In Vitro Digestion-In Situ Absorption Setup Employing a Physiologically Relevant Value of the Membrane Surface Area/Volume Ratio for Evaluating Performance of Lipid-Based Formulations: A Comparative Study with an In Vitro Digestion-Permeation Model.

The aim of this study is to establish and test an in vitro digestion-in situ absorption model that can mimic in vivo drug flux by employing a physiologically relevant value of the membrane surface area (S)/volume (V) ratio for accurate prediction of oral drug absorption from lipid-based formulations (LBFs). Three different types of LBFs (Type IIIA-MC, Type IIIA-LC, and Type IV) loaded with cinnarizine (CNZ), a lipophilic weak base with borderline permeability, and a control suspension were prepared. Subsequently, a simultaneous in vitro digestion-permeation experiment was conducted using a side-by-side diffusion cell with a dialysis membrane having a low S/V value. During digestion, CNZ partially precipitated for Type IV, while it remained solubilized in the aqueous phase for Type IIIA-MC and Type IIIA-LC in the donor compartment. However, in vitro drug fluxes for Type IIIA-MC and Type IIIA-LC were lower than those for Type IV due to the reduced free fraction of CNZ in the donor compartment. In pharmacokinetic studies, a similar improvement in in vivo oral exposure relative to suspension was observed, regardless of the LBFs used. Consequently, a poor correlation was found between in vitro permeation and areas under the plasma concentration-time curve (AUCoral) (R2 = 0.087). A luminal concentration measurement study revealed that this discrepancy was attributed to the extremely high absorption rate of CNZ in the gastrointestinal tract compared to that across a dialysis membrane evaluated by the in vitro digestion-permeation model, i.e., the absorption of CNZ in vivo was completed regardless of the extent of the free fraction, owing to the rapid removal of CNZ from the intestine. Subsequently, we aimed to predict the oral absorption of CNZ from the same formulations using a model that demonstrated high drug flux by employing the physiologically relevant S/V value and rat jejunum segment as an absorption sink (for replicating in vivo intestinal permeability). Predigested formulations were injected into the rat intestinal loop, and AUCloop values were calculated from the plasma concentration-time profiles. A better correlation was found between AUCloop and AUCoral (R2 = 0.72), although AUCloop underestimated AUCoral for Type IV due to the precipitation of CNZ during the predigestion process. However, this result indicated the importance of mimicking the in vivo drug absorption rate in the predictive model. The method presented herein is valuable for the development of LBFs.

A preliminary study of rapid-fire high-throughput metabolite analysis using nano-flow injection/Q-TOFMS.

In this study, we demonstrated nano-flow injection analysis (nano-FIA) with quadrupole time-of-flight mass spectrometry (Q-TOFMS) for 17 highly polar intermediates produced during glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway (PPP). We optimized the analytical conditions for nano-flow injection/Q-TOFMS, and set the flow rate and ion source temperature to 1000 nL/min and 150 °C, respectively. Under optimal conditions, a single run was finished within 3 min, and the RSD value of 50 sequential injections was 4.2%. The method also showed quantitativity of four stable-isotope-labeled compounds (r2 > 0.99), demonstrating its robustness, high repeatability, and specificity. In addition, we compared three sample-preparation methods for rodent blood samples and found that protein precipitation with threefold methanol was the most effective. Finally, we applied the method to plasma samples from the serotonin syndrome (SS) model and control rats, the results of which were evaluated by principal component analysis (PCA). The two groups showed clearly separated PCA score plots, suggesting that the method could successfully catch the differences in metabolic profiles between SS and control rats. The results obtained from our new method were further validated by using the established gas chromatography/tandem mass spectrometry method, which demonstrated that there were good correlations between the two methods (R = 0.902 and 0.958 for lactic acid and malic acid, respectively, each at p < 0.001), thus proving the validity of our method. The method described here enables high-throughput analysis of metabolites and will be of use for the rapid analysis of metabolic profiles. Graphical abstract.

Optimal inter-batch normalization method for GC/MS/MS-based targeted metabolomics with special attention to centrifugal concentration.

This study investigated the optimal inter-batch normalization method for gas chromatography/tandem mass spectrometry (GC/MS/MS)-based targeted metabolome analysis of rodent blood samples. The effect of centrifugal concentration on inter-batch variation was also investigated. Six serum samples prepared from a mouse and 2 quality control (QC) samples from pooled mouse serum were assigned to each batch, and the 3 batches were analyzed by GC/MS/MS at different days. The following inter-batch normalization methods were applied to metabolome data: QC-based methods with quadratic (QUAD)- or cubic spline (CS)-fitting, total signal intensity (TI)-based method, median signal intensity (MI)-based method, and isotope labeled internal standard (IS)-based method. We revealed that centrifugal concentration was a critical factor to cause inter-batch variation. Unexpectedly, neither the QC-based normalization methods nor the IS-based method was able to normalize inter-batch variation, though MI- or TI-based normalization methods were effective in normalizing inter-batch variation. For further validation, 6 disease model rat and 6 control rat plasma were evenly divided into 3 batches, and analyzed as different batches. Same as the results above, MI- or TI-based methods were able to normalize inter-batch variation. In particular, the data normalized by TI-based method showed similar metabolic profiles obtained from their intra-batch analysis. In conclusion, the TI-based normalization method is the most effective to normalize inter-batch variation for GC/MS/MS-based metabolome analysis. Graphical abstract.

Arabidopsis SBT5.2 and SBT1.7 subtilases mediate C-terminal cleavage of flg22 epitope from bacterial flagellin.

Plants initiate specific defense responses by recognizing conserved epitope peptides within the flagellin proteins derived from bacteria. Proteolytic cleavage of epitope peptides from flagellin by plant apoplastic proteases is thought to be crucial for the perception of the epitope by the plant receptor. However, the identity of the plant proteases involved in this process remains unknown. Here, we establish an efficient identification system for the target proteases in Arabidopsis apoplastic fluid; the method employs native two-dimensional electrophoresis followed by an in-gel proteolytic assay using a fluorescence-quenching peptide substrate. We designed a substrate to specifically detect proteolytic activity at the C-terminus of the flg22 epitope in flagellin and identified two plant subtilases, SBT5.2 and SBT1.7, as specific proteases responsible for the C-terminal cleavage of flg22. In the apoplastic fluid of Arabidopsis mutant plants deficient in these two proteases, we observe a decrease in the C-terminal cleavage of the flg22 domain from flagellin, leading to a decrease in the efficiency of flg22 epitope liberation. Consequently, defensive reactive oxygen species (ROS) production is delayed in sbt5.2 sbt1.7 double-mutant leaf disks compared to wild type following flagellin exposure.

Genomic analysis of an ultrasmall freshwater green alga, Medakamo hakoo.

Ultrasmall algae have attracted the attention of biologists investigating the basic mechanisms underlying living systems. Their potential as effective organisms for producing useful substances is also of interest in bioindustry. Although genomic information is indispensable for elucidating metabolism and promoting molecular breeding, many ultrasmall algae remain genetically uncharacterized. Here, we present the nuclear genome sequence of an ultrasmall green alga of freshwater habitats, Medakamo hakoo. Evolutionary analyses suggest that this species belongs to a new genus within the class Trebouxiophyceae. Sequencing analyses revealed that its genome, comprising 15.8 Mbp and 7629 genes, is among the smallest known genomes in the Viridiplantae. Its genome has relatively few genes associated with genetic information processing, basal transcription factors, and RNA transport. Comparative analyses revealed that 1263 orthogroups were shared among 15 ultrasmall algae from distinct phylogenetic lineages. The shared gene sets will enable identification of genes essential for algal metabolism and cellular functions.

Peptide ligand-mediated trade-off between plant growth and stress response.

Deciding whether to grow or to divert energy to stress responses is a major physiological trade-off for plants surviving in fluctuating environments. We show that three leucine-rich repeat receptor kinases (LRR-RKs) act as direct ligand-perceiving receptors for PLANT PEPTIDE CONTAINING SULFATED TYROSINE (PSY)-family peptides and mediate switching between two opposing pathways. By contrast to known LRR-RKs, which activate signaling upon ligand binding, PSY receptors (PSYRs) activate the expression of various genes encoding stress response transcription factors upon depletion of the ligands. Loss of PSYRs results in defects in plant tolerance to both biotic and abiotic stresses. This ligand-deprivation-dependent activation system potentially enables plants to exert tuned regulation of stress responses in the tissues proximal to metabolically dysfunctional damaged sites where ligand production is impaired.

An infant case of pseudohypoaldosteronism type1A caused by a novel NR3C2 variant.

Pseudohypoaldosteronism type1A (PHA1A) is the renal form of pseudohypoaldosteronism with autosomal dominant inheritance. PHA1A is caused by haploinsufficiency of the mineralocorticoid receptor, which is encoded by NR3C2. We encountered an infant who was diagnosed with PHA1A due to hyponatremia, hyperkalemia, and poor weight gain in the neonatal period. She carried a novel heterozygous mutation (NM_000901.5: c.1757 + 1 G > C) in the splice donor site of IVS-2 in NR3C2.

Metabolome analysis of the serotonin syndrome rat model: Abnormal muscular contraction is related to metabolic alterations and hyper-thermogenesis.

AIMS: Serotonin syndrome (SS) is an adverse outcome of selective serotonin reuptake inhibitors, though its mechanism is not understood and there is no specific clinical biomarker. In this article, metabolic profiles of the SS model rats and causes of metabolome disruption were investigated. MAIN METHODS: Gas chromatography-tandem mass spectrometry (GC/MS/MS)-based metabolomics, clinical biomarker measurements and qRT-PCR analysis for UCP-3 in skeletal muscles were performed. KEY FINDINGS: Metabolome analysis demonstrated that 55, 22, 49 and 41 of those were significantly altered in plasma, liver, gastrocnemius muscle, and trapezius, respectively. In particular, lactic acid significantly accumulated in the gastrocnemius muscle of the model, while the branched chain amino acids were not consumed in the trapezius, suggesting site differences in abnormal muscular contractions in the model. This result was supported by UCP-3 expression analysis. Alteration of the urea cycle was observed in the liver of the model, attributed mainly to catabolism of proteins and/or amino acids from excess skeletal muscle activity, which was supported by plasma BUN: BUN levels in the model were significantly higher than those in the control. In contrast, almost all metabolites including amino acids and TCA-cycle intermediates significantly increased in plasma of the model, suggesting these were not consumed in some parts of the muscle due to acceleration of anaerobic respiration. SIGNIFICANCE: Metabolic profiling revealed that abnormal muscular contractions occurred in specific skeletal muscles and enhanced energy production by up-regulation of anaerobic respiration, followed by excess expression of UCP-3, which contributes to the hyper-thermogenesis observed in the SS model.