Newsletter 01/2026 edited by Loredano Pollegioni

D-AAs AND PATHOLOGIES:

Sources and Metabolism of D-Amino Acids and Their Roles as Biomarkers in Kidney Disease: A Review

Zha Z, Wan Y, Wu X, Lei E, Lu T, Zhang X, Liu J

Med Sci Monit. 2026 Jan 16;32:e950486. doi: 10.12659/MSM.950486.

D-amino acids in the human body originate mainly from intestinal microbiota, with additional contributions from diet and limited endogenous synthesis. The kidney plays a central role in maintaining systemic D-amino acid balance through stereoselective reabsorption and excretion, making D-amino acid profiles informative indicators of renal function. Clinical studies link specific alterations, such as increased urinary excretion of D-serine, to kidney diseases including diabetic nephropathy, IgA nephropathy, and chronic kidney disease. This review summarizes the sources, metabolism, and biomarker potential of D-amino acids in renal physiology and pathology.

Microbiota-derived D-amino acids in intestinal homeostasis and inflammatory bowel disease

Miyamoto K, Sujino T

Inflamm Regen. 2026 Jan 6;46:3. doi: 10.1186/s41232-025-00403-3.

Microbiota-derived D-amino acids (D-AAs) are emerging as important postbiotic metabolites that regulate mucosal barrier integrity, immune responses, and microbial ecology in the gut. This review summarizes current knowledge on the biosynthesis and functions of D-AAs, with emphasis on their roles in intestinal immunity and inflammatory bowel disease (IBD). Disruption of D-AA metabolism or host sensing, such as through altered D-amino acid oxidase activity, is associated with dysbiosis and exacerbated intestinal inflammation. The authors highlight the potential of D-AAs as non-invasive biomarkers and therapeutic targets for IBD.

Structure-based virtual screening and post-docking analysis of a D-amino acid oxidase inhibitor targeting the molecular surface

Kato Y, Fukui K

J Biomol Struct Dyn. 2025 Dec 25:1-18. doi: 10.1080/07391102.2025.2606809.

A multi-step structure-based virtual screening of a 160,000-compound library identified novel inhibitors targeting a surface region of D-amino acid oxidase distinct from the active site. Using consensus docking followed by molecular dynamics, clustering, and MM/GBSA analysis, one compound was found to exhibit significant in vitro DAAO inhibition and stable surface binding. Post-docking refinement revealed convergent low-energy binding poses and highlighted effective clustering strategies for pose selection. This integrated workflow demonstrates a robust approach for discovering non-classical, surface-binding enzyme inhibitors.

First exposure to second-generation antipsychotics alters gut microbiota and metabolic profiles in patients with glucose-lipid metabolism disorders

Wu Y, Zang S, Wu Z, Huang J

Front Psychiatry. 2025 Dec 8;16:1722760. doi: 10.3389/fpsyt.2025.1722760.

This self-controlled study investigated metabolic side effects of second-generation antipsychotics (SGAs) by integrating gut microbiota and fecal metabolomic analyses in patients with schizophrenia before and after 3 months of treatment. SGAs significantly altered gut microbial composition, increasing Escherichia and Bifidobacterium while decreasing Faecalibacterium and Blautia, and were associated with changes in lipid-related metabolites and amino acid metabolic pathways, including D-amino acid metabolism. Key metabolites such as oleamide and stearamide were reduced and correlated with microbial shifts, with ornithine emerging as a central metabolic node. These findings suggest that SGA-induced metabolic disturbances are closely linked to changes in gut microbiota and their metabolic functions.

Integrated salivary microbiome and metabolome profiling reveals ecological and functional alterations in severe early childhood caries

Liu Y, Yu S, Wang X, Ye D, Aili M, Fu X, Zou J, Ma Q

J Transl Med. 2025 Dec 8;24:53. doi: 10.1186/s12967-025-07541-9.

This study integrated salivary microbiome and metabolomic profiling to characterize ecological and functional changes associated with severe early childhood caries (S-ECC). Children with S-ECC showed enrichment of cariogenic bacteria and widespread metabolic alterations, particularly in amino acid–related pathways including D-amino acid metabolism. Correlation analyses linked key pathogens with specific metabolites implicated in caries progression. These findings reveal distinct microbial–metabolic signatures of S-ECC and suggest potential biomarkers and therapeutic targets for early detection and intervention.

Effects of risperidone on amino acid metabolism, glucose, and kidney function in healthy adults: A pilot randomized controlled trial

Oshima M, Toyama T, Nakade Y, Miyagi S, Ogura H, Nakagawa S, Yuasa T, Koshino A, Horikoshi K, Minami T, Sako K, Tsuge S, Tamai A, Nishioka R, Miyagawa T, Ito K, Kitajima S, Mizushima I, Hara A, Sakai N, Shimizu M, Tokumaru T, Tsubomoto M, Kikuchi M, Kinoshita M, Nakada M, Mita M, Iwata Y, Wada T

PLoS One. 2025 Dec 5;20(12):e0324222. doi: 10.1371/journal.pone.0324222.

This pilot randomized controlled trial evaluated the effects of short-term risperidone administration on glucose metabolism, D-amino acid profiles, and kidney function in healthy adults. Risperidone did not significantly change insulin resistance, eGFR, or urinary albumin compared with controls. However, it significantly increased plasma D-serine levels and the urinary D-serine/creatinine ratio. These findings suggest that short-term risperidone modulates D-serine metabolism without causing acute renal or metabolic adverse effects.

D-amino acid oxidase suppresses hepatocellular carcinoma via oxidizing D-amino acids

Li Z, Fu C, Chen Y, Fang L, Liu W, Zhao J, Zhao J, Meng R, Pu R, Shi Y, Chen H, He Y, Jin Y, Ding Y, Cao G

J Transl Med. 2025 Nov 26;23:1359. doi: 10.1186/s12967-025-07399-x.

This study identifies D-amino acid oxidase (DAAO) as a key suppressor of hepatocellular carcinoma (HCC) progression and drug resistance using patient-derived organoids, cell lines, and in vivo models. DAAO expression was reduced in advanced HCC, sorafenib-resistant organoids, and tumors with stemness and EMT features, and low DAAO levels correlated with poor patient survival. Restoring DAAO expression inhibited tumor growth, migration, and invasion, and induced apoptosis through H₂O₂ generation, an effect enhanced by D-alanine supplementation. Combined DAAO restoration and D-amino acid supplementation also suppressed tumor growth in mice and reversed sorafenib resistance, highlighting a promising therapeutic strategy for HCC.

Thiazetidine-Based D-Amino Acid Oxidase Inhibitors: A Synergistic Approach Combining Design, Synthesis, Electronic Profiling, and In Silico Evaluation

Choudhury B, Balamurali MM, Chanda K

ChemistrySelect. 2025 10 (47), art. no. e06375. doi: 10.1002/slct.202506375.

This study reports a one-pot, ultrasound-assisted synthesis of disubstituted thiazetidine derivatives and evaluates their electronic properties and biological activity. The compounds were shown to competitively inhibit D-amino acid oxidase, with IC₅₀ values and binding constants determined by fluorimetric assays. Computational modeling, along with pharmacophore and pharmacokinetic analyses, provided insights into binding modes and druggability. These results identify thiazetidines as promising DAO inhibitors with potential relevance for schizophrenia-related neuropathology.

D-AAs AND CHIRALITY:

Quantum-elevated chiral discrimination for biomolecules

Yang Y, Hu X, Du W, Wu S, Yang P, Bao G, Zhang W

Sci Adv. 2026 Jan 16;12:eaea8201. doi: 10.1126/sciadv.aea8201.

Chiral discrimination is essential for analyzing enantiomeric biomolecules, but conventional optical methods suffer from weak signals and shot-noise–limited sensitivity. This study demonstrates quantum-enhanced chiral discrimination using polarization-entangled, quantum noise–squeezed light as sensitive and biocompatible probes. The approach achieves a 5 decibel improvement beyond the shot noise limit in distinguishing L- and D-amino acids in solution. This nondestructive technique offers a powerful platform for high-sensitivity chiral analysis in chemistry, biology, and medicine.

Mirror Life: Bridging Chirality, Ethics, and the Foundations of Life Creation

Li Y-Y, Moradialvand M, Asbridge L-A, Frazier KB, Tay FR, Makvandi P

Advanced Materials. 2026 38 , art. no. e10791. doi: 10.1002/adma.202510791.

This review discusses advances in the synthesis and engineering of mirror-image biomolecules that enable the development of orthogonal biological systems composed of D-amino acids and L-nucleotides. Mirror organisms hold promise for applications such as protease-resistant therapeutics, stable genetic materials, biosensors, and advanced biomaterials. At the same time, their unnatural chirality raises important biosafety concerns, including immune evasion and ecological persistence. The authors highlight both the transformative potential and the risks associated with constructing mirror-life architectures.

ENZYMES ACTIVE ON D-AAs:

Effect of high hydrostatic pressure on D-amino acid oxidase activity and stability

BMorales-Muralles M, Serrano SJ, Makhatadze G, Reyes-De-Corcuera JI

J Biol Eng. 2026 Jan 19. doi: 10.1186/s13036-025-00604-x.

This study investigated how high hydrostatic pressure (HHP) influences the stability and activity of pig kidney D-amino acid oxidase (DAAO). Enzyme activity decreased with pressure at 20 °C, while at 40 °C activity remained largely unchanged due to compensation between increased catalytic rate and inactivation; however, at 50–300 MPa higher temperatures accelerated inactivation. Unlike many other oxidases, pressure did not protect DAAO from thermal inactivation, and pressures above 100 MPa instead promoted inactivation. These results support the idea that small internal cavity size in DAAO limits pressure-induced stabilization, highlighting cavity volume as a key determinant of enzyme pressure sensitivity.

Peptidoglycan LD-Transpeptidases

Gastrell S, Vollmer W

Antibiotics (Basel). 2025 Dec 1;14(12):1210. doi: 10.3390/antibiotics14121210.

LD-transpeptidases (LDTs) are conserved peptidoglycan crosslinking enzymes that become especially important when classical penicillin-binding proteins are inactive and play key roles in β-lactam resistance in several major pathogens. They generate alternative LD-crosslinks, incorporate non-canonical D-amino acids into peptidoglycan, and in Gram-negative bacteria can tether outer membrane proteins to maintain envelope integrity and regulate virulence. Recent discoveries of new LDT subfamilies have expanded understanding of their functional diversity. These features make LDTs promising targets for the development of novel antibacterial therapies.

Identification and characterization of RacX, a new broad-specificity amino acid racemase from a novel taxon within the order Flavobacteriales

Hu L, Tan X-Y, Ye Y-Q, Liu X-Y, Li Y-Z, Wang J-Y, Zhang T-R, Du Z-J, Ye M-Q

Appl Environ Microbiol. 2025 Dec 22:e0201525. doi: 10.1128/aem.02015-25.

This paper reports the discovery of a halophilic bacterium, Halocola ammonii DA487ᵀ, and the identification of a broad-specificity amino acid racemase (RacX) with high catalytic efficiency and activity toward many amino acids. RacX shows a strong intrinsic bias toward D-amino acid production, which can be rationally altered by single-residue mutation, and functions stably under neutral to alkaline and high-salt conditions. Structural and mutational analyses revealed key residues involved in catalysis and substrate binding. These properties highlight RacX as a promising, engineerable biocatalyst for industrial D-amino acid synthesis.

Investigation of the metabolic role of D-amino acid aminotransferase in Arabidopsis thaliana using a column-switching two-dimensional high-performance liquid chromatography system

Sekine M, Katane M, Miyamoto T, Saitoh Y, Homma H, Sakai-Kato K

J Pharm Biomed Anal Open. 2026 7, art. no. 100098. doi: 10.1016/j.jpbao.2025.100098

This study investigates the physiological role of Arabidopsis thaliana D-amino acid aminotransferase (AtDAAT) using AtDAAT-deficient plants grown with exogenous D-aspartate. A validated two-dimensional HPLC method enabled selective quantification of multiple D- and L-amino acid enantiomers. AtDAAT-deficient plants showed accumulation of D-aspartate and reduced levels of D-glutamate, D-alanine, and D-valine, confirming AtDAAT’s role in D-Asp metabolism. Increased levels of all L-amino acids further suggest involvement of additional amino acid–metabolizing enzymes.

Cofactor-Independent Amino Acid Epimerases with Catalytic Serines Instead of Cysteines

Lamer T, van Belkum MJ, Chen P, Perov I, Heard BL, Wijewardane A, Lemieux MJ, Vederas JC

J Mol Biol. 2025, 437 (21), art. no. 169375. doi: 10.1016/j.jmb.2025.169375.

This study identifies and characterizes a new class of cofactor-independent amino acid epimerases that use paired catalytic serine residues instead of cysteines. These DapF-SS enzymes catalyze diaminopimelic acid epimerization with kinetics and mechanisms similar to classical cysteine-based DapF enzymes, yet serine-to-cysteine mutations severely impair activity. Thousands of related sequences were found across diverse bacteria, and some exhibit alternative substrate specificities. The serine-based architecture confers activity under oxidative conditions, suggesting an evolutionary advantage over cysteine-dependent counterparts.

D-AAs AND BIOTECHNOLOGY:

Switching Enantioselectivity in Phenylalanine Ammonia Lyase for the Synthesis of Electron-Deficient Aromatic D-Amino Acids

Buslov I, Desmons S, Wang W, Massaad LE, Hu X

Angewandte Chemie – International Edition. 2025, 64 (43), art. no. e202511739. doi: 10.1002/anie.202511739.

This study reports rational engineering of phenylalanine ammonia lyase to enable highly enantioselective synthesis of D-aromatic amino acids via asymmetric hydroamination. A single active-site mutation (L205F) conferred >99% enantiomeric excess toward D-products from electron-deficient aryl acrylates and supported gram-scale synthesis of valuable D-amino acid derivatives. Structural and mutational analyses revealed key roles of the MIO cofactor and residues L205, Y64, and K397 in controlling stereoselectivity. These findings establish PALs as programmable biocatalysts for tailored D- or L-amino acid synthesis.

Development of a highly functional thermostable D-amino acid oxidase for efficient L-phosphinothricin biosynthesis using ProteinMPNN-guided engineering

Yang K, Huang Y, Yao L, Huang B, Zeng W, Huang J

Int J Biol Macromol. 2026 Jan;338(Pt 2):149737. doi: 10.1016/j.ijbiomac.2025.149737.

This paper reports the engineering of a highly improved D-amino acid oxidase (DAAO) variant using a ProteinMPNN-guided mutagenesis strategy. The resulting enzyme shows greatly enhanced catalytic efficiency toward D-glufosinate and dramatically increased thermostability, along with an expanded substrate scope. The variant efficiently converted high concentrations of substrate with excellent stereospecificity at preparative scale. These results demonstrate an effective approach for creating robust biocatalysts for industrial chiral amino acid synthesis.

Metabolically Labeling Near-Infrared-II Excited Fluorophores on Bacterial Outer Membrane Vesicles for Targeted Imaging and Photoimmunotherapy of Gram-Negative Bacterial Peritonitis

Li N, Li Z, Zou J, Wu F, Wang M, Xiong J, Wu G, Chen, G, Tan X, Yang Q

Anal Chem. 2026 Jan 23. doi: 10.1021/acs.analchem.5c06887.

Gram-negative bacterial infections are difficult to treat due to their outer membrane, but outer membrane vesicles (OMVs) offer a promising platform for immunotherapy. This study reports the development of near-infrared II–excitable fluorophore-labeled OMVs (IR-FCTP-OMVs) for deep-tissue imaging and targeted photoimmunotherapy of Gram-negative infections. The engineered OMVs exhibit selective bacterial targeting, strong bactericidal activity under NIR-II irradiation, and the ability to activate both innate and adaptive immune responses. In an E. coli–induced peritonitis model, IR-FCTP-OMVs significantly reduced bacterial burden and inflammation, highlighting their diagnostic and therapeutic potential.

Reactive oxygen species promotion drives auranofin’s antiviral activity against hepatitis E virus

Tiller K, Williams ST, Wang B, Tian D, Meng X-J, Weger-Lucarelli J

J Virol. 2026 Jan 20;100:e0191725. doi: 10.1128/jvi.01917-25.

This work identifies promotion of reactive oxygen species (ROS) as a host-directed antiviral mechanism against hepatitis E virus (HEV). The FDA-approved drug auranofin and D-amino acid oxidase both induced ROS accumulation and showed dose-dependent antiviral activity against multiple HEV genotypes, including a ribavirin-resistant mutant. Blocking ROS reversed these antiviral effects, confirming a causal role for ROS in viral inhibition. These findings support repurposing auranofin, alone or combined with ribavirin, as a potential therapeutic strategy for HEV.

In vivo chemogenetic generation of intraneuronal HO

Maltsev DI, Solotenkov MA, Elesina EA, Dolbeneva AA, Fedotova AA, Morozova KI, Fedotov IV, Lanin AA, Fedotov AB, Korzhenevskii DA, Moshchenko AA, Brazhe NA, Podgorny OV, Belousov VV

Free Radic Biol Med. 2026 Jan 5;245:329-342. doi: 10.1016/j.freeradbiomed.2026.01.006.

This study establishes a method to induce and monitor oxidative stress in the brain in vivo using recombinant D-amino acid oxidase (DAAO). Viral expression of DAAO together with the H₂O₂ biosensor HyPer7 in hippocampal neurons, combined with fiber photometry, confirmed controlled intraneuronal H₂O₂ generation after D-norvaline administration. Chemogenetically induced H₂O₂ production was associated with reduced electron loading in the mitochondrial electron transport chain. These results validate DAAO/D-norvaline as a practical approach for modeling chronic oxidative stress in the brain.

Ready-to-use cryopreservation of undifferentiated induced pluripotent stem cells (iPSCs) without detachment from culture plates using D-proline and a synthetic polymer

Morita K, Kawasaki S, Yashiro T, Futai R, Kin C, Nakahashi A, Amo H, Kitayama Y, Aoi T, Koyanagi-Aoi M, Maruyama T

Biochem Eng Journal. 2026 227, art. no. 110041. doi: 10.1016/j.bej.2025.110041.

This study reports a xeno-free freezing medium containing D-proline and a synthetic block copolymer that enables efficient cryopreservation of 2D-cultured iPSCs without cell detachment. The new formulation achieved ≥70% cell recovery after thawing, far outperforming commercially available media. Cryopreserved cells maintained stable viability for at least three months and preserved pluripotency. These results establish a practical approach for in situ cryopreservation of adherent iPSC cultures.

A Tumor-Selective Self-Assembling Network of Poly(amino acid) Induces Cascading Bystander Cytotoxicity through Microvesicle Fission Amplification

Sun J, Ma Y, Sun J, Ding J, Chen X

J Am Chem Soc. 2026 Jan 21;148:2148-2164. doi: 10.1021/jacs.5c09790.

This study introduces an enzyme-responsive poly(D-amino acid) copolymer that self-assembles within tumors to form a positively charged network, enabling localized generation of cytotoxic microvesicles. Alkaline phosphatase–triggered assembly induces membrane disruption, organelle damage, and potent tumor-selective cell death, while initiating a self-sustaining microvesicle fission cascade that amplifies cytotoxic signaling. The generated microvesicles propagate tumor-killing activity across multiple generations. In vivo, this strategy achieves strong tumor growth inhibition and metastasis suppression, demonstrating a new paradigm for localized, EV-mediated cancer therapy.

A fluorescent probe for enantioselective recognition of amino acids in solutions, cells and zebrafishes

Fu S-W, Huang Z-X, Ni C-Z, Zhang F-Q, Li Y, Zhu Y-Y, Sun Q, Gu S-X

Dyes and Pigments. 2025 239, art. no. 112739. doi: 10.1016/j.dyepig.2025.112739.

This study reports binaphthyl-based chiral fluorescent probes that display strong enantioselective responses toward amino acid enantiomers. The (S)- and (R)-probes preferentially recognize L- and D-amino acids, respectively, with exceptionally high fluorescence enhancement for several amino acids. The probes enabled chiral fluorescence imaging of selected amino acids in living cells and zebrafish. These findings demonstrate a promising strategy for detecting amino acid chirality in biological systems relevant to racemization-associated diseases.

Production Enhancement of Surface-Expressed D-hydantoinase and D-carbamoylase in E. coli by Developing a Novel Coculture Process for the Efficient Biotransformation of D-p-hydroxyphenylglycine

Poureini F, Babaeipour V, Khalilzadeh R, Sajedi RH

Protein J. 2025 Dec 29. doi: 10.1007/s10930-025-10312-6.

This study developed a bacterial surface display system in Escherichia coli to co-express D-hydantoinase and D-carbamoylase for efficient whole-cell production of D-p-hydroxyphenylglycine (D-PHPG). Culture medium composition and induction conditions were systematically optimized, resulting in high cell density and elevated surface expression of both enzymes. Scale-up in a stirred-tank bioreactor further enhanced enzyme activities and expression levels. The optimized process achieved a 95% substrate conversion and 90% D-PHPG yield, demonstrating an effective strategy for industrial D-amino acid production.

Chiral inversion mutagenesis identifies geometrically constrained residues within self-associating low-complexity domains

Beckner RL, Carter C, Liszczak G

bioRxiv [Preprint]. 2025 Dec 19:2025.12.17.694949. doi: 10.64898/2025.12.17.694949.

This study investigates how backbone stereochemistry influences self-association of protein low-complexity domains (LCDs), beyond side-chain identity. Using synthetic Chiral Inversion Mutagenesis (ChIM), targeted L-to-D amino acid substitutions were introduced to identify Cα stereocenters under geometric constraint. Applied to LCDs from Emerin and neurofilament light chain, chiral inversions produced strongly position-dependent effects on self-association. The results reveal key structural features governing LCD interactions and establish ChIM as a powerful strategy to probe LCD biochemistry.

Living Carbonylative Polymerization of Vinylarenes Using P-Chirogentic Pd Catalyst: A Route to Functionalized Polyketone with Chirality Recognition

Wang SY, Ren BH, Song YH, Guo LJ, Chen SY, Lu XB, Liu Y

Angew Chem Int Ed Engl. 2026 Jan 22;65:e20391. doi: 10.1002/anie.202520391.

This investigation reports the design of P-chirogenic [P,O]-ligands enabling highly enantioselective Pd-catalyzed copolymerization of vinylarenes with carbon monoxide. The resulting catalysts achieve up to 99% isotacticity, perfect alternating insertion, and head-to-tail regularity, while exhibiting living polymerization behavior and very high molecular weights. The synthesized enantioenriched polyketones can form block architectures and include fluoro-containing films with unexpected chiral recognition toward L- and D-amino acids. These findings highlight a powerful strategy for producing functional chiral polyketones for applications in separation, sensing, and advanced materials.

Technetium(I) Complexes Bearing L- or D‑Amino Acids as Tumor-Seeking Agents

Silva RDM, Matos CP, Pinto CIG, Gano L, Guerreiro JF, Mendes F, Correia JDG

ACS Omega. 2025 Dec 3;10(49):60396-60415. doi: 10.1021/acsomega.5c07584

This work reports the synthesis and evaluation of novel technetium-99m tricarbonyl complexes bearing pendant L- or D-amino acids as potential SPECT radiotracers targeting amino acid transport in cancer. All amino acid–containing complexes showed higher cellular uptake than an amino-acid–free control, with the histidine- and tryptophan-containing tracers performing best. A β-isomer of the histidine complex displayed markedly enhanced cellular uptake and moderate tumor accumulation in lung cancer xenografts. These findings highlight amino acid–conjugated metal complexes, particularly those containing pyrazolyl-diamine units, as promising leads for further development of cancer imaging agents.

D-amino acids enhance the biocidal effect of free nitrous acid to realize sulfide control in sewers

Zhang W, Li R, Xia J, Sun J, Liang Z, Jiang F

Water Res. 2026 Feb 15;290:125087. doi: 10.1016/j.watres.2025.125087.

This paper shows that a mixture of D-amino acids synergistically enhances the effectiveness of low-dose free nitrous acid (FNA) for sulfide control in sewer biofilms. Co-application of D-amino acids and FNA achieved sustained suppression of sulfide production, reduced biofilm biomass and viable cells, and markedly lowered FNA consumption compared to FNA alone. Microscopy analyses revealed that D-amino acids disrupt the biofilm matrix, improving FNA penetration to sulfate-reducing bacteria. This combined strategy enables efficient sulfide control at much lower FNA dosages, offering a more sustainable and environmentally friendly approach.

D-AAs IN BACTERIA:

Metabolomics aided by machine learning decodes adaptive remodeling of Bacillus biofilms in response to pasteurization stress

Liang L, Wang P, Zhao X, Wang Z, Xu B, Ji Q, Wu, D, Chen Y

NPJ Sci Food. 2026 Jan 20. doi: 10.1038/s41538-026-00712-y.

Bacillus species challenge low-temperature dairy products because their spores and biofilms can survive pasteurization, and heat treatment may even enhance biofilm formation. Using untargeted metabolomics and random forest analysis, this study identified strain-specific metabolic mechanisms underlying heat-induced biofilm enhancement in raw milk isolates. In one strain, heat stress relieved metabolic inhibition and activated biofilm genes by altering amino acid and nucleoside metabolism, while another strain reduced the production of multiple biofilm-inhibitory metabolites through metabolic network restructuring. These findings reveal key metabolic drivers of heat-adaptive biofilm formation and suggest potential metabolic targets to improve dairy safety.

The combinatorial deletion of mycobacterial D,D-carboxypeptidases is readily tolerated in Mycobacterium smegmatis

Ealand CS, Moodley D, Ismail ZS, Maphatsoe M, Campbell L, Jacobs O, Kana BD

Access Microbiol. 2025 Dec 19;7(12):001074.v4. doi: 10.1099/acmi.0.001074.v4.

This study investigated the physiological roles of low-molecular-weight penicillin-binding proteins with D,D-carboxypeptidase activity in Mycobacterium smegmatis. Combinatorial deletion of D,D-CPase homologues had minimal effects on viability, biofilm formation, stress tolerance, and antibiotic susceptibility, with only minor changes in morphology and permeability. Peptidoglycan remodeling, assessed using a fluorescent D-amino acid probe, was not detectably altered, although mutant strains showed increased frequencies of unresolved septa. Overall, the results indicate that these non-essential D,D-CPases play a limited role in mycobacterial physiology, with a possible involvement in cell division.

Inducing an energy management crisis to combat bacterial biofilms: The mechanism of D-proline and sodium nitroprusside

Quan Q, Zhao B, An Q, Chen L, Liu X

Water Res. 2026 Feb 15;290:125049. doi: 10.1016/j.watres.2025.125049.

This investigation shows that D-proline and the nitric oxide donor sodium nitroprusside strongly inhibit biofilm formation of Roseobacter sp. SY3 and reduce membrane biofouling. Both agents suppress cell growth, decrease extracellular polymeric substances, and alter surface properties, leading to thinner biofilms and improved membrane flux. Transcriptomic and physiological analyses revealed that the treatments cause severe intracellular ATP depletion and downregulation of energy-generating pathways. The inhibitors induce an “energy management crisis,” explaining their potent antibiofilm activity and supporting their use as non-bactericidal antifouling strategies.

In vivo tracking and survival analysis of lactobacillus strains using fluorescent D-amino acids

Wei TT, Liu Y, Zhou Y

Food Sci Biotechnol. 2026 Jan 2;35:167-177. doi: 10.1007/s10068-025-02028-1.

This study developed a fluorescent D-amino acid–based labeling strategy to non-invasively track probiotic viability and behavior in the mouse gastrointestinal tract. Lactobacillus rhamnosus GG and Lactobacillus plantarum Lpc-115 were efficiently labeled without affecting viability, enabling real-time imaging of intestinal transit and persistence. L. rhamnosus showed stronger fluorescence and higher survival rates than L. plantarum. This genome-independent method provides a scalable tool for evaluating probiotic survival, functionality, and host interactions in vivo.

Differences in D-amino acid metabolism mediated by D-amino acid dehydrogenase in autotrophic and heterotrophic cultures of the Sulfur-Oxidizing Bacterium Starkeya novella

Tanigawa M, Usui H, Koike M, Tokuhisa M, Hirato Y, Matsushita S, Suzuki Y, Ukiya M, Nishimura K

Indian J Biochemistry and Biophysics. 2025 62 (9), 955 – 960. doi: 10.56042/ijbb.v62i9.9907.

This study investigates electron transfer from D-amino acid dehydrogenase (DAD) to respiratory components in the sulfur-oxidizing bacterium Starkeya novella. During heterotrophic growth, electrons generated from D-amino acid oxidation by DAD reduce cytochromes and drive oxygen consumption, indicating a role in respiration and ATP synthesis. Under autotrophic conditions, DAD-derived electrons instead reduce NAD⁺, supporting carbon fixation. These findings demonstrate that DAD serves distinct metabolic functions depending on the growth mode of S. novella.

Investigating GERMs: How Genotype, Environment, and Rhizosphere Microbiome interactions underlie heat response in maize and sorghum

Korth N, Borrero I, Rumley K, Woodley AL, Choudoir MJ, Gage JL

bioRxiv [Preprint]. 2025 Dec 10:2025.12.10.693489. doi: 10.64898/2025.12.10.693489.

This investigation applied a Genotype × Environment × Rhizosphere Microbiome (GERMs) framework to investigate how plant genotype, environment, and microbiome interactions influence cereal heat tolerance. A systems-level metatranscriptomic approach revealed that both plant gene expression and active microbial functions are shaped by host genotype and heat stress, with microbiome activity contributing to plant resilience. Integration of plant and microbial data identified D-amino acid metabolism as a potential mechanism underlying synergistic heat-stress responses. These findings highlight the rhizosphere microbiome as an active determinant of plant adaptation to high temperatures.

The XRE family protein DbuR is a transcriptional repressor of the dbu operon in Pseudomonas putida

Fulton RL, Downs DM

Appl Environ Microbiol. 2025 Dec 23;91(12):e0171525. doi: 10.1128/aem.01715-25.

This study investigates regulation of the dbu operon in Pseudomonas putida, which encodes enzymes involved in D-branched-chain amino acid catabolism. D-BCAAs induce dbu gene expression, while derepression of the operon also enables catabolism of D-tyrosine, revealing an expanded metabolic capability. The divergently transcribed gene dbuR encodes an XRE-family transcriptional repressor of the operon, although D-BCAA–dependent induction is only partially DbuR-dependent. These findings provide new insight into the regulatory complexity and metabolic versatility of P. putidarelevant to its use as a bioengineering chassis.

Enterococcus faecalis persists and replicates intracellularly within neutrophils

Stocks CJ, da Silva RAG, Antypas H, Jeyabalan N, Wong SL, Kline KA

Infect Immun. 2026 Jan 13;94:e0036425. doi: 10.1128/iai.00364-25.

This paper shows that Enterococcus faecalis can survive and replicate within neutrophils rather than being eliminated, creating an intracellular niche that may promote chronic wound infection. In murine and human models, infected neutrophils harbored viable bacteria for up to 24 hours and exhibited prolonged survival. Fluorescent D-amino acid labeling demonstrated active bacterial peptidoglycan synthesis and intracellular replication during early infection. These findings reveal an unexpected intracellular lifestyle of E. faecalis that may contribute to persistence and inflammation in chronic wounds.

D-AAs IN PEPTIDES AND PROTEINS:

Kinetic investigation on D-amino acid containing peptides and carboxypeptidase Y

Putman JI, Patel A, Olds M, Aziz A, Asokan-Sheeja H, Dong H, Johnson-Winters KL, Armstrong DW

Arch Biochem Biophys. 2026 Feb;776:110699. doi: 10.1016/j.abb.2025.110699.

This work examined how carboxypeptidase Y differentially hydrolyzes peptides with C-terminal L- versus D-amino acids. Steady-state kinetics showed that peptides ending in L-amino acids are cleaved two to five orders of magnitude faster than those containing D-amino acids. This large rate difference enables selective digestion of L-only peptides while leaving D-amino-acid–containing peptides intact for enrichment and detection. The findings provide a practical strategy for identifying D-amino acids in peptides without requiring chiral standards or inducing racemization.

AI-Based D-Amino Acid Substitution for Optimizing Antimicrobial Peptides to Treat Multidrug-Resistant Bacterial Infection

Zhao Y, Kong Q, Gong H, Li L, Fu J, Wan B, Wang P, Li X, Wang Y, Zhang J, Yu Y, Yang X, Zuo X, Wang H, Li Y

Adv Sci (Weinh). 2026 Jan 14:e18522. doi: 10.1002/advs.202518522.

D-amino acid substitution can improve the stability of antimicrobial peptides (AMPs), but the lack of general design rules limits efficient optimization. This study developed ADAPT, an AI-based predictor trained on curated datasets to evaluate the functional impact of D-amino acid substitutions and enable high-throughput AMP screening. Using this pipeline, most generated variants showed enhanced antibacterial activity, with dR2-1 displaying broad-spectrum potency, low toxicity, and high stability. The results establish an effective AI-driven framework for designing clinically promising, D-amino-acid–substituted AMPs.

A Novel D-Amino Acid-Composed GRPR-Targeted Peptide in Glioma

Ling X, Shi J, Zhao Y, Li Z, Chen L, Liu Y, Wang L, Li H, Wang X, Fan D, Li D

Mol Pharm. 2026 Jan 6. doi: 10.1021/acs.molpharmaceut.5c00756.

This study developed a D-amino acid–based bombesin analog, BBN(D), to improve the in vivo stability of GRPR-targeted imaging probes for glioma. Two tracers, BBN(D)-Cy5.5 for near-infrared fluorescence imaging and [⁶⁸Ga]Ga-DOTA-BBN(D) for PET/CT, showed high purity, strong GRPR-specific binding, and excellent tumor accumulation in subcutaneous and intracranial glioma models. BBN(D)-based probes demonstrated enhanced tumor uptake, blood–brain barrier penetration, and favorable imaging contrast compared with conventional BBN probes. These results indicate that BBN(D) is a promising platform for stable and specific glioma imaging in preclinical and potentially clinical settings.

Peptide chirality and opioid receptor modulation: Hepatoprotective effect of D-Met-enkephalin in acetaminophen-induced liver injury

Turčić P, Martinić R, Štambuk N, Weitner T, Momčilović M, Boban-Blagaić A, Kos M, Stojković R

Acta Pharm. 2026 Jan 13;75:659-672. doi: 10.2478/acph-2025-0036.

This study evaluated the biological activity of D-Met-enkephalin, the enantiomer of the neuroprotective peptide L-Met-enkephalin, in a mouse model of acetaminophen-induced liver injury. D-Met-enkephalin showed dose-dependent hepatoprotective effects comparable to the L-form, as indicated by reduced liver enzyme levels and tissue necrosis. Structural analyses suggested that glycine residues help preserve a functional conformation despite chirality inversion. Opioid receptor blockade reduced protection, indicating that the D-enantiomer retains opioid receptor–mediated activity.

Atom-level backbone engineering preserves peptide function while enhancing stability

He M, Cheng KF, Vu A, Torres MDT, Zanos S, Mughrabi IT, de la Fuente-Nunez C, Al-Abed Y

bioRxiv [Preprint]. 2025 Nov 18:2025.10.06.678421. doi: 10.1101/2025.10.06.678421.

This study systematically compares four backbone engineering strategies for improving peptide stability using bradykinin as a model. Although D-amino acid substitution and N-methylation greatly increased proteolytic resistance, they substantially reduced receptor binding and in vivo activity. In contrast, azapeptide incorporation preserved native-like affinity and physiological function while still enhancing stability. These results highlight the trade-off between stability and bioactivity and identify azapeptides as a promising, underexplored scaffold for therapeutic peptide design.

A stable all-D peptide ligand inspires broad-spectrum affinity and high performance in the purification of COVID-19 vaccines

Ma J, Dong X, Yu L, Sun Y, Shi Q

J Chromatogr A. 2026 Jan 11;1766:466577. doi: 10.1016/j.chroma.2025.466577.

This paper reports the development of a fully D-amino acid version of an affinity peptide (D-HWK) to overcome stability limitations of conventional peptide ligands. D-HWK showed higher binding affinity than its L-counterpart for wild-type and variant SARS-CoV-2 RBDs and spike proteins, supported by more stable binding conformations in molecular simulations. When immobilized on chromatography media, D-HWK enabled efficient and mild purification of wild-type and Omicron vaccines with broad binding conditions. Its strong resistance to enzymatic degradation and stable performance over multiple cycles highlight D-HWK as a robust ligand for broad-spectrum affinity chromatography in vaccine manufacturing.

Modifying the PSD-95 inhibitor NA-1 for brain delivery through proteolytic stabilization and conjugation to the potentially brain homing peptide BR1

Kolberg HG, Andresen AK, Thaysen M, Øie Solbak SM, Jensen C, Brodin B, Gammelgaard B, Kristensen M

Int J Pharm. 2026 Jan 5;687:126434. doi: 10.1016/j.ijpharm.2025.126434.

This study evaluated whether D-amino acid substitution and BR1 conjugation could improve the stability and brain delivery of the stroke therapeutic peptide NA-1. D-amino acid substitution markedly enhanced plasma stability but reduced receptor binding and partially decreased cellular uptake. Conjugation of BR1 to L-NA-1 increased accumulation in whole-brain lysates but did not improve parenchymal penetration, suggesting vascular retention rather than enhanced blood–brain barrier transport. Overall, the results highlight a trade-off between stability and target affinity, and indicate that BR1 does not promote transendothelial brain delivery of NA-1.

Inhibition of human Nav1.4 by D-amino acid modified μ-CnIIIC

Wu R, Wang Z, Quan Y, He Z, Zhao Y, Wang Y, Wang J, Ma Z

Bioorg Chem. 2026 Jan 7;170:109484. doi: 10.1016/j.bioorg.2026.109484.

This study examined whether D-amino acid substitution can improve the potency and stability of the conotoxin μ-CnIIIC against the human sodium channel NaV1.4. Replacing the N-terminal pyroglutamic acid with D-arginine produced dR-μ-CnIIIC, which showed significantly enhanced inhibitory activity and stronger binding interactions with NaV1.4 compared to the wild-type peptide. D-amino acid modification also markedly increased serum stability and resistance to proteolysis. These findings demonstrate a rational strategy to optimize peptide inhibitors of sodium channels and potentially other conotoxin-based therapeutics.

Functional D- and L‑Naphthalenediimide-Peptides: Microwave-Driven Synthesis, Supramolecular Aggregation, and Multiphoton Fluorescence Lifetime Imaging Microscopy in Living Cells

Giuffrida SG, Calatayud DG, Cortezon-Tamarit F, Ge H, Mirabello. V, Răsădean DM, Pourzand C, Botchway SW, Estrela P, Pantoş GD, Eggleston IM, Pascu SI

ACS Bio Med Chem Au. 2025 Jul 18;5:947-965. doi: 10.1021/acsbiomedchemau.5c00064.

This study describes the microwave-assisted synthesis of novel peptide-linked optical imaging probes incorporating the GRPR-targeting L-[7,13]-bombesin fragment and both L- and D-amino acid–substituted naphthalenediimide conjugates. The probes exhibit tunable fluorescence, water solubility, and stereochemistry-dependent self-assembly, while remaining non-toxic under imaging conditions. Advanced microscopy revealed that peptide stereochemistry and conjugate architecture strongly influence cellular uptake and subcellular localization, with a BODIPY-labeled derivative preferentially localizing to the endoplasmic reticulum. These results provide design principles for developing stable, biologically active peptide-based imaging probes for cancer biomarker detection.

Receptor-Mediated Shuttling of a D-Amino Acid Peptide Achieves High Nanomolar Cytosolic Concentrations

List M, Beck-Sickinger AG

J Am Chem Soc. 2026 Jan 14;148:308-315. doi: 10.1021/jacs.5c12876.

This study investigates receptor-mediated shuttling via CMKLR1 as a strategy for delivering therapeutic peptides into the cytosol. Using the D-amino acid peptide PMIγ, fluorescence correlation spectroscopy showed that biologically relevant cytosolic concentrations can be achieved even without endosomal escape enhancers. Addition of a pH-responsive escape peptide further increased delivery but caused toxicity and was rapidly degraded. These results demonstrate the potential of receptor-mediated transport for cytosolic peptide delivery and provide quantitative insights into intracellular dosing.

D-AAs AND ANALYTICAL METHODS (AND FOODS):

Two-Dimensional LC-MS/MS Determination of Chiral Amino Acids in Real-World Samples

Ishii C, Hamase K

Methods Mol Biol. 2026;2994:231-244. doi: 10.1007/978-1-0716-5023-3_12.

This chapter highlights the growing importance of chiral amino acid analysis as D-amino acids emerge as biologically active molecules and potential biomarkers. Because these compounds are typically present at trace levels, highly sensitive and selective analytical approaches are required. The authors summarize the design and development of multidimensional liquid chromatography, particularly two-dimensional LC–MS/MS systems. Applications to diverse biological and complex samples demonstrate the utility of these methods for accurate chiral amino acid determination.

Quantitative analysis of D/L-serine and D/L-proline in serum by isotope-coded derivatization using an original chiral resolution labeling reagent

Ozaki M, Yamada Y, Hirose T, Shimotsuma M, Ikeda A, Kawase T, Tsuji A, Tomonaga S, Matsui M, Kuranaga T, Kakeya H

J Pharm and Biomed Anal Open. 2026 7, art. no. 100099. doi: 10.1016/j.jpbao.2025.100099.

In this study an isotope-coded derivatization LC–MS method for accurate quantification of D/L-serine and D/L-proline in human serum is reported. The approach enables reliable chiral separation, high stability of labeled samples, and good analytical performance in terms of recovery, reproducibility, and sensitivity. Using this method, serum levels of D-serine and D-proline were found to be higher in Alzheimer’s disease patients than in controls. These results support the potential of these D-amino acids as minimally invasive biomarkers for early AD diagnosis.

Enantiomeric separation of 1-methyl-tryptophan on a mixed-mode stationary phase via pre-column derivatization and in vivo assessment of chiral inversion in rats

Onozato M, Fu S, Takaura T, Sakamoto T, Fukushima T

J Chromatography Open. 2025 8, art. no. 100257. doi: 10.1016/j.jcoa.2025.100257.

This study developed an LC–MS/MS method with chiral derivatization for effective enantiomeric separation of D- and L-1-methyl-tryptophan. The method achieved superior resolution using a mixed-mode stationary phase compared with a conventional reversed-phase column. Pharmacokinetic analysis in rats revealed chiral inversion of D-MT to L-MT, but not vice versa. Inhibition studies suggested that D-amino acid oxidase contributes to this enantiomeric conversion.

Temperature Dependence of D-Amino-Acid Selectivity of L-Tryptophan Probed by Ultraviolet Photodissociation Spectroscopy

Kitahashi K, Fujihara A

Mass Spectrom (Tokyo). 2025 ;14:A0178. doi: 10.5702/massspectrometry.A0178.

This study examines how temperature influences chiral recognition of amino acids by L-tryptophan using gas-phase ultraviolet photodissociation spectroscopy. At cryogenic temperature (8 K), protonated L-tryptophan–amino acid clusters show distinct spectra for different amino acids and their enantiomers, indicating effective molecular recognition. In contrast, spectral differences are greatly reduced at 100 K due to hot bands and entropic effects. These results demonstrate that deep cryogenic cooling is essential for reliable differentiation of D- and L-amino acids using this approach.

INFORMATION

The D-amino acids International Research Center “DAAIR“ has been established in Gerenzano (Varese, Italy) in 2019 with the aim to support and perform scientific research projects and activities on the field of D-amino acids. The Center, located inside the Fondazione Istituto Insubrico Ricerca per la Vita, is aimed to represent a pole of excellence at international level for dissemination and research involving the D-amino acids (Director Silvia Sacchi).

Info@d-aminoacids.com

director@d-aminoacids.com

D-AMINO ACIDS INTERNATIONAL RESEARCH CENTER

INFO@D-AMINOACIDS.COM