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Integrating hotspot dynamics and centers of diversity: a review of Indo-Australian Archipelago biogeographic evolution and conservation.

The Indo-Australian Archipelago (IAA) is the world's preeminent marine biodiversity hotspot, distinguished by its exceptional species richness in tropical shallow waters. This biodiversity has spurred extensive research into its evolutionary and biogeographic origins. Two prominent theoretical frameworks dominate explanations for the IAA's biodiversity: the "centers-of hypotheses" and the "hopping hotspot hypothesis". The "centers-of hypotheses" posits that specific regions serve as key sources of IAA biodiversity, either through the accumulation and overlap of species from external areas or via elevated rates of local speciation. In contrast, the "hopping hotspot hypothesis" asserts that biodiversity hotspots are dynamic, shifting across geological timescales in response to tectonic and environmental changes. This review synthesizes these contrasting perspectives into an integrated framework, the "Dynamic Centers Hypothesis," which proposes that as biodiversity hotspots migrate over time, the IAA's role in generating and sustaining biodiversity has evolved, with varying contributions from different sources dominating distinct historical phases. By synthesizing the evidence for both hypotheses and incorporating recent findings, including fossil and phylogeography data, we propose the "Dynamic Centers Hypothesis" as a comprehensive and unifying explanation for the IAA's biodiversity. The review further explores biogeographic delineation, aligning tropical marine realms with the IAA's evolutionary trajectory, from its Tethyan roots to its modern Indo-West Pacific dominance. Looking forward, advances in DNA barcoding and genomics are uncovering vast cryptic diversity, revolutionizing our comprehension of IAA phylogeographic history. These discoveries underscore the imperative for a multidimensional conservation framework, integrating phylogenetic, and functional diversity, to preserve this biodiversity hotspot amid escalating global change.

Biogeography↗

Cloning and validating systems for high throughput molecular recording.

Molecular recording technologies record and store information about cellular history. Lineage tracing is one form of molecular recording and produces information describing cellular trajectories during mammalian development, differentiation and maintenance of adult stem cell niches, and tumor evolution. Our molecular recorder technology utilizes CRISPR-Cas9 barcode editing to generate mutations in genomically integrated, engineered DNA cassettes, which are read out by single-cell RNA sequencing and used to produce high-resolution lineage trees. Here, we describe optimized cloning and validation procedures to construct the molecular recorder lineage tracing system. We include information on considerations of technology design, cloning procedures, the generation of lineage tracing cell lines, and time course experiments to assess their performance.

Cloning, Molecular↗

Optical coding of mammalian cells using semiconductor quantum dots.

Cell-based assays are widely used to screen compounds and study complex phenotypes. Few methods exist, however, for multiplexing cellular assays or labeling individual cells in a mixed cell population. We developed a generic encoding method for cells that is based on peptide-mediated delivery of quantum dots (QDs) into live cells. The QDs are nontoxic and photostable and can be imaged using conventional fluorescence microscopy or flow cytometry systems. We created unique fluorescent codes for a variety of mammalian cell types and show that our encoding method has the potential to create > 100 codes. We demonstrate that QD cell codes are compatible with most types of compound screening assays including immunostaining, competition binding, reporter gene, receptor internalization, and intracellular calcium release. A multiplexed calcium assay for G-protein-coupled receptors using QDs is demonstrated. The ability to spectrally encode individual cells with unique fluorescent barcodes should open new opportunities in multiplexed assay development and greatly facilitate the study of cell/cell interactions and other complex phenotypes in mixed cell populations.

Animals↗

Metazoan OXPHOS gene families: evolutionary forces at the level of mitochondrial and nuclear genomes.

Mitochondrial and nuclear DNAs contribute to encode the whole mitochondrial protein complement. The two genomes possess highly divergent features and properties, but the forces influencing their evolution, even if different, require strong coordination. The gene content of mitochondrial genome in all Metazoa is in a frozen state with only few exceptions and thus mitochondrial genome plasticity especially concerns some molecular features, i.e. base composition, codon usage, evolutionary rates. In contrast the high plasticity of nuclear genomes is particularly evident at the macroscopic level, since its redundancy represents the main feature able to introduce genetic material for evolutionary innovations. In this context, genes involved in oxidative phosphorylation (OXPHOS) represent a classical example of the different evolutionary behaviour of mitochondrial and nuclear genomes. The simple DNA sequence of Cytochrome c oxidase I (encoded by the mitochondrial genome) seems to be able to distinguish intra- and inter-species relations between organisms (DNA Barcode). Some OXPHOS subunits (cytochrome c, subunit c of ATP synthase and MLRQ) are encoded by several nuclear duplicated genes which still represent the trace of an ancient segmental/genome duplication event at the origin of vertebrates.

Animals↗

A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors.

Endogenous small RNAs (miRNAs) regulate gene expression by mechanisms conserved across metazoans. While the number of verified human miRNAs is still expanding, only few have been functionally annotated. To perform genetic screens for novel functions of miRNAs, we developed a library of vectors expressing the majority of cloned human miRNAs and created corresponding DNA barcode arrays. In a screen for miRNAs that cooperate with oncogenes in cellular transformation, we identified miR-372 and miR-373, each permitting proliferation and tumorigenesis of primary human cells that harbor both oncogenic RAS and active wild-type p53. These miRNAs neutralize p53-mediated CDK inhibition, possibly through direct inhibition of the expression of the tumor-suppressor LATS2. We provide evidence that these miRNAs are potential novel oncogenes participating in the development of human testicular germ cell tumors by numbing the p53 pathway, thus allowing tumorigenic growth in the presence of wild-type p53.

Cells, Cultured↗

Systematic discovery of pathogen effector functions across human pathogens and pathways.

Pathogens deploy effector proteins to exploit host cell biology, and most effector open reading frames (ORFs) are rapidly evolving and lack functional annotation. We developed the effector ORFeome (eORFeome), a scalable functional genomics platform encompassing 3,835 effector ORFs from diverse viruses, bacteria, and parasites. High-throughput barcoded screens across nuclear factor κB (NF-κB), apoptosis, p53, cGAS-STING, and major histocompatibility complex class I (MHC class I) pathways revealed novel pathway-modulating functions for hundreds of uncharacterized eORFs, unexpected activities of known effectors, and distinct pathway-specific functions encoded by single ORFs. Illustrating the power of this approach, we identified HHV6A U14 as a p53 antagonist, HHV7 U21 as a dual-function STING antagonist and MHC-I antigen display inhibitor, and adenoviral 13.6K/i-leader protein as a de novo-evolved TAP inhibitor that suppresses MHC-I display. These results establish a general framework for systematic effector annotation, uncover new mechanisms of host-pathogen interaction across kingdoms, and highlight pathogen effectors as a versatile toolkit for rewiring and probing human cellular pathways.

Humans↗

Multiome Perturb-seq unlocks scalable discovery of integrated perturbation effects on the transcriptome and epigenome.

Single-cell CRISPR screens link genetic perturbations to transcriptional states, but high-throughput methods connecting these induced changes to their regulatory foundations are limited. Here, we introduce Multiome Perturb-seq, extending single-cell CRISPR screens to simultaneously measure perturbation-induced changes in gene expression and chromatin accessibility. We apply Multiome Perturb-seq in a CRISPRi screen of 13 chromatin remodelers in human RPE-1 cells, achieving efficient assignment of sgRNA identities to single nuclei via an improved method for capturing barcode transcripts from nuclear RNA. We organize expression and accessibility measurements into coherent programs describing the integrated effects of perturbations on cell state, finding that ARID1A and SUZ12 knockdowns induce programs enriched for developmental features. Modeling of perturbation-induced heterogeneity connects accessibility changes to changes in gene expression, highlighting the value of multimodal profiling. Overall, our method provides a scalable and simply implemented system to dissect the regulatory logic underpinning cell state. A record of this paper's transparent peer review process is included in the supplemental information.

Humans↗

Single-cell glycome and transcriptome profiling enabled by a library of anti-glycan antibodies.

Glycans play critical roles in cellular processes and clinical applications, but they remain difficult to study due to a shortage of well-characterized anti-glycan reagents and high-throughput technologies for glycome profiling, especially ones capable of single-cell resolution. To meet these needs, we generated a database of 650 anti-glycan antibody sequences, recombinantly expressed a library of 154 antibodies, and extensively characterized their binding properties using glycan microarrays. In addition to providing valuable information and resources for the field, the sequence database and microarray data also enabled development of "Glycomic-seq" (Glycome profiling via multiplexed immunoglobulins combined with sequencing), a DNA-barcoded anti-glycan antibody platform that enables high-throughput, single-cell profiling of both RNA and cell-surface glycan expression. Using Glycomic-seq, we profiled two isogenic colorectal cancer cell lines. The results revealed various glycans associated with cancer stem cells and metastasis, demonstrating the power of integrating glycomic information with multi-omic efforts to discover biomarkers and therapeutic targets.

Polysaccharides↗

Emerging Principles in Spatial Functional Genomics.

Spatial transcriptomic and proteomic atlases have enabled mapping of gene programs within intact tissues, but these measurements remain largely descriptive and do not define the mechanisms controlling tissue biology. Pooled CRISPR screening provides scalable causal interrogation of gene function but remains largely confined to dissociated systems that lack spatial context. In vivo spatial functional genomics (SFG) bridges these approaches by integrating genetic perturbations with in situ transcriptomic and proteomic readouts to measure gene function within intact tissue ecosystems. By preserving spatial organization, SFG enables interpretation of perturbations through effects on cell-cell interactions, diffusible signals, multicellular niches, and tissue architecture. Here, we outline key design axes of SFG: perturbation strategy, barcoding strategy, and phenotypic readout. We discuss computational challenges, including spatial autocorrelation, neighborhood dependence, and context-aware null modeling, and highlight how SFG reveals non-cell-autonomous, architecture-dependent mechanisms of gene function, advancing toward predictive models of tissue organization and gene function.

Genomics↗

Vector-based RNAi approaches for stable, inducible and genome-wide screens.

RNA interference (RNAi) has revolutionized the study of biology and offers numerous applications in basic biology as well as in drug discovery research. Since the discovery of RNAi, several tools have been developed to enable loss-of-function studies in mammalian systems. The efficacy of RNAi is dependent on specific and versatile RNAi triggers that have evolved to enable transient, stable and in-vivo applications. Recently developed genome-wide short hairpin RNA (shRNA) and microRNA-adapted short hairpin RNA (shRNAmir) libraries incorporate advances in shRNA design and molecular 'barcodes' to enable more complex RNAi screens and the opportunity to progress to more complex genetics in whole animals.

Animals↗

Trichoderma biodiversity in China: evidence for a North to South distribution of species in East Asia.

Towards assessing the biodiversity and biogeography of Trichoderma, we have analyzed the occurrence of Trichoderma species in soil and litter from four areas in China: North (Hebei province), South-East (Zhejiang province), West (Himalayan, Tibet) and South-West (Yunnan province). One hundred and thirty five isolates were grouped according to tentative morphological identification. A representative 64 isolates were verified at the species level by the oligonucleotide barcode program TrichO Key v.1.0 and the custom BLAST server TrichoBLAST, using sequences of the ITS1 and 2 region of the rRNA cluster and from the longest intron of the tef1 (translation elongation factor 1-alpha) gene. Eleven known species (Trichoderma asperellum, T. koningii, T. atroviride, T. viride, T. velutinum, T. cerinum, T. virens, T. harzianum, T. sinensis, T. citrinoviride, T. longibrachiatum) and two putative new species (T. sp. C1, and T. sp. C2), distinguished from known species both by morphological characters and phylogenetic analysis, were identified. A significant difference in the occurrence of these species was found between the North (Hebei) and South-West (Yunnan) areas, which correlates with previously reported species distributions in Siberia and South-East Asia, respectively. As in previous studies, T. harzianum accounted for almost half of the biodiversity; although, in this study, it was exclusively found in the North, and was predominantly represented by an ITS1 and 2 haplotype, which has so far been rarely found elsewhere. This study therefore reveals a North-South gradient in species distribution in East Asia, and identifies Northern China as a potential center of origin of a unique haplotype of T. harzianum.

China↗

Differences in straggling rates between two genera of dove lice (Insecta: Phthiraptera) reinforce population genetic and cophylogenetic patterns.

Differences in dispersal abilities have been implicated for causing disparate evolutionary patterns between Columbicola and Physconelloides lice (Insecta: Phthiraptera). However, no study has documented straggling (when lice are found on atypical hosts) rates within these lineages. We used the fact that the Galapagos Hawk, Buteo galapagoensis (Gould) (Falconiformes) feeds on the Galapagos Dove Zenaida galapagoensis Gould (Columbiformes) within an ecologically simplified setting. The Galapagos Dove is the only typical host of Columbicola macrourae (Wilson) and Physconelloides galapagensis (Kellogg and Huwana) in Galapagos. We quantitatively sampled and found these lice on both bird species. A DNA barcoding approach confirmed that stragglers were derived from Galapagos doves. We also collected a Bovicola sp. louse, likely originating from a goat (Capra hircus). On hawks, C. macrourae was significantly more prevalent than P. galapagensis. On doves, the two lice were equally prevalent and abundant. Differences in prevalence on hawks was a function of differences in straggling rate between lice, and not a reflection of their relative representation within the dove population. This provides further evidence that differences in dispersal abilities may drive differences in the degree of cospeciation in Columbicola and Phyconelloides lice, which have become model systems in evolutionary biology.

Animals↗

A revision of the interrelationships of Schistosoma including the recently described Schistosoma guineensis.

In light of the recently described human schistosome Schistosoma guineensis and recent phylogenetic studies of the genus Schistosoma, a revision of the interrelationships of the members of this genus is needed. This paper adds to previous phylogenetic studies on the family Schistosomatidae and offers the most up to date and robust phylogeny of the group based on complete small and large nuclear subunit rRNA genes and partial mitochondrial cox1, incorporating most of the 21 species of Schistosoma. Our findings show that the group retains the same topology as that resolved in previous studies except Schistosoma margrebowiei was resolved as the sister taxon to all others in the Schistosoma haematobium species group and S. guineensis was placed as sister species to both Schistosoma bovis and Schistosoma curassoni. The S. haematobium species group contains eight species of which many are of significant medical and veterinary importance. Additionally, many of these species have been shown to hybridise both in the wild and experimentally, making the correct identification and recognition of species very important. A pairwise comparison of cox1 among Schistosoma species suggests this gene alone would fail as a reliable barcode for species identification. Phylogenetic results clearly treat Schistosoma intercalatum and S. guineensis as separate taxa with each more closely related evolutionarily to S. haematobium than to each other. The study also highlights the problems associated with wrongly attributed sequences on public databases such as GenBank.

Africa South of the Sahara↗

An enhanced multisegment RT-PCR method for influenza A virus sequencing: Improved performance and reduced preparation time over traditional methods.

Influenza A viruses (IAVs) remain a major global health threat, affecting both human and animal populations. Whole-genome sequencing is essential for monitoring viral evolution, zoonotic transmission, and emerging variants. However, conventional RT-PCR methods often result in incomplete gene coverage, amplification biases, and reduced sequencing accuracy, particularly in clinical samples. We developed a robust In-house method for IAV full-genome sequencing using the Oxford Nanopore Technologies (ONT) long-read sequencing platform. This method integrates an in-house multisegment Reverse Transcription PCR (RT-PCR) method with a streamlined 2-pool primer design targeting all eight IAV gene segments. RNA extracted from clinical and stock virus samples was reverse-transcribed and amplified using Superscript IV-based chemistry, followed by magnetic bead purification to ensure high-quality amplicons. Sequencing libraries were prepared with the Native Barcoding Kit 24 (SQK-NBD114.24) and sequenced on R10.4.1 flow cells on the MinION MK1C device. Data analysis using the Iterative Refinement Meta-Assembler (IRMA) confirmed improved read depth, uniform coverage, and complete genome recovery. Compared to conventional methods, our In-House Multisegment 2-Pool (IH-MS2P) RT-PCR method generated higher numbers of matched read counts, minimized chimeric artifacts, and delivered superior genome coverage across human, swine, and avian isolates. This optimized RT-PCR method provides a high-performance, time-efficient, and portable solution for influenza genomics, demonstrating robust applicability even with clinical samples of low RNA yield.

Influenza A virus↗

Phage Immunoprecipitation and Sequencing-a Versatile Technique for Mapping the Antibody Reactome.

Characterizing the antibody reactome for circulating antibodies provide insight into pathogen exposure, allergies, and autoimmune diseases. This is important for biomarker discovery, clinical diagnosis, and prognosis of disease progression, as well as population-level insights into the immune system. The emerging technology phage display immunoprecipitation and sequencing (PhIP-seq) is a high-throughput method for identifying antigens/epitopes of the antibody reactome. In PhIP-seq, libraries with sequences of defined lengths and overlapping segments are bioinformatically designed using naturally occurring proteins and cloned into phage genomes to be displayed on the surface. These libraries are used in immunoprecipitation experiments of circulating antibodies. This can be done with parallel samples from multiple sources, and the DNA inserts from the bound phages are barcoded and subjected to next-generation sequencing for hit determination. PhIP-seq is a powerful technique for characterizing the antibody reactome that has undergone rapid advances in recent years. In this review, we comprehensively describe the history of PhIP-seq and discuss recent advances in library design and applications.

Humans↗

A robust toolkit for functional profiling of the yeast genome.

Study of mutant phenotypes is a fundamental method for understanding gene function. The construction of a near-complete collection of yeast knockouts (YKO) and the unique molecular barcodes (or TAGs) that identify each strain has enabled quantitative functional profiling of Saccharomyces cerevisiae. By using these TAGs and the SGA reporter, MFA1pr-HIS3, which facilitates conversion of heterozygous diploid YKO strains into haploid mutants, we have developed a set of highly efficient microarray-based techniques, collectively referred as dSLAM (diploid-based synthetic lethality analysis on microarrays), to probe genome-wide gene-chemical and gene-gene interactions. Direct comparison revealed that these techniques are more robust than existing methods in functional profiling of the yeast genome. Widespread application of these tools will elucidate a comprehensive yeast genetic network.

Gene Expression Profiling↗

ChemPerturb-seq screen identifies a small molecule cocktail enhancing human beta cell survival after subcutaneous transplantation.

Traditional chemical screens have focused on a single assay per screen, making them labor intensive and costly. Here, we combined a chemical screen with single-cell RNA sequencing (scRNA-seq) to perform Chemical Perturb-seq (ChemPerturb-seq), enabling a systematic analysis of the molecular changes of human beta cells upon individual small molecule treatments. Using this platform, we performed an in vivo barcoded screen and discovered a small molecule cocktail, including beta-lipotropin 61-91, insulin growth factor-1, and prostaglandin E2, with which preconditioning human beta cells and primary islets significantly enhanced function and survival when transplanted subcutaneously to female, but not to male, mice. We identified two additional molecules, serotonin and histamine, that promote islet function when transplanted subcutaneously to male mice using ChemPerturb-seq. Such small molecule cocktails could be applied to improve the current FDA-approved islet transplantation procedure. Finally, we developed an artificial intelligence (AI)-powered website, ChemPerturbDB, which provides user-friendly open access analysis of the extensive ChemPerturb-seq dataset.

Humans↗