Integrative plasma and fecal metabolomics identify functional metabolites in adenoma-colorectal cancer progression and as early diagnostic biomarkers.

Changes in plasma and fecal metabolomes in colorectal cancer (CRC) progression (normal-adenoma-CRC) remain unclear. Here, plasma and fecal samples were collected from four independent cohorts of 1,251 individuals (422 CRC, 399 colorectal adenoma [CRA], and 430 normal controls [NC]). By metabolomic profiling, signature plasma and fecal metabolites with consistent shift across NC, CRA, and CRC are identified, including CRC-enriched oleic acid and CRC-depleted allocholic acid. Oleic acid exhibits pro-tumorigenic effects in CRC cells, patient-derived organoids, and two murine CRC models, whereas allocholic acid has opposing effects. By integrative analysis, we found that oleic acid or allocholic acid directly binds to α-enolase or farnesoid X receptor-1 in CRC cells, respectively, to modulate cancer-associated pathways. Clinically, we establish a panel of 17 plasma metabolites that accurately diagnoses CRC in a discovery and three validation cohorts (AUC = 0.848-0.987). Overall, we characterize metabolite signatures, mechanistic significance, and diagnostic potential of plasma and fecal metabolomes in CRC.

A comparison of antibiotic resistance genes and mobile genetic elements in wild and captive Himalayan vultures.

As the most widely distributed scavenger birds on the Qinghai-Tibetan Plateau, Himalayan vultures (Gyps himalayensis) feed on the carcasses of various wild and domestic animals, facing the dual selection pressure of pathogens and antibiotics and are suitable biological sentinel species for monitoring antibiotic resistance genes (ARGs). This study used metagenomic sequencing to comparatively investigate the ARGs and mobile genetic elements (MGEs) of wild and captive Himalayan vultures. Overall, the resistome of Himalayan vultures contained 414 ARG subtypes resistant to 20 ARG types, with abundances ranging from 0.01 to 1,493.60 ppm. The most abundant resistance type was beta-lactam (175 subtypes), followed by multidrug resistance genes with 68 subtypes. Decreases in the abundance of macrolide-lincosamide-streptogramin (MLS) resistance genes were observed in the wild group compared with the zoo group. A total of 75 genera (five phyla) of bacteria were predicted to be the hosts of ARGs in Himalayan vultures, and the clinical (102 ARGs) and high-risk ARGs (35 Rank I and 56 Rank II ARGs) were also analyzed. Among these ARGs, twenty-two clinical ARGs, nine Rank I ARG subtypes, sixteen Rank II ARG subtypes were found to differ significantly between the two groups. Five types of MGEs (128 subtypes) were found in Himalayan vultures. Plasmids (62 subtypes) and transposases (44 subtypes) were found to be the main MGE types. Efflux pump and antibiotic deactivation were the main resistance mechanisms of ARGs in Himalayan vultures. Decreases in the abundance of cellular protection were identified in wild Himalayan vultures compared with the captive Himalayan vultures. Procrustes analysis and the co-occurrence networks analysis revealed different patterns of correlations among gut microbes, ARGs, and MGEs in wild and captive Himalayan vultures. This study is the first step in describing the characterization of the ARGs in the gut of Himalayan vultures and highlights the need to pay more attention to scavenging birds.

Broad-spectrum ginsentides are principal bioactives in unraveling the cure-all effects of ginseng.

Stress and illness connection is complex and involves multiple physiological systems. Panax ginsengs, reputed for their broad-spectrum "cure-all" effect, are widely prescribed to treat stress and related illnesses. However, the identity of ginseng's "cure-all" medicinal compounds that relieve stress remains unresolved. Here, we identify ginsentides as the principal bioactives that coordinate multiple systems to restore homeostasis in response to stress. Ginsentides are disulfide-rich, cell-penetrating and proteolytic-stable microproteins. Using affinity-enrichment mass spectrometry target identification together with in vitro, ex vivo and in vivo validations, we show that highly purified or synthetic ginsentides promote vasorelaxation by producing nitric oxide through endothelial cells via intracellular PI3K/Akt signaling pathway, alleviate α1-adrenergic receptor overactivity by reversing phenylephrine-induced constriction of aorta, decrease monocyte adhesion to endothelial cells via CD166/ESAM/CD40 and inhibit P2Y12 receptors to reduce platelet aggregation. Orally administered ginsentides were effective in animal models to reduce ADP-induced platelet aggregation, to prevent collagen and adrenaline-induced pulmonary thrombosis as well as anti-stress behavior of tail suspension and forced swimming tests in mice. Together, these results strongly suggest that ginsentides are the principal panacea compounds of ginsengs because of their ability to target multiple extra- and intra-cellular proteins to reverse stress-induced damages.

Ultrafast Biomimetic Oxidative Folding of Cysteine-rich Peptides and Microproteins in Organic Solvents.

Disulfides in peptides and proteins are essential for maintaining a properly folded structure. Their oxidative folding is invariably performed in an aqueous-buffered solution. However, this process is often slow and can lead to misfolded products. Here, we report a novel concept and strategy that is bio-inspired to mimic protein disulfide isomerase (PDI) by accelerating disulfide exchange rates many thousand-fold. The proposed strategy termed organic oxidative folding is performed under organic solvents to yield correctly folded cysteine-rich microproteins instantaneously without observable misfolded or dead-end products. Compared to conventional aqueous oxidative folding strategies, enormously large rate accelerations up to 113,200-fold were observed. The feasibility and generality of the organic oxidative folding strategy was successfully demonstrated on 15 cysteine-rich microproteins of different hydrophobicity, lengths (14 to 58 residues), and numbers of disulfides (2 to 5 disulfides), producing the native products in a second and in high yield.

Lysosomal-Cleavable Peptide Linkers in Antibody-Drug Conjugates.

Antibody-drug Conjugates (ADCs) are a powerful therapeutic modality for cancer treatment. ADCs are multi-functional biologics in which a disease-targeting antibody is conjugated to an effector payload molecule via a linker. The success of currently used ADCs has been largely attributed to the development of linker systems, which allow for the targeted release of cytocidal payload drugs inside cancer cells. Many lysosomal proteases are over expressed in human cancers. They can effectively cleave a variety of peptide sequences, which can be exploited for the design of ADC linker systems. As a well-established linker, valine-citrulline-p-aminobenzyl carbamate (ValCitPABC) is used in many ADCs that are already approved or under preclinical and clinical development. Although ValCitPABC and related linkers are readily cleaved by cathepsins in the lysosome while remaining reasonably stable in human plasma, many studies have shown that they are susceptible to carboxylesterase 1C (Ces1C) in mouse and rat plasma, which hinders the preclinical evaluation of ADCs. Furthermore, neutropenia and thrombocytopenia, two of the most commonly observed dose-limiting adverse effects of ADCs, are believed to result from the premature hydrolysis of ValCitPABC by human neutrophil elastase. In addition to ValCitPABC, the GGFG tetrapeptidyl-aminomethoxy linker is also cathepsin-cleavable and is used in the highly successful ADC drug, DS8201a. In addition to cathepsin-cleavable linkers, there is also growing interest in legumain-sensitive linkers for ADC development. Increasing plasma stability while maintaining lysosomal cleavability of ADC linkers is an objective of intensive current research. This review reports recent advances in the design and structure-activity relationship studies of various peptide/peptidomimetic linkers in this field.

Comparative analysis of gut DNA viromes in wild and captive Himalayan vultures.

Introduction: Himalayan vultures (Gyps hinalayensis) are widely distributed on the Qinghai-Tibetan Plateau and play a crucial role in maintaining the ecological balance by feeding on decayed corpses of wild and domestic animals. Large-scale culture and metagenomics studies have broadened our understanding of viral diversity in animals' gastrointestinal tracts. However, despite the importance of gut viral communities in regulating bacterial diversity and performing symbiotic functions, no gut viral study has been conducted on Himalayan vultures. Furthermore, the impact of captivity on the gut virome of these vultures remains unknown. Methods: In this study, metagenomic sequencing methods targeting DNA of virus-like particles enriched from feces were used to characterize the gut DNA viromes of wild and captive Himalayan vultures. Results: In total, 22,938 unique viral operational taxonomic units (vOTUs) were identified and assigned to 140 viral genera in 41 viral families. These families included viruses associated with bacteria, animals, plants, insects, and archaea. Phage communities, including Siphoviridae, Microviridae, Myoviridae, Inoviridae, and Herelleviridae, dominated the gut virome of Himalayan vultures. Wild vultures exhibited higher viral richness and diversity compared with those in captivity. The functional capacity of the gut virome was characterized by identifying 93 KEGG pathways, which were significantly enriched in metabolism and genetic information processing. Abundant auxiliary metabolic genes, such as carbohydrate-active enzyme, and antibiotic resistance genes, were also found in the vultures' gut virome. Discussion: Our findings reveal the complex and diverse viral community present in the gut virome of Himalayan vultures, which varies between wild, and captive states. The DNA virome dataset establishes a baseline for the vultures' gut virome and will serve as a reference for future virus isolation and cultivation. Understanding the impact of captivity on the gut virome contributes to our knowledge of vultures' response to captivity and aids in optimizing their rehabilitation and implementing protective measures.

Four additional natural 7-deazaguanine derivatives in phages and how to make them.

Bacteriophages and bacteria are engaged in a constant arms race, continually evolving new molecular tools to survive one another. To protect their genomic DNA from restriction enzymes, the most common bacterial defence systems, double-stranded DNA phages have evolved complex modifications that affect all four bases. This study focuses on modifications at position 7 of guanines. Eight derivatives of 7-deazaguanines were identified, including four previously unknown ones: 2'-deoxy-7-(methylamino)methyl-7-deazaguanine (mdPreQ1), 2'-deoxy-7-(formylamino)methyl-7-deazaguanine (fdPreQ1), 2'-deoxy-7-deazaguanine (dDG) and 2'-deoxy-7-carboxy-7-deazaguanine (dCDG). These modifications are inserted in DNA by a guanine transglycosylase named DpdA. Three subfamilies of DpdA had been previously characterized: bDpdA, DpdA1, and DpdA2. Two additional subfamilies were identified in this work: DpdA3, which allows for complete replacement of the guanines, and DpdA4, which is specific to archaeal viruses. Transglycosylases have now been identified in all phages and viruses carrying 7-deazaguanine modifications, indicating that the insertion of these modifications is a post-replication event. Three enzymes were predicted to be involved in the biosynthesis of these newly identified DNA modifications: 7-carboxy-7-deazaguanine decarboxylase (DpdL), dPreQ1 formyltransferase (DpdN) and dPreQ1 methyltransferase (DpdM), which was experimentally validated and harbors a unique fold not previously observed for nucleic acid methylases.

A Cascade Enzymatic Reaction Scheme for Irreversible Transpeptidative Protein Ligation.

Enzymatic peptide ligation holds great promise in the study of protein functions and development of protein therapeutics. Owing to their high catalytic efficiency and a minimal tripeptide recognition motif, peptidyl asparaginyl ligases (PALs) are particularly useful tools for bioconjugation. However, as an inherent limitation of transpeptidases, PAL-mediated ligation is reversible, requiring a large excess of one of the ligation partners to shift the reaction equilibrium in the forward direction. Herein, we report a method to make PAL-mediated intermolecular ligation irreversible by coupling it to glutaminyl cyclase (QC)-catalyzed pyroglutamyl formation. In this method, the acyl donor substrate of PALs is designed to have glutamine at the P1' position of the Asn-P1'-P2' tripeptide PAL recognition motif. Upon ligation with an acyl acceptor substrate, the acyl donor substrate releases a leaving group in which the exposed N-terminal glutamine is cyclized by QC, quenching the Gln Nα-amine in a lactam. Using this method, PAL-mediated ligation can achieve near-quantitative yields even at an equal molar ratio between the two ligation partners. We have demonstrated this method for a wide range of applications, including protein-to-protein ligations. We anticipate that this cascade enzymatic reaction scheme will make PAL enzymes well suited for numerous new uses in biotechnology.

Draft Genome Sequence of Fusobacterium vincentii CNGBCC1850030, Isolated from Healthy Human Feces.

Fusobacterium vincentii usually inhabits the oral cavity and plays an important role in periodontal diseases. Here, we report the draft genome sequence of F. vincentii strain CNGBCC1850030, isolated from healthy human feces.

Substrate-binding glycine residues are major determinants for hydrolase and ligase activity of plant legumains.

Peptide asparaginyl ligases (PALs) are useful tools for precision modifications of proteins and live-cell surfaces by ligating peptides after Asn/Asp (Asx). They share high sequence and structural similarity to plant legumains that are generally known as asparaginyl endopeptidases (AEPs), thus making it challenging to identify PALs from AEPs. In this study, we investigate 875 plant species from algae to seed plants with available sequence data in public databases to identify new PALs. We conducted evolutionary trace analysis on 1500 plant legumains, including eight known PALs, to identify key residues that could differentiate ligases and proteases, followed by recombinant expression and functional validation of 16 novel legumains. Previously, we showed that the substrate-binding sequences flanking the catalytic site can strongly influence the enzymatic direction of a legumain and which we named as ligase-activity determinants (LADs). Here, we show that two conserved substrate-binding Gly residues of LADs are critical, but negative determinants for ligase activity. Our results suggest that specific glycine residues are molecular determinants to identify PALs and AEPs as two different legumain subfamilies, accounting for c. 1% and 88%, respectively.

Arabidopsis NPF2.13 functions as a critical transporter of bacterial natural compound tunicamycin in plant-microbe interaction.

Metabolites including antibiotics, enzymes, and volatiles produced by plant-associated bacteria are key factors in plant-microbiota interaction that regulates various plant biological processes. There should be crucial mediators responsible for their entry into host plants. However, less is known about the identities of these plant transporters. We report that the Arabidopsis Nitrate Transporter1 (NRT1)/NPF protein NPF2.13 functions in plant uptake of tunicamycin (TM), a natural antibiotic produced by several Streptomyces spp., which inhibits protein N-glycosylation. Loss of NPF2.13 function resulted in enhanced TM tolerance, whereas NPF2.13 overexpression led to TM hypersensitivity. Transport assays confirmed that NPF2.13 is a H+ /TM symporter and the transport is not affected by other substrates like nitrate. NPF2.13 exclusively showed TM transport activity among tested NPFs. Tunicamycin uptake from TM-producing Streptomyces upregulated the expression of nitrate-related genes including NPF2.13. Moreover, nitrate allocation to younger leaves was promoted by TM in host plants. Tunicamycin could also benefit plant defense against the pathogen. Notably, the TM effects were significantly repressed in npf2.13 mutant. Overall, this study identifies NPF2.13 protein as an important TM transporter in plant-microbe interaction and provides insights into multiple facets of NPF proteins in modulating plant nutrition and defense by transporting exterior bacterial metabolites.

Structural basis for proenzyme maturation, substrate recognition, and ligation by a hyperactive peptide asparaginyl ligase.

Peptide ligases are versatile enzymes that can be utilized for precise protein conjugation for bioengineering applications. Hyperactive peptide asparaginyl ligases (PALs), such as butelase-1, belong to a small class of enzymes from cyclotide-producing plants that can perform site-specific, rapid ligation reactions after a target peptide asparagine/aspartic acid (Asx) residue binds to the active site of the ligase. How PALs specifically recognize their polypeptide substrates has remained elusive, especially at the prime binding side of the enzyme. Here we report crystal structures that capture VyPAL2, a catalytically efficient PAL from Viola yedoensis, in an activated state, with and without a bound substrate. The bound structure shows one ligase with the N-terminal polypeptide tail from another ligase molecule trapped at its active site, revealing how Asx inserts in the enzyme's S1 pocket and why a hydrophobic residue is required at the P2' position. Besides illustrating the anchoring role played by P1 and P2' residues, these results uncover a role for the Gatekeeper residue at the surface of the S2 pocket in shifting the nonprime portion of the substrate and, as a result, the activity toward ligation or hydrolysis. These results suggest a picture for proenzyme maturation in the vacuole and will inform the rational design of peptide ligases with tailored specificities.

PAL-Mediated Ligation for Protein and Cell-Surface Modification.

Peptidyl Asx-specific ligases (PALs) effect peptide ligation by catalyzing transpeptidation reactions at Asn/Asp-peptide bonds. Owing to their high efficiency and mild aqueous reaction conditions, these ligases have emerged as powerful biotechnological tools for protein manipulation in recent years. PALs are enzymes of the asparaginyl endopeptidase (AEP) superfamily but have predominant transpeptidase activity as opposed to typical AEPs which are predominantly hydrolases. Butelase-1 and VyPAL2, two PALs discovered by our teams, have been used successfully in a wide range of applications, including macrocyclization of synthetic peptides and recombinant proteins, protein N- or C-terminal modification, and cell-surface labeling. As shown in numerous reports, PAL-mediated ligation is highly efficient at Asn junctions. Although considerably less efficient, Asp-specific ligation has also been shown to be practically useful under suitable conditions. Herein, we describe the methods of using VyPAL2 for protein macrocyclization and labeling at an Asp residue as well as for protein dual labeling through orthogonal Asp- and Asn-directed ligations. We also describe a method for cell-surface protein modification using butelase-1, demonstrating its advantageous features over previous methods.

Multi-omics analyses of serum metabolome, gut microbiome and brain function reveal dysregulated microbiota-gut-brain axis in bipolar depression.

The intricate processes of microbiota-gut-brain communication in modulating human cognition and emotion, especially in the context of mood disorders, have remained elusive. Here we performed faecal metagenomic, serum metabolomics and neuroimaging studies on a cohort of 109 unmedicated patients with depressed bipolar disorder (BD) patients and 40 healthy controls (HCs) to characterise the microbial-gut-brain axis in BD. Across over 12,000 measured metabolic features, we observed a large discrepancy (73.54%) in the serum metabolome between BD patients and HCs, spotting differentially abundant microbial-derived neuroactive metabolites including multiple B-vitamins, kynurenic acid, gamma-aminobutyric acid and short-chain fatty acids. These metabolites could be linked to the abundance of gut microbiota presented with corresponding biosynthetic potentials, including Akkermansia muciniphila, Citrobacter spp. (Citrobacter freundii and Citrobacter werkmanii), Phascolarctobacterium spp., Yersinia spp. (Yersinia frederiksenii and Yersinia aleksiciae), Enterobacter spp. (Enterobacter cloacae and Enterobacter kobei) and Flavobacterium spp. Based on functional neuroimaging, BD-related neuroactive microbes and metabolites were discovered as potential markers associated with BD-typical features of functional connectivity of brain networks, hinting at aberrant cognitive function, emotion regulation, and interoception. Our study combines gut microbiota and neuroactive metabolites with brain functional connectivity, thereby revealing potential signalling pathways from the microbiota to the gut and the brain, which may have a role in the pathophysiology of BD.

Asparaginyl Endopeptidase-Mediated Protein C-Terminal Hydrazinolysis for the Synthesis of Bioconjugates.

Asparaginyl endopeptidases (AEPs) are cysteinyl enzymes naturally catalyzing the hydrolysis and transpeptidation reactions at Asx-Xaa bonds. These reactions go by a common acyl-enzyme thioester intermediate, which is either attacked by water (for a protease-AEP) or by a peptidic amine nucleophile (for a ligase-AEP) to form the respective hydrolysis or aminolysis product. Herein, we show that hydrazine and hydroxylamine, two α-effect nucleophiles, are capable of resolving the thioester intermediate to yield peptide and protein products containing a C-terminal hydrazide and hydroxamic acid functionality, respectively. The hydrazinolysis reaction exhibits very high efficiency and can be completed in minutes at a low enzyme-to-substrate ratio. We further show the utility of the so-formed asparaginyl hydrazide in native chemical ligation and hydrazone conjugation. Using an EGFR-targeting affibody as a model protein, we have showcased our methodology in the preparation of a number of protein ligation or conjugation products, which are decorated with various functional moieties. The ZEGFR affibody-doxorubicin conjugate shows high selective binding and cytotoxicity toward the EGFR-positive A431 cells. Our results demonstrate the advantages of AEP-mediated protein hydrazinolysis as a simple and straightforward strategy for the precision manufacturing of protein bioconjugates.

Seasonal variations in the composition and functional profiles of gut microbiota reflect dietary changes in plateau pikas.

Seasonal variations in gut microbiota of small mammals and how they are influenced by environmental variables are relatively poorly understood. We sampled 162 wild plateau pikas (Ochotona curzoniae) in 4 seasons over 2 and a half years and recorded the air temperature, precipitation, and nutrient content in edible vegetation at the sampling site. After conducting 16S rRNA and shotgun metagenomic sequencing, we found that the highest alpha diversity, the relative abundance of Firmicutes, and the simplest co-occurrence network occurred in winter, whereas the highest relative abundance of Proteobacteria and the most complex network structure were observed in spring. The highest relative abundance of Verrucomicrobiota and Spirochaetota was seen in summer and autumn, respectively. Air temperature, precipitation, and the contents of crude protein, crude fiber, and polysaccharide in vegetation had significant effects on the seasonal changes in gut microbiota. Diet contributed more to microbial variation than climatic factors. Metagenomic analysis revealed that the amino acid metabolism pathway and axillary activity enzymes were most abundant in summer, while abundance of carbohydrate-binding modules and carbohydrate esterases was highest in spring. These microbial variations were related to the changes in dietary nutrition, indicating that gut microbiota of plateau pika contribute to the efficient use of food resources. This study provides new evidence of how external environmental factors affect the intestinal environment of small mammals.

Site-Specific Protein Modifications by an Engineered Asparaginyl Endopeptidase from Viola canadensis.

Asparaginyl endopeptidases (AEPs) or legumains are Asn/Asp (Asx)-specific proteases that break peptide bonds, but also function as peptide asparaginyl ligases (PALs) that make peptide bonds. This ligase activity can be used for site-specific protein modifications in biochemical and biotechnological applications. Although AEPs are common, PALs are rare. We previously proposed ligase activity determinants (LADs) of these enzymes that could determine whether they catalyze formation or breakage of peptide bonds. LADs are key residues forming the S2 and S1' substrate-binding pockets flanking the S1 active site. Here, we build on the LAD hypothesis with the engineering of ligases from proteases by mutating the S2 and S1' pockets of VcAEP, an AEP from Viola canadensis. Wild type VcAEP yields <5% cyclic product from a linear substrate at pH 6.5, whereas the single mutants VcAEP-V238A (Vc1a) and VcAEP-Y168A (Vc1b) targeting the S2 and S1' substrate-binding pockets yielded 34 and 61% cyclic products, respectively. The double mutant VcAEP-V238A/Y168A (Vc1c) targeting both the S2 and S1' substrate-binding pockets yielded >90% cyclic products. Vc1c had cyclization efficiency of 917,759 M-1s-1, which is one of the fastest rates for ligases yet reported. Vc1c is useful for protein engineering applications, including labeling of DARPins and cell surface MCF-7, as well as producing cyclic protein sfGFP. Together, our work validates the importance of LADs for AEP ligase activity and provides valuable tools for site-specific modification of proteins and biologics.

The legumain McPAL1 from Momordica cochinchinensis is a highly stable Asx-specific splicing enzyme.

Legumains, also known as asparaginyl endopeptidases (AEPs), cleave peptide bonds after Asn/Asp (Asx) residues. In plants, certain legumains also have ligase activity that catalyzes biosynthesis of Asx-containing cyclic peptides. An example is the biosynthesis of MCoTI-I/II, a squash family-derived cyclic trypsin inhibitor, which involves splicing to remove the N-terminal prodomain and then N-to-C-terminal cyclization of the mature domain. To identify plant legumains responsible for the maturation of these cyclic peptides, we have isolated and characterized a legumain involved in splicing, McPAL1, from Momordica cochinchinensis (Cucurbitaceae) seeds. Functional studies show that recombinantly expressed McPAL1 displays a pH-dependent, trimodal enzymatic profile. At pH 4 to 6, McPAL1 selectively catalyzed Asp-ligation and Asn-cleavage, but at pH 6.5 to 8, Asn-ligation predominated. With peptide substrates containing N-terminal Asn and C-terminal Asp, such as is found in precursors of MCoTI-I/II, McPAL1 mediates proteolysis at the Asn site and then ligation at the Asp site at pH 5 to 6. Also, McPAL1 is an unusually stable legumain that is tolerant of heat and high pH. Together, our results support that McPAL1 is a splicing legumain at acidic pH that can mediate biosynthesis of MCoTI-I/II. We purport that the high thermal and pH stability of McPAL1 could have applications for protein engineering.

Sympatric Yaks and Plateau Pikas Promote Microbial Diversity and Similarity by the Mutual Utilization of Gut Microbiota.

Interactions between species provide the basis for understanding coexisting mechanisms. The plateau pika (Ochotona curzoniae) and the yak (Bos grunniens) are considered competitors because they have shared habitats and consumed similar food on the Qinghai-Tibetan Plateau for more than 1 million years. Interestingly, the population density of plateau pikas increases with yak population expansion and subsequent overgrazing. To reveal the underlying mechanism, we sequenced the fecal microbial 16S rDNA from both sympatric and allopatric pikas and yaks. Our results indicated that sympatry increased both gut microbial diversity and similarity between pikas and yaks. The abundance of Firmicutes, Proteobacteria, Cyanobacteria, and Tenericutes decreased, while that of Verrucomicrobia increased in sympatric pikas. As for sympatric yaks, Firmicutes, Bacteroidetes, and Spirochaetes significantly increased, while Cyanobacteria, Euryarchaeota, and Verrucomicrobia significantly decreased. In sympatry, plateau pikas acquired 2692 OTUs from yaks, and yaks obtained 453 OTUs from pikas. The predominant horizontally transmitted bacteria were Firmicutes, Bacteroidetes, Verrucomicrobia, and Proteobacteria. These bacteria enhanced the enrichment of pathways related to prebiotics and immunity for pikas, such as heparin sulfate, heparin, chitin disaccharide, chondroitin-sulfate-ABC, and chondroitin-AC degradation pathways. In yaks, the horizontally transmitted bacteria enhanced pathways related to hepatoprotection, xenobiotic biodegradation, and detoxification. Our results suggest that horizontal transmission is a process of selection, and pikas and yaks tend to develop reciprocity through the horizontal transmission of gut microbiota.

Nγ -Hydroxyasparagine: A Multifunctional Unnatural Amino Acid That is a Good P1 Substrate of Asparaginyl Peptide Ligases.

Peptidyl asparaginyl ligases (PALs) are powerful tools for peptide macrocyclization. Herein, we report that a derivative of Asn, namely Nγ -hydroxyasparagine or Asn(OH), is an unnatural P1 substrate of PALs. By Asn(OH)-mediated cyclization, we prepared cyclic peptides as new matrix metalloproteinase 2 (MMP2) inhibitors displaying the hydroxamic acid moiety of Asn(OH) as the key pharmacophore. The most potent cyclic peptide (Ki =2.8±0.5 nM) was built on the hyperstable tetracyclic scaffold of rhesus theta defensin-1. The Asn(OH) residue in the cyclized peptides can also be readily oxidized to Asp. By this approach, we synthesized several bioactive Asp-containing cyclic peptides (MCoTI-II, kB2, SFTI, and integrin-targeting RGD peptides) that are otherwise difficult targets for PAL-catalyzed cyclization owing to unfavorable kinetics of the P1-Asp substrates. This study demonstrates that substrate engineering is a useful strategy to expand the application of PAL ligation in the synthesis of therapeutic cyclic peptides.