Inhibition of influenza A virus and SARS-CoV-2 infection or co-infection by griffithsin and griffithsin-based bivalent entry inhibitor.

Outbreaks of acute respiratory viral diseases, such as influenza and COVID-19 caused by influenza A virus (IAV) and SARS-CoV-2, pose a serious threat to global public health, economic security, and social stability. This calls for the development of broad-spectrum antivirals to prevent or treat infection or co-infection of IAV and SARS-CoV-2. Hemagglutinin (HA) on IAV and spike (S) protein on SARS-CoV-2, which contain various types of glycans, play crucial roles in mediating viral entry into host cells. Therefore, they are key targets for the development of carbohydrate-binding protein-based antivirals. This study demonstrated that griffithsin (GRFT) and the GRFT-based bivalent entry inhibitor GL25E (GRFT-L25-EK1) showed broad-spectrum antiviral effects against IAV infection in vitro by binding to HA in a carbohydrate-dependent manner and effectively protected mice from lethal IAV infection. Although both GRFT and GL25E could inhibit infection of SARS-CoV-2 Omicron variants, GL25E proved to be significantly more effective than GRFT and EK1 alone. Furthermore, GL25E effectively inhibited in vitro co-infection of IAV and SARS-CoV-2 and demonstrated good druggability, including favorable safety and stability profiles. These findings suggest that GL25E is a promising candidate for further development as a broad-spectrum antiviral drug for the prevention and treatment of infection or co-infection from IAV and SARS-CoV-2.IMPORTANCEInfluenza and COVID-19 are highly contagious respiratory illnesses caused by the influenza A virus (IAV) and SARS-CoV-2, respectively. IAV and SARS-CoV-2 co-infection exacerbates damage to lung tissue and leads to more severe clinical symptoms, thus calling for the development of broad-spectrum antivirals for combating IAV and SARS-CoV-2 infection or co-infection. Here we found that griffithsin (GRFT), a carbohydrate-binding protein, and GL25E, a recombinant protein consisting of GRFT, a 25 amino acid linker, and EK1, a broad-spectrum coronavirus inhibitor, could effectively inhibit IAV and SARS-CoV-2 infection and co-infection by targeting glycans on HA of IAV and spike (S) protein of SARS-CoV-2. GL25E is more effective than GRFT because GL25E can also interact with the HR1 domain in SARS-CoV-2 S protein. Furthermore, GL25E possesses favorable safety and stability profiles, suggesting that it is a promising candidate for development as a drug to prevent and treat IAV and SARS-CoV-2 infection or co-infection.

Intestinal changes in permeability, tight junction and mucin synthesis in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid­ß (Aß) in the brain. The gut/brain axis may serve a role in AD pathogenesis. The present study investigated deposition of Aß in the intestinal epithelium and its potential effects on intestinal barrier function in a transgenic mouse model of AD. To investigate alterations in the structure and functionality of the intestinal mucosal barrier in AD model mice, hematoxylin and eosin staining for Paneth cell count, Alcian blue­periodic acid Schiff staining for goblet cells, immunohistochemistry and immunofluorescence for mucin (MUC)2 and wheat germ agglutin expression, transmission electron microscopy for mucosal ultrastructure, FITC­labeled dextran assay for intestinal permeability, quantitative PCR for goblet cell precursor expression and western blot analysis for tight junction proteins, MUC2 and inflammatory cytokine detection were performed. The results showed that AD model mice exhibited excessive Aß deposition in the intestinal epithelium, which was accompanied by increased intestinal permeability, inflammatory changes and decreased expression of tight junction proteins. These alterations in the intestinal barrier led to an increased proliferation of goblet and Paneth cells and increased mucus synthesis. Dysfunction of gut barrier occurs in AD and may contribute to its etiology. Future therapeutic strategies to reverse AD pathology may involve early manipulation of gut physiology and its microbiota.

Structure, function, and pathology of Neurexin-3.

Neurexin-3 is primarily localized in the presynaptic membrane and forms complexes with various ligands located in the postsynaptic membrane. Neurexin-3 has important roles in synapse development and synapse functions. Neurexin-3 mediates excitatory presynaptic differentiation by interacting with leucine-rich-repeat transmembrane neuronal proteins. Meanwhile, neurexin-3 modulates the expression of presynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors and γ-aminobutyric acid A receptors by interacting with neuroligins at excitatory and inhibitory synapses. Numerous studies have documented the potential contribution of neurexin-3 to neurodegenerative and neuropsychiatric disorders, such as Alzheimer's disease, addiction behaviors, and other diseases, which raises hopes that understanding the mechanisms of neurexin-3 may hold the key to developing new strategies for related illnesses. This review comprehensively covers the literature to provide current knowledge of the structure, function, and clinical role of neurexin-3.

Decreased expression of protein O-linked mannose ß-1,2-N-acetylglucosaminyltransferase 1 contributes to Alzheimer's disease-like pathologies.

Alzheimer's disease (AD) is pathologically characterized by senile plaques and neurofibrillary tangles composed of ß-amyloid peptide (Aß) and tau hyperphosphorylation, respectively. Mannosylation, a particular type of posttranslational modification, may be involved in the pathogenesis of AD. However, its underlying mechanism remains unclear. Protein O-linked mannose ß-1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) catalyzes the formation of the N-acetylglucosamine ß-1,2-Man linkage of O-mannosylglycan, which can increase the protein posttranslational mannosylation level. The defective POMGnT1 gene leads to the hypomannosylation of proteins, which may cause cognitive decline in aged people. This study aimed to investigate whether POMGnT1 participated in the pathogenesis of AD and explore its underlying role using AD mouse and cell models. In this study, the expression of POMGnT1 was measured in AD models [ß-amyloid precursor protein (APP)/presenilin-1 (PS1) transgenic mice, an AD mouse model; N2a cells stably transfected with Swedish mutant APP (N2a/APP), an AD cell model]. The results revealed that the expression of POMGnT1 decreased in AD mouse and cell models. In addition, POMGnT1-overexpressing N2a/APP cells were built by retroviral transfection. POMGnT1 overexpression may lower Aß levels by reducing APP production and downregulating ß- and γ-secretase activities. It also promoted clearance of Aß by upregulating insulin-degrading enzymes and ameliorated tau hyperphosphorylation. Hence, it was concluded that POMGnT1 was involved in the pathogenic process of AD. The decreased expression of POMGnT1 contributes to AD-like pathologies.NEW & NOTEWORTHY This study explored the role of mannosylation in the pathogenesis of AD through a mannosyltransferase-POMGnT1. Results demonstrated that target gene overexpression could ameliorate pathologies of Aß and tau hyperphosphorylation. This study is the first to examine the relationship between mannosylation and AD.

Analysis of the expression and distribution of protein O-linked mannose ß1,2-N-acetylglucosaminyltransferase 1 in the normal adult mouse brain.

Introduction: Protein O-linked mannose ß1,2-N-acetylglucosaminyltransferase 1 (POMGNT1) is crucial for the elongation of O-mannosyl glycans. Mutations in POMGNT1 cause muscle-eye-brain (MEB) disease, one of the main features of which is anatomical aberrations in the brain. A growing number of studies have shown that defects in POMGNT1 affect neuronal migration and distribution, disrupt basement membranes, and misalign Cajal-Retzius cells. Several studies have examined the distribution and expression of POMGNT1 in the fetal or neonatal brain for neurodevelopmental studies in the mouse or human brain. However, little is known about the neuroanatomical distribution and expression of POMGNT1 in the normal adult mouse brain. Methods: We analyzed the expression of POMGNT1 mRNA and protein in the brains of various neuroanatomical regions and spinal cords by western blotting and RT-qPCR. We also detected the distribution profile of POMGnT1 in normal adult mouse brains by immunohistochemistry and double-immunofluorescence. Results: In the present study, we found that POMGNT1-positive cells were widely distributed in various regions of the brain, with high levels of expression in the cerebral cortex and hippocampus. In terms of cell type, POMGNT1 was predominantly expressed in neurons and was mainly enriched in glutamatergic neurons; to a lesser extent, it was expressed in glial cells. At the subcellular level, POMGNT1 was mainly co-localized with the Golgi apparatus, but expression in the endoplasmic reticulum and mitochondria could not be excluded. Discussion: The present study suggests that POMGNT1, although widely expressed in various brain regions, may has some regional and cellular specificity, and the outcomes of this study provide a new laboratory basis for revealing the possible involvement of POMGNT1 in normal physiological functions of the brain from a morphological perspective.

Integration of transcriptome and targeted metabolome profiling reveals hormone related genes involved in the growth of Bletilla striata.

Bletilla striata (Thunb.) Reichb.f. (BS) is a traditional Chinese medicine with numerous beneficial effects. In our previous study, Aspergillus flavus was isolated from B. striata. To explore the physiological and molecular mechanisms of Aspergillus flavus elicitor (1-G4) that promoted Bletilla striata growth, in this study, we performed the determination of growth indexes and transcriptomics and metabolomics analysis under 5% and 10% 1-G4 conditions. Results showed that 1-G4 elicitor could significantly promote the growth and development of B. striata. With the increasing concentration of 1-G4 elicitor, the contents of SA, ICAld, and ME-IAA significantly increased while the IP and ACC contents decreased dramatically. A total of 1657 DEGs (763 up-regulated and 894 down-regulated) between the control (CK) and 5% elicitor (CK vs G5) and 2415 DEGs (1208 up-regulated and 1207 down-regulated) between the control and 10% elicitor (CK vs G10) were identified. Further, we found that 22, 38, and 2 unigenes were involved in ME-IAA, IP, and ACC, respectively. It was indicated that these unigenes might be involved in B. striata growth. Overall, the current study laid a theoretical foundation for the effective utilization of endophytic fungi and the optimization of germplasm resources of B. striata.

High-throughput optofluidic screening for improved microbial cell factories via real-time micron-scale productivity monitoring.

The industrial synthetic biology sector has made huge investments to achieve relevant miniaturized screening systems for scalable fermentation. Here we present the first example of a high-throughput (>103 genotypes per week) perfusion-based screening system to improve small-molecule secretion from microbial strains. Using the Berkeley Lights Beacon® system, the productivity of each strain could be directly monitored in real time during continuous culture, yielding phenotypes that correlated strongly (r2 > 0.8, p < 0.0005) with behavior in industrially relevant bioreactor processes. This method allows a much closer approximation of a typical fed-batch fermentation than conventional batch-like droplet or microplate culture models, in addition to rich time-dependent data on growth and productivity. We demonstrate these advantages by application to the improvement of high-productivity strains using whole-genome random mutagenesis, yielding mutants with substantially improved (by up to 85%) peak specific productivities in bioreactors. Each screen of ∼5 × 103 mutants could be completed in under 8 days (including 5 days involving user intervention), saving ∼50-75% of the time required for conventional microplate-based screening methods.

Challenging the workhorse: Comparative analysis of eukaryotic micro-organisms for expressing monoclonal antibodies.

For commercial protein therapeutics, Chinese hamster ovary (CHO) cells have an established history of safety, proven capability to express a wide range of therapeutic proteins and high volumetric productivities. Expanding global markets for therapeutic proteins and increasing concerns for broadened access of these medicines has catalyzed consideration of alternative approaches to this platform. Reaching these objectives likely will require an order of magnitude increase in volumetric productivity and a corresponding reduction in the costs of manufacture. For CHO-based manufacturing, achieving this combination of targeted improvements presents challenges. Based on a holistic analysis, the choice of host cells was identified as the single most influential factor for both increasing productivity and decreasing costs. Here we evaluated eight wild-type eukaryotic micro-organisms with prior histories of recombinant protein expression. The evaluation focused on assessing the potential of each host, and their corresponding phyla, with respect to key attributes relevant for manufacturing, namely (a) growth rates in industry-relevant media, (b) adaptability to modern techniques for genome editing, and (c) initial characterization of product quality. These characterizations showed that multiple organisms may be suitable for production with appropriate engineering and development and highlighted that yeast in general present advantages for rapid genome engineering and development cycles.

Rewriting yeast central carbon metabolism for industrial isoprenoid production.

A bio-based economy has the potential to provide sustainable substitutes for petroleum-based products and new chemical building blocks for advanced materials. We previously engineered Saccharomyces cerevisiae for industrial production of the isoprenoid artemisinic acid for use in antimalarial treatments. Adapting these strains for biosynthesis of other isoprenoids such as ß-farnesene (C15H24), a plant sesquiterpene with versatile industrial applications, is straightforward. However, S. cerevisiae uses a chemically inefficient pathway for isoprenoid biosynthesis, resulting in yield and productivity limitations incompatible with commodity-scale production. Here we use four non-native metabolic reactions to rewire central carbon metabolism in S. cerevisiae, enabling biosynthesis of cytosolic acetyl coenzyme A (acetyl-CoA, the two-carbon isoprenoid precursor) with a reduced ATP requirement, reduced loss of carbon to CO2-emitting reactions, and improved pathway redox balance. We show that strains with rewired central metabolism can devote an identical quantity of sugar to farnesene production as control strains, yet produce 25% more farnesene with that sugar while requiring 75% less oxygen. These changes lower feedstock costs and dramatically increase productivity in industrial fermentations which are by necessity oxygen-constrained. Despite altering key regulatory nodes, engineered strains grow robustly under taxing industrial conditions, maintaining stable yield for two weeks in broth that reaches >15% farnesene by volume. This illustrates that rewiring yeast central metabolism is a viable strategy for cost-effective, large-scale production of acetyl-CoA-derived molecules.

Non-natural cinnamic acid derivatives as substrates of cinnamate 4-hydroxylase.

Cinnamate 4-hydroxylase (C4H), a monooxygenase in the plant phenylpropanoid pathway, was assayed for its ability to hydroxylate 29 substrate analogues. Nine of the tested analogues with various aromatic side chains, including 3-coumaric acid, were metabolized by C4H. Seven products from these reactive analogues were characterized using LC/MS, 1H NMR and 13C NMR spectroscopic analysis. For example, caffeic acid was the product of 3-coumaric acid. The products 4-hydroxy-2-chlorocinnamic acid and 4-hydroxy-2-ethoxycinnamic acid are novel compounds that have not been previously reported. The kinetic parameters of C4H towards these analogues were determined.

Metabolic engineering of the phenylpropanoid pathway in Saccharomyces cerevisiae.

Flavonoids are valuable natural products derived from the phenylpropanoid pathway. The objective of this study was to create a host for the biosynthesis of naringenin, the central precursor of many flavonoids. This was accomplished by introducing the phenylpropanoid pathway with the genes for phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides, 4-coumarate:coenzyme A (CoA) ligase (4CL) from Arabidopsis thaliana, and chalcone synthase (CHS) from Hypericum androsaemum into two Saccharomyces cerevisiae strains, namely, AH22 and a pad1 knockout mutant. Each gene was cloned and inserted into an expression vector under the control of a separate individual GAL10 promoter. Besides its PAL activity, the recombinant PAL enzyme showed tyrosine ammonia lyase activity, which enabled the biosynthesis of naringenin without introducing cinnamate 4-hydroxylase (C4H). 4CL catalyzed the conversion of both trans-cinnamic acid and p-coumaric acid to their corresponding CoA products, which were further converted to pinocembrin chalcone and naringenin chalcone by CHS. These chalcones were cyclized to pinocembrin and naringenin. The yeast AH22 strain coexpressing PAL, 4CL, and CHS produced approximately 7 mg liter(-1) of naringenin and 0.8 mg liter(-1) of pinocembrin. Several by-products, such as 2',4',6'-trihydroxydihydrochalcone and phloretin, were also identified. Precursor feeding studies indicated that metabolic flux to the engineered flavonoid pathway was limited by the flux to the precursor l-tyrosine.

Optimization of an in vivo plant P450 monooxygenase system in Saccharomyces cerevisiae.

Cytochrome P450s are heme-thiolate oxygenases involved in a wide number of reactions such as epoxidation, hydroxylation, and demethylation. Heterologously expressed eukaryotic P450s are potentially useful biocatalysts for stereospecific oxygenation reactions under mild conditions. Numerous factors, such as intracellular pH, cytochrome P450, cytochrome P450 reductase, NADPH, and oxygen concentration all influence the in vivo activity. To systematically examine these factors, we selected ferulate 5-hydroxylase (F5H), a plant P450, with the Saccharomyces cerevisiae WAT11 strain as an expression host. Two media compositions and two cultivation procedures were investigated to optimize the in vivo activity of F5H. We modified a previously published selective growth medium (Pompon et al. [1996] Methods Enzymol 272:51-64) that increased the specific growth rate and cell yield of the host strain. A cultivation procedure with separate growth and induction stages that each contained selective media resulted in a 45% increase of whole cell F5H specific activity. In a medium designed for simultaneous growth and induction, we observed a 2.6-fold higher specific F5H activity, but substantially lower cell yield. Surprisingly, in this medium the higher specific F5H activity did not correlate with a higher P450 concentration. The effects of addition of the first committed heme precursor, delta-aminolevulinic acid, and Fe(III) at the beginning of induction period were also studied for our two-stage procedure. A small, but significant (P < 0.05) increase in whole cell F5H activity was observed following ALA addition.