanine mutagenesis showed the importance of E390 8.49b for all three signal transduction pathways, for the CGRP receptor, but mutations of other residues largely just altered ERK1/2 activation. Thus, ICL1 may modulate GPCR bias via interactions with ICL2, ICL4 and the Gβ subunit., Competing Interests: AJ and NJR were employees of Sosei Heptares at the time the work was performed. Neither are now employed by Sosei Heptares. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Winfield, Barkan, Routledge, Robertson, Harris, Jazayeri, Simms, Reynolds, Poyner and Ladds.)

Pitt-Hopkins syndrome (PTHS) is a neurodevelopmental disorder characterized by intellectual disability, specific facial features, and marked autonomic nervous system dysfunction, especially with disturbances of regulating respiration and intestinal mobility. It is caused by variants in the transcription factor TCF4. Heterogeneity in the clinical and molecular diagnostic criteria and care practices has prompted a group of international experts to establish guidelines for diagnostics and care. For issues, for which there was limited information available in international literature, we collaborated with national support groups and the participants of a syndrome specific international conference to obtain further information. Here, we discuss the resultant consensus, including the clinical definition of PTHS and a molecular diagnostic pathway. Recommendations for managing particular health problems such as dysregulated respiration are provided. We emphasize the need for integration of care for physical and behavioral issues. The recommendations as presented here will need to be evaluated for improvements to allow for continued optimization of diagnostics and care., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The canonical CGRP receptor is a complex between calcitonin receptor-like receptor (CLR), a family B G-protein-coupled receptor (GPCR) and receptor activity-modifying protein 1 (RAMP1). A third protein, receptor component protein (RCP) is needed for coupling to Gs. CGRP can interact with other RAMP-receptor complexes, particularly the AMY1 receptor formed between the calcitonin receptor (CTR) and RAMP1. Crystal structures are available for the binding of CGRP 27-37 [D 31 ,P 34 ,F 35 ] to the extracellular domain (ECD) of CLR and RAMP1; these show that extreme C-terminal amide of CGRP interacts with W84 of RAMP1 but the rest of the analogue interacts with CLR. Comparison with the crystal structure of a fragment of the allied peptide adrenomedullin bound to the ECD of CLR/RAMP2 confirms the importance of the interaction of the ligand C-terminus and the RAMP in determining pharmacology specificity, although the RAMPs probably also have allosteric actions. A cryo-electron microscope structure of calcitonin bound to the full-length CTR associated with Gs gives important clues as to the structure of the complete receptor and suggests that the N-terminus of CGRP makes contact with His 5.40b , high on TM5 of CLR. However, it is currently not known how the RAMPs interact with the TM bundle of any GPCR. Major challenges remain in understanding how the ECD and TM domains work together to determine ligand specificity, and how G-proteins influence this and the role of RCP. It seems likely that allosteric mechanisms are particularly important as are the dynamics of the receptors.

Background: Pitt-Hopkins syndrome (PTHS; MIM# 610954) is a genetically determined entity mainly caused by mutations in TransCription Factor 4 (TCF4). We have developed a new way to collect information on (ultra-)rare disorders through a web-based database which we call 'waihonapedia' (waihona [meaning treasure in Hawaiian] encyclopaedia)., Methods: We have built a waihonapedia system in a collaboration between physicians, social scientists, and parent support groups. The system consists of an initial extensive questionnaire for background cross-sectional data, and subsequent follow-up using small questionnaires, with a particular focus on behavioural aspects. The system was built to be used through the internet, ensuring a secure environment, respecting privacy for participants, and acting automated to allow for low costs and limiting human mistakes in data handling. Recruitment of participants is through the patient support groups. In addition, as a sub-study, we used the data from the waihonapedia system to compare the two proposed diagnostic classification systems for PTHS., Results: We present here the results of the initial, cross-sectional questionnaire in which early development, physical health, cognition and behaviour are interrogated, and to which modules specific for PTHS were added on epilepsy and breathing patterns. We describe 101 individuals with a molecularly confirmed diagnosis of PTHS. Comparison of the two classification systems aimed at helping the clinical diagnosis was performed in 47 of the present PTHS individuals, with disappointing results for both. Internationally accepted clinical diagnostic criteria are needed., Conclusion: The present cross-sectional data on the natural history of PTHS have yielded useful information which will further increase when follow-up data will be added. No doubt this will improve both care and research.

Background: Muscle fibrosis characterizes degenerated muscles in muscular dystrophies and in late onset myopathies. Fibrotic muscles often exhibit thickening of the extracellular matrix (ECM). The molecular regulation of this process is not fully understood. In oculopharyngeal muscular dystrophy (OPMD), an expansion of an alanine tract at the N-terminus of poly(A)-binding protein nuclear 1 (PABPN1) causes muscle symptoms. OPMD patient muscle degeneration initiates after midlife, while at an earlier age carriers of alanine expansion mutant PABPN1 (expPABPN1) are clinically pre-symptomatic. OPMD is characterized by fibrosis in skeletal muscles but the causative molecular mechanisms are not fully understood., Methods: We studied the molecular processes that are involved in OPMD pathology using cross-species mRNA expression profiles in muscles from patients and model systems. We identified significant dysregulation of the ECM functional group, among which the procollagen C-endopeptidase enhancer 1 gene (PCOLCE) was consistently down-regulated across species. We investigated PCOLCE subcellular localization in OPMD muscle samples and OPMD model systems to investigate any functional relevance of PCOLCE down-regulation in this disease., Results: We found that muscle degeneration in OPMD is associated with PCOLCE down-regulation. In addition to its known presence at the ECM, we also found PCOLCE within the nucleus of muscle cells. PCOLCE sub-cellular localization changes during myoblast cell fusion and is disrupted in cells expressing mutant expPABPN1. Our results show that PCOLCE binds to soluble PABPN1 and co-localizes with aggregated PABPN1 with a preference for the mutant protein. In muscle biopsies from OPMD patients we find that extracellular PCOLCE is depleted with its concomitant enrichment within the nuclear compartment., Conclusions: PCOLCE regulates collagen processing at the ECM. Depletion of extracellular PCOLCE is associated with the expression of expPABPN1 in OPMD patient muscles. PCOLCE is also localized within the nucleus where it binds to PABPN1, suggesting that PCOLCE shuttles between the ECM and the nucleus. PCOLCE preferentially binds to expPABPN1. Nuclear-localized PCOLCE is enriched in muscle cells expressing expPABPN1. We suggest that nuclear entrapment of PCOLCE and its extracellular depletion represents a novel molecular mechanism in late-onset muscle fibrosis.

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease caused by an alanine tract expansion mutation in poly(A) binding protein nuclear 1 (expPABPN1). To model OPMD in a myogenic and physiological context, we generated mouse myoblast cell clones stably expressing either human wild type (WT) or expPABPN1 at low levels. Transgene expression is induced on myotube differentiation and results in formation of insoluble nuclear PABPN1 aggregates that are similar to those observed in patients with OPMD. Quantitative analysis of PABPN1 in myotube cultures revealed that expPABPN1 accumulation and aggregation is greater than that of the WT protein. We found that aggregation of expPABPN1 is more affected than WT PABPN1 by inhibition of proteasome activity. Consistent with this, in myotube cultures expressing expPABPN1, deregulation of the proteasome was identified as the most significantly perturbed pathway. Differences in the accumulation of soluble WT and expPABPN1 were consistent with differences in ubiquitination and rate of protein turnover. This study demonstrates, for the first time to our knowledge, that, in myotubes, the ratio of soluble/insoluble expPABPN1 is significantly lower compared with that of the WT protein. We suggest that this difference can contribute to muscle weakness in OPMD., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Replicating episomal vectors (REV) are in principle able to provide long-term transgene expression in the absence of integration into the target cell genome. The scaffold/matrix attachment region (S/MAR) located 5' of the human beta-interferon gene (IFNB1) has been shown to confer a stable episomal replication and retention function within plasmid vectors when stably transfected and selected in mammalian cells. The minimal requirement for the IFNB1 S/MAR to function in DNA replication and episomal retention is transcription through this element. We used the erythroid beta-globin locus control region-beta-globin gene (betaLCR-HBB) microlocus cassette as a model to assess tissue-specific expression from within an IFNB1 S/MAR-based plasmid REV. The betaLCR-HBB plus S/MAR combination constructs provided either high or low levels of transcription through the S/MAR element. Our results show that the betaLCR-HBB microlocus is able to reproducibly and stably express at full physiological levels on an episome copy number basis. In addition, our data show that even low levels of transcription from betaLCR-HBB through the S/MAR element are sufficient to allow efficient episomal replication and retention. These data provide the principles upon which generic and flexible expression cassette-S/MAR-based REVs can be designed for a wide range of applications.

The mechanisms by which mutations within the 5' untranslated region (UTR) of the human beta-globin gene (HBB) cause thalassaemia are currently not well understood. We present here the first comprehensive comparative functional analysis of four 'silent' mutations in the human beta-globin 5'UTR, namely: +10(-T), +22(G --> A), +33(C --> G) and +(40-43)(-AAAC), which are present in patients with beta-thalassaemia intermedia. Expression of these genes under the control of the beta-globin locus control region in stable transfected murine erythroleukaemia cells showed that all four mutations decreased steady state levels of mRNA to 61.6%, 68%, 85.2% and 70.6%, respectively, compared with the wildtype gene. These mutations did not interfere with either mRNA transport from the nucleus to the cytoplasm, 3' end processing or mRNA stability. Nuclear run-on experiments demonstrated that mutations +10(-T) and +33(C --> G) reduced the rate of transcription to a degree that fully accounted for the observed lower level of mRNA accumulation, suggesting a disruption of downstream promoter sequences. Interestingly, mutation +22(G --> A) decreased the rate of transcription to a low degree, indicating the existence of a mechanism that acts post-transcriptionally. Generally, our data demonstrated the significance of functionally analysing mutants of this type in the presence of a full complement of transcriptional regulatory elements within a stably integrated chromatin context in an erythroid cell environment.

Our previous studies on nascent transcription across the human beta-globin gene cluster revealed the presence of intergenic transcripts in addition to the expected genic transcripts. We now show that transcription into the beta-globin locus control region (LCR) begins within an ERV9 endogenous retroviral long terminal repeat upstream of DNase I hypersensitive site 5. However, in a transgenic mouse, which has the human beta-globin LCR but lacks the ERV9 LTR, transcription begins upstream of the transgenic locus. We postulate that in this transgenic mouse nearby endogenous mouse promoters are activated by the LCR. Intergenic transcription is also detected across the whole transgenic globin gene locus independently of the stage of erythroid development. Intergenic transcription in the beta-globin cluster is erythroid specific; however, it can be induced in nonerythroid cells by several means: by transinduction with a plasmid transcribing part of the cluster, by exogenous addition of transcription factors, and by treatment with the histone deacetylase inhibitor trichostatin A.

The ribosomal frameshifting signal present in the genomic RNA of the coronavirus infectious bronchitis virus (IBV) contains a classic hairpin-type RNA pseudoknot that is believed to possess coaxially stacked stems of 11 bp (stem 1) and 6 bp (stem 2). We investigated the influence of stem 1 length on the frameshift process by measuring the frameshift efficiency in vitro of a series of IBV-based pseudoknots whose stem 1 length was varied from 4 to 13 bp in single base-pair increments. Efficient frameshifting depended upon the presence of a minimum of 11 bp; pseudoknots with a shorter stem 1 were either non-functional or had reduced frameshift efficiency, despite the fact that a number of them had a stem 1 with a predicted stability equal to or greater than that of the wild-type IBV pseudoknot. An upper limit for stem 1 length was not determined, but pseudoknots containing a 12 or 13 bp stem 1 were fully functional. Structure probing analysis was carried out on RNAs containing either a ten or 11 bp stem 1; these experiments confirmed that both RNAs formed pseudoknots and appeared to be indistinguishable in conformation. Thus the difference in frameshifting efficiency seen with the two structures was not simply due to an inability of the 10 bp stem 1 construct to fold into a pseudoknot. In an attempt to identify other parameters which could account for the poor functionality of the shorter stem 1-containing pseudoknots, we investigated, in the context of the 10 bp stem 1 construct, the influence on frameshifting of altering the slippery sequence-pseudoknot spacing distance, loop 2 length, and the number of G residues at the bottom of the 5'-arm of stem 1. For each parameter, it was possible to find a condition where a modest stimulation of frameshifting was observable (about twofold, from seven to a maximal 17 %), but we were unable to find a situation where frameshifting approached the levels seen with 11 bp stem 1 constructs (48-57 %). Furthermore, in the next smaller construct (9 bp stem 1), changing the bottom four base-pairs to G.C (the optimal base composition) only stimulated frameshifting from 3 to 6 %, an efficiency about tenfold lower than seen with the 11 bp construct. Thus stem 1 length is a major factor in determining the functionality of this class of pseudoknot and this has implications for models of the frameshift process., (Copyright 1999 Academic Press.)