Methods for Investigating the UPR in Filamentous Fungi

Filamentous fungi have a high-capacity secretory system and are therefore widely exploited for the industrial production of native and heterologous proteins. However, in most cases, the yields of nonfungal proteins are significantly lower than those obtained for fungal proteins. One well-studied bottleneck appears to be the result of slow or aberrant folding of heterologous proteins in the ER during the early stages of secretion within the endoplasmic reticulum, leading to stress responses in the host, including the unfolded protein response (UPR). Most of the key elements constituting the signal transduction pathway of the UPR in Saccharomyces cerevisiae have been identified in filamentous fungi, including the central activation mechanism of the pathway, that is, the stress-induced splicing of an unconventional (nonspliceosomal) intron in orthologs of the HAC1 mRNA. This splicing event relieves a translational block in the HAC1 mRNA, allowing for the translation of the bZIP transcription factor Hac1p that regulates the expression of UPR target genes. The UPR is involved in regulating the folding, yield, and delivery of secretory proteins and that has consequences for fungal lifestyles, including virulence and biotechnology. The recent releases of genome sequences of several species of filamentous fungi and the availability of DNA arrays, GeneChips, and deep sequencing methodologies have provided an unprecedented resource for exploring expression profiles in response to secretion stresses. Furthermore, genome-wide investigation of translation profiles through polysome analyses is possible, and here, we outline methods for the use of such techniques with filamentous fungi and, principally, Aspergillus niger. We also describe methods for the batch and controlled cultivation of A. niger and for the replacement and study of its hacA gene, which provides either a UPR-deficient strain or a constitutively activated UPR strain for comparative analysis with its wild type. Although we focus on A. niger, the utility of the hacA-deletion strategy is also described for use in investigating the virulence of the plant pathogen Alternaria brassicicola

Expanding the ku70 toolbox for filamentous fungi: establishment of complementation vectors and recipient strains for advanced gene analyses

Mutants with a defective non-homologous-end-joining (NHEJ) pathway have boosted functional genomics in filamentous fungi as they are very efficient recipient strains for gene-targeting approaches, achieving homologous recombination frequencies up to 100%. For example, deletion of the ku70 homologous gene kusA in Aspergillus niger resulted in a recipient strain in which deletions of essential or non-essential genes can efficiently be obtained. To verify that the mutant phenotype observed is the result of a gene deletion, a complementation approach has to be performed. Here, an intact copy of the gene is transformed back to the mutant, where it should integrate ectopically into the genome. However, ectopic complementation is difficult in NHEJ-deficient strains, and the gene will preferably integrate via homologous recombination at its endogenous locus. To circumvent that problem, we have constructed autonomously replicating vectors useful for many filamentous fungi which contain either the pyrG allele or a hygromycin resistance gene as selectable markers. Under selective conditions, the plasmids are maintained, allowing complementation analyses; once the selective pressure is removed, the plasmid becomes lost and the mutant phenotype prevails. Another disadvantage of NHEJ-defective strains is their increased sensitivity towards DNA damaging conditions such as radiation. Thus, mutant analyses in these genetic backgrounds are limited and can even be obscured by pleiotropic effects. The use of sexual crossings for the restoration of the NHEJ pathway is, however, impossible in imperfect filamentous fungi such as A. niger. We have therefore established a transiently disrupted kusA strain as recipient strain for gene-targeting approaches

Social media in cardiology: Reasons to learn how to use it

Social media has changed the way we learn, educate, and interact with our peers. The dynamic nature of social media and their immediate availability through our portable devices (smartphones, tablets, smartwatches, etc.) is quickly transforming the way we participate in society. The scope of these digital tools is broad as they deal with many different aspects: Teaching and learning, case discussion, congresses coverage, peer to peer interaction, research are examples worth mentioning. The scientific societies considered more innovative, are promoting these tools between their members. These new concepts need to be known by the cardiologists to stay updated, as countless information is moving rapidly through these channels. We summarize the main reasons why learning how to use these tools to be part of the conversation is essential for the cardiologist in training or fully stablished

Increased H3K27 trimethylation contributes to cone survival in a mouse model of cone dystrophy

Inherited retinal diseases (IRDs) are a heterogeneous group of blinding disorders, which result in dysfunction or death of the light-sensing cone and rod photoreceptors. Despite individual IRDs (Inherited retinal disease) being rare, collectively, they affect up to 1:2000 people worldwide, causing a significant socioeconomic burden, especially when cone-mediated central vision is affected. This study uses the Pde6c$^{cpfl1}$ mouse model of achromatopsia, a cone-specific vision loss IRD (Inherited retinal disease), to investigate the potential gene-independent therapeutic benefits of a histone demethylase inhibitor GSK-J4 on cone cell survival. We investigated the effects of GSK-J4 treatment on cone cell survival in vivo and ex vivo and changes in cone-specific gene expression via single-cell RNA sequencing. A single intravitreal GSK-J4 injection led to transcriptional changes in pathways involved in mitochondrial dysfunction, endoplasmic reticulum stress, among other key epigenetic pathways, highlighting the complex interplay between methylation and acetylation in healthy and diseased cones. Furthermore, continuous administration of GSK-J4 in retinal explants increased cone survival. Our results suggest that IRD (Inherited retinal disease)-affected cones respond positively to epigenetic modulation of histones, indicating the potential of this approach in developing a broad class of novel therapies to slow cone degeneration

Myocardial infarction with non-obstructive coronary arteries: A comprehensive review and future research directions

Acute coronary syndromes constitute a variety of myocardial injury presentations that include a subset of patients presenting with myocardial infarction with non-obstructive coronary arteries (MINOCA). This acute coronary syndrome differs from type 1 myocardial infarction (MI) regarding patient characteristics, presentation, physiopathology, management, treatment, and prognosis. Two-thirds of MINOCA subjects present ST-segment elevation; MINOCA patients are younger, are more often female and tend to have fewer cardiovascular risk factors. Moreover, MINOCA is a working diagnosis, and defining the aetiologic mechanism is relevant because it affects patient care and prognosis. In the absence of relevant coronary artery disease, myocardial ischaemia might be triggered by an acute event in epicardial coronary arteries, coronary microcirculation, or both. Epicardial causes of MINOCA include coronary plaque disruption, coronary dissection, and coronary spasm. Microvascular MINOCA mechanisms involve microvascular coronary spasm, takotsubo syndrome (TTS), myocarditis, and coronary thromboembolism. Coronary angiography with non-significant coronary stenosis and left ventriculography are first-line tests in the differential study of MINOCA patients. The diagnostic arsenal includes invasive and non-invasive techniques. Medical history and echocardiography can help indicate vasospasm or thrombosis, if one finite coronary territory is affected, or specify TTS if apical ballooning is present. Intravascular ultrasound, optical coherence tomography, and provocative testing are encouraged. Cardiac magnetic resonance is a cornerstone in myocarditis diagnosis. MINOCA is not a benign diagnosis, and its polymorphic forms differ in prognosis. MINOCA care varies across centres, and future multi-centre clinical trials with standardized criteria may have a positive impact on defining optimal cardiovascular care for MINOCA patients

Bianchi Type I Cosmology in Generalized Saez-Ballester Theory via Noether Gauge Symmetry

In this paper, we investigate the generalized Saez-Ballester scalar-tensor theory of gravity via Noether gauge symmetry (NGS) in the background of Bianchi type I cosmological spacetime. We start with the Lagrangian of our model and calculate its gauge symmetries and corresponding invariant quantities. We obtain the potential function for the scalar field in the exponential form. For all the symmetries obtained, we determine the gauge functions corresponding to each gauge symmmetry which include constant and dynamic gauge. We discuss cosmological implications of our model and show that it is compatible with the observational data.Comment: 13 pages, 2 figures, accepted for publication in 'European Physical Journal C

The contribution of rat studies to contemporary knowledge of Major Depressive Disorder: Results from citation analysis

Funding: This study was financed by Animalfree Research—Switzerland, a grant from the Johns Hopkins Center for Alternatives to Animal Testing (CAAT) and by Portuguese National Funds through FCT—Fundação para a Ciência e a Tecnologia, within the CFCUL Unit funding UIDB/00678/2020. TM thanks partial support by CEAUL (funded by FCT—Fundação para a Ciência e a Tecnologia, Portugal, through the project UID/MAT/00006/2019). FP thanks FCT/MCTES for the financial support to CESAM (UIDP/50017/2020 and UIDB/50017/2020), through national funds. Open access publication costs were covered by Animalfree Research-Switzerland and by funding provided by the Ketty and Leif Hjordt Foundation.Major depressive disorder (MDD) is the most severe depression type and one of the leading causes of morbidity worldwide. Animal models are widely used to understand MDD etiology, pathogenesis, and treatment, but the efficacy of this research for patients has barely been systematically evaluated. Such evaluation is important given the resource consumption and ethical concerns incurred by animal use. We used the citation tracking facilities within Web of Science and Scopus to locate citations of original research papers on rats related to MDD published prior to 2013—to allow adequate time for citations—identified in PubMed and Scopus by relevant search terms. Resulting citations were thematically coded in eight categories, and descriptive statistics were calculated. 178 publications describing relevant rat studies were identified. They were cited 8,712 times. More than half (4,633) of their citations were by other animal studies. 794 (less than 10%) were by human medical papers. Citation analysis indicates that rat model research has contributed very little to the contemporary clinical understanding of MDD. This suggests a misuse of limited funding hence supporting a change in allocation of research and development funds targeting this disorder to maximise benefits for patients.Publisher PDFPeer reviewe

Evolutionary Rate Covariation Identifies New Members of a Protein Network Required for Drosophila melanogaster Female Post-Mating Responses

Seminal fluid proteins transferred from males to females during copulation are required for full fertility and can exert dramatic effects on female physiology and behavior. In Drosophila melanogaster, the seminal protein sex peptide (SP) affects mated females by increasing egg production and decreasing receptivity to courtship. These behavioral changes persist for several days because SP binds to sperm that are stored in the female. SP is then gradually released, allowing it to interact with its female-expressed receptor. The binding of SP to sperm requires five additional seminal proteins, which act together in a network. Hundreds of uncharacterized male and female proteins have been identified in this species, but individually screening each protein for network function would present a logistical challenge. To prioritize the screening of these proteins for involvement in the SP network, we used a comparative genomic method to identify candidate proteins whose evolutionary rates across the Drosophila phylogeny co-vary with those of the SP network proteins. Subsequent functional testing of 18 co-varying candidates by RNA interference identified three male seminal proteins and three female reproductive tract proteins that are each required for the long-term persistence of SP responses in females. Molecular genetic analysis showed the three new male proteins are required for the transfer of other network proteins to females and for SP to become bound to sperm that are stored in mated females. The three female proteins, in contrast, act downstream of SP binding and sperm storage. These findings expand the number of seminal proteins required for SP's actions in the female and show that multiple female proteins are necessary for the SP response. Furthermore, our functional analyses demonstrate that evolutionary rate covariation is a valuable predictive tool for identifying candidate members of interacting protein networks. © 2014 Findlay et al

First Transcriptome of the Testis-Vas Deferens-Male Accessory Gland and Proteome of the Spermatophore from Dermacentor variabilis (Acari: Ixodidae)

Ticks are important vectors of numerous human diseases and animal diseases. Feeding stimulates spermatogenesis, mating and insemination of male factors that trigger female reproduction. The physiology of male reproduction and its regulation of female development are essentially a black box. Several transcriptomes have catalogued expression of tick genes in the salivary glands, synganglion and midgut but no comprehensive investigation has addressed male reproduction and mating. Consequently, a new global approach using transcriptomics, proteomics, and quantitative gene expression is needed to understand male reproduction and stimulation of female reproduction