Intranasally administered human MSC-derived extracellular vesicles inhibit NLRP3-p38/MAPK signaling after TBI and prevent chronic brain dysfunction.

Traumatic brain injury (TBI) leads to lasting brain dysfunction with chronic neuroinflammation typified by nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 (NLRP3) inflammasome activation in microglia. This study probed whether a single intranasal (IN) administration of human mesenchymal stem cell-derived extracellular vesicles (hMSC-EVs) naturally enriched with activated microglia-modulating miRNAs can avert chronic adverse outcomes of TBI. Small RNA sequencing confirmed the enrichment of miRNAs capable of modulating activated microglia in hMSC-EV cargo. IN administration of hMSC-EVs into adult mice ninety minutes after the induction of a unilateral controlled cortical impact injury resulted in their incorporation into neurons and microglia in both injured and contralateral hemispheres. A single higher dose hMSC-EV treatment also inhibited NLRP3 inflammasome activation after TBI, evidenced by reduced NLRP3, apoptosis-associated speck-like protein containing a CARD, activated caspase-1, interleukin-1 beta, and IL-18 levels in the injured brain. Such inhibition in the acute phase of TBI endured in the chronic phase, which could also be gleaned from diminished NLRP3 inflammasome activation in microglia of TBI mice receiving hMSC-EVs. Proteomic analysis and validation revealed that higher dose hMSC-EV treatment thwarted the chronic activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway by IL-18, which decreased the release of proinflammatory cytokines. Inhibition of the chronic activation of NLRP3-p38/MAPK signaling after TBI also prevented long-term cognitive and mood impairments. Notably, the animals receiving higher doses of hMSC-EVs after TBI displayed better cognitive and mood function in all behavioral tests than animals receiving the vehicle after TBI. A lower dose of hMSC-EV treatment also partially improved cognitive and mood function. Thus, an optimal IN dose of hMSC-EVs naturally enriched with activated microglia-modulating miRNAs can inhibit the chronic activation of NLRP3-p38/MAPK signaling after TBI and prevent lasting brain dysfunction.

gpGrouper: A Peptide Grouping Algorithm for Gene-Centric Inference and Quantitation of Bottom-Up Proteomics Data.

In quantitative mass spectrometry, the method by which peptides are grouped into proteins can have dramatic effects on downstream analyses. Here we describe gpGrouper, an inference and quantitation algorithm that offers an alternative method for assignment of protein groups by gene locus and improves pseudo-absolute iBAQ quantitation by weighted distribution of shared peptide areas. We experimentally show that distributing shared peptide quantities based on unique peptide peak ratios improves quantitation accuracy compared with conventional winner-take-all scenarios. Furthermore, gpGrouper seamlessly handles two-species samples such as patient-derived xenografts (PDXs) without ignoring the host species or species-shared peptides. This is a critical capability for proper evaluation of proteomics data from PDX samples, where stromal infiltration varies across individual tumors. Finally, gpGrouper calculates peptide peak area (MS1) based expression estimates from multiplexed isobaric data, producing iBAQ results that are directly comparable across label-free, isotopic, and isobaric proteomics approaches.