A rapid 2AB-UHPLC method based on magnetic beads extraction for N-glycan analysis of recombinant monoclonal antibody.

N-glycosylation is one of the major post-translational modifications, with significant effects on the mechanism of action, the efficacy, and the safety of antibody drugs or glycoproteins. With the growing application of therapeutic antibodies, routinely monitoring N-glycosylation becomes increasingly important during cell culture process development and quality control. However, the current pretreatment methods for N-glycan analysis are time- and labor-consuming. The purification procedure of enzymatically released glycans could also partly affect the accuracy of results due to its complexity. In this study, a rapid ultra-high performance liquid chromatography method based on magnetic bead extraction and 2-AB fluorescent labeling was developed and compared against three popular pretreatment methods for N-glycan profiling (two were solid phase extraction and the other was acetone precipitation). The method's repeatability results showed that magnetic bead extraction has higher precision (% relative standard deviation (RSD), 0.121.06%) than solid phase extraction (SPE) (%RSD, 0.38-8.02%) and acetone precipitation (%RSD, 0.42-8.58%). This robust pretreatment method also maximized the retention of some low abundance oligosaccharides, and may thus provide a rapid and high-throughput workflow option for N-Glycan analysis in the biopharmaceutical industry.

Low endotoxin recovery and its impact on endotoxin detection.

Endotoxin exists on the outer membrane of Gram-negative bacteria and poses risks to human health by triggering a series of immune responses. Therefore, its accurate detection is essential. The Limulus amoebocyte lysate (LAL) test is the most pharmacopeia-recognized and popular technique for endotoxin detection. Despite its wide industry adoption, the low endotoxin recovery (LER) phenomenon can compromise the LAL test's reliability. This review summarizes the possible reasons attributing to the LER phenomenon from three different perspectives: the endotoxin standards used in hold time study, protein active pharmaceutical ingredients, and excipients. Potential mechanisms and strategies to mitigate the LER phenomenon are also discussed as presented by different research groups.

Effect of different sample treatment methods on Low Endotoxin Recovery Phenomenon.

Endotoxin is a kind of lipopolysaccharide that exits on the cell wall of Gram-negative bacteria. It can cause fever, shock or even death when is delivered into human body. So, it is necessary to control the endotoxin contamination for biopharmaceutical products that are mainly administered by intravenous route. Limulus Amebocyte Lysate (LAL)-based tests are usually used to detect endotoxin content in biologics formulations. However, an undesirable phenomenon called "Low Endotoxin Recovery (LER)" often occurs in formulation buffers that usually contain chelating component, such as sodium citrate, and amphiphilic surfactant, such as Tween-20. The occurrence of this LER phenomenon may interfere with endotoxin detection and cause false negative results. In this study, we compared the effect of different sample treatment methods on endotoxin detection and found that the LER phenomenon was better controlled under the conditions of low pH (pH = 5.0), low temperature (2-8 °C) and in the presence of divalent cations in the solution. In addition, although the endotoxin activity was found to have decreased due to LER phenomenon, the particle size distribution of endotoxin determined by dynamic light scattering (DLS) in LER solution did not change obviously, which is different from previous hypothesis about LER phenomenon in literature that the particle size of endotoxin aggregates would decrease under LER conditions. These findings provide some insights into different sample treatment methods for endotoxin detection and give a better understanding and solution on minimizing the LER phenomenon.

Factor analysis of temperament and personality traits in bipolar patients: Correlates with comorbidity and disorder severity.

BACKGROUND: Temperament and personality traits have been suggested as endophenotypes for bipolar disorder based on several lines of evidence, including heritability. Previous work suggested an anxious-reactive factor identified across temperament and personality inventories that produced significant group discrimination and could potentially be useful in genetic analyses. We have attempted to further characterize this factor structure in a sample of bipolar patients. METHODS: A sample of 1195 subjects with bipolar I disorder was evaluated, all with complete data available. Dimension reduction across two inventories identified 18 factors explaining 39% of the variance. RESULTS: The two largest factors reflected affective instability and general anxiety/worry, respectively. Subsequent analyses of the clinical features associated with bipolar disorder revealed specificity for the factors in a predictable pattern. Cluster analysis of the factors identified a subgroup defined by a strong lack of general anxiety and low affective instability represented by the first two factors. The remaining subjects could be distinguished into two clusters by the presence of either more positive characteristics, including persistence/drive, spirituality, expressivity, and humor, or more negative characteristics of depression and anxiety. LIMITATIONS: These analyses involved bipolar I subjects only and must be extended to other bipolar spectrum diagnoses, unaffected relatives, and individuals at risk. CONCLUSIONS: These results suggest that temperament and personality measures access latent traits associated with important clinical features of bipolar disorder. By translating clinical variables into quantitative traits, we may identify subgroups of bipolar patients with distinct clinical profiles, thereby facilitating both individual treatment strategies and genetic analyses.

Nitric oxide-driven hypoxia initiates synovial angiogenesis, hyperplasia and inflammatory lesions in mice.

BACKGROUND: Rheumatoid arthritis (RA) is an inflammatory articular disease with cartilage and bone damage due to hyperplasic synoviocyte invasion and subsequent matrix protease digestion. Although monoclonal antibodies against tumor necrosis factor alpha (TNFα) have been approved for clinical use in patients with RA, desired therapeutic regimens suitable for non-responders are still unavailable because etiological initiators leading to RA remain enigmatic and unidentified. METHODOLOGY/PRINCIPAL FINDINGS: Bacteria-induced arthritis (BIA) that simulates collagen-induced arthritis (CIA) is developed in mice upon daily live bacterial feeding. The morphological lesions of paw erythema and edema together with the histological alterations of synovial hyperplasia and lymphocytic infiltration emerge as the early-phase manifestations of BIA and CIA. Bacteria- or collagen-mediated global upregulation of pro-inflammatory cytokines is accompanied by the burst of nitric oxide (NO). Elevation of the serum NO level is correlated with decline of the blood oxygen saturation percentage (SpO2), reflecting a hypoxic consequence during development towards arthritis. NO-driven hypoxia is further evident from a positive relationship between NO and lactic acid (LA), an end product from glycolysis. Upregulation of hypoxia inducible factor 1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) validates hypoxia-induced angiogenesis in the inflamed synovium of modeling mice. Administration of the NO donor compound sodium nitroprusside (SNP) causes articular inflammation by inducing synovial hypoxia. Anti-bacteria by the antibiotic cefotaxime and/or the immunosuppressant rapamycin or artesunate that also inhibits nitric oxide synthase (NOS) can abrogate NO production, mitigate hypoxia, and considerably ameliorate or even completely abort synovitis, hence highlighting that NO may serve as an initiator of inflammatory arthritis. CONCLUSIONS/SIGNIFICANCE: Like collagen, bacteria also enable synovial lesions via upregulating pro-inflammatory cytokines, triggering NO production, driving hypoxic responses, and inducing synovial angiogenesis and hyperplasia, suggesting that sustained infection might be, in part, responsible for the onset of synovitis and arthritis in mice.

The autoimmune disease-associated PTPN22 variant promotes calpain-mediated Lyp/Pep degradation associated with lymphocyte and dendritic cell hyperresponsiveness.

A variant of the PTPN22-encoded Lyp phosphatase (Lyp620W) confers risk for autoimmune disease, but the mechanisms underlying this association remain unclear. We show here that mice expressing the Lyp variant homolog Pep619W manifest thymic and splenic enlargement accompanied by increases in T-cell number, activation and positive selection and in dendritic- and B-cell activation. Although Ptpn22 (Pep) transcript levels were comparable in Pep619W and wild-type Pep619R mice, Pep protein levels were dramatically reduced in the mutant mice, with Pep619W protein being more rapidly degraded and showing greater association with and in vitro cleavage by calpain 1 than Pep619R. Similarly, levels of the Lyp620W variant were decreased in human T and B cells, and its calpain binding and cleavage were increased relative to wild-type Lyp620R. Thus, calpain-mediated degradation with consequently reduced Lyp/Pep expression and lymphocyte and dendritic cell hyperresponsiveness represents a mechanism whereby Lyp620W may increase risk for autoimmune disease.

Artesunate potentiates antibiotics by inactivating heme-harbouring bacterial nitric oxide synthase and catalase.

BACKGROUND: A current challenge of coping with bacterial infection is that bacterial pathogens are becoming less susceptible to or more tolerant of commonly used antibiotics. It is urgent to work out a practical solution to combat the multidrug resistant bacterial pathogens. FINDINGS: Oxidative stress-acclimatized bacteria thrive in rifampicin by generating antibiotic-detoxifying nitric oxide (NO), which can be repressed by artesunate or an inhibitor of nitric oxide synthase (NOS). Suppressed bacterial proliferation correlates with mitigated NO production upon the combined treatment of bacteria by artesunate with antibiotics. Detection of the heme-artesunate conjugate and accordingly declined activities of heme-harbouring bacterial NOS and catalase indicates that artesunate renders bacteria susceptible to antibiotics by alkylating the prosthetic heme group of hemo-enzymes. CONCLUSIONS: By compromising NO-mediated protection from antibiotics and triggering harmful hydrogen peroxide burst, artesunate may serve as a promising antibiotic synergist for killing the multidrug resistant pathogenic bacteria.

N-WASp is required for Schwann cell cytoskeletal dynamics, normal myelin gene expression and peripheral nerve myelination.

Schwann cells elaborate myelin sheaths around axons by spirally wrapping and compacting their plasma membranes. Although actin remodeling plays a crucial role in this process, the effectors that modulate the Schwann cell cytoskeleton are poorly defined. Here, we show that the actin cytoskeletal regulator, neural Wiskott-Aldrich syndrome protein (N-WASp), is upregulated in myelinating Schwann cells coincident with myelin elaboration. When N-WASp is conditionally deleted in Schwann cells at the onset of myelination, the cells continue to ensheath axons but fail to extend processes circumferentially to elaborate myelin. Myelin-related gene expression is also severely reduced in the N-WASp-deficient cells and in vitro process and lamellipodia formation are disrupted. Although affected mice demonstrate obvious motor deficits these do not appear to progress, the mutant animals achieving normal body weights and living to advanced age. Our observations demonstrate that N-WASp plays an essential role in Schwann cell maturation and myelin formation.

Production of artemisinin by genetically-modified microbes.

Artemisinin, an endoperoxidized sesquiterpene originally extracted from the medicinal plant Artemisia annua L., is a potent malaria-killing agent. Due to the urgent demand and short supply of this new antimalarial drug, engineering enhanced production of artemisinin by genetically-modified or transgenic microbes is currently being explored. Cloning and expression of the artemisinin biosynthetic genes in Saccharomyces cerevisiae and Escherichia coli have led to large-scale microbial production of the artemisinin precursors such as amorpha-4,11-diene and artemisinic acid. Although reconstruction of the complete biosynthetic pathway toward artemisinin in transgenic yeast and bacteria has not been achieved, artemisinic acid available from these transgenic microbes facilitates the subsequent partial synthesis of artemisinin by either chemical or biotransformational process, thereby providing an attractive strategy alternative to the direct extraction of artemisinin from A.annua L. In this review, we update the current trends and summarize the future prospects on genetic engineering of the microorganisms capable of accumulating artemisinin precursors through heterologous and functional expression of the artemisinin biosynthetic genes.