Abrin and ricin are toxic proteins produced by plants. Both proteins are composed of two subunits, an A-chain and a B-chain. The A-chain is responsible for the enzymatic activity, which causes toxicity. The B-chain binds to glycoproteins on the cell surface to direct the A-chain to its target. Both toxins depurinate 28S rRNA, making it impossible to differentiate these toxins based on only their enzymatic activity. We developed an analytical workflow for both ricin and abrin using a single method and sample. We have developed a novel affinity enrichment technique based on the ability of the B-chain to bind a glycoprotein, asialofetuin. After the toxin is extracted with asialofetuin-coated magnetic beads, an RNA substrate is added. Then, depurination is detected by a benchtop matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometer to determine the presence or absence of an active toxin. Next, the beads are subjected to tryptic digest. Toxin fingerprinting is done on a benchtop MALDI-TOF MS. We validated the assay through sensitivity and specificity studies and determined the limit of detection for each toxin as nanogram level for enzymatic activity and μg level for toxin fingerprinting. We examined potential cross-reactivity from proteins that are near neighbors of the toxins and examined potential false results in the presence of white powders.

SARS-CoV-2 spike (S) proteins undergo extensive glycosylation, aiding in proper folding, enhancing stability, and evading host immune surveillance. In this study, we used mass spectrometric analysis to elucidate the N-glycosylation characteristics and disulfide bonding of recombinant spike proteins derived from the SARS-CoV-2 Omicron variant (B.1.1.529) in comparison with the D614G spike variant. Furthermore, we conducted microsecond-long molecular dynamics simulations on spike proteins to resolve how the different N-glycans impact spike conformational sampling in the two variants. Our findings reveal that the Omicron spike protein maintains an overall resemblance to the D614G spike variant in terms of site-specific glycan processing and disulfide bond formation. Nonetheless, alterations in glycans were observed at certain N-glycosylation sites. These changes, in synergy with mutations within the Omicron spike protein, result in increased surface accessibility of the macromolecule, including the ectodomain, receptor-binding domain, and N-terminal domain. Additionally, mutagenesis and pull-down assays reveal the role of glycosylation of a specific sequon (N149); furthermore, the correlation of MD simulation and HDX-MS identified several high-dynamic areas of the spike proteins. These insights contribute to our understanding of the interplay between structure and function, thereby advancing effective vaccination and therapeutic strategies.

Botulism is a paralytic disease due to the inhibition of acetylcholine exocytosis at the neuromuscular junction, which can be lethal if left untreated. Botulinum neurotoxins (BoNTs) are produced by some spore-forming Clostridium bacteria. The current confirmatory assay to test for BoNTs in clinical specimens is the gold-standard mouse bioassay. However, an Endopep-MS assay method has been developed to detect BoNTs in clinical samples using benchtop mass spectrometric detection. This work demonstrates the validation of the Endopep-MS method for clinical specimens with the intent of method distribution in public health laboratories. The Endopep-MS assay was validated by assessing the sensitivity, robustness, selectivity, specificity, and reproducibility. The limit of detection was found to be equivalent to or more sensitive than the mouse bioassay. Specificity studies determined no cross-reactivity between the different serotypes and no false positives from an exclusivity panel of culture supernatants of enteric disease organisms and non-toxigenic strains of Clostridium. Inter-serotype specificity testing with 19 BoNT subtypes was 100% concordant with the expected results, accurately determining the presence of the correct serotype and the absence of incorrect serotypes. Additionally, a panel of potential interfering substances was used to test selectivity. Finally, clinical studies included clinical specimen stability and reproducibility, which was found to be 99.9% from a multicenter evaluation study. The multicenter validation study also included a clinical validation study, which yielded a 99.4% correct determination rate. Use of the Endopep-MS method will improve the capacity and response time for laboratory confirmation of botulism in public health laboratories.

BACKGROUND: Lipid profiling is central for coronary artery disease (CAD) risk assessment. Nonadherence or unreported use of lipid-lowering drugs, particularly statins, can significantly complicate the association between lipid profile measures and CAD clinical outcomes. By combining medication history evaluation with statin analysis in plasma, we determined the effects of inaccurately reported statin use on lipid profile measures and their association with CAD risk. METHODS: We compared medication history of statin use with statin concentration measurements, by liquid chromatography-tandem mass spectrometry, in 690 participants undergoing coronary angiography (63 ± 11 years of age). Nominal logistic regression was employed to model CAD diagnosis with statin measurements, phenotypic, and lipid profile characteristics. RESULTS: Medication history of statin use was confirmed by statin assay for 81% of the patients. Surprisingly, statins were detected in 46% of patients without statin use records. Nonreported statin use was disproportionately higher among older participants. Stratifying samples by statin history resulted in underestimated LDL-lipid measures. Apolipoprotein B concentrations had a significant inverse CAD association, which became nonsignificant upon re-stratification using the statin assay data. CONCLUSIONS: Our study uncovered prominent discrepancies between medication records and actual statin use measured by mass spectrometry. We showed that inaccurate statin use assessments may lead to overestimation and underestimation of LDL levels in statin user and nonuser categories, exaggerating the reverse epidemiology association between LDL levels and CAD diagnosis. Combining medication history and quantitative statin assay data can significantly improve the design, analysis, and interpretation of clinical and epidemiological studies.

Rationale: Hydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide precise analysis of a protein's conformational dynamics across varied states, such as heat-denatured versus native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX-MS, but one challenge for complete sequence coverage is N-glycosylation sites. The deuteration of peptides post-translationally modified by asparagine-bound glycans (glycopeptides) has not always been identified in previous reports of HDX-MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein. Methods: We detected deuterated glycopeptides with a Tribrid Orbitrap Eclipse mass spectrometer performing data-dependent acquisition. An MS scan was used to identify precursor ions; if high-energy collision-induced dissociation MS/MS of the precursor indicated oxonium ions diagnostic for complex glycans, then electron transfer low-energy collision-induced dissociation MS/MS scans of the precursor identified the modified asparagine residue and the glycan's mass. As in traditional HDX-MS, the identified glycopeptides were then analyzed at the MS level in samples labeled with D2O. Results: We report HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric prefusion form, which has 22 predicted N-glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their average isotopic mass shifts from deuteration. Inclusion of the deuterated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural rearrangements. Conclusion: Inclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors. Published 2024. This article is a U.S. Government work and is in the public domain in the USA.

This manuscript describes the use of an analytical assay that combines transfection of mammalian cells and isotope dilution mass spectrometry (IDMS) for accurate quantification of antigen expression. Expired mRNA COVID-19 vaccine material was stored at 4 °C, room temperature (∼25 °C), and 56 °C over a period of 5 weeks. The same vaccine was also exposed to 5 freeze-thaw cycles. Every week, the spike protein antigenic expression in mammalian (BHK-21) cells was evaluated. Housekeeping proteins, β-actin and GAPDH, were simultaneously quantified to account for the variation in cell counts that occurs during maintenance and growth of cell cultures. Data show that vaccine stored at elevated temperatures results in reduced spike protein expression. Also, maintaining the vaccine in ultracold conditions or exposing the vaccine to freeze-thaw cycles had less effect on the vaccine's ability to produce the antigen in mammalian cells. We describe the use of IDMS as an antibody-free means to accurately quantify expressed protein from mammalian cells transfected with mRNA vaccine.

N-glycosylation plays an important role in the structure and function of membrane and secreted proteins. The spike protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is heavily glycosylated and the major target for developing vaccines, therapeutic drugs and diagnostic tests. The first major SARS-CoV-2 variant carries a D614G substitution in the spike (S-D614G) that has been associated with altered conformation, enhanced ACE2 binding, and increased infectivity and transmission. In this report, we used mass spectrometry techniques to characterize and compare the N-glycosylation of the wild type (S-614D) or variant (S-614G) SARS-CoV-2 spike glycoproteins prepared under identical conditions. The data showed that half of the N-glycosylation sequons changed their distribution of glycans in the S-614G variant. The S-614G variant showed a decrease in the relative abundance of complex-type glycans (up to 45%) and an increase in oligomannose glycans (up to 33%) on all altered sequons. These changes led to a reduction in the overall complexity of the total N-glycosylation profile. All the glycosylation sites with altered patterns were in the spike head while the glycosylation of three sites in the stalk remained unchanged between S-614G and S-614D proteins.Competing Interest StatementThe authors have declared no competing interest.

N-Glycosylation plays an important role in the structure and function of membrane and secreted proteins. Viral proteins used in cell entry are often extensively glycosylated to assist in protein folding, provide stability, and shield the virus from immune recognition by its host (described as a "glycan shield"). The SARS-CoV-2 spike protein (S) is a prime example, having 22 potential sites of N-glycosylation per protein protomer, as predicted from the primary sequence. In this report, we conducted mass spectrometric analysis of the N-glycosylation profiles of recombinant spike proteins derived from four common SARS-CoV-2 variants classified as Variant of Concern, including Alpha, Beta, Gamma, and Delta along with D614G variant spike as a control. Our data reveal that the amino acid substitutions and deletions between variants impact the abundance and type of glycans on glycosylation sites of the spike protein. Some of the N-glycosylation sequons in S show differences between SARS-CoV-2 variants in the distribution of glycan forms. In comparison with our previously reported site-specific glycan analysis on the S-D614G and its ancestral protein, glycan types on later variants showed high similarity on the site-specific glycan content to S-D614G. Additionally, we applied multiple digestion methods on each sample, and confirmed the results for individual glycosylation sites from different experiment conditions to improve the identification and quantification of glycopeptides. Detailed site-specific glycan analysis of a wide variety of SARS-CoV-2 variants provides useful information toward the understanding of the role of protein glycosylation on viral protein structure and function and development of effective vaccines and therapeutics.

The advent of mRNA vaccine technology has been vital in rapidly creating and manufacturing COVID-19 vaccines at an industrial scale. To continue to accelerate this leading vaccine technology, an accurate method is needed to quantify antigens produced by the transfection of cells with a mRNA vaccine product. This will allow monitoring of protein expression during mRNA vaccine development and provide information on how changes to vaccine components affects the expression of the desired antigen. Developing novel approaches that allow for high-throughput screening of vaccines to detect changes in antigen production in cell culture prior to in vivo studies could aid vaccine development. We have developed and optimized an isotope dilution mass spectrometry method to detect and quantify the spike protein expressed after transfection of baby hamster kidney cells with expired COVID-19 mRNA vaccines. Five peptides of the spike protein are simultaneously quantified and provide assurance that protein digestion in the region of the target peptides is complete since results between the five peptides had a relative standard deviation of less than 15 %. In addition, two housekeeping proteins, actin and GAPDH, are quantified in the same analytical run to account for any variation in cell growth within the experiment. IDMS allows a precise and accurate means to quantify protein expression by mammalian cells transfected with an mRNA vaccine.

Designing studies for lipid-metabolism-related biomarker discovery is challenging because of the high prevalence of various statin and fibrate usage for lipid-lowering therapies. When the statin and fibrate use is determined based on self-reports, patient adherence to the prescribed statin dose regimen remains unknown. A potentially more accurate way to verify a patient's medication adherence is by direct analytical measurements. Current analytical methods are prohibitive because of the limited panel of drugs per test and large sample volume requirement that is not available from archived samples. A 4-min-long method was developed for the detection of seven statins and three fibrates using 10 µL of plasma analyzed via reverse-phase liquid chromatography and tandem mass spectrometry. The method was applied to the analysis of 941 archived plasma samples collected from patients before cardiac catheterization. When statin use was self-reported, statins were detected in 78.6% of the samples. In the case of self-reported atorvastatin use, the agreement with detection was 90.2%. However, when no statin use was reported, 42.4% of the samples had detectable levels of statins, with a similar range of concentrations as the samples from the self-reported statin users. The method is highly applicable in population studies designed for biomarker discovery or diet and lifestyle intervention studies, where the accuracy of statin or fibrate use may strongly affect the statistical evaluation of the biomarker data.

Large epidemiological studies often require sample transportation and storage, presenting unique considerations when applying advanced lipidomics techniques. The goal of this study was to acquire lipidomics data on plasma and serum samples stored at potential preanalytical conditions (e.g., thawing, extracting, evaporating), systematically monitoring lipid species for a period of one month. Split aliquots of 10 plasma samples and 10 serum samples from healthy individuals were kept in three temperature-related environments: refrigerator, laboratory benchtop, or heated incubator. Samples were analyzed at six different time points over 28 days using a Bligh & Dyer lipid extraction protocol followed by direct infusion into a lipidomics platform using differential mobility with tandem mass spectrometry. The observed concentration changes over time were evaluated relative to method and inter-individual biological variability. In addition, to evaluate the effect of lipase enzyme levels on concentration changes during storage, we compared corresponding fasting and post-prandial plasma samples collected from 5 individuals. Based on our data, a series of low abundance free fatty acid (FFA), diacylglycerol (DAG), and cholesteryl ester (CE) species were identified as potential analytical markers for degradation. These FFA and DAG species are typically produced by endogenous lipases from numerous triacylglycerols (TAGs), and certain high abundance phosphatidylcholines (PCs). The low concentration CEs, which appeared to increase several fold, were likely mass-isobars from oxidation of other high concentration CEs. Although the concentration changes of the high abundant TAG, PC, and CE precursors remained within method variability, the concentration trends of FFA, DAG, and oxidized CE products should be systematically monitored over time to inform analysts about possible pre-analytical biases due to degradation in the study sample sets.

Inhalation anthrax has three clinical stages: early-prodromal, intermediate-progressive, and late-fulminant. We report the comprehensive characterization of anthrax toxins, including total protective antigen (PA), total lethal factor (LF), total edema factor (EF), and their toxin complexes, lethal toxin and edema toxin in plasma, during the course of inhalation anthrax in 23 cynomolgus macaques. The toxin kinetics were predominantly triphasic with an early rise (phase-1), a plateau/decline (phase-2), and a final rapid rise (phase-3). Eleven animals had shorter survival times, meanstandard deviation of 58.77.6 hours (fast progression), 11 animals had longer survival times, 11334.4 hours (slow progression), and one animal survived. Median (lower-upper quartile) LF levels at the end-of-phase-1 were significantly higher in animals with fast progression [138 (54.9-326) ng/mL], than in those with slow progression [23.8 (15.6-26.3) ng/mL] (p = 0.0002), and the survivor (11.1 ng/mL). The differences were also observed for other toxins and bacteremia. Animals with slow progression had an extended phase-2 plateau, with low variability of LF levels across all time points and animals. Characterization of phase-2 toxin levels defined upper thresholds; critical levels for exiting phase-2 and entering the critical phase-3, 342 ng/mL (PA), 35.8 ng/mL (LF), and 1.10 ng/mL (EF). The thresholds were exceeded earlier in animals with fast progression (38.57.4 hours) and later in animals with slow progression (78.715.2 hours). Once the threshold was passed, toxin levels rose rapidly in both groups to the terminal stage. The time from threshold to terminal was rapid and similar; 20.87.4 hours for fast and 19.97.5 hours for slow progression. The three toxemic phases were aligned with the three clinical stages of anthrax for fast and slow progression which showed that anthrax progression is toxin- rather than time-dependent. This first comprehensive evaluation of anthrax toxins provides new insights into disease progression.

Aberrations in lipid and lipoprotein metabolic pathways can lead to numerous diseases, including cardiovascular disease, diabetes, neurological disorders, and cancer. The integration of quantitative lipid and lipoprotein profiling of human plasma may provide a powerful approach to inform early disease diagnosis and prevention. In this study, we leveraged data-driven quantitative targeted lipidomics and proteomics to identify specific molecular changes associated with different metabolic risk categories, including hyperlipidemic, hypercholesterolemic, hypertriglyceridemic, hyperglycemic, and normolipidemic conditions. Based on the quantitative characterization of serum samples from 146 individuals, we have determined individual lipid species and proteins that were significantly up- or down-regulated relative to the normolipidemic group. Then, we established protein-lipid topological networks for each metabolic category and linked dysregulated proteins and lipids with defined metabolic pathways. To evaluate the differentiating power of integrated lipidomics and proteomics data, we have built an artificial neural network model that simultaneously and accurately categorized the samples from each metabolic risk category based on the determined lipidomics and proteomics profiles. Together, our findings provide new insights into molecular changes associated with metabolic risk conditions, suggest new condition-specific associations between apolipoproteins and lipids, and may inform new biomarker discovery in lipid metabolism-associated disorders.

Bacillus anthracis has traditionally been considered the etiologic agent of anthrax. However, anthrax-like illness has been documented in welders and other metal workers infected with Bacillus cereus group spp. harboring pXO1 virulence genes that produce anthrax toxins. We present 2 recent cases of severe pneumonia in welders with B. cereus group infections and discuss potential risk factors for infection and treatment options, including antitoxin.

We propose the hypothesis that small high-density lipoprotein (HDL) particles reduce the risk of Alzheimer's disease (AD) by virtue of their capacity to exchange lipids, affecting neuronal membrane composition and vascular and synaptic functions. Concentrations of small HDLs in cerebrospinal fluid (CSF) and plasma were measured in 180 individuals ≥60 years of age using ion mobility methodology. Small HDL concentrations in CSF were positively associated with performance in three domains of cognitive function independent of apolipoprotein E (APOE) ε4 status, age, sex, and years of education. Moreover, there was a significant correlation between levels of small HDLs in CSF and plasma. Further studies will be aimed at determining whether specific components of small HDL exchange across the blood, brain, and CSF barriers, and developing approaches to exploit small HDLs for therapeutic purposes.

N-glycosylation plays an important role in the structure and function of membrane and secreted proteins. The spike protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is heavily glycosylated and the major target for developing vaccines, therapeutic drugs and diagnostic tests. The first major SARS-CoV-2 variant carries a D614G substitution in the spike (S-D614G) that has been associated with altered conformation, enhanced ACE2 binding, and increased infectivity and transmission. In this report, we used mass spectrometry techniques to characterize and compare the N-glycosylation of the wild type (S-614D) or variant (S-614G) SARS-CoV-2 spike glycoproteins prepared under identical conditions. The data showed that half of the N-glycosylation sequons changed their distribution of glycans in the S-614G variant. The S-614G variant showed a decrease in the relative abundance of complex-type glycans (up to 45%) and an increase in oligomannose glycans (up to 33%) on all altered sequons. These changes led to a reduction in the overall complexity of the total N-glycosylation profile. All the glycosylation sites with altered patterns were in the spike head while the glycosylation of three sites in the stalk remained unchanged between S-614G and S-614D proteins.

Ricin is a toxic protein derived from the castor bean plant (Ricinus communis) and has potential for bioterrorism or criminal use. Therefore, sensitive and rapid analytical methods are needed for its confirmatory detection in environmental samples. Our laboratory previously reported on the development of a confirmatory method to detect ricin involving antibody capture of ricin followed by mass spectrometric detection of ricin's enzymatic activity and of tryptic fragments unique to ricin. Here, we describe a novel ricin capture method of magnetic beads coated with 4-aminophenyl-1-thiol-β-galactopyranoside, using ricin's lectin characteristics. The assay has been adapted for use on a simple, benchtop MALDI-TOF MS mass spectrometer common in clinical microbiology laboratories. Validation of the novel assay includes establishment of a limit of detection, and an examination of assay selectivity. The limit of detection of the enzymatic activity method is 8 ng/mL and 500 ng/mL for the confirmatory tryptic fragment assay. The assay is highly selective with no cross-reactivity from near neighbors and highly specific with a panel of 19 cultivars all testing positive. Additionally, there were no interferences found during testing of a panel of white powders. This allows for a confirmatory detection method for ricin in laboratories lacking expensive, sophisticated mass spectrometers.

The emergence and subsequent global outbreak of the novel coronavirus SARS-CoV-2 prompted our laboratory to launch efforts to develop methods for SARS-CoV-2 antigen detection and quantification. We present an isotope dilution mass spectrometry method (IDMS) for rapid and accurate quantification of the primary antigens, spike and nucleocapsid proteins. This IDMS method utilizes liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze sample tryptic digests for detection and quantification of selected conserved peptides of SARS-CoV-2 spike and nucleocapsid proteins. The IDMS method has the necessary attributes to be successfully utilized for accurate quantification in SARS-CoV-2 protein-based vaccines and as targets of rapid diagnostic tests. Absolute quantification was achieved by quantifying and averaging 5 peptides for spike protein (3 peptides in the S1 subunit and 2 peptides in the S2 subunit) and 4 peptides for nucleocapsid protein. The overall relative standard deviation of the method was 3.67% for spike protein and 5.11% for nucleocapsid protein. IDMS offers speed (5 h total analysis time), sensitivity (LOQ; 10 fmol/µL) and precision for quantification of SARS-CoV-2 spike and nucleocapsid proteins.

Clostridioides difficile BI/NAP1/ribotype 027 is an epidemic hypervirulent strain found worldwide, including in Latin America. We examined the genomes and exoproteomes of two multilocus sequence type (MLST) clade 2 C. difficile strains considered hypervirulent: ICC-45 (ribotype SLO231/UK[CE]821), isolated in Brazil, and NAP1/027/ST01 (LIBA5756), isolated during a 2010 outbreak in Costa Rica. C. difficile isolates were cultured and extracellular proteins were analyzed using high-performance liquid chromatography-tandem mass spectrometry. Genomic analysis revealed that these isolates shared most of the gene composition. Only 83 and 290 NAP1/027 genes were considered singletons in ICC-45 and NAP1/027, respectively. Exoproteome analysis revealed 197 proteins, of which 192 were similar in both strains. Only five proteins were exclusive to the ICC-45 strain. These proteins were involved with catalytic and binding functions and indirectly interacted with proteins related to pathogenicity. Most proteins, including TcdA, TcdB, flagellin subunit, and cell surface protein, were overrepresented in the ICC-45 strain; 14 proteins, including mature S-layer protein, were present in higher proportions in LIBA5756. Data are available via ProteomeXchange with identifier PXD026218. These data show close similarity between the genome and proteins in the supernatant of two strains with hypervirulent features isolated in Latin America and underscore the importance of epidemiological surveillance of the transmission and emergence of new strains.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic of coronavirus disease 2019 (COVID-19). The spike protein expressed on the surface of this virus is highly glycosylated and plays an essential role during the process of infection. We conducted a comprehensive mass spectrometric analysis of the N-glycosylation profiles of the SARS-CoV-2 spike proteins using signature ions-triggered electron-transfer/higher-energy collision dissociation (EThcD) mass spectrometry. The patterns of N-glycosylation within the recombinant ectodomain and S1 subunit of the SARS-CoV-2 spike protein were characterized using this approach. Significant variations were observed in the distribution of glycan types as well as the specific individual glycans on the modification sites of the ectodomain and subunit proteins. The relative abundance of sialylated glycans in the S1 subunit compared to the full-length protein could indicate differences in the global structure and function of these two species. In addition, we compared N-glycan profiles of the recombinant spike proteins produced from different expression systems, including human embryonic kidney (HEK 293) cells and Spodoptera frugiperda (SF9) insect cells. These results provide useful information for the study of the interactions of SARS-CoV-2 viral proteins and for the development of effective vaccines and therapeutics.

Avian influenza viruses, such as A(H5N1) and A(H7N9), are primary public health concerns due to their pandemic potential. Influenza vaccines represent the most effective response to this threat especially with timely provision. The current pandemic response timelines require a substantial period for strain-specific reference antigen and sera preparation for use with single-radial immunodiffusion (SRID), the accepted vaccine potency assay. To address this time lag, the isotope dilution mass spectrometry (IDMS) method was developed to quantify the absolute hemagglutinin (HA, the main influenza antigen) amount in the vaccine without the need for purified, inactivated, and calibrated virus reference antigens. However, an additional challenge in determining potency is to differentiate between vaccine antigens in their most potent form from other less potent, stressed antigen forms. The limited trypsin digestion (LTD) method has been developed and does not require strain-specific full-length reference antigens or antibodies; instead, stressed HA is selectively degraded, leaving the more potent form to be measured. LTD, followed by precipitation and IDMS, allows for efficient differentiation between potent and significantly less potent HA for vaccine release and potency testing across the vaccine's shelf life. In this study, we tested the LTD-IDMS assay on A(H5N1) vaccine material that had been stressed by low pH, heat, and multiple freeze-thaw cycles. The results showed that the LTD-IDMS method effectively quantified the potent HA in A(H5N1) vaccine material with results comparable to SRID. As such, it shows great promise to complement and potentially replace SRID in a pandemic when strain-specific reagents may not be readily available.

Mesocestoides corti (syn. vogae) is a useful model for developmental studies of platyhelminth parasites of the Cestoda class, such as Taenia spp. or Echinococcus spp. It has been used in studies to characterize cestode strobilation, i.e. the development of larvae into adult worms. So far, little is known about the initial molecular events involved in cestode strobilation and, therefore, we carried out a study to characterize newly synthesized (NS) proteins upon strobilation induction. An approach based on bioorthogonal noncanonical amino acid tagging and mass spectrometry was used to label, isolate, identify, and quantify NS proteins in the initial steps of M. corti strobilation. Overall, 121 NS proteins were detected exclusively after induction of strobilation, including proteins related to development pathways, such as insulin and notch signaling. Metabolic changes that take place in the transition from the larval stage to adult worm were noted in special NS protein subsets related to developmental processes, such as focal adhesion, cell leading edge, and maintenance of location. The data shed light on mechanisms underlying early steps of cestode strobilation and enabled identification of possible developmental markers. We also consider the use of developmental responsive proteins as potential drug targets for developing novel anthelmintics. BIOLOGICAL SIGNIFICANCE: Larval cestodiases are life-threatening parasitic diseases that affect both man and domestic animals worldwide. Cestode parasites present complex life cycles, in which they undergo major morphological and physiological changes in the transition from one life-stage to the next. One of these transitions occurs during cestode strobilation, when the mostly undifferentiated and non-segmented larval or pre-adult form develops into a fully segmented and sexually differentiated (strobilated) adult worm. Although the proteomes of bona fide larvae and strobialted adults have been previously characterized for a few cestode species, little is still known about the dynamic of protein synthesis during the early steps of cestode strobilation. Now, the assessment of newly synthesized (NS) proteins within the first 48 h of strobilation the model cestode M. corti allowed to shed light on molecular mechanisms that are triggered by strobilation induction. The functional analyses of this repertoire of over a hundred NS proteins pointed out to changes in metabolism and activation of classical developmental signaling pathways in early strobilation. Many of the identified NS proteins may become valuable cestode developmental markers and their involvement in vital processes make them also good candidate targets for novel anthelmintic drugs.

BACKGROUND: Inhalational anthrax is rare and clinical experience limited. Expert guidelines recommend treatment with combination antibiotics including protein synthesis-inhibitors to decrease toxin production and increase survival, although evidence is lacking. METHODS: Rhesus macaques exposed to an aerosol of Bacillus anthracis spores were treated with ciprofloxacin, clindamycin, or ciprofloxacin + clindamycin after becoming bacteremic. Circulating anthrax lethal factor and protective antigen were quantitated pretreatment and 1.5 and 12 hours after beginning antibiotics. RESULTS: In the clindamycin group, 8 of 11 (73%) survived demonstrating its efficacy for the first time in inhalational anthrax, compared to 9 of 9 (100%) with ciprofloxacin, and 8 of 11 (73%) with ciprofloxacin + clindamycin. These differences were not statistically significant. There were no significant differences between groups in lethal factor or protective antigen levels from pretreatment to 12 hours after starting antibiotics. Animals that died after clindamycin had a greater incidence of meningitis compared to those given ciprofloxacin or ciprofloxacin + clindamycin, but numbers of animals were very low and no definitive conclusion could be reached. CONCLUSION: Treatment of inhalational anthrax with clindamycin was as effective as ciprofloxacin in the nonhuman primate. Addition of clindamycin to ciprofloxacin did not enhance reduction of circulating toxin levels.

The poly-gamma-glutamic acid (PGA) capsule produced by Bacillus anthracis is composed entirely of D-isomer glutamic acid, whereas nonpathogenic Bacillus species produce mixed D-, L-isomer PGAs. To determine if B. anthracis PGA confers a pathogenic advantage over other PGAs, we compared the responses of human innate immune cells to B. anthracis PGA and PGAs from nonpathogenic B. subtilis subsp. chungkookjang and B. licheniformis Monocytes and immature dendritic cells (iDCs) responded differentially to the PGAs, with B. anthracis PGA being least stimulatory and B. licheniformis PGA most stimulatory. All three elicited IL-8 and IL-6 from monocytes, but B. subtilis PGA also elicited IL-10 and TNF-alpha, whereas B. licheniformis PGA elicited all those plus IL-1beta. Similarly, all three PGAs elicited IL-8 from iDCs, but B. subtilis PGA also elicited IL-6, and B. licheniformis PGA elicited those plus IL-12p70, IL-10, IL-1beta, and TNF-alpha. Only B. licheniformis PGA induced dendritic cell maturation. TLR assays also yielded differential results. B. subtilis PGA and B. licheniformis PGA both elicited more TLR2 signal than B. anthracis PGA, but only responses to B. subtilis PGA were affected by a TLR6 neutralizing Ab. B. licheniformis PGA elicited more TLR4 signal than B. anthracis PGA, whereas B. subtilis PGA elicited none. B. anthracis PGA persisted longer in high m.w. form in monocyte and iDC cultures than the other PGAs. Reducing the m.w. of B. anthracis PGA reduced monocytes' cytokine responses. We conclude that B. anthracis PGA is recognized less effectively by innate immune cells than PGAs from nonpathogenic Bacillus species, resulting in failure to induce a robust host response, which may contribute to anthrax pathogenesis.

BACKGROUND: The causative agents for the current national outbreak of electronic-cigarette, or vaping, product use-associated lung injury (EVALI) have not been established. Detection of toxicants in bronchoalveolar-lavage (BAL) fluid from patients with EVALI can provide direct information on exposure within the lung. METHODS: BAL fluids were collected from 51 patients with EVALI in 16 states and from 99 healthy participants who were part of an ongoing study of smoking involving nonsmokers, exclusive users of e-cigarettes or vaping products, and exclusive cigarette smokers that was initiated in 2015. Using the BAL fluid, we performed isotope dilution mass spectrometry to measure several priority toxicants: vitamin E acetate, plant oils, medium-chain triglyceride oil, coconut oil, petroleum distillates, and diluent terpenes. RESULTS: State and local health departments assigned EVALI case status as confirmed for 25 patients and as probable for 26 patients. Vitamin E acetate was identified in BAL fluid obtained from 48 of 51 case patients (94%) in 16 states but not in such fluid obtained from the healthy comparator group. No other priority toxicants were found in BAL fluid from the case patients or the comparator group, except for coconut oil and limonene, which were found in 1 patient each. Among the case patients for whom laboratory or epidemiologic data were available, 47 of 50 (94%) had detectable tetrahydrocannabinol (THC) or its metabolites in BAL fluid or had reported vaping THC products in the 90 days before the onset of illness. Nicotine or its metabolites were detected in 30 of 47 of the case patients (64%). CONCLUSIONS: Vitamin E acetate was associated with EVALI in a convenience sample of 51 patients in 16 states across the United States. (Funded by the National Cancer Institute and others.).

CDC, the Food and Drug Administration (FDA), state and local health departments, and multiple public health and clinical partners are investigating a national outbreak of e-cigarette, or vaping, product use-associated lung injury (EVALI). Based on data collected as of October 15, 2019, 86% of 867 EVALI patients reported using tetrahydrocannabinol (THC)-containing products in the 3 months preceding symptom onset (1). Analyses of THC-containing product samples by FDA and state public health laboratories have identified potentially harmful constituents in these products, such as vitamin E acetate, medium chain triglyceride oil (MCT oil), and other lipids (2,3) (personal communication, D.T. Heitkemper, FDA Forensic Chemistry Center, November 2019). Vitamin E acetate, in particular, might be used as an additive in the production of e-cigarette, or vaping, products; it also can be used as a thickening agent in THC products (4). Inhalation of vitamin E acetate might impair lung function (5-7).

Cystic hydatid disease (CHD) is a worldwide neglected zoonotic disease caused by Echinococcus granulosus. The parasite is well adapted to its host by producing protective molecules that modulate host immune response. An unexplored issue associated with the parasite's persistence in its host is how the organism can survive the oxidative stress resulting from parasite endogenous metabolism and host defenses. Here, we used hydrogen peroxide (H2O2) to induce oxidative stress in E. granulosus protoescoleces (PSCs) to identify molecular pathways and antioxidant responses during H2O2 exposure. Using proteomics, we identified 550 unique proteins; including 474 in H2O2-exposed PSCs (H-PSCs) samples and 515 in non-exposed PSCs (C-PSCs) samples. Larger amounts of antioxidant proteins, including GSTs and novel carbonyl detoxifying enzymes, such as aldo-keto reductase and carbonyl reductase, were detected after H2O2 exposure. Increased concentrations of caspase-3 and cathepsin-D proteases and components of the 26S proteasome were also detected in H-PSCs. Reduction of lamin-B and other caspase-substrate, such as filamin, in H-PSCs suggested that molecular events related to early apoptosis were also induced. We present data that describe proteins expressed in response to oxidative stress in a metazoan parasite, including novel antioxidant enzymes and targets with potential application to treatment and prevention of CHD.

Mycoplasma hyopneumoniae is the etiological agent of enzootic pneumonia (EP), a widespread disease that causes major economic losses to the pig industry. The swine host response plays an important role in the outcome of M. hyopneumoniae infections. The whole proteome of newborn pig trachea (NPTr) epithelial cells infected with the M. hyopneumoniae pathogenic strain 7448 was analyzed using an LC-MS/MS approach to shed light on intracellular processes triggered in response to the pathogen. Overall, 853 swine protein species were identified, 156 of which were differentially represented in response to M. hyopneumoniae 7448 infection in comparison with non-infected control cells. These differentially represented proteins were categorized by function. Fifty-seven of them were assigned to the immune system and/or response to stimulus functional subcategories. Comparative expression analysis of these immune-related proteins in NPTr cells infected with attenuated or non-pathogenic mycoplasmas (M. hyopneumoniae J strain and M. flocculare, respectively) revealed proteins whose abundance was altered only in response to the pathogenic M. hyopneumoniae 7448 strain. Among these proteins, calcium homeostasis and endoplasmic reticulum stress-related biomarkers were detected, providing evidence of molecular mechanisms that might lead to swine cell apoptosis.

Botulinum neurotoxins (BoNTs) are a family of protein toxins consisting of seven known serotypes (BoNT/A-BoNT/G) and multiple subtypes within the serotypes, and all of which cause the disease botulism-a disease of great public health concern. Accurate detection of BoNTs in human clinical samples is therefore an important public health goal. To achieve this goal, our laboratory developed a mass spectrometry-based assay detecting the presence of BoNT via its enzymatic activity on a peptide substrate. Recently, publications reported the use of new peptide substrates to detect BoNT/A and /B with improved results over other peptide substrates. However, the authors did not provide results of their peptide substrate on multiple subtypes of BoNT. In this work, we describe the results of testing the new substrates with multiple BoNT/A and /B subtypes and find that the substrates cannot detect many subtypes of BoNT/A and /B.