Amino Acid Availability Research Articles

Recent advances in development of electrochemical biosensors for the detection of biogenic amines.

Biogenic amines (BAs) are widely found in food as a consequence of diverse factors including free amino acid availability, microbial production of decarboxylases, and variations in processing and storage conditions. Hence, BAs are considered as an important marker for determining the freshness and quality of food. Owing to the documentation of BAs in different dietary products, their numerous negative impacts on human health have reported to be a serious concern in past few decades. Therefore, the quantification of these chemical species in food becomes crucial as it can immensely contributes toward control of new episodes on food intoxication in humans. In this line, various chromatographic and colorimetric methods have been developed to detect BAs. However, these methods are in use from a longer time, still are limited by high cost, lengthy procedures, huge infrastructure and skilled personnel requirements that hinder their on-field application. In pursuit of a reliable method offering accurate detection of BAs, this review presents the state-of-the-art of electrochemical strategies for BAs sensing in food. The core of the review discusses about the widely employed electrochemical transducers, such as amperometric, potentiometric, impedimetric and conductometric-based BAs biosensors with significant findings of research work conducted previously. The application of electrochemical sensors to analyze BAs in different fields including food systems (fermented and non-fermented types) and smart packaging systems has been reviewed. Moreover, existing challenges and further available prospects for the development of rapid, facile, and sensitive electrochemical strategies for on-site determination of BAs have also been discussed.

Azolla as a New Dietary Source in Broiler Feed: a Physiological and Production Study.

The high requirement of the broiler to protein in the diet needs more knowledge about protein quality, availability of amino acids, prices or cost, the minimum concentration of anti-nutritional factors (ANFs) and gastrointestinal tract (GIT) health. Azolla is a floating fern with high protein content, vitamins (Vit B12, Vit A, and growth promoters), essential amino acids, and minerals (phosphor, calcium, ferrous and potassium). Azolla can fix nitrogen from the atmosphere through the presence of blue-green algae (Anabaena azollae) in their leaves cavities. This study investigated the physiological and production effect of including different levels of Azolla in broiler feed. Four hundred broiler Ross chicks were randomly divided into four equal groups (n=100). The Control group received a regular diet without any supplementation. Animals in the treatment 1 group (T1) received 15% Azolla in their regular diet formulation, the treatment 2 group (T2) received 30% Azolla in their diet formulation, and the treatment 3 group (T3) received 45% Azolla. The animals had ad libitum access to food and water. Body weights and food conversion ratio (FCR) were monitored weekly, and blood samples were collected at the end of the experiment for assessment of lipid profile (cholesterol, triglyceride, HDL, LDL and VLDL), blood proteins (total protein and albumin) and activity of liver enzymes (GOT, GPT and ALP). The results showed an increasing body weight significantly (P≤0.5) in the T3 group compared with the control along with a significant decrease in FCR at an exact quantity of feed for each group (3 Kg). The results showed no change in physiological parameters (lipid profile, blood protein and liver enzymes activity) except for the levels of GOT, which decreased significantly (P≤0.5). In conclusion, the inclusion of Azolla at 15, 30 and 45% in broiler feed has a good effect on body weight and FCR, with no adverse effect on the normal physiology of broilers. Also, supplementation of Azolla can decrease production costs in the broiler industry by more than 30%.

Fat metabolism in laying hens on the background of different lipid components in the diet

The mechanisms of the effects of different oils with different fatty acid profiles on poultry performance remain poorly understood. Therefore, the aim of this study was to investigate the duodenal activity of digestive enzymes and the digestibility of nutrients in the diets of laying hens fed different oils in the same diet (sunflower, soybean, rapeseed and linseed oils, 2.6% of the total diet). The test was conducted on 10 laying hens of the Hysex white cross with chronic duodenal (7 birds) or ileal (3 birds) fistulas. Different oils influenced lipase and alkaline phosphatase activity in duodenal chyme. The highest lipase and alkaline phosphatase activity was found when soybean oil was administered. At the same time, there was a significant (P<0.05) decrease in lipase activity by 35.6-37.6% only when all other oils were administered. Crude fat digestibility was at the highest level when using rapeseed oil. Introduction of this oil into the diet also resulted in the highest digestibility of crude fiber and crude protein and significantly higher availability of amino acids: lysine, threonine, serine, glycine, alanine, histidine and cystine (P<0.05 compared to the control period with the introduction of sunflower oil). In summary, a positive correlation was found between duodenal protease activity and crude protein digestibility with rapeseed oil (r=0.52) and sunflower oil (r=0.54). It was concluded that unrefined sunflower and rapeseed oils are the most physiologically effective oils in diets for laying hens.

Responses of the Macroalga Ulva prolifera Müller to Ocean Acidification Revealed by Complementary NMR- and MS-Based Omics Approaches.

Ocean acidification (OA) is a dramatic perturbation of seawater environments due to increasing anthropogenic emissions of CO2. Several studies indicated that OA frequently induces marine biota stress and a reduction of biodiversity. Here, we adopted the macroalga Ulva prolifera as a model and applied a complementary multi-omics approach to investigate the metabolic profiles under normal and acidified conditions. Our results show that U. prolifera grows at higher rates in acidified environments. Consistently, we observed lower sucrose and phosphocreatine concentrations in response to a higher demand of energy for growth and a higher availability of essential amino acids, likely related to increased protein biosynthesis. In addition, pathways leading to signaling and deterrent compounds appeared perturbed. Finally, a remarkable shift was observed here for the first time in the fatty acid composition of triglycerides, with a decrease in the relative abundance of PUFAs towards an appreciable increase of palmitic acid, thus suggesting a remodeling in lipid biosynthesis. Overall, our studies revealed modulation of several biosynthetic pathways under OA conditions in which, besides the possible effects on the marine ecosystem, the metabolic changes of the alga should be taken into account considering its potential nutraceutical applications.

Metabolomics elucidating the effect of water activity on the thermal resistance of Salmonella in wheat flour

With mounting evidence indicating an enhanced thermal resistance of Salmonella at lower aw, the effectiveness of thermal treatment in wheat flour decontamination is challenged. Therefore, this study was carried out to evaluate the thermal resistance of three Salmonella strains, including Enteritidis (ATCC 13076), Typhimurium (ATCC 14028) and Newport (ATCC 6962), at 65 °C in wheat flour at three aw levels (0.33, 0.53 and 0.69), and to explore the mechanisms of the difference in thermal resistance via nuclear magnetic resonance (NMR)-based metabolomics. The results showed that except for the insignificant difference between the reductions of S. Newport at 0.53 and 0.69 flour aw (P > 0.05), a remarkable decreasing trend in Salmonella cell reduction with decreasing flour aw was observed after the 20-min thermal treatment. By comparing the metabolic profiles of each strain recovered from the lower-aw (0.33 or 0.53) flour with that from the aw-0.69 flour, the metabolic differences implying more efficient misfolded protein degradation, higher availability of amino acids as osmoprotectants, larger throughput of energy production by ATP synthase as well as wiser glucose allocation in the metabolic network were suspected to contribute to the strains’ enhanced thermal resistance. Overall, the study adds to the evidence for the effect of lower aw in increasing the thermal resistance of Salmonella in wheat flour. Meanwhile, the identified discriminative metabolic pathways may be artificially modified in the future to help ease Salmonella inhibition during cooking or any types of thermal treatments.

SLC7A8 is a key amino acids supplier for the metabolic programs that sustain homeostasis and activation of type 2 innate lymphoid cells

Group 2 innate lymphoid cells (ILC2) are innate counterparts of T helper 2 (Th2) cells that maintain tissue homeostasis and respond to injuries through rapid interleukin (IL)-5 and IL-13 secretion. ILC2s depend on availability of arginine and branched-chain amino acids for sustaining cellular fitness, proliferation, and cytokine secretion in both steady state and upon activation. However, the contribution of amino acid transporters to ILC2 functions is not known. Here, we found that ILC2s selectively express Slc7a8, encoding a transporter for arginine and large amino acids. Slc7a8 was expressed in ILC2s in a tissue-specific manner in steady state and was further increased upon activation. Genetic ablation of Slc7a8 in lymphocytes reduced the frequency of ILC2s, suppressed IL-5 and IL-13 production upon stimulation, and impaired type 2 immune responses to helminth infection. Consistent with this, Slc7a8-deficient ILC2s also failed to induce cytokine production and recruit eosinophils in a model of allergic lung inflammation. Mechanistically, reduced amino acid availability due to Slc7a8 deficiency led to compromised mitochondrial oxidative phosphorylation, as well as impaired activation of mammalian target of rapamycin and c-Myc signaling pathways. These findings identify Slc7a8 as a key supplier of amino acids for the metabolic programs underpinning fitness and activation of ILC2s.

Funneliformis constrictum modulates polyamine metabolism to enhance tolerance of Zea mays L. to salinity

The mechanisms underlie increased stress tolerance in plants of salinity stress in plants by arbuscular mycorrhizal fungi (AMF) are poorly understood, particularly the role of polyamine metabolism. The current study was conducted to investigate how inoculation with the AMF, Funneliformis constrictum, affects maize plant tolerance to salt stress. To this end, we investigated the changes in photosynthesis, redox status, primary metabolites (amino acids) and secondary metabolism (phenolic and polyamine metabolism). Control and inoculated maize plants were grown using different concentrations of diluted seawater (0%, 10%, 20% and 40%). Results revealed that treatment with 10% seawater had a beneficial effect on AMF and its host growth. However, irrigation with 20% and 40% significantly reduced plant growth and biomass. As seawater concentration increased, the plants' reliance on mycorrhizal fungi increased resulting in enhanced growth and photosynthetic pigments contents. Under higher seawater concentrations, inoculation with AMF reduced salinity induced oxidative stress and supported redox homeostasis by reducing H2O2 and MDA levels as well as increasing antioxidant-related enzymes activities (e.g., CAT, SOD, APX, GPX, POX, GR, and GSH). AMF inoculation increased amino acid contents in shoots and roots under control and stress conditions. Amino acids availability provides a route for polyamines biosynthesis, where AMF increased polyamines contents (Put, Spd, Spm, total Pas) and their metabolic enzymes associated (ADC, SAMDC, Spd synthase, and Spm synthase), particularly under 40% seawater irrigation. Consistently, the transcription of genes, involved in polyamine metabolism was also up regulated in salinity-stressed plants. AMF further increased the expression in genes involved in polyamine biosynthesis (ODC, SAMDC, SPDS2 and decreased expression of those in catabolic biosynthesis (ADC and PAO). Overall, inoculation with Funneliformis constrictum could be adopted as a practical strategy to alleviate salinity stress.

Apparent digestibility coefficients of animal feed ingredients for olive flounder (Paralichthys olivaceus)

Apparent digestibility coefficients (ADCs) of dry matter, crude protein, crude lipid, nitrogen-free extract, energy and essential amino acids in animal-based feed ingredients were determined for olive flounder (Paralichthys olivaceus). A reference diet (RF) was formulated to contain 1.0% chromic oxide (Cr2O3) as an inert indicator. Nine test diets were formulated to contain RF and one of the feed ingredients (pollock meal [PM], jack mackerel meal [JMM], anchovy meal [AM], cod meal [CM], sardine meal [SM], sand eel meal [SEM], tuna meal [TM], meat meal [MM] and squid liver meal [SLM]) at a 7:3 ratio in each diet designated as PM, JMM, AM, CM, SM, SEM, TM, MM and SLM, respectively. Olive flounder, averaging 150 ± 8.0 g, were stocked at a density of 25 fish per tank in 400-L fiberglass tanks attached with fecal collection columns. Feces were collected from triplicate groups of fish one time a day for four weeks. Dry matter and crude protein ADCs of CM and SEM were significantly higher than the other tested ingredients. Lipid ADCs of JMM, CM and SEM were significantly higher than the other test ingredients. Energy ADCs of CM and SEM were significantly higher than that of the other tested ingredients. The availability of amino acids in CM was generally higher than the other animal protein sources. PM exhibited the lowest amino acid availability among the treatments. Interestingly, MM exhibited significantly higher nutrient digestibility than several marine-based ingredients. However, CM and SEM are seeming to be highly digestible and effective to use in olive flounder diet compared to the other tested ingredients. Overall, the results of this study provide information about the bioavailability of nutrients and energy in animal feedstuffs to apply when formulating cost-effective practical feeds for olive flounder.

The Genetic Variability of Members of the SLC38 Family of Amino Acid Transporters (SLC38A3, SLC38A7 and SLC38A9) Affects Susceptibility to Type 2 Diabetes and Vascular Complications

Type 2 Diabetes (T2D) is a metabolic disease associated with long-term complications, with a multifactorial pathogenesis related to the interplay between genetic and modifiable risk factors, of which nutrition is the most relevant. In particular, the importance of proteins and constitutive amino acids (AAs) in disease susceptibility is emerging. The ability to sense and respond to changes in AA supplies is mediated by complex networks, of which AA transporters (AATs) are crucial components acting also as sensors of AA availability. This study explored the associations between polymorphisms in selected AATs genes and T2D and vascular complications in 433 patients and 506 healthy controls. Analyses revealed significant association of SLC38A3-rs1858828 with disease risk. Stratification of patients based on presence/absence of vascular complications highlighted significant associations of SLC7A8-rs3783436 and SLC38A7-rs9806843 with diabetic retinopathy. Additionally, the SLC38A9-rs4865615 resulted associated with chronic kidney disease. Notably, these genes function as AAs sensors, specifically glutamine, leucine, and arginine, linked to the main nutrient signaling pathway mammalian target of rapamycin complex 1 (mTORC1). Thus, their genetic variability may contribute to T2D by influencing the ability to properly transduce a signal activating mTORC1 in response to AA availability. In this scenario, the contribution of dietary AAs supply to disease risk may be relevant.

Construction of multi-strain microbial consortia producing amylase, serine and proline for enhanced bioconversion of food waste into lipopeptides

The availability of precursor amino acids affects the production of lipopeptides. Its yield reached to 517.73 mg/L in the co-culture of Bacillus amyloliquefaciens HM618 with recombinant Corynebacterium glutamicum producing high-level serine. In the three-strain artificial consortium (TSAC) containing strains HM618 and engineered C. glutamicum producing serine and proline, the levels of turin A, fengycin, and surfactin reached 294.60, 171.12, and 108.90 mg/L, respectively. The total lipopeptide (574.62 mg/L) in TSAC was 1.97-fold higher than that in the pure culture strain HM618. To efficiently utilize starch in food waste (FW), engineered Pichia pastoris, which produces amylase, was introduced into TSAC to construct a four-strain artificial consortium (FSAC) for the bioconversion of FW into lipopeptides. Under an FW medium, the two-strain co-culture, TSAC, and FSAC had significantly enhanced lipopeptide production and amylase activity. Therefore, a mutualistic symbiosis in the artificial consortia of strains HM618 and engineered strains supplying amino acid precursors is beneficial to improve the lipopeptides co-production.

A cell-based chemical-genetic screen for amino acid stress response inhibitors reveals torins reverse stress kinase GCN2 signaling

mTORC1 and GCN2 are serine/threonine kinases that control how cells adapt to amino acid availability. mTORC1 responds to amino acids to promote translation and cell growth while GCN2 senses limiting amino acids to hinder translation via eIF2α phosphorylation. GCN2 is an appealing target for cancer therapies because malignant cells can harness the GCN2 pathway to temper the rate of translation during rapid amino acid consumption. To isolate new GCN2 inhibitors, we created cell-based, amino acid limitation reporters via genetic manipulation of Ddit3 (encoding the transcription factor CHOP). CHOP is strongly induced by limiting amino acids and in this context, GCN2-dependent. Using leucine starvation as a model for essential amino acid sensing, we unexpectedly discovered ATP-competitive PI3 kinase-related kinase inhibitors, including ATR and mTOR inhibitors like torins, completely reversed GCN2 activation in a time-dependent way. Mechanistically, via inhibiting mTORC1-dependent translation, torins increased intracellular leucine, which was sufficient to reverse GCN2 activation and the downstream integrated stress response including stress-induced transcriptional factor ATF4 expression. Strikingly, we found that general translation inhibitors mirrored the effects of torins. Therefore, we propose that mTOR kinase inhibitors concurrently inhibit different branches of amino acid sensing by a dual mechanism involving direct inhibition of mTOR and indirect suppression of GCN2 that are connected by effects on the translation machinery. Collectively, our results highlight distinct ways of regulating GCN2 activity.

The Efficient Antiviral Response of A549 Cells Is Enhanced When Mitochondrial Respiration Is Promoted.

When exposed to a viral infection, the attacked cells promptly set up defense mechanisms. As part of the antiviral responses, the innate immune interferon pathway and associated interferon-stimulated genes notably allow the production of proteins bearing antiviral activity. Numerous viruses are able to evade the interferon response, highlighting the importance of controlling this pathway to ensure their efficient replication. Several viruses are also known to manipulate the metabolism of infected cells to optimize the availability of amino acids, nucleotides, and lipids. They then benefit from a reprogramming of the metabolism that favors glycolysis instead of mitochondrial respiration. Given the increasingly discussed crosstalk between metabolism and innate immunity, we wondered whether this switch from glycolysis to mitochondrial respiration would be beneficial or deleterious for an efficient antiviral response. We used a cell-based model of metabolic reprogramming. Interestingly, we showed that increased mitochondrial respiration was associated with an enhanced interferon response following polyriboinosinic:polyribocytidylic acid (poly:IC) stimulation. This suggests that during viral infection, the metabolic reprogramming towards glycolysis is also part of the virus’ strategies to inhibit the antiviral response.

Effects of broiler chicken age and dietary protease on the standardised ileal digestibility of amino acids in seeds from two lupin species

Abstract The extent to which lupin seeds can replace soybean meal in diets for broiler chickens is limited, and one of the reasons for this may be a lack of careful consideration given to the age-related amino acid availability differences in the feed formulation process. This study aimed to determine and compare standardised ileal digestibility (SID) coefficients of amino acids (AA) in raw seeds of the Lupinus luteus (YL) and the Lupinus angustifolius (NLL) cultivars as sole sources of AA in the diet of broiler chickens aged 14 or 28 days. An additional purpose was to check the influence of exogenous mono-component protease added to lupin-based assay diets on AA SID in birds at both ages. Each assay diet was offered to six replicatecages. On both sampling days, the contents from the lower half of the ileum were collected for determination of the apparent digestibility values. The SID coefficients were calculated using the age-appropriate basal ileal endogenous AA losses determined from birds fed a N-free diet. Results indicated a substantial advantage of YL over the NLL in SID of Met, Cys, His, Leu, Gly, Asp, Glu, Pro, Tyr and the average of total AA for 14-d-old chickens. With the exception of Cys and Tyr, there was no significant difference between these Lupinus species in the SID of AA in 28-d-old birds. The significant age-related differences within species were found only for the seeds of NLL, where the higher SID for Met, His, Ala, Pro, and the averages of total and indispensable AA were noted in birds aged 28 d. The protease increased SID of most AA from L. luteus in older broilers, whereas for L. angustifolius its efficacy was more pronounced in younger birds, giving the SID values similar to those determined at 28 d on an enzyme-unsupplemented diet. In conclusion, the SID coefficients of indispensable AA determined at 14 or 28 days of age for yellow lupin should not be applied to the precise formulation of starter-type feeds containing seeds of NLL.

MTOR- and LARP1-dependent regulation of TOP mRNA poly(A) tail and ribosome loading

Translation of 5' terminal oligopyrimidine (TOP) mRNAs encoding the protein synthesis machinery is strictly regulated by an amino-acid-sensing mTOR pathway. However, its regulatory mechanism remains elusive. Here, we demonstrate that TOP mRNA translation positively correlates with its poly(A) tail length under mTOR active/amino-acid-rich conditions, suggesting that TOP mRNAs are post-transcriptionally controlled by poly(A) tail-length regulation. Consistent with this, the tail length of TOP mRNAs dynamically fluctuates in response to amino acid availability. The poly(A) tail shortens under mTOR active/amino-acid-rich conditions, whereas the long-tailed TOP mRNAs accumulate under mTOR inactive/amino-acid-starved (AAS) conditions. An RNA-binding protein, LARP1, is indispensable for the process. LARP1 interacts with non-canonical poly(A) polymerases and induces post-transcriptional polyadenylation of the target. Our findings illustrate that LARP1 contributes to the selective accumulation of TOP mRNAs with long poly(A) tails under AAS, resulting in accelerated ribosomal loading onto TOP mRNAs for the resumption of translation after AAS.

The use of non-model Drosophila species to study natural variation in TOR pathway signaling.

Nutrition and growth are strongly linked, but not much is known about how nutrition leads to growth. To understand the connection between nutrition through the diet, growth, and proliferation, we need to study the phenotypes resulting from the activation and inhibition of central metabolic pathways. One of the most highly conserved metabolic pathways across eukaryotes is the Target of Rapamycin (TOR) pathway, whose primary role is to detect the availability of nutrients and to either induce or halt cellular growth. Here we used the model organism Drosophila melanogaster (D. mel.) and three non-model Drosophila species with different dietary needs, Drosophila guttifera (D. gut.), Drosophila deflecta (D. def.), and Drosophila tripunctata (D. tri.), to study the effects of dietary amino acid availability on fecundity and longevity. In addition, we inhibited the Target of Rapamycin (TOR) pathway, using rapamycin, to test how the inhibition interplays with the nutritional stimuli in these four fruit fly species. We hypothesized that the inhibition of the TOR pathway would reverse the phenotypes observed under conditions of overfeeding. Our results show that female fecundity increased with higher yeast availability in all four species but decreased in response to TOR inhibition. The longevity data were more varied: most species experienced an increase in median lifespan in both genders with an increase in yeast availability, while the lifespan of D. mel. females decreased. When exposed to the TOR inhibitor rapamycin, the life spans of most species decreased, except for D. tri, while we observed a major reduction in fecundity across all species. The obtained data can benefit future studies on the evolution of metabolism by showing the potential of using non-model species to track changes in metabolism. Particularly, our data show the possibility to use relatively closely related Drosophila species to gain insight on the evolution of TOR signaling.

Systemically engineering Bacillus amyloliquefaciens for increasing its antifungal activity and green antifungal lipopeptides production.

The biosynthesis of antifungal lipopeptides iturin and fengycin has attracted broad interest; however, there is a bottleneck in its low yield in wild strains. Because the key metabolic mechanisms in the lipopeptides synthesis pathway remain unclear, genetic engineering approaches are all ending up with a single or a few gene modifications. The aim of this study is to develop a systematic engineering approach to improve the antifungal activity and biosynthesis of iturin and fengycin in Bacillus amyloliquefaciens. First, blocking the carbon overflow metabolic pathway to increase precursor supply of the branched-chain amino acids by knockout of bdh, disrupting sporulation to extend the stage for producing antifungal lipopeptides by deletion of kinA, blocking of siderophore synthesis to enhance the availability of amino acids and fatty acids by deletion of dhbF, and increasing Spo0A∼P by deletion of rapA, could improve the antifungal activity by 24%, 10%, 13% and 18%, respectively. Second, the double knockout strain ΔbdhΔkinA, triple knockout strain ΔbdhΔkinAΔdhbF and quadruple knockout strain ΔkinAΔbdhΔdhbFΔrapA could improve the antifungal activity by 38%, 44% and 53%, respectively. Finally, overexpression of sfp in ΔkinAΔbdhΔdhbFΔrapA further increased the antifungal activity by 65%. After purifying iturin and fengycin as standards for quantitative analysis of lipopeptides, we found the iturin titer was 17.0 mg/L in the final engineered strain, which was 3.2-fold of the original strain. After fermentation optimization, the titer of iturin and fengycin reached 31.1 mg/L and 175.3 mg/L in flask, and 123.5 mg/L and 1200.8 mg/L in bioreactor. Compared to the original strain, the iturin and fengycin titer in bioreactor increased by 22.8-fold and 15.9-fold in the final engineered strain, respectively. This study may pave the way for the commercial production of green antifungal lipopeptides, and is also favorable for understanding the regulatory and biosynthetic mechanism of iturin and fengycin.

A Rag GTPase dimer code defines the regulation of mTORC1 by amino acids

Amino acid availability controls mTORC1 activity via a heterodimeric Rag GTPase complex that functions as a scaffold at the lysosomal surface, bringing together mTORC1 with its activators and effectors. Mammalian cells express four Rag proteins (RagA–D) that form dimers composed of RagA/B bound to RagC/D. Traditionally, the Rag paralogue pairs (RagA/B and RagC/D) are referred to as functionally redundant, with the four dimer combinations used interchangeably in most studies. Here, by using genetically modified cell lines that express single Rag heterodimers, we uncover a Rag dimer code that determines how amino acids regulate mTORC1. First, RagC/D differentially define the substrate specificity downstream of mTORC1, with RagD promoting phosphorylation of its lysosomal substrates TFEB/TFE3, while both Rags are involved in the phosphorylation of non-lysosomal substrates such as S6K. Mechanistically, RagD recruits mTORC1 more potently to lysosomes through increased affinity to the anchoring LAMTOR complex. Furthermore, RagA/B specify the signalling response to amino acid removal, with RagB-expressing cells maintaining lysosomal and active mTORC1 even upon starvation. Overall, our findings reveal key qualitative differences between Rag paralogues in the regulation of mTORC1, and underscore Rag gene duplication and diversification as a potentially impactful event in mammalian evolution.

Extracellular microbial proteases with specificity for plant proteins in food fermentation

Plant-based food products are generating a growing interest as part of the ongoing transition to a primarily plant-based diet, which makes demands to the quality, functionality, and health properties of plant proteins. Microbes used for traditional food fermentations such as lactic acid bacteria (LAB) and fungi (yeasts and molds) carry out enzymatic changes on their protein substrates by which technological and sensorial characteristics can be improved. The literature on extracellular proteases targeting plant proteins, on the other hand, is scattered with only a narrow representation of plants even for traditionally plant-based products. Therefore, this review aims to explore the current state of knowledge regarding the application potential of microbial extracellular proteases targeting plant proteins, with a focus on traditional applied food microbes. Plant proteins are targeted by proteolytic microbes of both animal and plant origins, and their proteases show a wide range of activities. Extracellular microbial proteases can hydrolyze specific protein-based allergens and even reduce the toxicity of plant proteins. Additionally, microbial assisted proteolysis can improve plant protein digestibility by increasing availability of peptides and amino acids. This catabolic process will change the organoleptic characteristics of fermented plant proteins, and the release of bioactive peptides can provide additional functionalities to the plant matrix. The proteolytic activity is determined by the microbial strain, and it can be quite substrate selective, which is why proteases may be overlooked by the prevalent use of casein as substrate in proteolytic screenings. The synergetic effects of LAB and fungal species consortia can facilitate and steer plant protein hydrolysis by which co-fermentation may increase or change the properties of plant protein hydrolysates. Microbes do not necessarily require extracellular proteases because endogenous proteases in a plant-matrix may meet the microbial amino acid requirements. However, extracellular proteases have the potential to provide central properties to diverse food-matrixes by which the full proteolytic potential of food microbes needs to be explored in order to facilitate the development of high-quality plant-based food products.

Ascorbic Acid (Vitamin C) as a Cosmeceutical to Increase Dermal Collagen for Skin Antiaging Purposes: Emerging Combination Therapies.

Ascorbic acid (AA) is an essential nutrient and has great potential as a cosmeceutical that protects the health and beauty of the skin. AA is expected to attenuate photoaging and the natural aging of the skin by reducing oxidative stress caused by external and internal factors and by promoting collagen gene expression and maturation. In this review, the biochemical basis of AA associated with collagen metabolism and clinical evidence of AA in increasing dermal collagen and inhibiting skin aging were discussed. In addition, we reviewed emerging strategies that have been developed to overcome the shortcomings of AA as a cosmeceutical and achieve maximum efficacy. Because extracellular matrix proteins, such as collagen, have unique amino acid compositions, their production in cells is influenced by the availability of specific amino acids. For example, glycine residues occupy 1/3 of amino acid residues in collagen protein, and the supply of glycine can be a limiting factor for collagen synthesis. Experiments showed that glycinamide was the most effective among the various amino acids and amidated amino acids in stimulating collagen production in human dermal fibroblasts. Thus, it is possible to synergistically improve collagen synthesis by combining AA analogs and amino acid analogs that act at different stages of the collagen production process. This combination therapy would be useful for skin antiaging that requires enhanced collagen production.

Phenotypic response of yeast metabolic network to availability of proteinogenic amino acids.

Genome-scale metabolism can best be described as a highly interconnected network of biochemical reactions and metabolites. The flow of metabolites, i.e., flux, throughout these networks can be predicted and analyzed using approaches such as flux balance analysis (FBA). By knowing the network topology and employing only a few simple assumptions, FBA can efficiently predict metabolic functions at the genome scale as well as microbial phenotypes. The network topology is represented in the form of genome-scale metabolic models (GEMs), which provide a direct mapping between network structure and function via the enzyme-coding genes and corresponding metabolic capacity. Recently, the role of protein limitations in shaping metabolic phenotypes have been extensively studied following the reconstruction of enzyme-constrained GEMs. This framework has been shown to significantly improve the accuracy of predicting microbial phenotypes, and it has demonstrated that a global limitation in protein availability can prompt the ubiquitous metabolic strategy of overflow metabolism. Being one of the most abundant and differentially expressed proteome sectors, metabolic proteins constitute a major cellular demand on proteinogenic amino acids. However, little is known about the impact and sensitivity of amino acid availability with regards to genome-scale metabolism. Here, we explore these aspects by extending on the enzyme-constrained GEM framework by also accounting for the usage of amino acids in expressing the metabolic proteome. Including amino acids in an enzyme-constrained GEM of Saccharomyces cerevisiae, we demonstrate that the expanded model is capable of accurately reproducing experimental amino acid levels. We further show that the metabolic proteome exerts variable demands on amino acid supplies in a condition-dependent manner, suggesting that S. cerevisiae must have evolved to efficiently fine-tune the synthesis of amino acids for expressing its metabolic proteins in response to changes in the external environment. Finally, our results demonstrate how the metabolic network of S. cerevisiae is robust towards perturbations of individual amino acids, while simultaneously being highly sensitive when the relative amino acid availability is set to mimic a priori distributions of both yeast and non-yeast origins.