Extracellular vesicles from mouse trophoblast cells: Effects on neural progenitor cells and potential participants in the placenta-brain axis†.

The fetal brain of the mouse is thought to be dependent upon the placenta as a source of serotonin (5-hydroxytryptamine; 5-HT) and other factors. How factors reach the developing brain remains uncertain but are postulated here to be part of the cargo carried by placental extracellular vesicles (EV). We have analyzed the protein, catecholamine, and small RNA content of EV from mouse trophoblast stem cells (TSC) and TSC differentiated into parietal trophoblast giant cells (pTGC), potential primary purveyors of 5-HT. Current studies examined how exposure of mouse neural progenitor cells (NPC) to EV from either TSC or pTGC affect their transcriptome profiles. The EV from trophoblast cells contained relatively high amounts of 5-HT, as well as dopamine and norepinephrine, but there were no significant differences between EV derived from pTGC and from TSC. Content of miRNA and small nucleolar (sno)RNA, however, did differ according to EV source, and snoRNA were upregulated in EV from pTGC. The primary inferred targets of the microRNA (miRNA) from both pTGC and TSC were mRNA enriched in the fetal brain. NPC readily internalized EV, leading to changes in their transcriptome profiles. Transcripts regulated were mainly ones enriched in neural tissues. The transcripts in EV-treated NPC that demonstrated a likely complementarity with miRNA in EV were mainly up- rather than downregulated, with functions linked to neuronal processes. Our results are consistent with placenta-derived EV providing direct support for fetal brain development and being an integral part of the placenta-brain axis.

Effect of pH and protein composition on proteolysis of goat milk proteins by pepsin and pancreatin.

The roles of protein composition, pH and enzymes in goat milk protein hydrolysis is still unclear and the proteolysis of low abundant goat milk proteins has received limited attention. The aim of this study was to study the impact of protein composition and proteolytic conditions on goat milk protein hydrolysis in a simplified digestion model. Both whole milk and infant formula were hydrolyzed at pH 2 and 4, using pepsin as well as pepsin combined with pancreatin. Intact proteins were separated from digests using spin filters, followed by bottom-up proteomics of the separated proteins. Results show that under all conditions, caseins are hydrolyzed quickly. Goat casein hydrolysis in infant formula was slightly faster than in goat whole milk, possibly due to less casein coagulation during pepsin hydrolysis at both pH 2 and 4. Several low abundant immunoactive goat milk proteins, especially immunoglobulins, GLYCAM-1 and osteopontin, resisted proteolysis more than high abundant proteins, independent of the pH and enzyme used for hydrolysis. Fast hydrolysis of casein and slow hydrolysis of immunoactive proteins may indicate a good balance between protein utilization and protection of the infant by goat milk proteins.

Characterization of the extracellular vesicles, ultrastructural morphology, and intercellular interactions of multiple clinical isolates of the brain-eating amoeba, Naegleria fowleri.

Introduction: As global temperatures rise to unprecedented historic levels, so too do the latitudes of habitable niches for the pathogenic free-living amoeba, Naegleria fowleri. This opportunistic parasite causes a rare, but >97% fatal, neurological infection called primary amoebic meningoencephalitis. Despite its lethality, this parasite remains one of the most neglected and understudied parasitic protozoans. Methods: To better understand amoeboid intercellular communication, we elucidate the structure, proteome, and potential secretion mechanisms of amoeba-derived extracellular vesicles (EVs), which are membrane-bound communication apparatuses that relay messages and can be used as biomarkers for diagnostics in various diseases. Results and Discussion: Herein we propose that N. fowleri secretes EVs in clusters from the plasma membrane, from multivesicular bodies, and via beading of thin filaments extruding from the membrane. Uptake assays demonstrate that EVs are taken up by other amoebae and mammalian cells, and we observed a real-time increase in metabolic activity for mammalian cells exposed to EVs from amoebae. Proteomic analysis revealed >2,000 proteins within the N. fowleri-secreted EVs, providing targets for the development of diagnostics or therapeutics. Our work expands the knowledge of intercellular interactions among these amoebae and subsequently deepens the understanding of the mechanistic basis of PAM.

Multi-Omic, Histopathologic, and Clinicopathologic Effects of Once-Weekly Oral Rapamycin in a Naturally Occurring Feline Model of Hypertrophic Cardiomyopathy: A Pilot Study.

Hypertrophic cardiomyopathy (HCM) remains the single most common cardiomyopathy in cats, with a staggering prevalence as high as 15%. To date, little to no direct therapeutical intervention for HCM exists for veterinary patients. A previous study aimed to evaluate the effects of delayed-release (DR) rapamycin dosing in a client-owned population of subclinical, non-obstructive, HCM-affected cats and reported that the drug was well tolerated and resulted in beneficial LV remodeling. However, the precise effects of rapamycin in the hypertrophied myocardium remain unknown. Using a feline research colony with naturally occurring hereditary HCM (n = 9), we embarked on the first-ever pilot study to examine the tissue-, urine-, and plasma-level proteomic and tissue-level transcriptomic effects of an intermittent low dose (0.15 mg/kg) and high dose (0.30 mg/kg) of DR oral rapamycin once weekly. Rapamycin remained safe and well tolerated in cats receiving both doses for eight weeks. Following repeated weekly dosing, transcriptomic differences between the low- and high-dose groups support dose-responsive suppressive effects on myocardial hypertrophy and stimulatory effects on autophagy. Differences in the myocardial proteome between treated and control cats suggest potential anti-coagulant/-thrombotic, cellular remodeling, and metabolic effects of the drug. The results of this study closely recapitulate what is observed in the human literature, and the use of rapamycin in the clinical setting as the first therapeutic agent with disease-modifying effects on HCM remains promising. The results of this study establish the need for future validation efforts that investigate the fine-scale relationship between rapamycin treatment and the most compelling gene expression and protein abundance differences reported here.

Dermal thirdhand smoke exposure induces oxidative damage, initiates skin inflammatory markers, and adversely alters the human plasma proteome.

BACKGROUND: Thirdhand smoke (THS) exposure correlated with significant metabolism of carcinogenic chemicals and the potential to cause detrimental health effects. Human harm research of THS exposure is limited to one other study and overall, there is a general lack of knowledge of the human health responses to THS exposure. METHODS: This was a clinical investigation to evaluate the health effects of 3-h dermal THS exposure from urine and plasma. 10 healthy, non-smoking subjects were recruited for dermal exposure for 3 h exposed to clothing impregnated with filtered clean air or THS. Exposures to clean air or THS occurred 20-30 days apart. FINDINGS: In THS-exposed group, there was a significant elevation of urinary 8-OHdG, 8-isoprostane, protein carbonyls. The THS 3-h exposure identified proteomics pathways of inflammatory response (p=2.18 × 10-8), adhesion of blood cells (p=2.23 × 10-8), atherosclerosis (p=2.78 × 10-9), and lichen planus (p=1.77 × 10-8). Nine canonical pathways were significantly activated including leukocyte extravasation signaling (z-score=3.0), and production of nitric oxide and reactive oxygen in macrophages (z-score=2.1). The THS 22-h proteomics pathways revealed inflammation of organ (p=3.09 × 10-8), keratinization of the epidermis (p=4.0 × 10-7), plaque psoriasis (p=5.31 × 10-7), and dermatitis (p=6.0 × 10-7). Two activated canonical pathways were production of nitric oxide and reactive oxygen in macrophages (z-score=2.646), and IL-8 signaling (z-score=2.0). INTERPRETATION: This is a clinical study demonstrating that acute dermal exposure to THS mimics the harmful effects of cigarette smoking, alters the human plasma proteome, initiates mechanisms of skin inflammatory disease, and elevates urinary biomarkers of oxidative harm. FUNDING: Funding was provided by the Tobacco Related Disease Research Program (TRDRP) 24RT-0037 TRDRP, 24RT-0039 TRDRP, and 28PT-0081 TRDRP.

Identification of Endogenous Peptides in Nasal Swab Transport Media used in MALDI-TOF-MS Based COVID-19 Screening.

Mass spectrometry (MS) based diagnostic detection of 2019 novel coronavirus infectious disease (COVID-19) has been postulated to be a useful alternative to classical PCR based diagnostics. These MS based approaches have the potential to be both rapid and sensitive and can be done on-site without requiring a dedicated laboratory or depending on constrained supply chains (i.e., reagents and consumables). Matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) MS has a long and established history of microorganism detection and systemic disease assessment. Previously, we have shown that automated machine learning (ML) enhanced MALDI-TOF-MS screening of nasal swabs can be both sensitive and specific for COVID-19 detection. The underlying molecules responsible for this detection are generally unknown nor are they required for this automated ML platform to detect COVID-19. However, the identification of these molecules is important for understanding both the mechanism of detection and potentially the biology of the underlying infection. Here, we used nanoscale liquid chromatography tandem MS to identify endogenous peptides found in nasal swab saline transport media to identify peptides in the same the mass over charge (m/z) values observed by the MALDI-TOF-MS method. With our peptidomics workflow, we demonstrate that we can identify endogenous peptides and endogenous protease cut sites. Further, we show that SARS-CoV-2 viral peptides were not readily detected and are highly unlikely to be responsible for the accuracy of MALDI based SARS-CoV-2 diagnostics. Further analysis with more samples will be needed to validate our findings, but the methodology proves to be promising.

Glycopeptide Biomarkers in Serum Haptoglobin for Hepatocellular Carcinoma Detection in Patients with Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is rising in prevalence in the United States and is a growing cause of hepatocellular carcinomas (HCCs). Site-specific glycan heterogeneity on glycoproteins has been shown as a potential diagnostic biomarker for HCC. Herein, we have performed a comprehensive screening of site-specific N-glycopeptides in serum haptoglobin (Hp), a reporter molecule for aberrant glycosylation in HCC, to characterize glycopeptide markers for NASH-related HCCs. In total, 70 NASH patients (22 early HCC, 15 advanced HCC, and 33 cirrhosis cases) were analyzed, with Hp purified from 20 µL of serum in each patient, and 140 sets of mass spectrometry (MS) data were collected using liquid chromatography coupled with electron-transfer high-energy collisional dissociation tandem MS (LC-EThcD-MS/MS) for quantitative analysis on a novel software platform, Byos. Differential quantitation analysis revealed that five N-glycopeptides at sites N184 and N241 were significantly elevated during the progression from NASH cirrhosis to HCC (p < 0.05). Receiver operating characteristic (ROC) curve analysis demonstrated that the N-glycopeptides at sites N184 and N241 bearing a monofucosylated triantennary glycan A3G3F1S3 had the best diagnostic performance in detection of early NASH HCC, area under the curve (AUC) = 0.733 and 0.775, respectively, whereas α-fetoprotein (AFP) had an AUC of 0.692. When combined with AFP, the two panels improved the sensitivity for early NASH HCC from 59% (AFP alone) to 73% while maintaining a specificity of 70%, based on the optimal cutoff. Two-dimensional (2-D) scatter plots of the AFP value and N-glycopeptides showed that these N-glycopeptide markers detected 58% of AFP-negative HCC patients as distinct from cirrhosis. These site-specific N-glycopeptides could serve as potential markers for early detection of HCC in patients with NASH-related cirrhosis.

A monoclonal antibody specific for prophase phosphorylation of histone deacetylase 1: a readout for early mitotic cells.

Histone deacetylases (HDACs) are modification enzymes that regulate a plethora of biological processes. HDAC1, a crucial epigenetic modifier, is deregulated in cancer and subjected to a variety of post-translational modifications. Here, we describe the generation of a new monoclonal antibody that specifically recognizes a novel highly dynamic prophase phosphorylation of serine 406-HDAC1, providing a powerful tool for detecting early mitotic cells.

Electron induced dissociation of singly deprotonated peptides.

Dissociation of singly charged species is more challenging compared with that of multiply charged precursor ions because singly charged ions are generally more stable. In collision activated dissociation (CAD), singly charged ions also gain less kinetic energy in a fixed electric field compared with multiply charged species. Furthermore, ion-electron and ion-ion reactions that frequently provide complementary and more extensive fragmentation compared with CAD typically require multiply charged precursor ions. Here, we investigate electron induced dissociation (EID) of singly deprotonated peptides and compare the EID fragmentation patterns with those observed in negative ion mode CAD. Fragmentation induced upon electron irradiation and collisional activation is not specific and results in the formation of a wide range of product ions, including b-, y-, a-, x-, c-, and z-type ions. Characteristic amino acid side chain losses are detected in both techniques. However, differences are also observed between EID and CAD spectra of the same species, including formation of odd-electron species not seen in CAD, in EID. Furthermore, EID frequently results in more extensive fragmentation compared with CAD. For modified peptides, EID resulted in retention of sulfonation and phosphorylation, allowing localization of the modification site. The observed differences are likely due to both vibrational and electronic excitation in EID, whereas only the former process occurs in CAD.

Structural analysis of the RZZ complex reveals common ancestry with multisubunit vesicle tethering machinery.

The RZZ complex recruits dynein to kinetochores. We investigated structure, topology, and interactions of the RZZ subunits (ROD, ZWILCH, and ZW10) in vitro, in vivo, and in silico. We identify neuroblastoma-amplified gene (NAG), a ZW10 binder, as a ROD homolog. ROD and NAG contain an N-terminal beta propeller followed by an alpha solenoid, which is the architecture of certain nucleoporins and vesicle coat subunits, suggesting a distant evolutionary relationship. ZW10 binding to ROD and NAG is mutually exclusive. The resulting ZW10 complexes (RZZ and NRZ) respectively contain ZWILCH and RINT1 as additional subunits. The X-ray structure of ZWILCH, the first for an RZZ subunit, reveals a novel fold distinct from RINT1's. The evolutionarily conserved NRZ likely acts as a tethering complex for retrograde trafficking of COPI vesicles from the Golgi to the endoplasmic reticulum. The RZZ, limited to metazoans, probably evolved from the NRZ, exploiting the dynein-binding capacity of ZW10 to direct dynein to kinetochores.

Identification and verification of novel rodent postsynaptic density proteins.

The postsynaptic density (PSD) is a cellular structure specialized in receiving and transducing synaptic information. Here we describe the identification of 452 proteins isolated from biochemically purified PSD fractions of rat and mouse brains using nanoflow HPLC coupled to electrospray tandem mass spectrometry (LC-MS/MS). Fluorescence microscopy and Western blotting were used to verify that many of the novel proteins identified exhibit subcellular distributions consistent with those of PSD-localized proteins. In addition to identifying most previously described PSD components, we also detected proteins involved in signaling to the nucleus as well as regulators of ADP-ribosylation factor signaling, ubiquitination, RNA trafficking, and protein translation. These results suggest new mechanisms by which the PSD helps regulate synaptic strength and transmission.