Role of site-directed mutagenesis and adjuvants in the stability and potency of anthrax protective antigen.

Anthrax is a zoonotic infection caused by the gram-positive, aerobic, spore-forming bacterium Bacillus anthracis. Depending on the origin of the infection, serious health problems or mortality is possible. The virulence of B. anthracis is reliant on three pathogenic factors, which are secreted upon infection: protective antigen (PA), lethal factor (LF), and edema factor (EF). Systemic illness results from LF and EF entering cells through the formation of a complex with the heptameric form of PA, bound to the membrane of infected cells through its receptor. The currently available anthrax vaccines have multiple drawbacks, and recombinant PA is considered a promising second-generation vaccine candidate. However, the inherent chemical instability of PA through Asn deamidation at multiple sites prevents its use after long-term storage owing to loss of potency. Moreover, there is a distinct possibility of B. anthracis being used as a bioweapon; thus, the developed vaccine should remain efficacious and stable over the long-term. Second-generation anthrax vaccines with appropriate adjuvant formulations for enhanced immunogenicity and safety are desired. In this article, using protein engineering approaches, we have reviewed the stabilization of anthrax vaccine candidates that are currently licensed or under preclinical and clinical trials. We have also proposed a formulation to enhance recombinant PA vaccine potency via adjuvant formulation.

Vanadium: History, chemistry, interactions with α-amino acids and potential therapeutic applications.

In the last 30 years, since the discovery that vanadium is a cofactor found in certain enzymes of tunicates and possibly in mammals, different vanadium-based drugs have been developed targeting to treat different pathologies. So far, the in vitro studies of the insulin mimetic, antitumor and antiparasitic activity of certain compounds of vanadium have resulted in a great boom of its inorganic and bioinorganic chemistry. Chemical speciation studies of vanadium with amino acids under controlled conditions or, even in blood plasma, are essential for the understanding of the biotransformation of e.g. vanadium antidiabetic complexes at the physiological level, providing clues of their mechanism of action. The present article carries out a bibliographical research emphaticizing the chemical speciation of the vanadium with different amino acids and reviewing also some other important aspects such as its chemistry and therapeutical applications of several vanadium complexes.

Short-chain fatty acids reprogram metabolic profiles with the induction of reactive oxygen species production in human colorectal adenocarcinoma cells.

Short-chain fatty acids (SCFAs) exhibit anticancer activity in cellular and animal models of colon cancer. Acetate, propionate, and butyrate are the three major SCFAs produced from dietary fiber by gut microbiota fermentation and have beneficial effects on human health. Most previous studies on the antitumor mechanisms of SCFAs have focused on specific metabolites or genes involved in antitumor pathways, such as reactive oxygen species (ROS) biosynthesis. In this study, we performed a systematic and unbiased analysis of the effects of acetate, propionate, and butyrate on ROS levels and metabolic and transcriptomic signatures at physiological concentrations in human colorectal adenocarcinoma cells. We observed significantly elevated levels of ROS in the treated cells. Furthermore, significantly regulated signatures were involved in overlapping pathways at metabolic and transcriptomic levels, including ROS response and metabolism, fatty acid transport and metabolism, glucose response and metabolism, mitochondrial transport and respiratory chain complex, one-carbon metabolism, amino acid transport and metabolism, and glutaminolysis, which are directly or indirectly linked to ROS production. Additionally, metabolic and transcriptomic regulation occurred in a SCFAs types-dependent manner, with an increasing degree from acetate to propionate and then to butyrate. This study provides a comprehensive analysis of how SCFAs induce ROS production and modulate metabolic and transcriptomic levels in colon cancer cells, which is vital for understanding the mechanisms of the effects of SCFAs on antitumor activity in colon cancer.

Dual Heterozygous Mutations in CYP21A2 and CYP11B1 in a Case of Nonclassic Congenital Adrenal Hyperplasia.

Background/Objective: Nonclassic congenital adrenal hyperplasia (NCCAH) may be overlooked or mistaken for polycystic ovarian syndrome. Unlike congenital adrenal hyperplasia (CAH), the enzymatic activities of 21-hydroxylase or 11ß-hydroxylase in NCCAH are not completely lost. In this case, NCCAH presented in a patient with CYP21A2 and CYP11B1 heterozygous mutations, one of which is a variant of unknown significance in CYP11B1. Case Report: A 30-year-old woman presented with a chief complaint of irregular menses and hirsutism. Previous medical history was significant for a prolactin level of 34.7 ng/mL (reference range, 2.0-23.0 ng/mL), a total serum testosterone level of 77 ng/dL (reference range, 25-125 ng/dL, not sex-specific), and a 2-mm × 3-mm pituitary lesion. An adrenocorticotrophic hormone stimulation test increased the 17-hydroxyprogesterone level from 444 ng/dL at baseline to 837 ng/dL at 60 minutes (baseline female reference range and stimulated reference ranges are 10-300 ng/dL and <1000 ng/dL, respectively). Gene sequencing revealed a heterozygous pathogenic CYP21A2 variant and a heterozygous, previously undescribed variant of unknown significance in CYP11B1. Discussion: Unlike CAH, NCCAH presents more subtly and later in life, and salt wasting and hypertension are not typically seen. Although mutations in CYP11B1 that cause steroid 11ß-hydroxylase deficiency more commonly lead to the CAH phenotype, cases have been reported of CYP11B1 mutations leading to NCCAH, depending on the location of the mutations. Conclusion: This patient's case demonstrates physical examination and laboratory findings suggestive of NCCAH. Our case adds to the database of described mutations in CYP11B1 and suggests that heterozygous mutations in 2 different genes may present phenotypically as NCCAH.

In silico study reveals binding potential of rotenone at multiple sites of pulmonary surfactant proteins: A matter of concern.

Rotenone is a broad-spectrum pesticide employed in various agricultural practices all over the world. Human beings are exposed to this chemical through oral, nasal, and dermal routes. Inhalation of rotenone exposes bio-molecular components of lungs to this chemical. Biophysical activity of lungs is precisely regulated by pulmonary surfactant to facilitate gaseous exchange. Surfactant proteins (SPs) are the fundamental components of pulmonary surfactant. SPs like SP-A and SP-D have antimicrobial activities providing a crucial first line of defense against infections in lungs whereas SP-B and SP-C are mainly involved in respiratory cycle and reduction of surface tension at air-water interface. In this study, molecular docking analysis using AutoDock Vina has been conducted to investigate binding potential of rotenone with the four SPs. Results indicate that, rotenone can bind with carbohydrate recognition domain (CRD) of SP-A, N-, and C- terminal peptide of SP-B, SP-C, and CRD of SP-D at multiples sites via several interaction mediators such as H bonds, C-H bonds, alkyl bonds, pi-pi stacked, Van der Waals interaction, and other. Such interactions of rotenone with SPs can disrupt biophysical and anti-microbial functions of SPs in lungs that may invite respiratory ailments and pathogenic infections.

Long-term intake of ginger protease-degraded collagen hydrolysate reduces blood lipid levels and adipocyte size in mice.

Collagen hydrolysate has various beneficial effects, such as bone strengthening, joint/skin protection and lipid metabolism regulation. In this study, the anti-obesity activity of ginger protease-degraded collagen hydrolysate (GDCH) was evaluated in BALB/c mice fed diets containing 14% casein (control group) or 10% casein +4% GDCH (GDCH group) for 10 weeks. In the GDCH group, triglyceride (TG) and cholesterol (CHO) levels in blood and adipocyte size in white adipose tissue were significantly decreased compared with those of the control group. Further, gene expression related to fatty acid synthesis, such as acetyl-CoA carboxylase, fatty acid synthase and stearoyl-CoA desaturase, was decreased in the liver and white adipose tissue of GDCH-fed mice. On the other hand, single oral administration of GDCH did not result in decrease in blood TG and CHO compared with vehicle and casein in ICR mice pre-administered soybean oil. These results suggest that the GDCH-induced decreases in tissue and blood lipids occur through long-term alterations in lipid metabolism, not transient inhibition of lipid absorption. The lipid-lowering effects exhibited by partial substitution of casein with GDCH imply the possibility that daily supplementation of GDCH contributes to prevention/attenuation of obesity and hyperlipidemia.

The concentration of brain homogenates with the Amicon Ultra Centrifugal filters.

Accurately measuring the brain concentration of a neurotherapeutic is critical in determining its pharmacokinetic profile in vivo. Biologics are potential therapeutics for neurologic diseases and biologics fused to an antibody targeting a transcytosis receptor at the Blood-Brain Barrier, designated as antibody-biologic fusion proteins, are Blood-Brain Barrier penetrating neurotherapeutics. The use of sandwich immunosorbent assays to measure concentrations of antibody-biologic fusion proteins in brain homogenates has become increasingly popular. The raw brain homogenate contains many proteins and other macromolecules that can cause a matrix effect, potentially interfering with the limit of detection of such assays and reduce the overall sample signal. Further, the low sample loading volumes while running these assays can reduce the sample signal. Our aim was therefore to optimize the existing tissue sample preparation and processing to concentrate the sample to elevate the signal of the analyte. Here, we present a protocol for concentrating and increasing the signal of transferrin receptor antibody-biologic fusion proteins in mouse brain homogenates using the Amicon Ultra Centrifugal filters. • The presented method uses the Amicon Ultra Centrifugal filters to concentrate mouse brain tissue homogenates. • The concentrated brain tissue homogenates are then assayed using standard sandwich enzyme-linked immunosorbent assay (ELISA) protocols. • This method improves upon the traditional brain homogenization procedure and ELISA measurements for antibody-biologic fusion proteins by effectively concentrating brain tissue homogenates.

Phytoconstituents from ten natural herbs as potent inhibitors of main protease enzyme of SARS-COV-2: In silico study.

Background: Lack of treatment of novel Coronavirus disease led to the search of specific antivirals that are capable to inhibit the replication of the virus. The plant kingdom has demonstrated to be an important source of new molecules with antiviral potential. Purpose: The present study aims to utilize various computational tools to identify the most eligible drug candidate that have capabilities to halt the replication of SARS-COV-2 virus by inhibiting Main protease (Mpro) enzyme. Methods: We have selected plants whose extracts have inhibitory potential against previously discovered coronaviruses. Their phytoconstituents were surveyed and a library of 100 molecules was prepared. Then, computational tools such as molecular docking, ADMET and molecular dynamic simulations were utilized to screen the compounds and evaluate them against Mpro enzyme. Results: All the phytoconstituents showed good binding affinities towards Mpro enzyme. Among them laurolitsine possesses the highest binding affinity i.e. -294.1533 kcal/mol. On ADMET analysis of best three ligands were simulated for 1.2 ns, then the stable ligand among them was further simulated for 20 ns. Results revealed that no conformational changes were observed in the laurolitsine w.r.t. protein residues and low RMSD value suggested that the Laurolitsine-protein complex was stable for 20 ns. Conclusion: Laurolitsine, an active constituent of roots of Lindera aggregata, was found to be having good ADMET profile and have capabilities to halt the activity of the enzyme. Therefore, this makes laurolitsine a good drug candidate for the treatment of COVID-19.

Sex-specific metabolic alterations in the type 1 diabetic brain of mice revealed by an integrated method of metabolomics and mixed-model.

Type 1 diabetes (T1D) can cause brain region-specific metabolic disorders, but whether gender influences T1D-related brain metabolic changes is rarely reported. Therefore, here we examined metabolic changes in six different brain regions of male and female mice under normal and T1D conditions using an integrated method of NMR-based metabolomics and linear mixed-model, and aimed to explore sex-specific metabolic changes from normal to T1D. The results demonstrate that metabolic differences occurred in all brain regions between two genders, while the hippocampal metabolism is more likely to be affected by T1D. At the 4th week after streptozotocin treatment, brain metabolic disorders mainly occurred in the cortex and hippocampus in female T1D mice, but the striatum and hippocampus in male T1D mice. In addition, anaerobic glycolysis was significantly altered in male mice, mainly in the striatum, midbrain, hypothalamus and hippocampus, but not in female mice. We also found that female mice exhibited a hypometabolism status relative to male mice from normal to T1D. Collectively, this study suggests that T1D affected brain region-specific metabolic alterations in a sex-specific manner, and may provide a metabolic view on diabetic brain diseases between genders.

Gene interactions observed with the HDL-c blood lipid, intakes of protein, sugar and biotin in relation to circulating homocysteine concentrations in a group of black South Africans.

BACKGROUND: Elevated homocysteine (Hcy) is associated with several pathologies. Gene-diet interactions related to Hcy might be used to customize dietary advice to reduce disease incidence. To explore this possibility, we investigated interactions between anthropometry, biochemical markers and diet and single-nucleotide polymorphisms (SNPs) in relation to Hcy concentrations. Five SNPs of Hcy-metabolizing enzymes were analyzed in 2010 black South Africans. RESULTS: Hcy was higher with each additional methylenetetrahydrofolate reductase (MTHFR) C677T minor allele copy, but was lower in methionine synthase (MTR) 2756AA homozygotes than heterozygotes. Individuals harboring cystathionine ß synthase (CBS) 833 T/844ins68 had lower Hcy concentrations than others. No interactive effects were observed with any of the anthropometrical markers. MTHFR C677T and CBS T833C/844ins68 homozygote minor allele carriers presented with lower Hcy as high density lipoprotein cholesterol (HDL-c) increased. Hcy concentrations were negatively associated with dietary protein and animal protein intake in the TT and TC genotypes, but positively in the CC genotype of CBS T833C/844ins68. Hcy was markedly higher in TT homozygotes of MTHFR C677T as added sugar intake increased. In CBS T833C/844ins68 major allele carriers, biotin intake was negatively associated with Hcy; but positively in those harboring the homozygous minor allele. CONCLUSIONS: The Hcy-SNP associations are modulated by diet and open up the possibility of invoking dietary interventions to treat hyperhomocysteinemia. Future intervention trials should further explore the observed gene-diet and gene-blood lipid interactions.

Palladium(II) complexes of biorelevant ligands. Synthesis, structures, cytotoxicity and rich DNA/HSA interaction studies.

In this work, a pair of new palladium(II) complexes, [Pd(Gly)(Phe)] and [Pd(Gly)(Tyr)], (where Gly is glycine, Phe is phenylalanine, and Tyr is tyrosine) were synthesized and characterized by UV-Vis, FT-IR, elemental analysis, 1H-NMR, and conductivity measurements. The detailed 1H NMR and infrared spectral studies of these Pd(II) complexes ascertain the mode of binding of amino acids to palladium through nitrogen of -NH2 and oxygen of -COO- groups as bidentate chelates. The Pd(II) complexes have been tested for in vitro cytotoxicity activities against cancer cell line of K562. Interactions of these Pd(II) complexes with CT-DNA and human serum albumin were identified through absorption/emission titrations and gel electrophoresis which indicated significant binding proficiency. The binding distance (r) between these synthesized complexes and HSA based on Forster's theory of non-radiation energy transfer were calculated. Alterations of HSA secondary structure induced by complexes were confirmed by FT-IR measurements. The results of emission quenching at three temperatures have revealed that the quenching mechanism of these Pd(II) complexes with CT-DNA and HSA were the static and dynamic quenching mechanism, respectively. Binding constants (Kb), binding site number (n), and the corresponding thermodynamic parameters were calculated and revealed that the hydrogen binding and hydrophobic forces played a major role when Pd(II) complexes interacted with DNA and HSA, respectively. We bid that [Pd(Gly)(Phe)] and [Pd(Gly)(Tyr)] complexes exhibit the groove binding with CT-DNA and interact with the main binding pocket of HSA. The complexes follow the binding affinity order of [Pd(Gly)(Tyr)] > [Pd(Gly)(Phe)] with CT-DNA- and HSA-binding.

Investigation on the interaction of newly designed potential antibacterial Zn(II) complexes with CT-DNA and HSA.

Two Zn(II) complexes of formula [Zn(bpy)(Gly)]NO3 (I) and [Zn(phen)(Gly)]NO3 (II) (where bpy = 2,2'-bipyridine, phen = 1,10-phenanthroline and Gly = glycine) were synthesized and characterized by elemental analysis, molar conductance measurements, UV-vis, FT-IR, and 1H NMR spectra. The interaction ability of these complexes with calf thymus DNA was monitored using spectroscopic methods, including UV-vis absorption spectroscopy, ethidium bromide displacement, Fourier transform infrared, and electrophoretic mobility assay. Further, the human serum albumin interactions of complexes I and II were investigated using UV-vis absorption spectroscopy, fluorescence quenching, circular dichroism, and Fourier transform infrared. The results obtained from these analyses indicated that both complexes interact effectively with CT-DNA and HSA. The binding constant (Kb), the Stern-Volmer constant (Ksv), and the number of binding sites (n) at different temperatures were determined for CT-DNA and HSA. Also, the negative ΔH° and ΔS° values showed that both hydrogen bonds and van der Waals forces played major roles in the association of CT-DNA-Zn(II) and HSA-Zn(II) complex formation. The displacement experiments suggested that Zn(II)-complexes primarily bound to Sudlow's site II of HSA. The distance between the donor (HSA) and the acceptor (Zn(II) complexes) was estimated on the basis of the Forster resonance energy transfer (FRET) and the alteration of HSA secondary structure induced by the compounds were confirmed by FT-IR spectroscopy. The complexes follow the binding affinity order of I > II with DNA and II > I with HSA. Finally, Antibacterial activity of complexes I and II have been screened against gram positive and gram negative bacteria.

A comprehensive evaluation of constraining amino acid biosynthesis in compartmented models for metabolic flux analysis.

Recent advances in the availability and applicability of genetic tools for non-conventional yeasts have raised high hopes regarding the industrial applications of such yeasts; however, quantitative physiological data on these yeasts, including intracellular flux distributions, are scarce and have rarely aided in the development of novel yeast applications. The compartmentation of eukaryotic cells adds to model complexity. Model constraints are ideally based on biochemical evidence, which is rarely available for non-conventional yeast and eukaryotic cells. A small-scale model for 13C-based metabolic flux analysis of central yeast carbon metabolism was developed that is universally valid and does not depend on localization information regarding amino acid anabolism. The variable compartmental origin of traced metabolites is a feature that allows application of the model to yeasts with uncertain genomic and transcriptional backgrounds. The presented test case includes the baker's yeast Saccharomyces cerevisiae and the methylotrophic yeast Hansenula polymorpha. Highly similar flux solutions were computed using either a model with undefined pathway localization or a model with constraints based on curated (S. cerevisiae) or computationally predicted (H. polymorpha) localization information, while false solutions were found with incorrect localization constraints. These results indicate a potentially adverse effect of universally assuming Saccharomyces-like constraints on amino acid biosynthesis for non-conventional yeasts and verify the validity of neglecting compartmentation constraints using a small-scale metabolic model. The model was specifically designed to investigate the intracellular metabolism of wild-type yeasts under various growth conditions but is also expected to be useful for computing fluxes of other eukaryotic cells.

Obstructive sleep apnea is associated with altered midbrain chemical concentrations.

Obstructive sleep apnea (OSA) is accompanied by altered structure and function in cortical, limbic, brainstem, and cerebellar regions. The midbrain is relatively unexamined, but contains many integrative nuclei which mediate physiological functions that are disrupted in OSA. We therefore assessed the chemistry of the midbrain in OSA in this exploratory study. We used a recently developed accelerated 2D magnetic resonance spectroscopy (2D-MRS) technique, compressed sensing-based 4D echo-planar J-resolved spectroscopic imaging (4D-EP-JRESI), to measure metabolites in the midbrain of 14 OSA (mean age±SD:54.6±10.6years; AHI:35.0±19.4; SAO2 min:83±7%) and 26 healthy control (50.7±8.5years) subjects. High-resolution T1-weighted scans allowed voxel localization. MRS data were processed with custom MATLAB-based software, and metabolite ratios calculated with respect to the creatine peak using a prior knowledge fitting (ProFit) algorithm. The midbrain in OSA showed decreased N-acetylaspartate (NAA; OSA:1.24±0.43, Control:1.47±0.41; p=0.03; independent samples t-test), a marker of neuronal viability. Increased levels in OSA over control subjects appeared in glutamate (Glu; OSA:1.23±0.57, Control:0.98±0.33; p=0.03), ascorbate (Asc; OSA:0.56±0.28, Control:0.42±0.20; (50.7±8.5years; p=0.03), and myo-inositol (mI; OSA:0.96±0.48, Control:0.72±0.35; p=0.03). No differences between groups appeared in γ-aminobutyric acid (GABA) or taurine. The midbrain in OSA patients shows decreased NAA, indicating neuronal injury or dysfunction. Higher Glu levels may reflect excitotoxic processes and astrocyte activation, and higher mI is also consistent with glial activation. Higher Asc levels may result from oxidative stress induced by intermittent hypoxia in OSA. Additionally, Asc and Glu are involved with glutamatergic processes, which are likely upregulated in the midbrain nuclei of OSA patients. The altered metabolite levels help explain dysfunction and structural deficits in the midbrain of OSA patients.

10-Gingerol as an inducer of apoptosis through HTR1A in cumulus cells: In-vitro and in-silico studies.

OBJECTIVES: Cumulus cells play a crucial role as essential mediators in the maturation of ova. Ginger contains 10-gingerol, which induces apoptosis in colon cancer cells. Based on this hypothesis, this study aimed to determine whether 10-gingerol is able to induce apoptosis in normal cells, namely, cumulus cells. METHODS: This study used an in vitro analysis by culturing Cumulus cells in M199 containing 10-gingerol in various concentrations (12, 16, and 20 µM) and later detected early apoptotic activity using an Annexin V-FITC detection kit. RESULT: The in vitro data revealed that the number of apoptosis cells increased along with the period of incubation as follows: 12 µM (63.71% ± 2.192%); 16 µM (74.51% ± 4.596%); and 20 µM (78.795% ± 1.435%). The substance 10-gingerol induces apoptosis in cumulus cells by inhibiting HTR1A functions and inactivating GSK3B and AKT-1. CONCLUSIONS: These findings indicate that further examination is warranted for 10-gingerol as a contraception agent.

Function and conformation analyses of an aspartate substitution of the invariant glycine in the integrin ßI domain α1-α1' helix.

We showed that the αLß2 integrin with the non-functional mutation G150D cannot be induced with Mg/EGTA to express the mAb KIM127 epitope, which reports the leg-extended conformation. We extended the study to the αIIbß3, an integrin without an αI domain. The equivalent mutation, i.e. G161D, also resulted in an expressible, but non-adhesive αIIbß3 integrin. An NMR study of synthetic peptides spanning the α1-α1' helix of the ß3 I domain shows that both wild-type and mutant peptides are α-helical. However, whereas in the wild-type peptide this helix is continuous, the mutant presents a discontinuity, or kink, precisely at the site of mutation G161D. Our results suggest that the mutation may lock integrin heterodimers in a bent conformation that prevents integrin activation via conformational extension.

Impact of plasma transaminase levels on the peripheral blood glutamate levels and memory functions in healthy subjects.

BACKGROUND & AIMS: Blood aspartate aminotransferase (AST) and alanine transaminase (ALT) levels are the most frequently reliable biomarkers of liver injury. Although AST and ALT play central roles in glutamate production as transaminases, peripheral blood levels of AST and ALT have been regarded only as liver injury biomarkers. Glutamate is a principal excitatory neurotransmitter, which affects memory functions in the brain. In this study, we investigated the impact of blood transaminase levels on blood glutamate concentration and memory. METHODS: Psychiatrically, medically, and neurologically healthy subjects (n = 514, female/male: 268/246) were enrolled in this study through local advertisements. Plasma amino acids (glutamate, glutamine, glycine, d-serine, and l-serine) were measured using a high performance liquid chromatography system. The five indices, verbal memory, visual memory, general memory, attention/concentration, and delayed recall of the Wechsler Memory Scale-Revised were used to measure memory functions. RESULTS: Both plasma AST and ALT had a significant positive correlation with plasma glutamate levels. Plasma AST and ALT levels were significantly negatively correlated with four of five memory functions, and plasma glutamate was significantly negatively correlated with three of five memory functions. Multivariate analyses demonstrated that plasma AST, ALT, and glutamate levels were significantly correlated with memory functions even after adjustment for gender and education. CONCLUSIONS: As far as we know, this is the first report which could demonstrate the impact of blood transaminase levels on blood glutamate concentration and memory functions in human. These findings are important for the interpretation of obesity-induced metabolic syndrome with elevated transaminases and cognitive dysfunction.

Glutamine up-regulates MAPK phosphatase-1 induction via activation of Ca2+→ ERK cascade pathway.

The non-essential amino acid L-glutamine (Gln) displays potent anti-inflammatory activity by deactivating p38 mitogen activating protein kinase and cytosolic phospholipase A2 via induction of MAPK phosphatase-1 (MKP-1) in an extracellular signal-regulated kinase (ERK)-dependent way. In this study, the mechanism of Gln-mediated ERK-dependency in MKP-1 induction was investigated. Gln increased ERK phosphorylation and activity, and phosphorylations of Ras, c-Raf, and MEK, located in the upstream pathway of ERK, in response to lipopolysaccharidein vitro and in vivo. Gln-induced dose-dependent transient increases in intracellular calcium ([Ca2+]i) in MHS macrophage cells. Ionomycin increased [Ca2+]i and activation of Ras → ERK pathway, and MKP-1 induction, in the presence, but not in the absence, of LPS. The Gln-induced pathways involving Ca2+→ MKP-1 induction were abrogated by a calcium blocker. Besides Gln, other amino acids including L-phenylalanine and l-cysteine (Cys) also induced Ca2+ response, activation of Ras → ERK, and MKP-1 induction, albeit to a lesser degree. Gln and Cys were comparable in suppression against 2, 4-dinitrofluorobenzene-induced contact dermatitis. Gln-mediated, but not Cys-mediated, suppression was abolished by MKP-1 small interfering RNA. These data indicate that Gln induces MKP-1 by activating Ca2+→ ERK pathway, which plays a key role in suppression of inflammatory reactions.

Comparison of unsupervised classification methods for brain tumor segmentation using multi-parametric MRI.

Tumor segmentation is a particularly challenging task in high-grade gliomas (HGGs), as they are among the most heterogeneous tumors in oncology. An accurate delineation of the lesion and its main subcomponents contributes to optimal treatment planning, prognosis and follow-up. Conventional MRI (cMRI) is the imaging modality of choice for manual segmentation, and is also considered in the vast majority of automated segmentation studies. Advanced MRI modalities such as perfusion-weighted imaging (PWI), diffusion-weighted imaging (DWI) and magnetic resonance spectroscopic imaging (MRSI) have already shown their added value in tumor tissue characterization, hence there have been recent suggestions of combining different MRI modalities into a multi-parametric MRI (MP-MRI) approach for brain tumor segmentation. In this paper, we compare the performance of several unsupervised classification methods for HGG segmentation based on MP-MRI data including cMRI, DWI, MRSI and PWI. Two independent MP-MRI datasets with a different acquisition protocol were available from different hospitals. We demonstrate that a hierarchical non-negative matrix factorization variant which was previously introduced for MP-MRI tumor segmentation gives the best performance in terms of mean Dice-scores for the pathologic tissue classes on both datasets.

Interaction of storage carbohydrates and other cyclic fluxes with central metabolism: A quantitative approach by non-stationary 13C metabolic flux analysis.

13C labeling experiments in aerobic glucose limited cultures of Saccharomyces cerevisiae at four different growth rates (0.054; 0.101, 0.207, 0.307 h-1) are used for calculating fluxes that include intracellular cycles (e.g., storage carbohydrate cycles, exchange fluxes with amino acids), which are rearranged depending on the growth rate. At low growth rates the impact of the storage carbohydrate recycle is relatively more significant than at high growth rates due to a higher concentration of these materials in the cell (up to 560-fold) and higher fluxes relative to the glucose uptake rate (up to 16%). Experimental observations suggest that glucose can be exported to the extracellular space, and that its source is related to storage carbohydrates, most likely via the export and subsequent extracellular breakdown of trehalose. This hypothesis is strongly supported by 13C-labeling experimental data, measured extracellular trehalose, and the corresponding flux estimations.