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SUPT16H encodes the large subunit of the FAcilitate Chromatin Transcription (FACT) complex, which functions as a nucleosome organizer during transcription. We identified two individuals from unrelated families carrying de novo missense variants in SUPT16H. The probands exhibit global developmental delay, intellectual disability, epilepsy, facial dysmorphism and brain structural abnormalities. We used Drosophila to characterize two variants: p.T171I and p.G808R. Loss of the fly ortholog, dre4, causes lethality at an early developmental stage. RNAi-mediated knockdown of dre4 in either glia or neurons causes severely reduced eclosion and longevity. Tissue-specific knockdown of dre4 in the eye or wing leads to the loss of these tissues, whereas overexpression of SUPT16H has no dominant effect. Moreover, expression of the reference SUPT16H significantly rescues the loss-of-function phenotypes in the nervous system as well as wing and eye. In contrast, expression of SUPT16H p.T171I or p.G808R rescues the phenotypes poorly, indicating that the variants are partial loss-of-function alleles. While previous studies argued that the developmental arrest caused by loss of dre4 is due to impaired ecdysone production in the prothoracic gland, our data show that dre4 is required for proper cell growth and survival in multiple tissues in a cell-autonomous manner. Altogether, our data indicate that the de novo loss-of-function variants in SUPT16H are indeed associated with developmental and neurological defects observed in the probands.

PPFIA3encodes the Protein-Tyrosine Phosphatase, Receptor-Type, F Polypeptide-Interacting Protein Alpha-3 (PPFIA3), which is a member of the LAR protein-tyrosine phosphatase-interacting protein (liprin) family involved in synaptic vesicle transport and presynaptic active zone assembly. The protein structure and function are well conserved in both invertebrates and vertebrates, but human diseases related to PPFIA3 dysfunction are not yet known. Here, we report 14 individuals with rare mono-allelicPPFIA3variants presenting with features including developmental delay, intellectual disability, hypotonia, autism, and epilepsy. To determine the pathogenicity ofPPFIA3variantsin vivo, we generated transgenic fruit flies expressing either human PPFIA3 wildtype (WT) or variant protein using GAL4-UAS targeted gene expression systems. Ubiquitous expression with Actin-GAL4 showed that thePPFIA3variants had variable penetrance of pupal lethality, eclosion defects, and anatomical leg defects. Neuronal expression with elav-GAL4 showed that thePPFIA3variants had seizure-like behaviors, motor defects, and bouton loss at the 3rdinstar larval neuromuscular junction (NMJ). Altogether, in the fly overexpression assays, we found that thePPFIA3variants in the N-terminal coiled coil domain exhibited stronger phenotypes compared to those in the C-terminal region. In the loss-of-function fly assay, we show that the homozygous loss of flyLiprin-α leads to embryonic lethality. This lethality is partially rescued by the expression of humanPPFIA3WT, suggesting human PPFIA3 protein function is partially conserved in the fly. However, thePPFIA3variants failed to rescue lethality. Altogether, the human and fruit fly data reveal that the rarePPFIA3variants are dominant negative loss-of-function alleles that perturb multiple developmental processes and synapse formation.

Eukaryotic initiation factor-4A2 (EIF4A2) is an ATP-dependent RNA helicase and a member of the DEAD-box protein family that recognizes the 5' cap structure of mRNAs, allows mRNA to bind to the ribosome, and plays an important role in microRNA-regulated gene repression. Here, we report on 15 individuals from 14 families presenting with global developmental delay, intellectual disability, hypotonia, epilepsy, and structural brain anomalies, all of whom have extremely rare de novo mono-allelic or inherited bi-allelic variants in EIF4A2. Neurodegeneration was predominantly reported in individuals with bi-allelic variants. Molecular modeling predicts these variants would perturb structural interactions in key protein domains. To determine the pathogenicity of the EIF4A2 variants in vivo, we examined the mono-allelic variants in Drosophila melanogaster (fruit fly) and identified variant-specific behavioral and developmental defects. The fruit fly homolog of EIF4A2 is eIF4A, a negative regulator of decapentaplegic (dpp) signaling that regulates embryo patterning, eye and wing morphogenesis, and stem cell identity determination. Our loss-of-function (LOF) rescue assay demonstrated a pupal lethality phenotype induced by loss of eIF4A, which was fully rescued with human EIF4A2 wild-type (WT) cDNA expression. In comparison, the EIF4A2 variant cDNAs failed or incompletely rescued the lethality. Overall, our findings reveal that EIF4A2 variants cause a genetic neurodevelopmental syndrome with both LOF and gain of function as underlying mechanisms.

The aim of this research is to design and synthesize an efficient catalyst to enhance high value-added products, such as aromatic hydrocarbons and phenols, from the catalytic fast pyrolysis (CFP) of different types of forestry and agricultural residues. All three biomasses (rape straw, wheat straw, and bamboo powder) had no aromatic production via thermal pyrolysis alone; however, the aromatic selectivity and monocyclic aromatic selectivity were largely enhanced using ZSM-5, with suitable silica-alumina ratios and Ni loadings. Specifically, for rape straw, the optimum catalyst was 15 wt.% Ni/ZSM-5 (silica-aluminum ratios = 85), and the selectivity of aromatic hydrocarbons was achieved at 39%, of which 71% were monocyclic aromatic hydrocarbons. For wheat straw, the optimum catalyst was 10 wt.% Ni/ZSM-5 (silica-aluminum ratios = 18), and the selectivity of aromatic hydrocarbons was 67%, of which 55% were monocyclic aromatic hydrocarbons. For bamboo powder, the optimum catalyst was 10 wt.% Ni/ZSM-5 (silica-aluminum ratios = 18), and the selectivity of aromatic hydrocarbons was achieved at 21%, of which 80% were monocyclic aromatic hydrocarbons. Meanwhile, biomass types have significant effects on the pyrolyzed product distribution due to their different components. Cellulose and hemicellulose promoted the production of aromatic hydrocarbons, while lignin enhanced the production of phenols. The promotion of phenol by Ni was better and more efficient than that by the molecular sieve.

Raw bio-oil was pretreated and tested for hydrodeoxygenation (HDO) using three types of the commercial catalysts (HT-36, HT2300, and HT951T) to improve physio-chemical properties and enhance hydrocarbon yields. The three catalysts prompted different levels of hydrodeoxygenation, and the organic phase products (OLPs) yields were 25.30, 27.83, and 13.05 wt%, respectively. Moreover, OLPs had lower water content, total acid numbers (TAN), and O content as well as higher heating value (HHV), C, and H contents. For the three catalysts, HT-36 had the best HDO effects, resulting in 34.8% hydrocarbon production with improved HHV, water content value and TAN as well as element contents. The different level of HDO depended on the catalyst components, structure, and morphology. This research is beneficial for the selection and preparation of effective catalysts for bio-oil upgrading.

Individuals with autism spectrum disorder (ASD) exhibit an increased burden of de novo mutations (DNMs) in a broadening range of genes. While these studies have implicated hundreds of genes in ASD pathogenesis, which DNMs cause functional consequences in vivo remains unclear. We functionally test the effects of ASD missense DNMs using Drosophila through “humanization” rescue and overexpression-based strategies. We examine 79 ASD variants in 74 genes identified in the Simons Simplex Collection and find 38% of them to cause functional alterations. Moreover, we identify GLRA2 as the cause of a spectrum of neurodevelopmental phenotypes beyond ASD in 13 previously undiagnosed subjects. Functional characterization of variants in ASD candidate genes points to conserved neurobiological mechanisms and facilitates gene discovery for rare neurodevelopmental diseases.

Hydrodeoxygenation is an essential process for producing liquid transportation fuels. In this study, the effects of CoMo/γ-Al2O3 catalysts form and loading ratio on the hydrodeoxygenation upgrading of bio-oil were investigated in a batch reactor. Raw bio-oil was first oxidized with hydrogen peroxides and oxone to obtain the oxidized bio-oil with reduced levels of aldehydes and ketones, increasing the organic liquid yield during hydrodeoxygenation by suppressing the coke formation. CoMo/γ-Al2O3 was selected as the catalyst because of its low cost and commercial availability. The effect of the reduction and sulfidation of CoMo/γ-Al2O3 catalyst on the hydrodeoxygenation of the oxidized bio-oil was compared. The effect of the catalyst loading ratio on bio-oil hydrodeoxygenation using sulfided CoMo/γ-Al2O3 catalysts was also investigated. The research results showed that the sulfided CoMo/γ-Al2O3 catalyst facilitated the formation of hydrocarbons, while the reduced CoMo/γ-Al2O3 catalyst produced more phenols in the organic liquids. Moreover, a high sulfided catalyst loading ratio promoted the formation of hydrocarbons.

SummaryIndividuals with autism spectrum disorders (ASD) exhibit an increased burden ofde novovariants in a broadening range of genes. We functionally tested the effects of ASD missense variants usingDrosophilathrough ‘humanization’ rescue and overexpression-based strategies. We studied 79 ASD variants in 74 genes identified in the Simons Simplex Collection and found 38% of them caused functional alterations. Moreover, we identifiedGLRA2as the cause of a spectrum of neurodevelopmental phenotypes beyond ASD in eight previously undiagnosed subjects. Functional characterization of variants in ASD candidate genes point to conserved neurobiological mechanisms and facilitates gene discovery for rare neurodevelopmental diseases.

Determining what role newly discovered genes play in the body is an important part of genetics. This task requires a lot of extra information about each gene, such as the specific cells where the gene is active, or what happens when the gene is deleted. To answer these questions, researchers need tools and methods to manipulate genes within a living organism. The fruit fly Drosophila is useful for such experiments because a toolbox of genetic techniques is already available. Gene editing in fruit flies allows small pieces of genetic information to be removed from or added to anywhere in the animal’s DNA. Another tool, known as GAL4-UAS, is a two-part system used to study gene activity. The GAL4 component is a protein that switches on genes. GAL4 alone does very little in Drosophila cells because it only recognizes a DNA sequence called UAS. However, if a GAL4-producing cell is also engineered to contain a UAS-controlled gene, GAL4 will switch the gene on. Lee et al. used gene editing to insert a small piece of DNA, containing the GAL4 sequence followed by a ‘stop’ signal, into many different fly genes. The insertion made the cells where each gene was normally active produce GAL4, but – thanks to the stop signal – rendered the rest of the original gene non-functional. This effectively deleted the proteins encoded by each gene, giving information about the biological processes they normally control. Lee et al. went on to use their insertion approach to make a Drosophila genetic library. This is a collection of around 1,000 different strains of fly, each carrying the GAL4/stop combination in a single gene. The library allows any gene in the collection to be studied in detail simply by combining the GAL4 with different UAS-controlled genetic tools. For example, introducing a UAS-controlled marker would pinpoint where in the body the original gene was active. Alternatively, adding UAS-controlled human versions of the gene would create humanized flies, which are a valuable tool to study potential disease-causing genes in humans. This Drosophila library is a resource that contributes new experimental tools to fly genetics. Insights gained from flies can also be applied to more complex animals like humans, especially since around 65% of genes are similar across humans and Drosophila. As such, Lee et al. hope that this resource will help other researchers shed new light on the role of many different genes in health and disease.

We generated a library of ~1000 Drosophila stocks in which we inserted a construct in the intron of genes allowing expression of GAL4 under control of endogenous promoters while arresting transcription with a polyadenylation signal 3’ of the GAL4. This allows numerous applications. First, ~90% of insertions in essential genes cause a severe loss-of-function phenotype, an effective way to mutagenize genes. Interestingly, 12/14 chromosomes engineered through CRISPR do not carry second-site lethal mutations. Second, 26/36 (70%) of lethal insertions tested are rescued with a single UAS-cDNA construct. Third, loss-of-function phenotypes associated with many GAL4 insertions can be reverted by excision with UAS-flippase. Fourth, GAL4 driven UAS-GFP/RFP reports tissue and cell-type specificity of gene expression with high sensitivity. We report the expression of hundreds of genes not previously reported. Finally, inserted cassettes can be replaced with GFP or any DNA. These stocks comprise a powerful resource for assessing gene function., eLife digest Determining what role newly discovered genes play in the body is an important part of genetics. This task requires a lot of extra information about each gene, such as the specific cells where the gene is active, or what happens when the gene is deleted. To answer these questions, researchers need tools and methods to manipulate genes within a living organism. The fruit fly Drosophila is useful for such experiments because a toolbox of genetic techniques is already available. Gene editing in fruit flies allows small pieces of genetic information to be removed from or added to anywhere in the animal’s DNA. Another tool, known as GAL4-UAS, is a two-part system used to study gene activity. The GAL4 component is a protein that switches on genes. GAL4 alone does very little in Drosophila cells because it only recognizes a DNA sequence called UAS. However, if a GAL4-producing cell is also engineered to contain a UAS-controlled gene, GAL4 will switch the gene on. Lee et al. used gene editing to insert a small piece of DNA, containing the GAL4 sequence followed by a ‘stop’ signal, into many different fly genes. The insertion made the cells where each gene was normally active produce GAL4, but – thanks to the stop signal – rendered the rest of the original gene non-functional. This effectively deleted the proteins encoded by each gene, giving information about the biological processes they normally control. Lee et al. went on to use their insertion approach to make a Drosophila genetic library. This is a collection of around 1,000 different strains of fly, each carrying the GAL4/stop combination in a single gene. The library allows any gene in the collection to be studied in detail simply by combining the GAL4 with different UAS-controlled genetic tools. For example, introducing a UAS-controlled marker would pinpoint where in the body the original gene was active. Alternatively, adding UAS-controlled human versions of the gene would create humanized flies, which are a valuable tool to study potential disease-causing genes in humans. This Drosophila library is a resource that contributes new experimental tools to fly genetics. Insights gained from flies can also be applied to more complex animals like humans, especially since around 65% of genes are similar across humans and Drosophila. As such, Lee et al. hope that this resource will help other researchers shed new light on the role of many different genes in health and disease.

Analysis of long-term care for the elderly people can lose the status quo, field research in elderly care hospital in Henan Province, Zhengzhou Erqi apartments for the elderly and community health services involved in Luohe disabled elderly long-term care model in which community health services mode of long-term care, community health service station stationed pension agency model, social home care model, discuss community health services long-term care system in the role in urban disabled elderly; proposes should accelerate China's national conditions of long-term care service system construction, promote the combination of community health services long-term care service system, the establishment of the basic framework..

We demonstrate the versatility of a collection of insertions of the transposon Minos-mediated integration cassette (MiMIC), in Drosophila melanogaster. MiMIC contains a gene-trap cassette and the yellow+ marker flanked by two inverted bacteriophage ΦC31 integrase attP sites. MiMIC integrates almost at random in the genome to create sites for DNAmanipulation. The attP sites allow the replacement of the intervening sequence of the transposon with any other sequence through recombinase-mediated cassette exchange (RMCE). We can revert insertions that function as gene traps and cause mutant phenotypes to revert to wild type by RMCE and modify insertions to control GAL4 or QF overexpression systems or perform lineage analysis using the Flp recombinase system. Insertions in coding introns can be exchanged with protein-tag cassettes to create fusion proteins to follow protein expression and perform biochemical experiments. The applications of MiMIC vastly extend the D. melanogaster toolkit.

Epidermal growth factor receptor pathway substrate clone 15 (Eps15) is a protein implicated in endocytosis, endosomal protein sorting, and cytoskeletal organization. Its role is, however, still unclear, because of reasons including limitations of dominant-negative experiments and apparent redundancy with other endocytic proteins. We generated Drosophila eps15-null mutants and show that Eps15 is required for proper synaptic bouton development and normal levels of synaptic vesicle (SV) endocytosis. Consistent with a role in SV endocytosis, Eps15 moves from the center of synaptic boutons to the periphery in response to synaptic activity. The endocytic protein, Dap160/intersectin, is a major binding partner of Eps15, and eps15 mutants phenotypically resemble dap160 mutants. Analyses of eps15 dap160 double mutants suggest that Eps15 functions in concert with Dap160 during SV endocytosis. Based on these data, we hypothesize that Eps15 and Dap160 promote the efficiency of endocytosis from the plasma membrane by maintaining high concentrations of multiple endocytic proteins, including dynamin, at synapses.

Previously, we described a large collection of Minos-Mediated Integration Cassettes (MiMICs) that contain two phiC31 recombinase target sites and allow the generation of a new exon that encodes a protein tag when the MiMIC is inserted in a codon intron (Nagarkar-Jaiswal et al., 2015). These modified genes permit numerous applications including assessment of protein expression pattern, identification of protein interaction partners by immunoprecipitation followed by mass spec, and reversible removal of the tagged protein in any tissue. At present, these conversions remain time and labor-intensive as they require embryos to be injected with plasmid DNA containing the exon tag. In this study, we describe a simple and reliable genetic strategy to tag genes/proteins that contain MiMIC insertions using an integrated exon encoding GFP flanked by FRT sequences. We document the efficiency and tag 60 mostly uncharacterized genes.

Lysosomal storage is the most common cause of neurodegenerative brain disease in preadulthood. However, the underlying cellular mechanisms that lead to neuronal dysfunction are unknown. Here, we report that loss of Drosophila benchwarmer (bnch), a predicted lysosomal sugar carrier, leads to carbohydrate storage in yolk spheres during oogenesis and results in widespread accumulation of enlarged lysosomal and late endosomal inclusions. At the bnch larval neuromuscular junction, we observe similar inclusions and find defects in synaptic vesicle recycling at the level of endocytosis. In addition, loss of bnch slows endosome-to-lysosome trafficking in larval garland cells. In adult bnch flies, we observe age-dependent synaptic dysfunction and neuronal degeneration. Finally, we find that loss of bnch strongly enhances tau neurotoxicity in a dose-dependent manner. We hypothesize that, in bnch, defective lysosomal carbohydrate efflux leads to endocytic defects with functional consequences in synaptic strength, neuronal viability, and tau neurotoxicity.

The isolation of chemically induced mutations in forward genetic screens is one of the hallmarks of Drosophila genetics. However, mapping the corresponding loci and identifying the molecular lesions associated with these mutations are often difficult and labor-intensive. Two mapping methods are most often used in flies: meiotic recombination mapping with marked chromosomes and deficiency mapping. The availability of the fly genome sequence allows the establishment and usage of molecular markers. Single-nucleotide polymorphisms have therefore recently been used to map several genes. Here we show that thousands of molecularly mapped P element insertions in fly strains that are publicly available provide a powerful alternative method to single-nucleotide polymorphism mapping. We present a strategy that allows mapping of lethal mutations, as well as viable mutations with visible phenotypes, with minimal resources. The most important unknown in using recombination rates to map at high resolution is how accurately recombination data correlate with molecular maps in small intervals. We therefore surveyed distortions of recombination rates in intervals P elements in Drosophila.

Ginseng acidic polysaccharide WGPA isolated from the root of Panax ginseng C. A. Meyer was fractionated into WGPA-A and WGPA-N by anion-exchange chromatography. The antifatigue activity of ginseng acidic polysaccharide WGPA has been reported in our previous research. This present study was designed to identify its active component and elucidate the mechanism for preventing chronic fatigue syndrome (CFS). WGPA, WGPA-A and WGPA-N were orally administered to mice once daily for 15 days. The effects of these compounds on physiological biomarkers of oxidative stress and on the morphology of the mitochondria in striated skeletal muscle were assessed. The results of forced swimming test-induced indicated that WGPA and WGPA-A could lengthen the swimming time, while WGPA-N could not. In addition, malondialdehyde and lactate dehydrogenase levels in serum were enhanced; while those of superoxide dismutase and glutathione peroxidase were lowered. Interestingly, the structural degeneration of mitochondria were all ameliorated. These findings suggested that WGPA-A is the active component of WGPA, it might have potential therapeutic effects for CFS and the oxidative stress might be involved in the pathogenesis. Our results also provided essential data for a better understanding of the antifatigue effects of P. ginseng extracts.

We constructed Drosophila melanogaster bacterial artificial chromosome libraries with 21-kilobase and 83-kilobase inserts in the P[acman] system. We mapped clones representing 12-fold coverage and encompassing more than 95% of annotated genes onto the reference genome. These clones can be integrated into predetermined attP sites in the genome using UC31 integrase to rescue mutations. They can be modified through recombineering, for example, to incorporate protein tags and assess expression patterns.

Summary Notch signaling is broadly used to regulate cell-fate decisions. We have identified a gene, rumi, with a temperature-sensitive Notch phenotype. At 28°C–30°C, rumi clones exhibit a full-blown loss of Notch signaling in all tissues tested. However, at 18°C only a mild Notch phenotype is evident. In vivo analyses reveal that the target of Rumi is the extracellular domain of Notch. Notch accumulates intracellularly and at the cell membrane of rumi cells but fails to be properly cleaved, despite normal binding to Delta. Rumi is an endoplasmic reticulum-retained protein with a highly conserved CAP10 domain. Our studies show that Rumi is a protein O -glucosyltransferase, capable of adding glucose to serine residues in Notch EGF repeats with the consensus C 1 -X-S-X-P-C 2 sequence. These data indicate that by O -glucosylating Notch in the ER, Rumi regulates its folding and/or trafficking and allows signaling at the cell membrane.

During sensory organ precursor (SOP) specification, a single cell is selected from a proneural cluster of cells. Here, we present evidence that Senseless (Sens), a zinc-finger transcription factor, plays an important role in this process. We show that Sens is directly activated by proneural proteins in the presumptive SOPs and a few cells surrounding the SOP in most tissues. In the cells that express low levels of Sens, it acts in a DNA-binding-dependent manner to repress transcription of proneural genes. In the presumptive SOPs that express high levels of Sens, it acts as a transcriptional activator and synergizes with proneural proteins. We therefore propose that Sens acts as a binary switch that is fundamental to SOP selection.