Optimized quantification of fatty acids in complex media using advanced analytical techniques.

RATIONALE: Various medium formulations contain essential fatty acids at concentrations ranging from 10 to 100 mg/L. Accurate and precise lipid measurement in media is crucial for monitoring media quality and conducting studies on lipids in the context of cell culture. This study employed two-dimensional gas chromatography (GC × GC) analyses to offer enhanced resolution, sensitivity, and separation performance compared to GC. METHODS: Quantification of fatty acid methyl esters (FAMEs) in a medium was conducted using GC × GC combined with a high-resolution mass spectrometer and flame ionization detector, considering potential interference from nonionic surfactant Tween 80, which was precipitated and removed by optimizing the concentration of cobalt thiocyanate (CTA) solution during pretreatment. This advanced analytical approach enabled identification of cis and trans isomers of identical molecular weights and determination of the location and number of double bonds in the same carbon number structure. RESULTS: Our analysis identified 36 FAMEs within the C6-C24 region, and a 5% CTA solution was optimal for efficient removal of Tween 80 during lipid extraction. Additionally, this advanced method minimized FAME contamination and loss during pretreatment, thereby significantly reducing the sample volume required to detect trace levels of FAMEs. This improvement led to a fatty acid recovery rate of 106% while maintaining the average relative standard deviation for the target FAMEs of about 3%. CONCLUSIONS: Our research paves the way for future investigation into medium quality control and the role of fatty acids in cell culture. This offers the possibility for economical and effective trace quantification of fatty acids in complex media.

CELLULAR RNA INTERACTS WITH MAVS TO PROMOTE ANTIVIRAL SIGNALING.

Immune signaling needs to be well-regulated to promote clearance of pathogens, while preventing aberrant inflammation. Interferons (IFNs) and antiviral genes are activated by the detection of viral RNA by RIG-I-like receptors (RLRs). Signal transduction downstream of RLRs proceeds through a multi-protein complex organized around the central adaptor protein MAVS. Recent work has shown that protein complex function can be modulated by RNA molecules providing allosteric regulation or acting as molecular guides or scaffolds. Thus, we hypothesized that RNA plays a role in organizing MAVS signaling platforms. Here, we show that MAVS, through its central intrinsically disordered domain, directly interacts with the 3' untranslated regions of cellular mRNAs. Importantly, elimination of RNA by RNase treatment disrupts the MAVS signalosome, including newly identified regulators of RLR signaling, and inhibits phosphorylation of the transcription factor IRF3. This supports the hypothesis that RNA molecules scaffold proteins in the MAVS signalosome to induce IFNs. Together, this work uncovers a function for cellular RNA in promoting signaling through MAVS and highlights a generalizable principle of RNA regulatory control of cytoplasmic immune signaling complexes.

Comparative analysis of organic chemical compositions in airborne particulate matter from Ulaanbaatar, Beijing, and Seoul using UPLC-FT-ICR-MS and artificial neural network.

This paper presents comparative study on the composition and sources of PM2.5 in Ulaanbaatar, Beijing, and Seoul. Ultrahigh performance liquid chromatography (UPLC) combined with ultrahigh resolution mass spectrometry (UHR-MS) were employed to analyze 85 samples collected in winter. The obtained 340 spectra were interpreted with artificial neural network (ANN). PM2.5 mass concentrations in Ulaanbaatar were significantly higher than those in Beijing and Seoul. ANN based interpretation of UPLC UHR-MS data showed that aliphatic/lipid derived organo­sulfur compounds, polycyclic aromatic and organo­oxygen compounds were characteristic to Ulaanbaatar. Whereas, aliphatic/lipid-derived organo­oxygen compounds were major components in Beijing and Seoul. Aromatic organo­nitrogen compounds were the main contributors to differentiating the spectra obtained from Beijing from the other cities. Based on two-dimensional gas chromatography/high resolution mass spectrometric (GCxGC/HRMS) data, it was determined that the concentrations of the polycyclic aromatic hydrocarbon (PAH) and polycyclic aromatic sulfur heterocycle (PASH) containing sulfur were highest in Ulaanbaatar, followed by Beijing and Seoul. Coal/biomass combustion was identified as the primary source of contamination in Ulaanbaatar, while petroleum combustion was the main contributor to PM2.5 in Beijing and Seoul. The conclusion that diesel-powered heavy-duty trucks and buses are the main contributors to NOx emissions in Beijing is consistent with previous reports. This study provides a more comprehensive understanding of the composition and sources of PM2.5 in the three cities, with a focus on the differences in their atmospheric pollution profiles based on the UPLC UHR-MS and ANN analysis. It is notable that this study is the first to utilize this method on a large-scale sample set, providing a more detailed and molecular-level understanding of the compositional differences among PM2.5. Overall, the study contributes to a better understanding of the sources and composition of PM2.5 in Northeast Asia, which is essential for developing effective strategies to reduce air pollution and improve public health.

Advanced assessment through intact glycopeptide analysis of Infliximab's biologics and biosimilar.

Characterization of therapeutic monoclonal antibodies (mAbs) represents a major challenge for analytical sciences due to their heterogeneity associated with post-translational modifications (PTMs). The protein glycosylation requires comprehensive identification, which could influence on the mAbs' structure and their function. Here, we demonstrated high-resolution tandem mass spectrometry with an ultra-high-performance liquid chromatography for characterization and comparison between biologics and biosimilar of infliximab at an advanced level. Comparing the N- and O-glycopeptides profiles, a total of 49 and 54 glycopeptides was identified for each product of the biologics and biosimilar, respectively. We also discovered one novel N-glycosylation site at the light chain from both biopharmaceuticals and one novel O-glycopeptide at the heavy chain from only biosimilar. Site-specific glycopeptide analysis process will be a robust and useful technique for evaluating therapeutic mAbs and complex glycoprotein products.

Signaling from the RNA sensor RIG-I is regulated by ufmylation.

The RNA-binding protein RIG-I is a key initiator of the antiviral innate immune response. The signaling that mediates the antiviral response downstream of RIG-I is transduced through the adaptor protein MAVS and results in the induction of type I and III interferons (IFNs). This signal transduction occurs at endoplasmic reticulum (ER)­mitochondrial contact sites, to which RIG-I and other signaling proteins are recruited following their activation. RIG-I signaling is highly regulated to prevent aberrant activation of this pathway and dysregulated induction of IFN. Previously, we identified UFL1, the E3 ligase of the ubiquitin-like modifier conjugation system called ufmylation, as one of the proteins recruited to membranes at ER­mitochondrial contact sites in response to RIG-I activation. Here, we show that UFL1, as well as the process of ufmylation, promote IFN induction in response to RIG-I activation. We found that following RNA virus infection, UFL1 is recruited to the membrane-targeting protein 14­3-3ε and that this complex is then recruited to activated RIG-I to promote downstream innate immune signaling. Importantly, we found that 14­3-3ε has an increase in UFM1 conjugation following RIG-I activation. Additionally, loss of cellular ufmylation prevents the interaction of 14­3-3ε with RIG-I, which abrogates the interaction of RIG-I with MAVS and thus the downstream signal transduction that induces IFN. Our results define ufmylation as an integral regulatory component of the RIG-I signaling pathway and as a posttranslational control for IFN induction.

FTO Suppresses STAT3 Activation and Modulates Proinflammatory Interferon-Stimulated Gene Expression.

Signaling initiated by type I interferon (IFN) results in the induction of hundreds of IFN-stimulated genes (ISGs). The type I IFN response is important for antiviral restriction, but aberrant activation of this response can lead to inflammation and autoimmunity. Regulation of this response is incompletely understood. We previously reported that the mRNA modification m6A and its deposition enzymes, METTL3 and METTL14 (METTL3/14), promote the type I IFN response by directly modifying the mRNA of a subset of ISGs to enhance their translation. Here, we determined the role of the RNA demethylase fat mass and obesity-associated protein (FTO) in the type I IFN response. FTO, which can remove either m6A or cap-adjacent m6Am RNA modifications, has previously been associated with obesity and body mass index, type 2 diabetes, cardiovascular disease, and inflammation. We found that FTO suppresses the transcription of a distinct set of ISGs, including many known pro-inflammatory genes, and that this regulation requires its catalytic activity but is not through the actions of FTO on m6Am. Interestingly, depletion of FTO led to activation of the transcription factor STAT3, whose role in the type I IFN response is not well understood. This activation of STAT3 increased the expression of a subset of ISGs. Importantly, this increased ISG induction resulting from FTO depletion was partially ablated by depletion of STAT3. Together, these results reveal that FTO negatively regulates STAT3-mediated signaling that induces proinflammatory ISGs during the IFN response, highlighting an important role for FTO in suppression of inflammatory genes.

The small GTPase RAB1B promotes antiviral innate immunity by interacting with TNF receptor-associated factor 3 (TRAF3).

Innate immune detection of viral nucleic acids during viral infection activates a signaling cascade that induces type I and type III IFNs as well as other cytokines, to generate an antiviral response. This signaling is initiated by pattern recognition receptors, such as the RNA helicase retinoic acid-inducible gene I (RIG-I), that sense viral RNA. These sensors then interact with the adaptor protein mitochondrial antiviral signaling protein (MAVS), which recruits additional signaling proteins, including TNF receptor-associated factor 3 (TRAF3) and TANK-binding kinase 1 (TBK1), to form a signaling complex that activates IFN regulatory factor 3 (IRF3) for transcriptional induction of type I IFNs. Here, using several immunological and biochemical approaches in multiple human cell types, we show that the GTPase-trafficking protein RAB1B up-regulates RIG-I pathway signaling and thereby promotes IFN-ß induction and the antiviral response. We observed that RAB1B overexpression increases RIG-I-mediated signaling to IFN-ß and that RAB1B deletion reduces signaling of this pathway. Additionally, loss of RAB1B dampened the antiviral response, indicated by enhanced Zika virus infection of cells depleted of RAB1B. Importantly, we identified the mechanism of RAB1B action in the antiviral response, finding that it forms a protein complex with TRAF3 to facilitate the interaction of TRAF3 with mitochondrial antiviral signaling protein. We conclude that RAB1B regulates TRAF3 and promotes the formation of innate immune signaling complexes in response to nucleic acid sensing during RNA virus infection.

Molecular-level investigation of soils contaminated by oil spilled during the Gulf War.

In this study, molecular-level chemical compositions of soils contaminated by oil spilled during the Gulf War were studied. Two soil samples, respectively collected at 0.1 m and between 0.5 and 1 m below the surface from an oil spill site, were extracted with organic solvents and water. The extracts were analyzed via ultrahigh resolution FT-ICR and two-dimensional gas chromatography/high resolution mass spectrometry. The data showed that the spilled oil was significantly affected by vaporization due to high surface temperatures in the desert. The data obtained with (+) atmospheric pressure photo ionization (APPI) and (-) electrospray ionization (ESI) coupled with ultrahigh resolution-mass spectrometry (UHR-MS) indicated that the degradation of aromatic compounds and increase in oxygen-containing classes occurred in the following order: surface soil > below surface soil > crude oil. The oxygenated compounds were confirmed by principal component analysis. The score and loading plots of Ox and SOx showed that they were the major contributors to differentiate the samples. However, a comparison with previously reported oceanic oil spills showed that less significant degradation occurred even after almost 30 years. Our data can provide an information basis for designing a strategy for clean-up and restoration efforts of Gulf War oil spills.

Validation of Reduced Reagent Volumes in the Implementation of the Quantifiler® Trio Quantification Kit.

The Quantifiler® Trio Quantification Kit has been developed to quantify the total amount of amplifiable and human male DNA in samples and to estimate the extent of DNA degradation. To minimize the cost of DNA quantification, we evaluated kit performance using a reduced volume of reagents (1/10-volume) using DNA samples of varying types and concentrations. Our results demonstrated concordance between the manufacturer's method and the low-volume method for DNA quantification, DNA degradation index estimation, and human male DNA quantification. We confirmed the practical utility of the low-volume method with 109 casework samples by evaluating short tandem repeat (STR) profiling success with respect to DNA quantity and quality. We also defined a cutoff value for DNA quantity to ensure reliable STR results. Using a reduced volume of reagents, 10 times more reactions per kit are possible; accordingly, this method reduces the cost of DNA quantification, while maintaining performance.

Biology of Acinetobacter baumannii: Pathogenesis, Antibiotic Resistance Mechanisms, and Prospective Treatment Options.

Acinetobacter baumannii is undoubtedly one of the most successful pathogens responsible for hospital-acquired nosocomial infections in the modern healthcare system. Due to the prevalence of infections and outbreaks caused by multi-drug resistant A. baumannii, few antibiotics are effective for treating infections caused by this pathogen. To overcome this problem, knowledge of the pathogenesis and antibiotic resistance mechanisms of A. baumannii is important. In this review, we summarize current studies on the virulence factors that contribute to A. baumannii pathogenesis, including porins, capsular polysaccharides, lipopolysaccharides, phospholipases, outer membrane vesicles, metal acquisition systems, and protein secretion systems. Mechanisms of antibiotic resistance of this organism, including acquirement of ß-lactamases, up-regulation of multidrug efflux pumps, modification of aminoglycosides, permeability defects, and alteration of target sites, are also discussed. Lastly, novel prospective treatment options for infections caused by multi-drug resistant A. baumannii are summarized.

Current use of autologous adipose tissue-derived stromal vascular fraction cells for orthopedic applications.

Autologous adipose stromal vascular fractions (SVFs) containing adipose tissue-derived stem cells (ASCs) are currently being used in clinical settings for various orthopedic applications for human patients. Due to its potential capability of regenerating cartilage, bone, and tendons, autologous adipose SVFs are being tried in treating patients with osteoarthritis (OA), chondromalacia, meniscus tear, osteonecrosis of the femoral head, and tendon injuries. Here, we have reviewed available human clinical studies with regard to patient applications of autologous adipose SVF containing ASCs, specifically assessing effectiveness and safety in the field of orthopedic disorders. All studies reviewed in this article presents potential benefits of autologous adipose SVF in various orthopedic applications without any serious side effects.

ER-to-Golgi blockade of nascent desmosomal cadherins in SERCA2-inhibited keratinocytes: Implications for Darier's disease.

Darier's disease (DD) is an autosomal dominantly inherited skin disorder caused by mutations in sarco/endoplasmic reticulum Ca2+ -ATPase 2 (SERCA2), a Ca2+ pump that transports Ca2+ from the cytosol to the endoplasmic reticulum (ER). Loss of desmosomes and keratinocyte cohesion is a characteristic feature of DD. Desmosomal cadherins (DC) are Ca2+ -dependent transmembrane adhesion proteins of desmosomes, which are mislocalized in the lesional but not perilesional skin of DD. We show here that inhibition of SERCA2 by 2 distinct inhibitors results in accumulation of DC precursors in keratinocytes, indicating ER-to-Golgi transport of nascent DC is blocked. Partial loss of SERCA2 by siRNA has no such effect, implicating that haploinsufficiency is not sufficient to affect nascent DC maturation. However, a synergistic effect is revealed between SERCA2 siRNA and an ineffective dose of SERCA2 inhibitor, and between an agonist of the ER Ca2+ release channel and SERCA2 inhibitor. These results suggest that reduction of ER Ca2+ below a critical level causes ER retention of nascent DC. Moreover, colocalization of DC with ER calnexin is detected in SERCA2-inhibited keratinocytes and DD epidermis. Collectively, our data demonstrate that loss of SERCA2 impairs ER-to-Golgi transport of nascent DC, which may contribute to DD pathogenesis.

Analysis of stress, musculoskeletal disorders, and fatigue among broadcasting actors.

[Purpose] The purpose of the study was to assess the health effects of broadcasting actors through a comprehensive research on their job stress, psychosocial stress, and fatigue and to investigate those factors having an impact on their health condition to present a basis for comparative studies and effective human resource management in the future. [Subjects and Methods] A survey was performed to analyze the relevance of the general features, job stress, psychosocial stress, and fatigue. [Results] Analysis of job stress, one of the characteristics of individuals, revealed that 32.4% of the subjects with less than 5 years of service, 55.5% of those with 6 to 10 years of service, and 52.4% of those with more than 10 years of service showed a high level of stress. Analysis of psychosocial stress, another characteristic of individuals, revealed that 13.4% of the nonsmokers had a high level of psychosocial stress, while 37.7% of smokers had a high level of psychosocial stress based on analysis of chronic disease and psychosocial health. [Conclusion] Based on this study of the stress and fatigue of broadcasting actors, it is expected that improvements can be made to promote their mental health conditions and, organizational safety and to promote effective human resource management.

Why cannot a ß-lactamase gene be detected using an efficient molecular diagnostic method?

OBJECTIVE: Fast detection of ß-lactamase (bla) genes can minimize the spread of antibiotic resistance. Although several molecular diagnostic methods have been developed to detect limited bla gene types, these methods have significant limitations, such as their failure to detect almost all clinically available bla genes. We have evaluated a further refinement of our fast and accurate molecular method, developed to overcome these limitations, using clinical isolates. METHODS: We have recently developed the efficient large-scale bla detection method (large-scaleblaFinder) that can detect bla gene types including almost all clinically available 1,352 bla genes with perfect specificity and sensitivity. Using this method, we have evaluated a further refinement of this method using clinical isolates provided by International Health Management Associates, Inc. (Schaumburg, Illinois, USA). Results were interpreted in a blinded manner by researchers who did not know any information on bla genes harbored by these isolates. RESULTS: With only one exception, the large-scaleblaFinder detected all bla genes identified by the provider using microarray and multiplex PCR. In one of the Escherichia coli test isolates, a blaDHA-1 gene was detected using the multiplex PCR assay but it was not detected using the large-scaleblaFinder. CONCLUSION: The truncation of a blaDHA-1 gene is an important reason for an efficient molecular diagnostic method (large-scaleblaFinder) not to detect the bla gene.

Complex Class 1 Integron Carrying qnrB62 and blaVIM-2 in a Citrobacter freundii Clinical Isolate.

The coexistence of qnrB62 and blaVIM-2 was detected in a Citrobacter clinical isolate. The reduced fluoroquinolone susceptibility is attributable to qnrB62, mutations of quinolone-resistance-determining regions, and an efflux pump or pumps. The genetic context surrounding chromosomal qnrB62 was a novel complex class 1 integron (In1184::ISCR1::qnrB62) containing a unique gene array (blaVIM-2-aacA4'-8-gucD). An 18-nucleotide deletion at the 3' end of the pspA gene [pspA(Δ18)], upstream of qnrB62, and an inverted repeat region (IRR2) were detected in In1184::ISCR1::qnrB62, indicating past transposition events.

Highly sensitive wavelength-dependent nonaqueous capillary electrophoresis for simultaneous screening of various synthetic organic dyes.

A novel multi-wavelength nonaqueous capillary electrophoresis (MW-NACE) technique based on wavelength-dependent laser-induced fluorescence (LIF) detection was investigated for the simultaneous screening of various synthetic organic dyes. Multi-wavelength excitation light sources were utilized to excite different organic dyes [e.g., 543 nm for crystal violet (CV), methyl violet B (MVB), methyl violet B base (MBB), rhodamine 6G (R6G), and rhodamine B base (RBB); 635 nm for nile blue A (NBA) and methylene blue (MB)] simultaneously. Using a nonaqueous buffer system composed of 15 mM sodium borate and 835 mM acetic acid in 100% ethanol (pH=5.4), all dyes were analyzed within 15 min with excellent resolution (R≥4.0) under an electric field of 500 V/cm. Calibration curves showed excellent linearity with square of correlation coefficients (r(2)) greater than 0.9908 over wide dynamic ranges of 0.4-50 µM for CV, 0.8-50 µM for MVB, 1.5-50 µM for MBB, 0.08-5 nM for R6G, 0.06-10 µM for MB, 0.02-10 µM for NBA, and 0.13-10 pM for RBB. The detection limits (S/N=3) of 40 fM to 0.5 µM were 10-200,000 times lower than those of previous detection methods. While adjacent peaks were not well distinguished with baseline separation in a single capillary, the devised technique was faster and more sensitive than conventional aqueous and nonaqueous CE approaches, thereby enabling the quantitative analysis of various dyes based on wavelength-dependent fluorescence detection with different excitation wavelengths.

Risk Factors for Musculoskeletal Symptoms Among Korean Broadcast Actors.

BACKGROUND: Musculoskeletal diseases (MSDs) are functional disabilities in the musculoskeletal area that occur when continuous damage to the muscles or tissues is caused by performing a repetitive task. These diseases are usually found in the waist, shoulder, neck, arm, and wrist. MSD is also referred to as cumulative trauma disorder, repetitive strain injury, occupational overuse syndrome, and visual display terminal, depending on the country. The condition is now commonly referred to as work-related musculoskeletal disorder. OBJECTIVES: The aim of this study was to develop a prevention plan against musculoskeletal disease and to provide better health care to broadcast actors by understanding the association between musculoskeletal symptoms and working conditions. The results of the study can be utilized to maintain effective systematic resources to treat such diseases. METHODS: A survey was conducted in Seoul between January 1 and May 10, 2014 with broadcast actors working in the South Korean entertainment industry. FINDINGS: Tests with respect to musculoskeletal symptoms indicated that the study participants were likely to experience having musculoskeletal symptoms in the shoulders, waist, neck, leg/foot, hand/wrist/finger, and arm/elbow. Most of the participants reported pain on both sides of their shoulders and in their legs/feet or on the right side of the arm/elbow and in hand/wrist/finger. Pain lasted between 1 and 7 days, with an incidence of 33.8% in the neck, 36% in the shoulders, 33.3% in the arm/elbow, 47.4% in the hand/wrist/finger, 34.7% in the waist, and 39.3% in the leg/foot. CONCLUSIONS: This study should prove useful in determining systematic and effective resources to prevent broadcast actors from developing MSD in the future.

Mucosal pemphigus vulgaris anti-Dsg3 IgG is pathogenic to the oral mucosa of humanized Dsg3 mice.

There are two major clinical subsets of pemphigus vulgaris (PV)-mucosal PV (mPV) and mucocutaneous PV (mcPV). The mPV subset exhibits anti-human desmoglein (Dsg) 3 autoantibodies that fail to recognize murine Dsg3 (mDsg3); thus, passive transfer experiments of mPV IgG into wild-type (WT) mice have been unsuccessful at inducing disease. We therefore generated a fully humanized Dsg3 (hDSG3) murine model utilizing a hDsg3 transgenic animal crossed to the mDsg3 knockout line. Expression of hDsg3 in the mucosa rescues the mDsg3 knockout phenotype. Well-characterized mPV sera bind mucosal epithelia from the hDsg3 mice, but not mucosal tissues from WT mice, as detected by indirect immunofluorescence (IF). The majority of mPV sera preferentially recognize hDsg3 compared with mDsg3 by immunoprecipitation as well. Passive transfer of mPV IgG into adult hDsg3 mice, but not WT mice, induces suprabasilar acantholysis in mucosal tissues, thus confirming the pathogenicity of mPV anti-hDsg3 IgG in vivo. Human anti-hDsg3 antibodies are detected in perilesional mucosa as well as in sera of recipient mice by IF. These findings suggest that the Dsg3 epitopes targeted by pathogenic mPV IgG are human specific. This hDsg3 mouse model will be invaluable in studying the clinical transition from mPV to mcPV.

The Thomsen-Friedenreich antigen-binding lectin jacalin interacts with desmoglein-1 and abrogates the pathogenicity of pemphigus foliaceus autoantibodies in vivo.

Pemphigus foliaceus (PF) is an autoimmune skin blistering disease mediated by pathogenic autoantibodies against the desmosomal core glycoprotein desmoglein-1 (Dsg1). This study demonstrated that the O-glycan-specific plant lectin jacalin binds Dsg1 and inhibits the interaction of Dsg1/PF IgG. N-glycosylation is not involved in the interaction of Dsg1/jacalin or Dsg1/PF IgG. Subcutaneous injection of jacalin into neonatal mice drastically reduced PF IgG deposition at the epidermal cell surface and blocked PF IgG-induced skin blisters, both clinically and histologically. Interestingly, another plant lectin, peanut agglutinin, which shares the same carbohydrate specificity toward the O-linked carbohydrate structure known as Thomsen-Friedenreich antigen (TF antigen, Galß1-3GalNAcα-O-Ser/Thr), also bound Dsg1 and blocked the skin blistering. In contrast, the plant lectin vicia villosa-B4 (VVL-B4), which shares the carbohydrate specificity toward the O-linked monosaccharide known as Thomsen-nouveau antigen (GalNAc-α1-O-Ser/Thr), did not bind Dsg1 and did not show a protective effect against the disease induced by the autoantibodies. Collectively, these results suggest that the binding of jacalin to O-linked TF carbohydrate motifs on Dsg1 impairs the Dsg1/PF autoantibody interactions and abrogates its pathogenicity in vivo. TF-specific binding ligands may have a potential therapeutic value for PF.