Biology subjects

Rose, S.

Publications and source records attributed to Rose, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Social attention and coordinated movement produce dissociable spectral and network changes during dyadic improvisational dance

Dance is increasingly delivered as an intervention for social and affective functioning, but a live dyadic encounter confounds several distinct ingredients - social attention, verbal engagement, coordinated movement, and in-the-moment generation - so its neural effects cannot be assigned to any one of them. We recorded dual-brain mobile EEG from adult human participants of both sexes (16 female, 2 male) and a dance instructor across six five-minute conditions that vary these ingredients independently: solitary rest, direct eye gaze, conversation, teacher-led and participant-led mirroring, and free improvisation, with concurrent head-mounted accelerometry. Referencing each condition to eye gaze - socially engaging but nearly motionless - rather than to solitary rest partitioned the response. The components proved dissociable. Nonverbal social attention alone reorganized the power spectrum, flattening the aperiodic exponent (g = -0.97) and desynchronizing alpha-band oscillations (g = -0.90), while leaving inter-regional connectivity unchanged (amplitude g = +0.15; phase g = +0.11). Verbal engagement added high-frequency amplitude coupling (g = +2.61) without further spectral change. Coordinated movement added a decrease in theta/alpha coupling involving the frontoparietal-control network (g = -0.59), the signature of an externally directed state, alongside a further spectral shift (g = -0.61). Movement-vigor covariates, corticokinematic coherence, and aperiodic-adjusted power indicated that the spectral effects were cortical rather than myogenic. Four weeks of improvisational dance training increased empathic responsiveness to others' distress relative to a movie control (g = 0.82). Coordinated social movement thus acts on the brain componentially, with mutual attention rather than motor load establishing the engaged state.

neuroscience↗

bioRxiv2026

Dendritic cell PD-L2 restrains intratumoral CD8+ T cell immunity

Blockade of inhibitory PD-1 signaling on T cells is a cornerstone of cancer immunotherapy, with current strategies targeting PD-1 or its ligand PD-L1. However, PD-1 engages an alternative ligand, PD-L2, whose role in tumor immunity remains poorly defined. Here, we show that PD-L2 is upregulated on intratumoral CCR7+ conventional dendritic cells (cDCs) in both mouse and human melanoma. Using genetic mouse models enabling selective ablation of PD-L1 or PD-L2 in cDCs, we identify a division of labor between these ligands: PD-L1 controls the size of the progenitor CD8 T cell pool in tumor-draining lymph nodes by modulating stem-like CD8 T cells, whereas PD-L2 limits progenitor exhausted CD8 T cell differentiation within the tumor microenvironment. Loss of PD-L2 in cDCs enhances cytotoxic CD8 T cell responses and suppresses tumor growth, particularly in tumors enriched for CCR7+ cDC1s. Consistent with this, increased CCR7+ cDC abundance is associated with poor prognosis in human cancers. Spatial transcriptomic analyses reveal co-localization of CCR7+ cDC1s and Tpex within CCL19hi niches, where cancer-associated fibroblasts serve as the predominant source of CCL19. Finally, intratumoral GM-CSF drives PD-L2 expression on CCR7+ cDCs, with Tpex and NK cells as major sources. Together, these findings establish cDC-associated PD-L1 and PD-L2 as spatially and functionally distinct checkpoints governing CD8 T cell differentiation. Our results suggest that the abundance of CCR7+ cDC1s may guide the choice between anti-PD-1 and anti-PD-L1 therapies and support the development of PD-L2-directed blockade.

immunology↗

bioRxiv2026

Amplitude without phase: interpersonal movement coordination grades inter-brain coupling

Interpersonal movement coordination is a foundational feature of human sociality, scaffolding communication, affiliation, and joint action from infancy onward. Whether this behavioral coupling is accompanied by genuine coupling between brains, rather than one manufactured by shared sensory input or motion artifact, has proven difficult to establish. We recorded dual-brain EEG with synchronized head-mounted accelerometry from participant-partner dyads (16 dyads, 155 dyad-condition recordings) across six conditions grading interpersonal coordination from stillness to fully reciprocal joint movement, using improvisational dance to elicit sustained, naturalistic, gradable coordination. Windowed cross-correlation confirmed graded, partner-specific movement coordination, including a lead-lag signature that reversed with who led. Against a pseudo-dyad surrogate, inter-brain synchrony took one specific form: amplitude-envelope coupling in the high-beta and gamma bands, robust to a leakage-corrected measure, with no phase-based or slow-band coupling. Within dyads, movement synchrony predicted the strength of this amplitude coupling (standardized beta = 0.54 in gamma), an association that strengthened monotonically with frequency, held under a dyad fixed-effects estimator, and was undiminished when the amount of movement was entered into the model. Movement synchrony and inter-brain coupling also co-fluctuated within interactions, above surrogate. Two control analyses excluded the leading alternatives: the coupling was not sensorimotor rhythm-tracking, since corticokinematic coherence was present but non-localized and not movement-selective, and not a shared movement-frequency rhythm linking the brains. These results identify interpersonal movement coordination as an organizing variable for inter-brain coupling and specify that coupling as fast-band amplitude co-modulation, not phase synchrony. Brains couple when bodies coordinate, not when they merely move together.

neuroscience↗

bioRxiv2026

The basal cell state maintains pancreatic cancers by controlling an immunosuppressive circuit

Intra-tumoral heterogeneity is a cardinal feature of solid tumors, yet how distinct cancer cell states functionally contribute to malignant and stromal diversity in situ remains poorly understood. Using mouse models to lineage-trace or genetically ablate the two predominant cancer cell states in autochthonous pancreatic ductal adenocarcinoma (PDAC), we discover that basal cancer cells are highly plastic, whereas classical cancer cells exhibit limited plasticity. Strikingly, ablation of the basal, but not the classical, state induced rapid and durable tumor collapse, driven by loss of immunosuppressive cancer-associated fibroblasts, macrophage repolarization, and reprogramming of the tumor cytokine milieu, culminating in tumor destruction by cytotoxic lymphocytes. Knockout of a single cytokine, GM-CSF, specifically in basal cells recapitulated macrophage repolarization and lymphocyte recruitment observed upon basal state ablation and shrank tumors. These results reveal the basal cell state controls an immunosuppressive cell circuit critical for PDAC maintenance, motivating therapeutic targeting of the basal cells.

cancer biology↗

bioRxiv2025

Novel polymer fixed-target microfluidic platforms with an ultra-thin moisture barrier for serial macromolecular crystallography

The advent of ultrabright fourth generation X-ray light sources, including X-ray free-electron lasers (XFELs) and diffraction limited synchrotrons, has significantly advanced the field of serial macromolecular protein crystallography (SX). SX experiments demand a continuous supply of fresh microcrystalline sample, ideally while minimizing overall sample consumption. Here, we introduce a novel, robust, and user-friendly polymer film technology that can be assembled in various configurations to encapsulate protein microcrystals and provide sample support for SX. This system provides an efficient hydration barrier over extended durations while maintaining an exceptionally low X-ray background. We have validated this technology by assessing hydration retention under both ambient and ultra-high vacuum conditions, and by evaluating its mechanical stability under XFEL pulses. Furthermore, we have demonstrated the effectiveness of this approach in two room-temperature serial crystallography studies to determine the structure of a 24 kDa Rapid Encystment Phenotype (REP24) protein from Franciscella tularensis.

biophysics↗

bioRxiv2025

Genetic risk in endolysosomal network genes correlates with endolysosomal dysfunction across neural cell types in Alzheimer's disease

Late-onset Alzheimer disease (LOAD) has a complex genomic architecture. LOAD risk variants suggest multiple pathways, including the endolysosomal network (ELN), contribute to the pathobiology of Alzheimer disease (AD). Whether genetic risk in specific pathways correlates with corresponding biological dysfunction remains largely unknown. We developed an endolysosomal pathway-specific polygenic risk score (ePRS) using 14 well-established AD risk alleles implicating ELN genes. We investigated the association between ePRS and AD neuropathology, then examined cell-specific endolysosomal morphology and transcriptomic profiles in post-mortem dorsolateral prefrontal cortex samples from donors stratified by ePRS burden. We found that the ePRS was significantly associated with AD diagnosis and neuropathological measures, comparable to a pathway-agnostic PRS despite representing far fewer loci. High ePRS correlated with increased neuronal endosome volume, number and perinuclear aggregation independent of AD pathology. Single-nucleus RNA sequencing revealed cell type-specific transcriptomic changes associated with ePRS status, influencing glutamatergic signaling, protein homeostasis, responses to DNA damage and immune function. Neurons, astrocytes, oligodendrocytes, and microglia each showed varied gene expression patterns associated with ePRS burden. Together, these results provide evidence that AD genetic risk variants harboring ELN genes correlate with endolysosomal dysfunction in human brain tissue. These findings suggest that pathway-specific genetic risk contributes to corresponding cellular pathology in AD and nominates candidate mechanisms by which ELN AD variants contribute to pathogenesis.

neuroscience↗

bioRxiv2025

Combining MicroED and native mass spectrometry for structural discovery of enzyme-biosynthetic inhibitor complexes

With the goal of accelerating the discovery of small molecule-protein complexes, we leverage fast, low-dose, event based electron counting microcrystal electron diffraction (MicroED) data collection and native mass spectrometry. This approach resolves structures of the epoxide-based cysteine protease inhibitor, and natural product, E-64, and its biosynthetic analogs bound to the model cysteine protease, papain. The combined structural power of MicroED and the analytical capabilities of native mass spectrometry (ED-MS) allows assignment of papain structures bound to E-64-like ligands with data obtained from crystal slurries soaked with mixtures of known inhibitors, and crude biosynthetic reactions. ED-MS further discriminates the highest-affinity ligand soaked into microcrystals from a broad inhibitor cocktail, and identifies multiple similarly high-affinity ligands soaked into microcrystals simultaneously. This extends to libraries of printed ligands dispensed directly onto TEM grids and later soaked with papain microcrystal slurries. ED-MS identifies papain binding to its preferred natural products, by showing that two analogues of E-64 outcompete others in binding to papain crystals, and by detecting papain bound to E-64 and an analogue from crude biosynthetic reactions, without purification. This illustrates the utility of ED-MS for natural product ligand discovery and for structure-based screening of small molecule binders to macromolecular targets.

biophysics↗

bioRxiv2025

Ambient temperature structural studies of Penicillin-binding Protein 2a of Methicillin-Resistant Staphylococcus aureus with XFELs and synchrotrons

Penicillin-binding protein 2a (PBP2a) is a transpeptidase responsible for the {beta}-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA), posing significant challenges to antibiotic therapy. PBP2as unique structural features, including its highly flexible active site and allosteric regulation, enable it to maintain catalytic activity even in the presence of {beta}-lactam antibiotics. Despite extensive characterization using cryogenic crystallography, key questions remain about its dynamic properties and conformational changes under near-physiological conditions. Room-temperature crystallography methods, particularly serial femtosecond X-ray crystallography (SFX) at XFELs, provide a powerful approach to capture these dynamics. Unlike cryogenic conditions that can constrain protein flexibility, SFX enables the study of conformational variability and interaction networks closer to physiological states. Here, we present the first room-temperature structures of PBP2a obtained using SFX to uncover insights into the enzymes flexibility, allosteric communication, and catalytic mechanisms. These findings are built upon optimized large-scale production and crystallization protocols for PBP2a, ensuring high-quality microcrystals suitable for SFX data collection. The room-temperature structures reveal novel interaction patterns, including unique salt bridge networks and dynamic structural elements absent in cryogenic studies. Furthermore, comparative analyses highlight how environmental conditions influence the conformational states of PBP2a, providing new perspectives on its resistance mechanisms. By integrating structural data from EuXFEL and LCLS, this study not only enhances our understanding of PBP2as functional dynamics but also underscores the value of room-temperature crystallography in studying antibiotic resistance. These insights could guide the design of next-generation {beta}-lactam antibiotics capable of overcoming PBP2a-mediated resistance.

biochemistry↗

bioRxiv2024

The nucleating agent crystallophore induces instant protein crystallization

The rapid preparation of homogeneous suspensions of micro- or nano-crystals is a crucial step in serial crystallography. We show how additives, such as the crystallophore (TbXo4) that acts as a molecular glue by promoting protein-protein interactions, can facilitate sample preparation for both serial synchrotron crystallography (SSX) and micro electron diffraction (3D ED). This lanthanide complex was used here for its nucleating properties to crystallize hen egg white lysozyme. SAXS monitoring indicates that crystals formed in a few minutes in low salt conditions that would not lead to spontaneous nucleation. Resulting micro- and nano-crystals were successfully used to determine the structure of the lysozyme-TbXo4 complex by SSX and 3D ED, illustrating the diffraction quality of the produced crystals and the usefulness of such compounds in the sample preparation pipeline for serial crystallography.

biophysics↗

bioRxiv2023

A chromosome-level genome assembly of Solanum chilense, a tomato wild relative associated with resistance to salinity and drought

Solanum chilense is a wild relative of tomato reported to exhibit resistance to biotic and abiotic stresses. There is potential to improve tomato cultivars via breeding with wild relatives, a process greatly accelerated by suitable genomic and genetic resources. In this study we generated a high-quality, chromosome-level, de novo assembly for the S. chilense accession LA1972 using a hybrid assembly strategy with [~]180 Gbp of Illumina short reads and [~]50 Gbp long PacBio reads. Further scaffolding was performed using Bionano optical maps and 10x Chromium(R) reads. The resulting sequences were arranged into 12 pseudomolecules using Hi-C sequencing. This resulted in a 901 Mbp assembly, with a completeness of 95%, as determined by Benchmarking with Universal Single-Copy Orthologs (BUSCO). Sequencing of RNA from multiple tissues resulting in [~]219 Gbp of reads was used to annotate the genome assembly with an RNA-Seq guided gene prediction, and for a de novo transcriptome assembly. This chromosome-level, high-quality reference genome for S. chilense accession LA1972 will support future breeding efforts for more sustainable tomato production. Gene sequences related to drought and salt resistance were compared between S. chilense and S. lycopersicum to identify amino acid variations with high potential for functional impact. These variants were subsequently analysed in 84 resequenced tomato lines across 12 different related species to explore the variant distributions. We identified a set of 7 putative impactful amino acid variants some of which may also impact on fruit development for example the ethylene-responsive transcription factor WIN1 and ethylene-insensitive protein 2. These variants could be tested for their ability to confer functional phenotypes to cultivars that have lost these variants.

bioinformatics↗

bioRxiv2022

Electrophysiological Markers of Memory Consolidation in the Human Brain when Memories are Reactivated during Sleep

Human accomplishments depend on learning, and effective learning depends on consolidation. Consolidation is the process whereby new memories are gradually stored in an enduring way in the brain so that they can be available when needed. For factual or event knowledge, consolidation is thought to progress during sleep as well as during waking states, and to be mediated by interactions between hippocampal and neocortical networks. However, consolidation is difficult to observe directly, but rather is inferred through behavioral observations. Here, we investigated overnight memory change by measuring electrical activity in and near the hippocampus. Electroencephalographic (EEG) recordings were made in five patients from electrodes implanted to determine whether a surgical treatment could relieve their seizure disorders. One night, while each patient slept in a hospital monitoring room, we recorded electrophysiological responses to 10-20 specific sounds that were presented very quietly, to avoid arousal. Half of the sounds had been associated with objects and their precise spatial locations that patients learned before sleep. After sleep, we found systematic improvements in spatial recall, replicating prior results. We assume that when the sounds were presented during sleep, they reactivated and strengthened corresponding spatial memories. Notably, the sounds also elicited oscillatory intracranial EEG activity, including increases in theta, sigma, and gamma EEG bands. Gamma responses, in particular, were consistently associated with the degree of improvement in spatial memory exhibited after sleep. We thus conclude that this electrophysiological activity in the hippocampus and adjacent medial temporal cortex reflects sleep-based enhancement of memory storage. Significance StatementSleep contributes to memory consolidation, we presume, because memories are replayed during sleep. Understanding this aspect of consolidation can help with optimizing normal learning in many contexts, and with treating memory disorders and other diseases. Here, we systematically manipulated sleep-based processing using targeted memory reactivation; brief sounds coupled with pre-sleep learning were quietly presented again during sleep, producing (a) recall improvements for specific spatial memories associated with those sounds, and (b) physiological responses in the sleep EEG. Neural activity in the hippocampus and adjacent medial temporal cortex was thus found in association with memory consolidation during sleep. These findings advance understanding of consolidation by linking beneficial memory changes during sleep to both memory reactivation and specific patterns of brain activity.

neuroscience↗

bioRxiv2021

Subtype transcriptomic profiling of myeloid cells in Alzheimer Disease brain illustrates the diversity in active microglia phenotypes

Microglia contribute to Alzheimers Disease (AD) progression and are candidate therapeutic targets. Human microglia exhibit an array of transcriptional phenotypes implying that accurate manipulation of microglial function will require clarity of their molecular states and context dependent regulation. To increase the number of microglia analyzed per subject we employed fluorescence activated nuclei sorting prior to single-nucleus RNA-seq on human prefrontal cortices. We observed microglia phenotypes previously unrecognized in human brain gene expression studies and mapped their transcriptomic relationships by trajectory inference. Three clusters were enriched for endolysosomal pathways, one of which showed differential expression of AD GWAS genes in addition to genes implicated in nucleic acid detection and interferon signaling. Analysis of the "homeostatic" microglia cluster revealed a uniquely AD subcluster. Our study demonstrates the value of deeply profiling microglia to explore the biological implications of microglia transcriptomic diversity.

neuroscience↗

bioRxiv2021

Pre-copulatory reproductive behaviours are preserved in Drosophila melanogaster infected with bacteria.

Reproduction and immunity are crucial traits that determine an animals fitness. Terminal investment hypothesis predicts that reproductive investment should increase in the face of a mortality risk caused by infection. However, due to competitive allocation of energetic resources, individuals fighting infections are expected to decrease reproductive efforts. While there is evidence for both hypotheses, the factors that determine the choice between these strategies are poorly understood. Here, we assess the impact of bacterial infection on pre-copulatory behaviours in the fruit fly Drosophila melanogaster. We found that male flies infected with six different bacteria, including pathogenic and non-pathogenic strains, show no significant differences in courtship intensity and mating success. Similarly, bacterial infections did not affect sexual receptivity in female flies. Our data suggest that pre-copulatory reproductive behaviours remain preserved in infected animals, despite the huge metabolic cost of infection.

animal behavior and cognition↗

bioRxiv2021

Molecular dissection of Carbapenem-resistant Acinetobacter baumannii circulating in Indian hospitals usingWhole Genome Sequencing

ObjectivesCarbapenem-resistant Acinetobacter baumannii (CRAB) has acquired worldwide recognition as a serious nosocomial infection. It poses a concern to hospitalized patients because of the limited therapeutic options available. Thus, we investigated the molecular epidemiology and antibiotic resistance profiles of A. baumannii isolates in India. Materials and MethodsWe characterized 306 retrospective A. baumannii clinical isolates collected from 18 centers across 10 states and 1 Union Territory of India between 2015 and 2019. Molecular epidemiology, and carbapenem resistance were studied by Whole Genome Sequencing. ResultsA total of 105 different Sequence Types (STs) were identified including 48 reported STs and 57 Novel STs. 99 isolates were classified into Clonal Complex 451 (CC451) among which ST848 and ST1956 were the common STs. Carbapenemase resistance was confirmed in all the isolates with the presence of intrinsic blaOXA-51-like genes, and the acquired blaOXA-23 and blaNDM-1 genes. ConclusionMost of the isolates were grouped under clonal complex 451. ST1053 caused an outbreak in Northern India during 2018 and 2019. Novel MLST alleles and STs were also detected, underlining an evolutionary divergence in India. The carbapenem-resistance was dominated by OXA-type carbapenemases and further surveillance of these carbapenem-resistant A. baumannii and antimicrobial stewardship should be strengthened.

genomics↗

bioRxiv2021

High-Resolution Genomic Profiling of Carbapenem-Resistant Klebsiella pneumoniae Isolates: A Multicentric Retrospective Indian Study

summaryWe report insights into genome sequences of Indian K. pneumoniae isolates, highlighting the presence of high-risk international clones and genetic pools different from those predominating in other regions. Identification of multidrug-resistant and hypervirulent K. pneumoniae elicits public health concerns. BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) is a threat to public health in India due to its high dissemination, mortality, and limited treatment options. Its genomic variability is reflected in the diversity of sequence types, virulence factors, and antimicrobial resistance (AMR) mechanisms. This study aims to characterize the clonal relationships and genetic mechanisms of resistance and virulence in CRKP isolates in India. Materials and MethodsWe characterized 344 retrospective K. pneumoniae clinical isolates collected from 8 centers across India collected in 2013-2019. Susceptibility to antibiotics was tested with VITEK 2. Capsular types, MLST, virulence genes, AMR determinants, plasmid replicon types, and a single-nucleotide polymorphism (SNP) phylogeny were inferred from their whole genome sequences. ResultsPhylogenetic analysis of the 325 Klebsiella isolates that passed QC revealed 3 groups: K. pneumoniae sensu stricto (n=307), K. quasipneumoniae (n=17), and K. varicolla (n=1). Sequencing and capsular diversity analysis of the 307 K. pneumoniae sensu stricto isolates revealed 28 sequence types, 26 K-locus types, and 11 O-locus types, with ST231, KL51, and O1V2 being predominant. blaOXA-48-like and blaNDM-1/5 were present in 73.2% and 24.4% of isolates respectively. The major plasmid replicon types associated with carbapenase genes were IncF (51.0%), and Col group (35.0%). ConclusionOur study documents for the first time the genetic diversity of K- and O-antigens circulating in India. The results demonstrate the practical applicability of genomic surveillance and its utility in tracking the population dynamics of CRKP. It alerts us to the urgency for longitudinal surveillance of these virulent and transmissible lineages. FundingThis work was supported by Official Development Assistance (ODA) funding from the National Institute of Health Research [grant number 16_136_111]. This research was commissioned by the National Institute of Health Research using Official Development Assistance (ODA) funding. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health. Conflict of InterestThe authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.

genomics↗

bioRxiv2021

P53 is a direct regulator of the immune co-stimulatory molecule CD80

Increasing evidence indicates oncogenes and tumor suppressors not only influence cell fitness but can also control the immunophenotype of cells. Here, we examined how 34 commonly mutated genes in colorectal cancer (CRC) may influence the expression of 8 key immunomodulatory proteins. To do this, we employed a functional genomics approach utilizing Pro-Code/CRISPR libraries for high-dimensional analysis. We introduced a library of 102 Pro-Code/gRNA combinations, targeting each of the 34 genes, in CT26 cells, a CRC cell model, and measured the expression of each of the immunomodulatory proteins by CyTOF mass cytometry. Notably, cells carrying a Pro-Code/CRISPR targeting the Trp53 lost expression of the immune co-stimulatory molecule CD80. Validation confirmed that Trp53 knockout resulted in the loss of CD80 and that activation of P53, through DNA damage or stabilization, resulted in CD80 upregulation. P53 ChIP-seq identified the CD80 promoter as a direct target of P53. CD80 regulation by P53 was identified in other cells, including normal epithelial cells and macrophages. Functionally, CD80 reduction caused by P53 loss led to a reduced capacity for CRC to prime antigen-specific T cells. These studies establish CD80, a canonical co-stimulatory molecule, as a direct target of the tumor suppressor and DNA damage response gene, P53.

immunology↗

bioRxiv2020

Genome analysis of clinical pneumococcal isolates to decipher sequence types, antibiotic resistance mechanisms and virulence gene profiles

IntroductionStreptococcus pneumoniae is a common cause of meningitis and pneumonia. Various studies have explored the relationship between genome and phenotypic characteristics like antibiotic resistance. MethodsFour pneumococcal isolates from clinical specimens were included. DNA was extracted and genomes were sequenced in 2x101bp layout. Standard genome analysis tools and pipelines were employed to understand phenotypic characteristics. ResultsThere was almost perfect agreement of genome-based predictions and phenotypic methods for antibiotic resistance. Novel sequence type (ST) was identified in one of the isolates. In total 29 virulence genes were common for all the isolates.

microbiology↗

bioRxiv2019

Optimization and validation of a quadruplex real-time PCR assay for the diagnosis of diphtheria

Diphtheria is caused by toxigenic strains of Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis. For diagnostic purposes, species identification and detection of toxigenic strains (diphtheria toxin (tox)-positive strains) is typically performed using end-point PCR. A faster quadruplex real-time PCR (qPCR) was recently developed (De Zoysa et al. J Med Microbiol. 2016 65(12):1521-1527). Here, we present an improvement of the quadruplex method, in which a 16S rRNA gene target was added as an internal processing control, providing confirmation of the presence of bacterial DNA in the assays. This improved qPCR method was validated using 36 bacterial isolates and 16 clinical samples. The method allows detection of the tox gene and distinguishing C. diphtheriae (including the newly described species C. belfantii) from C. ulcerans and C. pseudotuberculosis. Complete diagnostic specificity, sensitivity and experimental robustness of the method to temperature and reagent concentration variations were demonstrated. The lower limit of detection for C. diphtheriae, C. ulcerans and tox targets was 1.86 genome copies per 5 L reaction volume. Finally, the method was successfully used on two distinct qPCR technologies (LightCycler 480, Roche Diagnostics and Rotor-Gene Q, Qiagen) and in two laboratories (Institut Pasteur, Paris, France and Public Health England - National Infection Service, London, UK). This work describes validation of the improved qPCR quadruplex method and supports its implementation for the biological diagnosis of diphtheria.

microbiology↗