Bounds on the Ultrasensitivity of Biochemical Reaction Cascades.

The ultrasensitivity of a dose response function can be quantifiably defined using the generalized Hill coefficient of the function. We examined an upper bound for the Hill coefficient of the composition of two functions, namely the product of their individual Hill coefficients. We proved that this upper bound holds for compositions of Hill functions, and that there are instances of counterexamples that exist for more general sigmoidal functions. Additionally, we tested computationally other types of sigmoidal functions, such as the logistic and inverse trigonometric functions, and we provided computational evidence that in these cases the inequality also holds. We show that in large generality there is a limit to how ultrasensitive the composition of two functions can be, which has applications to understanding signaling cascades in biochemical reactions.

Carbon monoxide-releasing nanomotors based on endogenous biochemical reactions for tumor therapy.

The lack of selective release ability in the tumor microenvironment and the limited efficacy of monotherapy are important factors that limit the current use of carbon monoxide (CO) donors for tumor therapy. Herein, inspired by endogenous biochemical reactions in vivo, one kind of CO-releasing nanomotor was designed for the multimodal synergistic treatment of tumor. Specifically, glucose oxidase (GOx) and 5-aminolevulinic acid (5-ALA) were co-modified onto metal-organic framework material (MIL-101) to obtain MIL-GOx-ALA nanomotors (M-G-A NMs), which exhibit excellent biocompatibility and degradation ability in tumor microenvironment. Subsequently, the released 5-ALA generates CO in the tumor microenvironment through an endogenous reaction and further acts on mitochondria to release large amounts of reactive oxygen species (ROS), which directly kill tumor cells. Furthermore, the produced ROS and the degradation products of M-G-A NMs can also provide the reaction substrate for the Fenton reaction, thereby enhancing chemodynamic therapy (CDT) and inducing apoptosis of tumor cells. Both in vitro and in vivo experimental data confirm the successful occurrence of the above process, and the combination of CO gas therapy/enhanced CDT can effectively inhibit tumor growth. This CDT-enhancing agent designed based on endogenous biochemical reactions has good prospects for tumor treatment application.

Variante da Subunidade Beta 3 da Proteína G ( GNB3 ) Está Associada a Alterações Bioquímicas em Pacientes Brasileiros com Hipertensão / G Protein Subunit Beta 3 (GNB3) Variant Is Associated with Biochemical Changes in Brazilian Patients with Hypertension

Resumo Fundamento Genes e suas variantes associadas a fatores ambientais contribuem para o desenvolvimento do fenótipo hipertenso. O gene da subunidade beta 3 da proteína G ( GNB3 ) está envolvido no processo de sinalização intracelular e suas variantes têm sido relacionadas à suscetibilidade à hipertensão arterial. Objetivo Determinar a associação da variante GNB3 (rs5443:C>T) com a hipertensão arterial, parâmetros bioquímicos, idade e obesidade em indivíduos hipertensos e normotensos de Ouro Preto, Minas Gerais. Método A identificação das variantes foi realizada por PCR em tempo real, utilizando o sistema TaqMan®, em amostras de 310 pacientes (155 hipertensos e 155 normotensos). Análises bioquímicas (função renal, perfil lipídico e glicemia) foram realizadas a partir do soro por meio de espectrofotometria UV/Vis e eletrodo íon-seletivo. Foi utilizado um modelo de regressão logística múltipla para identificar fatores associados à hipertensão arterial. A análise das variáveis contínuas com distribuição normal foi realizada usando o teste t de Student não pareado; dados não normais foram analisados usando o teste de Mann-Whitney. Valores de p < 0,05 foram considerados significativos. Resultados A variante rs5443:C>T não esteve associada à hipertensão arterial na população avaliada (p = 0,88). Em relação às medidas bioquímicas, o alelo T esteve associado a níveis elevados de triglicerídeos, glicose e ácido úrico em indivíduos hipertensos (p < 0,05). Conclusão Os presentes resultados mostram a importância do diagnóstico genético para prevenir as causas e consequências de doenças e sugerem que a variante GNB3 rs5443:C>T pode estar associada a alterações no perfil bioquímico em indivíduos hipertensos.

Abstract Background Genes and their variants associated with environmental factors contribute to the development of the hypertensive phenotype. The G protein beta 3 subunit gene (GNB3) is involved in the intracellular signaling process, and its variants have been related to susceptibility to arterial hypertension. Objective To determine the association of the GNB3 variant (rs5443:C>T) with arterial hypertension, biochemical parameters, age, and obesity in hypertensive and normotensive individuals from Ouro Preto, Minas Gerais, Brazil. Method The identification of variants was performed by real-time PCR, using the TaqMan® system, in 310 samples (155 hypertensive and 155 normotensive). Biochemical analyses (renal function, lipid profile and glycemia) were performed from the serum using UV/Vis spectrophotometry and ion-selective electrode. A multiple logistic regression model was used to identify factors associated with arterial hypertension. The analysis of continuous variables with normal distribution was performed using the unpaired Student's t test; non-normal data were analyzed using Mann-Whitney. P < 0.05 was considered significant. Results The rs5443:C>T variant was not associated with arterial hypertension in the evaluated population (p = 0.88). Regarding biochemical measures, the T allele was associated with high levels of triglycerides, glucose and uric acid in hypertensive individuals (p < 0.05). Conclusion These results show the importance of genetic diagnosis to prevent the causes and consequences of diseases and imply that the GNB3 rs5443:C>T variant may be associated with changes in the biochemical profile in hypertensive individuals.

Proton transfer reactions: From photochemistry to biochemistry and bioenergetics.

The present Review is an attempt by projecting the basic knowledge on photochemical proton transfer to achieve consistent understanding of proton motions in biocatalysis, photobiocatalysis, operation of selective proton channels and systems of photosynthesis and cellular respiration. The basic mechanisms of proton transfer are in active research in the electronic excited states of organic molecules. This allows observing the reactions directly in real time, providing their dynamic and thermodynamic description and coupling with structural and energetic variables. These achievements lay the background for understanding the proton transfers in biochemical reactions, where such ultrafast events are not only 'optically silent' but are hidden under much slower rate-limiting steps, such as protein conformational changes, substrate binding and product release. The mechanistic description of biocatalytic and transmembrane proton transport is shown as a multi-step proton migration that is available for modeling in photochemical reactions. For explaining the formation of transmembrane proton gradients, a simple 'proton lift' concept is presented that may be the basis of further research and analysis.

G protein subunit Beta 3 (GNB3) variant is associated with biochemical changes in brazilian patients with Hypertension

Background: Genes and their variants associated with environmental factors contribute to the development of the hypertensive phenotype. The G protein beta 3 subunit gene (GNB3) is involved in the intracellular signaling process, and its variants have been related to susceptibility to arterial hypertension. Objective: To determine the association of the GNB3 variant (rs5443:C>T) with arterial hypertension, biochemical parameters, age, and obesity in hypertensive and normotensive individuals from Ouro Preto, Minas Gerais, Brazil. Method: The identification of variants was performed by real-time PCR, using the TaqMan® system, in 310 samples (155 hypertensive and 155 normotensive). Biochemical analyses (renal function, lipid profile and glycemia) were performed from the serum using UV/Vis spectrophotometry and ion-selective electrode. A multiple logistic regression model was used to identify factors associated with arterial hypertension. The analysis of continuous variables with normal distribution was performed using the unpaired Student’s t test; non-normal data were analyzed using Mann Whitney. P < 0.05 was considered significant. Results: The rs5443:C>T variant was not associated with arterial hypertension in the evaluated population (p = 0.88). Regarding biochemical measures, the T allele was associated with high levels of triglycerides, glucose and uric acid in hypertensive individuals (p < 0.05). Conclusion: These results show the importance of genetic diagnosis to prevent the causes and consequences of diseases and imply that the GNB3 rs5443:C>T variant may be associated with changes in the biochemical profile in hypertensive individuals.

G protein subunit Beta 3 (GNB3) variant is associated with biochemical changes in brazilian patients with hypertension

Fundamento Genes e suas variantes associadas a fatores ambientais contribuem para o desenvolvimento do fenótipo hipertenso. O gene da subunidade beta 3 da proteína G ( GNB3 ) está envolvido no processo de sinalização intracelular e suas variantes têm sido relacionadas à suscetibilidade à hipertensão arterial. Objetivo Determinar a associação da variante GNB3 (rs5443:C>T) com a hipertensão arterial, parâmetros bioquímicos, idade e obesidade em indivíduos hipertensos e normotensos de Ouro Preto, Minas Gerais. Método A identificação das variantes foi realizada por PCR em tempo real, utilizando o sistema TaqMan®, em amostras de 310 pacientes (155 hipertensos e 155 normotensos). Análises bioquímicas (função renal, perfil lipídico e glicemia) foram realizadas a partir do soro por meio de espectrofotometria UV/Vis e eletrodo íon-seletivo. Foi utilizado um modelo de regressão logística múltipla para identificar fatores associados à hipertensão arterial. A análise das variáveis contínuas com distribuição normal foi realizada usando o teste t de Student não pareado; dados não normais foram analisados usando o teste de Mann-Whitney. Valores de p < 0,05 foram considerados significativos. Resultados A variante rs5443:C>T não esteve associada à hipertensão arterial na população avaliada (p = 0,88). Em relação às medidas bioquímicas, o alelo T esteve associado a níveis elevados de triglicerídeos, glicose e ácido úrico em indivíduos hipertensos (p < 0,05). Conclusão Os presentes resultados mostram a importância do diagnóstico genético para prevenir as causas e consequências de doenças e sugerem que a variante GNB3 rs5443:C>T pode estar associada a alterações no perfil bioquímico em indivíduos hipertensos.

Identification and biological characterization of a Streptococcus parasuis strain / 中华微生物学和免疫学杂志

Objective:To identify a strain isolated from the cerebrospinal fluid of a patient and to investigate its biological characteristics.Methods:The strain was analyzed by several methods including Gram staining, biochemical identification, 16S rRNA and recN gene sequencing, average nucleotide identity (ANI), antibiotic susceptibility testing and detection of drug resistance and virulence genes. Results:The strain was Gram-positive cocci and formed α-hemolytic colonies on the blood plate. It was identified as Streptococcus parasuis by 16S rRNA, recN gene and whole-genome sequencing. It was sensitive to multiple antibiotics and carried the genes encoding a variety of virulence factors such as adhesion. Conclusions:Streptococcus parasuis could cause human infection and be identified by whole-genome sequencing.

Mechanistic insight into allosteric activation of human pyruvate carboxylase by acetyl-CoA.

Pyruvate carboxylase (PC) catalyzes the two-step carboxylation of pyruvate to produce oxaloacetate, playing a key role in the maintenance of metabolic homeostasis in cells. Given its involvement in multiple diseases, PC has been regarded as a potential therapeutic target for obesity, diabetes, and cancer. Albeit acetyl-CoA has been recognized as the allosteric regulator of PC for over 60 years, the underlying mechanism of how acetyl-CoA induces PC activation remains enigmatic. Herein, by using time-resolved cryo-electron microscopy, we have captured the snapshots of PC transitional states during its catalytic cycle. These structures and the biochemical studies reveal that acetyl-CoA stabilizes PC in a catalytically competent conformation, which triggers a cascade of events, including ATP hydrolysis and the long-distance communication between the two reactive centers. These findings provide an integrated picture for PC catalysis and unveil the unique allosteric mechanism of acetyl-CoA in an essential biochemical reaction in all kingdoms of life.

Insights into Interactions of Human Cytochrome P450 17A1: A Review.

Cytochrome P450s are a widespread and vast superfamily of hemeprotein monooxygenases that metabolize physiologically essential chemicals necessary for most species' survival, ranging from protists to plants to humans. They catalyze the synthesis of steroid hormones, cholesterol, bile acids, and arachidonate metabolites and the degradation of endogenous compounds, such as steroids, fatty acids, and other catabolizing compounds as an energy source and detoxifying xenobiotics, such as drugs, procarcinogens, and carcinogens. The human CYP17A1 is one of the cytochrome P450 genes located at the 10q chromosome. The gene expression occurs in the adrenals and gonads, with minor amounts in the brain, placenta, and heart. This P450c17 cytochrome gene is a critical steroidogenesis regulator which performs two distinct activities: 17 alpha-hydroxylase activity (converting pregnenolone to 17- hydroxypregnenolone and progesterone to 17-hydroxyprogesterone; these precursors are further processed to provide glucocorticoids and sex hormones) and 17, 20-lyase activity (which converts 17-hydroxypregnenolone to DHEA). Dozens of mutations within CYP17A1 are found to cause 17-alpha-hydroxylase and 17, 20-lyase deficiency. This condition affects the function of certain hormone-producing glands, resulting in high blood pressure levels (hypertension), abnormal sexual development, and other deficiency diseases. This review highlights the changes in CYP17A1 associated with gene-gene interaction, drug-gene interaction, chemical-gene interaction, and its biochemical reactions; they have some insights to correlate with the fascinating functional characteristics of this human steroidogenic gene. The findings of our theoretical results will be helpful to further the design of specific inhibitors of CYP17A1.

Distinguishing between models of mammalian gene expression: telegraph-like models versus mechanistic models.

Two-state models (telegraph-like models) have a successful history of predicting distributions of cellular and nascent mRNA numbers that can well fit experimental data. These models exclude key rate limiting steps, and hence it is unclear why they are able to accurately predict the number distributions. To answer this question, here we compare these models to a novel stochastic mechanistic model of transcription in mammalian cells that presents a unified description of transcriptional factor, polymerase and mature mRNA dynamics. We show that there is a large region of parameter space where the first, second and third moments of the distributions of the waiting times between two consecutively produced transcripts (nascent or mature) of two-state and mechanistic models exactly match. In this region: (i) one can uniquely express the two-state model parameters in terms of those of the mechanistic model, (ii) the models are practically indistinguishable by comparison of their transcript numbers distributions, and (iii) they are distinguishable from the shape of their waiting time distributions. Our results clarify the relationship between different gene expression models and identify a means to select between them from experimental data.

Leveraging biochemical reactions to unravel functional impacts of cancer somatic variants affecting protein interaction interfaces.

Background: Considering protein mutations in their biological context is essential for understanding their functional impact, interpretation of high-dimensional datasets and development of effective targeted therapies in personalized medicine. Methods: We combined the curated knowledge of biochemical reactions from Reactome with the analysis of interaction-mediating 3D interfaces from Mechismo. In addition, we provided a software tool for users to explore and browse the analysis results in a multi-scale perspective starting from pathways and reactions to protein-protein interactions and protein 3D structures. Results: We analyzed somatic mutations from TCGA, revealing several significantly impacted reactions and pathways in specific cancer types. We found examples of genes not yet listed as oncodrivers, whose rare mutations were predicted to affect cancer processes similarly to known oncodrivers. Some identified processes lack any known oncodrivers, which suggests potentially new cancer-related processes (e.g. complement cascade reactions). Furthermore, we found that mutations perturbing certain processes are significantly associated with distinct phenotypes (i.e. survival time) in specific cancer types (e.g. PIK3CA centered pathways in LGG and UCEC cancer types), suggesting the translational potential of our approach for patient stratification. Our analysis also uncovered several druggable processes (e.g. GPCR signalling pathways) containing enriched reactions, providing support for new off-label therapeutic options. Conclusions: In summary, we have established a multi-scale approach to study genetic variants based on protein-protein interaction 3D structures. Our approach is different from previously published studies in its focus on biochemical reactions and can be applied to other data types (e.g. post-translational modifications) collected for many types of disease.

Understanding the action of muscle iron concentration on dark cutting: An important aspect affecting consumer confidence of purchasing meat.

We investigated the association of muscle iron concentration, in addition to ultimate pH (pHU), on dark meat formation in sheep of different breeds fed forage-based diets. At 1 h simulated display, redness of meat (a*-value) increased (P < .0001) by about 3 units as the iron concentration increased from 10 to 22 mg/kg of meat, whereas the a*-value decreased by 2 units as pHU increased from 5.5 to 6.2 in fresh meat (P < .0001). After 90 days storage the corresponding responses were about 2 units increase for iron concentration and about 1 unit decrease for pHU, respectively. The results clearly show that increased muscle iron concentration was strongly associated with reduced dark cutting in fresh and stored meat evaluated at 1 h simulated display. We conclude that it may be desirable to measure iron concentration, along with pHU, for evaluation of the potential for carcasses to produce dark cutting meat, and for the meat to turn brown during display.

Adsorption of amino acids at clay surfaces and implication for biochemical reactions: Role and impact of surface charges.

Almost all clay minerals carry an abundance of surface charges. The role and impacts of surface charges during adsorption of amino acids and biochemical reactions are of great importance while currently remain elusive, which are to be tackled in this study by first-principles density functional calculations. A wide range of surface charges (-0.42˜ + 0.42 C m-2) have been considered. Distribution of different amino acid isomers and their interaction with clay minerals rely strongly on the sign and amount of surface charges. Zwitterionic structures remain stable for all negative surface charges and become dominant when negative surface charges are abundant (σ ≤ -0.28 C m-2), whereas only very high positive surface charges (σ ≥ +0.35 C m-2) can stabilize zwitterionic glycine. Increase of surface charges pronouncedly enhances the interactions of amino acids with clay minerals, which favors their gathering at clay surfaces and condensation to protein fragments. The superior binding of amino acids by negatively rather than positively charged clay minerals is due to stronger H bonding and electrostatic interactions. The biochemical reactions are greatly accelerated at higher surface charges and zwitterion formation becomes almost barrierless; however, the reverse reactions of forming canonical isomers have so moderate activation barriers that can occur facilely and get ready for the condensation to protein fragments. Accordingly, clay minerals, even in the anhydrous state, should be the suitable birthplace for life, where surface charges play a central role.

Computing Weakly Reversible Deficiency Zero Network Translations Using Elementary Flux Modes.

We present a computational method for performing structural translation, which has been studied recently in the context of analyzing the steady states and dynamical behavior of mass-action systems derived from biochemical reaction networks. Our procedure involves solving a binary linear programming problem where the decision variables correspond to interactions between the reactions of the original network. We call the resulting network a reaction-to-reaction graph and formalize how such a construction relates to the original reaction network and the structural translation. We demonstrate the efficacy and efficiency of the algorithm by running it on 508 networks from the European Bioinformatics Institutes' BioModels database. We also summarize how this work can be incorporated into recently proposed algorithms for establishing mono- and multistationarity in biochemical reaction systems.

Methods of phenotypic identification of non-tuberculous mycobacteria.

Non-tuberculous mycobacteria (NTM) are composed of mycobacterial species other than the Mycobacterium tuberculosis complex. Initially thought to be mere contaminants when isolated from clinical specimens, literature is increasing by the day showing NTM as proven pathogens. Due to the difference in antimicrobial susceptibility of different species, it becomes imperative for the microbiology laboratory to identify them to the species level. Molecular methods are available for rapid and accurate identification, but in a resource limited nation, phenotypic methods, albeit time consuming, are of paramount importance. By means of this article, the authors intend provide a concise summary of the basic biochemical reactions which can be done to identify most commonly isolated NTM.

Introduction of a new platform for parameter estimation of kinetically complex environmental systems.

A modeling framework (ReKinSim - Reaction Kinetics Simulator) is introduced, within which biogeochemical reactions in environmental systems can be described and inversely fitted to experimental data. Three key features of this simulation environment are: (1) a generic mathematical tool for solving sets of unlimited, arbitrary, non-linear ordinary differential equations; (2) no limitation to the number or type of reactions or other influential dynamics (e.g., isotope fractionation or small-scale mass-transfer limitations); (3) an easy to use and flexible module for nonlinear data-fitting. It allows users to easily define any kinetic model by a set of biogeochemical reactions relevant to the experimental application and to obtain the values of the kinetic parameters by fitting of the model to data. By allowing users to include the environmentally related processes and solving them along with the chemical kinetics, ReKinSim helps the user to elucidate the extent that these processes are controlled by factors other than kinetics. The novelty of the presented program primary lays in its unique combination of flexibility, computational efficiency and user-friendliness. ReKinSim's usability is showcased by four case studies of varying complexity, and compared against a set of currently available modeling tools.

Utility of Adenosine Monophosphate Detection System for Monitoring the Activities of Diverse Enzyme Reactions.

Adenosine monophosphate (AMP) is a key cellular metabolite regulating energy homeostasis and signal transduction. AMP is also a product of various enzymatic reactions, many of which are dysregulated during disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. In this study, we demonstrate the versatility of an enzyme-coupled assay that quantifies the amount of AMP produced by any enzymatic reaction regardless of its substrates. We successfully implemented it to enzyme reactions that use adenosine triphosphate (ATP) as a substrate (aminoacyl tRNA synthetase and DNA ligase) by an elaborate strategy of removing residual ATP and converting AMP produced into ATP; so it can be detected using luciferase/luciferin and generating light. We also tested this assay to measure the activities of AMP-generating enzymes that do not require ATP as substrate, including phosphodiesterases (cyclic adenosine monophosphate) and Escherichia coli DNA ligases (nicotinamide adenine dinucleotide [NAD+]). In a further elaboration of the AMP-Glo platform, we coupled it to E. coli DNA ligase, enabling measurement of NAD+ and enzymes that use NAD+ like monoadenosine and polyadenosine diphosphate-ribosyltransferases. Sulfotransferases use 3'-phosphoadenosine-5'-phosphosulfate as the universal sulfo-group donor and phosphoadenosine-5'-phosphate (PAP) is the universal product. PAP can be quantified by converting PAP to AMP by a Golgi-resident PAP-specific phosphatase, IMPAD1. By coupling IMPAD1 to the AMP-Glo system, we can measure the activities of sulfotransferases. Thus, by utilizing the combinations of biochemical enzymatic conversion of various cellular metabolites to AMP, we were able to demonstrate the versatility of the AMP-Glo assay.

Model reduction of the intracellular-signaling subsystem of apoptosis.

In recent few decades numerical treatment has become a standard tool in the system analysis and investigation of complex chemical reactions networks of reacting flows. The information about certain networks of biochemical reactions constantly increases. This leads to detailed descriptions of biochemical processes as a system of differential equations of high complexity and dimension. Nowadays methods, which are able automatically reduce the system dimension and complexity, are highly desirable. Recently several methods were developed for model reduction in combustion and chemical kinetics aiming at automatic numerical treatment and constructing the reduced system. The reduced system represents reliable description reproducing the detailed system behavior accurately enough. In this work the method of qualitative ODEs system analysis and the global quasi-linearization method (GQL) for kinetic mechanism reduction of combustion models are applied to the biochemical reaction network of the apoptosis. It is shown that the original model of the apoptosis can be essentially simplified firstly by using linear system integrals (9 dimensions) of the ODEs system, secondly the results of GQL analysis reveals the possibility of a further reduction (4 dimensions). It means that the final system dimension reaches 15 compare to the original 28 without any noticeable accuracy losses.

Mathematical modeling and simulation of the evolution of plaques in blood vessels.

In this paper, a model is developed for the evolution of plaques in arteries, which is one of the main causes for the blockage of blood flow. Plaque rupture and spread of torn-off material may cause closures in the down-stream vessel system and lead to ischemic brain or myocardial infarctions. The model covers the flow of blood and its interaction with the vessel wall. It is based on the assumption that the penetration of monocytes from the blood flow into the vessel wall, and the accumulation of foam cells increasing the volume, are main factors for the growth of plaques. The dynamics of the vessel wall is governed by a deformation gradient, which is given as composition of a purely elastic tensor, and a tensor modeling the biologically caused volume growth. An equation for the evolution of the metric is derived quantifying the changing geometry of the vessel wall. To calculate numerically the solutions of the arising free boundary problem, the model system of partial differential equations is transformed to an ALE (Arbitrary Lagrangian-Eulerian) formulation, where all equations are given in fixed domains. The numerical calculations are using newly developed algorithms for ALE systems. The results of the simulations, obtained for realistic system parameters, are in good qualitative agreement with observations. They demonstrate that the basic modeling assumption can be justified. The increase of stresses in the vessel wall can be computed. Medical treatment tries to prevent critical stress values, which may cause plaque rupture and its consequences.

Typing Discrepancy Between Phenotypic and Molecular Characterization Revealing an Emerging Biovar 9 Variant of Smooth Phage-Resistant B. abortus Strain 8416 in China.

A newly isolated smooth colony morphology phage-resistant strain 8416 isolated from a 45-year-old cattle farm cleaner with clinical features of brucellosis in China was reported. The most unusual phenotype was its resistance to two Brucella phages Tbilisi and Weybridge, but sensitive to Berkeley 2, a pattern similar to that of Brucella melitensis biovar 1. VITEK 2 biochemical identification system found that both strain 8416 and B. melitensis strains shared positive ILATk, but negative in other B. abortus strains. However, routine biochemical and phenotypic characteristics of strain 8416 were most similar to that of B. abortus biovar 9 except CO2 requirement. In addition, multiple PCR molecular typing assays including AMOS-PCR, B. abortus special PCR (B-ab PCR) and a novel sub-biovar typing PCR, indicated that strain 8416 may belong to either biovar 3b or 9 of B. abortus. Surprisingly, further MLVA typing results showed that strain 8416 was most closely related to B. abortus biovar 3 in the Brucella MLVA database, primarily differing in 4 out of 16 screened loci. Therefore, due to the unusual discrepancy between phenotypic (biochemical reactions and particular phage lysis profile) and molecular typing characteristics, strain 8416 could not be exactly classified to any of the existing B. abortus biovars and might be a new variant of B. abortus biovar 9. The present study also indicates that the present phage typing scheme for Brucella sp. is subject to variation and the routine Brucella biovar typing needs further studies.