Biological Studies Research Articles

Enrollment on upfront high-risk neuroblastoma trials by race, ethnicity, and poverty status: A report from the Children's Oncology Group.

It is not clear whether trial access disparities exist in the Children's Oncology Group (COG). Here, we leverage a cohort of children with high-risk neuroblastoma (HR-NBL) enrolled on the COG ANBL00B1 neuroblastoma biology study to examine subsequent enrollment to upfront COG therapeutic trials by race, ethnicity, and proxied poverty status. Among 1917 children with HR-NBL enrolled on ANBL00B1, 696 (36.3%) subsequently enrolled on an upfront therapeutic trial with no difference by race, ethnicity, or proxied poverty status. In neuroblastoma, trial access disparities are not comparable to adult oncology, and efforts to advance equity should prioritize other mechanisms of survival disparities.

An improved chromosome-level genome assembly and annotation of Echeneis naucrates

Echeneis naucrates, as known as live sharksucker, is famous for the behavior of attaching to hosts using a highly modified dorsal fin with oval-shaped sucking disc. Here, we generated an improved high-quality chromosome-level genome assembly of E. naucrates using Illumina short reads, PacBio long reads and Hi-C data. Our assembled genome spans 572.85 Mb with a contig N50 of 23.19 Mb and is positioned to 24 pseudo-chromosomes. Additionally, at least one telomere was identified for 23 out of 24 chromosomes. Furthermore, we identified a total of 22,161 protein-coding genes, of which 21,402 genes (96.9%) were annotated successfully with functions. The combination of ab initio predictions and Repbase-based searches revealed that 15.57% of the assembled E. naucrates genome was identified as repetitive sequences. The completeness of the genome assembly and the gene annotation were estimated to be 97.5% and 95.4% with BUSCO analyses. This work enhances the utility of the live sharksucker genome and provides a valuable groundwork for the future study of genomics, biology and adaptive evolution in this species.

Oxidovanadium(V) Schiff Base Complexes Derived from Chiral 3-amino-1,2-propanediol Enantiomers: Synthesis, Spectroscopic Studies, Catalytic and Biological Activity

Oxidovanadium(V) complexes, [(+)VOL1-5] and [(–)VOL1-5], with chiral tetradentate Schiff bases, which are products of monocondensation of S(‒)-3-amino-1,2-propanediol or R(+)-3-amino-1,2-propanediol with salicylaldehyde derivatives, have been synthesized. Different spectroscopic methods, viz. 1H and 51V NMR, IR, UV-Vis, and circular dichroism, as well as elemental analysis, have been used for their detailed characterization. Furthermore, the epoxidation of styrene, cyclohexene, and two monoterpenes, S(‒)-limonene and (‒)-α-pinene, using two oxidants, aqueous 30% H2O2 or tert-butyl hydroperoxide (TBHP) in decane, has been studied with catalytic amounts of all complexes. Finally, biological cytotoxicity studies have also been performed with these oxidovanadium(V) compounds for comparison with cis-dioxidomolybdenum(VI) Schiff base complexes with the same chiral ligands, as well as to determine the cytoprotection against the oxidative damage caused by 30% H2O2 in the HT-22 hippocampal neuronal cells in the range of their 10–100 μM concentration.

Synthesis, Biological and Molecular Docking Studies of Thiazole-Thiadiazole derivatives as potential Anti-Tuberculosis Agents.

Tuberculosis remains a global health threat, with increasing infection rates and mortality despite existing anti-TB drugs. The present work focuses on the research findings regarding the development and evaluation of thiadiazole-linked thiazole derivatives as potential anti-tuberculosis agents. We present the synthesis data and confirm the compound structures using spectroscopic techniques. The current study reports twelve thiazole-thiadiazole compounds (5a-5l) for their anti-tuberculosis and related bioactivities. This paper emphasizes compounds 5g, 5i, and 5l, which exhibited promising MIC values, leading to further in silico and interaction analysis. Pharmacophore mapping data included in the present analysis identified tubercular ThyX as potential drug targets. The compounds were evaluated for anti-tubercular activity using standard methods, revealing significant MIC values, particularly compound 5l, with the best MIC value of 7.1285 µg/ml. Compounds 5g and 5i also demonstrated moderate to good MIC values against M. tuberculosis (H37Ra). Structural inspection of the docked poses revealed interactions such as hydrogen bonds, halogen bonds, and interactions containing Pi electron cloud, shedding light on conserved interactions with residues like Arg 95, Cys 43, His 69, and Arg 87 from the tubercular ThyX enzyme.

Maternal-Fetal Exposure to Antibiotics: Levels, Mother-to-Child Transmission, and Potential Health Risks.

Due to its widespread applications in various fields, antibiotics are continuously released into the environment and ultimately enter the human body through diverse routes. Meanwhile, the unreasonable use of antibiotics can also lead to a series of adverse outcomes. Pregnant women and developing fetuses are more susceptible to the influence of external chemicals than adults. The evaluation of antibiotic exposure levels through questionnaire surveys or prescriptions in medical records and biomonitoring-based data shows that antibiotics are frequently prescribed and used by pregnant women around the world. Antibiotics may be transmitted from mothers to their offspring through different pathways, which then adversely affect the health of offspring. However, there has been no comprehensive review on antibiotic exposure and mother-to-child transmission in pregnant women so far. Herein, we summarized the exposure levels of antibiotics in pregnant women and fetuses, the exposure routes of antibiotics to pregnant women, and related influencing factors. In addition, we scrutinized the potential mechanisms and factors influencing the transfer of antibiotics from mother to fetus through placental transmission, and explored the adverse effects of maternal antibiotic exposure on fetal growth and development, neonatal gut microbiota, and subsequent childhood health. Given the widespread use of antibiotics and the health threats posed by their exposure, it is necessary to comprehensively track antibiotics in pregnant women and fetuses in the future, and more in-depth biological studies are needed to reveal and verify the mechanisms of mother-to-child transmission, which is crucial for accurately quantifying and evaluating fetal health status.

Structures, mechanisms and applications of RNA-centric CRISPR-Cas13.

Prokaryotes are equipped with a variety of resistance strategies to survive frequent viral attacks or invading mobile genetic elements. Among these, CRISPR-Cas surveillance systems are abundant and have been studied extensively. This Review focuses on CRISPR-Cas type VI Cas13 systems that use single-subunit RNA-guided Cas endonucleases for targeting and subsequent degradation of foreign RNA, thereby providing adaptive immunity. Notably, distinct from single-subunit DNA-cleaving Cas9 and Cas12 systems, Cas13 exhibits target RNA-activated substrate RNase activity. This Review outlines structural, biochemical and cell biological studies toward elucidation of the unique structural and mechanistic principles underlying surveillance effector complex formation, precursor CRISPR RNA (pre-crRNA) processing, self-discrimination and RNA degradation in Cas13 systems as well as insights into suppression by bacteriophage-encoded anti-CRISPR proteins and regulation by endogenous accessory proteins. Owing to its programmable ability for RNA recognition and cleavage, Cas13 provides powerful RNA targeting, editing, detection and imaging platforms with emerging biotechnological and therapeutic applications.

Fluorescent Schiff Bases with Phenothiazine and Coumarin Moieties: Synthesis, Characterization, Photophysical, Electrochemical, Computational and Biological Studies.

We present the design and synthesis of four new schiff bases viz., two each of phenothiazine and coumarin derivatives. The structures were proposed based on FT-IR, 1H NMR, 13C NMR and mass spectral data. The photophysical, solvatochromic and electrochemical studies of all the compounds were carried out. Furthermore, theoretical studies such as density functional theory (DFT) were carried out to gain a better understanding of the compounds' intramolecular charge transfer properties and electronic structures. Good agreement was noticed between the HOMO-LUMO energy gap obtained from DFT studies and that calculated from absorption threshold wavelengths. All the compounds showed Stokes shifts in the range of 6345-11,405cm-1. These findings showed that the new schiff bases could be considered as attractive candidates for use in the development of OLEDs, organic electrical devices and optoelectronic devices. Newly synthesized compounds were tested for biological activities. When compared to conventional standards, gallic acid and indomethacin, the schiff bases showed better antioxidant and antiinflammatory activities, respectively.

The complete mitochondrial genome of the sand bubbler crab Scopimera longidactyla Shen 1932 (Decapoda: Dotillidae) and its phylogenetic analysis

The sand bubbler crab, Scopimera longidactyla Shen, 1932 (Arthropoda: Malacostraca: Decapoda: Thoracotremata: Dotillidae), is commonly found along tropical and subtropical sandy shores of China, Korea, and Taiwan. Ecologically, it plays an important role in the productivity of sandy shores through their feeding and burrowing activities. In this study, the first complete mitochondrial genome (mitogenome) of S. longidactyla was analyzed using next-generation sequencer. Its mitogenome, circular in structure, spans 15,965 bp with a GC content of 29.97%, consisting of 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and one putative control region. Its mitogenome arrangement and composition are identical to its two congeners, S. globosa and S. intermedia. Phylogenetic analysis fully supports for the monophyly of the genus Scopimera and the sister relationship between S. longidactyla and S. globosa. The complete mitogenome of S. longidactyla and its phylogenetic implications will provide valuable insights for further studies in phylogenetic and evolutionary biology.

Establishment of Transgene-Free Porcine Induced Pluripotent Stem Cells.

Although protocols to generate authentic transgene-free mouse and human induced pluripotent stem cells (iPSCs) are now well established, standard methods for reprogramming porcine somatic cells still suffer from low efficiency and transgene retention. The Basic Protocol describes reprogramming procedures to establish transgene-free porcine iPSCs (PiPSCs) from porcine fibroblasts. This method uses episomal plasmids encoding POU5F1, SOX2, NANOG, KLF4, SV40LT, c-MYC, LIN28A, and microRNA-302/367, combined with an optimized medium, to establish PiPSC lines. Support protocols describe the establishment and characterization of clonal PiPSC lines, as well as the preparation of feeder cells and EBNA1 mRNA. This optimized, step-by-step approach tailored to this species enables the efficient derivation of PiPSCs in ∼4 weeks. The establishment of transgene-free PiPSCs provides a new and valuable model for studies of larger mammalian species' development, disease, and regenerative biology. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Reprogramming of porcine fibroblasts with episomal plasmids Support Protocol 1: Preparation of mouse embryonic fibroblasts for feeder layer Support Protocol 2: Preparation of in vitro-transcribed EBNA1 mRNA Support Protocol 3: Establishment of clonal porcine induced pluripotent stem cell (PiPSC) lines Support Protocol 4: PiPSC characterization: Genomic DNA PCR and RT-PCR Support Protocol 5: PiPSC characterization: Immunostaining.

Macromolecules with predominant β-pleated sheet proteins in extracellular vesicles released from Raphanus sativus L. var. caudatus Alef microgreens induce DNA damage-mediated apoptosis in HCT116 colon cancer cells

Plant-derived bioactive macromolecules (i.e., proteins, lipids, and nucleic acids) were prepared as extracellular vesicles (EVs). Plant-derived EVs are gaining pharmaceutical research interest because of their bioactive components and delivery properties. The spherical nanosized EVs derived from Raphanus sativus L. var. caudatus Alef microgreens previously showed antiproliferative activity in HCT116 colon cancer cells from macromolecular compositions (predominantly proteins). To understand the mechanism of action, the biological activity studies, i.e., antiproliferation, cellular biochemical changes, DNA conformational changes, DNA damage, apoptotic nuclear morphological changes, apoptosis induction, and apoptotic pathways, were determined by neutral red uptake assay, synchrotron radiation-based Fourier transform infrared microspectroscopy, circular dichroism spectroscopy, comet assay, 4′,6-diamidino-2-phenylindole (DAPI) staining, flow cytometry, and caspase activity assay, respectively. EVs inhibited HCT116 cell growth in concentration- and time-dependent manners, with a half-maximal inhibitory concentration of 675.4 ± 33.8 μg/ml at 48 h and a selectivity index of 1.5 ± 0.076. HCT116 treated with EVs mainly changed the cellular biochemical compositions in the nucleic acids and carbohydrates region. The DNA damage caused no changes in DNA conformation. The apoptotic nuclear morphological changes were associated with the increased apoptotic cell population. The apoptotic cell death was induced by both extrinsic and intrinsic pathways. EVs have potential as antiproliferative bioparticles.

New nanometric metal complexes based on thiazole derivative as potential antitumor and antimicrobial agents: Full structural elucidation, docking simulation and biological activity studies

Novel Mn(II), Ni(II), Zr(IV) and Cd(II) nanosized complexes of thiazole Schiff base derivative (HL) were synthesized for further medicinal applications. The inspected complexes were fully characterized using elemental and thermal analyses in addition to infrared spectroscopy (IR) and nuclear magnetic resonance (1HNMR). Electronic absorption spectra, X-ray powder diffraction (XRD), transmittance electron microscopy (TEM), molar conductance and magnetic moment were also measured and explained. The brought about data revealed that the ligand–metal chelation happened through the N atom of N = N group, and the ortho-dehydrogenated phenolic O atom to form the chelates where the metal binds with one molecule of the parent ligand. All applied spectroscopic and analytical techniques confirmed tetrahedral geometry in Mn(II) and Ni(II) complexes and octahedral geometry for Cd(II) and Zr(IV) complexes. The ligand, Mn(II), Ni(II), and Zr(IV) chelates' XRD patterns indicated an amorphous character where the Cd(II) complex's nature is crystalline. Transmission electron microscopy (TEM) images of the prepared compounds showed that the surface morphology is uniform and homogeneous. Also, the metal ions distribute through the complexes' surface in a nanosized nature. The metal complexes have been simulated by docking in the co-crystal structures of the liver cancer and breast cancer proteins with PDB IDs, 2JW2 and 3S7S, successively. Antibacterial, antifungal and anticancer activities for the ligand that has been produced and its complexes were achieved. The most encouraging antitumor results were acquired by Ni(II) chelate which exhibited an IC50 of 23.52 µg/ml contra MCF-7 cells (human breast cancer) which is lower than the reference 5-Fluorouracil (5-FU, IC50 = 28.00 µg/ml).

Does intraspecific variation in Trissolcus japonicus affect its response to non-target hosts?

AbstractHost specificity is a key criterion for the implementation of any biological control program. In parasitoid wasps, host specificity is mediated by their response to chemical cues directly and indirectly associated with their hosts during foraging. Although intraspecific variation in foraging behaviour is documented, it is rarely integrated into risk assessment studies of potential biological control agents, as is the case for Trissolcus japonicus (Ashmead) (Hymenoptera: Scelionidae), an Asian egg parasitoid and biological control agent of the invasive Halyomorpha halys (Stål) (Hemiptera: Pentatomidae). In the present study, we compared the behavioural response of T. japonicus females of an adventive line present in Switzerland and an Asian line (Beijing USDA), recently mass released in Italy, to cues of its target host H. halys and non-target host Pentatoma rufipes (L.) (Hemiptera: Pentatomidae). We observed minimal differences in host acceptance, exploitation and suitability in no-choice tests. In contrast, the behavioural response to host contact kairomones left on leaf substrates significantly differed between the two lines. While both lines preferred chemical footprints of H. halys in comparison to P. rufipes, females of the Beijing USDA line responded more strongly to host kairomones. The observed intraspecific variation in behavioural traits highlights the relevance of considering genetically distinct parasitoid lines and their host foraging behaviour in biological risk assessment studies. However, the implications of intraspecific variation in parasitoid foraging behaviour for biological control of a pest and non-target effects in the field remain challenging to predict.

Identification, characterization and biological studies of Kinesine spindle protein inhibitor, benzo[b] [1, 4]oxazine-6-carboxamide derivative, via 3D-pharmacophore virtual screening

Identification, characterization and biological studies of Kinesine spindle protein inhibitor, benzo[b] [1, 4]oxazine-6-carboxamide derivative, via 3D-pharmacophore virtual screening

Two new rhodamine-based fluorescent probes with thiophen/selenophene acrylate group for selective detection of Cys in living cells

Cysteine (Cys), one of the biological thiols, has long been an important sensing target. In this work, two new rhodamine-based fluorescent probes with thiophen/selenophene acrylate group (Rh-S and Rh-Se) have been obtained and fully characterized. Their sensing behaviors have been researched by UV-Vis absorption and fluorescence spectra, which display high sensitivity and good selectivity for the detection of Cys with low detection limit (0.268/0.270 μM) and fast response time (30/40 min), and exhibit a "turn on" response toward Cys. The possible sensing mechanism has been proposed and confirmed by HRMS and 1H NMR. Biological studies reveal that probe Rh-S has low cytotoxicity and good biocompatibility, thereby could be further used to determine Cys in living cells.

Phytochemical profiling, antidiabetic, antiobesity, and antibacterial activities of Cometes abyssinica

This study aims to complete my chemical and biological studies on Cometes abyssinica (CA). The chemical profile was reported by LC-ESI-TOF-MS and GC-MS analyses. Antidiabetic (α-Glucosidase and a-amylase inhibitory activity assay), antiobesity (Lipase inhibitory activity assay), and antibacterial activity (agar well diffusion assay) were determined. The GC-MS analysis led to the identification of 27 hydrocarbons representing 93.4% of the total hydrocarbon content in hexane extract. 11-Octadecenoic acid, methyl ester (28.07%), citronellol (9.31%), hexadecanoic acid, methyl ester (7.64%), phytol (5.93%), and 10-epi-γ-eudesmol (4.91%) were the major components. The LC-ESI-TOF-MS analyses of aqueous-methanolic extract led to the identification of 99 secondary metabolites including phenolics, flavonoids, alkaloids, and vitamins. The percentages of inhibition of the n-hexane, chloroform, ethyl acetate, and methanol extracts of CA were evaluated against α-glucosidase, α-amylase, and pancreatic lipase enzymes at different concentrations. Hexane extract showed the most powerful α-glucosidase and α-amylase inhibitory activity with IC50 value of 0.22 and 0.27 mg/ml comparing with acarbose (IC50 0.43 and 0.38), respectively. Among all CA extracts, only ethyl acetate extract revealed potent antiobesity as a lipase inhibitory activity with IC50 value of 0.079 mg/ml comparing with orlistat (IC50 0.11 mg/ml). CA extracts showed mild-weak antibacterial effect against Pseudomonas aeruginosa and Bacillus cereus. KEY WORDS: Cometes abyssinica, Chemical constituents, α-Amylase inhibitors, α-Glucosidase inhibitors, Lipase inhibitors Bull. Chem. Soc. Ethiop. 2024, 38(4), 1051-1067.                                                           DOI: https://dx.doi.org/10.4314/bcse.v38i4.19

Synthesis, Identification and Biological Activity Study of Some New Spiro-Isatin Derivatives

     Spiro-isatin derivatives have been the subject of extensive research due to their diverse pharmacological properties and potential therapeutic applications. These molecules are characterized by a spirocyclic structure that includes an isatin moiety and one or more heterocyclic rings, resulting in a unique and versatile molecular architecture. In this work, a series of new spiro-isatin derivatives B1-12 were synthesized over two steps. The first step included a condensation reaction between isatin and various aromatic amines, affording the corresponding Schiff bases A1-12 in high yields (up to 96%). In the second step, A1-12 underwent a reaction with glycine, resulting in the formation of B1-12 with yields varying between 65 and 92%. The structures of the compounds were confirmed by FT-IR and 1H NMR spectroscopy. A few of the produced compounds were examined for antibacterial and antioxidant properties. 

Emerging roles of long non-coding RNAs in human epilepsy.

Genome-scale biological studies conducted in the post-genomic era have revealed that two-thirds of human genes do not encode proteins. Most functional non-coding RNA transcripts in humans are products of long non-coding RNA (lncRNA) genes, an abundant but still poorly understood class of human genes. As a result of their fundamental and multitasking regulatory roles, lncRNAs are associated with a wide range of human diseases, including neurological disorders. Approximately 40% of lncRNAs are specifically expressed in the brain, and many of them exhibit distinct spatiotemporal patterns of expression. Comparative genomics approaches have determined that 65%-75% of human lncRNA genes are primate-specific and hence can be posited as a contributing potential cause of the higher-order complexity of primates, including human, brains relative to those of other mammals. Although lncRNAs present important mechanistic examples of epileptogenic functions, the human/primate specificity of lncRNAs questions their relevance in rodent models. Here, we present an in-depth review that supports the contention that human lncRNAs are direct contributors to the etiology and pathogenesis of human epilepsy, as a means to accelerate the integration of lncRNAs into clinical practice as potential diagnostic biomarkers and therapeutic targets. Meta-analytically, the major finding of our review is the commonality of lncRNAs in epilepsy and cancer pathogenesis through mitogen-activated protein kinase (MAPK)-related pathways. In addition, neuroinflammation may be a relevant part of the common pathophysiology of cancer and epilepsy. LncRNAs affect neuroinflammation-related signaling pathways such as nuclear factor kappa- light- chain- enhancer of activated B cells (NF-κB), Notch, and phosphatidylinositol 3- kinase/ protein kinase B (Akt) (PI3K/AKT), with the NF-κB pathway being the most common. Besides the controversy over lncRNA research in non-primate models, whether neuroinflammation is triggered by injury and/or central nervous system (CNS) toxicity during epilepsy modeling in animals or is a direct consequence of epilepsy pathophysiology needs to be considered meticulously in future studies.

Synthesis, characterization, and antibacterial studies of some of first transition series metals and zinc complexes with mixed ligands of trimethoprim-isatin and nitrogen base

Complexes of mixed-ligand Schiff bases [trimetho-prim (TMP) with isatin] (L1) and nitrogen bases [2,2′-bipyridine (bipy)] (L2), [1,10-phenanthroline (phen) (L3)] with metal chlorides: M = Cr3+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+ were prepared and characterized by metal content determination (%), FT-IR, electronic, nuclear magnetic resonance, mass spectroscopy, magnetic susceptibility, conductance measurements and elemental analysis (CHN). The bacterial activity of the ligands and their metal compounds was evaluated via various microorganisms. Infrared spectra of Schiff base showed an azomethine peak shifted in complexes that coincide synthesis and coordination of Schiff base through the nitrogen atom of azomethine C=N) and the oxygen atom of the carbonyl group of L1 with metal ions. L2, L3 (bipy, phen) coordinated through the di imine nitrogen atoms. Conductance dimensions suggested the non-conductance nature of all the compounds except Cr(III) complexes, this outcome fits in with the molar conductance value estimated for 1:1 electrolyte in DMF. Magnetic susceptibility analysis and electronic spectra indicated an octahedral geometry for all complexes. Biological study for prepared compounds have been evaluated towards pathogenic microbes Staphylococcus aureus and Bacillus subtilis. Some compounds have stronger antibacterial action than Schiff base towards tested microbes. KEY WORDS: Antimicrobial, Heterocyclic compounds, Schiff base, Trimethoprim drug, 2,2-Bipyridyl, 1,10-Phenanthroline Bull. Chem. Soc. Ethiop. 2024, 38(4), 1013-1025.                                                            DOI: https://dx.doi.org/10.4314/bcse.v38i4.16

MiRSNP rs188493331: A key player in genetic control of microRNA-induced pathway activation in hypertrophic scars and keloids.

Our study aims to delineate the miRSNP-microRNA-gene-pathway interactions in the context of hypertrophic scars (HS) and keloids. We performed a computational biology study involving differential expression analysis to identify genes and their mRNAs in HS and keloid tissues compared to normal skin, identifying key hub genes and enriching their functional roles, comprehensively analyzing microRNA-target genes and related signaling pathways through bioinformatics, identifying MiRSNPs, and constructing a pathway-based network to illustrate miRSNP-miRNA-gene-signaling pathway interactions. Our results revealed a total of 429 hub genes, with a strong enrichment in signaling pathways related to proteoglycans in cancer, focal adhesion, TGF-β, PI3K/Akt, and EGFR tyrosine kinase inhibitor resistance. Particularly noteworthy was the substantial crosstalk between the focal adhesion and PI3K/Akt signaling pathways, making them more susceptible to regulation by microRNAs. We also identified specific miRNAs, including miRNA-1279, miRNA-429, and miRNA-302e, which harbored multiple SNP loci, with miRSNPs rs188493331 and rs78979933 exerting control over a significant number of miRNA target genes. Furthermore, we observed that miRSNP rs188493331 shared a location with microRNA302e, microRNA202a-3p, and microRNA20b-5p, and these three microRNAs collectively targeted the gene LAMA3, which is integral to the focal adhesion signaling pathway. The study successfully unveils the complex interactions between miRSNPs, miRNAs, genes, and signaling pathways, shedding light on the genetic factors contributing to HS and keloid formation.

Nanobody in a Double "Y"-Shaped Assembly: A Promising Candidate for Lateral Flow Immunoassays.

Derived from camelid heavy-chain antibodies, nanobodies (Nbs) are the smallest natural antibodies and are an ideal tool in biological studies because of their simple structure, high yield, and low cost. Nbs possess significant potential for developing highly specific and user-friendly diagnostic assays. Despite offering considerable advantages in detection applications, knowledge is limited regarding the exclusive use of Nbs in lateral flow immunoassay (LFIA) detection. Herein, we present a novel double "Y" architecture, achieved by using the SpyTag/SpyCatcher and Im7/CL7 systems. The double "Y" assemblies exhibited a significantly higher affinity for their epitopes, as particularly evident in the reduced dissociation rate. An LFIA employing double "Y" assemblies was effectively used to detect the severe acute respiratory syndrome coronavirus-2 N protein, with a detection limit of at least 500 pg/mL. This study helps broaden the array of tools available for the development of Nb-based diagnostic techniques.