Identification of Key lncRNAs in Gout Under Copper Death and Iron Death Mechanisms: A Study Based on ceRNA Network Analysis and Random Forest Algorithm.

This study focused on identifying potential key lncRNAs associated with gout under the mechanisms of copper death and iron death through ceRNA network analysis and Random Forest (RF) algorithm, which aimed to provide new insights into the molecular mechanisms of gout, and potential molecular targets for future therapeutic strategies of gout. Initially, we conducted an in-depth bioinformatics analysis of gout microarray chips to screen the key cuproptosis-related genes (CRGs) and key ferroptosis-related genes (FRGs). Using these data, we constructed a key ceRNA network for gout. Finally, key lncRNAs associated with gout were identified through the RF algorithm combined with ROC curves, and validated using the Comparative Toxicogenomics Database (CTD). We successfully identified NLRP3, LIPT1, and DBT as key CRGs associated with gout, and G6PD, PRKAA1, LIG3, PHF21A, KLF2, PGRMC1, JUN, PANX2, and AR as key FRGs associated with gout. The key ceRNA network identified four downregulated key lncRNAs (SEPSECS-AS1, LINC01054, REV3L-IT1, and ZNF883) along with three downregulated mRNAs (DBT, AR, and PRKAA1) based on the ceRNA theory. According to CTD validation inference scores and biological functions of target mRNAs, we identified a potential gout-associated lncRNA ZNF883/hsa-miR-539-5p/PRKAA1 regulatory axis. This study identified the key lncRNA ZNF883 in the context of copper death and iron death mechanisms related to gout for the first time through the application of ceRNA network analysis and the RF algorithm, thereby filling a research gap in this field and providing new insights into the molecular mechanisms of gout. We further found that lncRNA ZNF883 might function in gout patients by regulating PRKAA1, the mechanism of which was potentially related to uric acid reabsorption in the proximal renal tubules and inflammation regulation. The proposed lncRNA ZNF883/hsa-miR-539-5p/PRKAA1 regulatory axis might represent a potential RNA regulatory pathway for controlling the progression of gout disease. This discovery offered new molecular targets for the treatment of gout, and had significant implications for future therapeutic strategies in managing the gout.

Efficient L-valine production using systematically metabolic engineered Klebsiella oxytoca.

L-Valine, a branched-chain amino acid with diversified applications, is biosynthesized with α-acetolactate as the key precursor. In this study, the metabolic flux in Klebsiella oxytoca PDL-K5, a Risk Group 1 organism producing 2,3-butanediol as the major fermentation product, was rearranged to L-valine production by introducing exogenous L-valine biosynthesis pathway and blocking endogenous 2,3-butanediol generation at the metabolic branch point α-acetolactate. After further enhancing L-valine efflux, strengthening pyruvate polymerization and selecting of key enzymes for L-valine synthesis, a plasmid-free K. oxytoca strain VKO-9 was obtained. Fed-batch fermentation with K. oxytoca VKO-9 in a 7.5 L fermenter generated 122 g/L L-valine with a yield of 0.587 g/g in 56 h. In addition, repeated fed-batch fermentation was conducted to prevent precipitation of L-valine due to oversaturation. The average concentration, yield, and productivity of produced L-valine in three cycles of repeated fed-batch fermentation were 81.3 g/L, 0.599 g/g, and 3.39 g/L/h, respectively.

A novel role for astrocytic fragmented mitochondria in regulating morphine addiction.

Chronic morphine exposure causes the development of addictive behaviors, accompanied by an increase in neuroinflammation in the central nervous system. While previous researches have shown that astrocytes contribute to brain diseases, the role of astrocyte in morphine addiction through induced neuroinflammation remain unexplored. Here we show that morphine-induced inflammation requires the crosstalk among neuron, astrocyte, and microglia. Specifically, astrocytes respond to morphine-induced neuronal activation by increasing glycolytic metabolism. The dysregulation of glycolysis leads to an increased in the generation of mitochondrial reactive oxygen species and causes excessive mitochondrial fragmentation in astrocytes. These fragmented, dysfunctional mitochondria are consequently released into extracellular environment, leading to activation of microglia and release of inflammatory cytokines. We also found that blocking the nicotinamide adenine dinucleotide salvage pathway with FK866 could inhibit astrocytic glycolysis and restore the mitochondrial homeostasis and effectively attenuate neuroinflammatory responses. Importantly, FK866 reversed morphine-induced addictive behaviors in mice. In summary, our findings illustrate an essential role of astrocytic immunometabolism in morphine induced neural and behavioral plasticity, providing a novel insight into the interactions between neurons, astrocytes, and microglia in the brain affected by chronic morphine exposure.

Characterization of cerebrovascular changes in mice treated with alcohol by photoacoustic imaging.

Alcohol has complex effects on cerebrovascular health. Monitoring the pathology of alcohol induced cerebrovascular changes in vivo is essential for understanding the mechanism and developing potential treatment strategies. Here, photoacoustic imaging was employed to examine cerebrovascular changes in mice under the treatment of alcohol at different doses. By analyzing the association of cerebrovascular structure, hemodynamics, neuronal function and corresponding behavior, we found that alcohol affected brain function and behavior in a dose-dependent manner. Low dose of alcohol increased cerebrovascular blood volume and activated neurons, without addictive behaviors and cerebrovascular structure changes. With the dose increased, cerebrovascular blood volume gradually decreased, triggering obviously progressive effects on the immune microenvironment, cerebrovascular structure and addictive behavior. These findings will provide further insights into the characterization of the biphasic effects of alcohol.

LINE-1 repression in Epstein-Barr virus-associated gastric cancer through viral-host genome interaction.

Long INterspersed Element 1 (LINE-1 or L1) acts as a major remodeling force in genome regulation and evolution. Accumulating evidence shows that virus infection impacts L1 expression, potentially impacting host antiviral response and diseases. The underlying regulation mechanism is unclear. Epstein-Barr virus (EBV), a double-stranded DNA virus linked to B-cell and epithelial malignancies, is known to have viral-host genome interaction, resulting in transcriptional rewiring in EBV-associated gastric cancer (EBVaGC). By analyzing publicly available datasets from the Gene Expression Omnibus (GEO), we found that EBVaGC has L1 transcriptional repression compared with EBV-negative gastric cancer (EBVnGC). More specifically, retrotransposition-associated young and full-length L1s (FL-L1s) were among the most repressed L1s. Epigenetic alterations, especially increased H3K9me3, were observed on FL-L1s. H3K9me3 deposition was potentially attributed to increased TASOR expression, a key component of the human silencing hub (HUSH) complex for H3K9 trimethylation. The 4C- and HiC-seq data indicated that the viral DNA interacted in the proximity of the TASOR enhancer, strengthening the loop formation between the TASOR enhancer and its promoter. These results indicated that EBV infection is associated with increased H3K9me3 deposition, leading to L1 repression. This study uncovers a regulation mechanism of L1 expression by chromatin topology remodeling associated with viral-host genome interaction in EBVaGC.

Clinical implications of miR-223 in allergic conjunctivitis and related factors affecting disease recurrence.

This study aims to explore the clinical implications of miR-223 in allergic conjunctivitis (AC) and the related factors affecting disease recurrence. 47 AC patients and 58 healthy controls were enrolled to measure miR-223 expression level, serum level of inflammatory mediators, and the correlation between miR-223 and inflammatory mediators. Subsequently, AC patients were followed up for six months to record disease recurrence and explore the risk factors affecting disease recurrence. Compared to the healthy controls, the miR-223 level was lower, while inflammatory cytokines and immunoglobulins levels were higher in AC patients. There was a negative correlation of miR-223 with inflammatory cytokines and immunoglobulins. Also, miR-223 was evidently lower in AC recurrence patients than those without recurrence. Moreover, family history, pet-keeping, and other allergic histories were among the risk factors contributing to AC recurrence. These results indicate that miR-223 plays an important role in the pathology of allergic conjunctivitis.

An ancient interleukin-16-like molecule regulates hemocyte proliferation via integrin ß1 in invertebrates.

Interleukins (ILs) are cytokines with crucial functions in innate and adaptive immunity. IL genes are only found in vertebrates, except for IL-16, which has been cloned in some arthropod species. However, the function of this gene in invertebrates is unknown. In the present study, an IL-16-like gene (EsIL-16) was identified from the Chinese mitten crab Eriocheir sinensis. EsIL-16 was predicted to encode a precursor (proEsIL-16) that shares similarities with pro-IL-16 proteins from insects and vertebrates. We show that caspase-3 processes proEsIL-16 into an approximately 144-kDa N-terminal prodomain with nuclear import activity and an approximately 34-kDa mature peptide that might be secreted into the extracellular region. EsIL-16 mRNA could be detected in all analyzed tissues and was significantly upregulated after immune challenge both in vitro and in vivo. T7 phage display library screening suggested potential binding activity between EsIL-16 and integrin, which was confirmed by coimmunoprecipitation assay. Interestingly, EsIL-16 promoted cell proliferation via integrin ß1 in primary cultured crab hemocytes and Drosophila S2 cells. Furthermore, the interaction between EsIL-16 and integrin ß1 was necessary to efficiently protect the host from bacterial infection. To our knowledge, this study revealed integrin ß1 as a receptor for IL-16 and the function of this interaction in hemocyte proliferation in invertebrates for the first time. These results provide new insights into the regulation of innate immune responses in invertebrates and shed the light on the evolution of ILs within the animal kingdom.

Distinct vitellogenin domains differentially regulate immunological outcomes in invertebrates.

The classical role of Vitellogenin (Vg) is providing energy reserves for developing embryos, but its roles appear to extend beyond this nutritional function, and its importance in host immune defense is garnering increasing research attention. However, Vg-regulated immunological functions are dependent on three different domains within different species and remain poorly understood. In the present study, we confirmed three conserved VG domains-LPD_N, DUF1943, and VWD-in the Chinese mitten crab (Eriocheir sinensis), highlighting functional similarities of Vg in vertebrates and invertebrates. Of these three domains, DUF1943 and VWD showed definitive bacterial binding activity via interaction with the signature components on microbial surfaces, but this activity was not exhibited by the LPD_N domain. Antibacterial assays indicated that only the VWD domain inhibits bacterial proliferation, and this function may be conserved between different species due to the conserved amino acid residues. To further explore the relationship between Vg and polymeric immunoglobulin receptor (pIgR), we expressed EspIgR and the three E. sinensis Vg (EsVg) domains in HEK293T cells, and coimmunoprecipitation assay demonstrated that only the DUF1943 domain interacts with EspIgR. Subsequent experiments demonstrated that EsVg regulates hemocyte phagocytosis by binding with EspIgR through the DUF1943 domain, thus promoting bacterial clearance and protecting the host from bacterial infection. To the best of our knowledge, our work is the first to report distinct domains in Vg inducing different immunological outcomes in invertebrates, providing new evidence that pIgR acts as a phagocytic receptor for Vg.

Vitellogenin receptor expression in ovaries controls innate immunity in the Chinese mitten crab (Eriocheir sinensis) by regulating vitellogenin accumulation in the hemolymph.

The vitellogenin receptor (Vgr), which is specific for vitellogenin (Vtg), recognises and transports Vtg into the ovaries. Accumulating evidence suggests that Vtg also performs an immune defence function and plays critical roles in innate immunity in oviparous animals. However, whether Vgr is involved in innate immunity in the Chinese mitten crab (Eriocheir sinensis) is unknown. In this study, we obtained a 3009 nucleotide partial cDNA of the E. sinensis vitellogenin receptor gene (Es-vgr) encoding an open reading frame of 1003 amino acid residues. Bioinformatics analysis showed that the domains of Es-vgr were conserved during evolution. Quantitative real-time PCR and western blotting revealed that the highest Es-vgr expression levels occurred in the ovary, and expression was specific. Comparison of the expression levels of Es-vgr and the Vtg gene (Es-vtg1) at different ovary developmental stages suggested that there may be some regulatory relationship between them. Bacterial challenge induced high-level expression of antimicrobial peptide genes and reduced Es-vgr expression in ovaries, resulting in massive accumulation of Vtg in the hemolymph. The survival rate of crabs increased significantly after injection with recombinant Es-vtg1 protein following bacterial infection. Collectively, these results demonstrate that Es-vgr plays critical roles in antimicrobial function by regulating the accumulation of Vtg in the hemolymph.

[Construction and verification of Lactococcus lactis NZ9000 genome-scale metabolic model].

With the advent of the post-genomic era, metabolic engineering of microorganisms plays an increasingly important role in industrial production. The genome-scale metabolic model (GSMM) integrates all known metabolic information in the organism to provide an optimal platform for global understanding of the metabolic state of the organism and rational guidance for metabolic engineering. As a model strain, Lactococcus lactis NZ9000 plays an important role in industrial fermentation, but there is still no specific genome-scale metabolic model for it. Based on genomic function annotation and comparative genomics, we constructed the first genome-scale metabolic model iWK557 of L. lactis NZ9000, which contains 557 genes, 668 metabolites, and 840 reactions, and further verified at both qualitative and quantitative levels, to provide a good tool for rationally guiding metabolic engineering.

Composition, source, and influencing factors of white spots formation in soybean paste.

White spots are commonly found in bean-based fermented food, which will significantly lower the product quality. This study aimed to analyze the composition of white spots and further reveal the source and influencing factors of white spots in bean-based fermented food using soybean paste as study model. The results showed that white spots were mainly composed of 40.96% free tyrosine and 37.94% tyrosine in combination form. During soybean paste fermentation, tyrosine was found to be produced by the actions of proteolytic enzymes secreted by Aspergillus oryzae 3.042 instead of the microbial metabolism and the excessive accumulation of tyrosine in soybean paste led to the formation of white spots. Among all influencing factors, high temperature treatment favored the formation of white spots. The existence of soy peptone and phenylalanine would postpone the precipitation of tyrosine while promoting the aggregation of the tyrosine precipitation. Field emission scanning electron microscope analysis showed that tyrosine would accumulate around the soybean protein particles and treatment at 120 °C would disrupt the structure of tyrosine-protein complex. Based on the above results, we proposed that treatment of soybean paste at temperature lower than 80 °C was the current practically applicable method to control the formation of white spots in soybean paste. PRACTICAL APPLICATION: This study developed a new idea to understand the composition and formation of white spots in soybean paste, which would provide guidance for prevention and control of white spots during the production of soybean paste for manufacturers and researchers.

Nacre-biomimetic graphene oxide paper intercalated by phytic acid and its ultrafast fire-alarm application.

A novel nacre-like flame-retardant paper based on graphene oxide (GO), and phytic acid (PTA) is fabricated via evaporation-induced self-assembly. This facile method is time saving and low energy consuming. A facile approach is proposed to improve thermal oxidative stability of GO paper by in situ phosphorus doping during flame exposure. Then fire-alarm system is designed based on the high-temperature thermal reduction characteristic of GO. The GO paper functionalized with PTA (GO-PTA) can provide ultrasensitive, reliable and longtime fire early-warning signal. Fire alarm can be triggered at approximately 0.50 s when GO-PTA samples are attacked by fire. Phosphorus atoms are in situ doped into graphene layers during fire exposure, endowing GO-PTA paper with outstanding thermal oxidative stability, and thus alarm duration time of GO is greatly improved. The work develops advanced fire detection and early-warning sensors that provide reliable and continuous signals, which provide more available time for fire evacuation.

Residue analysis of gibberellic acid isomer (iso-GA3) in brewing process and its toxicity evaluation in mice.

Gibberellic acid (GA3) is an efficient plant growth regulator, which could speed up barley germination in the brewing industry. However, the residue of GA3 in malt gets denatured into an isomer, termed iso-GA3. In this study, the concentration of iso-GA3 and the conversion rate of GA3 to iso-GA3 during the brewing process was studied by high performance liquid chromatography and the potential toxicity of iso-GA3 was evaluated in ICR mice. The concentration of iso-GA3 increased in the saccharification and wort boiling processes while its concentration was stable during fermentation. The maximum conversion rates of GA3 to iso-GA3 in Canadian malt, Australian malt SCO and Australian malt FAQ were 88%, 87% and 87%, respectively. In the acute oral toxicity study, the median lethal dose (LD50) of iso-GA3 was 2.82 g/kg body weight (BW). In the 28-day repeated dose toxicity study, the iso-GA3 could cause weight loss in mice. And the mice of high-dose group showed a slight decrease in food consumption. Moreover, inflammation and cell necrosis were found in kidney and liver tissue, which were alleviated during the recovery phase. These results establish a practical reference for food safety in products, in which GA3 is added as a food additive.

Theoretical Study on Sandwich-Like Transition-Metal-Cyclooctatetraene Clusters and One-Dimensional Infinite Molecular Wires.

Using density functional theory calculations, we investigated the structure and electronic properties of cyclooctatetraene (C8H8, COT)-ligand mono- or bi-transition-metal (M) sandwich clusters, M n (COT) n+1 (M = Sc, Ti, Cr, Mn, n = 1, 2) or (COT)M1(COT)M2(COT), as well as their one-dimensional infinite molecular wires. All the sandwich M-COT clusters and molecular wires are rather stable with their binding energies ranging from 3.20 to 7.48 eV per transition-metal atom. Superior to M n Bz n+1 complexes, most sandwich M-COT complexes are in their high spin states with ultrahigh magnetic moments. Moreover, one-dimensional infinite molecular wires, [Cr(COT)]∞, [(COT)V(COT)Ti]∞ and [(COT)Sc(COT)Cr]∞, are predicted to be ferromagnetic half-metals. Our findings suggest that such M-COT sandwich complexes may be potential candidates for applications in spintronics.

Isomerization of Gibberellic Acid During the Brewing Process.

Gibberellic acid (GA3) was added to three types of beer barley, and the chemical changes to GA3 during the beer brewing process were studied using HPLC. The results demonstrated that the GA3 concentration decreased throughout the malting, mashing, and boiling processes and that no GA3 was detected in the congress wort. A new substance, herein called Substance A, was detected by HPLC analysis using a C18 column; this substance exhibited retention characteristics different from GA3. The concentration of Substance A increased throughout the malting, mashing, and boiling processes. Mass spectrometry revealed that Substance A has the same molecular weight as GA3 and nuclear magnetic resonance studies determined that Substance A is a structural isomer of GA3. PRACTICAL APPLICATION: This study developed a new idea to understand GA3 behavior during the brewing, which provided a practical reference for food safety in beer and other fields using GA3 as a food additive.

Adjustable Thermal Expansion Properties in Zr2MoP2O12/ZrO2 Ceramic Composites.

Zr2MoP2O12/ZrO2 composites were successfully synthesized by the solid state method in attempt to fabricate the near-zero thermal expansion ceramics. The phase composition, micromorphology and thermal expansion behavior of the Zr2MoP2O12/ZrO2 composites with different mass ratios were investigated using X-ray diffraction, scanning electron microscopy and thermal mechanical analysis. Results indicate that Zr2MoP2O12/ZrO2 composites can be prepared by pre-sintering at 500°C for 3 h and then sintering at 1050°C for 6 h. The resulting Zr2MoP2O12/ZrO2 composites consisted of orthorhombic Zr2MoP2O12 and monoclinic ZrO2. With increasing content of Zr2MoP2O12, the Zr2MoP2O12/ZrO2 ceramics became more compact and the coefficient of thermal expansion decreased gradually. Zr2MoP2O12/ZrO2 composites show an adjustable coefficient of thermal expansion (CTE) from 5.57 × 10-6 K-1 to -5.73 × 10-6 K-1 by changing the mass ratio of Zr2MoP2O12 and ZrO2. The Zr2MoP2O12/ZrO2 composite with a mass ratio of 2:1 showed near zero thermal expansion, and the average linear thermal expansion coefficient is measured to be 0.0065 × 10-6 K-1 in the temperature range from 25 to 700°C.

Synthesis of Zr2WP2O12/ZrO2 Composites with Adjustable Thermal Expansion.

Zr2WP2O12/ZrO2 composites were fabricated by solid state reaction with the goal of tailoring the thermal expansion coefficient. XRD, SEM and TMA were used to investigate the composition, microstructure, and thermal expansion behavior of Zr2WP2O12/ZrO2 composites with different mass ratio. Relative densities of all the resulting Zr2WP2O12/ZrO2 samples were also tested by Archimedes' methods. The obtained Zr2WP2O12/ZrO2 composites were comprised of orthorhombic Zr2WP2O12 and monoclinic ZrO2. As the increase of the Zr2WP2O12, the relative densities of Zr2WP2O12/ZrO2 ceramic composites increased gradually. The coefficient of thermal expansion of the Zr2WP2O12/ZrO2 composites can be tailored from 4.1 × 10-6 K-1 to -3.3 × 10-6 K-1 by changing the content of Zr2WP2O12. The 2:1 Zr2WP2O12/ZrO2 specimen shows close to zero thermal expansion from 25 to 700°C with an average linear thermal expansion coefficient of -0.09 × 10-6 K-1. These adjustable and near zero expansion ceramic composites will have great potential application in many fields.

Genome-Wide Analysis of CCA1-Like Proteins in Soybean and Functional Characterization of GmMYB138a.

Plant CIRCADIAN CLOCK ASSOCIATED1 (CCA1)-like proteins are a class of single-repeat MYELOBLASTOSIS ONCOGENE (MYB) transcription factors generally featured by a highly conserved motif SHAQK(Y/F)F, which play important roles in multiple biological processes. Soybean is an important grain legume for seed protein and edible vegetable oil. However, essential understandings regarding CCA1-like proteins are very limited in soybean. In this study, 54 CCA1-like proteins were identified by data mining of soybean genome. Phylogenetic analysis indicated that soybean CCA1-like subfamily showed evolutionary conservation and diversification. These CCA1-like genes displayed tissue-specific expression patterns, and analysis of genomic organization and evolution revealed 23 duplicated gene pairs. Among them, GmMYB138a was chosen for further investigation. Our protein-protein interaction studies revealed that GmMYB138a, but not its alternatively spliced isoform, interacts with a 14-3-3 protein (GmSGF14l). Although GmMYB138a was predominately localized in nucleus, the resulting complex of GmMYB138a and GmSGF14l was almost evenly distributed in nucleus and cytoplasm, supporting that 14-3-3s interact with their clients to alter their subcellular localization. Additionally, qPCR analysis suggested that GmMYB138a and GmSGF14l synergistically or antagonistically respond to drought, cold and salt stresses. Our findings will contribute to future research in regard to functions of soybean CCA1-like subfamily, especially regulatory mechanisms of GmMYB138a in response to abiotic stresses.

Discovery of N-(3,5-bis(1-pyrrolidylmethyl)-4-hydroxybenzyl)-4-methoxybenzenesulfamide (sulcardine) as a novel anti-arrhythmic agent.

AIM: To investigate the anti-arrhythmic effects of sulfamide analogues of changrolin and to characterize the sulfate of compound 6f (sulcardine sulfate, Sul) as a novel anti-arrhythmic agent. METHODS: The anti-arrhythmic effects of compounds were studied against aconitine-induced arrhythmias in rats and ouabain-induced arrhythmias in guinea pigs. The effects of Sul on transmembrane action potentials were investigated in isolated rabbit sinoatrial nodes and guinea-pig papillary muscles using intracellular recording. With a whole-cell recording technique, the effects of Sul on sodium current, calcium current, and potassium currents were examined in isolated single guinea-pig ventricular myocytes. RESULTS: In aconitine-induced arrhythmias of rats, sulfamide analogues of changrolin (4, 5, and 6a-6p) exhibited various anti-arrhythmic activities. The sulfate of compound 6f (Sul) increased the amount of aconitine required to induce arrhythmias in each treated animal. The ED50 value of Sul in rats was 196 mg/kg. In ouabain-induced arrhythmias of guinea pigs, 25, 50, and 100 mg/kg doses of Sul increased the dose of ouabain required to induce VP, VT, and VF in a dose-dependent manner. In papillary preparations, Sul produced a concentration-dependent decrease in APA and V(max), prolonged APD(90) and ERP, whereas RP was unaffected. In the spontaneously beating sinus nodes, Sul reduced APA and V(max) in a concentration-dependent manner. The whole-cell recording studies revealed that Sul produced a reversible reduction in I(Na) (IC50=26.9 µmol/L) and I(Ca,L)(IC50=69.2 µmol/L), whereas the inward rectifier (I(K1)) and the delayed rectifier potassium currents (I(K)) were unaffected. CONCLUSION: As a multi-ion channel blocker, Sul may have potent efficacy in anti-atrial and ventricular arrhythmias.

Liquid chromatography/tandem mass spectrometry for the determination of magnesium lithospermate B in beagle dog serum.

A sensitive and specific isocratic liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the quantification of magnesium lithospermate B (MLB) in beagle dog serum with silibinin as internal standard. The serum samples were treated by special liquid-liquid extraction, and the analytes were determined using electrospray negative ionization mass spectrometry in the selective monitoring mode, with sufficient sensitivity to allow analysis of dog serum samples generated following administration of a clinically relevant dose. The calibration curve for MLB was linear over the range 8-2048 ng/ml with coefficients of correlation >0.999. The intra- and inter-day precisions (CV) of analysis were <7%, and accuracy ranged from 90 to 106%. This quantitation method was successfully applied to a pharmacokinetic study of i.h. administration of MLB with dosages of 3, 6, 12 mg/kg in beagle dogs.