Genome-wide screen identifies new set of genes for improved heterologous laccase expression in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is widely used as a host cell for recombinant protein production due to its fast growth, cost-effective culturing, and ability to secrete large and complex proteins. However, one major drawback is the relatively low yield of produced proteins compared to other host systems. To address this issue, we developed an overlay assay to screen the yeast knockout collection and identify mutants that enhance recombinant protein production, specifically focusing on the secretion of the Trametes trogii fungal laccase enzyme.

Identification of four latent classes of acute respiratory distress syndrome using PaO/FO ratio: an observational cohort study.

Biological phenotypes in patients with the acute respiratory distress syndrome (ARDS) have previously been described. We hypothesized that the trajectory of PaO/FO ratio could be used to identify phenotypes of ARDS. We used a retrospective cohort analysis of an ARDS database to identify latent classes in the trajectory of PaO/FO ratio over time.

Mandatory vs. optional split-dose bowel preparation for morning colonoscopies: a pragmatic noninferiority randomized controlled trial.

BACKGROUND : We compared the effectiveness of optional split-dose bowel preparation (SDBP) with mandatory SDBP for morning colonoscopies in usual clinical practice. METHODS : Adult patients undergoing outpatient early morning (8:00 AM-10:30 PM) and late morning (10:30 AM-12:00 PM) colonoscopies were included. Written bowel preparation instructions were provided based on randomization: one group were instructed to take their bowel preparation (4 L polyethylene glycol solution) as a split dose (mandatory), while the comparator group was allowed the choice of SDBP or single-dose bowel preparation administered entirely on the day before (optional).

Biodiversity patterns diverge along geographic temperature gradients.

Models applying space-for-time substitution, including those projecting ecological responses to climate change, generally assume an elevational and latitudinal equivalence that is rarely tested. However, a mismatch may lead to different capacities for providing climatic refuge to dispersing species. We compiled community data on zooplankton, ectothermic animals that form the consumer basis of most aquatic food webs, from over 1200 mountain lakes and ponds across western North America to assess biodiversity along geographic temperature gradients spanning nearly 3750 m elevation and 30° latitude.

Local stressors mask the effects of warming in freshwater ecosystems.

Climate warming is a ubiquitous stressor in freshwater ecosystems, yet its interactive effects with other stressors are poorly understood. We address this knowledge gap by testing the ability of three contrasting null models to predict the joint impacts of warming and a range of other aquatic stressors using a new database of 296 experimental combinations. Despite concerns that stressors will interact to cause synergisms, we found that net impacts were usually best explained by the effect of the stronger stressor alone (the dominance null model), especially if this stressor was a local disturbance associated with human land use.

Clionamines stimulate autophagy, inhibit Mycobacterium tuberculosis survival in macrophages, and target Pik1.

The pathogen Mycobacterium tuberculosis (Mtb) evades the innate immune system by interfering with autophagy and phagosomal maturation in macrophages, and, as a result, small molecule stimulation of autophagy represents a host-directed therapeutics (HDTs) approach for treatment of tuberculosis (TB). Here we show the marine natural product clionamines activate autophagy and inhibit Mtb survival in macrophages. A yeast chemical-genetics approach identified Pik1 as target protein of the clionamines.

A scalable Drosophila assay for clinical interpretation of human PTEN variants in suppression of PI3K/AKT induced cellular proliferation.

Gene variant discovery is becoming routine, but it remains difficult to usefully interpret the functional consequence or disease relevance of most variants. To fill this interpretation gap, experimental assays of variant function are becoming common place. Yet, it remains challenging to make these assays reproducible, scalable to high numbers of variants, and capable of assessing defined gene-disease mechanism for clinical interpretation aligned to the ClinGen Sequence Variant Interpretation (SVI) Working Group guidelines for 'well-established assays'.

Ist2 recruits the lipid transporters Osh6/7 to ER-PM contacts to maintain phospholipid metabolism.

ER-plasma membrane (PM) contacts are proposed to be held together by distinct families of tethering proteins, which in yeast include the VAP homologues Scs2/22, the extended-synaptotagmin homologues Tcb1/2/3, and the TMEM16 homologue Ist2. It is unclear whether these tethers act redundantly or whether individual tethers have specific functions at contacts. Here, we show that Ist2 directly recruits the phosphatidylserine (PS) transport proteins and ORP family members Osh6 and Osh7 to ER-PM contacts through a binding site located in Ist2's disordered C-terminal tethering region.

MAPS: machine-assisted phenotype scoring enables rapid functional assessment of genetic variants by high-content microscopy.

Background: Genetic testing is widely used in evaluating a patient's predisposition to hereditary diseases. In the case of cancer, when a functionally impactful mutation (i.e.

Climate warming moderates the impacts of introduced sportfish on multiple dimensions of prey biodiversity.

Human-assisted introductions of exotic species are a leading cause of anthropogenic change in biodiversity; however, context dependencies and interactions with co-occurring stressors impede our ability to predict their ecological impacts. The legacy of historical sportfish stocking in mountainous regions of western North America creates a unique, natural quasiexperiment to investigate factors moderating invasion impacts on native communities across broad geographic and environmental gradients. Here we synthesize fish stocking records and zooplankton relative abundance for 685 mountain lakes and ponds in the Cascade and Canadian Rocky Mountain Ranges, to reveal the effects of predatory sportfish introduction on multiple taxonomic, functional and phylogenetic dimensions of prey biodiversity.

Proteomic analysis reveals the direct recruitment of intrinsically disordered regions to stress granules in .

Stress granules (SGs) are stress-induced membraneless condensates that store non-translating mRNA and stalled translation initiation complexes. Although metazoan SGs are dynamic compartments where proteins can rapidly exchange with their surroundings, yeast SGs seem largely static. To gain a better understanding of yeast SGs, we identified proteins that sediment after heat shock using mass spectrometry.

Sentinel interaction mapping - a generic approach for the functional analysis of human disease gene variants using yeast.

Advances in sequencing technology have led to an explosion in the number of known genetic variants of human genes. A major challenge is to now determine which of these variants contribute to diseases as a result of their effect on gene function. Here, we describe a generic approach using the yeast to quickly develop gene-specific assays that can be used to quantify the level of function of a genetic variant.

A Premalignant Cell-Based Model for Functionalization and Classification of Variants.

As sequencing becomes more economical, we are identifying sequence variations in the population faster than ever. For disease-associated genes, it is imperative that we differentiate a sequence variant as either benign or pathogenic, such that the appropriate therapeutic interventions or surveillance can be implemented. is a frequently mutated tumor suppressor that has been linked to the PTEN hamartoma tumor syndrome.

Multi-model functionalization of disease-associated PTEN missense mutations identifies multiple molecular mechanisms underlying protein dysfunction.

Functional variomics provides the foundation for personalized medicine by linking genetic variation to disease expression, outcome and treatment, yet its utility is dependent on appropriate assays to evaluate mutation impact on protein function. To fully assess the effects of 106 missense and nonsense variants of PTEN associated with autism spectrum disorder, somatic cancer and PTEN hamartoma syndrome (PHTS), we take a deep phenotypic profiling approach using 18 assays in 5 model systems spanning diverse cellular environments ranging from molecular function to neuronal morphogenesis and behavior. Variants inducing instability occur across the protein, resulting in partial-to-complete loss-of-function (LoF), which is well correlated across models.

Multiscale drivers of phytoplankton communities in north-temperate lakes.

Multiple factors operating across different spatial and temporal scales affect β-diversity, the variation in community composition among sites. Disentangling the relative influence of co-occurring ecological drivers over broad biogeographic gradients and time is critical to developing mechanistic understanding of community responses to natural environmental heterogeneity as well as predicting the effects of anthropogenic change. We partitioned taxonomic β-diversity in phytoplankton communities across 75 north-temperate lakes and reservoirs in Alberta, Canada, using data-driven, spatially constrained null models to differentiate between spatially structured, spatially independent, and spuriously correlated associations with a suite of biologically relevant environmental variables.

pH Biosensing by PI4P Regulates Cargo Sorting at the TGN.

Phosphoinositides, diacylglycerolpyrophosphate, ceramide-1-phosphate, and phosphatidic acid belong to a unique class of membrane signaling lipids that contain phosphomonoesters in their headgroups having pK values in the physiological range. The phosphomonoester headgroup of phosphatidic acid enables this lipid to act as a pH biosensor as changes in its protonation state with intracellular pH regulate binding to effector proteins. Here, we demonstrate that binding of pleckstrin homology (PH) domains to phosphatidylinositol 4-phosphate (PI4P) in the yeast trans-Golgi network (TGN) is dependent on intracellular pH, indicating PI4P is a pH biosensor.

A nuclear role for the DEAD-box protein Dbp5 in tRNA export.

Dbp5 is an essential DEAD-box protein that mediates nuclear mRNP export. Dbp5 also shuttles between nuclear and cytoplasmic compartments with reported roles in transcription, ribosomal subunit export, and translation; however, the mechanism(s) by which nucleocytoplasmic transport occurs and how Dbp5 specifically contributes to each of these processes remains unclear. Towards understanding the functions and transport of Dbp5 in , alanine scanning mutagenesis was used to generate point mutants at all possible residues within a GFP-Dbp5 reporter.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster.

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates.

Mechanisms of PTPσ-Mediated Presynaptic Differentiation.

Formation of synapses between neurons depends in part on binding between axonal and dendritic cell surface synaptic organizing proteins, which recruit components of the developing presynaptic and postsynaptic specializations. One of these presynaptic organizing molecules is protein tyrosine phosphatase σ (PTPσ). Although the protein domains involved in adhesion between PTPσ and its postsynaptic binding partners are known, the mechanisms by which it signals into the presynaptic neuron to recruit synaptic vesicles and other necessary components for regulated transmitter release are not well understood.

A Novel Mutation in Brain Tumor Causes Both Neural Over-Proliferation and Neurodegeneration in Adult .

A screen for neuroprotective genes in led to the identification of a mutation that causes extreme, progressive loss of adult brain neuropil in conjunction with massive brain overgrowth. We mapped the mutation to the () locus, which encodes a tripartite motif-NCL-1, HT2A, and LIN-41 (TRIM-NHL) RNA-binding protein with established roles limiting stem cell proliferation in developing brain and ovary. However, a neuroprotective role for in the adult brain has not been described previously.

Viral proteins as a potential driver of histone depletion in dinoflagellates.

Within canonical eukaryotic nuclei, DNA is packaged with highly conserved histone proteins into nucleosomes, which facilitate DNA condensation and contribute to genomic regulation. Yet the dinoflagellates, a group of unicellular algae, are a striking exception to this otherwise universal feature as they have largely abandoned histones and acquired apparently viral-derived substitutes termed DVNPs (dinoflagellate-viral-nucleoproteins). Despite the magnitude of this transition, its evolutionary drivers remain unknown.

Mito-Nuclear Interactions Affecting Lifespan and Neurodegeneration in a Model of Leigh Syndrome.

Proper mitochondrial activity depends upon proteins encoded by genes in the nuclear and mitochondrial genomes that must interact functionally and physically in a precisely coordinated manner. Consequently, mito-nuclear allelic interactions are thought to be of crucial importance on an evolutionary scale, as well as for manifestation of essential biological phenotypes, including those directly relevant to human disease. Nonetheless, detailed molecular understanding of mito-nuclear interactions is still lacking, and definitive examples of such interactions are sparse.

Fat storage-inducing transmembrane (FIT or FITM) proteins are related to lipid phosphatase/phosphotransferase enzymes.

Fat storage-inducing transmembrane (FIT or FITM) proteins have been implicated in the partitioning of triacylglycerol to lipid droplets and the budding of lipid droplets from the ER. At the molecular level, the sole relevant interaction is that FITMs directly bind to triacyglycerol and diacylglycerol, but how they function at the molecular level is not known. has two FITM homologues: Scs3p and Yft2p.

Genetic variability affects absolute and relative potencies and kinetics of the anesthetics isoflurane and sevoflurane in Drosophila melanogaster.

Genetic variability affects the response to numerous xenobiotics but its role in the clinically-observed irregular responses to general anesthetics remains uncertain. To investigate the pharmacogenetics of volatile general anesthetics (VGAs), we developed a Serial Anesthesia Array apparatus to expose multiple Drosophila melanogaster samples to VGAs and behavioral assays to determine pharmacokinetic and pharmacodynamic properties of VGAs. We studied the VGAs isoflurane and sevoflurane in four wild type strains from the Drosophila Genetic Reference Panel, two commonly used laboratory strains (Canton S and w ), and a mutant in Complex I of the mitochondrial electron transport chain (ND23 ).