Selected Amino Acids Research Articles

Tracing of Amino Acids Dynamics in Cell Lines Based on 18O Stable Isotope Labeling.

Metabolite levels and turnover rates are necessary to understand metabolomic dynamics in a living organism fully. Amino acids can play distinct roles in various cellular processes, and their abnormal levels are associated with pathological conditions, including cancer. Therefore, their levels, especially turnover rates, may provide enormous information about a phenotype. 13C- or 13C,15N-labeled amino acids have also been commonly used to trace amino acid metabolism. This study presented a new methodology based on 18O labeling for amino acids that relied on monitoring mass isotopologues to calculate the turnover rates of amino acids. The method optimization studies were carried over for selective amino acid monitoring. This methodology provides a rapid, robust, and simple GC-MS method for analyzing the fluxes of amino acid metabolism. The developed method was applied to fetal human colon (FHC) and human colon carcinoma (Caco-2) cell lines to determine cancer-induced shifts in the turnover rates of amino acids. These results defined metabolic reprogramming in Caco-2 cells through increased glutamate and serine turnovers and sharply decreased turnovers of aspartate, threonine, and methionine, therefore pointing to some metabolic vulnerabilities in the metabolism of cancerous cells. The simple mechanism of the developed methodology, the availability of affordable 18O-enriched water, and the ease of application can open a new arena in fluxomics analysis.

Enhancing newborn screening sensitivity and specificity for missed NICCD using selected amino acids and acylcarnitines

PurposeTo enhance the detection rate of Neonatal Intrahepatic Cholestasis caused by Citrin Deficiency (NICCD) through newborn screening (NBS), we analyzed the metabolic profiles of missed patients and proposed a more reliable method for early diagnosis.MethodsIn this retrospective study, NICCD patients were classified into “Newborn Screening” (64 individuals) and “Missed Screening” (52 individuals) groups. Metabolic profiles were analyzed using the non-derivatized MS/MS Kit, and genetic mutations were identified via next-generation sequencing and confirmed by Sanger sequencing. Receiver Operating Characteristic (ROC) analysis evaluated the predictive value of amino acids and acylcarnitines in dried blood spots (DBS) for identifying missed patients including 40 missed patients and 17,269 healthy individuals, with additional validation using 12 missed patients and 454 healthy controls.ResultsThe age of diagnosis was significantly higher in the “Missed Screening” group compared to the “Newborn Screening” group (74.50 vs. 18.00 days, P < 0.001). ROC analysis revealed that citrulline had excellent diagnostic accuracy for missed patients, with an AUC of 0.970 and a cut-off value of 17.57 µmol/L. Additionally, glycine, phenylalanine, ornithine, and C8 were significant markers, each with an AUC greater than 0.70. A combination of these markers achieved an AUC of 0.996 with a cut-off value of 0.00195. Validation demonstrated a true positive rate of 91.67% and a true negative rate of 96.48%. Common SLC25A13 mutations in both groups were c.852_855del, IVS16ins3kb, and c.615 + 5G > A.ConclusionsCombining multiple metabolic markers during NBS significantly improves sensitivity and specificity for detecting missed NICCD cases. However, the relationship between genetic mutations and missed cases remains unclear.

Comparative Analysis of Drosophila Bam and Bgcn Sequences and Predicted Protein Structural Evolution.

The protein encoded by the Drosophila melanogaster gene bag of marbles (bam) plays an essential role in early gametogenesis by complexing with the gene product of benign gonial cell neoplasm (bgcn) to promote germline stem cell daughter differentiation in males and females. Here, we compared the AlphaFold2 and AlphaFold Multimer predicted structures of Bam protein and the Bam:Bgcn protein complex between D. melanogaster, D. simulans, and D. yakuba, where bam is necessary in gametogenesis to that in D. teissieri, where it is not. Despite significant sequence divergence, we find very little evidence of significant structural differences in high confidence regions of the structures across the four species. This suggests that Bam structure is unlikely to be a direct cause of its functional differences between species and that Bam may simply not be integrated in an essential manner for GSC differentiation in D. teissieri. Patterns of positive selection and significant amino acid diversification across species is consistent with the Selection, Pleiotropy, and Compensation (SPC) model, where detected selection at bam is consistent with adaptive change in one major trait followed by positively selected compensatory changes for pleiotropic effects (in this case perhaps preserving structure). In the case of bam, we suggest that the major trait could be genetic interaction with the endosymbiotic bacteria Wolbachia pipientis. Following up on detected signals of positive selection and comparative structural analysis could provide insight into the distribution of a primary adaptive change versus compensatory changes following a primary change.

Methane Hydrate-in-Oil Systems in the Presence of Natural Amino Acid-Equilibrium Phase Condition Measurements.

This experimental study reports the thermodynamic influence of three different amino acids on methane hydrate in oil-dominated systems, namely, glycine, proline, and alanine. To thoroughly examine the effect of selected amino acids on methane (CH4) hydrate formation compared to the commercial inhibitor monoethylene glycol (MEG) in the presence of oil, the hydrate liquid-vapor equilibrium (H-Lw-Lo-V) curve is used to measure amino acid aqueous solutions. All experiments are performed at a concentration of 10 wt % by using the isochoric T-cycle technique in a high-pressure reactor cell at the selected range of pressures with temperatures of 4.0-9.0 MPa and 276.5-286.0 K, respectively. Results show that all studied amino acids inhibit hydrate formation of methane; the inhibition trend shows as glycine > alanine > proline in both systems; in the brine water system, the inhibition performance was higher than in the pure water system due to the presence of NaCl. Glycine showed the highest inhibition strength in both systems with an average reduced temperature in pure and brine water of 0.92 and 1.75 K, respectively, at 10 wt %, making the inhibition performance of glycine comparable to the commercial inhibitor MEG. The inhibition effect is attributed to the amino acid's hydrogen bonding energies and side group alkyl chain. Calculating the dissociation enthalpies of methane hydrates in the presence of amino acids using the Clausius-Clapeyron equation implies that the amino acids do not occupy the cage structures during methane hydrate formation.

Measuring 15N and 13C Enrichment Levels in Sparsely Labeled Proteins Using High-Resolution and Tandem Mass Spectrometry.

Isotope labeling of both 15N and 13C in selected amino acids in a protein, known as sparse labeling, is an alternative to uniform labeling and is particularly useful for proteins that must be expressed using mammalian cells, including glycoproteins. High levels of enrichment in the selected amino acids enable multidimensional heteronuclear NMR measurements of glycoprotein three-dimensional structure. Mass spectrometry provides a means to quantify the degree of enrichment. Mass spectrometric measurements of tryptic peptides of a selectively labeled glycoprotein expressed in HEK293 cells revealed complicated isotope patterns which consisted of many overlapping isotope patterns from intermediately labeled peptides, which complicates the determination of the label incorporation. Two challenges are uncovered by these measurements. Metabolic scrambling of amino groups can reduce the 15N content of enriched amino acids or increase the 15N in nontarget amino acids. Also, undefined, unlabeled medium components may dilute the enrichment level of labeled amino acids. The impact of this unexpected metabolic scrambling was overcome by simulating isotope patterns for all isotope-labeled peptide states and generating linear combinations to fit to the data. This method has been used to determine the percent incorporation of 15N and 13C labels and has identified several metabolic scrambling effects that were previously undetected in NMR experiments. Ultrahigh mass resolution is also utilized to obtain isotopic fine structure, from which enrichment levels of 15N and 13C can be assigned unequivocally. Finally, tandem mass spectrometry can be used to confirm the location of heavy isotope labels in the peptides.

In silico sequential mutagenesis of the Carbohydrate Binding Module Family 32 (CBM32) enhances ligand binding affinity

Alginate lyase is a promising target for genetic modification for its degrading biofilm, contributing to bacterial proliferation and antimicrobial resistance. Apart from the main enzyme, the carbohydrate binding module (CBM) component can also be modified to enhance alginate lyase’s activity. This study aimed to perform sequential in silico mutagenesis, molecular docking of selected amino acid residues of Vibrio splendidus CBM32 and performed molecular dynamics (MD) simulations of the mutated structure to validate its ligand-binding efficacy. Seven residues were selected for mutagenesis based on the predicted bonds that formed between the CBM32 and the glucuronic acid ligand (LGU9). Four of seven sequential residue substitutions increased the ligand binding affinity cumulatively from -5.4 Kcal/mol to -6.9 Kcal/mol. The mutated CBM32 had similar MolProbity scores to the original V. splendidus CBM32 structure. From the post-MD simulation analysis, the mutated CBM32 had higher structural stability in a solvent system, a greater number of hydrogen bonds formed with ligand but a lower solvent-accessible surface area than the original structure. The sequential mutagenesis process significantly increased the ligand binding affinity of CBM32 while incurring a minimal change in the overall CBM32 structure. The information on these substituted residues would be beneficial for designing subsequent in vitro mutagenesis and enzymatic assays.

Investigations on the complexation and binding mechanism of bovine serum albumin with Ag-doped TiO2 nanoparticles.

It is essential to study the interactions between nanoparticles and proteins to better understand the biological interactions of nanoparticles. In this study, we studied the protein adsorption mode on the surface of Ag-doped TiO2 nanoparticles (NPs) using a model protein, bovine serum albumin (BSA). The mechanism of binding BSA to the Ag-doped TiO2 NPs was studied by applying fluorescence quenching, absorbance measurements, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy techniques. The strong binding between BSA and Ag-doped TiO2 NPs was confirmed by a high value of binding constant (K = 2.65 × 105 L mol-1). We also studied the thermal stability of BSA in the presence of the Ag-doped TiO2 NPs. Thermodynamic parameters indicated that the adsorption of BSA on the Ag-doped TiO2 NPs was a spontaneous, natural and exothermic process. The effect of Ag-doped TiO2 NPs on the transportation function of BSA was also studied using a fluorescence spectroscopic technique. Fluorescence spectroscopic data suggested the existence of a strong interaction between BSA and the surface of the Ag-doped TiO2 NPs, which indicated that the binding affinities of some selected amino acids in BSA changed. This, in turn, clearly confirms that the Ag-doped TiO2 NPs affect the transportation capability of BSA in blood.

Natural selection on apical membrane antigen 1 (AMA1) of an emerging zoonotic malaria parasite Plasmodium inui

Apical membrane antigen 1 (AMA1) of malaria parasites plays an important role in host cell invasion. Antibodies to AMA1 can inhibit malaria merozoite invasion of erythrocytes while vaccine-induced specific cytotoxic T cell responses to this protein are associated with clinical protection. Polymorphisms in AMA1 of Plasmodium falciparum (PfAMA1) and P. vivax (PvAMA1) are of concern for vaccine development. To date, little is known about sequence diversity in ama1 of P. inui (Piama1), an emerging zoonotic malaria parasite. In this study, 80 complete Piama1 coding sequences were obtained from 57 macaques in Thailand that defined 60 haplotypes clustering in two phylogenetic lineages. In total, 74 nucleotide substitutions were identified and distributed unevenly across the gene. Blockwise analysis of the rates of synonymous (dS) and nonsynonymous (dN) nucleotide substitutions did not show a significant deviation from neutrality among Thai isolates. However, significantly negative Tajima’s D values were detected in domain I and the loop region of domain II, implying purifying selection. Codon-based analysis of dN/dS has identified 12 and 14 codons under positive and negative selections, respectively. Meanwhile, 85 amino acid substitutions were identified among 80 Thai and 11 non-Thai PiAMA1 sequences. Of these, 48 substituted residues had a significant alteration in physicochemical properties, suggesting positive selection. More than half of these positively selected amino acids (32 of 48) corresponded to the predicted B-cell or T-cell epitopes, suggesting that selective pressure could be mediated by host immunity. Importantly, 14 amino acid substitutions were singletons and predicted to be deleterious that could be subject to ongoing purifying selection or elimination. Besides genetic drift and natural selection, intragenic recombination identified in domain II could generate sequence variation in Piama1. It is likely that malarial ama1 exhibits interspecies differences in evolutionary histories. Knowledge of the sequence diversity of the Piama1 locus further provides an evolutionary perspective of this important malaria vaccine candidate.

Amino-Acid-Encoded Supramolecular Nanostructures for Persistent Bioluminescence Imaging of Tumor.

Bioluminescence imaging (BLI) is a powerful technique for noninvasive monitoring of biological processes and cell transplantation. Nonetheless, the application of D-luciferin, which is widely employed as a bioluminescent probe, is restricted in long-term in vivo tracking due to its short half-life. This study presents a novel approach using amino acid-encoded building blocks to accumulate and preserve luciferin within tumor cells, through a supramolecular self-assembly strategy. The building block platform called Cys(SEt)-X-CBT (CXCBT, with X representing any amino acid) utilizes a covalent-noncovalent hybrid self-assembly mechanism to generate diverse luciferin-containing nanostructures in tumor cells after glutathione reduction. These nanostructures exhibit efficient tumor-targeted delivery as well as sequence-dependent well-designed morphologies and prolonged bioluminescence performance. Among the selected amino acids (X = Glu, Lys, Leu, Phe), Cys(SEt)-Lys-CBT (CKCBT) exhibits the superior long-lasting bioluminescence signal (up to 72h) and good biocompatibility. This study demonstrates the potential of amino-acid-encoded supramolecular self-assembly as a convenient and effective method for developing BLI probes for long-term biological tracking and disease imaging.

Evolution of a biological thermocouple by adaptation of cytochrome c oxidase in a subterrestrial metazoan, Halicephalobus mephisto

In this study, we report a biological temperature-sensing electrical regulator in the cytochrome c oxidase of the Devil Worm, Halicephalobus mephisto. This extremophile metazoan was isolated 1.3 km underground in a South African goldmine, where it adapted to heat and potentially to hypoxia, making its mitochondrial sequence a likely target of adaptational change. We obtained the complete mitochondrial genome sequence of this organism and show through dN/dS analysis evidence of positive selection in H. mephisto cytochrome c oxidase subunits. Seventeen of these positively selected amino acid substitutions were located in proximity to the H- and K-pathway proton channels of the complex. Surprisingly, the H. mephisto cytochrome c oxidase completely shuts down at low temperatures (20 °C), leading to a 4.8-fold reduction in the transmembrane proton gradient (ΔΨm) compared to optimal temperature (37 °C). Direct measurement of oxygen consumption found a corresponding 4.6-fold drop at 20 °C compared to 37 °C. Correspondingly, the lifecycle of H. mephisto takes four times longer at low temperature than at higher. This elegant evolutionary adaptation creates a finely-tuned mitochondrial temperature sensor, allowing this ectothermic organism to maximize its reproductive success across varying environmental temperatures.

Chemical composition and sensory profile of Spirogyra neglecta (Hassall) Kützing

SummaryThe proximate composition, selected amino acid composition, and chemical constituents of Spirogyra neglecta (Hassall) Kützing, a freshwater algae collected from Phrae province, Thailand was investigated. It was found to be a good source of protein (29.42% dry wt. basis) and fibre (6.70% dry wt. basis). The total amino acid content was 2611.85 mg/100 g edible. The chemical constituents were characterised by GC–MS, which demonstrated that it contained phytol (62.10%), ethyl 5,8,11,14,17‐icosapentaenoate (8.55%) among others. Based on its chemical composition results, it could serve as a potential ingredient with high nutritional values in the food and nutraceutical industry. Furthermore, the descriptive sensory profile consisting of 17 descriptors, namely, green colour, glossy, filamentous, aroma (algae, grassy, fishy, and earthy), flavour (algae, grassy, fishy, and earthy), sweet, bitter, and texture (fibrousness and softness), mouthfeel (entire mouth‐coating), and aftertaste (astringent), were successfully developed to allow characterisation of the sensory quality of this algae.

Replicative δ Polymerases from Plants and Animals Possess Very Similar Polymerase, Proofreading and Regulatory Domains

In eukaryotes, genome replication starts with the initiation of replication by the primases, followed by the synthesis of the leading- and lagging-strands by two different replicative DNA polymerases (pols), viz. ε and δ, respectively. The polymerase and proofreading (PR) active sites of the δ pols are analyzed from various animal and plant sources by multiple sequence alignment (MSA). The animal and plant δ pols are found to possess almost identical polymerase and PR domains. However, the BLASTp analysis has shown only 56.57% identity between the plant (Arabidopsis thaliana) and animal (human) δ pols. The template-binding pair (–YG-), the catalytic amino acid (K) and the nucleotide selection amino acid (Q) are found to be the same in both plant and animal δ pols. The δ pols from plant and animal sources contain a typical Mg2+-binding motif, (-YGDTD-) in the polymerase domain and 2 possible Zn2+-binding motifs (ZBMs) in their carboxy terminal domain (CTD). One of the ZBMs binds to the 4Fe-4S cluster and is suggested to be involved in the regulation of replication. Interestingly, the invariant –SLYPS- and -YGDTD- motifs which are found in the δ pols are not found in the other replicative pol ε. Furthermore, both animal and plant δ pols use the same PR exonuclease active site amino acids, and thus, belong to the DEDD(Y)-superfamily of exonucleases, as found in other DNA pols. Besides, many specialized, conserved sequence motifs are also identified and discussed.

Site-directed mutagenesis and molecular docking simulations identified the crucial substrate binding site asparagine 101 of xyloglucan endotransglycosylase/hydrolase (XTH) in woody plants

Xyloglucan endotransglycosylase/hydrolase (XTH) is a key enzyme in plant cell wall remodeling. Previous investigations on the angiosperm Populus tomentosa identified 11 PtoXTH proteins with the XTH catalytic motif. Surprisingly, only two (PtoXTH27 and PtoXTH34) exhibited xyloglucan endotransglucosylase (XET) activity, suggesting the presence of additional key substrate binding or catalytic activity sites within XTH proteins. To test this hypothesis, we performed site-directed mutagenesis on selected amino acids in PtoXTH27 and PtoXTH34 and characterized their biochemical properties. Molecular docking simulations and biochemical analyses revealed that N101 is a critical substrate binding site, while Q80, M81, and V180 are essential functional residues influencing XET activity in XTH proteins. Notably, XET-deficient PtoXTH26 regained activity following site-directed mutation, underscoring the significance of these residues. Expanding our study to the gymnosperm Larix kaempferi, we identified two XTH proteins: LkXTH1, which exhibited both the key substrate binding site and XET activity, and LkXTH2, which lacked XET activity. Intriguingly, LkXTH2 regained XET activity after site-directed mutation, highlighting the versatility of the newly identified substrate binding site across different woody plants subphyla. This research provides a theoretical basis for the rapid identification of enzymatically active members within the XTH gene family in woody plants. Furthermore, our findings offer insights into the targeted engineering of XTH enzymes, paving the way for advancements in plant biotechnology and crop improvement.

Evolution of Key Oxygen-Sensing Genes Is Associated with Hypoxia Tolerance in Fishes.

Low dissolved oxygen (hypoxia) is recognized as a major threat to aquatic ecosystems worldwide. Because oxygen is paramount for the energy metabolism of animals, understanding the functional and genetic drivers of whole-animal hypoxia tolerance is critical to predicting the impacts of aquatic hypoxia. In this study, we investigate the molecular evolution of key genes involved in the detection of and response to hypoxia in ray-finned fishes: the prolyl hydroxylase domain (PHD)-hypoxia-inducible factor (HIF) oxygen-sensing system, also known as the EGLN (egg-laying nine)-HIF oxygen-sensing system. We searched fish genomes for HIFA and EGLN genes, discovered new paralogs from both gene families, and analyzed protein-coding sites under positive selection. The physicochemical properties of these positively selected amino acid sites were summarized using linear discriminants for each gene. We employed phylogenetic generalized least squares to assess the relationship between these linear discriminants for each HIFA and EGLN and hypoxia tolerance as reflected by the critical oxygen tension (Pcrit) of the corresponding species. Our results demonstrate that Pcrit in ray-finned fishes correlates with the physicochemical variation of positively selected sites in specific HIFA and EGLN genes. For HIF2A, two linear discriminants captured more than 90% of the physicochemical variation of these sites and explained between 20% and 39% of the variation in Pcrit. Thus, variation in HIF2A among fishes may contribute to their capacity to cope with aquatic hypoxia, similar to its proposed role in conferring tolerance to high-altitude hypoxia in certain lineages of terrestrial vertebrates.

Identification by HSQC and quantification by qHNMR innovate pharmaceutical amino acid analysis

This study introduces a new NMR-based methodology for identification (ID) and quantification (purity, strength) assays of widely used amino acids. A detailed analysis of four amino acids and their available salts was performed with both a high-field (600 MHz) and a benchtop (60 MHz) NMR instrument. To assess sensitivity constraints, samples for 1H NMR analysis were initially prepared using only 10 mg of analyte and 1 mg of maleic acid (MA) as an internal calibrant (IC) and secondary chemical shift reference. The characteristic dispersion of the peak patterns indicating the presence or absence of a counterion (mostly chloride) was conserved at both high and low-field strength instruments, showing that the underlying NMR spectroscopic parameters, i.e., chemical shifts and coupling constants, are independent of the magnetic field strength. However, as the verbal descriptions of 1H NMR spectra are challenging in the context of reference materials and pharmaceutical monographs, an alternative method for the identification (ID) of amino acids is proposed that uses 13C NMR patterns from multiplicity-edited HSQC (ed-HSQC), which are both compound-specific and straightforward to document. For ed-HSQC measurements, the sample amount was increased to 30 mg of the analyte and several acquisition parameters were tested, including t1 increments used in the pulse program, number of scans, and repetition time. Excellent congruence with deviations <0.1 ppm was achieved for the 13C chemical shifts from 1D 13C NMR spectra (150 MHz) vs. those extracted from ed-HSQC (15 MHz traces). Finally, all samples of amino acid candidate reference materials were quantified by 1H qNMR (abs-qHNMR) at both 600 and 60 MHz. At high field, both IC and relative quantitations were performed, however, with the low-field instrument, only the IC method was used. The results showed that the analyzed reference material candidates were generally highly pure compounds. To achieve adequately low levels of uncertainty for such high-purity materials, the sample amounts were increased to 100 mg of analytes and 10 mg of the IC and replicates were analyzed for selected amino acids.

Replacing Cereal with Ultraprocessed Foods in Pig Diets Does Not Adverse Gut Microbiota, L-glutamate Uptake, or Serum Insulin

BackgroundUsing ultraprocessed food (UPF) to replace traditional feed ingredients offers a promising strategy for enhancing food production sustainability. ObjectiveTo analyze the impact of salty and sugary UPF on gut microbiota, amino acids uptake, and serum analytes in growing and finishing pig. MethodsThirty-six Swiss Large White male castrated pigs were assigned to 3 experimental diets: 1) standard (ST), 0% UPF; 2) 30% conventional ingredients replaced by sugary (SU) UPF; and 3) 30% conventional ingredients replaced by salty (SA) UPF. The next-generation sequencing was used to characterize the fecal microbiota. Transepithelial electrical resistance and the active uptake of selected amino acids in pig jejuna were also evaluated. Data were enriched with measurements of fecal volatile fatty acids and serum urea, minerals, and insulin. All data analyses were run in R v4.0.3. The packages phyloseq, vegan, microbiome, and microbiomeutilities were used for microbiota data analysis. The remaining data were analyzed by analysis of variance using linear mixed-effects regression models. ResultsThe UPF did not affect fecal microbiota abundance or biodiversity. The Firmicutes to Bacteroidetes ratio remained unaffected. SU-induced increase in the Anaerostipes genus suggested altered glucose metabolism, whereas SA increased the abundance of CAG-352 and p-2534-18B. No effects on fecal volatile fatty acids were observed. Assumptions of UPF negatively affecting small intestinal physiology were not supported by the measurements of transepithelial electrical resistance in pigs. Active amino acids uptake tests showed potential decrease in L-glutamate absorption in the SA compared with the SU diet. Blood serum analysis indicated no adverse effects on urea, calcium, magnesium, or potassium concentration but the SU group resulted in a lower blood serum insulin concentration at the time of blood collection. ConclusionsWhen incorporated at 30% into a standard growing finishing diet for pigs, UPF does not have detrimental effects on gut microbiota, intestinal integrity, and blood mineral homeostasis.

Select amino acids recover cytokine-altered ENaC function in human bronchial epithelial cells.

The airway epithelium plays a pivotal role in regulating mucosal immunity and inflammation. Epithelial barrier function, homeostasis of luminal fluid, and mucociliary clearance are major components of mucosal defense mechanisms. The epithelial sodium channel (ENaC) is one of the key players in controlling airway fluid volume and composition, and characteristic cytokines cause ENaC and barrier dysfunctions following pulmonary infections or allergic reactions. Given the limited understanding of the requisite duration and magnitude of cytokines to affect ENaC and barrier function, available treatment options for restoring normal ENaC activity are limited. Previous studies have demonstrated that distinct amino acids can modulate epithelial ion channel activities and barrier function in intestines and airways. Here, we have investigated the time- and concentration-dependent effect of representative cytokines for Th1- (IFN-γ and TNF-α), Th2- (IL-4 and IL-13), and Treg-mediated (TGF-β1) immune responses on ENaC activity and barrier function in human bronchial epithelial cells. When cells were exposed to Th1 and Treg cytokines, ENaC activity decreased gradually while barrier function remained largely unaffected. In contrast, Th2 cytokines had an immediate and profound inhibitory effect on ENaC activity that was subsequently followed by epithelial barrier disruption. These functional changes were associated with decreased membrane protein expression of α-, β-, and γ-ENaC, and decreased mRNA levels of β- and γ-ENaC. A proprietary blend of amino acids was developed based on their ability to prevent Th2 cytokine-induced ENaC dysfunction. Exposure to the select amino acids reversed the inhibitory effect of IL-13 on ENaC activity by increasing mRNA levels of β- and γ-ENaC, and protein expression of γ-ENaC. This study indicates the beneficial effect of select amino acids on ENaC activity in an in vitro setting of Th2-mediated inflammation suggesting these amino acids as a novel therapeutic approach for correcting this condition.

Unique myoglobin adaptation to endothermy and flight since the origin of birds.

Myoglobin (Mb) mediates oxygen diffusion and storage in muscle tissue and thus is important for the energy utilization and activity of animals. Birds generally have a high body temperature, and most species also possess the capability of powered flight. Both of these require high levels of aerobic metabolism. Within endothermic mammals, bats also independently evolved flight. Although the functional evolution of myoglobins in deep-diving amniote vertebrates has been well-studied, the functional evolution of myoglobin since the origins of both birds and bats is unclear. Here, with Mb-coding sequences from >200 extant amniote species, we reconstructed ancestral sequences to estimate the functional properties of myoglobin through amniote evolution. A dramatic change in net surface charge on myoglobin occurred during the origin of Aves, which might have been driven by positively selected amino acid substitutions that occurred on the lineage leading to all birds. However, in bats, no change in net surface charge occurred and instead, the Mb genes show evidence of strong purifying selection. The increased net surface charge on bird myoglobins implies an adaptation to flight-related endothermic and higher body temperatures, possibly by reducing harmful protein aggregations. Different from the findings of net surface charge, myoglobins of extant birds show lower stability compared with other amniotes, which probably accelerates the rate of oxygen utilization in muscles. In bats and other mammals, higher stability of Mb may be an alternative pathway for adaptation to endothermy, indicating divergent evolution of myoglobin in birds and bats.

Controlling DNA Fragments Translocation across Nanopores with the Synergic Use of Site-Directed Mutagenesis, pH-Dependent Charge Tuning, and Electroosmotic Flow.

Biological and solid-state nanopores are at the core of transformative techniques and nanodevices, democratizing the examination of matter and biochemical reactions at the single-molecule level, with low cost, portability, and simplicity in operation. One of the crucial hurdles in such endeavors is the fast analyte translocation, which limits characterization, and a rich number of strategies have been explored over the years to overcome this. Here, by site-directed mutagenesis on the α-hemolysin protein nanopore (α-HL), sought to replace selected amino acids with glycine, electrostatic binding sites were induced on the nanopore's vestibule and constriction region and achieved in the most favorable case a 20-fold increase in the translocation time of short single-stranded DNA (ssDNA) at neutral pH, with respect to the wild-type (WT) nanopore. We demonstrated an efficient tool of controlling the ssDNA translocation time, via the interplay between the nanopore-ssDNA surface electrostatic interactions and electroosmotic flow, all mediated by the pH-dependent ionization of amino acids lining the nanopore's translocation pathway. Our data also reveal the nonmonotonic, pH-induced alteration of ssDNA average translocation time. Unlike mildly acidic conditions (pH ∼ 4.7), at a pH ∼ 2.8 maintained symmetrically or asymmetrically across the WT α-HL, we evidenced the manifestation of a dominant electroosmotic flow, determining the speeding up of the ssDNA translocation across the nanopore by counteracting the ssDNA-nanopore attractive electrostatic interactions. We envision potential applications of the presented approach by enabling easy-to-use, real-time detection of short ssDNA sequences, without the need for complex biochemical modifications to the nanopore to mitigate the fast translocation of such sequences.

Identification of differentially expressed genes controlling the expression of flowering in Bambara groundnut (Vigna subterranea [L.] Verdc.)

Bambara groundnut flowering is a crucial developmental stage in the vegetative to reproductive period. The earliness to lateness of flowering is regulated by various interconnected genetic pathways encoded by genes. Deoxyribonucleic acid (DNA) of the selected accessions was extracted through leaf samples at 3 weeks old, using Dellaporta Miniprep for Plant DNA Isolation procedure. The high-quality DNA was sequenced using Diversity Arrays Technology (DArT) markers and single nucleotide polymorphisms (SNP’s) associated with flowering was identified. There is need to investigate the genetic make-up of the cleistogamous flower of Bambara through associated genes for improvement. This research work identified four markers associated with the flowering of Vigna subterranea and the role of variant identified genes in flowering. The identified markers from the sequence and the selected amino acid sequence were used as a query to search the legume protein database in Vigna radiata. The four markers with adequate information associated with flowering in the sequence were 24385352|F|0–28:T > C-28:T > C; 27641816|F|0–17:C > T-17:C > T; 24384204|F|0–24:C > T-24:C > T and 24346601|F|0–67:T > C-67:T > C and significant at P < 1.68 × 10−4 at chromosomes 7, 11, 4, and 5. The identified genes including histones, Polyketide, cyclase/dehydrase, Transcription factor MYC/MYB N-terminal, Rhamnogalacturonate lyase, DHHC-type zinc finger protein, Putative S-adenosyl-L-methionine-dependent methyltransferase, Ribosomal protein L2, D-galactoside/L-rhamnose binding SUEL lectin domain, Lipase GDSL, Histone deacetylase superfamily, Basic-leucine zipper domain, TUP1-like enhancer of split, Zinc finger ZZ-type, Homeodomain-like, Phosphatidylethanolamine-binding protein PEBP, Leucine-rich repeat which are tools in controlling flowering in Bambara groundnut. This study revealed that Bambara groundnut flowering is controlled by the interplay of genes.