Target Amino Acid Research Articles

Synthesis and physicochemical evaluation of tetrafluorinated GABA analogue

Synthesis of 2,2,3,3-tetraftluoro-γ-aminobutyric acid (tetraF-GABA) starting from commercially available 2,2,3,3-tetrafluorobutane-1,4-diol is described. The straightforward nine-step reaction sequence allowed the production of up to 20 g of the target amino acid in 41 % overall yield. Potentiometric titration revealed increased acidity of both ionizable moieties in the title compound (the pKa values were diminished by 1.6 and 4.0 units for the COOH and NH3+ groups, respectively, as compared to parent GABA). X-ray diffraction studies and DFT calculations demonstrated different preferential conformations in solid state and solution, although the fluorine gauche effect was observed in both cases.

Gene expression and anticancer evaluation of Kigelia africana (Lam.) Benth. Extracts using MDA-MB-231 and MCF-7 cell lines.

In recent years, a cancer research trend has shifted towards identifying novel therapeutic compounds from natural assets for the management of cancer. In this study, we aimed to assess the cytotoxic activity of Kigelia Africana (KA) extracts on breast cancer (MDA-MB-231 and MCF-7) and noncancerous kidney cells (HEK-293T) to develop an efficient anticancer medication. We used gas chromatography mass spectrometry (GC-MS to analyze the constituents of EKA and HKA extracts meanwhile the crystal violet and the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assays were used to examine the possible cytotoxic effects of plant extracts on our cancer cell lines along with non-cancerous control. The quantitative real-time PCR (RT-PCR) was run on cell samples to evaluate the differential expression of cell proliferative markers of cancer (BCL-2 and TP53). These phytochemicals have been reported to have binding affinity for some other growth factors and receptors as well which was evaluated by the in-silico molecular docking against Bcl2, EGFR, HER2, and TP53. Our Morphological observation showed a significant difference in the cell morphology and proliferation potential which was decreased under the effect of plant extracts treatment as compared to the control samples. The ethanol extract exhibited a marked antiproliferative activity towards MDA-MB-231 and MCF-7 cell lines with IC50 = 20 and 32 μg/mL, respectively. Quantitative RT-PCR gene expression investigation revealed that the IC50 concentration of ethanolic extract regulated the levels of mRNA expression of apoptotic genes. With the target and active binding site amino acids discovered in the molecular docking investigation, TP53/Propanoic acid, 3-(2, 3, 6-trimethyl-1, 4-dioxaspiro [4.4] non-7-yl)-, methyl ester (-7.1 kcal/mol) is the best-docked ligand. The use of this plant in folk remedies justifies its high in vitro anti-cancer capabilities. This work highlights the role of phytochemicals in the inhibition of cancer proliferation. Based on all these findings, it can be concluded that EKA extract has promising anti-proliferative effect on cancerous cells but more study is required in future to further narrow down the active ingredients of total crude extract with specific targets in cancer cells.

A Metabolomic Approach to Investigate the Effect of Phytonutrients on Proteostasis and Metabolic Pathways in Drosophila melanogaster

AbstractThe use of Drosophila melanogaster as a biological platform to study the effect of diet and food bioactives on the metabolome remains a highly unexplored subject. Aiming to establish alternative solutions for the investigation of nutritional interventions with bioactive natural products by employing LC-MS-based metabolomics approaches, we assessed the effect of a phytonutrient-rich extract from the endemic Mediterranean plant Cichorium spinosum (stamnagkàthi) on a Drosophila population. The extractʼs modulating effect on the proteostasis network and metabolism of young D. melanogaster flies was evaluated. Furthermore, an untargeted metabolomics approach, employing a C18 UPLC-ESI-Orbitrap-HRMS/MS platform, permitted the detection of several biomarkers in the metabolic profile of Drosophila’s tissues; while targeted amino acid quantification in Drosophila tissue was simultaneously performed by employing aTRAQ labeling and an ion-pairing UPLC-ESI-SWATH-HRMS/MS platform. The detected metabolites belong to different chemical classes, and statistical analysis with chemometrics tools was utilized to reveal patterns and trends, as well as to uncover potential class-distinguishing features and possible biomarkers. Our findings suggest that Drosophila can serve as a valuable in vivo model for investigating the role of bioactive phytoconstituents, like those found in C. spinosum’s decoction, on diverse metabolic processes. Additionally, the fruit fly represents a highly effective platform to investigate the molecular mechanisms underlying sex differences in diverse aspects of nutrition and physiology in higher metazoans.

Amino Acid Biosynthesis Inhibitors in Tuberculosis Drug Discovery.

According to the latest World Health Organization (WHO) report, an estimated 10.6 million people were diagnosed with tuberculosis (TB) in 2022, and 1.30 million died. A major concern is the emergence of multi-drug-resistant (MDR) and extensively drug-resistant (XDR) strains, fueled by the length of anti-TB treatment and HIV comorbidity. Innovative anti-TB agents acting with new modes of action are the only solution to counteract the spread of resistant infections. To escape starvation and survive inside macrophages, Mtb has evolved to become independent of the host by synthesizing its own amino acids. Therefore, targeting amino acid biosynthesis could subvert the ability of the mycobacterium to evade the host immune system, providing innovative avenues for drug discovery. The aim of this review is to give an overview of the most recent progress in the discovery of amino acid biosynthesis inhibitors. Among the hits discovered over the past five years, tryptophan (Trp) inhibitors stand out as the most advanced and have significantly contributed to demonstrating the feasibility of this approach for future TB drug discovery. Future efforts should be directed at prioritizing the chemical optimization of these hits to enrich the TB drug pipeline with high-quality leads.

Integration of targeted metabolome and transcript profiling of Pseudomonas syringae-triggered changes in defence-related phytochemicals in oat plants

Main conclusionA gene-to-metabolite approach afforded new insights regarding defence mechanisms in oat plants that can be incorporated into plant breeding programmes for the selection of markers and genes related to disease resistance.Monitoring metabolite levels and changes therein can complement and corroborate transcriptome (mRNA) data on plant–pathogen interactions, thus revealing mechanisms involved in pathogen attack and host defence. A multi-omics approach thus adds new layers of information such as identifying metabolites with antimicrobial properties, elucidating metabolomic profiles of infected and non-infected plants, and reveals pathogenic requirements for infection and colonisation. In this study, two oat cultivars (Dunnart and SWK001) were inoculated with Pseudomonas syringae pathovars, pathogenic and non-pathogenic on oat. Following inoculation, metabolites were extracted with methanol from leaf tissues at 2, 4 and 6 days post-infection and analysed by multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer system. Relatedly, mRNA was isolated at the same time points, and the cDNA analysed by quantitative PCR (RT-qPCR) for expression levels of selected gene transcripts associated with avenanthramide (Avn) biosynthesis. The targeted amino acids, hydroxycinnamic acids and Avns were successfully quantified. Distinct cultivar-specific differences in the metabolite responses were observed in response to pathogenic and non-pathogenic strains. Trends in aromatic amino acids and hydroxycinnamic acids seem to indicate stronger activation and flux through these pathways in Dunnart as compared to SWK001. A positive correlation between hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) gene expression and the abundance of Avn A in both cultivars was documented. However, transcript profiling of selected genes involved in Avn synthesis did not reveal a clear pattern to distinguish between the tolerant and susceptible cultivars.Graphical abstract

Anti-vitiligo effect of Thespesia populnea L. bark against Tyrosinase enzyme using In-silico model

Background: Siddha medicine is one among the popular traditional systems of medicine in treating various diseases. Vitiligo is a primary autoimmune de-pigmentary disorder. Generally, melanin pigment production is diminished in vitiligo. By improving tyrosinase activity, melanogenesis can be achieved in vitiligo. Hence the phytocomponents which bind with the target Tyrosinase enzyme, act as a potential treatment for vitiligo. Objective: The objective of this study is to find the lead molecules that bind with these core bio active amino acid residues namely His 190, His54, His63, His194, His38 and His216.These bioactive residues mediates the enzymatic action of tyrosinase enzyme and tends to enhance the action of tyrosinase enzyme which improves melanogenesis. Methods: Auto dock program was used for the molecular docking studies against Tyrosinase enzyme. Results: From reported data of the herb, the phytochemicals Myricetin reveals highiest of 4 interactions with the core active amino acid residues of the target Tyrosinase enzyme.Second highiest level is reached by the compounds such as Catechin, Apigenin and Cinnamic acid with the 3 interactions with the active site . Gallic acid and Quercetin reveal 2 interactions over the target enzyme. From the results of docking study of the herb, the leads such as Catechin, Myricetin, Apigenin and Cinnamic acid possess 3-4 interactions with core target amino acids of Tyrosinase enzyme and helps in treatment and management of vitiligo. Conclusion: These phytochemicals exhibit anti-vitiligo activity by harmonising the action of tyrosinase enzyme in vitiligo treatment. Further clinical trials need to be performed for identifying the efficacy and effectiveness of Thespesia populnea in the treatment and management of vitiligo.

Serum metabolomics analyses reveal biomarkers of osteoporosis and the mechanism of Quanduzhong capsules

With the aging of the population, the prevalence of osteoporosis (OP) is rising rapidly, making it an important public health concern. Early screening and effective treatment of OP are the primary challenges facing the management of OP today. Quanduzhong capsule (QDZ) is a single preparation composed of Eucommia ulmoides Oliv., which is included in the Pharmacopoeia of the People's Republic of China. It is used to treat OP in clinical practice, but its mechanisms are unclear. This study involved 30 patients with OP, 30 healthy controls (HC), and 28 OP patients treated with QDZ to identify potential biomarkers for the early diagnosis of OP and to investigate the potential mechanism of QDZ in treating OP. The serum samples were analyzed using targeted amino acid metabolomics. Significant differences in amino acid metabolism were identified between the OP cohort and the HC group, as well as between OP patients before and after QDZ treatment. Compared with HC, the serum levels of 14 amino acids in OP patients changed significantly. Kynurenine, arginine, citrulline, methionine, and their combinations are expected to be potential biomarkers for OP diagnosis. Notably, QDZ reversed the changes in levels of 10 amino acids in the serum of OP patients and significantly impacted numerous metabolic pathways during the treatment of OP. This study focuses on screening potential biomarkers for the early detection of OP, which offers a new insight into the mechanism study of QDZ in treating OP.

Enantioselective three-dimensional high-performance liquid chromatographic determination of amino acids in the Hayabusa2 returned samples from the asteroid Ryugu

The chirality of amino acids in extraterrestrial materials may provide an insight into the origin of the essential l-enantiopure amino acids in the terrestrial biosphere. In 2020, the Hayabusa2 mission succeeded in bringing back surface materials from the C-type asteroid (162173) Ryugu to the Earth. Amino acids were one of the targeted organic molecules to be studied in the Ryugu samples. To analyze the various structural isomers of amino acids, which were expected to be present, from the limited amount of the returned samples, the development of a highly-sensitive and selective analytical method was necessary. In the present study, a three-dimensional high-performance liquid chromatography (3D-HPLC) system has been developed for the enantioselective determination of five proteinogenic and three non-proteinogenic amino acids in the Ryugu samples, in which amino acids in the sample were separated by reversed-phase, anion-exchange and enantioselective columns after the fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiozole. The applicability of the analytical system to the extraterrestrial samples was evaluated by analyzing several types of carbonaceous meteorites before applying the system to the Ryugu samples. In the analysis of the Ryugu samples, all of the target amino acids were successfully determined quantitatively. Non-proteinogenic amino acids including 2-amino-n-butyric acid, isovaline and norvaline, rarely present in the terrestrial environment, were found as almost racemic mixtures with 47.1 to 55.2%l.

Showcasing water-based delivery of an amino acid targeting fingermark developer in a hydrogel

Most recommended methods for visualising fingermarks on paper rely on chemical developers that target and react with amino acids. Traditionally, these developers are sprayed onto paper substrates in solutions of per- and polyfluoroalkyl substances (PFAS), but now those same PFAS chemicals are undergoing phaseout or phasedown, which threatens to undermine forensic capabilities. This situation provides an opportunity to pivot towards greener approaches to fingermark visualisation. The ideal methodology would be a water-based treatment, as these provide superior safety for practitioners, combined with environmental sustainability. A major hurdle to implementing a water-based fingermark developer targeting amino acids is that water, as a universal solvent, can dissolve the eccrine components in fingermarks, as well as any optical or luminescent dyes that are created, causing the ridge detail to run or dissolve. This work circumvents this problem by delivering the amino acid developer alloxan in a hydrogel, which enables sharp fingermark ridge details to be observed despite it being a water-based treatment. Alloxan dissolved in a viscous hydrogel is shown here to react with the amino acids in fingerprint residues to form the coloured dye murexide, supported by optimisation and characterisation studies.

Amino Acids during Pregnancy and Offspring Cardiovascular-Kidney-Metabolic Health.

Amino acids are essential for normal pregnancy and fetal development. Disruptions in maternal amino acid metabolism have been associated with various adult diseases later in life, a phenomenon referred to as the developmental origins of health and disease (DOHaD). In this review, we examine the recent evidence highlighting the significant impact of amino acids on fetal programming, their influence on the modulation of gut microbiota, and their repercussions on offspring outcomes, particularly in the context of cardiovascular-kidney-metabolic (CKM) syndrome. Furthermore, we delve into experimental studies that have unveiled the protective effects of therapies targeting amino acids. These interventions have demonstrated the potential to reprogram traits associated with CKM in offspring. The discussion encompasses the challenges of translating the findings from animal studies to clinical applications, emphasizing the complexity of this process. Additionally, we propose potential solutions to overcome these challenges. Ultimately, as we move forward, future research endeavors should aim to pinpoint the most effective amino-acid-targeted therapies, determining the optimal dosage and mode of administration. This exploration is essential for maximizing the reprogramming effects, ultimately contributing to the enhancement of cardiovascular-kidney-metabolic health in offspring.

Metabolomic Markers in Attention-Deficit/Hyperactivity Disorder (ADHD) among Children and Adolescents-A Systematic Review.

Attention-Deficit/Hyperactivity Disorder (ADHD), characterized by clinical diversity, poses diagnostic challenges often reliant on subjective assessments. Metabolomics presents an objective approach, seeking biomarkers for precise diagnosis and targeted interventions. This review synthesizes existing metabolomic insights into ADHD, aiming to reveal biological mechanisms and diagnostic potentials. A thorough PubMed and Web of Knowledge search identified studies exploring blood/urine metabolites in ADHD-diagnosed or psychometrically assessed children and adolescents. Synthesis revealed intricate links between ADHD and altered amino acid metabolism, neurotransmitter dysregulation (especially dopamine and serotonin), oxidative stress, and the kynurenine pathway impacting neurotransmitter homeostasis. Sleep disturbance markers, notably in melatonin metabolism, and stress-induced kynurenine pathway activation emerged. Distinct metabolic signatures, notably in the kynurenine pathway, show promise as potential diagnostic markers. Despite limitations like participant heterogeneity, this review underscores the significance of integrated therapeutic approaches targeting amino acid metabolism, neurotransmitters, and stress pathways. While guiding future research, this overview of the metabolomic findings in ADHD suggests directions for precision diagnostics and personalized ADHD interventions.

Interaction of melanoidins, proline, and valine with sorbents of various nature

The isolation of pure amino acids from industrial solutions is one of the important tasks of production. Solutions of amino acids obtained as a result of chemical or microbiological treatment contain a number of impurities, mostly organic ones. The microbiological synthesis of proline is accompanied by the presence of valine, leucine, alanine, and coloured high-molecular components in the culture liquid that belong to the melanoidin group. Melanoidins of proline acid production are high-molecular compounds containing amino acid fragments, heterocyclic structures, and amide bonds. Melanoidins in solutions can exist in cationic, bipolar, and anionic forms. They have different molecular weights, molecular sizes, and isoelectric states. Various ion exchange methods can be used to isolate the target amino acid. It should be noted that the interaction of amino acids with ion-exchange materials is a complex process depending on the electrolytic properties of the sorbate and sorbent and accompanied by multiple side effects. It is necessary to know the specific features of interaction in the “amino acid - sorbent” system and the factors that affect the transport of sorbate ions in the sorbent phase when choosing an isolation method for the target product. The goal of this work was to determine the impact of pH on the sorption process of proline and valine by high-basic ion exchangers, to identify the features of their interactions, and to use the obtained results to optimise the conditions for the separation of amino acids. We studied the process of interaction of bipolar ions and anions of proline (Pro±, Pro-) and valine (Val±, Val-) with anionites AV-17-8 and AV-17-2P. It was established that the mechanism of amino acid ion exchange in granular sorbents depended on the рН value in the “anionite - external solution” system. Based on the studied interactions, we suggested the methods of the anion-exchange separation of proline and valine. The selected conditions of separation of proline and valine were on anionite AV-17-2P in ОН-form (рН of the initial solution was 6.6-6.7; the solution flow rate was 2 cm3/min). The results of separation of proline and valine indicated that even in a one-step sorption process, five to seven fractions with proline contained no valine ions, while the concentration of proline was 2.0-2.7 times higher as compared to the original solution.

Four new diterpenoids from the aerial parts of Leucas zeylanica (L.) R. Br.

Four new undescribed halimane- and labdane-type diterpenoids, named zeylleucapenoids E–H (1–4), along with four known analogues (5–8), were isolated from the aerial parts of Leucas zeylanica (L.) R. Br. Their structures were determined by comprehensive spectroscopic analysis and computational calculations. Compounds 1 and 2 are the highly modified halimane diterpenoids featuring a 6/6/6-fused tricyclic system with an unusual six-membered 6,11-ether ring. Compound 8 exhibits nontoxic effects for zebrafish embryo, while it displays efficient reduction against NO production in a dose-dependent manner and strongly suppresses the secretion of LPS-induced TNF-α and IL-6 cytokines in RAW264.7 macrophages. In addition, marked reductions of iNOS and COX-2 expression were observed. Molecular docking analysis indicated that 8 has high affinities with the target amino acid residues on protein-binding sites, which may be a possible mechanism contributing to the anti-inflammatory potential of this molecule.

Exploring 4-chloro chalcone: Synthesis, spectroscopic, chemical reactivity, topological, hirshfeld surface, drug-Likeness, molecular docking and assessment of electronic properties in diverse solvents

Chalcones are a significant class of naturally occurring chemical substances and their derivatives are extremely influential in the field of biological industries. In the current study, the title composite,4 - Chlorochalcone has undergone extensive investigation to understand its spectral properties using a combination of experimental and theoretical calculations. Fourier transform (FT-IR) infrared and FT-Raman characterizations provided valuable insights into the vibrational assignments (functional groups) in the header molecule, while ultraviolet–visible (UV–Vis) spectroscopy explored its electronic transitions and absorbance characteristics. The electronic properties were studied for the solvents viz., ethanol, methanol, DMSO, acetonitrile and benzene through theoretical-computational DFT calculations, revealing details about their molecular orbitals, electronic energies, and other energy parameters. Additionally, the NBO-natural bond orbital analysis shed light on the chemical bonding and electron distributions by donor and acceptor atoms. Furthermore, MEP- molecular electrostatic potential was analyzed for the various solvents and the calculated energy parameters were obtained. A drug-likeness evaluation was performed to assess its potential as a drug candidate based on its molecular properties. Moreover, the topological analysis and electron-hole distributions, contributed to a comprehensive understanding of electron density and localization within the molecule for the solvents viz., ethanol, methanol, DMSO, acetonitrile and benzene. Molecular docking investigations, as well as the Ramchandran plots, revealed that substituents targeting amino acids contribute to the antiviral action of the header compound. The potential of 4′-Chlorochalcone (ligand) as an antiviral agent offers promising prospects for further exploration and development in various therapeutic areas within the field of medicinal chemistry.

Enhancing Leukemia Treatment: The Role of Combined Therapies Based on Amino Acid Starvation.

Cancer cells demand amino acids beyond their usage as "building blocks" for protein synthesis. As a result, targeting amino acid acquisition and utilization has emerged as a pivotal strategy in cancer treatment. In the setting of leukemia therapy, compelling examples of targeting amino acid metabolism exist at both pre-clinical and clinical stages. This review focuses on summarizing novel insights into the metabolism of glutamine, asparagine, arginine, and tryptophan in leukemias, and providing a comprehensive discussion of perturbing their metabolism to improve the therapeutic outcomes. Certain amino acids, such as glutamine, play a vital role in the energy metabolism of cancer cells and the maintenance of redox balance, while others, such as arginine and tryptophan, contribute significantly to the immune microenvironment. Therefore, assessing the efficacy of targeting amino acid metabolism requires comprehensive strategies. Combining traditional chemotherapeutics with novel strategies to perturb amino acid metabolism is another way to improve the outcome in leukemia patients via overcoming chemo-resistance or promoting immunotherapy. In this review, we also discuss several ongoing or complete clinical trials, in which targeting amino acid metabolism is combined with other chemotherapeutics in treating leukemia.

Sporisorium reilianum polysaccharides improve DSS-induced ulcerative colitis by regulating intestinal barrier function and metabolites

This study investigated the regulatory effects of Sporisorium reilianum polysaccharides (SRPS) on metabolism and the intestinal barrier in mice with colitis induced by dextran sulfate sodium (DSS). SRPS were resistant to the digestion of saliva, gastric juices, and intestinal fluid. SRPS significantly reduced the disease activity index and inhibited DSS-induced colon shortening. The expression of proinflammatory cytokines in the colon was normal (P < 0.05). Acetic acid, propionic acid, butyric acid, isobutyric acid, and isovaleric acid contents increased. Moreover, 64 biomarker metabolites were affected, including 42 abnormal decreases and 22 abnormal increases caused by DSS, which targeted amino acid biosynthesis; tryptophan metabolism; protein digestion and absorption; aminoacyl-tRNA biosynthesis; and glycine, serine, and threonine metabolism. In addition, SRPS reduced goblet cell loss and increased mucin secretion. The short-chain fatty acid receptor GPR41 was activated, and zonula occludens-1 and occludin expression levels were upregulated. Epithelial cell apoptosis was inhibited by increased Bcl-2 and decreased Bax expression NLRP3, ASC, and caspase-1 protein levels decreased. Intestinal barrier damage improved, and colon inflammation was reduced. Thus, our preliminary findings reveal that SRPS regulates metabolism and has the potential to protect the intestinal barrier in ulcerative colitis mice.

Evaluation of Individual Variation of d-Amino Acids in Human Plasma by a Two-Dimensional LC-MS/MS System and Application to the Early Diagnosis of Chronic Kidney Disease.

For the discovery of sensitive biomarkers of kidney function focusing on chiral amino acids, a multiple heart-cutting two-dimensional (2D) liquid chromatography-mass spectrometry (LC-MS)/MS system has been designed/developed. As the target analytes, alanine (Ala), aspartic acid, glutamic acid (Glu), leucine (Leu), lysine, methionine, phenylalanine (Phe), proline (Pro), serine (Ser), and valine were selected considering the presence of their d-forms in mammals. The 2D LC-MS/MS system consisted of the nonenantioselective reversed-phase separation of the target amino acids, the separations of the d- and l-enantiomers, and detection using MS/MS. Using the method, the plasma chiral amino acids, precolumn derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, were isolated from other intrinsic substances, then determined without losing sensitivity by the fully automated whole-peak volume transfer operation from first to second dimension. In all of the tested plasma samples obtained from five healthy individuals and 15 patients with chronic kidney disease (CKD), the target chiral amino acids were determined without interference. In healthy individuals, the levels of all the tested d-amino acids were regulated in the low ranges. In contrast, the % d values of Glu, Leu, and Phe significantly increased with the progress of kidney dysfunction, besides the previously reported values of d-Ala, Pro, and Ser. Concerning Phe, the significant increase of the % d values (p < 0.05) was reported for the first time even in the mild CKD group compared to those of the healthy group; d-Phe might be a more sensitive marker than the previously reported d-forms. These results demonstrated the potential of these d-forms as the sensitive biomarkers of kidney function for the early diagnosis of CKD.

Multi-omics dataset of bovine mammary epithelial cells stimulated by ten different essential amino acids

Application of high-throughput sequencing and screening help to detect the transcriptional and metabolic discrepancies in organs provided with various levels of nutrients. The influences of individual essential amino acid (EAA) administration on transcriptomic and metabolomic profilings of bovine mammary epithelial cells (BMECs) were systematically investigated. A RNA sequencing and liquid chromatography-tandem mass spectrometry generated a comprehensive comparison of transcriptomics, non-targeted metabolomics and targeted amino acids profilings of BMECs with individual EAA stimulation by turn. The sequencing data and raw LC-MS/MS data of samples were presented in the databases of Gene Expression Omnibus, MetaboLights and Figshare for efficient reuse, including exploring the divergences in metabolisms between different EAAs and screening valuable genes and metabolites regulating casein synthesis.

Identification of tumor-suppressive miRNAs that target amino acid transporter LAT1 and exhibit anti-proliferative effects on cholangiocarcinoma cells

Amino acid transporter LAT1 is highly upregulated in various cancer types, including cholangiocarcinoma (CHOL), and contributes to the rapid proliferation of cancer cells and disease progression. However, the molecular mechanisms underlying the pathological upregulation of LAT1 remain largely unknown. This study pursued the possibility of miRNA-mediated regulation of the LAT1 expression in CHOL cells. Using online target prediction methods, we extracted five candidate miRNAs commonly predicted to regulate the LAT1 expression. Three of them, miR-194-5p, miR-122-5p, and miR-126-3p, were significantly downregulated in CHOL cancer compared to normal tissues. Correlation analysis revealed weak-to-moderate negative correlations between the expression of these miRNAs and LAT1 mRNA in CHOL cancer tissues. We selected miR-194-5p and miR-122-5p for further analyses and found that both miRNAs functionally target 3′UTR of LAT1 mRNA by a luciferase-based reporter assay. Transfection of the miRNA mimics significantly suppressed the LAT1 expression at mRNA and protein levels and inhibited the proliferation of CHOL cells, with a trend of affecting intracellular amino acids and amino acid-related signaling pathways. This study indicates that the decreased expression of these LAT1-targeting tumor-suppressive miRNAs contributes to the upregulation of LAT1 and the proliferation of CHOL cells, highlighting their potential for developing novel cancer therapeutics and diagnostics.

Production and secretion of recombinant spider silk in Bacillus megaterium

BackgroundSilk proteins have emerged as versatile biomaterials with unique chemical and physical properties, making them appealing for various applications. Among them, spider silk, known for its exceptional mechanical strength, has attracted considerable attention. Recombinant production of spider silk represents the most promising route towards its scaled production; however, challenges persist within the upstream optimization of host organisms, including toxicity and low yields. The high cost of downstream cell lysis and protein purification is an additional barrier preventing the widespread production and use of spider silk proteins. Gram-positive bacteria represent an attractive, but underexplored, microbial chassis that may enable a reduction in the cost and difficulty of recombinant silk production through attributes that include, superior secretory capabilities, frequent GRAS status, and previously established use in industry.ResultsIn this study, we explore the potential of gram-positive hosts by engineering the first production and secretion of recombinant spider silk in the Bacillus genus. Using an industrially relevant B. megaterium host, it was found that the Sec secretion pathway enables secretory production of silk, however, the choice of signal sequence plays a vital role in successful secretion. Attempts at increasing secreted titers revealed that multiple translation initiation sites in tandem do not significantly impact silk production levels, contrary to previous findings for other gram-positive hosts and recombinant proteins. Notwithstanding, targeted amino acid supplementation in minimal media was found to increase production by 135% relative to both rich media and unaltered minimal media, yielding secretory titers of approximately 100 mg/L in flask cultures.ConclusionIt is hypothesized that the supplementation strategy addressed metabolic bottlenecks, specifically depletion of ATP and NADPH within the central metabolism, that were previously observed for an E. coli host producing the same recombinant silk construct. Furthermore, this study supports the hypothesis that secretion mitigates the toxicity of the produced silk protein on the host organism and enhances host performance in glucose-based minimal media. While promising, future research is warranted to understand metabolic changes more precisely in the Bacillus host system in response to silk production, optimize signal sequences and promoter strengths, investigate the mechanisms behind the effect of tandem translation initiation sites, and evaluate the performance of this system within a bioreactor.