Current statistical methods for estimating the K_m and V_max of Michaelis-Menten kinetics

Global warming is leading to extended stratification in deep lakes, which may exacerbate phosphorus (P) limitation in the upper waters. Conversion of labile dissolved organic P (DOP) is a possible adaptive strategy to maintain primary production. To test this, the spatiotemporal distributions of various soluble P fractions and phosphomonesterase (PME)/phosphodiesterase (PDE) activities were investigated in Lake Fuxian during the stratification period and the transition capacity of organic P and its impact on primary productivity were evaluated. The results indicated that the DOP concentration (mean 0.20 ± 0.05 μmol L ^{− 1}) was significantly higher than that of dissolved inorganic P (DIP) (mean 0.08 ± 0.03 μmol L ^{− 1}) in the epilimnion and metalimnion, which were predominantly composed of orthophosphate monoester (monoester-P) and orthophosphate diesters (diester-P). The low ratio of diester-P / monoester-P and high activities of PME and PDE indicate DOP mineralization in the epilimnion and metalimnion. We detected a DIP threshold of approximately 0.19 μmol L ^{− 1}, corresponding to the highest total PME activity in the lake. Meta-analysis further demonstrated that DIP thresholds of PME activities were prevalent in oligotrophic (0.19 μmol L ^{− 1}) and mesotrophic (0.74 μmol L ^{− 1}) inland waters. In contrast to the phosphate-sensitive phosphatase PME, dissolved PDE was expressed independent of phosphate availability and its activity invariably correlated with chlorophyll a, suggesting the involvement of phytoplankton in DOP utilization. This study provides important field evidence for the DOP transformation processes and the strategy for maintaining primary productivity in P-deficient scenarios, which contributes to the understanding of P cycles and the mechanisms of system adaptation to future long-term P limitations in stratified waters.

Milk glycoproteins play various biological roles including antibacterial, antiviral activities, modulating immune responses in living organisms. Released N-glycans from milk glycoproteins act as growth substrates for infant-associated bifidobacteria, which are key members of the breastfed infant’s gut. To date, the mechanisms, and contributions of glycans to the biological activities of glycoproteins remain to be elucidated. Only by testing both the released glycans and the deglycosylated protein in their native (i.e., non-denatured) form, can the individual contribution to the biological activity of glycoproteins be elucidated. However, for conventional enzymatic and chemical deglycosylation strategies to work efficiently, glycoprotein denaturation is required, which alters the protein native shape, hindering further investigations of its biological roles. An endo-β-N-acetylglucosaminidase (EndoBI-1) from Bifidobacterium longum subsp. infantis ATCC 15697 (B. infantis) was characterized as having the ability to release N-glycans from bovine milk glycoproteins efficiently, without the denaturation. In this study, the activity of EndoBI-1 was compared to a commercial enzyme to release N-glycans, the peptide-N-glycosidase F (PNGase F), using dairy glycoproteins as the substrate. The kinetic evaluation showed that EndoBI-1 displayed higher activity on native glycoproteins than PNGase F, with 0.036 mg/mL×min and 0.012 mg/mL×min glycan release, respectively. EndoBI-1 released a broader array of glycan structures compared to PNGase F from native glycoproteins. Thirty-two and fifteen distinct compositions were released from the native glycoproteins by EndoBI-1 and PNGase F, respectively, as characterized by advanced mass spectrometry. EndoBI-1 can be considered a promising enzyme for the release of N-glycans and their protein backbone in the native form, which will enable effective glycan release and will facilitate subsequent investigations to reveal their contribution to glycoproteins’ biological roles.

Like many other scientific fields, an inverse technique is used to find unknown material properties (such as thermal conductivity, specific heat) or boundary conditions (e.g. heat flux, heat transfer coefficient) in the heat transfer problem. The role of the inverse technique is even more important if the properties or boundary conditions change with location and/or time. In this study, a Bayesian inference based inverse technique is used to find local heat transfer coefficient as well as local steam temperature at the inner surface of a steam header where the header was first heated by supplying steam followed by the cooling of the header due to the release of steam from the chamber. A finite volume based numerical model is developed to solve the forward unsteady heat conduction problem in the steam header. For inverse problem, a Markov chain Monte Carlo method is used to evaluate the posterior probability density functions (PPDFs) of unknown parameters in Bayesian framework, while a Metropolis-Hastings algorithm is used for random sample generation. Here PPDFs are used to extract the point estimates of the parameters and their credible intervals from their possible distributions. With the estimated parameters, the calculated outer surface temperatures agree well with experimental measurements in both heating and cooling process. At the end of the discharge process, the temperature distribution in the radial direction approaches flat profiles, but a steady state is only achieved at the bottom of the header. Our stochastic based Bayesian technique can be used to estimate unknown boundary conditions efficiently at an inaccessible location.

Erwinia amylovora is the causal agent of fire blight, a devastating disease of apples and pears. A previous study revealed that an E. amylovora uracil auxotroph was still virulent and can cause disease, suggesting that uracil can be obtained from the host environment. The E. amylovora genome contains a locus encoding for a uracil transporter belonging to the nucleobase cation symporter 2 family, displaying a high level of amino acid sequence similarity to the Escherichia coli UraA. Expression of E. amylovora UraA in nucleobase transporter-deficient E. coli strains, coupled with radiolabeled uptake studies reveal that E. amylovora UraA is a high affinity uracil transporter with a K_m of 0.57 μM. Both E. coli and E. amylovora carrying extra copies of E. amylovora UraA are sensitive to growth on the toxic analog 5-fluorouracil. An E. amylovora ΔuraA::Cam^r mutant is still able to grow and cause disease symptoms on immature pears and apples.

Nucleobase Cation Symporter 1 proteins are present in bacteria, fungi and among plants. Microbes often contain numerous NCS1 proteins that have evolved specific solute specificity profiles (solute transport and binding). Conversely, most members of Viridiplantae have a unique NCS1 that shows a broad, yet species –specific, solute specificity profile. Properties of NCS1 from Nicotiana sylvestris Speg. & Comes (NsNCS1) have been determined as part of an ongoing evolution-function analysis through heterologous expression studies in ncs1-deficient Saccharomyces cerevisiae Meyen ex E.C. Hansen as well as functional assays and subcellular localization studies in planta. NsNCS1 transports the purine adenine with high affinity and moves guanine, uracil, cytosine and hypoxanthine. Xanthine, uric acid and 5-flourocytosine act as competitive inhibitors for NsNCS1. When expressed in Arabidopsis, NsNCS1 locates to the chloroplast and facilitates the transport of guanine and 8-azaguanine. The NsNCS1 solute specificity profile is very similar, yet distinct, from AtNCS1 (A. thaliana (L.) Heynh. NCS1) and indicates that despite evolutionary divergence in the Eudicots - Nicotiana, an Astrid, and Arabidopsis, a Rosid, − little functional difference evolved. The alignment of select plant NCS1 amino acid sequences coupled with a detailed knowledge of each solute specificity profile facilitates identifying residues that may contribute to subtle solute discrimination. Such an evolution-function analysis will complement recent molecular model based site-directed mutagenesis analysis of select microbial and plant NCS1.

In neurodegenerative diseases such as Alzheimer's and Parkinson's, excessive irons as well as lactoferrin (Lf), but not transferrin (Tf), have been found in and around the affected regions of the brain. These evidences suggest that lactoferrin plays a critical role during neurodegenerative diseases, although Lf-mediated iron transport across blood-brain barrier (BBB) is negligible compared to that of transferrin in normal condition. However, the kinetics of lactoferrins and lactoferrin-mediated iron transport are still unknown.

To determine the kinetic rate constants of lactoferrin-mediated iron transport through BBB, a mass-action based ordinary differential equation model has been presented. A Bayesian framework is developed to estimate the kinetic rate parameters from posterior probability density functions. The iron transport across BBB is studied by considering both Lf- and Tf-mediated pathways for both normal and pathologic conditions.

Using the point estimates of kinetic parameters, our model can effectively reproduce the experimental data of iron transport through BBB endothelial cells. The robustness of the model and parameter estimation process are further verified by perturbation of kinetic parameters. Our results show that surge in high-affinity receptor density increases lactoferrin as well as iron in the brain.

Due to the lack of a feedback loop such as iron regulatory proteins (IRPs) for lactoferrin, iron can transport to the brain continuously, which might increase brain iron to pathological levels and can contribute to neurodegeneration.

This study provides an improved understanding of presence of lactoferrin and iron in the brain during neurodegenerative diseases.