Changes in Hole and Electron Injection under Electrical Stress and the Rapid Electroluminescence Loss in Blue Quantum-Dot Light-Emitting Devices.

Blue quantum dot light-emitting devices (QLEDs) suffer from fast electroluminescence (EL) loss when under electrical bias. Here, it is identified that the fast EL loss in blue QLEDs is not due to a deterioration in the photoluminescence quantum yield of the quantum dots (QDs), contrary to what is commonly believed, but rather arises primarily from changes in charge injection overtime under the bias that leads to a deterioration in charge balance. Measurements on hole-only and electron-only devices show that hole injection into blue QDs increases over time whereas electron injection decreases. Results also show that the changes are associated with changes in hole and electron trap densities. The results are further verified using QLEDs with blue and red QDs combinations, capacitance versus voltage, and versus time characteristics of the blue QLEDs. The changes in charge injection are also observed to be partially reversible, and therefore using pulsed current instead of constant current bias for driving the blue QLEDs leads to an almost 2.5× longer lifetime at the same initial luminance. This work systematically investigates the origin of blue QLEDs EL loss and provides insights for designing improved blue QDs paving the way for QLEDs technology commercialization.

Glial fibrillary acidic protein, neurofilament light, matrix metalloprotease 3 and fatty acid binding protein 4 as non-invasive brain tumor biomarkers.

BACKGROUND: Gliomas are aggressive malignant tumors, with poor prognosis. There is an unmet need for the discovery of new, non-invasive biomarkers for differential diagnosis, prognosis, and management of brain tumors. Our objective is to validate four plasma biomarkers - glial fibrillary acidic protein (GFAP), neurofilament light (NEFL), matrix metalloprotease 3 (MMP3) and fatty acid binding protein 4 (FABP4) - and compare them with established brain tumor molecular markers and survival. METHODS: Our cohort consisted of patients with benign and malignant brain tumors (GBM = 77, Astrocytomas = 26, Oligodendrogliomas = 23, Secondary tumors = 35, Meningiomas = 70, Schwannomas = 15, Pituitary adenomas = 15, Normal individuals = 30). For measurements, we used ultrasensitive electrochemiluminescence multiplexed immunoassays. RESULTS: High plasma GFAP concentration was associated with GBM, low GFAP and high FABP4 were associated with meningiomas, and low GFAP and low FABP4 were associated with astrocytomas and oligodendrogliomas. NEFL was associated with progression of disease. Several prognostic genetic alterations were significantly associated with all plasma biomarker levels. We found no independent associations between plasma GFAP, NEFL, FABP4 and MMP3, and overall survival. The candidate biomarkers could not reliably discriminate GBM from primary or secondary CNS lymphomas. CONCLUSIONS: GFAP, NEFL, FABP4 and MMP3 are useful for differential diagnosis and prognosis, and are associated with molecular changes in gliomas.

Discovery of novel glioma serum biomarkers by proximity extension assay.

BACKGROUND: Gliomas are among the most malignant tumors, with a very poor prognosis. Early diagnosis is highly desirable since it can help implement more effective treatments for smaller tumors, which have not yet extensively metastasized. Improving early diagnosis may facilitate access of patients to clinical trials and prepare them for the future availability of new disease-modifying treatments. METHODS: We analyzed retrospective samples collected at diagnosis (before therapy initiation), with PEA (Olink Proteomics), quantifying about 3000 proteins. We utilized 30 plasmas from gliomas (20 glioblastomas, 5 anaplastic astrocytomas, 5 anaplastic oligodendrogliomas) and 20 meningiomas (as controls). We then analyzed the data to identify proteins which either alone, or in combination, could discriminate gliomas from meningiomas, or correlate with clinical and molecular alterations. RESULTS: We identified 8 plasma proteins which were increased in gliomas vs. meningiomas (GFAP, NEFL, EDDM3B, PROK1, MMP3, CTRL, GP2, SPINT3) and 4 proteins which were decreased in gliomas vs. meningiomas (FABP4, ALDH3A1, IL-12B and OXT). Partition algorithms and logistic regression algorithms with two biomarkers (GFAP and FABP4) achieved sensitivity of 83% and 93% at 100% and 90% specificity, respectively. The strongest single marker was GFAP with an area under the ROC curve (AUC) of 0.86. The AUC for the GFAP-FABP4 combination was 0.98. CONCLUSION: PEA is a powerful new proteomic technology for biomarker discovery. GFAP and a handful of other plasma biomarkers may be useful for early glioma detection and probably, prognosis. STATEMENT: Detecting gliomas as early as possible is highly desirable since it can significantly improve the chances of effective treatments. Reliable glioma biomarkers can timely inform glioma patients about the efficacy of their prescribed treatment. Our results reveal some novel putative glioma markers that may prove valuable, when used alone or in combination, towards improved clinical care of gliomas. In order to better appreciate the potential usefulness of these markers, their performance needs to be further validated in a larger cohort of samples.

The optimal pH of AID is skewed from that of its catalytic pocket by DNA-binding residues and surface charge.

Activation-induced cytidine deaminase (AID) is a member of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family of cytidine deaminases. AID mutates immunoglobulin loci to initiate secondary antibody diversification. The APOBEC3 (A3) sub-branch mutates viral pathogens in the cytosol and acidic endosomal compartments. Accordingly, AID functions optimally near-neutral pH, while most A3s are acid-adapted (optimal pH 5.5-6.5). To gain a structural understanding for this pH disparity, we constructed high-resolution maps of AID catalytic activity vs pH. We found AID's optimal pH was 7.3 but it retained most (>70%) of the activity at pH 8. Probing of ssDNA-binding residues near the catalytic pocket, key for bending ssDNA into the pocket (e.g. R25) yielded mutants with altered pH preference, corroborating previous findings that the equivalent residue in APOBEC3G (H216) underlies its acidic pH preference. AID from bony fish exhibited more basic optimal pH (pH 7.5-8.1) and several R25-equivalent mutants altered pH preference. Comparison of pH optima across the AID/APOBEC3 family revealed an inverse correlation between positive surface charge and overall catalysis. The paralogue with the most robust catalytic activity (APOBEC3A) has the lowest surface charge and most acidic pH preference, while the paralogue with the most lethargic catalytic rate (AID) has the most positive surface charge and highest optimal pH. We suggest one possible mechanism is through surface charge dictating an overall optimal pH that is different from the optimal pH of the catalytic pocket microenvironment. These findings illuminate an additional structural mechanism that regulates AID/APOBEC3 mutagenesis.

Ancestral reconstruction reveals catalytic inactivation of activation-induced cytidine deaminase concomitant with cold water adaption in the Gadiformes bony fish.

BACKGROUND: Antibody affinity maturation in vertebrates requires the enzyme activation-induced cytidine deaminase (AID) which initiates secondary antibody diversification by mutating the immunoglobulin loci. AID-driven antibody diversification is conserved across jawed vertebrates since bony and cartilaginous fish. Two exceptions have recently been reported, the Pipefish and Anglerfish, in which the AID-encoding aicda gene has been lost. Both cases are associated with unusual reproductive behavior, including male pregnancy and sexual parasitism. Several cold water fish in the Atlantic cod (Gadinae) family carry an aicda gene that encodes for a full-length enzyme but lack affinity-matured antibodies and rely on antibodies of broad antigenic specificity. Hence, we examined the functionality of their AID. RESULTS: By combining genomics, transcriptomics, immune responsiveness, and functional enzymology of AID from 36 extant species, we demonstrate that AID of that Atlantic cod and related fish have extremely lethargic or no catalytic activity. Through ancestral reconstruction and functional enzymology of 71 AID enzymes, we show that this enzymatic inactivation likely took place relatively recently at the emergence of the true cod family (Gadidae) from their ancestral Gadiformes order. We show that this AID inactivation is not only concordant with the previously shown loss of key adaptive immune genes and expansion of innate and cell-based immune genes in the Gadiformes but is further reflected in the genomes of these fish in the form of loss of AID-favored sequence motifs in their immunoglobulin variable region genes. CONCLUSIONS: Recent demonstrations of the loss of the aicda gene in two fish species challenge the paradigm that AID-driven secondary antibody diversification is absolutely conserved in jawed vertebrates. These species have unusual reproductive behaviors forming an evolutionary pressure for a certain loss of immunity to avoid tissue rejection. We report here an instance of catalytic inactivation and functional loss of AID rather than gene loss in a conventionally reproducing vertebrate. Our data suggest that an expanded innate immunity, in addition to lower pathogenic pressures in a cold environment relieved the pressure to maintain robust secondary antibody diversification. We suggest that in this unique scenario, the AID-mediated collateral genome-wide damage would form an evolutionary pressure to lose AID function.

Patients with severe COVID-19 do not have elevated autoantibodies against common diagnostic autoantigens.

OBJECTIVES: Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative pathogen of coronavirus disease 2019 (COVID-19) presents occasionally with an aberrant autoinflammatory response, including the presence of elevated circulating autoantibodies in some individuals. Whether the development of autoantibodies against self-antigens affects COVID-19 outcomes remains unclear. To better understand the prognostic role of autoantibodies in COVID-19, we quantified autoantibodies against 23 markers that are used for diagnosis of autoimmune disease. To this end, we used serum samples from patients with severe [intensive care unit (ICU)] and moderate (ward) COVID-19, across two to six consecutive time points, and compared autoantibody levels to uninfected healthy and ICU controls. METHODS: Acute and post-acute serum (from 1 to 26 ICU days) was collected from 18 ICU COVID-19-positive patients at three to six time points; 18 ICU COVID-19-negative patients (sampled on ICU day 1 and 3); 21 ward COVID-19-positive patients (sampled on hospital day 1 and 3); and from 59 healthy uninfected controls deriving from two cohorts. Levels of IgG autoantibodies against 23 autoantigens, commonly used for autoimmune disease diagnosis, were measured in serum samples using MSD® U-PLEX electrochemiluminescence technology (MSD division Meso Scale Discovery®), and results were compared between groups. RESULTS: There were no significant elevations of autoantibodies for any of the markers tested in patients with severe COVID-19. CONCLUSIONS: Sample collections at longer time points should be considered in future studies, for assessing the possible development of autoantibody responses following infection with SARS-CoV-2.

Activation-induced cytidine deaminase can target multiple topologies of double-stranded DNA in a transcription-independent manner.

Activation-induced cytidine deaminase (AID) mutates immunoglobulin genes and acts genome-wide. AID targets robustly transcribed genes, and purified AID acts on single-stranded (ss) but not double-stranded (ds) DNA oligonucleotides. Thus, it is believed that transcription is the generator of ssDNA for AID. Previous cell-free studies examining the relationship between transcription and AID targeting have employed a bacterial colony count assay wherein AID reverts an antibiotic resistance stop codon in plasmid substrates, leading to colony formation. Here, we established a novel assay where kb-long dsDNA of varying topologies is incubated with AID, with or without transcription, followed by direct sequencing. This assay allows for an unselected and in-depth comparison of mutation frequency and pattern of AID targeting in the absence of transcription or across a range of transcription dynamics. We found that without transcription, AID targets breathing ssDNA in supercoiled and, to a lesser extent, in relaxed dsDNA. The most optimal transcription only modestly enhanced AID action on supercoiled dsDNA in a manner dependent on RNA polymerase speed. These data suggest that the correlation between transcription and AID targeting may reflect transcription leading to AID-accessible breathing ssDNA patches naturally occurring in de-chromatinized dsDNA, as much as being due to transcription directly generating ssDNA.

Influence of Encapsulation on the Efficiency and Positive Aging Behavior in Blue Quantum Dot Light-Emitting Devices.

Encapsulating blue quantum dot light-emitting devices (QLEDs) using an ultraviolet curable resin is known to lead to a significant increase in their efficiency. Some of this efficiency increase occurs immediately, whereas some of it proceeds over a period of time, typically over several tens of hours following the encapsulation, a behavior commonly referred to as positive aging. The root causes of this positive aging, especially in blue QLEDs, remain not well understood. Here, it is revealed that contrary to the expectation, the significant improvement in device efficiency during positive aging arises primarily from an improvement in electron injection across the QD/ZnMgO interface and not due to the inhibition of interface exciton quenching as is widely believed. The underlying changes are investigated by XPS measurements. Results show that the enhancement in device performance arises primarily from the reduction in O-related defects in both the QDs and ZnMgO at the QD/ZnMgO interface. After 51.5 h, the blue QLEDs reach the optimal performance, exhibiting an EQEmax of 12.58%, which is more than sevenfold higher than that in the control device without encapsulation. This work provides design principles for realizing high efficiency in blue QLEDs with oxide electron-transporting layers (ETLs) and provides a new understanding of the mechanisms underlying positive aging in these devices and thus offers a new starting point for both fundamental investigations and practical applications.

Inverted Solution-Processed Quantum Dot Light-Emitting Devices with Wide Band Gap Quantum Dot Interlayers.

Despite its benefits for facilitating device fabrication, utilization of a polymeric hole transport layer (HTL) in inverted quantum dots (QDs) light-emitting devices (IQLEDs) often leads to poor device performance. In this work, we find that the poor performance arises primarily from electron leakage, inefficient charge injection, and significant exciton quenching at the HTL interface in the inverted architecture and not due to solvent damage effects as is widely believed. We also find that using a layer of wider band gap QDs as an interlayer (IL) in between the HTL and the main QDs' emission material layer (EML) can facilitate hole injection, suppress electron leakage, and reduce exciton quenching, effectively mitigating the poor interface effects and resulting in high electroluminescence performance. Using an IL in IQLEDs with a solution-processed poly(9,9-dioctylfluorene-alt-N-(4-sec-butylphenyl)-diphenylamine) (TFB), HTL improves the efficiency by 2.85× (from 3 to 8.56%) and prolongs the lifetime by 9.4× (from 1266 to 11,950 h at 100 cd/m2), which, to the best of our knowledge, is the longest lifetime for an R-IQLED with a solution-coated HTL. Measurements on single-carrier devices reveal that while electron injection becomes easier as the band gap of the QDs decreases, hole injection surprisingly becomes more difficult, indicating that EMLs of QLEDs are more electron-rich in the case of red devices and more hole-rich in the case of blue devices. Ultraviolet photoelectron spectroscopy measurements verify that blue QDs have a shallower valence band energy than their red counterparts, corroborating these conclusions. The findings in this work, therefore, provide not only a simple approach for achieving high performance in IQLEDs with solution-coated HTLs but also novel insights into charge injection and its dependence on QDs' band gap as well as into different HTL interface properties of the inverted versus upright architecture.

Sensitive Serology Measurements in the Saliva of Individuals with COVID-19 Symptoms Using a Multiplexed Immunoassay.

BACKGROUND: There are numerous benefits to performing salivary serology measurements for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen for coronavirus disease 2019 (COVID-19). Here, we used a sensitive multiplex serology assay to quantitate salivary IgG against 4 SARS-CoV-2 antigens: nucleocapsid, receptor-binding domain, spike, and N-terminal domain. METHODS: We used single samples from 90 individuals with COVID-19 diagnosis collected at 0 to 42 days postsymptom onset (PSO) and from 15 uninfected control subjects. The infected individuals were segmented in 4 groups (0-7 days, 8-14 days, 15-21 days, and >21 days) based on days PSO, and values were compared to controls. RESULTS: Compared to controls, infected individuals showed higher levels of antibodies against all antigens starting from 8 days PSO. When applying cut-offs with at least 93.3% specificity at every time interval segment, nucleocapsid protein serology had the best sensitivity at 0 to 7 days PSO (60% sensitivity [35.75% to 80.18%], ROC area under the curve [AUC] = 0.73, P = 0.034). Receptor-binding domain serology had the best sensitivity at 8 to 14 days PSO (83.33% sensitivity [66.44%-92.66%], ROC AUC = 0.90, P < 0.0001), and all assays except for N-terminal domain had 92% sensitivity (75.03%-98.58%) at >14 days PSO. CONCLUSIONS: This study shows that our multiplexed immunoassay can distinguish infected from uninfected individuals and reliably (93.3% specificity) detect seroconversion (in 60% of infected individuals) as early as the first week PSO, using easy-to-collect saliva samples.

Evolutionary Comparative Analyses of DNA-Editing Enzymes of the Immune System: From 5-Dimensional Description of Protein Structures to Immunological Insights and Applications to Protein Engineering.

The immune system is unique among all biological sub-systems in its usage of DNA-editing enzymes to introduce targeted gene mutations and double-strand DNA breaks to diversify antigen receptor genes and combat viral infections. These processes, initiated by specific DNA-editing enzymes, often result in mistargeted induction of genome lesions that initiate and drive cancers. Like other molecules involved in human health and disease, the DNA-editing enzymes of the immune system have been intensively studied in humans and mice, with little attention paid (< 1% of published studies) to the same enzymes in evolutionarily distant species. Here, we present a systematic review of the literature on the characterization of one such DNA-editing enzyme, activation-induced cytidine deaminase (AID), from an evolutionary comparative perspective. The central thesis of this review is that although the evolutionary comparative approach represents a minuscule fraction of published works on this and other DNA-editing enzymes, this approach has made significant impacts across the fields of structural biology, immunology, and cancer research. Using AID as an example, we highlight the value of the evolutionary comparative approach in discoveries already made, and in the context of emerging directions in immunology and protein engineering. We introduce the concept of 5-dimensional (5D) description of protein structures, a more nuanced view of a structure that is made possible by evolutionary comparative studies. In this higher dimensional view of a protein's structure, the classical 3-dimensional (3D) structure is integrated in the context of real-time conformations and evolutionary time shifts (4th dimension) and the relevance of these dynamics to its biological function (5th dimension).

Structure-Based Design of First-Generation Small Molecule Inhibitors Targeting the Catalytic Pockets of AID, APOBEC3A, and APOBEC3B.

Activation-induced cytidine deaminase (AID) initiates antibody diversification by mutating immunoglobulin loci in B lymphocytes. AID and related APOBEC3 (A3) enzymes also induce genome-wide mutations and lesions implicated in tumorigenesis and tumor progression. The most prevalent mutation signatures across diverse tumor genomes are attributable to the mistargeted mutagenic activities of AID/A3s. Thus, inhibiting AID/A3s has been suggested to be of therapeutic benefit. We previously used a computational-biochemical approach to gain insight into the structure of AID's catalytic pocket, which resulted in the discovery of a novel type of regulatory catalytic pocket closure that regulates AID/A3s that we termed the "Schrodinger's CATalytic pocket". Our findings were subsequently confirmed by direct structural studies. Here, we describe our search for small molecules that target the catalytic pocket of AID. We identified small molecules that inhibit purified AID, AID in cell extracts, and endogenous AID of lymphoma cells. Analogue expansion yielded derivatives with improved potencies. These were found to also inhibit A3A and A3B, the two most tumorigenic siblings of AID. Two compounds exhibit low micromolar IC50 inhibition of AID and A3A, exhibiting the strongest potency for A3A. Docking suggests key interactions between their warheads and residues lining the catalytic pockets of AID, A3A, and A3B and between the tails and DNA-interacting residues on the surface proximal to the catalytic pocket opening. Accordingly, mutants of these residues decreased inhibition potency. The chemistry and abundance of key stabilizing interactions between the small molecules and residues within and immediately outside the catalytic pockets are promising for therapeutic development.

Characterization and Transcript Expression Analyses of Atlantic Cod Viperin.

Viperin is a key antiviral effector in immune responses of vertebrates including the Atlantic cod (Gadus morhua). Using cloning, sequencing and gene expression analyses, we characterized the Atlantic cod viperin at the nucleotide and hypothetical amino acid levels, and its regulating factors were investigated. Atlantic cod viperin cDNA is 1,342 bp long, and its predicted protein contains 347 amino acids. Using in silico analyses, we showed that Atlantic cod viperin is composed of 5 exons, as in other vertebrate orthologs. In addition, the radical SAM domain and C-terminal sequences of the predicted Viperin protein are highly conserved among various species. As expected, Atlantic cod Viperin was most closely related to other teleost orthologs. Using computational modeling, we show that the Atlantic cod Viperin forms similar overall protein architecture compared to mammalian Viperins. qPCR revealed that viperin is a weakly expressed transcript during embryonic development of Atlantic cod. In adults, the highest constitutive expression of viperin transcript was found in blood compared with 18 other tissues. Using isolated macrophages and synthetic dsRNA (pIC) stimulation, we tested various immune inhibitors to determine the possible regulating pathways of Atlantic cod viperin. Atlantic cod viperin showed a comparable pIC induction to other well-known antiviral genes (e.g., interferon gamma and interferon-stimulated gene 15-1) in response to various immune inhibitors. The pIC induction of Atlantic cod viperin was significantly inhibited with 2-Aminopurine, Chloroquine, SB202190, and Ruxolitinib. Therefore, endosomal-TLR-mediated pIC recognition and signal transducers (i.e., PKR and p38 MAPK) downstream of the TLR-dependent pathway may activate the gene expression response of Atlantic cod viperin. Also, these results suggest that antiviral responses of Atlantic cod viperin may be transcriptionally regulated through the interferon-activated pathway.

DNA/RNA hybrid substrates modulate the catalytic activity of purified AID.

Activation-induced cytidine deaminase (AID) converts cytidine to uridine at Immunoglobulin (Ig) loci, initiating somatic hypermutation and class switching of antibodies. In vitro, AID acts on single stranded DNA (ssDNA), but neither double-stranded DNA (dsDNA) oligonucleotides nor RNA, and it is believed that transcription is the in vivo generator of ssDNA targeted by AID. It is also known that the Ig loci, particularly the switch (S) regions targeted by AID are rich in transcription-generated DNA/RNA hybrids. Here, we examined the binding and catalytic behavior of purified AID on DNA/RNA hybrid substrates bearing either random sequences or GC-rich sequences simulating Ig S regions. If substrates were made up of a random sequence, AID preferred substrates composed entirely of DNA over DNA/RNA hybrids. In contrast, if substrates were composed of S region sequences, AID preferred to mutate DNA/RNA hybrids over substrates composed entirely of DNA. Accordingly, AID exhibited a significantly higher affinity for binding DNA/RNA hybrid substrates composed specifically of S region sequences, than any other substrates composed of DNA. Thus, in the absence of any other cellular processes or factors, AID itself favors binding and mutating DNA/RNA hybrids composed of S region sequences. AID:DNA/RNA complex formation and supporting mutational analyses suggest that recognition of DNA/RNA hybrids is an inherent structural property of AID.

Evaluation of the Relationship between the Incubation Time and Carotenoid Production in Rhodotorula Slooffiae and R. Mucilaginosa Isolated from Leather Tanning Wastewater .

OBJECTIVE(S): Carotenoids which are naturally synthesized by fungi such as yeasts can act as an antioxidant which is closely related to their ability to decrease the risk of a variety of degenerative diseases. In recent years, the increase of demand for carotenoids obtained from natural sources has promoted major efforts to improve carotenoid production from biological sources such as pigmented yeasts. The aim of this study was comparing incubation time and carotenoid production in Rhodotorula slooffiae and R. mucilaginosa isolated from leather tanning wastewater. MATERIALS AND METHODS: To isolate the carotenoid pigment, cells were suspended in acetone and broken using a homogenizer, followed by centrifugation and separation of supernatant. In order to study the effect of incubation time, samples were held at 30 ˚С in a shaker at 150 rpm for 24, 48, 72, 96, and 120 hr. For analytical evaluation, pigments were measured spectrophotometrically at 450 nm using the extinction coefficient E(1%) 450=2500. RESULTS: The results showed that the content of total carotenoid in R. slooffiae was the highest when samples were incubated for 72 hr. Overall, R. mucilaginosa had more potential to produce carotenoid. The best incubation periods for R. slooffiae and R. mucilaginosa were 72 hr and 48 hr, respectively. CONCLUSION: It seemed that the maximum rate of total carotenoid was not directly associated with the maximum amount of cell biomass and the type of carotenoid and their relative amount may vary depending on genus of yeast.

Effects of paraoxonase 1 activity and gene polymorphisms on long-term pulmonary complications of sulfur mustard-exposed veterans.

Sulfur mustard (SM) is an alkylating agent with prolonged adverse effects. The antioxidant paraoxonase 1 (PON1), an endogenous free radical scavenger, plays a protective role against oxidative stress. The possible roles of oxidative stress in the pathogenesis of SM, together with the antioxidant activity of PON1, are enough to warrant the analysis of PON1 polymorphisms and allelic variants in incapacitated veterans. PON1 55 L/M and 192 Q/R polymorphisms were assayed in 289 male veterans with severe pulmonary conditions, who were exposed to SM 20-25 years ago, and 66 gender-, age- and ethnic-matched healthy controls. As we showed previously the PON1 activity decreased significantly in veterans. However, PON1 55 L/M and 192 Q/R genotype distributions were not significantly different between the veterans and the controls. R and L allele carriers have also significantly higher basal and salt-stimulated PON1 activity than Q and M allele carriers. Paraoxonase and arylesterase activities in individuals with the QQ+(MM or LM) genotype were significantly lower than those with the (RR or QR)+LL genotype. Furthermore, basal and salt-stimulated paraoxonase activity in veterans with the (RR or QR)+LL genotype was significantly lower than that in the controls. A positive correlation has been determined between serum PON1 activity and pulmonary function test in QR/LL genotypes. Some of the veterans with RR+QR genotypes have also shown a novel missense change of Asn227Ser in exon 6 of the enzyme. This substitution is close to the binding domain of PON1 and so modifies enzyme activity.