Non-Necroptotic Roles of MLKL in Diet-Induced Obesity, Liver Pathology, and Insulin Sensitivity: Insights from a High-Fat, High-Fructose, High-Cholesterol Diet Mouse Model.

Chronic inflammation is a key player in metabolic dysfunction-associated fatty liver disease (MAFLD) progression. Necroptosis, an inflammatory cell death pathway, is elevated in MAFLD patients and mouse models, yet its role is unclear due to the diverse mouse models and inhibition strategies. In our study, we inhibited necroptosis by targeting mixed lineage kinase domain-like pseudokinase (MLKL), the terminal effector of necroptosis, in a high-fat, high-fructose, high-cholesterol (HFHFrHC) mouse model of diet-induced MAFLD. Despite the HFHFrHC diet upregulating MLKL (2.5-fold), WT mice livers showed no increase in necroptosis markers or associated proinflammatory cytokines. Surprisingly, Mlkl-/- mice experienced exacerbated liver inflammation without protection from diet-induced liver damage, steatosis, or fibrosis. In contrast, Mlkl+/- mice showed a significant reduction in these parameters that was associated with elevated Pparα and Pparγ levels. Both Mlkl-/- and Mlkl+/- mice on the HFHFrHC diet resisted diet-induced obesity, attributed to the increased beiging, enhanced oxygen consumption, and energy expenditure due to adipose tissue, and exhibited improved insulin sensitivity. These findings highlight the tissue-specific effects of MLKL on the liver and adipose tissue, and they suggest a dose-dependent effect of MLKL on liver pathology.

Non-Necroptotic Roles of MLKL in Diet-Induced Obesity, Liver Pathology, and Insulin Sensitivity: Insights from a High Fat, High Fructose, High Cholesterol Diet Mouse Model.

Chronic inflammation is a key player in metabolic dysfunction-associated fatty liver disease (MAFLD) progression. Necroptosis, an inflammatory cell death pathway, is elevated in MAFLD patients and mouse models, yet its role is unclear due to diverse mouse models and inhibition strategies. In our study, we inhibited necroptosis by targeting mixed lineage kinase domain like pseudokinase (MLKL), the terminal effector of necroptosis, in a high-fat, high-fructose, high-cholesterol (HFHFrHC) mouse model of diet-induced MAFLD mouse model. Despite HFHFrHC diet upregulating MLKL (2.5-fold), WT mice livers showed no increase in necroptosis markers or associated proinflammatory cytokines. Surprisingly, Mlkl -/- mice experienced exacerbated liver inflammation without protection from diet-induced liver damage, steatosis, or fibrosis. In contrast, Mlkl +/- mice showed significant reduction in these parameters that was associated with elevated Pparα and Pparγ levels. Both Mlkl -/- and Mlkl +/- mice on HFHFrHC diet resisted diet-induced obesity, attributed to increased beiging, enhanced oxygen consumption and energy expenditure due to adipose tissue, and exhibited improved insulin sensitivity. These findings highlight the tissue specific effects of MLKL on the liver and adipose tissue, and suggest a dose-dependent effect of MLKL on liver pathology.

Dietary Nitrate from Plant Foods: A Conditionally Essential Nutrient for Cardiovascular Health.

Under specific conditions, such as catabolic stress or systemic inflammation, endogenous nutrient production becomes insufficient and exogenous supplementation (for example, through dietary intake) is required. Herein, we propose consideration of a dietary nitrate from plant foods as a conditionally essential nutrient for cardiovascular health based on its role in nitric oxide homeostasis. Nitrate derived from plant foods may function as a conditionally essential nutrient, whereas nitrate obtained from other dietary sources, such as drinking water and cured/processed meats, warrants separate consideration because of the associated health risks. We have surveyed the literature and summarized epidemiological evidence regarding the effect of dietary nitrate on cardiovascular disease and risk factors. Meta-analyses and population-based observational studies have consistently demonstrated an inverse association of dietary nitrate with blood pressure and cardiovascular disease outcomes. Considering the available evidence, we suggest 2 different approaches to providing dietary guidance on nitrate from plant-based dietary sources as a nutrient: the Dietary Reference Intakes developed by the National Academies of Sciences, Engineering, and Medicine, and the dietary guidelines evaluated by the Academy of Nutrition and Dietetics. Ultimately, this proposal underscores the need for food-based dietary guidelines to capture the complex and context-dependent relationships between nutrients, particularly dietary nitrate, and health.

A Genetically Heterogeneous Rat Model with Divergent Mitochondrial Genomes.

We generated a genetically heterogenous rat model by a 4-way cross strategy using 4 inbred strains (Brown Norway [BN], Fischer 344 [F344], Lewis [LEW], and Wistar Kyoto [KY]) to provide investigators with a highly genetically diverse rat model from commercially available inbred rats. We made reciprocal crosses between males and females from the 2 F1 hybrids to generate genetically heterogeneous rats with mitochondrial genomes from either the BN (OKC-HETB, a.k.a "B" genotype) or WKY (OKC-HETW a.k.a "W" genotype) parental strains. These two mitochondrial genomes differ at 94 nucleotides, more akin to human mitochondrial genome diversity than that available in classical laboratory mouse strains. Body weights of the B and W genotypes were similar. However, mitochondrial genotype antagonistically affected grip strength and treadmill endurance in females only. In addition, mitochondrial genotype significantly affected multiple responses to a high-fat diet (HFD) and treatment with 17α-estradiol. Contrary to findings in mice in which males only are affected by 17α-estradiol supplementation, female rats fed a HFD beneficially responded to 17α-estradiol treatment as evidenced by declines in body mass, adiposity, and liver mass. Male rats, by contrast, differed in a mitochondrial genotype-specific manner, with only B males responding to 17α-estradiol treatment. Mitochondrial genotype and sex differences were also observed in features of brain-specific antioxidant response to a HFD and 17α-estradiol as shown by hippocampal levels of Sod2 acetylation, JNK, and FoxO3a. These results emphasize the importance of mitochondrial genotype in assessing responses to putative interventions in aging processes.

Development of a non-radiolabeled glucosyltransferase activity assay for C. difficile toxin A and B using ultra performance liquid chromatography.

Clostridium difficile infection (CDI) is the leading cause of gastroenteritis-associated death in the United States. The major virulent factors of C. difficile are toxin A (TcdA) and toxin B (TcdB). Toxicity is mediated by the glucosyltransferase domains on TcdA and TcdB wherein a glucose is transferred from UDP-glucose to Ras homolog family member A (RhoA) receptor. This modification results in disruption of critical cell signaling events. Vaccination against these toxins is considered the best way to combat the CDI. In order to produce non-toxic TcdA and TcdB antigens, their glucosyltransferase domains were genetically mutated to inactivate the toxin activity. We have developed a reverse phase ultra performance liquid chromatographic (RP-UPLC) method to measure this glucosyltransferase activity by separating RhoA and glucosylated RhoA. Glucosylated RhoA and RhoA have a retention time (RT) of 31.25 and 31.95min. We determine for the first time the glucosyltransferase kinetics (Km and kcat) of both full length TcdA and TcdB to RhoA and demonstrate that the genetically mutated TcdA and TcdB show no glucosyltransferase activity. Furthermore, two-dimensional electron microscopy (2D EM) data demonstrates that the overall global structures of mutated toxins do not change compared to native toxins.

Discovery and Characterization of Phage Display-Derived Human Monoclonal Antibodies against RSV F Glycoprotein.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants, the elderly and in immunosuppressed populations. The vast majority of neutralizing antibodies isolated from human subjects target the RSV fusion (F) glycoprotein, making it an attractive target for the development of vaccines and therapeutic antibodies. Currently, Synagis® (palivizumab) is the only FDA approved antibody drug for the prevention of RSV infection, and there is a great need for more effective vaccines and therapeutics. Phage display is a powerful tool in antibody discovery with the advantage that it does not require samples from immunized subjects. In this study, Morphosys HuCAL GOLD® phage libraries were used for panning against RSV prefusion and postfusion F proteins. Panels of human monoclonal antibodies (mAbs) against RSV F protein were discovered following phage library panning and characterized. Antibodies binding specifically to prefusion or postfusion F proteins and those binding both conformations were identified. 3B1 is a prototypic postfusion F specific antibody while 2E1 is a prototypic prefusion F specific antibody. 2E1 is a potent broadly neutralizing antibody against both RSV A and B strains. Epitope mapping experiments identified a conformational epitope spanning across three discontinuous sections of the RSV F protein, as well as critical residues for antibody interaction.

Cell confluency analysis on microcarriers by micro-flow imaging.

The productivity of cell culture-derived vaccines grown in anchorage-dependent animal cells is limited by bioreactor surface area. One way to increase the available surface area is by growing cells as monolayers on small spheres called microcarriers, which are approximately 100-250 µm in diameter. In order for microcarrier-based cell culture to be a success, it is important to understand the kinetics of cell growth on the microcarriers. Micro-flow imaging (MFI) is a simple and powerful technique that captures images and analyzes samples as they are drawn through a precision flow cell. In addition to providing size distribution and defect frequency data to compare microcarrier lots, MFI was used to generate hundreds of images to determine cell coverage and confluency on microcarriers. Same-day manual classification of these images provided upstream cell culture teams with actionable data that informed in-process decision making (e.g. time of infection). Additionally, an automated cell coverage algorithm was developed to increase the speed and throughput of the analyses.

Characterization of N-glycosylation profiles from mammalian and insect cell derived chikungunya VLP.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes severe arthralgia. The envelope of CHIKV is composed of 240 copies of two glycoproteins: E1 and E2. In this work, we have characterized the N-glycosylation patterns of CHIKV virus-like particles (VLPs), containing both E1 and E2 proteins, derived from mammalian and insect cells using hydrophilic interaction liquid chromatography (HILIC) with fluorescence (FL) and mass spectrometry (MS) detection. While HEK293 derived CHIKV VLPs contain oligomannose, hybrid and complex glycans, VLPs derived from SfBasic predominantly contain oligomannose glycans. This strong host dependence of N-glycosylation pattern resembles other alphaviruses such as SINV. The VLPs from HEK293 and SfBasic, with significantly different N-glycosylation profiles, are valuable reagents enabling future in-depth correlation studies between immunogenicity and glycosylation. In addition, the characterization tools presented here allow one to monitor glycosylation during vaccine process development and ensure process consistency.

Use of flow cytometry for characterization of human cytomegalovirus vaccine particles.

Despite a 40-year effort, an effective vaccine against human cytomegalovirus (HCMV) remains an unmet medical need. The discovery of potent neutralizing epitopes on the pentameric gH complex (gH/gL/UL128/130/131) has reenergized HCMV vaccine development. Our whole-virus vaccine candidate, currently in a Phase I clinical trial, is based on the attenuated AD169 strain with restored expression of the pentameric gH complex. Given the complexity of a whole-virus vaccine, improved analytical methods have been developed to better characterize heterogeneous viral particles released from infected cells during vaccine production. Here we show the utility of a commercial flow cytometer for the detection of individual HCMV particles, either via light scattering or using fluorescence after labeling of specific antigens. Rapid measurements requiring minimal material provide near real-time information on particle concentration, distributions of different particle types, and product purity. Additionally, utilizing immunoreagents has allowed us to characterize the distribution of key antigens across individual particles and particle types.

Soluble Human Cytomegalovirus gH/gL/pUL128-131 Pentameric Complex, but Not gH/gL, Inhibits Viral Entry to Epithelial Cells and Presents Dominant Native Neutralizing Epitopes.

Congenital infection of human cytomegalovirus (HCMV) is one of the leading causes of nongenetic birth defects, and development of a prophylactic vaccine against HCMV is of high priority for public health. The gH/gL/pUL128-131 pentameric complex mediates HCMV entry into endothelial and epithelial cells, and it is a major target for neutralizing antibody responses. To better understand the mechanism by which antibodies interact with the epitopes of the gH/gL/pUL128-131 pentameric complex resulting in viral neutralization, we expressed and purified soluble gH/gL/pUL128-131 pentameric complex and gH/gL from Chinese hamster ovary cells to >95% purity. The soluble gH/gL, which exists predominantly as (gH/gL)2 homodimer with a molecular mass of 220 kDa in solution, has a stoichiometry of 1:1 and a pI of 6.0-6.5. The pentameric complex has a molecular mass of 160 kDa, a stoichiometry of 1:1:1:1:1, and a pI of 7.4-8.1. The soluble pentameric complex, but not gH/gL, adsorbs 76% of neutralizing activities in HCMV human hyperimmune globulin, consistent with earlier reports that the most potent neutralizing epitopes for blocking epithelial infection are unique to the pentameric complex. Functionally, the soluble pentameric complex, but not gH/gL, blocks viral entry to epithelial cells in culture. Our results highlight the importance of the gH/gL/pUL128-131 pentameric complex in HCMV vaccine design and emphasize the necessity to monitor the integrity of the pentameric complex during the vaccine manufacturing process.

Label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group B outer membrane vesicle vaccine.

The development of a multivalent outer membrane vesicle (OMV) vaccine where each strain contributes multiple key protein antigens presents numerous analytical challenges. One major difficulty is the ability to accurately and specifically quantitate each antigen, especially during early development and process optimization when immunoreagents are limited or unavailable. To overcome this problem, quantitative mass spectrometry methods can be used. In place of traditional mass assays such as enzyme-linked immunosorbent assays (ELISAs), quantitative LC-MS/MS using multiple reaction monitoring (MRM) can be used during early-phase process development to measure key protein components in complex vaccines in the absence of specific immunoreagents. Multiplexed, label-free quantitative mass spectrometry methods using protein extraction by either detergent or 2-phase solvent were developed to quantitate levels of several meningococcal serogroup B protein antigens in an OMV vaccine candidate. Precision was demonstrated to be less than 15% RSD for the 2-phase extraction and less than 10% RSD for the detergent extraction method. Accuracy was 70 to 130% for the method using a 2-phase extraction and 90-110% for detergent extraction. The viability of MS-based protein quantification as a vaccine characterization method was demonstrated and advantages over traditional quantitative methods were evaluated. Implementation of these MS-based quantification methods can help to decrease the development time for complex vaccines and can provide orthogonal confirmation of results from existing antigen quantification techniques.

Development and application of a reversed-phase high-performance liquid chromatographic method for quantitation and characterization of a Chikungunya virus-like particle vaccine.

To effectively support the development of a Chikungunya (CHIKV) virus-like particle (VLP) vaccine, a sensitive and robust high-performance liquid chromatography (HPLC) method that can quantitate CHIKV VLPs and monitor product purity throughout the manufacturing process is needed. We developed a sensitive reversed-phase HPLC (RP-HPLC) method that separates capsid, E1, and E2 proteins in CHIKV VLP vaccine with good resolution. Each protein component was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS). The post-translational modifications on the viral glycoproteins E1 and E2 were further identified by intact protein mass measurements with liquid chromatography-mass spectrometry (LC-MS). The RP-HPLC method has a linear range of 0.51-12 µg protein, an accuracy of 96-106% and a precision of 12% RSD, suitable for vaccine product release testing. In addition, we demonstrated that the RP-HPLC method is useful for characterizing viral glycoprotein post-translational modifications, monitoring product purity during process development and assessing product stability during formulation development.

Organic solvent extraction of proteins and peptides from serum as an effective sample preparation for detection and identification of biomarkers by mass spectrometry.

A method to extract peptides and low molecular weight proteins from serum under denaturing conditions using acetonitrile containing 0.1% trifluoroacetic acid has been developed. The extraction procedure precipitates large, abundant proteins to simplify subsequent mass spectral analysis. This sample preparation method provides an efficient way to extract serum peptides, enabling them to be compared and identified using different mass spectrometry approaches. Surface-enhanced laser desorption/ionization-time of flight mass spectrometry analysis of mouse blood serum samples prepared by this method allowed detection of two markers which were significantly reduced in mice with B cell lymphoma tumor. One of these markers has been identified as apolipoprotein A-II.

The octapeptidic end of the C-terminal tail of histone H2A is cleaved off in cells exposed to carcinogenic nickel(II).

We have demonstrated previously that Ni(II) binds to the C-terminal -TESHHKAKGK motif of isolated bovine histone H2A. At physiological pH, the bound Ni(II) assists in hydrolysis of the E-S peptide bond in this motif that results in a cleavage of the terminal octapeptide SHHKAKGK off the histone's C-tail. To test if the hydrolysis could also occur in living cells, we cultured CHO (Chinese hamster ovary), NRK-52 (rat renal tubular epithelium), and HPL1D (human lung epithelium) cells with 0.1-1 mM Ni(II) for 3-7 days. As found by gel electrophoresis, Western blotting, and liquid chromatography/mass spectrometry, histones extracted from the cells contained a new fraction of histone H2A lacking the terminal octapeptide (q-H2A). The abundance of q-H2A increased with Ni(II) concentration and exposure time. It can be anticipated that the truncation of histone H2A may alter chromatin structure and affect gene expression. The present results provide evidence for novel mechanisms of epigenetic effects of Ni(II) that may be involved in nickel toxicity and carcinogenesis.

Quantitative proteomics employing primary amine affinity tags.

A proteomics-based method using stable isotope labeling to assess the relative abundance of peptides or proteins is described. Bradykinin and carbonic anhydrase were labeled with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate, a membrane impermeant reagent that is reactive with primary amines. Specificity of the label to primary amines was demonstrated using tandem mass spectrometry. Also, relative quantitation was achieved by secondary labeling with natural isotopic abundance and stable isotope-labeled methyl iodide. We believe this to be an effective stable isotope-labeling method for quantitative proteomics.