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A one-step analysis method was developed for four types of amino acids using a microfluidic paper-based analytical device fabricated from chromatography filtration paper and laminate films. Aminoacyl-tRNA synthetase was used to detect each amino acid. The obtained laminated paper-based analytical device (LPAD) contained four enzymatic reaction areas. Colorimetric detection was performed based on the molybdenum blue reaction. A model method for the simple, easy, and simultaneous detection of several amino acid concentrations was suggested, in contrast to the conventional methods such as HPLC or LC–MS. The method provided a selective quantification at the ranges of 3.6–100 μM for tryptophan, 10.1–100 μM for glycine, 5.9–100 μM for histidine and 5.6–100 μM for lysine with a detection limit of 1.1 μM, 3.3 μM, 1.9 μM and 1.8 μM, respectively. LPAD fabrication was considerably simple, and the subsequent detection process was easy and required a short period of time (within 15 min).

Purpose To study the metagenomics of the microbes isolated from the canaliculus of patients with infective canaliculitis. Methods A prospective study was performed on five consecutive canalicular samples obtained for the metagenomic analysis from the patients with infective canaliculitis who underwent non-incisional canalicular curettage at a tertiary care Dacryology service. The canalicular concretions were collected intraoperatively soon after a canalicular curettage and immediately transported on ice to the laboratory. Following DNA extraction and library preparation, a whole shotgun metagenome sequencing was performed on the Illumina™ platform. The downstream processing and bioinformatics of the samples were performed using multiple software packaged in SqueezeMeta™ pipeline or MG-RAST™ pipeline. Results The taxonomic hit distribution across the samples showed that bacteria were the most common isolates (mean—80.5%), followed by viruses (mean—0.74%), and archaea (0.01%). The five major phyla identified across the samples of infective canaliculitis were, Fusobacteria, Bacteroidetes, Proteobacteria, Actinobacteria, and Firmicutes. The prevalent organisms include Fusobacterium nucelatum, Fusobacterium periodonticum, Parvimonas micra, Prevotella oris, Selonomonas noxia, Pseudopropionobacterium propoinicum, Campylobacter showae, and Streptococcus anginosus, amongst few others. Actinomycetes israelii was noted in all the samples, though it was not the most abundant. The microbial gene mapping and protein prediction demonstrated proteins with known functions to range from 69.91% to 87.09% across the samples. The functional subsystem profiling demonstrated genes associated with carbohydrate, amino acid, and co-enzyme transport and metabolism, cell wall or cell membrane biogenesis, energy production and conversion, transcription, translation, and cellular communications. Conclusion This is the first whole metagenome sequencing of infective canaliculitis. Infected canaliculi harbor diverse microbial communities, including bacteria, viruses, and archaea. Functional analysis has provided newer insights into the ecosystem dynamics and strategies of microbial communities.

Amino acids are closely related to human health, and their rapid determination is important for the rapid diagnosis, timely treatment, and assessment of serious diseases. In this work, we propose a novel paper-based sample-processing device combined with isotope-dilution MS for the fast analysis of 11 amino acids from blood samples. By using an isoelectric focusing electrokinetic separation method, without the aid of carrier ampholytes and the addition of inhibitors, this approach uses only the characteristic of the isoelectric point of the target amino acids to achieve separation and purification from other coexisting components in the medium; it can meet the requirements for mass spectrometry detection. Driven by a DC voltage, a stable and sharp pH gradient (pH 3-10.5 over 5 mm) can be established in a glass-fiber paper-based fluidic channel with a MS-friendly electrolyte. Amphoteric species can be well separated from the complex blood matrix and concentrated into a narrow band in the channel within 2 min, which is 20 times faster than a commercial kit method. The method can be applied to both liquid and dry spot samples, and the cleaned sample band can be simply dissolved for direct IDMS detection in ESI MRM mode. This method is a promising strategy for the rapid MS-based detection of amino acids from serum without pre-separation

A direct fluorimetric method, employing μicro-analytical paper-based devices (μ-PADs) for the selective determination of histidine (HIS) is described. The suggested method exploits the fluorescence emission of histidine after its rapid reaction with o-phthalaldehyde (OPA) at a basic medium (pH = 10) on the surface of a paper device with the application of a UV lamp at 354 nm. The devices are inexpensive and are composed of chromatographic paper and wax barriers. The analytical protocol is easily applicable with minimal technical expertise and without the need of expensive experimental apparatus. The user has to add a test sample, illuminate the device with a UV lamp, and read the fluorescence of the sensing area using a simple imaging device such as a cell-phone camera. The method is free from common interferences likely to affect the measurement of histidine and is selective among all other amino acids. This analytical procedure was optimized and validated, paying special attention to its intended application. The detection limits are as low as 1.8 μM with very satisfactory precision ranging from 6.4% (intra-day) to 8.9% (inter-day). Random urine samples from adult volunteers (n = 5) were successfully analyzed and HIS content ranged between 260 and 1114 μmol L

We propose a printed 384-well microtiter paper-based fluorescent chemosensor array device (384-well microtiter PCAD) to simultaneously categorize and discriminate saccharides and sulfur-containing amino acids for food analysis. The 384-well microtiter PCAD requiring 1 μL/4 mm

Funding Information: The authors thank the Portuguese Fundaçao para a Ciência e a Tecnologia, which supports the Applied Molecular Biosciences Unit - UCIBIO, the European Commission (Water JPI project 196 (Water-Works2014 ERA-NET Co-funded Call): “Smart decentralized water management through a dynamic integration of technologies (Watintech)” and the Australian Research Council ( ARC LP190100329 ). Publisher Copyright: © 2023 Members of the genus Tetrasphaera are putative polyphosphate accumulating organisms (PAOs) that have been found in greater abundance than Accumulibacter in many full-scale enhanced biological phosphorus removal (EBPR) wastewater treatment plants worldwide. Nevertheless, previous studies on the effect of environmental conditions, such as pH, on the performance of EBPR have focused mainly on the response of Accumulibacter to pH changes. This study examines the impact of pH on a Tetrasphaera PAO enriched culture, over a pH range from 6.0 to 8.0 under both anaerobic and aerobic conditions, to assess its impact on the stoichiometry and kinetics of Tetrasphaera metabolism. It was discovered that the rates of phosphorus (P) uptake and P release increased with an increase of pH within the tested range, while PHA production, glycogen consumption and substrate uptake rate were less sensitive to pH changes. The results suggest that Tetrasphaera PAOs display kinetic advantages at high pH levels, which is consistent with what has been observed previously for Accumulibacter PAOs. The results of this study show that pH has a substantial impact on the P release and uptake kinetics of PAOs, where the P release rate was >3 times higher and the P uptake rate was >2 times higher at pH 8.0 vs pH 6.0, respectively. Process operational strategies promoting both Tetrasphaera and Accumulibacter activity at high pH do not conflict with each other, but lead to a potentially synergistic impact that can benefit EBPR performance. publishersversion published

The endolysosomal system not only is an integral part of the cellular catabolic machinery that processes and recycles nutrients for synthesis of biomaterials, but also acts as signaling hub to sense and coordinate the energy state of cells with growth and differentiation. Lysosomal dysfunction adversely influences vesicular transport-dependent macromolecular degradation and thus causes serious problems for human health. In mammalian cells, loss of the lysosome associated membrane proteins LAMP1 and LAMP2 strongly affects autophagy and cholesterol trafficking. Here we show that the previously uncharacterized Drosophila Lamp1 is a bona fide ortholog of vertebrate LAMP1 and LAMP2. Surprisingly and in contrast to lamp1 lamp2 double-mutant mice, Drosophila Lamp1 is not required for viability or autophagy, suggesting that fly and vertebrate LAMP proteins acquired distinct functions, or that autophagy defects in lamp1 lamp2 mutants may have indirect causes. However, Lamp1 deficiency results in an increase in the number of acidic organelles in flies. Furthermore, we find that Lamp1 mutant larvae have defects in lipid metabolism as they show elevated levels of sterols and diacylglycerols (DAGs). Because DAGs are the main lipid species used for transport through the hemolymph (blood) in insects, our results indicate broader functions of Lamp1 in lipid transport. Our findings make Drosophila an ideal model to study the role of LAMP proteins in lipid assimilation without the confounding effects of their storage and without interfering with autophagic processes. Abbreviations: aa: amino acid; AL: autolysosome; AP: autophagosome; APGL: autophagolysosome; AV: autophagic vacuole (i.e. AP and APGL/AL); AVi: early/initial autophagic vacuoles; AVd: late/degradative autophagic vacuoles; Atg: autophagy-related; CMA: chaperone-mediated autophagy; Cnx99A: Calnexin 99A; DAG: diacylglycerol; eMI: endosomal microautophagy; ESCRT: endosomal sorting complexes required for transport; FB: fat body; HDL: high-density lipoprotein; Hrs: Hepatocyte growth factor regulated tyrosine kinase substrate; LAMP: lysosomal associated membrane protein; LD: lipid droplet; LDL: low-density lipoprotein; Lpp: lipophorin; LTP: Lipid transfer particle; LTR: LysoTracker Red; MA: macroautophagy; MCC: Manders colocalization coefficient; MEF: mouse embryonic fibroblast MTORC: mechanistic target of rapamycin kinase complex; PV: parasitophorous vacuole; SNARE: soluble N-ethylmaleimide sensitive factor attachment protein receptor; Snap: Synaptosomal-associated protein; st: starved; TAG: triacylglycerol; TEM: transmission electron microscopy; TFEB/Mitf: transcription factor EB; TM: transmembrane domain; tub: tubulin; UTR: untranslated region.

Metallothionein (MT) is a family of low molecular weight, cysteine-rich proteins that regulate zinc homeostasis and have potential protective effects against oxidative stress and toxic metals. MT1 and MT2 gene knockout (MTKO) mice show shorter lifespans than wild-type (WT) mice. In this study, we aimed to investigate how MT gene deficiency accelerates aging. We performed comparative metabolomic analyses of plasma between MTKO and WT male mice at middle age (50-week-old) and advanced age (100-week-old) using liquid chromatography with time-of-flight mass spectrometry (LC-TOF-MS). The concentration of N6,N6,N6-trimethyl-L-lysine (TML), which is a metabolic intermediate in carnitine biosynthesis, was consistently higher in the plasma of MTKO mice compared to that of WT mice at middle and advanced age. Quantitative reverse transcription PCR (RT-PCR) analysis revealed remarkably lower mRNA levels of Tmlhe, which encodes TML dioxygenase, in the liver and kidney of male MTKO mice compared to that of WT mice. L-carnitine is essential for β-oxidation of long-chain fatty acids in mitochondria, the activity of which is closely related to aging. Our results suggest that reduced carnitine biosynthesis capacity in MTKO mice compared to WT mice led to metabolic disorders of fatty acids in mitochondria in MTKO mice, which may have caused shortened lifespans.

Regulators of G protein signaling (RGSs) are proteins that negatively regulate G protein signal transduction. In this study, seven putative RGSs were characterized in the nematode-trapping (NT) fungus, Arthrobotrys oligospora. Deleting Rgs genes significantly increased intracellular cAMP levels, and caused defects in mycelia growth, stress resistance, conidiation, trap formation, and nematocidal activity. In particular, the ΔAoFlbA mutant was unable to produce conidia and traps. Transcriptomic analysis showed that amino acid metabolic and biosynthetic processes were significantly enriched in the ΔAoFlbA mutant compared to WT. Interestingly, Gas1 family genes are significantly expanded in A. oligospora and other NT fungi that produce adhesive traps, and are differentially expressed during trap formation in A. oligospora. Disruption of two Gas1 genes resulted in defective conidiation, trap formation, and pathogenicity. Our results indicate that RGSs play pleiotropic roles in regulating A. oligospora mycelial growth, development, and pathogenicity. Further, AoFlbA is a prominent member and required for conidiation and trap formation, possibly by regulating amino acid metabolism and biosynthesis. Our results provide a basis for elucidating the signaling mechanism of vegetative growth, lifestyle transition, and pathogenicity in NT fungi.

One fundamental problem of protein biochemistry is to predict protein structure from amino acid sequence. The inverse problem, predicting either entire sequences or individual mutations that are consistent with a given protein structure, has received much less attention even though it has important applications in both protein engineering and evolutionary biology. Here, we ask whether 3D convolutional neural networks (3D CNNs) can learn the local fitness landscape of protein structure to reliably predict either the wild-type amino acid or the consensus in a multiple sequence alignment from the local structural context surrounding site of interest. We find that the network can predict wild type with good accuracy, and that network confidence is a reliable measure of whether a given prediction is likely going to be correct or not. Predictions of consensus are less accurate and are primarily driven by whether or not the consensus matches the wild type. Our work suggests that high-confidence mis-predictions of the wild type may identify sites that are primed for mutation and likely targets for protein engineering. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10867-021-09593-6.

A convenient and simple protocol has been developed for the synthesis of a series of new tetracyclic tetrahydroisoquinoline derivatives, 7,12-dihydro-6,12-methanodibenzo[c,f]-azocine-5-carboxylic acids by three component Petasis reaction with the use of aminoacetaldehyde acetals bearing substituted benzyl groups as the amine components followed by Pomeranz–Fritsch double cyclization reaction. By applying this method, several acids have been prepared in satisfactory yields. An unprecedented chemical behavior of a Petasis reaction product in diluted HCl solution leading to the formation of a phenylglycine derivative has been observed and the mechanism explaining such reactivity has been proposed.

As the global population grows, large quantities of agrochemicals are used to secure food demand and there is an urgent need to develop efficient and environmentally friendly pesticides and fertilizers. Compared with traditional pesticides, nanopesticide formulations can achieve better foliar wettability, chemical stability, and water dispersibility without or with the minimal use of organic solvents, in line with the development of sustainable agriculture.In this research, glycidyl methacrylate (GMA) was used as an intermediate and glycine methyl ester (GLY) was used as an organic nitrogen source to modify carboxymethyl cellulose (CMC) to synthesize a pesticide nanocarrier CMC-PGMA-GLY. A nanopesticide EB@CMC-PGMA-GLY (EB@CPG) was formed by the hydrophobic and electrostatic interactions of emamectin benzoate (EB), which had good water dispersion. The good affinity of the composite towards leaves led to the effective moistening of the leaf surface by the pesticide and through the pH control pesticide release. Biological experiments show that nanopesticides can maintain high insecticidal activity and have no toxicity to seed germination. The results of pot experiment showed that the nanocarriers could be used as an organic nitrogen fertilizer to increase the fresh weight of plants by 39.77%.This environmentally friendly pesticide carrier can be used as a fertilizer to provide sufficient nutrients for crops, opening a new method for the development of sustainable agriculture. © 2021 Society of Chemical Industry.

Metabolic syndrome (MetS) is a significant factor for cardiometabolic comorbidities in people living with HIV (PLWH) and a barrier to healthy aging. The long-term consequences of HIV-infection and combination antiretroviral therapy (cART) in metabolic reprogramming are unknown. In this study, we investigated metabolic alterations in well-treated PLWH with MetS to identify potential mechanisms behind the MetS phenotype using advanced statistical and machine learning algorithms. We included 200 PLWH from the Copenhagen Comorbidity in HIV-infection (COCOMO) study. PLWH were grouped into PLWH with MetS (n= 100) defined according to the International Diabetes Federation (IDF) consensus worldwide definition of the MetS or without MetS (n= 100). The untargeted plasma metabolomics was performed using ultra-high-performance liquid chromatography/mass spectrometry (UHPLC/MS/MS) and immune-phenotyping of Glut1 (glucose transporter), xCT (glutamate/cysteine transporter) and MCT1 (pyruvate/lactate transporter) by flow cytometry. We applied several conventional approaches, machine learning algorithms, and linear classification models to identify the biologically relevant metabolites associated with MetS in PLWH. Of the 877 identified biochemicals, 9% (76/877) differed significantly between PLWH with and without MetS (false discovery rate

The ability to maintain systemic metabolic homeostasis through various mechanisms represents a crucial strength of kidneys in the study of metabolic syndrome or aging. Moreover, age-associated kidney failure has been widely accepted. However, efforts to demonstrate aging-dependent renal metabolic rewiring have been limited. In the present study, we investigated aging-related renal metabolic determinants by integrating metabolomic and transcriptomic data sets from kidneys of young (3 months, n = 7 and 3 for respectively) and old (24 months, n = 8 and 3 for respectively) naive C57BL/6 male mice. Metabolite profiling analysis was conducted, followed by data processing via network and pathway analyses, to identify differential metabolites. In the aged group, the levels of glutathione and oxidized glutathione were significantly increased, but the levels of gamma-glutamyl amino acids, amino acids combined with the gamma-glutamyl moiety from glutathione by membrane transpeptidases, and circulating glutathione levels were decreased. In transcriptomic analysis, differential expression of metabolic enzymes is consistent with the hypothesis of aging-dependent rewiring in renal glutathione metabolism; pathway and network analyses further revealed the increased expression of immune-related genes in the aged group. Collectively, our integrative analysis results revealed that defective renal glutathione metabolism is a signature of renal aging. Therefore, we hypothesize that restraining renal glutathione metabolism might alleviate or delay age-associated renal metabolic deterioration, and aberrant activation of the renal immune system.

Recent improvements in mRNA display have enabled the selection of peptides that incorporate non‐natural amino acids, thus expanding the chemical diversity of macrocycles beyond what is accessible in nature. Such libraries have incorporated non‐natural amino acids at the expense of natural amino acids by reassigning their codons. Here we report an alternative approach to expanded amino‐acid diversity that preserves all 19 natural amino acids (no methionine) and adds 6 non‐natural amino acids, resulting in the highest sequence complexity reported to date. We have applied mRNA display to this 25‐letter library to select functional macrocycles that bind human STING, a protein involved in immunoregulation. The resulting STING‐binding peptides include a 9‐mer macrocycle with a dissociation constant (K D) of 3.4 nM, which blocks binding of cGAMP to STING and induces STING dimerization. This approach is generalizable to expanding the amino‐acid alphabet in a library beyond 25 building blocks., In vitro selection of macrocyclic peptides from a library that uses a 25‐amino‐acid alphabet has allowed the identification of peptides that block binding of human STING to its natural ligand and induce dimerization of STING monomers. Our method splits multiple degenerate codons and reassigns them to non‐natural amino acids.

The infectivity of HIV-1 requires its protease cleave multiple cut-sites with low sequence similarity. The diversity of cleavage sites has made it challenging to investigate the underlying sequence properties that determine binding and turnover of substrates by PR. We engineered a mutational scanning approach utilizing yeast display, flow cytometry, and deep sequencing to systematically measure the impacts of all individual amino acid changes at 12 positions in three different cut-sites (MA/CA, NC/p1, and p1/p6). The resulting fitness landscapes revealed common physical features that underlie cutting of all three cut-sites at the amino acid positions closest to the scissile bond. In contrast, positions more than two amino acids away from the scissile bond exhibited a strong dependence on the sequence background of the rest of the cut-site. We observed multiple amino acid changes in cut-sites that led to faster cleavage rates, including a preference for negative charge five and six amino acids away from the scissile bond at locations where the surface of protease is positively charged. Analysis of individual cut sites using full-length matrix-capsid proteins indicate that long-distance sequence context can contribute to cutting efficiency such that analyses of peptides or shorter engineered constructs including those in this work should be considered carefully. This work provides a framework for understanding how diverse substrates interact with HIV-1 protease and can be extended to investigate other viral proteases with similar properties.

Various protein properties are often illuminated using sequence comparisons of protein homologs. For example, in analyses of the pyruvate kinase multiple sequence alignment, the set of positions that changed during speciation (“phylogenetic” positions) were enriched for “rheostat” positions in human liver pyruvate kinase (hLPYK). (Rheostat positions are those which, when substituted with various amino acids, yield a range of functional outcomes). However, the correlation was moderate, which could result from multiple biophysical constraints acting on the same position during evolution and/or various sources of noise. To further examine this correlation, we here tested Zymomonas mobilis PYK (ZmPYK), which has

The beany flavor adversely influences consumer acceptance of soymilk (SM) products. Thus, in this work, the co-fermentation of isolated new yeasts (Kluyveromyces marxianus SP-1, Candida ethanolica ATW-1, and Pichia amenthionina Y) and Kluyveromyces marxianus K (a commercial yeast) along with an XPL-1 starter (including five strains of lactic acid bacteria (LAB)) was utilized to mend the beany flavor of fermented SM (FSM) beverages. Probiotic count, pH, titratable acidity, syneresis, water holding capacity, rheological characteristics, and sensory attributes were investigated. Furthermore, the free amino acids, nucleotides, and volatile compounds (VCs) were analyzed, also presenting the collected VC data by exploiting a principal component analysis (PCA) and a heatmap with a hierarchical cluster analysis. The co-fermentation with Kluyveromyces marxianus SP-1 and K remarkably enhanced the LAB strain growth and acid production, improving the rheological attributes, whereas that of yeast along with XPL-1 as a mullite starter could reduce the beany odor. PCA chart displayed that higher amounts of alcohols, ketones, acids, and esters that significantly improved the flavor quality of FSM beverages were generated throughout the co-fermentation process. The co-fermentation with Pichia amenthionina Y generated the highest acetoin (36.19%) and diacetyl (2.02%), thus improving the overall acceptance of FSM, as well as the sensory characteristics of FSM beverages with the highest umami, sweet, odorless amino acids, and umami nucleotides, and the lowest content of alcohol and inosine. Taken together, the co-fermentation of Pichia amenthionina Y along with XPL-1 within SM provides novel insights regarding the development of FSM and fermented beverages. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-022-00773-7.

DcsB, an enzyme produced from the d‐cycloserine biosynthetic gene cluster, displays moderate similarity to arginase in the sequence and three‐dimensional structure. Arginase is a ubiquitous enzyme hydrolyzing l‐arginine to generate l‐ornithine and urea, whereas DcsB hydrolyzes N (ω)‐hydroxy‐l‐arginine (l‐NOHA), an arginase inhibitor, to generate l‐ornithine and hydroxyurea. We determined the crystal structure of DcsB associated with l‐ornithine and that with the tetrahedral derivative of 2(S)‐amino‐6‐boronohexanoic acid, whose boron atom forms a covalent bond with an oxygen atom bridging two manganese ions at the active center. The substrate‐binding pocket of DcsB is narrower than that of arginase, suggesting that DcsB is unsuitable for the binding of l‐NOHA in an inhibitory manner. The transition state‐like structure demonstrated that Asp210 and Glu241 have a role to trap a positively charged ion near the dimanganese cluster. Kinetic analysis using the mutated DcsB showed that the enzyme employs different catalytic mechanisms under the neutral and alkaline pH conditions. Glu241 in DcsB is likely involved in the recognition of the hydroxyguanidino group of l‐NOHA, whereas Asp210, in cooperation with Glu241, seems to contribute to the reactivity toward the protonated l‐NOHA, which is a preferable species under the neutral pH conditions. After entering of the protonated l‐NOHA to the substrate‐binding pocket of DcsB, a hydronium ion may be trapped at the positive ion‐binding site. Then, the ion serves as a specific acid catalyst to facilitate the collapse of the tetrahedral intermediate of l‐NOHA.

The computed electrostatic and proton transfer properties are studied for 20 enzymes that represent all six major enzyme commission classes and a variety of different folds. The properties of aspartate, glutamate, and lysine residues that have been previously experimentally determined to be catalytically active are reported. The catalytic aspartate and glutamate residues studied here are strongly coupled to at least one other aspartate or glutamate residue and often to multiple other carboxylate residues with intrinsic pK (a) differences less than 1 pH unit. Sometimes these catalytic acidic residues are also coupled to a histidine residue, such that the intrinsic pK (a) of the acidic residue is higher than that of the histidine. All catalytic lysine residues studied here are strongly coupled to tyrosine or cysteine residues, wherein the intrinsic pK (a) of the anion‐forming residue is higher than that of the lysine. Some catalytic lysines are also coupled to other lysines with intrinsic pK (a) differences within 1 pH unit. Some evidence of the possible types of interactions that facilitate nucleophilicity is discussed. The interactions reported here provide important clues about how side chain functional groups that are weak Brønsted acids or bases for the free amino acid in solution can achieve catalytic potency and become strong acids, bases or nucleophiles in the enzymatic environment.

An increase in fish consumption, combined with a decrease in wild fish harvest, is driving the aquaculture industry at rapid pace. Today, farmed seafood accounts for about half of all global seafood demand for human consumption. As the aquaculture industry continues to grow, so does the market for aquafeed. Currently, some of the feed ingredients are coming from low-value forage fishes (fish meal) and terrestrial plants. The production of fish meal can't be increased as it would affect the sustainability and ecosystem of the ocean. Similarly, increasing the production of terrestrial plant-based feed leads to deforestation and increased freshwater use. Hence, alternative and environmentally sustainable sources of feed ingredients need to be developed. Microalgae biomasses represent potential feed source ingredients as the cell metabolites of these microorganisms contain a blend of essential amino acids, healthy triglycerides as fat, vitamins, and pigments. In addition to serving as bulk ingredient in aquafeed, their unique array of bioactive compounds can increase the survivability of farmed species, improve coloration and quality of fillet. Microalgae has the highest areal biomass productivities among photosynthetic organisms, including fodder crops, and thus has a high commercial potential. Also, microalgal production has a low water and arable-land footprint, making microalgal-based feed environmentally sustainable. This review paper will explore the potential of producing microalgae biomass as an ingredient of aquaculture feed. 2021 The Authors The authors would like to acknowledge the support of the Qatar National Research Fund (QNRF, a member of Qatar Foundation) for providing the funding (under grant MME01-0910-190028 ) for this study. Scopus

Reduction of A. muciniphila relative abundance in the gut microbiota is a widely accepted signature associated with obesity-related metabolic disorders. Using untargeted metabolomics profiling of fasting plasma, our study aimed at identifying metabolic signatures associated with beneficial properties of alive and pasteurized A. muciniphila when administrated to a cohort of insulin-resistant individuals with metabolic syndrome. Our data highlighted either shared or specific alterations in the metabolome according to the form of A. muciniphila administered with respect to a control group. Common responses encompassed modulation of amino acid metabolism, characterized by reduced levels of arginine and alanine, alongside several intermediates of tyrosine, phenylalanine, tryptophan, and glutathione metabolism. The global increase in levels of acylcarnitines together with specific modulation of acetoacetate also suggested induction of ketogenesis through enhanced beta-oxidation. Moreover, our data pinpointed some metabolites of interest considering their emergence as substantial compounds pertaining to health and diseases in the more recent literature.

The interactions of proteins with surfaces are important in both biological processes and biotechnologies. In contrast to decades of study regarding the biophysics of proteins in bulk solution, however, our mechanistic understanding of the biophysics of proteins interacting with surfaces remains largely qualitative. In response, we have set to explore quantitatively the thermodynamics of protein-surface interactions. In this work, we explore systematically the role of electrostatics in modulating the interaction between proteins and charged surfaces. In particular, we use electrochemistry to explore the extent to which a macroscopic, hydroxyl-coated surface held at a slightly negative potential affects the folding thermodynamics of surface-attached protein variants with different composition of charged amino acids. Doing so, we find that attachment to the surface generally leads to a net stabilization, presumably due to excluded volume effects that reduce the entropy of the unfolded state. The magnitude of this stabilization, however, is strongly correlated with the charged-residue content of the protein. In particular, we find statistically significant correlations with both the net charge of the protein, with greater negative charge leading to less stabilization by the surface, and with the number of arginines, with more arginines leading to greater stabilization. Such findings refine our understanding of protein-surface interactions, providing in turn a guiding rationale to achieve the functional deposition of proteins on artificial surfaces for implementation in, for example, protein-based biotechnologies.

HFE-7100 is a routine carrier solvent in amino acid-sensitive fingermark detection reagents such as ninhydrin and 1,2-indanedione/zinc chloride (IND/Zn). However, a potential EU ban on hydrofluoroethers may require reformulation of these treatments worldwide. Solstice® PF has shown promise as a replacement for HFE-7100 in the United Kingdom. However, the performance (and hence optimal formulation) of IND/Zn is impacted by differences in climate and substrate composition, necessitating assessments under local conditions for different regions. We present a series of preliminary investigations in an Australian context, using the IND/Zn formulation used by Australian forensic service providers. The general performance of Solstice® PF-based IND/Zn was comparable to that using HFE-7100 on three substrate types, three ageing periods (1, 7 and 30 days) and 5 donors. However, slight differences in colour and luminescence intensity, as well as increased ink diffusion, suggest chemical interactions with other reagent components that may affect stability. Specifically, Solstice® PF-based reagent formed a precipitate within a month of storage, though this did not affect performance over a 4 month period. HFE-7100-based IND/Zn was found to be marginally more effective than Solstice® PF when applied to incidental fingermarks. These results indicate that Solstice® PF is a satisfactory alternative carrier solvent to HFE-7100 in an Australian context, though users should be aware of possible limitations regarding compatibility with other evidence components (particularly inks) and shelf-life.

We recently identified FAM134B2, which is an N-terminal truncated reticulophagy receptor highly induced by starvation such as fasting of mice and treatment of mammalian cells with a starvation medium that does not contain amino acids, glucose and growth factors. However, which starvation signal mediates the induction of FAM134B2 is still obscure. In this study, we found that amino acid deficiency (AAD) could mimic the starvation condition to induce FAM134B2 expression. Unexpectedly, EIF2AK4/GCN2-mediated integrated signal response (ISR) and MTOR (mechanistic target of rapamycin kinase) signals, which constitute two major signaling pathways that respond to AAD, did not contribute to AAD-induced FAM134B2 induction. mRNA-seq and siRNA screenings identified two ISR-independent transcription factors, MEF2D (myocyte enhancer factor 2D) and NR4A1 (nuclear receptor subfamily 4 group A member 1), involved in AAD-induced FAM134B2 expression. AAD induces MEF2D, resulting in the induction of NR4A1, which in turn induces FAM134B2-mediated reticulophagy to maintain intracellular amino acid levels. In conclusion, the MEF2D-NR4A1-FAM134B2 cascade is a critical signal in amino acid homeostasis. ABBREVIATIONS: AAD: amino acid deficiency; APOC3: apolipoprotein C3; BACH1: BTB domain and CNC homolog 1; CEBP: CCAAT enhancer binding protein; DDIT3/CHOP: DNA damage inducible transcript 3; EBSS: Earle’s Balanced Salt Solution; EIF2AK4/GCN2: eukaryotic translation initiation factor 2 alpha kinase 4; ER: endoplasmic reticulum; HisOH: histidinol; ISR: integrated stress response; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF2D: myocyte enhancer factor 2D; MTOR: mechanistic target of rapamycin kinase; NR4A1: nuclear receptor subfamily 4 group A member 1; RETREG1/FAM134B: reticulophagy regulator 1; RTN2: reticulon 2, TF: transcription factor; TFEB: transcription factor EB; ZBTB10: zinc finger and BTB domain containing 10

Motivation Intrinsically disordered proteins (IDPs) are involved in numerous processes crucial for living organisms. Bias in amino acid composition of these proteins determines their unique biophysical and functional features. Distinct intrinsically disordered regions (IDRs) with compositional bias play different important roles in various biological processes. IDRs enriched in particular amino acids in human proteome have not been described consistently. Results We developed DisEnrich—the database of human proteome IDRs that are significantly enriched in particular amino acids. Each human protein is described using Gene Ontology (GO) function terms, disorder prediction for the full-length sequence using three methods, enriched IDR composition and ranks of human proteins with similar enriched IDRs. Distribution analysis of enriched IDRs among broad functional categories revealed significant overrepresentation of R- and Y-enriched IDRs in metabolic and enzymatic activities and F-enriched IDRs in transport. About 75% of functional categories contain IDPs with IDRs significantly enriched in hydrophobic residues that are important for protein–protein interactions. Availability and implementation The database is available at http://prodata.swmed.edu/DisEnrichDB/. Supplementary information Supplementary data are available at Bioinformatics Advances online.

Aim: The phosphorylation level of the furosemide-sensitive Na +-K +-2Cl − cotransporter (NKCC2) in the thick ascending limb (TAL) is used as a surrogate marker for NKCC2 activation and TAL function. However, in mice, analyses of NKCC2 phosphorylation with antibodies against phosphorylated threonines 96 and 101 (anti-pT96/pT101) give inconsistent results. We aimed (a) to elucidate these inconsistencies and (b) to develop a phosphoform-specific antibody that ensures reliable detection of NKCC2 phosphorylation in mice. Methods: Genetic information, molecular biology, biochemical techniques and mouse phenotyping was used to study NKCC2 and kidney function in two commonly used mouse strains (ie 129Sv and in C57BL/6 mice). Moreover, a new phosphoform-specific mouse NKCC2 antibody was developed and characterized. Results: Amino acids sequence alignment revealed that C57BL/6 mice have a strain-specific five amino acids deletion (ΔF97-T101) in NKCC2 that diminishes the detection of NKCC2 phosphorylation with previously developed pT96/pT101 NKCC2 antibodies. Instead, the antibodies cross-react with the phosphorylated thiazide-sensitive NaCl cotransporter (NCC), which can obscure interpretation of results. Interestingly, the deletion in NKCC2 does not impact on kidney function and/or expression of renal ion transport proteins as indicated by the analysis of the F2 generation of crossbred 129Sv and C57BL/6 mice. A newly developed pT96 NKCC2 antibody detects pNKCC2 in both mouse strains and shows no cross-reactivity with phosphorylated NCC. Conclusion: Our work reveals a hitherto unappreciated, but essential, strain difference in the amino acids sequence of mouse NKCC2 that needs to be considered when analysing NKCC2 phosphorylation in mice. The new pNKCC2 antibody circumvents this technical caveat.

Palladium(0)-catalyzed chemistry represents one of the most important methods for the construction of carbon-carbon bonds, which are ubiquitous in organic compounds. The first part of this thesis describes the palladium(0)-catalyzed diastereoselective Mizoroki-Heck reaction of Vince lactam-derived precursors for the formation of spiroindolines (paper I) and spirooxindoles, which are scaffolds present in several natural products with interesting biological activity. The scope of the spiroindoline synthesis was investigated by varying the substitution on the cyclopentenyl-tethered aniline spirocyclization precursor. The mechanistic reasons behind the high diastereoselectivity of the spirocyclization reaction was also investigated through density functional theory (DFT) calculations. Functionalization of the N-terminal amino acid of therapeutic peptides is a strategy that has been employed for the improvement of metabolic stability. In paper II, a palladium(0)-catalyzed carbonylation method employing ex situ carbon monoxide generation from Mo(CO)6 in a two-chamber system is used for the N-capping of amino acids using various aryl bromides and triflates. The second part of the thesis describes the synthesis and biological evaluation of angiotensin II type 2 receptor (AT2R) ligands. The AT2R is a G-protein coupled receptor belonging to the renin-angiotensin-aldosterone system (RAAS), which is most commonly associated with the hypertensive disorder caused by an exaggerated activation of the angiotensin II type 1 receptor (AT1R). However, activation of AT2R exerts different and sometimes completely opposing effects to AT1R and has been implicated in processes related to neuropathic pain, where the AT2R antagonist EMA401 has been in clinical trials for this indication. In papers III and IV, the AT2R antagonist C38 developed in our laboratory, is used as a model compound for the synthesis of analogs with the aim of expanding our knowledge regarding the structure-activity relationship of the C38 scaffold. Three general strategies were used; functionalization of the C38 phenyl ring, replacement of the benzyl imidazole of C38 with bicyclic amides and extension of the linker between the phenyl ring and imidazole by the inclusion of a carbonyl. Through these approaches, compounds with improved affinity towards AT2R and increased metabolic stability were identified, which might serve as tools for the continued study of the AT2R.

Purpose. To improve the technology of caring for giant miscanthus plantations and establish the features of its productivity formation with the use of different types of fertilizers, starting from the second year of crop vegetation. Methods. The research was carried out in the years 2020–2021 in the Bila Tserkva Experimental and Breeding Station of the Institute of Bioenergy Crops and Sugar Beet of the National Academy of Agrarian Sciences of Ukraine, which is located in the zone of unstable moisture of the Right Bank Forest Steppe of Ukraine. The design of the experiment included application of mineral fertilizers [ammonium nitrate + ammonium sulfate (N24 + S6 kg/ha a.i.), ammonium nitrate (N24 kg/ha a.i.)] in a row zone in spring, foliar application of humates (Vermisol, 8.0l/ha, Humifield BP-18, 0.4l/ha) and complex fertilizer with amino acids (Quantum Aminomax, 0.5l/ha). Mineral fertilizers were applied before the emergence of miscanthus sprouts; foliar application of fertilizers was carried out in the 5-leaf stage and the second time after two weeks. Results. Giant miscanthus plants in second year of vegetation formed from 18 to 37 shoots with an average weight of 76.3 to 111.0g, with the maximum weight of one plant reaching 4091g. In the next year of research, the number of stems per plant and their average weight increased by 1.5–2 times, and the maximum weight of one plant was 7016 g. Biomass yield of giant miscanthus in the third year of vegetation was 31.0–74.6t/ha, the estimated yield of solid biofuel was 19.2–41.6t/ha and energy 315.5–683.2GJ/ha. Conclusions. Application of mineral fertilizers (ammonium nitrate + ammonium sulfate, ammonium nitrate) in spring contributed to significant increase in the indicators of raw biomass yield, solid biofuel output, and energy yield per hectare. Foliar application of Humifield BP-18 has a significant positive effect on the productivity of giant miscanthus. The best indicators of crop productivity were obtained in treatments combining spring fertilization with ammonium nitrate and ammonium sulfate (N24 + S6) followed by two-time foliar application of humates (Vermisol, 8.0 l/ha, Humifield BP-18, 0.4l/ha) and complex fertilizer with amino acids Quantum Aminomax, 0.5l/ha., Мета. Удосконалити технологію догляду за плантаціями міскантусу гігантського та встановити особливості формування його продуктивності за використання різних видів добрив, починаючи з другого року вегетації культури. Методи. Дослідження виконували впродовж 2020–2021рр. в умовах Білоцерківської дослідно-селекційної станції Інституту біоенергетичних культур і цукрових буряків НААН України, що розташована в зоні нестійкого зволоження Правобережного Лісостепу України. Схема досліду передбачала внесення навесні в зону рядка мінеральних добрив [аміачна селітра + сульфат амонію (N24 + S6 кг/га д.р.), аміачна селітра (N24 кг/га д.р.)] та позакореневе підживлення гуматами (Вермісол, 8л/га, Гуміфілд ВР-18, 0,4л/га) і комплексним добривом з амінокислотами (Квантум Аміномакс 0,5л/га). Мінеральні добрива вносили локально в міжряддя до появи сходів міскантусу, позакореневе підживлення рослин гуматами та антистресантом проводили у фазі 5-ти листків культури та повторно через два тижні. Результати. Рослини міскантусу гігантського впродовж другого року вегетації формували від 18 до 37 пагонів із середньою масою від 76,3 до 111,0 г, максимальна маса однієї рослини при цьому досягала 4091г. На наступний рік досліджень кількість стебел на одну рослину та їх середня маса зростали у 1,5–2 рази, а максимальна маса однієї рослини становила 7016г. Урожайність біомаси міскантусу гігантського третього року вегетації становила 31,0–74,6т/га, розрахунковий вихід твердого біопалива – 19,2–41,6т/га, енергії – 315,5–683,2ГДж/га. Висновки. За весняного підживлення мінеральними добривами (аміачна селітра + сульфат амонію, аміачна селітра) істотно зростали показники врожайності сирої біомаси, виходу твердого біопалива та енергії з гектара площі. Суттєвий позитивний вплив на продуктивність міскантусу гігантського мало й позакореневе підживлення препаратом Гуміфілд ВР-18. Найліпші показники продуктивності культури отримано у варіантах поєднання весняного удобрення аміачною селітрою із сульфатом амонію (N24+S6) з наступним дворазовим позакореневим підживленням гуматами (Вермісол, 8л/га, Гуміфілд ВР-18, 0,4л/га) та комплексним добривом з амінокислотами Квантум Аміномакс, 0,5л/га.

This doctoral thesis reports on recent developments in photoelectron spectroscopy from the liquid phase, with a prime focus on the electronic structure of liquid water and aqueous solutions. The enabling technique is liquid-jet photoelectron spectroscopy (LJ-PES), which combines state-of-the-art high-pressure-compatible electron analyzers with an in-vacuo liquid microjet. This technique is often applied in conjunction with soft-X-ray photons delivered from synchrotron-radiation facilities. In this work, I will also present data obtained with a laboratory helium-discharge source, providing XUV photon energies. There were three main objectives at the beginning of my thesis: (1) the application of photoelectron circular dichroism (PECD) to the liquid phase, (2) exploring how electron scattering in aqueous solution can disturb native photoelectron peaks, and (3) the development of experimental methods that allow the determination of accurate energetics of solutions, which largely refers to the ability to determine accurate valence and core-level electron binding energies from both liquid water and solutes. In addition, I have participated in several other studies with associated topics, although not immediately relevant for above-mentioned topics (1) to (3). These studies will be briefly described at the end of this thesis in Chapter 3. My first project (1), the application of PECD to the liquid phase, with a particular emphasis on chiral (biologically relevant) molecules in water, has started at a time where PECD has been well established for isolated (gas-phase) molecules. PECD manifests as a forward-backward asymmetry of the photoelectron emission intensity from chiral molecules, measured with respect to the propagation direction of circularly polarized light (CPL). No analogous studies in liquid-phase environments have been reported to date and were not even possible with existing equipment. Challenged by this lack of tools, and in the light of the importance of chirality in aqueous-phase biological systems and the relevance in the context of chiral pharmaceuticals, I set out to construct and build a unique apparatus that would allow to explore PECD in liquid phase. Its realization would be an expansion of the LJ-PES technique, now extending to the detection of the forward-backward asymmetry using a specialized geometry when ionizing the liquid jet with CPL. The result is the EASI apparatus (acronym for Electronic structure from Aqueous Solutions and Interfaces) and the demonstration of the very first PECD measurements from the liquid phase, exemplified for a liquid fenchone beam and the amino acid alanine in aqueous solution. In both cases, PECD was explored upon carbon core-level (near-threshold) ionization. A technical article describing EASI, its concepts and performance, and another article on liquid-fenchone PECD resulting from these efforts have been published (Paper I and Paper V). Additional, not-yet- published results from aqueous-phase alanine will be presented briefly in the results section (Chapter 3.3). Project (2) is intimately connected with PECD measurements as it investigates the effect of electron scattering in solution at low-to-very-low kinetic energies, which is the energy range where PECD is expected to be largest. As it turns out, this is exactly the region where quasi- elastic electron (small-energy-loss vibrational) scattering occurs, which can severely disturb the native photoelectron peaks of interest. It is noted that the successful combination of tunable XUV light with the detection of low-kinetic-energy LJ-PE spectra had been hampered for experimental reasons. Thus, our study is the first to actually quantify the role and importance of these scattering effects in water and aqueous solutions, enabling the determination of a critical kinetic-energy value below which an undistorted photoelectron spectrum cannot be measured. Details, including mechanistic aspects of electron scattering and implications for future PES studies from liquids are described in Paper II. A prerequisite to characterize low-energy processes is the ability to detect even those electrons that have lost all their energy and thus are left with an almost zero kinetic energy. In the PE spectrum, such electrons accumulate at the low-energy cut-off, a quantity (barely considered previously) which is also of great importance for the novel route to determine accurate energetics from solutions, project (3). The concept underpinning project (3) is the application of PES methodology that has been commonly used for condensed-matter studies but barely considered for liquid water. In fact, almost all reported LJ-PES studies, since the beginning about 20 years ago, have adopted a molecular-physics description of liquid water. That is, aqueous-phase spectra have been essentially described in terms of molecular orbitals just as the respective gas-phase counterparts. Additional energy shifts and peak broadenings with respect to the gas-phase spectra are mainly described as a perturbation of the intrinsic molecular electronic structure, and have been assigned to liquid-water electronic screening as well as to hydrogen-bonding-induced effects on the orbital symmetry, respectively. Similarly, ionization energies have exclusively been referenced to gas-phase PE features, which are inevitably measured together in PE spectra from the liquid phase due to evaporation. A critical consequence of this approach, which implies referencing detected liquid-phase energies to known water gas-phase (ionization) energies, is that the measured energy value depends on particular experimental conditions due to additional unwanted electric fields present in a typical LJ-PES experiment. Briefly, the liquid-jet surface is charged by electrokinetic charging, from molecular surface dipoles, or upon ionization of solutions with insufficient electrical conductivity. Hence, under all practical experimental conditions, there will be an electric field between the liquid jet and the electron detector. Photoelectrons originating from ionization of water gas-phase molecules surrounding the liquid jet will vary in kinetic energy because of acceleration or deceleration under the influence of the present field, depending on the distance of birth from the jet. Disentangling these contributions is very complex and generally is not even attempted in typical LJ-PES experiments; instead, a predetermined ionization energy for prominent liquid features has been used to define the energy scale of a measured spectrum with possibly large associated errors of this approach. The new approach does not at all rely on the gas phase but rather uses the intrinsic liquid- phase low-energy cut-off as a reference. Then, with an accurately known photon energy, one can precisely determine both solute and solvent electron binding energies. We were thus able to determine the accurate energy value for liquid water’s leading orbital (HOMO), which has been controversially discussed in the literature. More importantly, though, the new method enables the measurement of water’s electronic-structure changes induced by solute for the first time, since this energy-referencing method does not require assuming solvent PE features as fixed in energy. This achievement has cumulated in the publication of Paper III and IV, and a summary will be provided in the results section (Chapter 3.2). In conjunction with the new referencing schemes, additional energy referencing with respect to the Fermi energy has been attempted, which promises an accurate determination of the work function of liquid water and aqueous solutions. At the end of this thesis (Chapter 4), I evaluate and discuss the challenge of characterizing the Fermi level of liquid water and aqueous solutions, thus bridging the gap between liquid- and solid-phase PES. Another study I was actively involved in, but less closely related to the main objectives of my thesis is the electronic-structure characterization of glucose as a function of pH. This particular LJ-PES study focused on the identification of electronic-structure changes of the anomeric center when scanning the pH range across the acidity constant, p𝐾a, by investigating the site- and energy-specific signature of the carbon 1s spectrum. I briefly explain the motivation of this study and ongoing related studies in the framework of chirality in aqueous phase in the results section (Chapter 3.4). The results of this study are published in Paper VI. The second side project was the investigation of non-local electronic relaxation processes upon 1s ionization of divalent and trivalent metal cations in water. This followed up on a long and ongoing interest of our group in identifying, for instance, intermolecular / interatomic Coulombic decay (ICD) processes in liquid water and aqueous solutions, and relates observed spectral information to hydrogen bonding (including nuclear dynamics) and general solution structure properties. The individual ICD features show a high selectivity for specific water ionization channels and are considered to reflect the hydration water molecules, which is possible due to the nature of ICD to involve molecules in vicinity of the excited system. A short summary of this published study (Paper VII) is also provided in the results section (Chapter 3.5). Another study that relates less closely to the main objectives of my thesis includes the development of a liquid flatjet, which essentially forms a flat leaf of two laminarly co-flowing aqueous solutions and thus a liquid–liquid interface. The motivation for those experiments is described in the results section (Chapter 3.6). For example, an application of a flatjet for the determination of chemical kinetics (exemplified for the luminol – hydrogen peroxide chemical reaction in water) – not using PES though – is explained, which summarizes results that we published in Paper VIII., Diese Doktorarbeit thematisiert die neuesten Entwicklungen im Bereich der Photoelektronen- Spektroskopie in der flüssigen Phase. Das Hauptaugenmerk liegt dabei auf den elektronischen Strukturen von flüssigem Wasser und wässrigen Lösungen. Das hierfür angewendete Verfahren ist die sogenannte Flüssigkeitsstrahl-Photoelektronen-Spektroskopie (Liquid-Jet Photoelectron Spectroscopy, LJ-PES), welches modernste Elektronenanalysatoren mit einem flüssigen mikrometergroßen Strahl im Vakuum kombiniert. Dieses Verfahren wird häufig in Verbindung mit weicher Röntgenstrahlung (Soft X-rays) angewendet, die von Elektronenspeicherringen (Synchrotrons) erzeugt wird. In dieser Arbeit werden zudem Daten gezeigt und diskutiert, die mit einer Helium- Plasmaquelle und somit mit ultravioletter Röntgenstrahlung (XUV) im Labor gemessen wurden. Am Anfang meiner Doktorarbeit standen drei Hauptziele: (1) Die Anwendung des sogenannten Photoelektronen-Zirkulardichroismus (Photoelectron Circular Dichroism, PECD) in flüssiger Phase, (2) Studien zur Elektronenstreuung in wässriger Lösung und damit verbundenen Störungen von nativen Photoelektronen-Signalen und (3) die Entwicklung experimenteller Methoden, die durch die Bestimmung genauer Valenz- und Innerschalen- Bindungsenergien von flüssigem Wasser sowie von gelösten Stoffen die Bestimmung der genauen (Bindungs-)Energien von Lösungen ermöglichen sollen. Darüber hinaus habe ich an mehreren anderen Studien mit verwandten Themen teilgenommen, die jedoch für die oben genannten Themen (1) bis (3) nicht unmittelbar relevant sind. Diese Studien werden am Ende dieser Arbeit kurz beschrieben. Das erste Projekt (1), die Anwendung von PECD in flüssiger Phase mit besonderem Schwerpunkt auf chiralen (biologisch relevanten) Molekülen im Wasser, begann, als PECD für isolierte (Gasphasen-)Moleküle bereits etabliert war. PECD manifestiert sich als eine Vorwärts-Rückwärts-Asymmetrie der Photoelektronen-Emissionsintensität von chiralen Molekülen, gemessen in Bezug auf die Ausbreitungsrichtung der Photonen des dafür genutzten zirkular polarisierten Lichtes (Circularly Polarized Light, CPL). Bisher wurden keine analogen Studien in wässriger Phase in der Literatur erwähnt. Darüber hinaus waren solche Studien mit den vorhandenen technischen Mitteln nicht durchführbar. Angesichts des Mangels einer entsprechenden experimentellen Methode und im Hinblick auf die Bedeutung der Chiralität für biologische Systeme in wässriger Phase sowie deren Relevanz im Zusammenhang mit chiralen Pharmazeutika konstruierte und baute ich eine einzigartige Apparatur, die es ermöglicht, PECD in flüssiger Phase zu untersuchen. Die Entwicklung dieser Apparatur und deren Funktionsweise im Rahmen meiner Doktorarbeit ist eine bedeutende Erweiterung der LJ-PES-Technik: Ein Flüssigkeitsstrahl wird mit CPL ionisiert und die Vorwärts-Rückwärts-Asymmetrie der Photoelektronen-Intensität wird unter Verwendung einer speziellen Geometrie detektiert. Das Ergebnis ist die EASI-Apparatur (Akronym für Electronic Structure from Aqueous Solutions and Interfaces) und die Demonstration der allerersten PECD-Messungen aus der flüssigen Phase am Beispiel eines flüssigen Fenchon-Jets und der Aminosäure Alanin in wässriger Lösung. In beiden Fällen wurde PECD nach einer Innerschalen-Ionisation von Kohlenstoff (nahe der Ionisationsschwelle) untersucht. Ein technisch-wissenschaftliches Paper, welches die Konzepte und Möglichkeiten von EASI beschreibt, sowie ein weiteres Paper über PECD von flüssigem Fenchon, sind hieraus hervorgegangen und veröffentlicht (Paper I und Paper V). Weitere, noch nicht publizierte Ergebnisse von Alanin in wässriger Phase werden darüber hinaus kurz im Ergebnisteil (Kapitel 3.3) vorgestellt. Projekt (2) ist sehr eng mit den PECD-Messungen verbunden, da es den Effekt der Elektronenstreuung in Lösungen bei sehr geringen kinetischen Energien untersucht und der beobachtbare PECD-Effekt in diesem unteren Energiebereich voraussichtlich am größten sein wird. Wie sich herausstellte, tritt in diesem Bereich quasi-elastische Elektronenstreuung (Vibrationsstreuung mit geringem Energieverlust) auf, die das native Photoelektronen-Signal signifikant stören kann. Erst die erfolgreiche Kombination von ultravioletter Röntgenstrahlung mit der Fähigkeit, emittierte Elektronen aus Flüssigkeitsstrahlen mit sehr niedrigen kinetischen Energien zu detektieren, ermöglichte solche Messungen. Somit ist unsere Studie die erste, die die Rolle und Bedeutung dieser Streueffekte quantifiziert und die die Bestimmung eines kritischen kinetischen Energiewertes ermöglicht. Unterhalb dieses Energiewertes kann ein unverzerrtes Photoelektronen-Spektrum nicht gemessen werden. Einzelheiten, einschließlich mechanistischer Aspekte der Elektronenstreuung und Implikationen für zukünftige Photoelektronen-Studien aus Flüssigkeiten, werden in Paper II ausführlich beschrieben. Eine Voraussetzung für die Charakterisierung solch niederenergetischer Prozesse ist die Fähigkeit auch solche Elektronen zu detektieren, die ihre gesamte Energie verloren haben und daher mit einer kinetischen Energie nahe Null gemessen werden. In einem Photoelektronen-Spektrum finden sich solche Elektronen an der sogenannten niederenergetischen Untergrenze (Low-Energy Cut-off). Diese Größe, die bisher in Flüssigkeiten kaum Beachtung fand, ist essenziell für die neuartige Methode zur Bestimmung genauer Energien aus Lösungen, was wiederum für Projekt (3) von großer Bedeutung ist. Das zugrunde liegende Konzept von Projekt (3) ist die Anwendung einer PES-Methodik, die üblicherweise für Studien an kondensierter Materie verwendet wird, jedoch für flüssiges Wasser kaum in Betracht gezogen wurde. Tatsächlich haben fast alle publizierten LJ-PES-Studien seit Beginn dieser Experimente vor etwa 20 Jahren eine molekularphysikalische Beschreibung von flüssigem Wasser verwendet. Dabei wurden Spektren in wässriger Phase im Wesentlichen in Form von Molekülorbitalen beschrieben, die aus der Gasphase bekannt waren. Auftretende Energieverschiebungen und Signalverbreiterungen in der Flüssigkeitsphase im Vergleich zur Gasphase wurden hauptsächlich als Störung der intrinsischen molekularen elektronischen Struktur beschrieben. Sie wurden zudem der Elektronen-Abschirmung in flüssigem Wasser sowie den durch Wasserstoffbrückenbindungen induzierten Effekten auf die Orbitalsymmetrie zugeordnet. In ähnlicher Weise wurden Ionisierungsenergien der flüssigen Phase ausschließlich auf Photoelektronen-Merkmale der Gasphase bezogen, die aufgrund der Verdampfung des Flüssigkeitsstrahls im Vakuum zwangsläufig zusammen mit den Photoelektronen-Merkmalen aus der flüssigen Phase gemessen werden. Jedoch ist der gemessene Energiewert der Gasphase, der als Referenz für die Energiewerte der Flüssigphase genutzt wurde, aufgrund von unerwünschten elektrischen Feldern, die typischerweise bei einem LJ-PES Experiment auftreten, stark abhängig von den experimentellen Bedingungen. Zusammenfassend gilt: Die Oberfläche des Flüssigkeitsstrahls wird durch elektrokinetische Aufladung von molekularen Oberflächendipolen oder bei der Ionisierung von Lösungen mit unzureichender elektrischer Leitfähigkeit aufgeladen. Daher besteht unter allen praktischen experimentellen Bedingungen ein elektrisches Feld zwischen dem Flüssigkeitsstrahl und dem Elektronen-Detektor. Die kinetische Energie der Photoelektronen, die aus der Ionisation der Wassermoleküle aus der den Flüssigkeitsstrahl umgebenden Gasphase stammen, variiert aufgrund von Abbremsung und Beschleunigung unter dem Einfluss des vorhandenen Feldes in Abhängigkeit der Entfernung zum Flüssigkeitsstrahl. Das Quantifizieren dieser Beiträge ist sehr komplex und wird im Allgemeinen in typischen LJ-PES-Experimenten nicht durchgeführt. Stattdessen wurde eine vorbestimmte Ionisationsenergie für die Photoelektronen-Signale von flüssigem Wasser verwendet, um die Energieskala eines gemessenen Spektrums mit möglichst kleinen experimentellen Fehlern zu definieren. Der neue Ansatz bezieht sich nicht auf die Gasphase, sondern verwendet die intrinsische niederenergetische Untergrenze der Flüssigphase als Referenz. Somit kann man mit einer genau bekannten Photonenenergie sowohl die Elektronenbindungsenergien des gelösten Stoffes als auch des Lösungsmittels genau bestimmen. Damit konnte der in der Literatur kontrovers diskutierte Energiewert für das höchste besetzte Molekülorbital (Highest Occupied Molecular Orbital, HOMO) von flüssigem Wasser bestimmt werden. Noch wichtiger ist jedoch, dass die neue Methode zum ersten Mal die Messung von Änderungen der elektronischen Struktur von Wasser, die durch gelöste Stoffe induziert werden, ermöglicht. Hintergrund dafür ist, dass diese Methode nicht die Annahme voraussetzt, dass die PE-Merkmale der gemessen Lösungsmittel-Photoelektronen-Spektren energiefixiert sein müssen. Die Ergebnisse dieser Methodik sind in Paper III und IV veröffentlicht und eine Zusammenfassung wird im Ergebniskapitel (Kapitel 3.2) dargestellt. In Verbindung mit der neuen Methode zur Referenzierung genauer Energien in wässrigen Lösungen wurde eine zusätzliche Energie-Referenzierung bezüglich der Fermi-Energie untersucht, die eine genaue Bestimmung der Austrittsarbeit von flüssigem Wasser und wässrigen Lösungen ermöglicht. Am Ende von Kapitel 3.2 und in Kapitel 4 bewerte und diskutiere ich die Hürden, die überwunden werden mussten, um das Fermi-Niveau in Bezug auf flüssigen Wasser zu charakterisieren und somit die Lücke zwischen Flüssig- und Festphasen-PES zu schließen. Eine weitere Studie, an der ich aktiv beteiligt war, die aber weniger eng mit den Hauptzielen dieser Doktorarbeit zusammenhängt, ist die Charakterisierung der elektronischen Struktur von Glucose als Funktion des pH-Werts. Diese spezielle LJ-PES-Studie konzentrierte sich auf die Identifizierung von Änderungen der elektronischen Struktur des anomeren Zentrums bei unterschiedlichen pH-Werten, nahe des p𝐾a-Wertes, indem die orts- und energiespezifische Signatur des Kohlenstoff-Innerschalen-Spektrums untersucht wurde. Die Ergebnisse dieser Studie sind in Paper VI publiziert. Darüber hinaus erläutere ich kurz die Motivation zu dieser Arbeit und zu weiteren, derzeit laufenden Arbeiten im Hinblick auf die Chiralität in wässriger Phase im Ergebniskapitel (Kapitel 3.4). Die Untersuchung nicht-lokaler elektronischer Relaxationsprozesse bei 1s-Ionisation von zwei- und dreiwertigen Metall-Kationen in Wasser, war eine weitere Studie, bei der ich eng involviert war. Dieses Projekt resultierte aus einem langen und anhaltenden Interesse unserer Forschungsgruppe, intermolekulare / interatomare Coulomb-Zerfallsprozesse (Interatomic / Intermolecular Coulombic Decay, ICD) in flüssigem Wasser und wässrigen Lösungen zu identifizieren und somit Rückschlüsse auf die Struktur der Wasserstoffbrückenbindungen (einschließlich Kerndynamik) und allgemein der molekularen Struktur von Wasser und wässrigen Lösungen aus den spektralen Informationen zu ziehen. Die einzelnen ICD-Merkmale zeigen eine sehr hohe Selektivität für spezifische Wasserionisationskanäle und man geht davon aus, dass diese Kanäle die Wassermoleküle der Hydratationshülle widerspiegeln, da es mittels ICD möglich ist, Moleküle in direkter Nähe des angeregten Systems mit einzubeziehen. Eine kurze Zusammenfassung dieser publizierten Studie (Paper VII) findet sich auch im Ergebnisteil dieser Arbeit (Kapitel 3.5). Eine weitere Studie, die weniger eng mit den Hauptzielen meiner Dissertation zusammenhängt, ist die Entwicklung eines flüssigen Flachstrahls, der im Wesentlichen ein flaches Blatt aus zwei laminar zusammenströmenden wässrigen Lösungen ist und so eine Grenzfläche von zwei (verschiedenen) Flüssigkeiten bildet. Die Motivation für Studien an diesem planaren Flüssigkeitssystem wird in Kapitel 3.6 beschrieben. Unter anderem wird eine Anwendung des Flachstrahls zur Bestimmung der chemischen Reaktionskinetik am Beispiel der chemischen Reaktion von Luminol mit wässriger Wasserstoffperoxid-Lösung gezeigt; allerdings nicht unter Verwendung von Photoelektronen-Spektroskopie. Die Ergebnisse dieser Studie wurden in Paper VIII veröffentlicht.

The biggest group of transporters, the solute carriers (SLCs), has more than 400 members, and about 30% of these are still orphan. In order to decipher their biological function and possible role in disease, there is a need for characterization of these. Around 25% of SLCs are estimated to have amino acids as substrates, including transporters belonging to the SLC38 family. The SLC38 members are sometimes referred to their alternative name: sodium-coupled neutral amino acid transporters (SNATs). One of these transporters, SNAT10 (or SLC38A10), has been characterized as a bidirectional transporter of glutamate, glutamine, alanine and aspartate, as well as having an efflux of serine, and is ubiquitously expressed in the body. However, its biological importance is not yet understood. The aim with this thesis was to characterize a mouse model deficient in SNAT10 protein in order to find the biological importance of this transporter. In paper I, this is done by using a series of behavioral tests, including the open field test, elevated plus maze, rotarod and Y-maze, among others. The SNAT10 knockout mouse was found to have an increased risk-taking behavior, but no motor or spatial working memory impairments. Furthermore, the knockout mouse was found to have a decreased body weight. In paper II, an additional behavioral characterization was performed by using the multivariate concentric square field™ (MCSF) test. The MCSF test is an arena with different zones associated to different behavioral traits, which generates a behavioral profile depending on where the mouse spends its time. The result from this test implies that the SNAT10 deficient mouse has a lower explorative behavior than its wild type littermates. In paper III, gene expression was studied in whole brain and some genes related to cell cycle regulation and p53 expression were found to be differentially expressed in the knockout brain. Additional gene expression was studied in kidney, liver, lung and muscle, but no changes were found. Plasma levels of histidine and threonine were altered in males, but no altered amino acid levels were found in knockout females, suggesting a possible sex-specific effect. These studies together imply that SNAT10 might be involved in processes related to risk-taking and explorative behavior in the open field and MCSF tests. SNAT10 deficiency also affected amino acid levels in plasma, indicating a disrupted amino acid homeostasis.

Predicting the polyproline type II (PPII) helix structure is crucial important in many research areas, such as the protein folding mechanisms, the drug targets, and the protein functions. However, many existing PPII helix prediction algorithms encode the protein sequence information in a single way, which causes the insufficient learning of protein sequence feature information. To improve the protein sequence encoding performance, this paper proposes a BERT-based PPII helix structure prediction algorithm (BERT-PPII), which learns the protein sequence information based on the BERT model. The BERT model’s CLS vector can fairly fuse sample’s each amino acid residue information. Thus, we utilize the CLS vector as the global feature to represent the sample’s global contextual information. As the interactions among the protein chains’ local amino acid residues have an important influence on the formation of PPII helix, we utilize the CNN to extract local amino acid residues’ features which can further enhance the information expression of protein sequence samples. In this paper, we fuse the CLS vectors with CNN local features to improve the performance of predicting PPII structure. Compared to the state-of-the-art PPIIPRED method, the experimental results on the unbalanced dataset show that the proposed method improves the accuracy value by 1% on the strict dataset and 2% on the less strict dataset. Correspondingly, the results on the balanced dataset show that the AUCs of the proposed method are 0.826 on the strict dataset and 0.785 on less strict datasets, respectively. For the independent test set, the proposed method has the AUC value of 0.827 on the strict dataset and 0.783 on the less strict dataset. The above experimental results have proved that the proposed BERT-PPII method can achieve a superior performance of predicting the PPII helix.

The present thesis describes the applicability of two different iridium(III) complexes for C-O and C-N bond forming reactions. The different projects described in this work are united by the use of an iridium catalyst bearing a functionalized N-heterocyclic carbene ligand. These catalysts operate through a hydrogen transfer mechanism to afford the desired products. This approach gives access to a variety of valuable products in a sustainable and atomeconomical manner, generating water as the only byproduct. In Chapter 1, a general introduction to catalysis, followed by a more detailed description of organometallic complexes and their use in hydrogen transfer reactions is described. An overview of different mechanistic studies that have been employed within this thesis is also provided. Chapter 2 describes the use of an alkoxy-functionalized iridium-NHC complex, previously reported by our group, on cyclodehydration reactions (Paper I). A range of cyclic ethers are obtained from the corresponding diol compounds. Mechanistic studies confirmed an unexplored hydrogen transfer pathway to be operating for the majority of the substrates. In Chapter 3, a new amino-functionalized iridium-NHC catalyst is designed and synthesized, aiming at improving the reactivity compared to previous reports. Then, its outstanding efficiency on carbon-nitrogen bond forming reactions is depicted in different protocols (Papers II, III and IV). Paper II achieves the base-free and selective mono-N-alkylation of amines with alcohols mostly at room temperature. Mechanistic studies are performed to gain a better understanding of the catalytic cycle by combining experimental and in silico studies. In Paper III, the use of this catalyst to access N-modified amino acids is described. Excellent group tolerance allowed for the synthesis of more than 100 compounds. Moreover, quantitative yields are obtained in all cases without the need of tedious derivatization/purification steps. In addition, convenient scalability, recyclability and deuterium labelling studies are also presented. Further applicability of these compounds as high-value building blocks for the synthesis of N-modified peptides is demonstrated. In Paper IV, the catalyst is used for the direct mono-N-alkylation of a range of aminosugars with alcohols. The method is developed to be applied on unprotected carbohydrates, that enables to access these important organic molecules in a direct, and thus sustainable, manner, avoiding unnecessary protection and deprotection steps.

There is a general consensus that a dietary protein intake of 0.8 g protein/kg/day will prevent symptoms of protein deficiency in young, healthy individuals. However, individuals in many physiological circumstances may benefit from higher rates of dietary protein intake. Stable isotope tracer methodology enables a variety of approaches to assessing the optimal dietary protein intake in humans. In this paper, we present an overview of a variety of tracer methods, with a discussion of necessary assumptions, as well as the clinical circumstances in which different methods may be preferable. Although we discuss the nontracer method of nitrogen balance, which has historically been used to estimate dietary protein requirements, this paper primarily focuses on tracer methods for estimating dietary protein and essential amino acid requirements under different physiological conditions. We will explain the following approaches: isotopic measurement of urea production; the arterial-venous tracer balance method; measurement of the fractional synthetic and breakdown rates of muscle protein; the indicator and the direct amino acid oxidation methods; and different approaches to measuring whole-body protein synthesis and breakdown. The advantages and limitations of each method are discussed in the context of the optimal approaches for use under different circumstances.

Malignant tumors are non-communicable diseases that affect human life health and quality of life. Anti-tumour-related research has also been the focus and difficulty in oncology research. With the rise of metabolomics, tumour biology, and the theory of tumour reprogramming, amino acid metabolic reprogramming has become a new target for antitumor research. Amino acids provide biomolecules such as nucleotides for tumour cell proliferation, invasion, and immune escape processes. They are also essential metabolites for immune cell activation and antitumor effects in the tumour microenvironment. Abnormal changes in amino acid metabolism are closely related to tumour development and immunity. Some essential proteins or critical enzymes in their metabolic pathways can be used for tumour diagnosis and prognosis assessment markers. Therefore, this paper reviews the effects of amino acid metabolism on tumour cell proliferation and the abnormal alterations of amino acid metabolism during the tumour metabolic cycle and analyzes and prospects the tumour therapeutic drugs targeting amino acid metabolism. This paper provides theoretical references for the in-depth study of the regulation of amino acid metabolism on tumour development and its possible therapeutic targets.

Small 30-nm vesicles containing the integral membrane protein Atg9 provide the initial membrane source for autophagy in yeast. Atg23 is an Atg9 binding protein that is required for Atg9 vesicle trafficking but whose exact function is unknown. In our recent paper, we explored the function of Atg23 using an approach combining cellular biology and biochemistry on purified protein. We determined that Atg23 is an elongated dimer spanning 320 Å in length. We also demonstrated that Atg23 is a membrane-binding and -tethering protein. Furthermore, we identified a series of amino acids residing in a putative coiled-coil region that when mutated prevent Atg23 dimer formation resulting in a stable Atg23 monomer. Last, we demonstrated that when monomeric Atg23 is expressed in yeast lacking Atg23, this leads to a loss of Atg23 puncta, a reduction in Atg9 puncta, a reduction in nonselective autophagy and a complete block in the cytoplasm-to-vacuole targeting (Cvt) pathway.

Cancer cells exhibit higher proliferation rates than normal cells, and as a consequence, a higher nutritional demand for metabolites such as amino acids. Such cells demonstrate high expression of amino acid transporters and are significantly dependent on the external uptake of amino acids. Moreover, some types of cancer cells exhibit oncogenic mutations that render them auxotrophic to certain amino acids. This metabolic difference between tumor and normal cells has been explored for developing anticancer drugs. Enzymes capable of depleting certain amino acids in the bloodstream can be employed to inhibit the proliferation of cancer cells and promote cell death. Certain microbial enzymes, such as l -asparaginase and l -amino acid oxidases, have been studied for this purpose. In this paper, we discuss the role of l -asparaginase, the only enzyme currently used as a chemotherapeutic agent. We also review the studies on a new potential antineoplastic agent, l -lysine α-oxidase, an enzyme of l -amino acid oxidase family.

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare congenital autosomal recessive metabolic disorder caused by pathogenic homozygous or compound heterozygous variants in the dopa decarboxylase (DDC) gene. Adeno-associated viral vector-mediated gene transfer of the human AADC gene into the putamina has become available. This systematic review on PubMed, Scopus databases, and other sources is aimed at describing the AADC whole phenotypic spectrum in order to facilitate its early diagnosis. Literature reviews, original articles, retrospective and comparative studies, large case series, case reports, and short communications were considered. A database was set up using Microsoft Excel to collect clinical, molecular, biochemical, and therapeutic data. By analysing 261 patients from 41 papers with molecular and/or biochemical diagnosis of AADC deficiency for which individuality could be determined with certainty, we found symptom onset to occur in the first 6 months of life in 93% of cases. Hypotonia and developmental delay are cardinal signs, reported as present in 73.9% and 72% of cases, respectively. Oculogyric crises were seen in 67% of patients while hypokinesia in 42% and ptosis in 26%. Dysautonomic features have been revealed in 53% and gastrointestinal symptoms in 19% of cases. With 37% and 30% of patients reported being affected by sleep and behavioural disorders, it seems to be commoner than previously acknowledged. Although reporting bias cannot be excluded, there is still a need for comprehensive clinical descriptions of symptoms at onset and during follow-up. In fact, our review suggests that most of the neurological and extraneurological symptoms and signs reported, although quite frequent in this condition, are not pathognomonic, and therefore, ADCC deficiency can remain an underdiscovered disorder.

Abstract Monotopic membrane-bound flavoproteins, sulfide:quinone oxidoreductases (SQRs), have a variety of physiological functions, including sulfide detoxification. SQR enzymes are classified into six groups. SQRs use the flavin adenine dinucleotide (FAD) cofactor to transfer electrons from sulfide to quinone. A type VI SQR of the photosynthetic purple sulfur bacterium, Thiocapsa roseopersicina (TrSqrF), has been previously characterized, and the mechanism of sulfide oxidation has been proposed. This paper reports the characterization of quinone binding site (QBS) of TrSqrF composed of conserved aromatic and apolar amino acids. Val331, Ile333, and Phe366 were identified near the benzoquinone ring of enzyme-bound decylubiquinone (dUQ) using the TrSqrF homology model. In silico analysis revealed that Val331 and Ile333 alternately connected with the quinone head group via hydrogen bonds, and Phe366 and Trp369 bound the quinones via hydrophobic interactions. TrSqrF variants containing alanine (V331A, I333A, F366A) and aromatic amino acid (V331F, I333F, F366Y), as well as a C-terminal α-helix deletion (CTD) mutant were generated. These amino acids are critical for quinone binding and, thus, catalysis. Spectroscopic analyses proved that all mutants contained FAD. I333F replacement resulted in the lack of the charge transfer complex. In summary, the interactions described above maintain the quinone molecule’s head in an optimal position for direct electron transfer from FAD. Surprisingly, the CTD mutant retained a relatively high level of specific activity while remaining membrane-anchored. This is a unique study because it focuses on the QBS and the oxidative stage of a type VI sulfide-dependent quinone reduction. Key points • V331, I333, F366, and W369 were shown to interact with decylubiquinone in T. roseopersicina SqrF • These amino acids are involved in proper positioning of quinones next to FAD • I333 is essential in formation of a charge transfer complex from FAD to quinone Graphical abstract

Peptides, short stretches of amino acids or small proteins that occupy a strategic position between proteins and amino acids, are readily accessible by chemical and biological methods. With ideal properties for forming high-affinity and specific interactions with host target proteins, they have established an important niche in the drug development spectrum complementing small molecule and biological therapeutics. Among the most successful biomedicines in use today, peptide-based drugs show great promise. This, coupled with recent advances in synthetic and nanochemical biology, has led to the creation of tailor-made peptide therapeutics for improved biocompatibility. This review presents an overview of the latest research on pathogen-derived, host-cell-interacting peptides. It also highlights strategies for using peptide-based therapeutics that address cellular transport challenges through the introduction of nanoparticles that serve as platforms to facilitate the delivery of peptide biologics and therapeutics for treating various inflammatory diseases. Finally, this paper describes future perspectives, specific pathogen-based peptides that can enhance specificity, efficiency, and capacity in functional peptide-based therapeutics, which are in the spotlight as new treatment alternatives for various diseases, and also presents verified sequences and targets that can increase chemical and pharmacological value.

This paper describes a mild strategy to promote amide arylations. Photoinduced oxidation of a Ni(II) aryl amido intermediate is proposed to facilitate the challenging C-N reductive elimination step at moderate temperatures. Notably, the mildly basic conditions employed facilitate access to a broad scope including protected amino acids, heterocycles, phenols, and sterically hindered substituents. Hence, this work presents an attractive strategy to enable late-stage functionalization of pre-existing amide moieties in commercial drugs and natural products.

The enzyme nicotine oxidoreductase (NicA2) is a member of the flavoprotein amine oxidase family that uses a cytochrome c protein (CycN) as its oxidant instead of dioxygen, which is the oxidant used by most other members of this enzyme family. We recently identified a potential binding site for CycN on the surface of NicA2 through rigid body docking [J. Biol. Chem. (2022) 298(8), 102251]. However, this potential binding interface has not been experimentally validated. In this paper, we used unnatural amino acid incorporation to probe the binding interface between NicA2 and CycN. Our results are consistent with a structural model of the NicA2-CycN complex predicted by protein-protein docking and AlphaFold, suggesting that this is the binding site for CycN on NicA2’s surface. Based on additional mutagenesis of potentially redox active residues in NicA2, we propose that electron transfer from NicA2’s flavin to CycN’s heme occurs without the assistance of a protein-derived wire.

Eriocheir sinensis is a unique freshwater crab found in China, which is well known for its rich nutrition and sweet and delicious taste. Free amino acids in Eriocheir sinensis are not only important nutrients but also are closely related to their unique taste and aroma. Therefore, the determination of the free amino acid contents in Eriocheir sinensis is of great significance for product quality evaluation, flavor research, authenticity, and origin identification. Herein we proposed an ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS)-based method for the determination of 17 free amino acids in Eriocheir sinensis. First, 5 g of the Eriocheir sinensis sample was weighed into a 50-mL polypropylene centrifuge tube. Then, 10 mL of extraction solvents was added to the centrifuge tube, and the resultant solution was mixed well using a vortex mixer. We compared a variety of solvents and finally selected 5%(v/v) perchloric acid aqueous solution as the optimum extraction solvent. The supernatant was transferred to another polypropylene centrifuge tube after centrifuging at 8000 r/min for 5 min. The extraction procedure was repeated according to the above-mentioned steps, and the extraction solution was combined with the supernatant. The extracts were then adjusted to pH 6.5 with 1 mol/L potassium hydroxide solution, and were diluted to 50 mL with water. After filtering by both qualitative filter paper and a 0.45-μm polyether sulfone syringe filter, the extracts were determined by UHPLC-HRMS. We compared three types of mobile phases and chose 0.1%(v/v) formic acid aqueous solution mixed with acetonitrile as the optimum one. Precise parent ion and ion source parameters were also optimized. The 17 analytes, viz. aspartic acid, threonine, serine, glutamic acid, proline, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, arginine, glycine, alanine, and histidine, were separated on an XDB-C

The YggS/PLPBP protein (also called COG0325 or PLPHP) is a conserved pyridoxal 5ʹ-phosphate (PLP)-binding protein present in all 3 domains of life. Recent studies have demonstrated that disruption or mutation of this protein has multifaceted effects in various organisms, including vitamin B6-dependent epilepsy in humans. In Escherichia coli, disruption of this protein—encoded by yggS—perturbs Thr-Ile/Val metabolism, one-carbon metabolism, coenzyme A synthesis, and vitamin B6 homeostasis. This protein is critical for maintaining low levels of pyridoxine 5ʹ-phosphate (PNP) in various organisms. In the yggS-deficient E. coli strain, inhibition of PLP-dependent enzymes, such as the glycine cleavage system by PNP, is the root cause of metabolic perturbation. Our data suggest that the YggS/PLPBP protein may be involved in the balancing of B6 vitamers by mediating efficient turnover of protein-bound B6 vitamers. This paper reviews recent findings on the function of the YggS/PLPBP protein.

Podrijetlo i odnosi među vrstama proučavaju se stoljećima. Kako je vrijeme prolazilo, pojavile su se mnoge nove tehnologije koje su povukle nova područja studija. Jedno od tih područja je bioinformatika, disciplina koja se bavi prikupljanjem i analizom bioloških podataka. Potpodručje bioinformatike koje se naziva filogenija usredotočuje se na odnose među organizmima. Koristi različite metode za izračunavanje razlika u genetskom materijalu vrsta, a zatim ih vizualizira u obliku filogenetskih stabala. U ovom radu implementirala sam dvije metode koje koriste udaljenosti sekvenci aminokiselina različitih podtipova HIV virusa za konstruiranje filogenetskog stabla: UPGMA i Metodu povezivanja susjeda. Udaljenost dviju sekvenci izračunata je primjenom Jukes-Cantorovog korekcijskog modela na broj vidljivih razlika. Stabla se vizualiziraju jednostavnom manipulacijom i ispisom znakova koji predstavljaju čvorove i grane. Na kraju uspoređujem svoju vizualizaciju s onom iz Biopython modula. The origin and relations between species have been studied for centuries. With the passage of time, many new technologies have emerged dragging along new fields of studies. One of those fields is bioinformatics, a discipline concerned with collecting and analyzing biological data. A subfield of bioinformatics called phylogeny focuses on relations between organisms. It uses different methods for calculating differences in the genetic material of species and then visualizes them in the form of phylogenetic trees. In this paper I have implemented two methods that use the distances of amino acid sequences of different HIV virus subtypes to construct a phylogenetic tree: UPGMA and Neighbor-Joining method. The distance of two sequences is calculated by applying Jukes-Cantor's correction model to the number of visible differences. Trees are visualized by simple manipulation and printing of characters that represent nodes and branches. In the end I compare my visualization to that of Biopython module.

Protein subcellular localization prediction is an important research area in bioinformatics, which plays an essential role in understanding protein function and mechanism. Many machine learning and deep learning algorithms have been employed for this task, but most of them do not use structural information of proteins. With the advances in protein structure research in recent years, protein contact map prediction has been dramatically enhanced. In this paper, we present GraphLoc, a deep learning model that predicts the localization of proteins at the subcellular level. The cores of the model are a graph convolutional neural network module and a multi-head attention module. The protein topology graph is constructed based on a contact map predicted from protein sequences, which is used as the input of the GCN module to take full advantage of the structural information of proteins. Multi-head attention module learns the weighted contribution of different amino acids to subcellular localization in different feature representation subspaces. Experiments on the benchmark dataset show that the performance of our model is better than others. The code can be accessed at https://github.com/GoodGuy398/GraphLoc . The proposed GraphLoc model consists of three parts. The first part is a graph convolutional network (GCN) module, which utilizes the predicted contact maps to construct protein graph, taking benefit of protein information accordingly. The second part is the multi-head attention module, which learns the weighted contribution of different amino acids in different feature representation subspace, and weighted average the feature map across all amino acid nodes. The last part is a fully connected layer that maps the flatten graph representation vector to another vector with a category number dimension, followed by a softmax layer to predict the protein subcellular localization.

Recently, we reviewed the important role of carbohydrates and lipids metabolism in different clinical aspects of multiple sclerosis (MS) disease. In the current paper, we aimed to review the contribution of amino acids and their major derivatives to different clinical outcomes of the disease, including etiology, pathogenesis, diagnosis, prognosis, and treatment. In this line, Thr (threonine), Phe (phenylalanine), Glu (glutamate), Trp (tryptophan), and Sero (serotonin) are the main examples of biomolecules that have been suggested for MS therapy. It has been concluded that different amino acids and their derivatives might be considered prominent tools for the clinical management of MS disease.

The purity of ionic liquids (IL) is important for its performance, so the methods able to quantify low concentration impurities are necessaries. Therefore, this paper aims to determine the purity and the anionic exchange efficiency for the preparation of amino acids ionic liquids (AAIL). For this, a Multiple-Injection Capillary Zone Electrophoresis (MICZE) method with direct UV detection was developed and optimized for iodide ( I − ) determination as impurity of AAIL synthesis, comparing this method with traditional injection modes. Furthermore, this paper aims to demonstrate the use of factorial design methods for the optimization of MICZE method. The method optimization allowed five consecutives I − injections of sample in a single run, with analysis time of less than 3 min, showing a larger analytical frequency, counting with 31 injections per hour. It was possible to determine the high purity of the prepared and analyzed AAIL, ranging from 89% to 95% and its overall ionic exchange yield varying from 55% to 87%.

Chronic kidney disease (CKD) has emerged as one of the most progressive diseases with increased mortality and morbidity. Metabolomics offers new insights into CKD pathogenesis and the discovery of new biomarkers for the early diagnosis of CKD. The aim of this cross-sectional study was to assess metabolomic profiling of serum and urine samples obtained from CKD patients. Untargeted metabolomics followed by multivariate and univariate analysis of blood and urine samples from 88 patients with CKD, staged by estimated glomerular filtration rate (eGFR), and 20 healthy control subjects was performed using ultra-high-performance liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry. Serum levels of Oleoyl glycine, alpha-lipoic acid, Propylthiouracil, and L-cysteine correlated directly with eGFR. Negative correlations were observed between serum 5-Hydroxyindoleacetic acid, Phenylalanine, Pyridoxamine, Cysteinyl glycine, Propenoylcarnitine, Uridine, and All-trans retinoic acid levels and eGFR. In urine samples, the majority of molecules were increased in patients with advanced CKD as compared with early CKD patients and controls. Amino acids, antioxidants, uremic toxins, acylcarnitines, and tryptophane metabolites were found in all CKD stages. Their dual variations in serum and urine may explain their impact on both glomerular and tubular structures, even in the early stages of CKD. Patients with CKD display a specific metabolomic profile. Since this paper represents a pilot study, future research is needed to confirm our findings that metabolites can serve as indicators of early CKD.

In this paper, we presented the design by computational tools of novel alkyl (2-alcoxy-2-hydroxypropanoyl)-L-tryptophanate derivatives, which can be potential inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). The molecular structure optimization of a group of 36 compounds was performed employing DFT-B3LYP calculations at the level 6-311G(d,p). Then, molecular docking calculations were performed using Autodock tools software, employing the Lamarckian genetic algorithm (LGA). Four parameters (binding, intermolecular and Van Der Waals hydrogen bonding desolvation energies, and HOMO-LUMO gap) were used to evaluate the potential as 11β-HSD1 inhibitors, which nominate L-tryptophan derivatives as the most promissory molecules. Finally, these molecules were obtained starting from the amino acid and pyruvic acid in a convergent methodology with moderate to low yields.