Specific long-term changes in anti-SARS-CoV-2 IgG modifications and antibody functions in mRNA, adenovector, and protein subunit vaccines.

Various vaccine platforms were developed and deployed against the COVID-19 disease. The Fc-mediated functions of IgG antibodies are essential in the adaptive immune response elicited by vaccines. However, the long-term changes of protein subunit vaccines and their combinations with messenger RNA (mRNA) vaccines are unknown. A total of 272 serum and plasma samples were collected from individuals who received first to third doses of the protein subunit Medigen, the mRNA (BNT, Moderna), or the adenovector AstraZeneca vaccines. The IgG subclass level was measured using enzyme-linked immunosorbent assay, and Fc-N glycosylation was measured using liquid chromatography coupled to tandem mass spectrometry. Antibody-dependent-cellular-phagocytosis (ADCP) and complement deposition (ADCD) of anti-spike (S) IgG antibodies were measured by flow cytometry. IgG1 and 3 reached the highest anti-S IgG subclass level. IgG1, 2, and 4 subclass levels significantly increased in mRNA- and Medigen-vaccinated individuals. Fc-glycosylation was stable, except in female BNT vaccinees, who showed increased bisection and decreased galactosylation. Female BNT vaccinees had a higher anti-S IgG titer than that of males. ADCP declined in all groups. ADCD was significantly lower in AstraZeneca-vaccinated individuals. Each vaccine produced specific long-term changes in Fc structure and function. This finding is critical when selecting a vaccine platform or combination to achieve the desired immune response.

Specific long-term changes in anti-SARS-CoV-2 IgG modifications and antibody functions in mRNA, adenovector, and protein subunit vaccines.

Various vaccine platforms were developed and deployed against the COVID-19 disease. The Fc-mediated functions of IgG antibodies are essential in the adaptive immune response elicited by vaccines. However, the long-term changes of protein subunit vaccines and their combinations with mRNA vaccines are unknown. A total of 272 serum and plasma samples were collected from individuals who received first to third doses of the protein subunit Medigen, the mRNA (BNT), or the adenovector AstraZeneca vaccines. The IgG subclass level was measured using enzyme-linked immunosorbent assay, and Fc-N glycosylation was measured using LC-MS/MS. Antibody-dependent phagocytosis (ADCP) and complement deposition (ADCD) of anti-spike (S) IgG antibodies were measured. IgG1 and 3 reached the highest anti-S IgG subclass level. IgG1, 2, and 4 subclass levels significantly increased in mRNA- and Medigen-vaccinated individuals. Fc-glycosylation was stable, except in female BNT vaccinees, who showed increased bisection and decreased galactosylation. Female BNT vaccinees had a higher anti-S IgG titer than that of males. ADCP declined in all groups. ADCD increased in Medigen-vaccinated individuals after the third dose. Each vaccine produced specific long-term changes in Fc structure and function. This finding is critical when selecting a vaccine platform or combination to achieve the desired immune response.

Effects of perfluorododecanoic acid on testicular function in mice.

Perfluorodecanoic acid (PFDoA) is a widely distributed environmental pollutant that can affect the functions of many organs. However, systematic evaluations of the effects of PFDoA on testicular functions are lacking. The aim of this study was to investigate the effects of PFDoA on mouse testicular functions, including spermatogenesis, testosterone synthesis, and stem Leydig cells (SLCs) in the interstitial tissue of the testis. PFDoA (0, 2, 5, 10 mg/kg/d) was administered via gavage to 2-month-old mice for 4 weeks. Serum hormone levels and sperm quality were assayed. Furthermore, to investigate the mechanisms by which PFDoA affects testosterone synthesis and spermatogenesis in vivo, the expression of StAR and P450scc in testicular tissue was measured by immunofluorescence staining and quantitative real-time PCR. In addition, the levels of SLC markers, including nestin and CD51, were studied. PFDoA decreased the luteinizing hormone concentration and sperm quality. Although the difference was not statistically significant, mean testosterone levels showed a downward trend. The expression of StAR, P450scc, CD51, and nestin was also suppressed in the PFDoA-treated groups compared with the control group. Our study suggested that PFDoA exposure can decrease testosterone biosynthesis, and even reduce the number of SLCs. These results indicated that PFDoA suppressed the main functions of testis, and further researches are required to identify strategies for preventing or reducing the effect of PFDoA on testicular function.

N6-Methyladenosine Modification of ANLN Enhances Hepatocellular Carcinoma Bone Metastasis.

Bones are categorized as the second most prevalent location of extra-hepatic metastasis in Hepatocellular Carcinoma (HCC), which is linked to an extremely poor prognosis due to limited therapeutic options. N6-methyladenosine (m6A) is a prominent modification involved in HCC, but the exact mechanisms on how m6A modifications induce HCC bone metastases (BM) remain unclear. The key modulators responsible for the abundant m6A RNA modification-induced HCC BM was found to be the METTL3 and YTHDF1. The expression of Anillin actin-binding protein (ANLN) was dramatically higher in HCC with BM tissues, and its messenger RNA (mRNA) stability was enhanced via m6A epitranscriptomic regulation by METTL3 and YTHDF1. High METTL3 and YTHDF1 expression along with nuclear ANLN protein was clinically correlated with BM in HCC patients. Furthermore, HCC BM was attributed to over-expression of nuclear ANLN forming a transcriptional complex with SP1 which enhanced KIF2C transcriptional activity to activate the mTORC1 pathway, therefore increased the expression of RANKL and disproportionated RANKL-OPG expression in bone microenvironment leading to malignant neoplasms invade bone tissue. In addition, inhibition of ANLN m6A modification by DZNeP attenuated HCC BM. This data provides meaningful understanding of the modulation and association of m6A epitranscriptomic-regulated BM in HCC, and moreover, defines potentially valuable therapeutic targets.

Factors influencing neutralizing antibody titers elicited by coronavirus disease 2019 vaccines.

The World Health Organization has highlighted the importance of an international standard (IS) for severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) neutralizing antibody titer detection to calibrate diagnostic techniques. We applied an IS to calibrate neutralizing antibody titers (NTs) (international units/mL) in response to coronavirus disease 2019 (COVID-19) vaccination. Moreover, the association between different factors and neutralizing antibodies was analyzed. A total of 1667 serum samples were collected from participants receiving different COVID-19 vaccines. Antibody titers were determined by a microneutralization assay using live viruses in a biosafety level 3 (BSL-3) laboratory and a commercial serological MeDiPro kit. The titer determined using the MeDiPro kit was highly correlated with the NT determined using live viruses and calibrated using IS. Fever and antipyretic analgesic treatment were related to neutralizing antibody responses in ChAdOx1-S and BNT162b2 vaccinations. Individuals with diabetes showed a low NT elicited by MVC-COV1901. Individuals with hypertension receiving the BNT162b2 vaccine had lower NTs than those without hypertension. Our study provided the international unit (IU) values of NTs in vaccinated individuals for the development of vaccines and implementation of non-inferiority trials. Correlation of the influencing factors with NTs can provide an indicator for selecting COVID-19 vaccines based on personal attributes.

Microwave resonant absorption of SARS-CoV-2 viruses.

Low power microwave can effectively deactivate influenza type A virus through the nonthermal structure-resonant energy transfer effect, at a frequency matching the confined-acoustic dipolar mode frequency of the virus. Currently, aerosol is considered the major route for SARS-CoV-2 transmission. For the potential microwave-based sterilization, the microwave-resonant frequency of SARS-CoV-2 must be unraveled. Here we report a microwave absorption spectroscopy study of the SARS-CoV-2 and HCoV-229E viruses through devising a coplanar-waveguide-based sensor. Noticeable microwave absorption can be observed, while we identified the resonant frequencies of the 1st and 2nd dipolar modes of SARS-CoV-2 virus as 4 and 7.5 GHz respectively. We further found that the resonant frequencies are invariant to the virus titer, and we also studied the microwave absorption of HCoV-229E in weak acidity medium to simulate the common pH value in fluid secretion. Our results suggest the possible radiation frequency for the recently proposed microwave sterilization devices to inactivate SARS-CoV-2 virus through a nonthermal mechanism so as to control the disease transmission in the post-pandemic era.

Molecular Virology of SARS-CoV-2 and Related Coronaviruses.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The global COVID-19 pandemic continues to threaten the lives of hundreds of millions of people, with a severe negative impact on the global economy. Although several COVID-19 vaccines are currently being administered, none of them is 100% effective. Moreover, SARS-CoV-2 variants remain an important worldwide public health issue. Hence, the accelerated development of efficacious antiviral agents is urgently needed. Coronavirus depends on various host cell factors for replication. An ongoing research objective is the identification of host factors that could be exploited as targets for drugs and compounds effective against SARS-CoV-2. In the present review, we discuss the molecular mechanisms of SARS-CoV-2 and related coronaviruses, focusing on the host factors or pathways involved in SARS-CoV-2 replication that have been identified by genome-wide CRISPR screening.

Quantifying Neutralizing Antibodies in Patients with COVID-19 by a Two-Variable Generalized Additive Model.

Considering the urgent demand for faster methods to quantify neutralizing antibody titers in patients with coronavirus (CoV) disease 2019 (COVID-19), developing an analytical model or method to replace the conventional virus neutralization test (NT) is essential. Moreover, a "COVID-19 immunity passport" is currently being proposed as a certification for people who travel internationally. Therefore, an enzyme-linked immunosorbent assay (ELISA) was designed to detect severe acute respiratory syndrome CoV 2 (SARS-CoV-2)-neutralizing antibodies in serum, which is based on the binding affinity of SARS-CoV-2 viral spike protein 1 (S1) and the viral spike protein receptor-binding domain (RBD) to antibodies. The RBD is considered the major binding region of neutralizing antibodies. Furthermore, S1 covers the RBD and several other regions, which are also important for neutralizing antibody binding. In this study, we assessed 144 clinical specimens, including those from patients with PCR-confirmed SARS-CoV-2 infections and healthy donors, using both the NT and ELISA. The ELISA results analyzed by spline regression and the two-variable generalized additive model precisely reflected the NT value, and the correlation between predicted and actual NT values was as high as 0.917. Therefore, our method serves as a surrogate to quantify neutralizing antibody titer. The analytic method and platform used in this study present a new perspective for serological testing of SARS-CoV-2 infection and have clinical potential to assess vaccine efficacy. IMPORTANCE Herein, we present a new approach for serological testing for SARS-CoV-2 antibodies using innovative laboratory methods that demonstrate a combination of biology and mathematics. The traditional virus neutralization test is the gold standard method; however, it is time-consuming and poses a risk to medical personnel. Thus, there is a demand for methods that rapidly quantify neutralizing antibody titers in patients with COVID-19 or examine vaccine efficacy at a biosafety level 2 containment facility. Therefore, we used a two-variable generalized additive model to analyze the results of the enzyme-linked immunosorbent assay and found the method to serve as a surrogate to quantify neutralizing antibody titers. This methodology has potential for clinical use in assessing vaccine efficacy.

Acyl-Coenzyme A Synthetase Long-Chain Family Member 4 Is Involved in Viral Replication Organelle Formation and Facilitates Virus Replication via Ferroptosis.

Enterovirus infections can cause severe complications, such as poliomyelitis, encephalitis, myocarditis, meningitis, neurological pulmonary edema, and even death. Here, we used genome-wide CRISPR screens to gain new insight into the mechanism by which enteroviruses co-opt host pathways to potentiate replication and propagation. We found that acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) is involved in viral replication organelle formation. ACSL4 is a key component of ferroptosis, an iron-dependent, nonapoptotic programmed cell death. Our results indicated that enteroviruses and coronaviruses can induce ferroptosis via ACSL4. Most importantly, ferroptosis inhibitors, including two FDA-approved drugs, rosiglitazone (ROSI; ACSL4 inhibitor) and pioglitazone (PIO; ACSL4 inhibitor), decreased the viral load of human enteroviruses and coronaviruses, suggesting that ACSL4 is a target for counteracting viral infection. IMPORTANCE We provide the first evidence for the role of ACSL4 in enterovirus replication organelle formation. Moreover, both enteroviruses and coronaviruses induce ferroptosis via ACSL4. These findings establish a novel regulatory mechanism for viral replication. The inhibition of ACSL4 by ferroptosis inhibitors can reduce viral yields of enteroviruses and coronaviruses, including SARS-CoV-2, implying that ACSL4-mediated ferroptosis is a promising therapeutic target for viral diseases.

Learning of 3D Graph Convolution Networks for Point Cloud Analysis.

Point clouds are among the popular geometry representations in 3D vision. However, unlike 2D images with pixel-wise layouts, such representations containing unordered data points which make the processing and understanding the associated semantic information quite challenging. Although a number of previous works attempt to analyze point clouds and achieve promising performances, their performances would degrade significantly when data variations like shift and scale changes are presented. In this paper, we propose 3D graph convolution networks (3D-GCN), which uniquely learns 3D kernels with graph max-pooling mechanisms for extracting geometric features from point cloud data across different scales. We show that, with the proposed 3D-GCN, satisfactory shift and scale invariance can be jointly achieved. We show that 3D-GCN can be applied to point cloud classification and segmentation tasks, with ablation studies and visualizations verifying the design of 3D-GCN.

Stability of SARS-CoV-2 Spike G614 Variant Surpasses That of the D614 Variant after Cold Storage.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) carrying the D614G mutation on the spike protein is the predominant circulating variant and is associated with enhanced infectivity. However, whether this dominant variant can potentially spread through the cold chain and whether the spike protein affects virus stability after cold storage remain unclear. To compare the infectivity of two SARS-CoV-2 variants, namely, SARS-CoV-2 variants with spike protein with the D614 mutation (S-D614) and G614 mutation (S-G614), after different periods of refrigeration (4°C) and freezing (-20°C). We also determined the integrity of the viral RNA and the ability of the spike protein to bind angiotensin-converting enzyme 2 (ACE2) after storage at these conditions. The results showed that SARS-CoV-2 was more stable and infectious after storage at -20°C than at 4°C. Particularly, the S-G614 variant was found to be more stable than the S-D614 variant. The spike protein of the S-G614 variant had better binding ability with the ACE2 receptor than that of the S-D614 variant after storage at -20°C for up to 30 days. Our findings revealed that SARS-CoV-2 remains stable and infectious after refrigeration or freezing, and their stability and infectivity up to 30 days depends on the spike variant. Stability and infectivity are related to each other, and the higher stability of S-G614 compared to that of S-D614 may contribute to rapid viral spread of the S-G614 variant.IMPORTANCE It has been observed that variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more stable and infectious after storage at -20°C than at 4°C. A SARS-CoV-2 S-D614G variant is currently the most dominant variant in circulation and is associated with enhanced infectivity. We compared the stability of two SARS-CoV-2 variants: the early S-D614 variant carrying the D614 spike protein and the new S-G614 variant carrying the G614 spike protein, stored at both 4°C and -20°C for different periods. We observed that SARS-CoV-2 remains stable and infectious after refrigeration or freezing, which further depends on the spike variant, that is, the ability of the spike protein to bind with the ACE2 receptor with higher efficiency. The high stability of the S-G614 variant also explains its rapid spread and infectivity. Therefore, precautions should be taken during and after handling food preserved under cold conditions.

Development of flexible electrochemical impedance spectroscopy-based biosensing platform for rapid screening of SARS-CoV-2 inhibitors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the cells through the binding of its spike protein (S-protein) to the cell surface-expressing angiotensin-converting enzyme 2 (ACE2). Thus, inhibition of S-protein-ACE2 binding may impede SARS-CoV-2 cell entry and attenuate the progression of Coronavirus disease 2019 (COVID-19). In this study, an electrochemical impedance spectroscopy-based biosensing platform consisting of a recombinant ACE2-coated palladium nano-thin-film electrode as the core sensing element was fabricated for the screening of potential inhibitors against S-protein-ACE2 binding. The platform could detect interference of small analytes against S-protein-ACE2 binding at low analyte concentration and small volume (0.1 µg/mL and ~1 µL, estimated total analyte consumption < 4 pg) within 21 min. Thus, a few potential inhibitors of S-protein-ACE2 binding were identified. This includes (2S,3aS,6aS)-1-((S)-N-((S)-1-Carboxy-3-phenylpropyl)alanyl)tetrahydrocyclopenta[b] pyrrole-2-carboxylic acid (ramiprilat) and (2S,3aS,7aS)-1-[(2S)-2-[[(2S)-1-Carboxybutyl]amino]propanoyl]-2,3,3a,4,5,6,7,7a-octahydroindole-2-carboxylic acid (perindoprilat) that reduced the binding affinity of S-protein to ACE2 by 72% and 67%; and SARS-CoV-2 in vitro infectivity to the ACE2-expressing human oral cavity squamous carcinoma cells (OEC-M1) by 36.4 and 20.1%, respectively, compared to the PBS control. These findings demonstrated the usefulness of the developed biosensing platform for the rapid screening of modulators for S-protein-ACE2 binding.

Insights into the Regulation on Proliferation and Differentiation of Stem Leydig Cells.

Male hypogonadism is a clinical syndrome caused by testosterone deficiency. Hypogonadism can be caused by testicular disease (primary hypogonadism) or hypothalamic-pituitary dysfunction (secondary hypogonadism). The present strategy for treating hypogonadism is the administration of exogenous testosterone. But exogenous testosterone is reported to have negative side effects including adverse cardiovascular events and disruption of physiological spermatogenesis probably due to its inability to mimic the physiological circadian rhythm of testosterone secretion in vivo. In recent years, a growing number of articles demonstrated that stem Leydig cells (SLCs) can not only differentiate into functional Leydig cells (LCs) in vivo to replace chemically disrupted LCs, but also secrete testosterone in a physiological pattern. The proliferation and differentiation of SLCs are regulated by various factors. However, the mechanisms involved in regulating the development of SLCs remain to be summarized. Factors involved in the regulation of SLCs can be divided into environmental pollutants, growth factors, cytokine and hormones. Environmental pollutants such as Perfluorooctanoic acid (PFOA) and Triphenyltin (TPT) could suppress SLCs proliferation or differentiation. Growth factors including FGF1, FGF16, NGF and activin A are essential for the maintenance of SLCs self-renewal and differentiation. Interleukin 6 family could inhibit differentiation of SLCs. Among hormones, dexamethasone suppresses SLCs differentiation, while aldosterone suppresses their proliferation. The present review focuses on new progress about factors regulating SLC's proliferation and differentiation which will undoubtedly deepen our insights into SLCs and help make better clinical use of them. Different factors affect on the proliferation and differentiation of stem Leydig cells. Firstly, each rat was intraperitoneally injected EDS so as to deplete Leydig cells from the adult testis. Secondly, the CD51+ or CD90+ cells from the testis of rats are SLCs, and the p75+ cells from human adult testes are human SLCs. These SLCs in the testis start to proliferate and some of them differentiate into LCs. Thirdly, during the SLCs regeneration period, researchers could explore different function of those factors (pollutants, growth factors, cytokines and hormones) towards SLCs.

Discovery of M Protease Inhibitors Encoded by SARS-CoV-2.

The coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a health threat worldwide. Viral main protease (Mpro, also called 3C-like protease [3CLpro]) is a therapeutic target for drug discovery. Herein, we report that GC376, a broad-spectrum inhibitor targeting Mpro in the picornavirus-like supercluster, is a potent inhibitor for the Mpro encoded by SARS-CoV-2, with a half-maximum inhibitory concentration (IC50) of 26.4 ± 1.1 nM. In this study, we also show that GC376 inhibits SARS-CoV-2 replication with a half-maximum effective concentration (EC50) of 0.91 ± 0.03 µM. Only a small portion of SARS-CoV-2 Mpro was covalently modified in the excess of GC376 as evaluated by mass spectrometry analysis, indicating that improved inhibitors are needed. Subsequently, molecular docking analysis revealed that the recognition and binding groups of GC376 within the active site of SARS-CoV-2 Mpro provide important new information for the optimization of GC376. Given that sufficient safety and efficacy data are available for GC376 as an investigational veterinary drug, expedited development of GC376, or its optimized analogues, for treatment of SARS-CoV-2 infection in human is recommended.

SARS-CoV-2 genomic surveillance in Taiwan revealed novel ORF8-deletion mutant and clade possibly associated with infections in Middle East.

Taiwan experienced two waves of imported infections with Coronavirus Disease 2019 (COVID-19). This study aimed at investigating the genomic variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Taiwan and compared their evolutionary trajectories with the global strains. We performed culture and full-genome sequencing of SARS-CoV-2 strains followed by phylogenetic analysis. A 382-nucleotides deletion in open reading frame 8 (ORF8) was found in a Taiwanese strain isolated from a patient on February 4, 2020 who had a travel history to Wuhan. Patients in the first wave also included several sporadic, local transmission cases. Genomes of 5 strains sequenced from clustered infections were classified into a new clade with ORF1ab-V378I mutation, in addition to 3 dominant clades ORF8-L84S, ORF3a-G251V and S-D614G. This highlighted clade also included some strains isolated from patients who had a travel history to Turkey and Iran. The second wave mostly resulted from patients who had a travel history to Europe and Americas. All Taiwanese viruses were classified into various clades. Genomic surveillance of SARS-CoV-2 in Taiwan revealed a new ORF8-deletion mutant and a virus clade that may be associated with infections in the Middle East, which contributed to a better understanding of the global SARS-CoV-2 transmission dynamics.

Comparison of hepatic resection and systemic treatment of breast cancer liver metastases: A propensity score matching study.

BACKGROUND: The role of surgery in breast cancer liver metastases (BCLM) remains elusive, and current application is limited. Our aim is to investigate whether hepatic resection (HR) of BCLM improves survival compared with non-hepatic resection (NHR) treatment. METHODS: Three hundred and eighty-four patients with BCLM from 2008 to 2018 were divided into two groups. Propensity score matching (PSM) analysis was used to compare the clinical outcomes. RESULTS: After PSM the mean overall survival (OS) and the 1, 3, and 5-year OS rates in HR group were 61.8 months, 92.6%, 54.7% and 54.7%, respectively; while for NHR group these values were 38.6 months, 79.2%, 45.6% and 21.9%, respectively (p < 0.007). Multivariate analysis indicated hormonal receptor status (p = 0.039) and hepatic resection (p = 0.032) were independent prognostic factors. CONCLUSION: Our study revealed that hepatectomy yields a survival benefit safely compared with medical treatments, especially for patients with positive hormonal receptors.

Novel Role for miR-1290 in Host Species Specificity of Influenza A Virus.

The role of microRNA (miRNA) in influenza A virus (IAV) host species specificity is not well understood as yet. Here, we show that a host miRNA, miR-1290, is induced through the extracellular signal-regulated kinase (ERK) pathway upon IAV infection and is associated with increased viral titers in human cells and ferret animal models. miR-1290 was observed to target and reduce expression of the host vimentin gene. Vimentin binds with the PB2 subunit of influenza A virus ribonucleoprotein (vRNP), and knockdown of vimentin expression significantly increased vRNP nuclear retention and viral polymerase activity. Interestingly, miR-1290 was not detected in either chicken cells or mouse animal models, and the 3' UTR of the chicken vimentin gene contains no binding site for miR-1290. These findings point to a host species-specific mechanism by which IAV upregulates miR-1290 to disrupt vimentin expression and retain vRNP in the nucleus, thereby enhancing viral polymerase activity and viral replication.

Monitoring the Disulfide Bonds of Folding Isomers of Synthetic CTX A3 Polypeptide Using MS-Based Technology.

Native disulfide formation is crucial to the process of disulfide-rich protein folding in vitro. As such, analysis of the disulfide bonds can be used to track the process of the folding reaction; however, the diverse structural isomers interfere with characterization due to the non-native disulfide linkages. Previously, a mass spectrometry (MS) based platform coupled with peptide demethylation and an automatic disulfide bond searching engine demonstrated the potential to screen disulfide-linked peptides for the unambiguous assignment of paired cysteine residues of toxin components in cobra venom. The developed MS-based platform was evaluated to analyze the disulfide bonds of structural isomers during the folding reaction of synthetic cardiotoxin A3 polypeptide (syn-CTX A3), an important medical component in cobra venom. Through application of this work flow, a total of 13 disulfide-linked peptides were repeatedly identified across the folding reaction, and two of them were found to contain cysteine pairings, like those found in native CTX A3. Quantitative analysis of these disulfide-linked peptides showed the occurrence of a progressive disulfide rearrangement that generates a native disulfide bond pattern on syn-CTX A3 folded protein. The formation of these syn-CTX A3 folded protein reaches a steady level in the late stage of the folding reaction. Biophysical and cell-based assays showed that the collected syn-CTX A3 folded protein have a ß-sheet secondary structure and cytotoxic activity similar to that of native CTX A3. In addition, the immunization of the syn-CTX A3 folded proteins could induce neutralization antibodies against the cytotoxic activity of native CTX A3. In contrast, these structure activities were poorly observed in the other folded isomers with non-native disulfide bonds. The study highlights the ability of the developed MS platform to assay isomers with heterogeneous disulfide bonds, providing insight into the folding mechanism of the bioactive protein generation.

Application of stable isotope dimethyl labeling for MRM based absolute antigen quantification of influenza vaccine.

Determining the precursor/product ion pair and optimal collision energy are the critical steps for developing a multiple reaction monitoring (MRM) assay using triple quadruple mass spectrometer for protein quantitation. In this study, a platform consisting of stable isotope dimethyl labeling coupled with triple-quadruple mass spectrometer was used to quantify the protein components of the influenza vaccines. Dimethyl labeling of both the peptide N-termini and the ϵ-amino group of lysine residues was achieved by reductive amination using formaldehyde and sodium cyanoborohydrate. Dimethylated peptides are known to exhibit dominant a1 ions under gas phase fragmentation in a mass spectrometer. These a1 ions can be predicted from the peptide N-terminal amino acids, and their signals do not vary significantly across a wide range of collision energies, which facilitates the determination of MRM transition settings for multiple protein targets. The intrinsic a1 ions provide sensitivity for acquiring MRM peaks that is superior to that of the typical b/y ions used for native peptides, and they also provided good linearity (R2 ≥ 0.99) at the detected concentration range for each peptide. These features allow for the simultaneous quantification of hemagglutinin and neuraminidase in vaccines derived from either embryo eggs or cell cultivation. Moreover, the low abundant ovalbumin residue originated from the manufacturing process can also be determined. The results demonstrate that the stable isotope dimethyl labeling coupled with MRM Mass spectrometry screening of a1 ions (i.e., SIDa-MS) can be used as a high-throughput platform for multiple protein quantification of vaccine products.

De novo protein sequencing, humanization and in vitro effects of an antihuman CD34 mouse monoclonal antibody.

QBEND/10 is a mouse immunoglobulin lambda-chain monoclonal antibody with strict specificity against human hematopoietic progenitor cell antigen CD34. Our in vitro study showed that QBEND/10 impairs the tube formation of human umbilical vein endothelial cells (HUVECs), suggesting that the antibody may be of potential benefit in blocking tumor angiogenesis. We provided a de novo protein sequencing method through tandem mass spectrometry to identify the amino acid sequences in the variable heavy and light chains of QBEND/10. To reduce immunogenicity for clinical applications, QBEND/10 was further humanized using the resurfacing approach. We demonstrate that the de novo sequenced and humanized QBEND/10 retains the biological functions of the parental mouse counterpart, including the binding kinetics to CD34 and blockage of the tube formation of the HUVECs.