Rapid lateral flow immunoassay for fluorescence detection of canine distemper virus (CDV).

Canine distemper virus (CDV) is a highly contagious and potentially lethal virus that affects dogs and other members of the Canidae family, including wolves, foxes, and coyotes. Here, we present a fluorescent lateral flow immunoassay (FLFA) platform for the detection of CDV, which utilizes fluorescent microspheres - fusion protein monoclonal antibody (mAb)-labeled monoclonal antibody. The assay detected CDV within 5 min, with a detection limit threshold of 3 × 102 TCID50/mL. Notably, the assay demonstrated no cross-reactivity with canine parvovirus, canine coronavirus, canine adenovirus, feline calicivirus, feline herpesvirus, or feline parvovirus. Field and clinical applicability of the assay was evaluated using 63 field samples, including 30 canine fecal samples, 18 swab samples, and 15 blood samples. The coincidence rate between the detection results of clinical samples obtained through FLFA and reverse transcription polymerase chain reaction (RT-PCR) was 96.83%. Thus, this assay offers a significant advancement for the rapid diagnosis of CDV at the point of care.

Chromosome level genome assembly and transcriptome analysis of E11 cells infected by tilapia lake virus.

The E11 cell line, derived from striped snakehead fish (Channa striata), possesses a distinctive feature: it is persistently infected with a C-type retrovirus. Notably, it exhibits high permissiveness to piscine nodavirus and the emerging tilapia lake virus (TiLV). Despite its popularity in TiLV research, the absence of genome assembly for the E11 cell line and Channa striata has constrained research on host-virus interactions. This study aimed to fill this gap by sequencing, assembling, and annotating the E11 cell line genome. Our efforts yielded a 600.5 Mb genome including 24 chromosomes with a BUSCO score of 98.8%. In addition, the complete proviral DNA sequence of snakehead retrovirus (SnRV) was identified in the E11 cell genome. Comparative genomic analysis between the E11 cell line and another snakehead species Channa argus revealed the loss of many immune-related gene families in the E11 cell genome, indicating a compromised immune response. We also conducted transcriptome analysis of mock- and TiLV-infected E11 cells, unveiling new perspectives on virus-virus and host-virus interactions. The TiLV infection suppressed the high expression of SnRV in E11 cells, and activated some other endogenous retroviruses. The protein-coding gene comparison revealed a pronounced up-regulation of genes involved in immune response, alongside a down-regulation of genes associated with specific metabolic processes. In summary, the genome assembly and annotation of the E11 cell line provide valuable resources to understand the SnRV and facilitate further studies on nodavirus and TiLV. The RNA-seq profiles shed light on the cellular mechanisms employed by fish cells in response to viral challenges, potentially guiding the development of therapeutic strategies against TiLV in aquaculture. This study also provides the first insights into the viral transcriptome profiles of endogenous SnRV and evading TiLV, enhancing our understanding of host-virus interactions in fish.

Probing the Mechanism of Isonitrile Formation by a Non-Heme Iron(II)-Dependent Oxidase/Decarboxylase.

The isonitrile moiety is an electron-rich functionality that decorates various bioactive natural products isolated from diverse kingdoms of life. Isonitrile biosynthesis was restricted for over a decade to isonitrile synthases, a family of enzymes catalyzing a condensation reaction between l-Trp/l-Tyr and ribulose-5-phosphate. The discovery of ScoE, a non-heme iron(II) and α-ketoglutarate-dependent dioxygenase, demonstrated an alternative pathway employed by nature for isonitrile installation. Biochemical, crystallographic, and computational investigations of ScoE have previously been reported, yet the isonitrile formation mechanism remains obscure. In the present work, we employed in vitro biochemistry, chemical synthesis, spectroscopy techniques, and computational simulations that enabled us to propose a plausible molecular mechanism for isonitrile formation. Our findings demonstrate that the ScoE reaction initiates with C5 hydroxylation of (R)-3-((carboxymethyl)amino)butanoic acid to generate 1, which undergoes dehydration, presumably mediated by Tyr96 to synthesize 2 in a trans configuration. (R)-3-isocyanobutanoic acid is finally generated through radical-based decarboxylation of 2, instead of the common hydroxylation pathway employed by this enzyme superfamily.

Sperm storage in the oviduct of the Chinese pond turtle Mauremys reevesii depends on oestrogen-based suppression of the TLR2/4 immune pathway.

The long-term storage of spermatozoa in the female reproductive tract is limited by the innate immune system. Oestrogen plays a role in regulating the innate immune system. Thus, exploring the expression of genes in the Toll-like receptor (TLR) 2/4 pathway and oestrogen receptors in the oviduct of Mauremys reevesii could contribute to our understanding of the mechanism of sperm storage. In this study, three parts of the oviduct (isthmus, uterus and vagina) in three mated and unmated female turtles were used to perform immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR). Immunohistochemistry revealed that the TLR2/4 protein was mainly distributed in epithelial tissues and glandular cell membranes, and that TLR2/4 levels in the oviduct were significantly decreased in mated compared with unmated turtles. Real-time qPCR indicated that TLR2/4, myeloid differentiation factor 88 (MyD88), interleukin 1 receptor associated kinase 4 (IRAK4), TNF receptor associated factor 6 (TRAF6), interferon regulatory factor 3 (IRF3) and interleukin 6 (IL6) mRNA expression was significantly higher in the oviduct of unmated than mated turtles, whereas the opposite was true for the expression of oestrogen receptor 1 (ESR1) and progesterone receptor (PGR). These results indicate that when spermatozoa are stored in the oviduct, an increase in oestrogen suppresses the immune response induced by the TLR2/4 pathway so that spermatozoa are not removed as a foreign substance, but stored until fertilisation. The findings of this study are relevant to our understanding of the relationship between sperm storage and the innate immune system in the oviduct of reptiles.

Biochemical and crystallographic investigations into isonitrile formation by a nonheme iron-dependent oxidase/decarboxylase.

The isonitrile moiety is found in marine sponges and some microbes, where it plays a role in processes such as virulence and metal acquisition. Until recently only one route was known for isonitrile biosynthesis, a condensation reaction that brings together a nitrogen atom of l-Trp/l-Tyr with a carbon atom from ribulose-5-phosphate. With the discovery of ScoE, a mononuclear Fe(II) α-ketoglutarate-dependent dioxygenase from Streptomyces coeruleorubidus, a second route was identified. ScoE forms isonitrile from a glycine adduct, with both the nitrogen and carbon atoms coming from the same glycyl moiety. This reaction is part of the nonribosomal biosynthetic pathway of isonitrile lipopeptides. Here, we present structural, biochemical, and computational investigations of the mechanism of isonitrile formation by ScoE, an unprecedented reaction in the mononuclear Fe(II) α-ketoglutarate-dependent dioxygenase superfamily. The stoichiometry of this enzymatic reaction is measured, and multiple high-resolution (1.45-1.96 Å resolution) crystal structures of Fe(II)-bound ScoE are presented, providing insight into the binding of substrate, (R)-3-((carboxylmethyl)amino)butanoic acid (CABA), cosubstrate α-ketoglutarate, and an Fe(IV)=O mimic oxovanadium. Comparison to a previously published crystal structure of ScoE suggests that ScoE has an "inducible" α-ketoglutarate binding site, in which two residues arginine-157 and histidine-299 move by approximately 10 Å from the surface of the protein into the active site to create a transient α-ketoglutarate binding pocket. Together, data from structural analyses, site-directed mutagenesis, and computation provide insight into the mode of α-ketoglutarate binding, the mechanism of isonitrile formation, and how the structure of ScoE has been adapted to perform this unusual chemical reaction.

Bacterial metabolism rescues the inhibition of intestinal drug absorption by food and drug additives.

Food and drug products contain diverse and abundant small-molecule additives (excipients) with unclear impacts on human physiology, drug safety, and response. Here, we evaluate their potential impact on intestinal drug absorption. By screening 136 unique compounds for inhibition of the key intestinal transporter OATP2B1 we identified and validated 24 potent OATP2B1 inhibitors, characterized by higher molecular weight and hydrophobicity compared to poor or noninhibitors. OATP2B1 inhibitors were also enriched for dyes, including 8 azo (R-N=N-R') dyes. Pharmacokinetic studies in mice confirmed that FD&C Red No. 40, a common azo dye excipient and a potent inhibitor of OATP2B1, decreased the plasma level of the OATP2B1 substrate fexofenadine, suggesting that FD&C Red No. 40 has the potential to block drug absorption through OATP2B1 inhibition in vivo. However, the gut microbiomes of multiple unrelated healthy individuals as well as diverse human gut bacterial isolates were capable of inactivating the identified azo dye excipients, producing metabolites that no longer inhibit OATP2B1 transport. These results support a beneficial role for the microbiome in limiting the unintended effects of food and drug additives in the intestine and provide a framework for the data-driven selection of excipients. Furthermore, the ubiquity and genetic diversity of gut bacterial azoreductases coupled to experiments in conventionally raised and gnotobiotic mice suggest that variations in gut microbial community structure may be less important to consider relative to the high concentrations of azo dyes in food products, which have the potential to saturate gut bacterial enzymatic activity.

Macrocyclic colibactin induces DNA double-strand breaks via copper-mediated oxidative cleavage.

Colibactin is an assumed human gut bacterial genotoxin, whose biosynthesis is linked to the clb genomic island that has a widespread distribution in pathogenic and commensal human enterobacteria. Colibactin-producing gut microbes promote colon tumour formation and enhance the progression of colorectal cancer via cellular senescence and death induced by DNA double-strand breaks (DSBs); however, the chemical basis that contributes to the pathogenesis at the molecular level has not been fully characterized. Here, we report the discovery of colibactin-645, a macrocyclic colibactin metabolite that recapitulates the previously assumed genotoxicity and cytotoxicity. Colibactin-645 shows strong DNA DSB activity in vitro and in human cell cultures via a unique copper-mediated oxidative mechanism. We also delineate a complete biosynthetic model for colibactin-645, which highlights a unique fate of the aminomalonate-building monomer in forming the C-terminal 5-hydroxy-4-oxazolecarboxylic acid moiety through the activities of both the polyketide synthase ClbO and the amidase ClbL. This work thus provides a molecular basis for colibactin's DNA DSB activity and facilitates further mechanistic study of colibactin-related colorectal cancer incidence and prevention.

Structure-activity-distribution relationship study of anti-cancer antimycin-type depsipeptides.

Small-molecule natural products have been an essential source of pharmaceuticals to treat human diseases, but very little is known about their behavior inside dynamic, live human cells. Here, we demonstrate the first structure-activity-distribution relationship (SADR) study of complex natural products, the anti-cancer antimycin-type depsipeptides, using the emerging bioorthogonal Stimulated Raman Scattering (SRS) Microscopy. Our results show that the intracellular enrichment and distribution of these compounds are driven by their potency and specific protein targets, as well as the lipophilic nature of compounds.

Isonitrile Formation by a Non-Heme Iron(II)-Dependent Oxidase/Decarboxylase.

The electron-rich isonitrile is an important functionality in bioactive natural products, but its biosynthesis has been restricted to the IsnA family of isonitrile synthases. We herein provide the first structural and biochemical evidence of an alternative mechanism for isonitrile formation. ScoE, a putative non-heme iron(II)-dependent enzyme from Streptomyces coeruleorubidus, was shown to catalyze the conversion of (R)-3-((carboxymethyl)amino)butanoic acid to (R)-3-isocyanobutanoic acid through an oxidative decarboxylation mechanism. This work further provides a revised scheme for the biosynthesis of a unique class of isonitrile lipopeptides, of which several members are critical for the virulence of pathogenic mycobacteria.

Enzymatic Synthesis of the Ribosylated Glycyl-Uridine Disaccharide Core of Peptidyl Nucleoside Antibiotics.

Muraymycins belong to a family of nucleoside antibiotics that have a distinctive disaccharide core consisting of 5-amino-5-deoxyribofuranose (ADR) attached to 6'- N-alkyl-5'- C-glycyluridine (GlyU). Here, we functionally assign and characterize six enzymes from the muraymycin biosynthetic pathway involved in the core assembly that starts from uridine monophosphate (UMP). The biosynthesis is initiated by Mur16, a nonheme Fe(II)- and α-ketoglutarate-dependent dioxygenase, followed by four transferase enzymes: Mur17, a pyridoxal-5'-phosphate (PLP)-dependent transaldolase; Mur20, an aminotransferase; Mur26, a pyrimidine phosphorylase; and Mur18, a nucleotidylyltransferase. The pathway culminates in glycosidic bond formation in a reaction catalyzed by an additional transferase enzyme, Mur19, a ribosyltransferase. Analysis of the biochemical properties revealed several noteworthy discoveries including that (i) Mur16 and downstream enzymes can also process 2'-deoxy-UMP to generate a 2-deoxy-ADR, which is consistent with the structure of some muraymycin congeners; (ii) Mur20 prefers l-Tyr as the amino donor source; (iii) Mur18 activity absolutely depends on the amine functionality of the ADR precursor consistent with the nucleotidyltransfer reaction occurring after the Mur20-catalyzed aminotransfer reaction; and (iv) the bona fide sugar acceptor for Mur19 is (5' S,6' S)-GlyU, suggesting that ribosyltransfer occurs prior to N-alkylation of GlyU. Finally, a one-pot, six-enzyme reaction was utilized to generate the ADR-GlyU disaccharide core starting from UMP.