FAM76B regulates PI3K/Akt/NF-κB-mediated M1 macrophage polarization by influencing the stability of PIK3CD mRNA.

Macrophage polarization is closely related to inflammation development, yet how macrophages are polarized remains unclear. In our study, the number of M1 macrophages was markedly increased in Fam76b knockout U937 cells vs. wild-type U937 cells, and FAM76B expression was decreased in M1 macrophages induced from different sources of macrophages. Moreover, Fam76b knockout enhanced the mRNA and protein levels of M1 macrophage-associated marker genes. These results suggest that FAM76B inhibits M1 macrophage polarization. We then further explored the mechanism by which FAM76B regulates macrophage polarization. We found that FAM76B can regulate PI3K/Akt/NF-κB pathway-mediated M1 macrophage polarization by stabilizing PIK3CD mRNA. Finally, FAM76B was proven to protect against inflammatory bowel disease (IBD) by inhibiting M1 macrophage polarization through the PI3K/Akt/NF-κB pathway in vivo. In summary, FAM76B regulates M1 macrophage polarization through the PI3K/Akt/NF-κB pathway in vitro and in vivo, which may inform the development of future therapeutic strategies for IBD and other inflammatory diseases.

Chlamydia trachomatis induces low-frequency, sustained CD4 T cell responses in most women, predominantly targeting chlamydial protease-like activity factor, CPAF.

BACKGROUND: Chlamydia trachomatis (CT) is a globally prevalent sexually transmitted infection (STI) that can result in pelvic inflammatory disease, ectopic pregnancy and infertility in women. Currently, there is no prophylactic vaccine. METHODS: This study examined T cell immunity in a cohort of women recently infected with CT. Participants were screened against peptides spanning 33 of 894 possible CT proteins, either ex vivo or using short-term cell lines (STCL). CT-specific T cells were characterized by IFN-γ ELISpot and flow cytometry. RESULTS: Ex vivo CT-specific T cells were rarely detected; however, following in vitro expanded CT-specific T cells were detected by IFN-γ ELISpot in 90% (27/30) of participants. Notably, over 50% of participants had T cell responses targeting chlamydial protease-like activity factor (CPAF). T cell epitopes were dispersed across the CPAF protein. Flow cytometry analysis of STCL found CT-specific cells, were mainly CD4+, produced IFN-γ and TNF-α and were sustained over 12 months. Ex vivo analysis suggested CT-specific T cells mostly exhibited a central memory phenotype. CONCLUSION: Our results indicate that CT infection elicits low-frequency, persistent CD4 T cell responses in most women and that the secreted protein, CPAF, is an immunoprevalent CT antigen. Altogether, these data support development and testing of CT vaccines that enhance CD4 T cells against CPAF.

Methyltransferase-like 3 promotes cervical cancer metastasis by enhancing cathepsin L mRNA stability in an N6-methyladenosine-dependent manner.

N6-methyladenosine (m6A) is a highly abundant RNA modification in eukaryotic cells. Methyltransferase-like 3 (METTL3), a major protein in the m6A methyltransferase complex, plays important roles in many malignancies, but its role in cervical cancer metastasis remains uncertain. Here, we found that METTL3 was significantly upregulated in cervical cancer tissue, and its upregulation was associated with a poor prognosis in cervical cancer patients. Knockdown of METTL3 significantly reduced cervical cancer cell migration and invasion. Conversely, METTL3 overexpression markedly promoted cervical cancer cell metastasis in vitro and in vivo. Furthermore, METTL3 mediated the m6A modification of cathepsin L (CTSL) mRNA at the 5'-UTR, and the m6A reader protein insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) bound to the m6A sites and enhanced CTSL mRNA stability. Our results indicated that METTL3 enhanced CTSL mRNA stability through an m6A-IGF2BP2-dependent mechanism, thereby promoting cervical cancer cell metastasis. These findings provide insights into a novel m6A modification pattern involved in cervical cancer development.

The MYBL2-CCL2 axis promotes tumor progression and resistance to anti-PD-1 therapy in ovarian cancer by inducing immunosuppressive macrophages.

BACKGROUND: An immunosuppressive tumor microenvironment in ovarian cancer facilitates tumor progression and resistance to immunotherapy. The function of MYB Proto-Oncogene Like 2 (MYBL2) in the tumor microenvironment remains largely unexplored. METHODS: A syngeneic intraovarian mouse model, flow cytometry analysis, and immunohistochemistry were used to explore the biological function of MYBL2 in tumor progression and immune escape. Molecular and biochemical strategies-namely RNA-sequencing, western blotting, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay, multiplex immunofluorescence, chromatic immunoprecipitation assay (CHIP) and luciferase assay-were used to reveal the mechanisms of MYBL2 in the OVC microenvironment. RESULTS: We found tumor derived MYBL2 indicated poor prognosis and selectively correlated with tumor associated macrophages (TAMs) in ovarian cancer. Mechanically, C-C motif chemokine ligand 2 (CCL2) transcriptionally activated by MYBL2 induced TAMs recruitment and M2-like polarization in vitro. Using a syngeneic intraovarian mouse model, we identified MYBL2 promoted tumor malignancyand increased tumor-infiltrating immunosuppressive macrophages. Cyclin-dependent kinase 2 (CDK2) was a known upstream kinase to phosphorylate MYBL2 and promote its transcriptional function. The upstream inhibitor of CDK2, CVT-313, reprogrammed the tumor microenvironment and reduced anti-PD-1 resistance. CONCLUSIONS: The MYBL2/CCL2 axis contributing to TAMs recruitment and M2-like polarization is crucial to immune evasion and anti-PD-1 resistance in ovarian cancer, which is a potential target to enhance the efficacy of immunotherapy.

Decomposition of variation of mixed variables by a latent mixed Gaussian copula model.

Many biomedical studies collect data of mixed types of variables from multiple groups of subjects. Some of these studies aim to find the group-specific and the common variation among all these variables. Even though similar problems have been studied by some previous works, their methods mainly rely on the Pearson correlation, which cannot handle mixed data. To address this issue, we propose a latent mixed Gaussian copula (LMGC) model that can quantify the correlations among binary, ordinal, continuous, and truncated variables in a unified framework. We also provide a tool to decompose the variation into the group-specific and the common variation over multiple groups via solving a regularized M-estimation problem. We conduct extensive simulation studies to show the advantage of our proposed method over the Pearson correlation-based methods. We also demonstrate that by jointly solving the M-estimation problem over multiple groups, our method is better than decomposing the variation group by group. We also apply our method to a Chlamydia trachomatis genital tract infection study to demonstrate how it can be used to discover informative biomarkers that differentiate patients.

Expression and functional analysis of fam76b in zebrafish.

FAM76B is nuclear speckle-localized protein with a molecular weight of 39 kDa. The amino sequence of FAM76B protein is highly conserved among species, suggesting that FAM76B has important biological functions. However, the biological function of FAM76B is currently still unclear. To explore the biological function of FAM76B, we firstly used zebrafish as the experimental model to study the distribution and expression level of Fam76b. The results indicated that fam76b is highly expressed in hematopoiesis and immune systems of zebrafish by real-time quantitative PCR, in situ hybridization and Tg(fam76b: eGFP) transgenic zebrafish. Then, the fam76b gene was knocked out by CRISPR/Cas9 in zebrafish and fam76b rescue in fam76b-/- zebrafish was performed using the TOL2 transposable system. fam76b gene knockout zebrafish exhibit reduced thymus, excessive inflammatory response, and increased mortality. FAM76B was further found to be involved in regulating the development of hematopoiesis and immune system, and participate in the process of inflammatory response. Our findings in the study lay the groundwork for elucidating the function of the new molecule Fam76b and provide new insights into the development of zebrafish hematopoietic and immune system.

Effect of thermal softening of double-lumen endobronchial tubes on postoperative sore throat in patients with prior SARS-CoV-2 infection: a randomized controlled trial.

BACKGROUND: The incidence of postoperative sore throat (POST) after tracheal intubation using double-lumen endobronchial tubes (DLTs) is higher in patients with prior severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection than in the general population. This prospective trial was conducted to determine whether thermal softening of DLTs could decrease the incidence of POST or other airway injuries in patients with prior SARS-CoV-2 infection. METHODS: A total of 120 patients with prior SARS-CoV-2 infection undergoing thoracoscopic surgery were randomly assigned to two groups (n = 60 each). In the thermal softening group, the distal portion of the DLT was placed in thermostatic saline (50 °C) for 10 min before endotracheal intubation. In the control group, the distal portion of the DLT was placed in room temperature saline for 10 min before endotracheal intubation. The incidence and severity of POST and hoarseness were assessed at 1, 6 and 24 h postoperatively. The primary outcomes were the incidence and severity of POST at 6 h postoperatively. The secondary outcomes were the incidence and severity of hoarseness, vocal cord and tracheal injuries, and hemodynamic changes in patients at intubation. RESULTS: The incidence of POST at 6 h postoperatively was greater in the control group than in the thermal softening group [41 (68%) vs. 22 (37%), P = 0.001]. The overall incidence of POST at 24 h postoperatively was greater in the control group than in the thermal softening group [46 (76%) vs. 24 (40%), P < 0.001]. The overall incidence of tracheal injuries was also greater in the control group than in the thermal softening group (P = 0.016). Vocal cord injuries occurred more frequently in the control group than in the thermal softening group (P = 0.006). CONCLUSION: Thermal softening of DLTs before intubation can reduce the incidence of POST and airway injuries in patients with prior SARS-CoV-2 infection undergoing DLT insertion. TRIAL REGISTRATION: This trial has been registered at www.chictr.org.cn (registration number: ChiCTR2200066821; registration date: December 19, 2022).

Reduced Endometrial Ascension and Enhanced Reinfection Associated With Immunoglobulin G Antibodies to Specific Chlamydia trachomatis Proteins in Women at Risk for Chlamydia.

BACKGROUND: Previous research revealed antibodies targeting Chlamydia trachomatis elementary bodies was not associated with reduced endometrial or incident infection in C. trachomatis-exposed women. However, data on the role of C. trachomatis protein-specific antibodies in protection are limited. METHODS: A whole-proteome C. trachomatis array screening serum pools from C. trachomatis-exposed women identified 121 immunoprevalent proteins. Individual serum samples were probed using a focused array. Immunoglobulin (Ig) G antibody frequencies and endometrial or incident infection relationships were examined using Wilcoxon rank sum test. The impact of the breadth and magnitude of protein-specific IgGs on ascension and incident infection were examined using multivariable stepwise logistic regression. Complementary RNA sequencing quantified C. trachomatis gene transcripts in cervical swab samples from infected women. RESULTS: IgG to pGP3 and CT_005 were associated with reduced endometrial infection; anti-CT_443, anti-CT_486, and anti-CT_123 were associated with increased incident infection. Increased breadth of protein recognition did not however predict protection from endometrial or incident infection. Messenger RNAs for immunoprevalent C. trachomatis proteins were highly abundant in the cervix. CONCLUSIONS: Protein-specific C. trachomatis antibodies are not sufficient to protect against ascending or incident infection. However, cervical C. trachomatis gene transcript abundance positively correlates with C. trachomatis protein immunogenicity. These abundant and broadly recognized antigens are viable vaccine candidates.

Progranulin deficiency promotes persistent neuroinflammation and causes regional pathology in the hippocampus following traumatic brain injury.

Traumatic brain injury (TBI) can be progressive and can lead to the development of a long-term complication termed chronic traumatic encephalopathy. The mechanisms underlying the progressive changes are still unknown; however, studies have suggested that microglia-mediated neuroinflammation in response to TBI may play a fundamental role. This study aimed to determine whether progranulin (PGRN), a major modulator of microglial activity, plays a role in the progressive damage following TBI. PGRN-deficient and wild-type mice were subjected to controlled cortical impact and were observed neuropathologically after 3 days, 7 days, and 5 months. Compared to sham and wild-type mice, the PGRN-deficient mice showed overall stronger microgliosis and astrocytosis. The astrocytosis involved broader areas than the microgliosis and was more prominent in the basal ganglia, hippocampus, and internal capsule in PGRN-deficient mice. Ongoing neuronal death was uniquely observed in the hippocampal CA3 region of PGRN-deficient mice at 5 months after TBI, accompanying the regional chronic microgliosis and astrocytosis involving the CA3 commissural pathway. In addition, there was M1 microglial polarization in the pericontusional area with activated TLR4/MyD88/NF-κB signaling; however, the hippocampus showed only mild M1 polarization 7 days after TBI. Lastly, Morris water maze tests showed PGRN-deficient mice had poorer spatial learning and memory 5 months after TBI than wild-type or sham mice. The data indicated the PGRN deficiency caused TBI progression by promoting persistent microgliosis with microglial polarization and astrocytosis, as well as regional pathology in the hippocampus. The study suggests that PGRN should be evaluated as a potential therapy for TBI.

Activity-Based Protein Profiling Probe for the Detection of Enzymes Catalyzing Polysorbate Degradation.

Polysorbates are nonionic surfactants that have been widely used in biotherapeutic formulations to prevent protein aggregation and denaturation. However, polysorbates are subject to degradation after prolonged storage if certain lipases are present in the biotherapeutic product. Because the degradation of polysorbates compromises the shelf life of biotherapeutics and leads to the formation of undesirable products such as protein aggregates and subvisible particles, it is important to identify the active enzymes that catalyze polysorbate hydrolysis. In this study, we developed a novel fluorophosphonate activity-based protein profiling (ABPP) probe (termed the REGN probe), which mimics the structure of polysorbate and targets lipases catalyzing polysorbate degradation. We demonstrated that the REGN probe could enrich certain lipases from Chinese hamster ovary (CHO) cell lysate by more than 100-fold compared with direct tryptic digestion. Furthermore, we found that the REGN probe had higher lipase enrichment efficiency than commercially available ABPP probes including fluorophosphonate-biotin (FP-biotin) and FP-desthiobiotin. Remarkably, the REGN probe can enrich several lipases that cannot be labeled by commercial probes, such as lysosomal acid lipase and cytosolic phospholipase A2. Additionally, we showed that lipases with abundances as low as 0.08 ppm in drug substances were detected by the REGN probe enrichment and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Collectively, we have developed a novel ABPP probe with higher enrichment efficiency and broader coverage for lipases compared with commercial probes, and this probe can be used to detect the trace level of lipases in biotherapeutic products and to facilitate their development and manufacturing.

Impact of the initial site of metastases on post-recurrence survival for neuroendocrine cervical cancer.

OBJECTIVE: To summarize the risk factors and emphasize the prognostic importance of the site of recurrent neuroendocrine cervical cancer (NECC). METHODS: We enrolled 88 patients who developed recurrence after radical surgery for pathological stage I-IVa primary NECC between January 2003 and 30 December 2020 and classified these cases into 7 groups based on the initial recurrence. The risk factors for post-recurrence survival (PRS) were analyzed by Kaplan-Meier and Cox regression methods. RESULTS: Among 88 NECC patients, nearly all patients (95.50%) experienced progression within 3 years. The time to progression was significantly longer in patients with lung recurrence than in patients without lung recurrence (p = 0.008). After the first recurrence, the median follow-up was 11.1 months (range 2.37-65.50 months), and the 5-year PRS was only 20.6%. The depth of invasion in the primary surgery, number of recurrent sites, abdominal organ recurrence were correlated with PRS by univariate analysis. Multivariate analyses revealed that the number of recurrent sites (p = 0.025) and abdominal organ recurrence (p = 0.031) were independent prognostic factors. Notably, the combination of immune checkpoint inhibitors and chemotherapy, with or without surgery, showed a 43.8% objective response rate in recurrent NECC. CONCLUSION: Patients with abdominal organ recurrence need more sophisticated therapy. The combination of immune therapy and chemotherapy might be an opportunity for recurrent NECC.

Maximizing hydrophobic peptide recovery in proteomics and antibody development using a mass spectrometry compatible surfactant.

Peptide loss due to surface absorption can happen at any step in a protein analysis workflow and is sometimes especially deleterious for hydrophobic peptides. In this study, we found the LC-MS compatible surfactant, n-Dodecyl-ß-D-maltoside (DDM), can maximize hydrophobic peptide recovery in various samples including single cell digests, mAb clinical PK samples, and mAb peptide mapping samples. In HeLa single cell proteomics analysis, more than half of all unique peptides identified were found only in DDM prepared samples, most of which had significantly higher hydrophobicities compared to peptides in control samples. In clinical PK studies, DDM enhanced hydrophobic complementarity-determining region (CDR) peptide signals significantly. The fold change of CDR peptides' intensity enhancement in DDM added samples compared to controls correlate with peptide retention time and hydrophobicity, providing guidance for surrogate peptide selection and peptide standard handling in PK studies. For peptide mapping analysis of mAbs, DDM can improve hydrophobic peptide signal and solution stability over 48 h in an autosampler at 4 °C, which can aid method qualification and transfer during drug development. Lastly, maximizing hydrophobic peptide recovery from samples dried in vacuo was achieved by DDM reconstitution, which provided higher signal for later eluting peaks and higher proteome coverage overall.

Generalized multi-SNP mediation intersection-union test.

To elucidate the molecular mechanisms underlying genetic variants identified from genome-wide association studies (GWAS) for a variety of phenotypic traits encompassing binary, continuous, count, and survival outcomes, we propose a novel and flexible method to test for mediation that can simultaneously accommodate multiple genetic variants and different types of outcome variables. Specifically, we employ the intersection-union test approach combined with the likelihood ratio test to detect mediation effect of multiple genetic variants via some mediator (e.g., the expression of a neighboring gene) on outcome. We fit high-dimensional generalized linear mixed models under the mediation framework, separately under the null and alternative hypothesis. We leverage Laplace approximation to compute the marginal likelihood of outcome and use coordinate descent algorithm to estimate corresponding parameters. Our extensive simulations demonstrate the validity of our proposed methods and substantial, up to 97%, power gains over alternative methods. Applications to real data for the study of Chlamydia trachomatis infection further showcase advantages of our methods. We believe our proposed methods will be of value and general interest in this post-GWAS era to disentangle the potential causal mechanism from DNA to phenotype for new drug discovery and personalized medicine.

Application of aromatic amide-derived atropisomers as chiral solvating agents for discrimination of optically active mandelic acid derivatives in 1 H nuclear magnetic resonance spectroscopy.

Non-biaryl atropisomers and their stereochemistry have attracted much attentions in the past years. However, application of the non-biaryl atropisomers as chiral solvating agents is yet to be explored. In this work, four aromatic amide-derived atropisomeric phosphine ligands (hosts) were used as chiral solvating agents to recognize various mandelic acid derivatives (guests) in 1 H nuclear magnetic resonance (NMR) spectroscopy. It is found that chiral center configurations of the four hosts have different effects on the enantiorecognition to the used guests. In addition, the host and guest interaction was further investigated by determination of the host-guest complex stoichiometry using the Job's method and density functional theory calculation, respectively. Moreover, chiral analysis accuracy of these hosts was evaluated through relationship between enantiomeric excess values of 4-chloromandelic acid provided by NMR and gravimetry, respectively.

Host Genetic Risk Factors for Chlamydia trachomatis-Related Infertility in Women.

BACKGROUND: Chlamydia trachomatis (Ct) infection ascending to the upper genital tract can cause infertility. Direct association of genetic variants as contributors is challenging because infertility may not be diagnosed until years after infection. Investigating the intermediate trait of ascension bridges this gap. METHODS: We identified infertility genome-wide association study (GWAS) loci using deoxyribonucleic acid from Ct-seropositive cisgender women in a tubal factor infertility study and Ct-infected cisgender women from a longitudinal pelvic inflammatory disease cohort with known fertility status. Deoxyribonucleic acid and blood messenger ribonucleic acid from 2 additional female cohorts with active Ct infection and known endometrial infection status were used to investigate the impact of infertility single-nucleotide polymorphisms (SNPs) on Ct ascension. A statistical mediation test examined whether multiple infertility SNPs jointly influenced ascension risk by modulating expression of mediator genes. RESULTS: We identified 112 candidate infertility GWAS loci, and 31 associated with Ct ascension. The SNPs altered chlamydial ascension by modulating expression of 40 mediator genes. Mediator genes identified are involved in innate immune responses including type I interferon production, T-cell function, fibrosis, female reproductive tract health, and protein synthesis and degradation. CONCLUSIONS: We identified Ct-related infertility loci and their potential functional effects on Ct ascension.

CRISPR/Cas9-mediated grna gene knockout leads to neurodevelopmental defects and motor behavior changes in zebrafish.

Progranulin (PGRN) is a secreted glycoprotein with multiple biological functions in early embryogenesis, anti-inflammation, and neurodegeneration. A good model for the functional study of PGRN is the zebrafish with knockdown or knockout of grn, the gene encoding PGRN. Morpholino oligonucleotides (MOs) and zinc finger nucleases have been used to generate zebrafish grn models, yet they have shown inconsistent phenotypes due to either the neurotoxicity of the MOs or possible genetic compensation responses during gene editing. In this study, we generated stable grna (one of the major grn homologues of zebrafish) knockout zebrafish by using CRISPR/Cas9-mediated genome editing. A grna sgRNA was designed to target the similar repeated sequence shared by exon 13, exon 15, and exon 19 in zebrafish. The F1 generation with the frameshift mutation of + 4 bp (the addition of 4 bp to exon15), which causes a premature termination, was obtained and subjected to morphological and behavioral evaluation. The grna knockout zebrafish showed neurodevelopmental defects, including spinal motor neurons with shorter axons, decreased sensory hair cells, thinning of the outer nuclear layer and thickening of the inner nuclear layer of the retina, decreased expression of rhodopsin in the cone cells, and motor behavior changes. Moreover, the phenotypes of grna knockout zebrafish could be rescued with the Tol2 system carrying the grna gene. The grna knockout zebrafish model generated in this study provides a useful tool to study PGRN function and has potential for high-throughput drug screening for disease therapy.

Simple and Sensitive Method for Deep Profiling of Host Cell Proteins in Therapeutic Antibodies by Combining Ultra-Low Trypsin Concentration Digestion, Long Chromatographic Gradients, and BoxCar Mass Spectrometry Acquisition.

Liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful tool for the analysis of host cell proteins (HCP) during antibody drug process development due to its sensitivity, selectivity, and adaptability. However, the enormous dynamic range between the therapeutic antibody and accompanying HCPs poses a significant challenge for LC-MS based detection of these low abundance impurities. To address this challenge, enrichment of HCPs via immunoaffinity, protein A, 2D-LC, or other strategies is typically performed. However, these enrichments are time-consuming and sometimes require a large quantity of sample. Here, we report a simple and sensitive strategy to analyze HCPs in therapeutic antibody samples without cumbersome enrichment by combining an ultra-low trypsin concentration during digestion under nondenaturing conditions, a long chromatographic gradient, and BoxCar acquisition (ULTLB) on a quadrupole-Orbitrap mass spectrometer. Application of this strategy to the NIST monoclonal antibody standard (NISTmAb) resulted in the identification of 453 mouse HCPs, which is a significant increase in the number of identified HCPs without enrichment compared to previous reports. Known amounts of HCPs were spiked into the purified antibody drug substance, demonstrating that the method sensitivity is as low as 0.5 ppm. Thus, the ULTLB method represents a sensitive and simple platform for deep profiling of HCPs in antibodies.

Determination of tautomeric preference of fenobam in solution by high-resolution NMR spectroscopy.

In this work, tautomeric preference of fenobam in solution was investigated by homonuclear and heteronuclear solution nuclear magnetic resonance (NMR) spectroscopy. 1 H-1 H nuclear Overhauser effect spectroscopy (NOESY) spectrum revealed that fenobam in liquid state exists exclusively in one of the two possible tautomeric structures, which was confirmed by 1 H-13 C HSQC and heteronuclear multiple bond correlation (HMBC) spectra. Moreover, difference between the two tautomeric structures was studied by theoretical calculations, which further proved the result obtained by the NMR experiments.

Combination of FAIMS, Protein A Depletion, and Native Digest Conditions Enables Deep Proteomic Profiling of Host Cell Proteins in Monoclonal Antibodies.

Host cell proteins (HCPs) are residual impurities generated by the expression cell line during the production of biopharmaceuticals. Although the majority of these contaminants are removed during purification, HCPs can represent a considerable risk to the efficacy and safety of a therapeutic protein if not actively monitored. The enzyme-linked immunosorbent assay (ELISA) is commonly used throughout production to monitor HCP levels but has limited ability to identify novel HCPs or provide detailed quantification. Liquid chromatography tandem mass spectrometry (LC-MS2) methods are increasingly being used in conjunction with established ELISA techniques to provide rapid adaptability to increasingly complex samples as well as highly quantitative and informative results. However, MS-based methods are still hindered by the large dynamic range between high abundance biopharmaceutical proteins and low abundance HCPs. Here, we propose a multifactorial approach designed to optimize HCP detection in purified monoclonal antibody samples with LC-MS2. By first depleting the sample of antibody on a protein A column, then specifically digesting HCPs while precipitating remaining antibody, and finally reducing spectral complexity through compensation voltage (CV) switching using high-field asymmetric waveform ion mobility spectrometry (FAIMS), we identified multiple-fold more HCPs in the NIST monoclonal antibody standard than any single established mass spectrometry technique reported in the literature. Our analyses consistently identified over 600 high confidence mouse HCPs, a multifold increase over established methods, while maintaining high reproducibility.

Multi-SNP mediation intersection-union test.

SUMMARY: Tens of thousands of reproducibly identified GWAS (Genome-Wide Association Studies) variants, with the vast majority falling in non-coding regions resulting in no eventual protein products, call urgently for mechanistic interpretations. Although numerous methods exist, there are few, if any methods, for simultaneously testing the mediation effects of multiple correlated SNPs via some mediator (e.g. the expression of a gene in the neighborhood) on phenotypic outcome. We propose multi-SNP mediation intersection-union test (SMUT) to fill in this methodological gap. Our extensive simulations demonstrate the validity of SMUT as well as substantial, up to 92%, power gains over alternative methods. In addition, SMUT confirmed known mediators in a real dataset of Finns for plasma adiponectin level, which were missed by many alternative methods. We believe SMUT will become a useful tool to generate mechanistic hypotheses underlying GWAS variants, facilitating functional follow-up. AVAILABILITY AND IMPLEMENTATION: The R package SMUT is publicly available from CRAN at https://CRAN.R-project.org/package=SMUT. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.