Midkine Attenuates Aß Fibril Assembly and AmyloidPlaque Formation.

Proteomic profiling of Alzheimer's disease (AD) brains has identified numerous understudied proteins, including midkine (MDK), that are highly upregulated and correlated with Aß since the early disease stage, but their roles in disease progression are not fully understood. Here we present that MDK attenuates Aß assembly and influences amyloid formation in the 5xFAD amyloidosis mouse model. MDK protein mitigates fibril formation of both Aß40 and Aß42 peptides in Thioflavin T fluorescence assay, circular dichroism, negative stain electron microscopy, and NMR analysis. Knockout of Mdkgene in 5xFAD increases amyloid formation and microglial activation. Further comprehensive mass spectrometry-based profiling of whole proteome and aggregated proteome in these mouse models indicates significant accumulation of Aß and Aß-correlated proteins, along with microglial components. Thus, our structural and mouse model studies reveal a protective role of MDK in counteracting amyloid pathology in Alzheimer's disease.

Identifying Sex-Specific Serum Patterns of Alzheimer's Mice through Deep TMT Profiling and a Concentration-Dependent Concatenation Strategy.

Alzheimer's disease (AD) is the most prevalent form of dementia, disproportionately affecting women in disease prevalence and progression. Comprehensive analysis of the serum proteome in a common AD mouse model offers potential in identifying possible AD pathology- and gender-associated biomarkers. Here, we introduce a multiplexed, nondepleted mouse serum proteome profiling via tandem mass-tag (TMTpro) labeling. The labeled sample was separated into 475 fractions using basic reversed-phase liquid chromatography (RPLC), which were categorized into low-, medium-, and high-concentration fractions for concatenation. This concentration-dependent concatenation strategy resulted in 128 fractions for acidic RPLC-tandem mass spectrometry (MS/MS) analysis, collecting ∼5 million MS/MS scans and identifying 3972 unique proteins (3413 genes) that cover a dynamic range spanning at least 6 orders of magnitude. The differential expression analysis between wild type and the commonly used AD model (5xFAD) mice exhibited minimal significant protein alterations. However, we detected 60 statistically significant (FDR < 0.05), sex-specific proteins, including complement components, serpins, carboxylesterases, major urinary proteins, cysteine-rich secretory protein 1, pregnancy-associated murine protein 1, prolactin, amyloid P component, epidermal growth factor receptor, fibrinogen-like protein 1, and hepcidin. The results suggest that our platform possesses the sensitivity and reproducibility required to detect sex-specific differentially expressed proteins in mouse serum samples.

CXCR6 orchestrates brain CD8+ T cell residency and limits mouse Alzheimer's disease pathology.

Neurodegenerative diseases, including Alzheimer's disease (AD), are characterized by innate immune-mediated inflammation, but functional and mechanistic effects of the adaptive immune system remain unclear. Here we identify brain-resident CD8+ T cells that coexpress CXCR6 and PD-1 and are in proximity to plaque-associated microglia in human and mouse AD brains. We also establish that CD8+ T cells restrict AD pathologies, including ß-amyloid deposition and cognitive decline. Ligand-receptor interaction analysis identifies CXCL16-CXCR6 intercellular communication between microglia and CD8+ T cells. Further, Cxcr6 deficiency impairs accumulation, tissue residency programming and clonal expansion of brain PD-1+CD8+ T cells. Ablation of Cxcr6 or CD8+ T cells ultimately increases proinflammatory cytokine production from microglia, with CXCR6 orchestrating brain CD8+ T cell-microglia colocalization. Collectively, our study reveals protective roles for brain CD8+ T cells and CXCR6 in mouse AD pathogenesis and highlights that microenvironment-specific, intercellular communication orchestrates tissue homeostasis and protection from neuroinflammation.

A novel role of RNase L in the development of nonalcoholic steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and affects about 25% of the population globally. NAFLD has the potential to cause significant liver damage in many patients because it can progress to nonalcoholic steatohepatitis (NASH) and cirrhosis, which substantially increases disease morbidity and mortality. Despite the key role of innate immunity in the disease progression, the underlying molecular and pathogenic mechanisms remain to be elucidated. RNase L is a key enzyme in interferon action against viral infection and displays pleiotropic biological functions such as control of cell proliferation, apoptosis, and autophagy. Recent studies have demonstrated that RNase L is involved in innate immunity. In this study, we revealed that RNase L contributed to the development of NAFLD, which further progressed to NASH in a time-dependent fashion after RNase L wild-type (WT) and knockout mice were fed with a high-fat and high-cholesterol diet. RNase L WT mice showed significantly more severe NASH, evidenced by widespread macro-vesicular steatosis, hepatocyte ballooning degeneration, inflammation, and fibrosis, although physiological and biochemical data indicated that both types of mice developed obesity, hyperglycemia, hypercholesterolemia, dysfunction of the liver, and systemic inflammation at different extents. Further investigation demonstrated that RNase L was responsible for the expression of some key genes in lipid metabolism, inflammation, and fibrosis signaling. Taken together, our results suggest that a novel therapeutic intervention for NAFLD may be developed based on regulating the expression and activity of RNase L.

A phycoerythrin isolated from Rhodomonas salina induces apoptosis via ERK/Bak and JNK/Caspase-3 pathway in A549 cells.

Rhodomonas salina, Cryptophyta, Rhodomonas genus, is a valuable source for live feed in aquaculture and for the production of phycoerythrin (PE). In this study, PE was extracted from Rhodomonas salina and characterized as having a molecular weight of approximately 24 kDa, an absorbance at 545 nm, and a purity of up to 6.61 (which meets reagent grade requirements with an OD545/OD280 ratio >4). The effects of PE on anticancer activity and its underlying mechanisms were evaluated to assess the immunomodulatory potential on the human lung cancer A549 cell line. Biochemical assays and western blot analysis were applied to confirm the immune mechanisms. The results showed that after 24 h of exposure to PE, the proliferation of A549 cells was significantly and dose-dependently decreased. PE also caused the generation of reactive oxygen species (ROS) and a decrease in mitochondrial membrane potential (MMP). The further results showed that PE can remarkably enhance the protein levels of cleaved caspase-3 and p53. Simultaneously, the BCL-2 family was also affected and had some changes, such as the dramatically enhance of Bim and Bak and the decrease of Bcl-2 level. However, it is interesting to note that there was no apparent alteration in Bax expression during the experiment. Furthermore, the biological mechanism for the potential of PE to induce apoptosis showed that the ERK/Bak and the JNK/caspase-3 signaling pathway were activated. This study provides evidence that the anticancer activity of PE in Rhodomonas salina may have potential for preventing cancer and serving as a novel immunostimulant in the pharmaceutical industry.

The evolving landscape of menstrual product advertisements in the United States: 2008-2018.

As menstrual product advertising evolves within the United States, it is important to understand how advertising messages, which have been shown to impact self-esteem and feelings of shame, may be influencing young people today. We analyzed menstrual product advertising over ten years (2008-2018) through a survey (n = 198) and focus groups (n = 21) with college and graduate student-aged adults. Three themes emerged: an emphasis on femininity and shame; the presence and role of men in the menstrual process; and racial, gender and body type inclusivity. Advertising shifts toward messages of inclusivity may positively influence young people's perceptions toward their bodies and menstruation.

Deep Single-Cell-Type Proteome Profiling of Mouse Brain by Nonsurgical AAV-Mediated Proximity Labeling.

Proteome profiling is a powerful tool in biological and biomedical studies, starting with samples at bulk, single-cell, or single-cell-type levels. Reliable methods for extracting specific cell-type proteomes are in need, especially for the cells (e.g., neurons) that cannot be readily isolated. Here, we present an innovative proximity labeling (PL) strategy for single-cell-type proteomics of mouse brain, in which TurboID (an engineered biotin ligase) is used to label almost all proteins in a specific cell type. This strategy bypasses the requirement of cell isolation and includes five major steps: (i) constructing recombinant adeno-associated viruses (AAVs) to express TurboID driven by cell-type-specific promoters, (ii) delivering the AAV to mouse brains by direct intravenous injection, (iii) enhancing PL labeling by biotin administration, (iv) purifying biotinylated proteins, followed by on-bead protein digestion, and (v) quantitative tandem-mass-tag (TMT) labeling. We first confirmed that TurboID can label a wide range of cellular proteins in human HEK293 cells and optimized the single-cell-type proteomic pipeline. To analyze specific brain cell types, we generated recombinant AAVs to coexpress TurboID and mCherry proteins, driven by neuron- or astrocyte-specific promoters and validated the expected cell expression by coimmunostaining of mCherry and cellular markers. Subsequent biotin purification and TMT analysis identified ∼10,000 unique proteins from a few micrograms of protein samples with excellent reproducibility. Comparative and statistical analyses indicated that these PL proteomes contain cell-type-specific cellular pathways. Although PL was originally developed for studying protein-protein interactions and subcellular proteomes, we extended it to efficiently tag the entire proteomes of specific cell types in the mouse brain using TurboID biotin ligase. This simple, effective in vivo approach should be broadly applicable to single-cell-type proteomics.

Alzheimer's disease-associated U1 snRNP splicing dysfunction causes neuronal hyperexcitability and cognitive impairment.

Recent proteome and transcriptome profiling of Alzheimer's disease (AD) brains reveals RNA splicing dysfunction and U1 small nuclear ribonucleoprotein (snRNP) pathology containing U1-70K and its N-terminal 40-KDa fragment (N40K). Here we present a causative role of U1 snRNP dysfunction to neurodegeneration in primary neurons and transgenic mice (N40K-Tg), in which N40K expression exerts a dominant-negative effect to downregulate full-length U1-70K. N40K-Tg recapitulates N40K insolubility, erroneous splicing events, neuronal degeneration and cognitive impairment. Specifically, N40K-Tg shows the reduction of GABAergic synapse components (e.g., the GABA receptor subunit of GABRA2), and concomitant postsynaptic hyperexcitability that is rescued by a GABA receptor agonist. Crossing of N40K-Tg and the 5xFAD amyloidosis model indicates that the RNA splicing defect synergizes with the amyloid cascade to remodel the brain transcriptome and proteome, deregulate synaptic proteins, and accelerate cognitive decline. Thus, our results support the contribution of U1 snRNP-mediated splicing dysfunction to AD pathogenesis.

Deep Profiling of Microgram-Scale Proteome by Tandem Mass Tag Mass Spectrometry.

Tandem mass tag (TMT)-based mass spectrometry (MS) enables deep proteomic profiling of more than 10,000 proteins in complex biological samples but requires up to 100 µg protein in starting materials during a standard analysis. Here, we present a streamlined protocol to quantify more than 9000 proteins with 0.5 µg protein per sample by 16-plex TMT coupled with two-dimensional liquid chromatography and tandem mass spectrometry (LC/LC-MS/MS). In this protocol, we optimized multiple conditions to reduce sample loss, including processing each sample in a single tube to minimize surface adsorption, increasing digestion enzymes to shorten proteolysis and function as carriers, eliminating a desalting step between digestion and TMT labeling, and developing miniaturized basic pH LC for prefractionation. By profiling 16 identical human brain tissue samples of Alzheimer's disease (AD), vascular dementia (VaD), and non-dementia controls, we directly compared this new microgram-scale protocol to the standard-scale protocol, quantifying 9116 and 10,869 proteins, respectively. Importantly, bioinformatics analysis indicated that the microgram-scale protocol had adequate sensitivity and reproducibility to detect differentially expressed proteins in disease-related pathways. Thus, this newly developed protocol is of general application for deep proteomics analysis of biological and clinical samples at sub-microgram levels.

The Extent to Which Menstruation-Related Issues Are Included in Graduate-Level Public Health Curricula.

Objectives: Menstruation is increasingly recognized as an issue in domestic and global public health. Public health graduates of U.S. schools of public health must have adequate competencies to address menstruation and its implications for health and well-being in their future endeavors in research, practice and policy. This study sought to understand the extent to which U.S. schools currently integrate menstruation-related content (menstrual health, menstrual hygiene, etc.) and related competencies into their curricula. Methods: We reviewed the course directories of the top 20 US schools of public health as ranked in 2018. Courses were selected based on inclusion of menstruation and adolescent health-related search terms. Syllabi were subsequently obtained and analyzed for inclusion of specific menstruation-related terms. Syllabi including these terms were further analyzed to determine the level of inclusion of menstruation-related topics in relation to public health competencies, and the area of specialization. Results: Of an estimated 5,000 courses assessed, 28 included menstruation-related topics. Most frequently, this inclusion was minimal (e.g., a single reading or assignment), and was limited in scope. Content was typically found within global health, environmental health, and maternal and child health. Conclusions: Given growing attention to menstruation domestically and globally, and the limited current inclusion of this issue in US schools of public health curricula, graduates may not be receiving adequate training on a critically important topic of relevance within population health. Schools should consider reviewing their curricula to assess whether there are opportunities to integrate menstruation-related content in relation to the relevant public health competencies.

High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry.

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing. The crucial quality control steps and improvements in the process specific for the 16-plex TMT analysis are highlighted. This multiplexed process offers a powerful tool for profiling a variety of complex samples such as cells, tissues, and clinical specimens. More than 10,000 proteins and posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination in highly complex biological samples from up to 16 different samples can be quantified in a single experiment, providing a potent tool for basic and clinical research.

Oral Ganglioside Supplement Improves Growth and Development in Patients with Ganglioside GM3 Synthase Deficiency.

Ganglioside GM3 synthase is a key enzyme involved in the biosynthesis of gangliosides. GM3 synthase deficiency (GM3D) causes an absence of GM3 and all downstream biosynthetic derivatives. The affected individuals manifest with severe irritability, intractable seizures, and profound intellectual disability. The current study is to assess the effects of an oral ganglioside supplement to patients with GM3D, particularly on their growth and development during early childhood. A total of 13 young children, 11 of them under 40 months old, received oral ganglioside supplement through a dairy product enriched in gangliosides, for an average of 34 months. Clinical improvements were observed in most children soon after the supplement was initiated. Significantly improved growth and development were documented in these subjects as average percentiles for weight, height, and occipitofrontal circumference increased in 1-2 months. Three children with initial microcephaly demonstrated significant catch-up head growth and became normocephalic. We also illustrated brief improvements in developmental and cognitive scores, particularly in communication and socialization domains through Vineland-II. However, all improvements seemed transient and gradually phased out after 12 months of supplementation. Gangliosides GM1 and GM3, although measureable in plasma during the study, were not significantly changed with ganglioside supplementation for up to 30 months. We speculate that the downstream metabolism of ganglioside biosynthesis is fairly active and the potential need for gangliosides in the human body is likely substantial. As we search for new effective therapies for GM3D, approaches to reestablish endogenous ganglioside supplies in the affected individuals should be considered.

A glucogalactomannan isolated from Agaricus bisporus induces apoptosis in macrophages through the JNK/Bim/caspase 3 pathway.

Agaricus bisporus is one of the most important edible and medicinal mushrooms in the world. It has been well known that Agaricus bisporus has an immunoregulatory role, but its active ingredients have not been completely identified. In this study, a glucogalactomannan named TJ3 was isolated and purified from Agaricus bisporus. TJ3 (827 kDa) is composed of mannose, galactose, glucose and xylose in the ratio 28.26 : 27.82 : 20.88 : 9.87 mainly joined by ß-linkages. Functional analysis of TJ3 revealed that it effectively induced apoptosis in RAW 264.7 cells, a mouse macrophage cell line. Cell apoptosis was determined by an Annexin V/PI staining assay. After treatment with TJ3 (2 µg mL-1) for 16 h, apoptosis was observed in 34% of the Raw cells (9% in the non-treated control cells). TJ3 treatment remarkably increased the production of cleaved caspase-3, PARP and Bim, and decreased the level of Bcl-2 although no obvious change in the level of Bax was observed. Interestingly, further elucidation of the molecular mechanism underlying the role of TJ3 in the induction of apoptosis showed that TJ3 activated the JNK signaling pathway through TLR4 and subsequently promoted the expression of Bim and activation of caspase-3. Our results demonstrate that TJ3 may be a novel active component in Agaricus bisporus responsible for its immunoregulatory role by the induction of macrophage apoptosis.

A galactomannoglucan derived from Agaricus brasiliensis: Purification, characterization and macrophage activation via MAPK and IκB/NFκB pathways.

In this study, a novel galactomannoglucan named as TJ2 was isolated from Agaricus brasiliensis with microwave extraction, macroporous resin, ion exchange resin and high resolution gel chromatography. TJ2 is composed of glucose, mannose and galactose in the ratio 99.2:0.2:0.6. Infrared spectra (IR), methylation analysis and nuclear magnetic resonance spectra indicated that TJ2 mainly contained a ß-(1→3) - linked glucopyranosyl backbone. Interestingly, TJ2 significantly promoted RAW264.7 cell proliferation, and was able to activate the cells to engulf E. coli. In addition, TJ2 induced the expression of Interleukin 1ß (IL-1ß), Interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and cyclooxygenase-2 (Cox-2) in the cells. TJ2 also promoted the production of nitric oxide (NO) by inducing the expression of inducible nitric oxide synthase (iNOS). Moreover, TJ2 is a potent inducer in activating the mitogen-activated protein kinase (MAPK) and inhibitor of nuclear factor-kappa B (IκB)/nuclear factor-kappa B (NFκB) pathways.

Quantification of monosialogangliosides in human plasma through chemical derivatization for signal enhancement in LC-ESI-MS.

Gangliosides are found in abundance in the central nervous system of vertebrates. Their metabolic disruption and dysfunction are associated with various neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. In order to improve our understanding of the etiology of these diseases, analytical ganglioside assays with sufficient specificity and sensitivity in relevant biological matrices are required. In the present work we have developed and validated a reverse-phase ultra-performance liquid chromatography (UPLC)/tandem mass spectrometry (MS) method for determining monosialogangliosides GM1, GM2, and GM3 present in human plasma. Compared with our previous method, this method enhanced, by 15 fold, MS responses of the analytes by employing 2-(2-Pyridilamino)-ethylamine (PAEA) & 4-(4, 6-Dimethoxy-1, 3, 5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM)-based derivatization. The analytes and internal standards were derivatized with PAEA&DMTMM after extraction from plasma using a protein precipitation procedure. They were then purified using liquid-liquid partitioning. When the samples were then analyzed by UPLC-MS/MS with a multiple reaction monitoring (MRM) mode, we achieved superior sensitivity and specificity. This method was evaluated for extraction recovery, calibration linearity, precision, accuracy, and lower limit of quantification (LLOQ). The validated method was successfully applied to monitor monosialoganglioside levels in the plasma from patients with GM3 synthase deficiency. With significantly increased sensitivity, we have, for the first time, detected a significant amount of GM3 in the affected patients.

A new liquid chromatography/tandem mass spectrometry method for quantification of gangliosides in human plasma.

Gangliosides are a family of glycosphingolipids characterized by mono- or polysialic acid-containing oligosaccharides linked through 1,3- and 1,4-ß glycosidic bonds with subtle differences in structure that are abundantly present in the central nervous systems of many living organisms. Their cellular surface expression and physiological malfunction are believed to be pathologically implicated in considerable neurological disorders, including Alzheimer and Parkinson diseases. Recently, studies have tentatively elucidated that mental retardation or physical stagnation deteriorates as the physiological profile of gangliosides becomes progressively and distinctively abnormal during the development of these typical neurodegenerative syndromes. In this work, a reverse-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay using standard addition calibration for determination of GM2, GM3, GD2, and GD3 in human plasma has been developed and validated. The analytes and internal standard were extracted from human plasma using a simple protein precipitation procedure. Then the samples were analyzed by reverse-phase ultra-performance liquid chromatography (UPLC)/MS/MS interfaced to mass spectrometry with electrospray ionization using a multiple reaction monitoring mode to obtain superior sensitivity and specificity. This assay was validated for extraction recovery, calibration linearity, precision, and accuracy. Our quick and sensitive method can be applied to monitor ganglioside levels in plasma from normal people and neurodegenerative patients.