Background/Objectives: Human milk is the optimal source of nutrition and protection against infection for infants. If breastfeeding is not possible, standard and hydrolyzed infant formulas (IF) are an alternative. Extensively hydrolyzed IFs (eHFs) contain bioactive peptides, but their activities have rarely been evaluated. The aim of this study was to characterize and compare the bioactive peptide profiles of different eHFs and standard IFs before and after in vitro digestion. Methods: Two forms, liquid and powder, of intact protein formula (iPF) and eHF were subjected to in vitro gastrointestinal digestion, mimicking a young infants gut (age 0-4 months) and an older infants gut (>6 months). Bioactive peptides of in vitro digested and undigested formulas were analysed with Liquid Chromatography-Mass Spectrometry (LC-MS). Results: In all samples, a variety of peptides with potential bioactive properties were found. Immuno-regulatory peptides, followed by antimicrobial and antioxidative peptides were most frequent, as were peptides promoting wound healing, increasing mucin secretion, regulating cholesterol metabolism, and preventing bacterial infection. Peptides typically found in yoghurt and colostrum were identified in some formula samples. Conclusions: The high amounts of bioactive peptides with various properties in eHFs and iPFs indicate a possible contribution to infection protection, healthy gut microbiomes, and immunological development of infants. eHFs showed similar compositions of bioactive peptides to iPFs, with intermittently increased peptide variety and quantity.

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.

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.

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.

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.

N-Myc is a key driver of neuroblastoma and neuroendocrine prostate cancer (NEPC). One potential way to circumvent the challenge of undruggable N-Myc is to target the protein homeostasis (proteostasis) system that maintains N-Myc levels. Here, we identify heat shock protein 70 (HSP70) as a top partner of N-Myc, which binds a conserved "SELILKR" motif and prevents the access of E3 ubiquitin ligase, STIP1 homology and U-box containing protein 1 (STUB1), possibly through steric hindrance. When HSP70's dwell time on N-Myc is increased by treatment with the HSP70 allosteric inhibitor, STUB1 is in close proximity with N-Myc and becomes functional to promote N-Myc ubiquitination on the K416 and K419 sites and forms polyubiquitination chains linked by the K11 and K63 sites. Notably, HSP70 inhibition significantly suppressed NEPC tumor growth, increased the efficacy of aurora kinase A (AURKA) inhibitors, and limited the expression of neuroendocrine-related pathways.