Spatial proteomics of Onchocerca volvulus with pleomorphic neoplasms shows local and systemic dysregulation of protein expression.

Onchocerca volvulus is the agent of onchocerciasis (river blindness) and targeted by WHO for elimination though mass drug administration with ivermectin. A small percentage of adult worms develop pleomorphic neoplasms (PN) that are positively associated with the frequency of ivermectin treatment. Worms with PN have a lower life expectancy and a better understanding about the proteins expressed in PN, and how PN affect protein expression in different tissues could help to elucidate the mechanisms of macrofilaricidal activity of ivermectin. Within a clinical trial of drug combinations that included ivermectin, we detected 24 (5.6%) O. volvulus females with PN by histology of paraffin embedded nodules. To assess the protein inventory of the neoplasms and to identify proteins that may be associated with tumor development, we used laser capture microdissection and highly sensitive mass spectrometry analysis. Neoplasm tissue from three female worms was analyzed, and compared to normal tissues from the body wall, uterus and intestine from the same worms, and to tissues from three females without PN. The healthy females showed all intact embryogenesis. In PN worms, 151 proteins were detected in the body wall, 215 proteins in the intestine, 47 proteins in the uterus and 1,577 proteins in the neoplasms. Only the uterus of one PN female with some stretched intrauterine microfilariae had an elevated number of proteins (601) detectable, while in the uteri of the healthy females 1,710 proteins were detected. Even in tissues that were not directly affected by PN (intestine, body wall), fewer proteins were detected compared to the corresponding tissue of the healthy controls. Immunolocalization of the calcium binding protein OvDig-1 (OVOC8391) confirmed the detection in PN by mass spectrometry. In conclusion we identified proteins that are potentially linked to the development of PN, and systemic dysregulation of protein expression may contribute to worm mortality. Author summary: Onchocerca volvulus , the causative agent of onchocerciasis (river blindness), is targeted for elimination by WHO. The primary strategy involves mass administration of ivermectin. A small proportion of adult female worms develop pleomorphic neoplasms (PN). Here, we used laser capture microdissection and highly sensitive mass spectrometry analysis to determine the protein inventory of PN to identify proteins that may be associated with tumor development. Neoplasm tissue from female worms was analyzed, and compared to normal tissue from the body wall, uterus and intestine from the same worms, and to tissues from females without PN. When compared, PN and healthy control (HC) worms display a different set of proteins, the PN tissue being the one with the highest number of proteins (1,390). From these, 594 were not present in any HC worm tissue. Despite the large number of proteins identified in PN tissue, their low abundance suggests also in PN dysregulation of protein expression. Immunolocalization of a calcium binding protein detected in PN confirmed the mass spectrometry results. In conclusion, we have developed a system to analyze the proteome of O. volvulus from nodule sections and identified proteins that are potentially linked to the development of PN and may contribute to worm mortality.

Enhanced Staphylococcus aureus protection by uncoupling of the α-toxin-ADAM10 interaction during murine neonatal vaccination.

Staphylococcus aureus remains a leading global cause of bacterial infection-associated mortality and has eluded prior vaccine development efforts. S. aureus α-toxin (Hla) is an essential virulence factor in disease, impairing the T cell response to infection. The anti-Hla antibody response is a correlate of human protective immunity. Here we observe that this response is limited early in human life and design a vaccine strategy to elicit immune protection against Hla in a neonatal mice. By targeted disruption of the interaction of Hla with its receptor ADAM10, we identify a vaccine antigen (HlaH35L/R66C/E70C, HlaHRE) that elicits an ~100-fold increase in the neutralizing anti-Hla response. Immunization with HlaHRE enhances the T follicular helper (TFH) cell response to S. aureus infection, correlating with the magnitude of the neutralizing anti-toxin response and disease protection. Furthermore, maternal HlaHRE immunization confers protection to offspring. Together, these findings illuminate a path for S. aureus vaccine development at the maternal-infant interface.

Experimental and Computational Evaluation of Lipidomic In-Source Fragmentation as a Result of Postionization with Matrix-Assisted Laser Desorption/Ionization.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can provide spatially resolved molecular information about a sample. Recently, a postionization approach (MALDI-2) has been commercially integrated with MALDI-MSI, allowing for bettered sensitivity and consequent improved spatial resolution. While advantages of MALDI-2 have previously been established, we demonstrate here statistically increased in-source fragmentation (ISF) results from postionization with a commercial instrument. Via lipid standard analyses, known MALDI ISF pathways (e.g., loss of trimethylamine) were statistically increased in MALDI-2 compared to MALDI-1 (65-172% increase in fragmentation). Gas phase molecular modeling with density functional theory estimated that the most-weighted virtual orbitals to excite within lipids involve ester and phosphate bonds. Protonated lipid excitation energies are furthermore red-shifted compared to those of other adduct types [e.g., 254 nm for protonated PC(16:0/18:1)] and approach the MALDI-2 laser energy (266 nm). Analysis of rat brain homogenate detected statistically more positive-ion mode peaks with MALDI-2 (1090) than that with MALDI-1 (719), where Kernel density estimations showed that the majority of this enhancement occurs with low m/z ions (i.e., m/z 75-500). Taken together with the lipid standard data, these observations may indicate ISF due to postionization. While artifact contributions from matrix blanks were also noted, both experimental and computational data sets suggest that the overall extent of ISF is statistically increased in MALDI-2 compared to MALDI-1.

The Impact of Milk on Gut Permeability, Fecal 16S rRNA Gene Microbiota Profiling, and Fecal Metabolomics in Children with Moderate Malnutrition in Sierra Leone: A Double-Blind, Randomized Controlled Trial.

BACKGROUND: Bovine milk is a beneficial ingredient in teh treatment of malnutrition. OBJECTIVES: Our objectives were to determine the effect of dietary milk protein and milk carbohydrate on the intestinal permeability, fecal 16S rRNA gene configuration, and fecal metabolomics of children with moderate malnutrition. METHODS: This was a randomized, double-blind, controlled trial among 413 children with wasting in rural Sierra Leone who received 1 of the following 4 supplementary foods, which differed in sources of protein and carbohydrate: milk protein and milk carbohydrate (MPMC), milk protein and vegetable carbohydrate (MPVC), vegetable protein and milk carbohydrate (VPMC), or a control group consuming entirely vegetable-based food (VPVC). After 4 wk, urine and stool were collected from participants enrolled with mid-upper arm circumference of <12.1 cm. Urine was analyzed for lactulose excretion (%L). Stool samples were subjected to both 16S rRNA gene analysis to assess ß-diversity and untargeted metabolomic abundance. RESULTS: Among the 386 children who completed permeability testing, the mean difference (95% CI) in %L excretion as compared with VPVC was 0.01 (-0.05, 0.07) for MPMC, 0.05 (-0.01, 0.11) for MPVC, and 0.01 (-0.05, 0.07) for VPMC. Of the 374 children who provided a stool sample that was analyzed, the ß-diversity among bacterial taxa was similar between dietary groups (P > 0.05 for all comparisons). No significant differences between dietary groups were seen among the 20 most abundant bacterial taxa. Among the 5769 unique metabolomic features identified, greater flavonoid levels in VPVC were seen. CONCLUSIONS: Abnormal intestinal permeability do not improve with 4 wk of supplementary feeding. Fecal rRNA do not differ with consumption of different diets. This trial was registered at clinicaltrials.gov as NCT04216043.

Evolution of the Umbilical Cord Blood Proteome Across Gestational Development.

Neonatal health is dependent on early risk stratification, diagnosis, and timely management of potentially devastating conditions, particularly in the setting of prematurity. Many of these conditions are poorly predicted in real-time by clinical data and current diagnostics. Umbilical cord blood may represent a novel source of molecular signatures that provides a window into the state of the fetus at birth. In this study, we comprehensively characterized the cord blood proteome of infants born between 24 to 42 weeks using untargeted mass spectrometry and functional enrichment analysis. We determined that the cord blood proteome at birth varies significantly across gestational development. Proteins that function in structural development and growth (e.g., extracellular matrix organization, lipid particle remodeling, and blood vessel development) are more abundant earlier in gestation. In later gestations, proteins with increased abundance are in immune response and inflammatory pathways, including complements and calcium-binding proteins. Furthermore, these data contribute to the knowledge of the physiologic state of neonates across gestational age, which is crucial to understand as we strive to best support postnatal development in preterm infants, determine mechanisms of pathology causing adverse health outcomes, and develop cord blood biomarkers to help tailor our diagnosis and therapeutics for critical neonatal conditions.

Oxytocin-induced birth causes sex-specific behavioral and brain connectivity changes in developing rat offspring.

Despite six decades of the use of exogenous oxytocin for management of labor, little is known about its effects on the developing brain. Motivated by controversial reports suggesting a link between oxytocin use during labor and autism spectrum disorders (ASDs), we employed our recently validated rat model for labor induction with oxytocin to address this important concern. Using a combination of molecular biological, behavioral, and neuroimaging assays, we show that induced birth with oxytocin leads to sex-specific disruption of oxytocinergic signaling in the developing brain, decreased communicative ability of pups, reduced empathy-like behaviors especially in male offspring, and widespread sex-dependent changes in functional cortical connectivity. Contrary to our hypothesis, social behavior, typically impaired in ASDs, was largely preserved. Collectively, our foundational studies provide nuanced insights into the neurodevelopmental impact of birth induction with oxytocin and set the stage for mechanistic investigations in animal models and prospective longitudinal clinical studies.

Development of a [89Zr]Zr-labeled Human Antibody using a Novel Phage-displayed Human scFv Library.

PURPOSE: Tax-interacting protein 1 (TIP1) is a cancer-specific radiation-inducible cell surface antigen that plays a role in cancer progression and resistance to therapy. This study aimed to develop a novel anti-TIP1 human antibody for noninvasive PET imaging in patients with cancer. EXPERIMENTAL DESIGN: A phage-displayed single-chain variable fragment (scFv) library was created from healthy donors' blood. High-affinity anti-TIP1 scFvs were selected from the library and engineered to human IgG1. Purified Abs were characterized by size exclusion chromatography high-performance liquid chromatography (SEC-HPLC), native mass spectrometry (native MS), ELISA, BIAcore, and flow cytometry. The labeling of positron emitter [89Zr]Zr to the lead Ab, L111, was optimized using deferoxamine (DFO) chelator. The stability of [89Zr]Zr-DFO-L111 was assessed in human serum. Small animal PET studies were performed in lung cancer tumor models (A549 and H460). RESULTS: We obtained 95% pure L111 by SEC-HPLC. Native MS confirmed the intact mass and glycosylation pattern of L111. Conjugation of three molar equivalents of DFO led to the optimal DFO-to-L111 ratio of 1.05. Radiochemical purity of 99.9% and specific activity of 0.37 MBq/µg was obtained for [89Zr]Zr-DFO-L111. [89Zr]Zr-DFO-L111 was stable in human serum over 7 days. The immunoreactive fraction in cell surface binding studies was 96%. In PET, preinjection with 4 mg/kg cold L111 before [89Zr]Zr-DFO-L111 (7.4 MBq; 20 µg) significantly (P < 0.01) enhanced the tumor-to-muscle standard uptake values (SUVmax) ratios on day 5 compared with day 2 postinjection. CONCLUSIONS: L111 Ab targets lung cancer cells in vitro and in vivo. [89Zr]Zr-DFO-L111 is a human antibody that will be evaluated in the first in-human study of safety and PET imaging.

Microglial REV-ERBα regulates inflammation and lipid droplet formation to drive tauopathy in male mice.

Alzheimer's disease, the most common age-related neurodegenerative disease, is characterized by tau aggregation and associated with disrupted circadian rhythms and dampened clock gene expression. REV-ERBα is a core circadian clock protein which also serves as a nuclear receptor and transcriptional repressor involved in lipid metabolism and macrophage function. Global REV-ERBα deletion has been shown to promote microglial activation and mitigate amyloid plaque formation. However, the cell-autonomous effects of microglial REV-ERBα in healthy brain and in tauopathy are unexplored. Here, we show that microglial REV-ERBα deletion enhances inflammatory signaling, disrupts lipid metabolism, and causes lipid droplet (LD) accumulation specifically in male microglia. These events impair microglial tau phagocytosis, which can be partially rescued by blockage of LD formation. In vivo, microglial REV-ERBα deletion exacerbates tau aggregation and neuroinflammation in two mouse tauopathy models, specifically in male mice. These data demonstrate the importance of microglial lipid droplets in tau accumulation and reveal REV-ERBα as a therapeutically accessible, sex-dependent regulator of microglial inflammatory signaling, lipid metabolism, and tauopathy.

An rRNA fragment in extracellular vesicles secreted by human airway epithelial cells increases the fluoroquinolone sensitivity of P. aeruginosa.

Lung infections caused by antibiotic-resistant strains of Pseudomonas aeruginosa are difficult to eradicate in immunocompromised hosts such as those with cystic fibrosis. We previously demonstrated that extracellular vesicles (EVs) secreted by primary human airway epithelial cells (AECs) deliver microRNA let-7b-5p to P. aeruginosa to suppress biofilm formation and increase sensitivity to beta-lactam antibiotics. In this study, we show that EVs secreted by AECs transfer multiple distinct short RNA fragments to P. aeruginosa that are predicted to target the three subunits of the fluoroquinolone efflux pump MexHI-OpmD, thus increasing antibiotic sensitivity. Exposure of P. aeruginosa to EVs resulted in a significant reduction in the protein levels of MexH (-48%), MexI (-50%), and OpmD (-35%). Moreover, EVs reduced planktonic growth of P. aeruginosa in the presence of the fluoroquinolone antibiotic ciprofloxacin by 20%. A mexGHI-opmD deletion mutant of P. aeruginosa phenocopied this increased sensitivity to ciprofloxacin. Finally, we found that a fragment of an 18S ribosomal RNA (rRNA) external transcribed spacer that was transferred to P. aeruginosa by EVs reduced planktonic growth of P. aeruginosa in the presence of ciprofloxacin, reduced the minimum inhibitory concentration of P. aeruginosa for ciprofloxacin by over 50%, and significantly reduced protein levels of both MexH and OpmD. In conclusion, an rRNA fragment secreted by AECs in EVs that targets the fluoroquinolone efflux pump MexHI-OpmD downregulated these proteins and increased the ciprofloxacin sensitivity of P. aeruginosa. A combination of rRNA fragments and ciprofloxacin packaged in nanoparticles or EVs may benefit patients with ciprofloxacin-resistant P. aeruginosa infections.NEW & NOTEWORTHY Human RNA fragments transported in extracellular vesicles interfere with Pseudomonas aeruginosa drug efflux pumps. A combination of rRNA fragments and ciprofloxacin packaged in nanoparticles or EVs may benefit patients with antibiotic-resistant P. aeruginosa infections.

Calpain activity is negatively regulated by a KCTD7-Cullin-3 complex via non-degradative ubiquitination.

Calpains are a class of non-lysosomal cysteine proteases that exert their regulatory functions via limited proteolysis of their substrates. Similar to the lysosomal and proteasomal systems, calpain dysregulation is implicated in the pathogenesis of neurodegenerative disease and cancer. Despite intensive efforts placed on the identification of mechanisms that regulate calpains, however, calpain protein modifications that regulate calpain activity are incompletely understood. Here we show that calpains are regulated by KCTD7, a cytosolic protein of previously uncharacterized function whose pathogenic mutations result in epilepsy, progressive ataxia, and severe neurocognitive deterioration. We show that KCTD7 works in complex with Cullin-3 and Rbx1 to execute atypical, non-degradative ubiquitination of calpains at specific sites (K398 of calpain 1, and K280 and K674 of calpain 2). Experiments based on single-lysine mutants of ubiquitin determined that KCTD7 mediates ubiquitination of calpain 1 via K6-, K27-, K29-, and K63-linked chains, whereas it uses K6-mediated ubiquitination to modify calpain 2. Loss of KCTD7-mediated ubiquitination of calpains led to calpain hyperactivation, aberrant cleavage of downstream targets, and caspase-3 activation. CRISPR/Cas9-mediated knockout of Kctd7 in mice phenotypically recapitulated human KCTD7 deficiency and resulted in calpain hyperactivation, behavioral impairments, and neurodegeneration. These phenotypes were largely prevented by pharmacological inhibition of calpains, thus demonstrating a major role of calpain dysregulation in KCTD7-associated disease. Finally, we determined that Cullin-3-KCTD7 mediates ubiquitination of all ubiquitous calpains. These results unveil a novel mechanism and potential target to restrain calpain activity in human disease and shed light on the molecular pathogenesis of KCTD7-associated disease.

Oncogenic deubiquitination controls tyrosine kinase signaling and therapy response in acute lymphoblastic leukemia.

Dysregulation of kinase signaling pathways favors tumor cell survival and therapy resistance in cancer. Here, we reveal a posttranslational regulation of kinase signaling and nuclear receptor activity via deubiquitination in T cell acute lymphoblastic leukemia (T-ALL). We observed that the ubiquitin-specific protease 11 (USP11) is highly expressed and associates with poor prognosis in T-ALL. USP11 ablation inhibits leukemia progression in vivo, sparing normal hematopoiesis. USP11 forms a complex with USP7 to deubiquitinate the oncogenic lymphocyte cell-specific protein-tyrosine kinase (LCK) and enhance its activity. Impairment of LCK activity leads to increased glucocorticoid receptor (GR) expression and glucocorticoids sensitivity. Genetic knockout of USP7 improved the antileukemic efficacy of glucocorticoids in vivo. The transcriptional activation of GR target genes is orchestrated by the deubiquitinase activity and mediated via an increase in enhancer-promoter interaction intensity. Our data unveil how dysregulated deubiquitination controls leukemia survival and drug resistance, suggesting previously unidentified therapeutic combinations toward targeting leukemia.

Zur and zinc increase expression of E. coli ribosomal protein L31 through RNA-mediated repression of the repressor L31p.

Bacteria can adapt in response to numerous stress conditions. One such stress condition is zinc depletion. The zinc-sensing transcription factor Zur regulates the way numerous bacterial species respond to severe changes in zinc availability. Under zinc sufficient conditions, Zn-loaded Zur (Zn2-Zur) is well-known to repress transcription of genes encoding zinc uptake transporters and paralogues of a few ribosomal proteins. Here, we report the discovery and mechanistic basis for the ability of Zur to up-regulate expression of the ribosomal protein L31 in response to zinc in E. coli. Through genetic mutations and reporter gene assays, we find that Zur achieves the up-regulation of L31 through a double repression cascade by which Zur first represses the transcription of L31p, a zinc-lacking paralogue of L31, which in turn represses the translation of L31. Mutational analyses show that translational repression by L31p requires an RNA hairpin structure within the l31 mRNA and involves the N-terminus of the L31p protein. This work uncovers a new genetic network that allows bacteria to respond to host-induced nutrient limiting conditions through a sophisticated ribosomal protein switching mechanism.

Bulk IgG glycosylation predicts COVID-19 severity and vaccine antibody response.

Although vaccination efforts have expanded, there are still gaps in our understanding surrounding the immune response to SARS-CoV-2. Measuring IgG Fc glycosylation provides insight into an infected individual's inflammatory state, among other functions. We set out to interrogate bulk IgG glycosylation changes from SARS-CoV-2 infection and vaccination, using plasma from mild or hospitalized COVID-19 patients, and from vaccinated individuals. Inflammatory glycans are elevated in hospitalized COVID-19 patients and increase over time, while mild patients have anti-inflammatory glycans that increase over time, including increased sialic acid correlating with RBD antibody levels. Vaccinated individuals with low RBD antibody levels and low neutralization have the same IgG glycan traits as hospitalized COVID-19 patients. In addition, a small vaccinated cohort reveals a decrease in inflammatory glycans associated with peak IgG concentrations and neutralization. This report characterizes the bulk IgG glycome associated with COVID-19 severity and vaccine responsiveness and can help guide future studies into SARS-CoV-2 protective immunity.

Revving an Engine of Human Metabolism: Activity Enhancement of Triosephosphate Isomerase via Hemi-Phosphorylation.

Triosephosphate isomerase (TPI) performs the 5th step in glycolysis, operates near the limit of diffusion, and is involved in "moonlighting" functions. Its dimer was found singly phosphorylated at Ser20 (pSer20) in human cells, with this post-translational modification (PTM) showing context-dependent stoichiometry and loss under oxidative stress. We generated synthetic pSer20 proteoforms using cell-free protein synthesis that showed enhanced TPI activity by 4-fold relative to unmodified TPI. Molecular dynamics simulations show that the phosphorylation enables a channel to form that shuttles substrate into the active site. Refolding, kinetic, and crystallographic analyses of point mutants including S20E/G/Q indicate that hetero-dimerization and subunit asymmetry are key features of TPI. Moreover, characterization of an endogenous human TPI tetramer also implicates tetramerization in enzymatic regulation. S20 is highly conserved across eukaryotic TPI, yet most prokaryotes contain E/D at this site, suggesting that phosphorylation of human TPI evolved a new switch to optionally boost an already fast enzyme. Overall, complete characterization of TPI shows how endogenous proteoform discovery can prioritize functional versus bystander PTMs.

SPT6 functions in transcriptional pause/release via PAF1C recruitment.

It is unclear how various factors functioning in the transcriptional elongation by RNA polymerase II (RNA Pol II) cooperatively regulate pause/release and productive elongation in living cells. Using an acute protein-depletion approach, we report that SPT6 depletion results in the release of paused RNA Pol II into gene bodies through an impaired recruitment of PAF1C. Short genes demonstrate a release with increased mature transcripts, whereas long genes are released but fail to yield mature transcripts, due to a reduced processivity resulting from both SPT6 and PAF1C loss. Unexpectedly, SPT6 depletion causes an association of NELF with the elongating RNA Pol II on gene bodies, without any observed functional significance on transcriptional elongation pattern, arguing against a role for NELF in keeping RNA Pol II in the paused state. Furthermore, SPT6 depletion impairs heat-shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause/release through PAF1C recruitment.

C16orf72/HAPSTR1 is a molecular rheostat in an integrated network of stress response pathways.

All cells contain specialized signaling pathways that enable adaptation to specific molecular stressors. Yet, whether these pathways are centrally regulated in complex physiological stress states remains unclear. Using genome-scale fitness screening data, we quantified the stress phenotype of 739 cancer cell lines, each representing a unique combination of intrinsic tumor stresses. Integrating dependency and stress perturbation transcriptomic data, we illuminated a network of genes with vital functions spanning diverse stress contexts. Analyses for central regulators of this network nominated C16orf72/HAPSTR1, an evolutionarily ancient gene critical for the fitness of cells reliant on multiple stress response pathways. We found that HAPSTR1 plays a pleiotropic role in cellular stress signaling, functioning to titrate various specialized cell-autonomous and paracrine stress response programs. This function, while dispensable to unstressed cells and nematodes, is essential for resilience in the presence of stressors ranging from DNA damage to starvation and proteotoxicity. Mechanistically, diverse stresses induce HAPSTR1, which encodes a protein expressed as two equally abundant isoforms. Perfectly conserved residues in a domain shared between HAPSTR1 isoforms mediate oligomerization and binding to the ubiquitin ligase HUWE1. We show that HUWE1 is a required cofactor for HAPSTR1 to control stress signaling and that, in turn, HUWE1 feeds back to ubiquitinate and destabilize HAPSTR1. Altogether, we propose that HAPSTR1 is a central rheostat in a network of pathways responsible for cellular adaptability, the modulation of which may have broad utility in human disease.

SF3B1 homeostasis is critical for survival and therapeutic response in T cell leukemia.

The production of noncanonical mRNA transcripts is associated with cell transformation. Driven by our previous findings on the sensitivity of T cell acute lymphoblastic leukemia (T-ALL) cells to SF3B1 inhibitors, we identified that SF3B1 inhibition blocks T-ALL growth in vivo with no notable associated toxicity. We also revealed protein stabilization of the U2 complex component SF3B1 via deubiquitination. Our studies showed that SF3B1 inhibition perturbs exon skipping, leading to nonsense-mediated decay and diminished levels of DNA damage response-related transcripts, such as the serine/threonine kinase CHEK2, and impaired DNA damage response. We also identified that SF3B1 inhibition leads to a general decrease in R-loop formation. We further demonstrate that clinically used SF3B1 inhibitors synergize with CHEK2 inhibitors and chemotherapeutic drugs to block leukemia growth. Our study provides the proof of principle for posttranslational regulation of splicing components and associated roles and therapeutic implications for the U2 complex in T cell leukemia.

Next-Generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire.

Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS, a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including entire variable regions in Ig light and heavy chains. Ig-MS uses recent advances in protein mass spectrometry (MS) for multiparametric readout of antibodies, with new metrics like Ion Titer (IT) and Degree of Clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without sequencing of B cells. We applied Ig-MS to plasma from subjects with severe and mild COVID-19 and immunized subjects after two vaccine doses, using the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 as the bait for antibody capture. Importantly, we report a new data type for human serology, that could use other antigens of interest to gauge immune responses to vaccination, pathogens, or autoimmune disorders.

Synergistic PIM kinase and proteasome inhibition as a therapeutic strategy for MYC-overexpressing triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the breast cancer subtype with the poorest clinical outcome. The PIM family of kinases has emerged as a factor that is both overexpressed in TNBC and associated with poor outcomes. Preclinical data suggest that TNBC with an elevated MYC expression is sensitive to PIM inhibition. However, clinical observations indicate that the efficacy of PIM inhibitors as single agents may be limited, suggesting the need for combination therapies. Our screening effort identifies PIM and the 20S proteasome inhibition as the most synergistic combination. PIM inhibitors, when combined with proteasome inhibitors, induce significant antitumor effects, including abnormal accumulation of poly-ubiquitinated proteins, increased proteotoxic stress, and the inability of NRF1 to counter loss in proteasome activity. Thus, the identified combination could represent a rational combination therapy against MYC-overexpressing TNBC that is readily translatable to clinical investigations.