Fluorescent human RPA to track assembly dynamics on DNA.

DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA). Almost all downstream DNA processes must first remodel/remove RPA or function alongside to access the ssDNA occluded under RPA. Formation of RPA-ssDNA complexes trigger the DNA damage checkpoint response and is a key step in activating most DNA repair and recombination pathways. Thus, in addition to protecting the exposed ssDNA, RPA functions as a gatekeeper to define functional specificity in DNA maintenance and genomic integrity. RPA achieves functional dexterity through a multi-domain architecture utilizing several DNA binding and protein-interaction domains connected by flexible linkers. This flexible and modular architecture enables RPA to adopt a myriad of configurations tailored for specific DNA metabolic roles. To experimentally capture the dynamics of the domains of RPA upon binding to ssDNA and interacting proteins we here describe the generation of active site-specific fluorescent versions of human RPA (RPA) using 4-azido-L-phenylalanine (4AZP) incorporation and click chemistry. This approach can also be applied to site-specific modifications of other multi-domain proteins. Fluorescence-enhancement through non-canonical amino acids (FEncAA) and Förster Resonance Energy Transfer (FRET) assays for measuring dynamics of RPA on DNA are also described. The fluorescent human RPA described here will enable high-resolution structure-function analysis of RPA-ssDNA interactions.

Boron neutron capture therapy delays the decline in neurological function in a mouse model of metastatic spinal tumors.

Metastatic spinal tumors are increasingly prevalent due to advancements in cancer treatment, leading to prolonged survival rates. This rising prevalence highlights the need for developing more effective therapeutic approaches to address this malignancy. Boron neutron capture therapy (BNCT) offers a promising solution by delivering targeted doses to tumors while minimizing damage to normal tissue. In this study, we evaluated the efficacy and safety of BNCT as a potential therapeutic option for spine metastases in mouse models induced by A549 human lung adenocarcinoma cells. The animal models were randomly allocated into three groups: untreated (n = 10), neutron irradiation only (n = 9), and BNCT (n = 10). Each mouse was administered 4-borono-L-phenylalanine (250 mg/kg) intravenously, followed by measurement of boron concentrations 2.5 h later. Overall survival, neurological function of the hindlimb, and any adverse events were assessed post irradiation. The tumor-to-normal spinal cord and blood boron concentration ratios were 3.6 and 2.9, respectively, with no significant difference observed between the normal and compressed spinal cord tissues. The BNCT group exhibited significantly prolonged survival rates compared with the other groups (vs. untreated, p = 0.0015; vs. neutron-only, p = 0.0104, log-rank test). Furthermore, the BNCT group demonstrated preserved neurological function relative to the other groups (vs. untreated, p = 0.0004; vs. neutron-only, p = 0.0051, multivariate analysis of variance). No adverse events were observed post irradiation. These findings indicate that BNCT holds promise as a novel treatment modality for metastatic spinal tumors.

Probing calmodulin-NO synthase interactions via site-specific infrared spectroscopy: an introductory investigation.

Calmodulin (CaM) binds to a linker between the oxygenase and reductase domains of nitric oxide synthase (NOS) to regulate the functional conformational dynamics. Specific residues on the interdomain interface guide the domain-domain docking to facilitate the electron transfer in NOS. Notably, the docking interface between CaM and the heme-containing oxygenase domain of NOS is isoform specific, which is only beginning to be investigated. Toward advancing understanding of the distinct CaM-NOS docking interactions by infrared spectroscopy, we introduced a cyano-group as frequency-resolved vibrational probe into CaM individually and when associated with full-length and a bi-domain oxygenase/FMN construct of the inducible NOS isoform (iNOS). Site-specific, selective labeling with p-cyano-L-phenylalanine (CNF) by amber suppression of CaM bound to the iNOS has been accomplished by protein coexpression due to the instability of recombinant iNOS protein alone. We introduced CNF at residue 108, which is at the putative CaM-heme (NOS) docking interface. CNF was also introduced at residue 29, which is distant from the docking interface. FT IR data show that the 108 site is sensitive to CaM-NOS complex formation, while insensitivity to its association with the iNOS protein or peptide was observed for the 29 site. Moreover, narrowing of the IR bands at residue 108 suggests the C≡N probe experiences a more limited distribution of environments, indicating side chain restriction apparent for the complex with iNOS. This initial work sets the stage for residue-specific characterizations of structural dynamics of the docked states of NOS proteins.

Systems engineering Escherichia coli for efficient production p-coumaric acid from glucose.

P-coumaric acid (p-CA), a pant metabolite with antioxidant and anti-inflammatory activity, is extensively utilized in biomedicine, food, and cosmetics industry. In this study, a synthetic pathway (PAL) for p-CA was designed, integrating three enzymes (AtPAL2, AtC4H, AtATR2) into a higher l-phenylalanine-producing strain Escherichia coli PHE05. However, the lower soluble expression and activity of AtC4H in the PAL pathway was a bottleneck for increasing p-CA titers. To overcome this limitation, the soluble expression of AtC4H was enhanced through N-terminal modifications. And an optimal mutant, AtC4HL373T/G211H, which exhibited a 4.3-fold higher kcat/Km value compared to the wild type, was developed. In addition, metabolic engineering strategies were employed to increase the intracellular NADPH pool. Overexpression of ppnk in engineered E. coli PHCA20 led to a 13.9-folds, 1.3-folds, and 29.1% in NADPH content, the NADPH/NADP+ ratio and p-CA titer, respectively. These optimizations significantly enhance p-CA production, in a 5-L fermenter using fed-batch fermentation, the p-CA titer, yield and productivity of engineered strain E. coli PHCA20 were 3.09 g/L, 20.01 mg/g glucose, and 49.05 mg/L/h, respectively. The results presented here provide a novel way to efficiently produce the plant metabolites using an industrial strain.

Systematic engineering enables efficient biosynthesis of L-phenylalanine in E. coli from inexpensive aromatic precursors.

BACKGROUND: L-phenylalanine is an essential amino acid with various promising applications. The microbial pathway for L-phenylalanine synthesis from glucose in wild strains involves lengthy steps and stringent feedback regulation that limits the production yield. It is attractive to find other candidates, which could be used to establish a succinct and cost-effective pathway for L-phenylalanine production. Here, we developed an artificial bioconversion process to synthesize L-phenylalanine from inexpensive aromatic precursors (benzaldehyde or benzyl alcohol). In particular, this work opens the possibility of L-phenylalanine production from benzyl alcohol in a cofactor self-sufficient system without any addition of reductant. RESULTS: The engineered L-phenylalanine biosynthesis pathway comprises two modules: in the first module, aromatic precursors and glycine were converted into phenylpyruvate, the key precursor for L-phenylalanine. The highly active enzyme combination was natural threonine aldolase LtaEP.p and threonine dehydratase A8HB.t, which could produce phenylpyruvate in a titer of 4.3 g/L. Overexpression of gene ridA could further increase phenylpyruvate production by 16.3%, reaching up to 5 g/L. The second module catalyzed phenylpyruvate to L-phenylalanine, and the conversion rate of phenylpyruvate was up to 93% by co-expressing PheDH and FDHV120S. Then, the engineered E. coli containing these two modules could produce L-phenylalanine from benzaldehyde with a conversion rate of 69%. Finally, we expanded the aromatic precursors to produce L-phenylalanine from benzyl alcohol, and firstly constructed the cofactor self-sufficient biosynthetic pathway to synthesize L-phenylalanine without any additional reductant such as formate. CONCLUSION: Systematical bioconversion processes have been designed and constructed, which could provide a potential bio-based strategy for the production of high-value L-phenylalanine from low-cost starting materials aromatic precursors.

Assessing the effectiveness of fluorinated and α-methylated 3-boronophenylalanine for improved tumor-specific boron delivery in boron neutron capture therapy.

A [10B]boron agent and a nuclear imaging probe for pharmacokinetic estimation form the fundamental pair in successful boron neutron capture therapy (BNCT). However, 4-[10B]borono-l-phenylalanine (BPA), used in clinical BNCT, has undesirable water solubility and tumor selectivity. Therefore, we synthesized fluorinated and α-methylated 3-borono-l-phenylalanine (3BPA) derivatives to realize improved water solubility, tumor targetability, and biodistribution. All 3BPA derivatives exhibited over 10 times higher water solubility than BPA. Treatment with α-methylated 3BPA derivatives resulted in decreased cell uptake via l-type amino acid transporter (LAT) 2 while maintaining LAT1 recognition, thereby significantly improving LAT1/LAT2 selectivity. Biodistribution studies showed that fluorinated α-methyl 3BPA derivatives exhibited reduced boron accumulation in nontarget tissues, including muscle, skin, and plasma. Consequently, these derivatives demonstrated significantly improved tumor-to-normal tissue ratios compared to 3BPA and BPA. Overall, fluorinated α-methyl 3BPA derivatives with the corresponding radiofluorinated compounds hold potential as promising agents for future BNCT/PET theranostics.

Comprehensive investigations of 2-phenylethanol production by the filamentous fungus Annulohypoxylon stygium.

2-Phenylethanol (2-PE) is an aromatic compound with a rose-like fragrance that is widely used in food and other industries. Yeasts have been implicated in the biosynthesis of 2-PE; however, few studies have reported the involvement of filamentous fungi. In this study, 2-PE was detected in Annulohypoxylon stygium mycelia grown in both potato dextrose broth (PDB) and sawdust medium. Among the 27 A. stygium strains investigated in this study, the strain "Jinjiling" (strain S20) showed the highest production of 2-PE. Under optimal culture conditions, the concentration of 2-PE was 2.33 g/L. Each of the key genes in Saccharomyces cerevisiae shikimate and Ehrlich pathways was found to have homologous genes in A. stygium. Upon the addition of L-phenylalanine to the medium, there was an upregulation of all key genes in the Ehrlich pathway of A. stygium, which was consistent with that of S. cerevisiae. A. stygium as an associated fungus provides nutrition for the growth of Tremella fuciformis and most spent composts of T. fuciformis contain pure A. stygium mycelium. Our study on the high-efficiency biosynthesis of 2-PE in A. stygium offers a sustainable solution by utilizing the spent compost of T. fuciformis and provides an alternative option for the production of natural 2-PE. KEY POINTS: • Annulohypoxylon stygium can produce high concentration of 2-phenylethanol. • The pathways of 2-PE biosynthesis in Annulohypoxylon stygium were analyzed. • Spent compost of Tremella fuciformis is a potential source for 2-phenylethanol.

Plant-Assisted Green Synthesis of MgO Nanoparticles as a Sustainable Material for Bone Regeneration: Spectroscopic Properties.

This work is devoted to magnesium oxide (MgO) nanoparticles (NPs) for their use as additives for bone implants. Extracts from four different widely used plants, including Aloe vera, Echeveria elegans, Sansevieria trifasciata, and Sedum morganianum, were evaluated for their ability to facilitate the "green synthesis" of MgO nanoparticles. The thermal stability and decomposition behavior of the MgONPs were analyzed by thermogravimetric analysis (TGA). Structure characterization was performed by X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDS), ultraviolet-visible spectroscopy (UV-Vis), dynamic light scattering (DLS), and Raman scattering spectroscopy (RS). Morphology was studied by scanning electron microscopy (SEM). The photocatalytic activity of MgO nanoparticles was investigated based on the degradation of methyl orange (MeO) using UV-Vis spectroscopy. Surface-enhanced Raman scattering spectroscopy (SERS) was used to monitor the adsorption of L-phenylalanine (L-Phe) on the surface of MgONPs. The calculated enhancement factor (EF) is up to 102 orders of magnitude for MgO. This is the first work showing the SERS spectra of a chemical compound immobilized on the surface of MgO nanoparticles.

Changes in the Antioxidant Potential of Camellia sinensis Cultures under the Influence of Phenolic Precursors.

The viability, productivity and survival of higher plants under the adverse factors influence are largely determined by the functional activity of the antioxidant system. The aim of our work was to investigate changes in formation of high-molecular (superoxide dismutase and peroxidase) and low-molecular (phenolics, including flavanols and proanthocyanidins) antioxidants in callus culture of Camellia sinensis under influence of phenolic precursors (L-phenylalanine-3 mM, trans-cinnamic acid-1 mM, naringenin-0.5 mM). According to the data obtained, the effect of precursors on tea callus cultures did not lead to significant increasing of superoxide dismutase and peroxidase activity in most cases. However, it led to the increased accumulation of the total phenolics content, as well as flavanols and proanthocyanidins contents. For C. sinensis callus cultures, the most promising regulator of phenolic compounds was L-phenylalanine, in the presence of which its content increased almost twice. Thus, the exogenous effect of various precursors is possible to use for the targeted regulation of certain phenolics classes accumulation in plant cells.

Transcriptomics and metabolomics analysis of L-phenylalanine overproduction in Escherichia coli.

BACKGROUND: Highly efficient production of L-phenylalanine (L-Phe) in E. coli has been achieved by multiple rounds of random mutagenesis and modification of key genes of the shikimate (SHIK) and L-Phe branch pathways. In this study, we performed transcriptomic (16, 24 and 48 h) and metabolomic analyses (8, 16, 24, 32,40, and 48 h) based on time sequences in an engineered E. coli strain producing L-Phe, aiming to reveal the overall changes of metabolic activities during the fermentation process. RESULTS: The largest biomass increase rate and the highest production rate were seen at 16 h and 24 h of fermentation, respectively reaching 5.9 h-1 and 2.76 g/L/h, while the maximal L-Phe titer of 60 g/L was accumulated after 48 h of fermentation. The DEGs and metabolites involved in the EMP, PP, TCA, SHIIK and L-Phe-branch pathways showed significant differences at different stages of fermentation. Specifically, the significant upregulation of genes encoding rate-limiting enzymes (aroD and yidB) and key genes (aroF, pheA and aspC) pushed more carbon flux toward the L-Phe synthesis. The RIA changes of a number of important metabolites (DAHP, DHS, DHQ, Glu and PPN) enabled the adequate supply of precursors for high-yield L-Phe production. In addition, other genes related to Glc transport and phosphate metabolism increased the absorption of Glc and contributed to rerouting the carbon flux into the L-Phe-branch. CONCLUSIONS: Transcriptomic and metabolomic analyses of an L-Phe overproducing strain of E. coli confirmed that precursor supply was not a major limiting factor in this strain, whereas the rational distribution of metabolic fluxes was achieved by redistributing the carbon flux (for example, the expression intensity of the genes tyrB, aspC, aroL and aroF/G/H or the activity of these enzymes is increased to some extent), which is the optimal strategy for enhancing L-Phe production.

Organocatalyzed Ring-Opening Polymerization of (S)-3-Benzylmorpholine-2,5-Dione.

A 3-benzylmorpholine-2,5-dione monomer is synthesized from the natural amino acid l-phenylalanine and characterized by means of nuclear magnetic resonance and infrared spectroscopy, electrospray ionization mass spectrometry, and elemental analysis. Subsequent to preliminary polymerization studies, a well-defined poly(ester amide) homopolymer is synthesized via ring-opening polymerization using a binary catalyst system comprising 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and a 1-(3,5-bis(trifluoromethyl)phenyl)-3-cyclohexylthiourea (TU) cocatalyst with a feed ratio of M/I/DBU/TU = 100/1/1/10. Kinetic studies reveal high controllability of the dispersities and molar masses up to conversions of almost 80%. Analysis by mass spectrometry hints toward excellent end-group fidelity at these conditions. In consequence, utilization of hydroxyl-functionalized poly(ethylene glycol) and poly(2-ethyl-2-oxazoline) as macroinitiators results in amphiphilic block copolymers. Bulk miscibility of the building blocks is indicated by differential scanning calorimetry investigations. As more and more promising new drugs are based on hydrophobic molecules featuring aromatic moieties, the novel polyesteramides seem highly promising materials to be used as potential drug delivery vehicles.

Comparative impact of exogenous phenylalanine on oenological isolates of Kluyveromyces marxianus and Saccharomyces cerevisiae.

AIMS: Kluyveromyces marxianus' high production of 2-phenylethyl acetate (2-PEA) via L-phenylalanine (Phe) catabolism makes it relevant for industries relying on the production of aroma compounds through fermentation processes. This study assessed the physiological impact of exogenous supplementation of Phe on cell viability, fermentation performance, and, by extension, on lipid and amino acid metabolism in a wine isolate of this yeast. METHODS AND RESULTS: The data showed that Phe exerted cytotoxic effects on K. marxianus IWBT Y885, which were minimal on Saccharomyces cerevisiae and impacted amino acid metabolism and aroma production. We demonstrated that K. marxianus strains fermented sugars more effectively in the absence of Phe. While lipid supplementation did not mitigate any deleterious effects of Phe, it supported viability maintenance and fermentation performance in the absence of Phe. Phe supplementation succeeded in augmenting the production of 2-PE and 2-PEA. CONCLUSIONS: The enhanced production of 2-PEA in K. marxianus suggests that this transesterification may be, at least in part, a compensatory detoxification mechanism for this yeast.

One-step synthesis of chiral molecularly imprinted polymer TiO2 nanoparticles for enantioseparation of phenylalanine in coated capillary electrochromatography.

A novel chiral molecularly imprinted polymer TiO2 nanoparticle was synthesized in one step for the enantioseparation of phenylalanine in coated capillary electrochromatography. To the author's knowledge, the chiral molecularly imprinted nanomaterials have still not been reported, to date. Chiral molecularly imprinted TiO2 nanomaterials (L-PHE@MIP(APTES-TEOS)@TiO2) were used as a chiral stationary phase to separate the phenylalanine enantiomers in coated capillary electrochromatography (CEC). The imprinted coating was prepared from L-phenylalanine (L-PHE) as the template, TiO2 nanoparticles (NPs) as the support substrate, 3-aminopropyltriethoxysilane (APTES) as the functional monomer, and tetraethyl silicate (TEOS) as the cross-linker. Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) were used for the characterization of the L-PHE@MIP(APTES-TEOS)@TiO2@capillary. Fourier transform infrared spectra (FT-IR), transmission electron microscopy (TEM), and thermogravimetric analysis (TGA) were employed for the characterization of the L-PHE@MIP(APTES-TEOS)@TiO2. The effects of the applied voltage, pH value, buffer concentration, and acetonitrile content were investigated  experimentally to determine the optimum conditions for CEC. The best resolution for  phenylalanine enantiomers by CEC reached a value of 3.48. In addition, the specific recognition effect of L-PHE@MIP(APTES-TEOS)@TiO2 on PHE enantiomers was studied by selective experiment. Finally, adsorption kinetic research, adsorption equilibrium isotherm study, and adsorption thermodynamic experiment were carried out to investigate the separation mechanism of PHE enantiomers with the L-PHE@MIP (APTES-TEOS)@TiO2@capillary, and the results were consistent with those of CEC experiments.

Enzymatic synthesis of halogen derivatives of L-phenylalanine and phenylpyruvic acid stereoselectively labeled with hydrogen isotopes in the side chain.

Halogenated, labeled with deuterium, tritium or doubly labeled with deuterium and tritium in the 3S position of the side chain isotopomers of L-phenylalanine and phenylpyruvic acid were synthesized. Isotopomers of halogenated L-phenylalanine were obtained by addition of ammonia from isotopically enriched buffer solution to the halogenated derivative of (E)-cinnamic acid catalyzed by phenylalanine ammonia lyase. Isotopomers of halogenated phenylpyruvic acid were obtained enzymatically by conversion of the appropriate isotopomer of halogenated L-phenylalanine in the presence of phenylalanine dehydrogenase. As a source of deuterium was used deuterated water, as a source of tritium was used a solution of highly diluted tritiated water. The labeling takes place in good yields and with high deuterium atom% abundance.

Decasubstituted Pillar[5]arene Derivatives Containing L-Tryptophan and L-Phenylalanine Residues: Non-Covalent Binding and Release of Fluorescein from Nanoparticles.

Sensitive systems with controlled release of drugs or diagnostic markers are attractive for solving the problems of biomedicine and antitumor therapy. In this study, new decasubstituted pillar[5]arene derivatives containing L-Tryptophan and L-Phenylalanine residues have been synthesized as pH-responsive drug nanocarriers. Fluorescein dye (Fluo) was loaded into the pillar[5]arene associates and used as a spectroscopic probe to evaluate the release in buffered solutions with pH 4.5, 7.4, and 9.2. The nature of the substituents in the pillar[5]arene structure has a huge influence on the rate of delivering. When the dye was loaded into the associates based on pillar[5]arene derivatives containing L-Tryptophan, the Fluo release occurs in the neutral (pH = 7.4) and alkaline (pH = 9.2) buffered solutions. When the dye was loaded into the associates based on pillar[5]arene with L-Phenylalanine fragments, the absence of release was observed in every pH evaluated. This happens as the result of different packing of the dye in the structure of the associate. This fact was confirmed by different fluorescence mechanisms (aggregation-caused quenching and aggregation-induced emission) and association constants. It was shown that the macrocycle with L-Phenylalanine fragments binds the dye more efficiently (lgKa = 3.92). The experimental results indicate that the pillar[5]arene derivatives with amino acids fragments have a high potential to be used as a pH-responsive drug delivery devices, especially for promoting the intracellular delivering, due to its nanometric size.

Adsorption and immobilization of ß-glucosidase from Thermoascus aurantiacus on macroporous cryogel by hydrophobic interaction.

Enzyme immobilization has been reported as a promising approach to improving parameters such as thermal stability, pH and reusability. In this study, a polyacrylamide cryogel functionalized with L-phenylalanine was prepared to be used in the adsorption of ß-glucosidase from Thermoascus aurantiacus, aiming at its separation and also its immobilization on the cryogel matrix. The enzyme was produced by solid state fermentation. First, the adsorption was studied as a function of the pH and the resulting yield (Y, %) and purification factor (PF, dimensionless) were determined (1.57-5.13 and 64.19-91.20, respectively). The PF and yield from eluate samples obtained at pH 3.0 were the highest (5.13 and 91.20, respectively). Then, ß-glucosidase was immobilized on the hydrophobic cryogel and the recovery activities (%) were determined as a function of temperature and in the presence of different saline solutions. The values ranged from 14.45 to 45.97. As expected, salt type and ionic strength affected the activity remained in the immobilized ß-glucosidase. The average bioreactor activity was 39.9 U/g of dry cryogel and its operational stability was measured, with no decrease in activity being observed during seven cycles. Kinetic parameters of free and immobilized enzyme were determined according to different models.

Investigation of the Impact of L-Phenylalanine and L-Tyrosine Pre-Treatment on the Uptake of 4-Borono-L-Phenylalanine in Cancerous and Normal Cells Using an Analytical Approach Based on SC-ICP-MS.

Boron has gained significant attention in medical research due to its B-10 isotope's high cross section for the reaction with thermal neutrons, generating ionizing particles that can eliminate cancer cells, propelling the development of boron neutron capture therapy (BNCT) for cancer treatment. The compound 4-borono-L-phenylalanine (BPA) has exhibited potential in BNCT clinical trials. Enhancing BPA uptake in cells involves proposing L-amino acid preloading. This study introduces a novel analytical strategy utilizing ICP-MS and single cell ICP-MS (SC-ICP-MS) to assess the effectiveness of L-tyrosine and L-phenylalanine preloading on human non-small cell lung carcinoma (A549) and normal Chinese hamster lung fibroblast (V79-4) models, an unexplored context. ICP-MS outcomes indicated that L-tyrosine and L-phenylalanine pre-treatment increased BPA uptake in V79-4 cells by 2.04 ± 0.74-fold (p = 0.000066) and 1.46 ± 0.06-fold (p = 0.000016), respectively. Conversely, A549 cells manifested heightened BPA uptake solely with L-tyrosine preloading, with a factor of 1.24 ± 0.47 (p = 0.028). BPA uptake remained higher in A549 compared to V79-4 regardless of preloading. SC-ICP-MS measurements showcased noteworthy boron content heterogeneity within A549 cells, signifying diverse responses to BPA exposure, including a subset with notably high BPA uptake. This study underscores SC-ICP-MS's utility in precise cellular boron quantification, validating cellular BPA uptake's heterogeneity.

Optimization of the Transwell® assay for the analysis of neutrophil chemotaxis using flow cytometry to refine the clinical investigation of immunodeficient patients.

Chemotaxis is the directed movement of neutrophils towards an infected site. This physiological process can be reproduced using a modified Boyden chamber, such as the Transwell® support. Different techniques can be used to count neutrophils after migration to the lower chamber of the holder. The present study supports the use of an optimized Transwell® assay coupled with a flow cytometry-based method (Sysmex XN-9000) to detect chemotaxis abnormalities. A reference interval of neutrophil's chemotaxis was determined as part of this work. A first step involves the extraction of neutrophils from whole blood. The migration of neutrophils from the upper to the lower support chamber is subsequently directed by a chemoattractant gradient using N-formyl-l-Methionyl-l-Leucyl-l-Phenylalanine (fMLP). Neutrophils collected in the lower chamber are finally counted by flow cytometry. The original protocol was optimized through the comparison of different parameters. The use of Polymorphprep®, in the extraction of neutrophils, showed an improvement of the neutrophils yield of 1.65 times (57.5% of recovery) compared to the extraction using the Ficoll-Hypaque® gradient. A solution containing 5% of Bovin Serum Albumin (BSA) was used to suspend the extracted neutrophils, stabilize their viability and preserve their integrity. The mechanical agitation of the Transwell® permeable supports during migration did not show an increase in neutrophil yield. A migration time of 1 h 30 was identified as the best time for collecting the largest number of neutrophils after migration. Finally, we demonstrated that scraping the bottom of the well after migration improved neutrophil collection from the lower chamber by 1.9-fold compared to a non-scraping method. In conclusion, our results support the use of Polymorphprep® and a 5% BSA solution in the suspension, without agitation of the medium. An incubation time of 1 h 30 was identified as optimal for neutrophil migration through the chamber. Scraping the bottom after neutrophil migration improved neutrophil collection yield. Normal adult values were obtained with directed migration equal to 32.4% ±13.41% on 15 men and 18 women.

Identification of a regulatory pathway of L-phenylalanine-induced GLP-1 secretion in the enteroendocrine L cells.

Glucagon like peptide-1 (GLP-1) is one of incretin hormone and is secreted when enteroendocrine L cells sense saccharides, amino acids, and fatty acids. Some amino acids have been shown to promote GLP-1 secretion from small intestinal enteroendocrine L cells. However, the molecular mechanisms that L-phenylalanine, a potent trigger of GLP-1 secretion, causes GLP-1 secretion from enteroendocrine L cells has not been elucidated. In this study, we used live-cell imaging to clarify the pathway by which L-phenylalanine activates enteroendocrine L cells. The results showed that L-phenylalanine was sensed by Gq-coupled receptor GPR142 and caused an increase in intracellular Ca2+ concentration. In addition, L-phenylalanine was taken up directly into the cell via Na+-dependent amino acid transporter, causing membrane depolarization and enhancing GLP-1 secretion. In summary, enteroendocrine L cells may regulate blood glucose levels in the body by detecting L-phenylalanine in the lumen and secreting GLP-1 via GPR142 and Na+-dependent amino acid transporters.

Efficient incorporation of p-azido-l-phenylalanine into the protein using organic solvents.

Azide, the most used photo-crosslinking group, facilitates the analysis of protein structure and function. This group is particularly useful when photochemically label antibodies and examine protein-protein interactions. The use of the expanded genetic code technique allows the special labeling of the functional azide group in proteins by adding the unnatural amino acid (UAA), p-azido-l-phenylalanine (AzF), in response to the amber codon during translation. However, a low UAA uptake rate due to mass transfer resistance in the cell membrane may lead to the early termination of the full-length protein. This study reports a general method for the efficient in vivo incorporation of AzF into the target protein by improving cell permeability using organic solvents. As expected, the yield of the full-length protein was significantly increased, which indicated that the AzF uptake was greatly improved due to the addition of organic solvents. Our method can serve as a good reference for improving the genetic incorporation of other kinds of UAAs into proteins.