Structure of a truncated human GlcNAc-1-phosphotransferase variant reveals the basis for its hyperactivity.

Mutations that cause loss of function of GlcNAc-1-phosphotransferase (PTase) lead to the lysosomal storage disorder mucolipidosis II. PTase is the key enzyme of the mannose 6-phosphate (M6P) targeting system that is responsible for tagging lysosomal hydrolases with the M6P moiety for their delivery to the lysosome. We had previously generated a truncated hyperactive form of PTase termed S1S3 which was shown to notably increase the phosphorylation level of secreted lysosomal enzymes and enhance their uptake by cells. Here, we report the 3.4 Å cryo-EM structure of soluble S1S3 lacking both transmembrane domains and cytosolic tails. The structure reveals a high degree of conservation of the catalytic core to full-length PTase. In this dimeric structure, the EF-hand of one protomer is observed interacting with the conserved region four of the other. In addition, we present a high-quality EM 3D map of the UDP-GlcNAc bound form of the full-length soluble protein showing the key molecular interactions between the nucleotide sugar donor and side chain amino acids of the protein. Finally, although the domain organization of S1S3 is very similar to that of the Drosophila melanogaster (fruit fly) PTase homolog, we establish that the latter does not act on lysosomal hydrolases.

Maternal nanoplastic ingestion induces an increase in offspring body weight through altered lipid species and microbiota.

The rapidly increasing prevalence of obesity and overweight, especially in children and adolescents, has become a serious societal issue. Although various genetic and environmental risk factors for pediatric obesity and overweight have been identified, the problem has not been solved. In this study, we examined whether environmental nanoplastic (NP) pollutants can act as environmental obesogens using mouse models exposed to NPs derived from polystyrene and polypropylene, which are abundant in the environment. We found abnormal weight gain in the progeny until 6 weeks of age following the oral administration of NPs to the mother during gestation and lactation. Through a series of experiments involving multi-omic analyses, we have demonstrated that NP-induced weight gain is caused by alterations in the lipid composition (lysophosphatidylcholine/phosphatidylcholine ratio) of maternal breast milk and he gut microbiota distribution of the progeny. These data indicate that environmental NPs can act as obesogens in childhood.

Three-dimensional label-free visualization of the interactions of PM2.5 with macrophages and epithelial cells using optical diffraction tomography.

Particulate matter ≤ 2.5 µm (PM2.5) poses health risks related to various diseases and infections. However, the interactions between PM2.5 and cells such as uptake and cell responses have not been fully investigated despite advances in bioimaging techniques, because the heterogeneous morphology and composition of PM2.5 make it challenging to employ labeling techniques, such as fluorescence. In this work, we visualized the interaction between PM2.5 and cells using optical diffraction tomography (ODT), which provides quantitative phase images by refractive index distribution. Through ODT analysis, the interactions of PM2.5 with macrophages and epithelial cells, such as intracellular dynamics, uptake, and cellular behavior, were successfully visualized without labeling techniques. ODT analysis clearly shows the behavior of phagocytic macrophages and nonphagocytic epithelial cells for PM2.5. Moreover, ODT analysis could quantitatively compare the accumulation of PM2.5 inside the cells. PM2.5 uptake by macrophages increased substantially over time, but uptake by epithelial cells increased only marginally. Our findings indicate that ODT analysis is a promising alternative approach to visually and quantitatively understanding the interaction of PM2.5 with cells. Therefore, we expect ODT analysis to be employed to investigate the interactions of materials and cells that are difficult to label.

Combined effects of microplastics and benz[a]anthracene on cardiotoxicity in zebrafish (Danio rerio) larvae: Size matters.

The size of microplastics (MPs) plays an important role in combined toxic effects including synergistic or antagonistic effects. However, the influence of the size of MPs on the combined toxicity of contaminants remains unclear. In this study, we employed a zebrafish model to investigate the effects of MP size on the combined toxicity of benz[a]anthracene (BaA), a representative polyaromatic hydrocarbon, using three different sizes of polystyrene MPs (PSMPs) (0.2, 1.0, and 10 µm). Treatment of all groups did not result in any mortality of the zebrafish larvae. However, small-sized PSMPs (0.2 µm) enhanced the toxic effect of BaA in larvae such as cardiac defect and disruption of vessel formation. Medium-sized PSMPs (1.0 µm) were boundary in terms of the combined toxic effect; however, large-sized PSMPs (10 µm) alleviated the cardiotoxicity of BaA, including cardiac defect, ROS levels, and cell death. The combined effects showed a correlation with the body burden of MPs and BaA in larvae according to particle size (in the order of 0.2 µm > 1.0 µm > 10 µm). The synergistic effects occurred likely because the small PSMPs facilitated the body burden of BaA, induced excessive ROS by Ahr-mediated activity, and caused cell death in the heart, resulting in increased heart defects in the larvae. In contrast, large PSMPs abated the combined toxic effect through decreased body burden, whereas medium PSMPs form a boundary in combined effects. Therefore, the combined toxic effects of MPs are dependent on their size, which plays an important role in the transport and accumulation of environmental pollutants.

A Simple and Rapid Methicillin-Resistant Staphylococcus aureus (MRSA) Screening Test Using a Mannose-Binding Lectin (MBL)-Conjugated Gold Nanoparticle Probe.

Rapid diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) is essential for guiding clinical treatment and preventing the spread of MRSA infections. Herein, we present a simple and rapid MRSA screening test based on the aggregation effect of mannose-binding lectin (MBL)-conjugated gold nanoparticles (AuNP), called the MRSA probe. Recombinant MBL protein is a member of the lectin family and part of the innate immune system. It can recognize wall teichoic acid (WTA) on the membrane of MRSA more specifically than that of methicillin-sensitive Staphylococcus aureus (MSSA) under optimized salt conditions. Thus, the MRSA probe can selectively bind to MRSA, and the aggregation of the probes on the surface of the target bacteria can be detected and analyzed by the naked eye within 5 min. To demonstrate the suitability of the method for real-world application, we tested 40 clinical S. aureus isolates (including 20 MRSA specimens) and recorded a sensitivity of 100%. In conclusion, the MRSA probe-based screening test with its excellent sensitivity has the potential for successful application in the microbiology laboratory.

Differential particle and ion kinetics of silver nanoparticles in the lungs and biotransformation to insoluble silver sulfide.

The measurement of nanoparticles (NPs) in a biological matrix is essential in various toxicity studies. However, the current knowledge has limitations in differentiating particulate and ionic forms and further identification of their biotransformation. Herein, we evaluate the biotransformation and differential lung clearance kinetics of particulate and ionic forms using PEGylated silver NPs (AgNP-PEGs; 47.51 nm) and PEGylated gold NPs (AuNP-PEGs; 11.76 nm). At 0, 3, and 6 h and 1, 3, 7, and 14 days after a single pharyngeal aspiration in mice at 25 µg/mouse, half of the lung is digested by proteinase K (PK) to separate particulates and ions, and the other half is subjected to the acid digestion method for comparison. The quantitative and qualitative evaluation of lung clearance kinetics suggests that AgNP-PEGs are quickly dissolved and transformed into insoluble silver sulfide (Ag2S), which shows a fast-clearing early phase (0 -6 h; particle T1/2: 4.8 h) and slow-clearing late phase (1 -14 days; particle T1/2: 13.20 days). In contrast, AuNP-PEGs were scarcely cleared or biotransformed in the lungs for 14 days. The lung clearance kinetics of AgNPs and biotransformation shown in this study can be informed by the PK digestion method and cannot be obtained using the acid digestion method.

Antioxidants prevent particulate matter-induced senescence of lung fibroblasts.

Particulate matter (PM) contributes to human diseases, particularly lung disease; however, the molecular mechanism of its action is yet to be determined. Herein, we found that prolonged PM exposure induced the cellular senescence of normal lung fibroblasts via a DNA damage-mediated response. This PM-induced senescence (PM-IS) was only observed in lung fibroblasts but not in A549 lung adenocarcinoma cells. Mechanistic analysis revealed that reactive oxygen species (ROS) activate the DNA damage response signaling axis, increasing p53 phosphorylation, ultimately leading to cellular senescence via an increase in p21 expression without affecting the p16-pRB pathway. A549 cells, instead, were resistant to PM-IS due to the PM-induced ROS production suppression. Water-soluble antioxidants, such as vitamin C and N-Acetyl Cysteine, were found to alleviate PM-IS by suppressing ROS production, implying that antioxidants are a promising therapeutic intervention for PM-mediated lung pathogenesis.

A review on the impacts of nanomaterials on neuromodulation and neurological dysfunction using a zebrafish animal model.

Nanomaterials have been widely employed from industrial to medical fields due to their small sizes and versatile characteristics. However, nanomaterials can also induce unexpected adverse effects on health. In particular, exposure of the nervous system to nanomaterials can cause serious neurological dysfunctions and neurodegenerative diseases. A number of studies have adopted various animal models to evaluate the neurotoxic effects of nanomaterials. Among them, zebrafish has become an attractive animal model for neurotoxicological studies due to several advantages, including the well-characterized nervous system, efficient genome editing, convenient generation of transgenic lines, high-resolution in vivo imaging, and an array of behavioral assays. In this review, we summarize recent studies on the neurotoxicological effects of nanomaterials, particularly engineered nanomaterials and nanoplastics, using zebrafish and discuss key findings with advantages and limitations of the zebrafish model in neurotoxicological studies.

Structure of the human GlcNAc-1-phosphotransferase αß subunits reveals regulatory mechanism for lysosomal enzyme glycan phosphorylation.

Vertebrates use the mannose 6-phosphate (M6P)-recognition system to deliver lysosomal hydrolases to lysosomes. Key to this pathway is N-acetylglucosamine (GlcNAc)-1-phosphotransferase (PTase) that selectively adds GlcNAc-phosphate (P) to mannose residues of hydrolases. Human PTase is an α2ß2γ2 heterohexamer with a catalytic core and several peripheral domains that recognize and bind substrates. Here we report a cryo-EM structure of the catalytic core of human PTase and the identification of a hockey stick-like motif that controls activation of the enzyme. Movement of this motif out of the catalytic pocket is associated with a rearrangement of part of the peripheral domains that unblocks hydrolase glycan access to the catalytic site, thereby activating PTase. We propose that PTase fluctuates between inactive and active states in solution, and selective substrate binding of a lysosomal hydrolase through its protein-binding determinant to PTase locks the enzyme in the active state to permit glycan phosphorylation. This mechanism would help ensure that only N-linked glycans of lysosomal enzymes are phosphorylated.

Fluorescent Polypropylene Nanoplastics for Studying Uptake, Biodistribution, and Excretion in Zebrafish Embryos.

Nanoplastics (NPs) are emerging environmental pollutants and are a significant concern for human health. The small size of NPs allows them to accumulate within and adversely affect various tissues by penetrating the gastrointestinal barrier. However, most toxicity studies on NPs have been based on commercial polystyrene nanoparticles. Among plastics, polypropylene (PP) is one of the most widely used, and it is continuously micronized in the environment. Although PP has high potential for forming NPs by weathering, little is known about the biological effects of polypropylene nanoplastics (PPNPs) due to a lack of particle models. Here, we present a simple and high-yield method for PPNP production by nonsolvent-induced phase separation. The synthesized PPNPs were spherical in shape, with an average diameter of 562.15 ± 118.47 nm and a high yield of over 84%. These PPNPs were fluorescently labeled by the combined swelling-diffusion method to study their biodistribution after exposure to developing zebrafish embryos (ZFEs). We found that the fluorescent PPNPs were internalized by ingestion, distributed in the intestine of developing ZFEs, and eventually excreted. This study will aid evaluations of the potential risks of environmentally relevant plastics at the nanoscale.

Maternal exposure to polystyrene nanoplastics causes brain abnormalities in progeny.

As global plastic production continues to grow, microplastics released from a massive quantity of plastic wastes have become a critical environmental concern. These microplastic particles are found in a wide range of living organisms in a diverse array of ecosystems. In this study, we investigated the biological effects of polystyrene nanoplastic (PSNP) on development of the central nervous system using cultured neural stem cells (NSCs) and mice exposed to PSNP during developmental stages. Our study demonstrates that maternal administration of PSNP during gestation and lactating periods altered the functioning of NSCs, neural cell compositions, and brain histology in progeny. Similarly, PSNP-induced molecular and functional defects were also observed in cultured NSCs in vitro. Finally, we show that the abnormal brain development caused by exposure to high concentrations of PSNP results in neurophysiological and cognitive deficits in a gender-specific manner. Our data demonstrate the possibility that exposure to high amounts of PSNP may increase the risk of neurodevelopmental defects.

Differential Clearance of Aß Species from the Brain by Brain Lymphatic Endothelial Cells in Zebrafish.

The failure of amyloid beta (Aß) clearance is a major cause of Alzheimer's disease, and the brain lymphatic systems play a crucial role in clearing toxic proteins. Recently, brain lymphatic endothelial cells (BLECs), a non-lumenized lymphatic cell in the vertebrate brain, was identified, but Aß clearance via this novel cell is not fully understood. We established an in vivo zebrafish model using fluorescently labeled Aß42 to investigate the role of BLECs in Aß clearance. We discovered the efficient clearance of monomeric Aß42 (mAß42) compared to oligomeric Aß42 (oAß42), which was illustrated by the selective uptake of mAß42 by BLECs and peripheral transport. The genetic depletion, pharmacological inhibition via the blocking of the mannose receptor, or the laser ablation of BLECs resulted in the defective clearance of mAß42. The treatment with an Aß disaggregating agent facilitated the internalization of oAß42 into BLECs and improved the peripheral transport. Our findings reveal a new role of BLECs in the differential clearance of mAß42 from the brain and provide a novel therapeutic strategy based on promoting Aß clearance.

Biomimetic Nanopillar-Based Biosensor for Label-Free Detection of Influenza A Virus.

Since the first emergence of influenza viruses, they have caused the flu seasonally worldwide. Precise detection of influenza viruses is required to prevent the spreading of the disease. Herein, we developed an optical biosensor using peptide-immobilized nanopillar structures for the label-free detection of influenza viruses. The spin-on-glass nanopillar structures were fabricated by nanoimprint lithography. A sialic acid-mimic peptide, which can specifically bind to hemagglutinin on the surface of the influenza virus, was immobilized onto the nanopillars via polymerized dopamine. The constructed nanopillar sensor enabled us to detect influenza A viruses in the range of 103-105 plaque-forming units through simple measurements of reflectance. Our findings suggest that biomimetic modification of nanopillar structures can be an alternative method for the immunodiagnosis of influenza viruses.

3D Hierarchical Nanotopography for On-Site Rapid Capture and Sensitive Detection of Infectious Microbial Pathogens.

Effective capture and rapid detection of pathogenic bacteria causing pandemic/epidemic diseases is an important task for global surveillance and prevention of human health threats. Here, we present an advanced approach for the on-site capture and detection of pathogenic bacteria through the combination of hierarchical nanostructures and a nuclease-responsive DNA probe. The specially designed hierarchical nanocilia and network structures on the pillar arrays, termed 3D bacterial capturing nanotopographical trap, exhibit excellent mechanical reliability and rapid (<30 s) and irreversible bacterial capturability. Moreover, the nuclease-responsive DNA probe enables the highly sensitive and extremely fast (<1 min) detection of bacteria. The bacterial capturing nanotopographical trap (b-CNT) facilitates the on-site capture and detection of notorious infectious pathogens (Escherichia coli O157:H7, Salmonella enteritidis, Staphylococcus aureus, and Bacillus cereus) from kitchen tools and food samples. Accordingly, the usefulness of the b-CNT is confirmed as a simple, fast, sensitive, portable, and robust on-site capture and detection tool for point-of-care testing.

Inactivation of the three GGA genes in HeLa cells partially compromises lysosomal enzyme sorting.

The Golgi-localized, gamma-ear containing, ADP-ribosylation factor-binding proteins (GGAs 1, 2, and 3) are multidomain proteins that bind mannose 6-phosphate receptors (MPRs) at the Golgi and play a role, along with adaptor protein complex 1 (AP-1), in the sorting of newly synthesized lysosomal hydrolases to the endolysosomal system. However, the relative importance of the two types of coat proteins in this process is still unclear. Here, we report that inactivation of all three GGA genes in HeLa cells decreased the sorting efficiency of cathepsin D from 97% to 73% relative to wild-type, with marked redistribution of the cation-independent MPR from peripheral punctae to the trans-Golgi network. In comparison, GNPTAB-/- HeLa cells with complete inactivation of the mannose 6-phosphate pathway sorted only 20% of the cathepsin D. We conclude that the residual sorting of cathepsin D in the GGA triple-knockout cells is mediated by AP-1.

Effect of elastin-like polypeptide incorporation on the adhesion maturation of mineral trioxide aggregates.

The aim of this study was to investigate the effects of the incorporation of elastin-like polypeptide (ELP) on the adhesion maturation of mineral trioxide aggregates (MTA). Two types of ELPs (V125 and V125E8) were genetically synthesized. V125 consisted of 125 repeating pentapeptides (Val-Pro-Gly-Xaa-Gly) and V125E8 was functionalized with octaglutamic acid in the C-terminus of V125; both were diluted to 10 wt% in solution. Three 1.5 mm diameter holes in dentin discs were filled with MTA mixed with either a solution of ELP or deionized water. Push-out bond strength tests were performed following storage in simulated body fluid (SBF) for 1, 2, 4, and 8 weeks (n = 12). The interface between dentin and MTA was observed via scanning electron microscopy (SEM). The maturation of MTA was evaluated with a stereoscopic microscope and SEM. The incorporation of a specific ELP (V125E8) significantly increased the bond strength of MTA to dentin with regard to every maturation period (p < 0.05). The bond strength of MTA also significantly increased with a longer maturation time irrespective of ELP incorporation (p < 0.05). V125E8-incorporated MTA exhibited a more intimate interface with dentin compared to the other groups. More spindle-shaped crystal structures and thicker crystals were observed in all MTA mixtures as the storage duration increased even though V125E8 exhibited fewer crystal structures on the surface. Within the limitations of this study, the incorporation of V125E8 increased the adhesive properties of MTA and the maturation of MTA occurred regardless of ELP incorporation.

Simple, rapid, and accurate malaria diagnostic platform using microfluidic-based immunoassay of Plasmodium falciparum lactate dehydrogenase.

This work reports on a rapid diagnostic platform for the detection of Plasmodium falciparum lactate dehydrogenase (PfLDH), a representative malaria biomarker, using a microfluidic microplate-based immunoassay. In this study, the microfluidic microplate made it possible to diagnose PfLDH with a small volume of sample (only 5 µL) and short time (< 90 min) compared to conventional immunoassays such as enzyme-linked immunosorbent assay (ELISA). Moreover, the diagnostic performance of PfLDH showed high sensitivity, specificity, and selectivity (i.e., 0.025 pg/µL in phosphate-buffered saline and 1 pg/µL in human serum). The microfluidic-based microplate sensing platform has the potential to adapt simple, rapid, and accurate diagnoses to the practical detection of malaria.

Disease-causing missense mutations within the N-terminal transmembrane domain of GlcNAc-1-phosphotransferase impair endoplasmic reticulum translocation or Golgi retention.

Transport of newly synthesized lysosomal enzymes to the lysosome requires tagging of these enzymes with the mannose 6-phosphate moiety by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase), encoded by two genes, GNPTAB and GNPTG. GNPTAB encodes the α and ß subunits, which are initially synthesized as a single precursor that is cleaved by Site-1 protease in the Golgi. Mutations in this gene cause the lysosomal storage disorders mucolipidosis II (MLII) and mucolipidosis III αß (MLIII αß). Two recent studies have reported the first patient mutations within the N-terminal transmembrane domain (TMD) of the α subunit of GlcNAc-1-phosphotransferase that cause either MLII or MLIII αß. Here, we demonstrate that two of the MLII missense mutations, c.80T>A (p.Val27Asp) and c.83T>A (p.Val28Asp), prevent the cotranslational insertion of the nascent GlcNAc-1-phosphotransferase polypeptide chain into the endoplasmic reticulum. The remaining four mutations, one of which is associated with MLII, c.100G>C (p.Ala34Pro), and the other three with MLIII αß, c.70T>G (p.Phe24Val), c.77G>A (p.Gly26Asp), and c.107A>C (p.Glu36Pro), impair retention of the catalytically active enzyme in the Golgi with concomitant mistargeting to endosomes/lysosomes. Our results uncover the basis for the disease phenotypes of these patient mutations and establish the N-terminal TMD of GlcNAc-1-phosphotransferase as an important determinant of Golgi localization.

Human GNPTAB stuttering mutations engineered into mice cause vocalization deficits and astrocyte pathology in the corpus callosum.

Stuttering is a common neurodevelopmental disorder that has been associated with mutations in genes involved in intracellular trafficking. However, the cellular mechanisms leading to stuttering remain unknown. Engineering a mutation in N-acetylglucosamine-1-phosphate transferase subunits α and ß (GNPTAB) found in humans who stutter into the mouse Gnptab gene resulted in deficits in the flow of ultrasonic vocalizations similar to speech deficits of humans who stutter. Here we show that other human stuttering mutations introduced into this mouse gene, Gnptab Ser321Gly and Ala455Ser, produce the same vocalization deficit in 8-day-old pup isolation calls and do not affect other nonvocal behaviors. Immunohistochemistry showed a marked decrease in staining of astrocytes, particularly in the corpus callosum of the Gnptab Ser321Gly homozygote mice compared to wild-type littermates, while the staining of cerebellar Purkinje cells, oligodendrocytes, microglial cells, and dopaminergic neurons was not significantly different. Diffusion tensor imaging also detected deficits in the corpus callosum of the Gnptab Ser321Gly mice. Using a range of cell type-specific Cre-drivers and a Gnptab conditional knockout line, we found that only astrocyte-specific Gnptab-deficient mice displayed a similar vocalization deficit. These data suggest that vocalization defects in mice carrying human stuttering mutations in Gnptab derive from abnormalities in astrocytes, particularly in the corpus callosum, and provide support for hypotheses that focus on deficits in interhemispheric communication in stuttering.

Poly-γ-Glutamic Acid Complexed With Alum Induces Cross-Protective Immunity of Pandemic H1N1 Vaccine.

The use of a good vaccine adjuvant may induce a higher immunogenicity profile of vaccine antigens. Here, we developed a new adjuvant by combining poly-γ-glutamic acid (γ-PGA) with alum (PGA/Alum) and investigated its ability to enhance the immunogenicity and the cross-reactive efficacy of pandemic H1N1 (pH1N1) influenza vaccine antigen. PGA/Alum enhanced antigen delivery to draining lymph nodes and antigen-specific immunogenicity in mice using OVA as a model antigen. It also greatly increased OVA-specific antibody production, cytotoxic T lymphocyte (CTL) activity, and antibody-dependent cellular cytotoxicity (ADCC). These abilities of PGA/Alum improved the protective efficacy of pH1N1 vaccine antigen by increasing hemagglutination-inhibition titers, enhancing ADCC and CTL activity, and speeding viral clearance following homologous viral challenge. Importantly, the cross-protective efficacy of pH1N1 vaccine against heterologous viruses [A/Puerto Rico/8/34 (H1N1) and A/Hong Kong/1/1968 (H3N2)] was significantly enhanced by PGA/Alum, and cross-reactive ADCC and CTL activities were observed. Together, our results strongly suggest that PGA/Alum may be a promising vaccine adjuvant for preventing influenza and other infectious diseases.