Integrated plasma proteomics identifies tuberculosis-specific diagnostic biomarkers.

BACKGROUNDNovel biomarkers to identify infectious patients transmitting Mycobacterium tuberculosis are urgently needed to control the global tuberculosis (TB) pandemic. We hypothesized that proteins released into the plasma in active pulmonary TB are clinically useful biomarkers to distinguish TB cases from healthy individuals and patients with other respiratory infections.METHODSWe applied a highly sensitive non-depletion tandem mass spectrometry discovery approach to investigate plasma protein expression in pulmonary TB cases compared to healthy controls in South African and Peruvian cohorts. Bioinformatic analysis using linear modeling and network correlation analyses identified 118 differentially expressed proteins, significant through 3 complementary analytical pipelines. Candidate biomarkers were subsequently analyzed in 2 validation cohorts of differing ethnicity using antibody-based proximity extension assays.RESULTSTB-specific host biomarkers were confirmed. A 6-protein diagnostic panel, comprising FETUB, FCGR3B, LRG1, SELL, CD14, and ADA2, differentiated patients with pulmonary TB from healthy controls and patients with other respiratory infections with high sensitivity and specificity in both cohorts.CONCLUSIONThis biomarker panel exceeds the World Health Organization Target Product Profile specificity criteria for a triage test for TB. The new biomarkers have potential for further development as near-patient TB screening assays, thereby helping to close the case-detection gap that fuels the global pandemic.FUNDINGMedical Research Council (MRC) (MR/R001065/1, MR/S024220/1, MR/P023754/1, and MR/W025728/1); the MRC and the UK Foreign Commonwealth and Development Office; the UK National Institute for Health Research (NIHR); the Wellcome Trust (094000, 203135, and CC2112); Starter Grant for Clinical Lecturers (Academy of Medical Sciences UK); the British Infection Association; the Program for Advanced Research Capacities for AIDS in Peru at Universidad Peruana Cayetano Heredia (D43TW00976301) from the Fogarty International Center at the US NIH; the UK Technology Strategy Board/Innovate UK (101556); the Francis Crick Institute, which receives funding from UKRI-MRC (CC2112); Cancer Research UK (CC2112); and the NIHR Biomedical Research Centre of Imperial College NHS.

Prevalence of persistent SARS-CoV-2 in a large community surveillance study.

Persistent SARS-CoV-2 infections may act as viral reservoirs that could seed future outbreaks1-5, give rise to highly divergent lineages6-8 and contribute to cases with post-acute COVID-19 sequelae (long COVID)9,10. However, the population prevalence of persistent infections, their viral load kinetics and evolutionary dynamics over the course of infections remain largely unknown. Here, using viral sequence data collected as part of a national infection survey, we identified 381 individuals with SARS-CoV-2 RNA at high titre persisting for at least 30 days, of which 54 had viral RNA persisting at least 60 days. We refer to these as 'persistent infections' as available evidence suggests that they represent ongoing viral replication, although the persistence of non-replicating RNA cannot be ruled out in all. Individuals with persistent infection had more than 50% higher odds of self-reporting long COVID than individuals with non-persistent infection. We estimate that 0.1-0.5% of infections may become persistent with typically rebounding high viral loads and last for at least 60 days. In some individuals, we identified many viral amino acid substitutions, indicating periods of strong positive selection, whereas others had no consensus change in the sequences for prolonged periods, consistent with weak selection. Substitutions included mutations that are lineage defining for SARS-CoV-2 variants, at target sites for monoclonal antibodies and/or are commonly found in immunocompromised people11-14. This work has profound implications for understanding and characterizing SARS-CoV-2 infection, epidemiology and evolution.

Lineage replacement and evolution captured by 3 years of the United Kingdom Coronavirus (COVID-19) Infection Survey.

The Office for National Statistics Coronavirus (COVID-19) Infection Survey (ONS-CIS) is the largest surveillance study of SARS-CoV-2 positivity in the community, and collected data on the United Kingdom (UK) epidemic from April 2020 until March 2023 before being paused. Here, we report on the epidemiological and evolutionary dynamics of SARS-CoV-2 determined by analysing the sequenced samples collected by the ONS-CIS during this period. We observed a series of sweeps or partial sweeps, with each sweeping lineage having a distinct growth advantage compared to their predecessors, although this was also accompanied by a gradual fall in average viral burdens from June 2021 to March 2023. The sweeps also generated an alternating pattern in which most samples had either S-gene target failure (SGTF) or non-SGTF over time. Evolution was characterized by steadily increasing divergence and diversity within lineages, but with step increases in divergence associated with each sweeping major lineage. This led to a faster overall rate of evolution when measured at the between-lineage level compared to within lineages, and fluctuating levels of diversity. These observations highlight the value of viral sequencing integrated into community surveillance studies to monitor the viral epidemiology and evolution of SARS-CoV-2, and potentially other pathogens.

Correction: Viral burden is associated with age, vaccination, and viral variant in a population-representative study of SARS-CoV-2 that accounts for time-since-infection-related sampling bias.

[This corrects the article DOI: 10.1371/journal.ppat.1011461.].

Viral burden is associated with age, vaccination, and viral variant in a population-representative study of SARS-CoV-2 that accounts for time-since-infection-related sampling bias.

In this study, we evaluated the impact of viral variant, in addition to other variables, on within-host viral burden, by analysing cycle threshold (Ct) values derived from nose and throat swabs, collected as part of the UK COVID-19 Infection Survey. Because viral burden distributions determined from community survey data can be biased due to the impact of variant epidemiology on the time-since-infection of samples, we developed a method to explicitly adjust observed Ct value distributions to account for the expected bias. By analysing the adjusted Ct values using partial least squares regression, we found that among unvaccinated individuals with no known prior exposure, viral burden was 44% lower among Alpha variant infections, compared to those with the predecessor strain, B.1.177. Vaccination reduced viral burden by 67%, and among vaccinated individuals, viral burden was 286% higher among Delta variant, compared to Alpha variant, infections. In addition, viral burden increased by 17% for every 10-year age increment of the infected individual. In summary, within-host viral burden increases with age, is reduced by vaccination, and is influenced by the interplay of vaccination status and viral variant.

scQCEA: a framework for annotation and quality control report of single-cell RNA-sequencing data.

BACKGROUND: Systematic description of library quality and sequencing performance of single-cell RNA sequencing (scRNA-seq) data is imperative for subsequent downstream modules, including re-pooling libraries. While several packages have been developed to visualise quality control (QC) metrics for scRNA-seq data, they do not include expression-based QC to discriminate between true variation and background noise. RESULTS: We present scQCEA (acronym of the single-cell RNA sequencing Quality Control and Enrichment Analysis), an R package to generate reports of process optimisation metrics for comparing sets of samples and visual evaluation of quality scores. scQCEA can import data from 10X or other single-cell platforms and includes functions for generating an interactive report of QC metrics for multi-omics data. In addition, scQCEA provides automated cell type annotation on scRNA-seq data using differential gene expression patterns for expression-based quality control. We provide a repository of reference gene sets, including 2348 marker genes, which are exclusively expressed in 95 human and mouse cell types. Using scRNA-seq data from 56 gene expressions and V(D)J T cell replicates, we show how scQCEA can be applied for the visual evaluation of quality scores for sets of samples. In addition, we use the summary of QC measures from 342 human and mouse shallow-sequenced gene expression profiles to specify optimal sequencing requirements to run a cell-type enrichment analysis function. CONCLUSIONS: The open-source R tool will allow examining biases and outliers over biological and technical measures, and objective selection of optimal cluster numbers before downstream analysis. scQCEA is available at https://isarnassiri.github.io/scQCEA/ as an R package. Full documentation, including an example, is provided on the package website.

An enrichment protocol and analysis pipeline for long read sequencing of the hepatitis B virus transcriptome.

Hepatitis B virus (HBV) is one of the smallest human DNA viruses and its 3.2 Kb genome encodes multiple overlapping open reading frames, making its viral transcriptome challenging to dissect. Previous studies have combined quantitative PCR and Next Generation Sequencing to identify viral transcripts and splice junctions, however the fragmentation and selective amplification used in short read sequencing precludes the resolution of full length RNAs. Our study coupled an oligonucleotide enrichment protocol with state-of-the-art long read sequencing (PacBio) to identify the repertoire of HBV RNAs. This methodology provides sequencing libraries where up to 25 % of reads are of viral origin and enable the identification of canonical (unspliced), non-canonical (spliced) and chimeric viral-human transcripts. Sequencing RNA isolated from de novo HBV infected cells or those transfected with 1.3 × overlength HBV genomes allowed us to assess the viral transcriptome and to annotate 5' truncations and polyadenylation profiles. The two HBV model systems showed an excellent agreement in the pattern of major viral RNAs, however differences were noted in the abundance of spliced transcripts. Viral-host chimeric transcripts were identified and more commonly found in the transfected cells. Enrichment capture and PacBio sequencing allows the assignment of canonical and non-canonical HBV RNAs using an open-source analysis pipeline that enables the accurate mapping of the HBV transcriptome.

Lactoferrin Inhibition of the Complex Formation between ACE2 Receptor and SARS CoV-2 Recognition Binding Domain.

The present investigation focuses on the analysis of the interactions among human lactoferrin (LF), SARS-CoV-2 receptor-binding domain (RBD) and human angiotensin-converting enzyme 2 (ACE2) receptor in order to assess possible mutual interactions that could provide a molecular basis of the reported preventative effect of lactoferrin against CoV-2 infection. In particular, kinetic and thermodynamic parameters for the pairwise interactions among the three proteins were measured via two independent techniques, biolayer interferometry and latex nanoparticle-enhanced turbidimetry. The results obtained clearly indicate that LF is able to bind the ACE2 receptor ectodomain with significantly high affinity, whereas no binding to the RBD was observed up to the maximum "physiological" lactoferrin concentration range. Lactoferrin, above 1 µM concentration, thus appears to directly interfere with RBD-ACE2 binding, bringing about a measurable, up to 300-fold increase of the KD value relative to RBD-ACE2 complex formation.

Variations in Aspects of Neural Precursor Cell Neurogenesis in a Human Model of HSV-1 Infection.

Encephalitis, the most significant of the central nervous system (CNS) diseases caused by Herpes simplex virus 1 (HSV-1), may have long-term sequelae in survivors treated with acyclovir, the cause of which is unclear. HSV-1 exhibits a tropism toward neurogenic niches in CNS enriched with neural precursor cells (NPCs), which play a pivotal role in neurogenesis. NPCs are susceptible to HSV-1. There is a paucity of information regarding the influence of HSV-1 on neurogenesis in humans. We investigated HSV-1 infection of NPCs from two individuals. Our results show (i) HSV-1 impairs, to different extents, the proliferation, self-renewing, and, to an even greater extent, migration of NPCs from these two subjects; (ii) The protective effect of the gold-standard antiherpetic drug acyclovir (ACV) varies with viral dose and is incomplete. It is also subject to differences in terms of efficacy of the NPCs derived from these two individuals. These results suggest that the effects of HSV-1 may have on aspects of NPC neurogenesis may vary among individuals, even in the presence of acyclovir, and this may contribute to the heterogeneity of cognitive sequelae across encephalitis survivors. Further analysis of NPC cell lines from a larger number of individuals is warranted.

Systems level profiling of chemotherapy-induced stress resolution in cancer cells reveals druggable trade-offs.

Cancer cells can survive chemotherapy-induced stress, but how they recover from it is not known. Using a temporal multiomics approach, we delineate the global mechanisms of proteotoxic stress resolution in multiple myeloma cells recovering from proteasome inhibition. Our observations define layered and protracted programs for stress resolution that encompass extensive changes across the transcriptome, proteome, and metabolome. Cellular recovery from proteasome inhibition involved protracted and dynamic changes of glucose and lipid metabolism and suppression of mitochondrial function. We demonstrate that recovering cells are more vulnerable to specific insults than acutely stressed cells and identify the general control nonderepressable 2 (GCN2)-driven cellular response to amino acid scarcity as a key recovery-associated vulnerability. Using a transcriptome analysis pipeline, we further show that GCN2 is also a stress-independent bona fide target in transcriptional signature-defined subsets of solid cancers that share molecular characteristics. Thus, identifying cellular trade-offs tied to the resolution of chemotherapy-induced stress in tumor cells may reveal new therapeutic targets and routes for cancer therapy optimization.

B Lymphocytes, but Not Dendritic Cells, Efficiently HIV-1 Trans Infect Naive CD4+ T Cells: Implications for the Viral Reservoir.

Insight into the establishment and maintenance of HIV-1 infection in resting CD4+ T cell subsets is critical for the development of therapeutics targeting the HIV-1 reservoir. Although the frequency of HIV-1 infection, as quantified by the frequency of HIV-1 DNA, is lower in CD4+ naive T cells (TN) than in the memory T cell subsets, recent studies have shown that TN harbor a large pool of replication-competent virus. Interestingly, however, TN are highly resistant to direct (cis) HIV-1 infection in vitro, in particular to R5-tropic HIV-1, as TN do not express CCR5. In this study, we investigated whether TN could be efficiently HIV-1 trans infected by professional antigen-presenting B lymphocytes and myeloid dendritic cells (DC) in the absence of global T cell activation. We found that B cells, but not DC, have a unique ability to efficiently trans infect TNin vitro In contrast, both B cells and DC mediated HIV-1 trans infection of memory and activated CD4+ T cells. Moreover, we found that TN isolated from HIV-1-infected nonprogressors (NP) harbor significantly disproportionately lower levels of HIV-1 DNA than TN isolated from progressors. This is consistent with our previous finding that antigen-presenting cells (APC) derived from NP do not efficiently trans infect CD4+ T cells due to alterations in APC cholesterol metabolism and cell membrane lipid raft organization. These findings support that B cell-mediated trans infection of TN with HIV-1 has a more profound role than previously considered in establishing the viral reservoir and control of HIV-1 disease progression.IMPORTANCE The latent human immunodeficiency virus type 1 (HIV-1) reservoir in persons on antiretroviral therapy (ART) represents a major barrier to a cure. Although most studies have focused on the HIV-1 reservoir in the memory T cell subset, replication-competent HIV-1 has been isolated from TN, and CCR5-tropic HIV-1 has been recovered from CCR5neg TN from ART-suppressed HIV-1-infected individuals. In this study, we showed that CCR5neg TN are efficiently trans infected with R5-tropic HIV-1 by B lymphocytes, but not by myeloid dendritic cells. Furthermore, we found that TN isolated from NP harbor no or significantly fewer copies of HIV-1 DNA than those from ART-suppressed progressors. These findings support that B cell-mediated trans infection of TN with HIV-1 has a more profound role than previously considered in establishing the viral reservoir and control of HIV-1 disease progression. Understanding the establishment and maintenance of the HIV-1 latent reservoir is fundamental for the design of effective treatments for viral eradication.

Discrete bHLH transcription factors play functionally overlapping roles in pigmentation patterning in flowers of Antirrhinum majus.

Floral pigmentation patterning is important for pollinator attraction as well as aesthetic appeal. Patterning of anthocyanin accumulation is frequently associated with variation in activity of the Myb, bHLH and WDR transcription factor complex (MBW) that regulates anthocyanin biosynthesis. Investigation of two classic mutants in Antirrhinum majus, mutabilis and incolorata I, showed they affect a gene encoding a bHLH protein belonging to subclade bHLH-2. The previously characterised gene, Delila, which encodes a bHLH-1 protein, has a bicoloured mutant phenotype, with residual lobe-specific pigmentation conferred by Incolorata I. Both Incolorata I and Delila induce expression of the anthocyanin biosynthetic gene DFR. Rosea 1 (Myb) and WDR1 proteins compete for interaction with Delila, but interact positively to promote Incolorata I activity. Delila positively regulates Incolorata I and WDR1 expression. Hierarchical regulation can explain the bicoloured patterning of delila mutants, through effects on both regulatory gene expression and the activity of promoters of biosynthetic genes like DFR that mediate MBW regulation. bHLH-1 and bHLH-2 proteins contribute to establishing patterns of pigment distribution in A. majus flowers in two ways: through functional redundancy in regulating anthocyanin biosynthetic gene expression, and through differences between the proteins in their ability to regulate genes encoding transcription factors.

Dendritic cells focus CTL responses toward highly conserved and topologically important HIV-1 epitopes.

BACKGROUND: During early HIV-1 infection, immunodominant T cell responses to highly variable epitopes lead to the establishment of immune escape virus variants. Here we assessed a type 1-polarized monocyte-derived dendritic cell (MDC1)-based approach to selectively elicit cytotoxic T lymphocyte (CTL) responses against highly conserved and topologically important HIV-1 epitopes in HIV-1-infected individuals from the Thailand RV254/SEARCH 010 cohort who initiated antiretroviral therapy (ART) during early infection (Fiebig stages I-IV). METHODS: Autologous MDC1 were used as antigen presenting cells to induce in vitro CTL responses against HIV-1 Gag, Pol, Env, and Nef as determined by flow cytometry and ELISpot assay. Ultra-conserved or topologically important antigens were respectively identified using the Epigraph tool and a structure-based network analysis approach and compared to overlapping peptides spanning the Gag proteome. FINDINGS: MDC1 presenting either the overlapping Gag, Epigraph, or Network 14-21mer peptide pools consistently activated and expanded HIV-1-specific T cells to epitopes identified at the 9-13mer peptide level. Interestingly, some CTL responses occurred outside known or expected HLA associations, providing evidence of new HLA-associated CTL epitopes. Comparative analyses demonstrated more sequence conservation among Epigraph antigens but a higher magnitude of CTL responses to Network and Gag peptide groups. Importantly, CTL responses against topologically constrained Gag epitopes contained in both the Network and Gag peptide pools were selectively enhanced in the Network pool-initiated cultures. INTERPRETATION: Our study supports the use of MDC1 as a therapeutic strategy to induce and focus CTL responses toward putative fitness-constrained regions of HIV-1 to prevent immune escape and control HIV-1 infection. FUNDING: A full list of the funding sources is detailed in the Acknowledgment section of the manuscript.

Evolution of Advanced Chronic Lymphoid Leukemia Unveiled by Single-Cell Transcriptomics: A Case Report.

Genetic and transcriptional heterogeneity of Chronic lymphocytic leukaemia (CLL) limits prevention of disease progression. Longitudinal single-cell transcriptomics represents the state-of-the-art method to profile the disease heterogeneity at diagnosis and to inform about disease evolution. Here, we apply single-cell RNA-seq to a CLL case, sampled at diagnosis and relapse, that was treated with FCR (Fludarabine, Cyclophosphamide, Rituximab) and underwent a dramatic decrease in CD19 expression during disease progression. Computational analyses revealed a major switch in clones' dominance during treatment. The clone that expanded at relapse showed 17p and 3p chromosomal deletions, and up-regulation of pathways related to motility, cytokine signaling and antigen presentation. Single-cell RNA-seq uniquely revealed that this clone was already present at low frequency at diagnosis, and it displays feature of plasma cell differentiation, consistent with a more aggressive phenotype. This study shows the benefit of single-cell profiling of CLL heterogeneity at diagnosis, to identify clones that might otherwise not be recognized and to determine the best treatment options.

Synthesis of non-nucleoside anti-viral cyclopropylcarboxacyl hydrazones and initial anti-HSV-1 structure-activity relationship studies.

The synthesis of a lead anti-viral cyclopropyl carboxy acyl hydrazone 4F17 (5) and three sequential arrays of structural analogues along with the initial assessment and optimization of the antiviral pharmacophore against the herpes simplex virus type 1 (HSV-1) are reported.

Interferon lambda 4 impacts the genetic diversity of hepatitis C virus.

Hepatitis C virus (HCV) is a highly variable pathogen that frequently establishes chronic infection. This genetic variability is affected by the adaptive immune response but the contribution of other host factors is unclear. Here, we examined the role played by interferon lambda-4 (IFN-λ4) on HCV diversity; IFN-λ4 plays a crucial role in spontaneous clearance or establishment of chronicity following acute infection. We performed viral genome-wide association studies using human and viral data from 485 patients of white ancestry infected with HCV genotype 3a. We demonstrate that combinations of host genetic variants, which determine IFN-λ4 protein production and activity, influence amino acid variation across the viral polyprotein - not restricted to specific viral proteins or HLA restricted epitopes - and modulate viral load. We also observed an association with viral di-nucleotide proportions. These results support a direct role for IFN-λ4 in exerting selective pressure across the viral genome, possibly by a novel mechanism.

Illumina and Nanopore methods for whole genome sequencing of hepatitis B virus (HBV).

Advancing interventions to tackle the huge global burden of hepatitis B virus (HBV) infection depends on improved insights into virus epidemiology, transmission, within-host diversity, drug resistance and pathogenesis, all of which can be advanced through the large-scale generation of full-length virus genome data. Here we describe advances to a protocol that exploits the circular HBV genome structure, using isothermal rolling-circle amplification to enrich HBV DNA, generating concatemeric amplicons containing multiple successive copies of the same genome. We show that this product is suitable for Nanopore sequencing as single reads, as well as for generating short-read Illumina sequences. Nanopore reads can be used to implement a straightforward method for error correction that reduces the per-read error rate, by comparing multiple genome copies combined into a single concatemer and by analysing reads generated from plus and minus strands. With this approach, we can achieve an improved consensus sequencing accuracy of 99.7% and resolve intra-sample sequence variants to form whole-genome haplotypes. Thus while Illumina sequencing may still be the most accurate way to capture within-sample diversity, Nanopore data can contribute to an understanding of linkage between polymorphisms within individual virions. The combination of isothermal amplification and Nanopore sequencing also offers appealing potential to develop point-of-care tests for HBV, and for other viruses.

Update in the classification and the role of intra-arterial stenting in the management of carotid body paragangliomas.

BACKGROUND: To review the Shamblin classification of carotid body paragangliomas (CBPs) and the role of intra-arterial stenting in their surgical management. METHODS: Retrospective case series of 20 patients with 28 CBPs that were surgically resected at our center. Intra-arterial stenting was performed in Shamblin II and II classes. RESULTS: The mean follow-up was 47.8 months. Five (17.9%) tumors were Shamblin class I, 15 (53.6%) were class II, and 8 (28.6%) were class III. Thirteen (68.4%) CBPs were associated with other paragangliomas. The internal carotid artery (ICA) was stented preoperatively in eight (28.6%) cases and occluded in four (14.3%) cases. The tumor extended to the jugular foramen in six cases (21.4%). Intraoperatively, there was an ICA injury in one case of Shamblin II CBP in the present era. CONCLUSIONS: The proposed classification enables the clinician to plan the management of the ICA and the right approach. Stenting of the ICA gives a chance for complete tumor removal with arterial preservation.

The Circulating Transcriptome as a Source of Biomarkers for Melanoma.

The circulating transcriptome is a valuable source of cancer biomarkers, which, with the exception of microRNAs (miRNAs), remains relatively unexplored. To elucidate which RNAs are present in plasma from melanoma patients and which could be used to distinguish cancer patients from healthy individuals, we used next generation sequencing (NGS), and validation was carried out by qPCR and/or ddPCR. We identified 442 different microRNAs in samples, eleven of which were differentially expressed (p < 0.05). Levels of miR-134-5p and miR-320a-3p were significantly down-regulated (p < 0.001) in melanoma samples (n = 96) compared to healthy controls (n = 28). Differentially expressed protein-encoding mRNA 5'-fragments were enriched for the angiopoietin, p21-activated kinase (PAK), and EIF2 pathways. Levels of ATM1, AMFR, SOS1, and CD109 gene fragments were up-regulated (p < 0.001) in melanoma samples (n = 144) compared to healthy controls (n = 41) (AUC = 0.825). Over 40% of mapped reads were YRNAs, a class of non-coding RNAs that to date has been little explored. Expression levels of RNY3P1, RNY4P1, and RNY4P25 were significantly higher in patients with stage 0 disease than either healthy controls or more advanced stage disease (p < 0.001). In conclusion, we have identified a number of novel RNA biomarkers, which, most importantly, we validated in multi-center retrospective and prospective cohorts, suggesting potential diagnostic use of these RNA species.

Single cell RNA-seq reveals profound transcriptional similarity between Barrett's oesophagus and oesophageal submucosal glands.

Barrett's oesophagus is a precursor of oesophageal adenocarcinoma. In this common condition, squamous epithelium in the oesophagus is replaced by columnar epithelium in response to acid reflux. Barrett's oesophagus is highly heterogeneous and its relationships to normal tissues are unclear. Here we investigate the cellular complexity of Barrett's oesophagus and the upper gastrointestinal tract using RNA-sequencing of single cells from multiple biopsies from six patients with Barrett's oesophagus and two patients without oesophageal pathology. We find that cell populations in Barrett's oesophagus, marked by LEFTY1 and OLFM4, exhibit a profound transcriptional overlap with oesophageal submucosal gland cells, but not with gastric or duodenal cells. Additionally, SPINK4 and ITLN1 mark cells that precede morphologically identifiable goblet cells in colon and Barrett's oesophagus, potentially aiding the identification of metaplasia. Our findings reveal striking transcriptional relationships between normal tissue populations and cells in a premalignant condition, with implications for clinical practice.