Oncogene. 2024 Aug 18. doi: 10.1038/s41388-024-03136-8. Online ahead of print.

ABSTRACT

Chemoresistance is an important cause of treatment failure in bladder cancer, and identifying genes that confer drug resistance is an important step toward developing new therapeutic strategies to improve treatment outcomes. In the present study, we show that gemcitabine plus cisplatin (GEM/DDP) therapy induces NF-κB signaling, which promotes p65-mediated transcriptional activation of OIP5. OIP5 recruits the E3 ubiquitin ligase TRIP12 to bind to and degrade the phosphatase PPP1CB, thereby enhancing the transcription factor activity of YBX1. This in turn upregulates drug-resistance-related genes under the transcriptional control of YBX1, leading to chemoresistance. Moreover, PPP1CB degradation can enhance the phosphorylation activity of IKKβ, triggering the NF-κB signaling cascade, which further stimulates OIP5 gene expression, thus forming a negative feedback regulatory loop. Consistently, elevated OIP5 expression was associated with chemoresistance and poor prognosis in patients with bladder cancer. Furthermore, we used a CRISPR/Cas9-based engineered gene circuit, which can monitor the progression of chemoresistance in real-time, to induce OIP5 knockout upon detection of increased NF-κB signaling. The gene circuit significantly inhibited tumor cell growth in vivo, underscoring the potential for synergy between gene therapy and chemotherapy in the treatment of cancer.

PMID:39155295 | DOI:10.1038/s41388-024-03136-8

Biol Direct. 2024 Aug 17;19(1):67. doi: 10.1186/s13062-024-00501-1.

ABSTRACT

The cell and molecular bases of arbuscular mycorrhizal (AM) symbiosis, a crucial plant-fungal interaction for nutrient acquisition, have been extensively investigated by coupling traditional RNA sequencing techniques of roots sampled in bulk, with methods to capture subsets of cells such as laser microdissection. These approaches have revealed central regulators of this complex relationship, yet the requisite level of detail to effectively untangle the intricacies of temporal and spatial development remains elusive.The recent adoption of single-cell RNA sequencing (scRNA-seq) techniques in plant research is revolutionizing our ability to dissect the intricate transcriptional profiles of plant-microbe interactions, offering unparalleled insights into the diversity and dynamics of individual cells during symbiosis. The isolation of plant cells is particularly challenging due to the presence of cell walls, leading plant researchers to widely adopt nuclei isolation methods. Despite the increased resolution that single-cell analyses offer, it also comes at the cost of spatial perspective, hence, it is necessary the integration of these approaches with spatial transcriptomics to obtain a comprehensive overview.To date, few single-cell studies on plant-microbe interactions have been published, most of which provide high-resolution cell atlases that will become crucial for fully deciphering symbiotic interactions and addressing future questions. In AM symbiosis research, key processes such as the mutual recognition of partners during arbuscule development within cortical cells, or arbuscule senescence and degeneration, remain poorly understood, and these advancements are expected to shed light on these processes and contribute to a deeper understanding of this plant-fungal interaction.

PMID:39154166 | DOI:10.1186/s13062-024-00501-1

Nat Commun. 2024 Aug 17;15(1):7092. doi: 10.1038/s41467-024-51259-z.

ABSTRACT

Mammalian TIP60 is a multi-functional enzyme with histone acetylation and histone dimer exchange activities. It plays roles in diverse cellular processes including transcription, DNA repair, cell cycle control, and embryonic development. Here we report the cryo-electron microscopy structures of the human TIP60 complex with the core subcomplex and TRRAP module refined to 3.2-Å resolution. The structures show that EP400 acts as a backbone integrating the motor module, the ARP module, and the TRRAP module. The RUVBL1-RUVBL2 hexamer serves as a rigid core for the assembly of EP400 ATPase and YL1 in the motor module. In the ARP module, an ACTL6A-ACTB heterodimer and an extra ACTL6A make hydrophobic contacts with EP400 HSA helix, buttressed by network interactions among DMAP1, EPC1, and EP400. The ARP module stably associates with the motor module but is flexibly tethered to the TRRAP module, exhibiting a unique feature of human TIP60. The architecture of the nucleosome-bound human TIP60 reveals an unengaged nucleosome that is located between the core subcomplex and the TRRAP module. Our work illustrates the molecular architecture of human TIP60 and provides architectural insights into how this complex is bound by the nucleosome.

PMID:39154037 | DOI:10.1038/s41467-024-51259-z

Sci Bull (Beijing). 2024 Aug 6:S2095-9273(24)00561-9. doi: 10.1016/j.scib.2024.02.042. Online ahead of print.

ABSTRACT

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease triggered by antigenic peptides with environmental and genetic risk factors. It has been shown that antigen-specific targeting could be a promising therapeutical strategy for RA by restoring immune tolerance to self-antigens without compromising normal immunity. Citrullination of antigens enhances antigenic properties and induces autoimmune responses. Here, we showed that citrullinated antigenic (citAg) vaccine ameliorated collagen-induced arthritis (CIA) with decreased T-helper 1 (Th1) and Th17 cells, downregulated proinflammatory cytokines including interlukin-6 and tumor necrosis factor-α, and inhibited antigen recall responses. B cell receptor (BCR) sequencing further revealed that citAg vaccine could dampen the dysregulated V(D)J recombination, restoring the immune repertoire. Taken together, the results demonstrated that citAg vaccine might have a therapeutic effect on RA.

PMID:39153908 | DOI:10.1016/j.scib.2024.02.042

Biochim Biophys Acta Bioenerg. 2024 Aug 14:149504. doi: 10.1016/j.bbabio.2024.149504. Online ahead of print.

ABSTRACT

Two-stage (e.g. light-dark) phosphorylation experiments showed that there is a stored 'high-energy' intermediate linking electron transport and phosphorylation. Large, artificial electrochemical proton gradients (protonmotive forces or pmfs) can also drive phosphorylation, a fact seen as strongly supportive of the chemiosmotic coupling hypothesis that a pmf is the 'high-energy' intermediate. However, in such experiments there is an experimental threshold (pmf >170 mV, equivalent to ΔpH ~2.8) below which no phosphorylation is in fact observed, and 220 mV are required to recreate in vivo rates. This leads to the correct question, which is then whether those values of the pmf generated by electron transport are large enough. Even the lower ones as required for any phosphorylation (leave alone those required to explain in vivo rates) are below the threshold [1, 2], whether measured directly with microelectrodes or via the use of membrane-permeant ions and/or acids/bases (which are always transporter substrates [3], so all such measurements are in fact artefactual). The single case that seemed large enough (220 mV) is now admitted to be a diffusion potential artefact [4]. Many other observables (inadequate bulk H+ in 'O2-pulse'-type experiments, alkaliphilic bacteria, dual-inhibitor titrations, uncoupler-binding proteins, etc.) are consistent with the view that values of the pmf, and especially of Δψ, are actually very low. A protet-based charge separation model [2], a protonic version analogous to how energy may be stored in devices called electrets, provides a high-energy intermediate that can explain the entire literature, including the very striking demonstration [5] that close proximity is required between electron transport and ATP synthase complexes for energy coupling between them to allow phosphorylation to occur. A chief purpose of this article is thus to summarise the extensive and self-consistent literature, much of which is of some antiquity and rarely considered by modern researchers, despite its clear message of the inadequacy of chemiosmotic coupling to explain these phenomena.

PMID:39153588 | DOI:10.1016/j.bbabio.2024.149504

Immunity. 2024 Aug 14:S1074-7613(24)00370-4. doi: 10.1016/j.immuni.2024.07.021. Online ahead of print.

ABSTRACT

Although the Bacille-Calmette-Guérin (BCG) vaccine is used to prevent tuberculosis, it also offers protection against a diverse range of non-mycobacterial infections. However, the underlying protective mechanisms in humans are not yet fully understood. Here, we surveyed at single-cell resolution the gene expression and chromatin landscape of human bone marrow, aspirated before and 90 days after BCG vaccination or placebo. We showed that BCG alters both the gene expression and epigenetic profiles of human hematopoietic stem and progenitor cells (HSPCs). Changes in gene expression occurred primarily within uncommitted stem cells. By contrast, changes in chromatin accessibility were most prevalent within differentiated progenitor cells at sites influenced by Kruppel-like factor (KLF) and early growth response (EGR) transcription factors and were highly correlated (r > 0.8) with the interleukin (IL)-1β secretion capacity of paired peripheral blood mononuclear cells (PBMCs). Our findings shed light on BCG vaccination's profound and lasting effects on HSPCs and its influence on innate immune responses and trained immunity.

PMID:39153479 | DOI:10.1016/j.immuni.2024.07.021

Neuron. 2024 Aug 14:S0896-6273(24)00540-3. doi: 10.1016/j.neuron.2024.07.018. Online ahead of print.

ABSTRACT

The globus pallidus externus (GPe) is a central component of the basal ganglia circuit that acts as a gatekeeper of cocaine-induced behavioral plasticity. However, the molecular and circuit mechanisms underlying this function are unknown. Here, we show that GPe parvalbumin-positive (GPePV) cells mediate cocaine responses by selectively modulating ventral tegmental area dopamine (VTADA) cells projecting to the dorsomedial striatum (DMS). Interestingly, GPePV cell activity in cocaine-naive mice is correlated with behavioral responses following cocaine, effectively predicting cocaine sensitivity. Expression of the voltage-gated potassium channels KCNQ3 and KCNQ5 that control intrinsic cellular excitability following cocaine was downregulated, contributing to the elevation in GPePV cell excitability. Acutely activating channels containing KCNQ3 and/or KCNQ5 using the small molecule carnosic acid, a key psychoactive component of Salvia rosmarinus (rosemary) extract, reduced GPePV cell excitability and impaired cocaine reward, sensitization, and volitional cocaine intake, indicating its therapeutic potential to counteract psychostimulant use disorder.

PMID:39153478 | DOI:10.1016/j.neuron.2024.07.018

Mol Cell. 2024 Aug 6:S1097-2765(24)00623-3. doi: 10.1016/j.molcel.2024.07.025. Online ahead of print.

ABSTRACT

Nuclear localization of the metabolic enzyme PKM2 is widely observed in various cancer types. We identify nuclear PKM2 as a non-canonical RNA-binding protein (RBP) that specifically interacts with folded RNA G-quadruplex (rG4) structures in precursor mRNAs (pre-mRNAs). PKM2 occupancy at rG4s prevents the binding of repressive RBPs, such as HNRNPF, and promotes the expression of rG4-containing pre-mRNAs (the "rG4ome"). We observe an upregulation of the rG4ome during epithelial-to-mesenchymal transition and a negative correlation of rG4 abundance with patient survival in different cancer types. By preventing the nuclear accumulation of PKM2, we could repress the rG4ome in triple-negative breast cancer cells and reduce migration and invasion of cancer cells in vitro and in xenograft mouse models. Our data suggest that the balance of folded and unfolded rG4s controlled by RBPs impacts gene expression during tumor progression.

PMID:39153475 | DOI:10.1016/j.molcel.2024.07.025

J Cell Mol Med. 2024 Aug;28(16):e18588. doi: 10.1111/jcmm.18588.

ABSTRACT

Huntington's disease (HD) is a gradually severe neurodegenerative ailment characterised by an increase of a specific trinucleotide repeat sequence (cytosine-adenine-guanine, CAG). It is passed down as a dominant characteristic that worsens over time, creating a significant risk. Despite being monogenetic, the underlying mechanisms as well as biomarkers remain poorly understood. Furthermore, early detection of HD is challenging, and the available diagnostic procedures have low precision and accuracy. The research was conducted to provide knowledge of the biomarkers, pathways and therapeutic targets involved in the molecular processes of HD using informatic based analysis and applying network-based systems biology approaches. The gene expression profile datasets GSE97100 and GSE74201 relevant to HD were studied. As a consequence, 46 differentially expressed genes (DEGs) were identified. 10 hub genes (TPM1, EIF2S3, CCN2, ACTN1, ACTG2, CCN1, CSRP1, EIF1AX, BEX2 and TCEAL5) were further differentiated in the protein-protein interaction (PPI) network. These hub genes were typically down-regulated. Additionally, DEGs-transcription factors (TFs) connections (e.g. GATA2, YY1 and FOXC1), DEG-microRNA (miRNA) interactions (e.g. hsa-miR-124-3p and has-miR-26b-5p) were also comprehensively forecast. Additionally, related gene ontology concepts (e.g. sequence-specific DNA binding and TF activity) connected to DEGs in HD were identified using gene set enrichment analysis (GSEA). Finally, in silico drug design was employed to find candidate drugs for the treatment HD, and while the possible modest therapeutic compounds (e.g. cortistatin A, 13,16-Epoxy-25-hydroxy-17-cheilanthen-19,25-olide, Hecogenin) against HD were expected. Consequently, the results from this study may give researchers useful resources for the experimental validation of Huntington's diagnosis and therapeutic approaches.

PMID:39153206 | DOI:10.1111/jcmm.18588

Arch Microbiol. 2024 Aug 17;206(9):382. doi: 10.1007/s00203-024-04107-z.

ABSTRACT

Respiratory tract infections (RTIs) have a significant impact on global health, especially among children and the elderly. The key bacterial pathogens Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus and non-fermenting Gram Negative bacteria such as Acinetobacter baumannii and Pseudomonas aeruginosa are most commonly associated with RTIs. These bacterial pathogens have evolved a diverse array of resistance mechanisms through horizontal gene transfer, often mediated by mobile genetic elements and environmental acquisition. Treatment failures are primarily due to antimicrobial resistance and inadequate bacterial engagement, which necessitates the development of alternative treatment strategies. To overcome this, our review mainly focuses on different virulence mechanisms and their resulting pathogenicity, highlighting different therapeutic interventions to combat resistance. To prevent the antimicrobial resistance crisis, we also focused on leveraging the application of artificial intelligence and machine learning to manage RTIs. Integrative approaches combining mechanistic insights are crucial for addressing the global challenge of antimicrobial resistance in respiratory infections.

PMID:39153075 | DOI:10.1007/s00203-024-04107-z

Br J Radiol. 2024 Aug 17:tqae164. doi: 10.1093/bjr/tqae164. Online ahead of print.

ABSTRACT

OBJECTIVE: We previously demonstrated the potential of radiomics for prediction of severe histological Placenta Accreta Spectrum (PAS) subtypes using T2-weighted MRI. We aim is to validate our model using an additional dataset. Secondly, we explore whether performance is improved using a new approach to develop a new multivariate radiomics model.

METHODS: Multi-centre retrospective analysis conducted between 2018-2023. Inclusion criteria: MRI performed for suspicion of PAS from ultrasound, clinical findings of PAS at laparotomy and/or histopathological confirmation. Radiomic features were extracted from T2-weighted MRI. The previous multivariate model was validated. Secondly, a 5-radiomic feature random forest classifier was selected from a randomised feature selection scheme to predict invasive placenta increta PAS cases. Prediction performance was assessed based on several metrics including Area Under the Curve (AUC) of the receiver operating characteristic curve (ROC), sensitivity and specificity.

RESULTS: We present 100 women (mean age 34.6 (±3.9) with PAS, 64 of whom had placenta increta. Firstly, we validated the previous multivariate model and found a Support Vector Machine classifier had a sensitivity of 0.620 (95% CI: 0.068; 1.0), specificity of 0.619 (95% CI: 0.059; 1.0), an AUC of 0.671 (95% CI: 0.440; 0.922) and accuracy of 0.602 (95% CI: 0.353; 0.817) for predicting placenta increta. From the new multivariate model, the best 5-feature subset selected via the random subset feature selection scheme comprised of 4 radiomic features and 1 clinical variable (number of previous caesareans). This clinical-radiomic model achieved an AUC of 0.713 (95% CI: 0.551; 0.854), accuracy of 0.695 (95% CI 0.563; 0.793), sensitivity of 0.843 (95% CI 0.682; 0.990) and specificity of 0.447 (95% CI 0.167; 0.667).

CONCLUSION: We validated our previous model and present a new multivariate radiomic model for prediction of severe placenta increta from a well-defined, cohort of PAS cases.

ADVANCES IN KNOWLEDGE: Radiomic features demonstrate good predictive potential for identifying placenta increta. This suggests radiomics may be a useful adjunct to clinicians caring for women with this high-risk pregnancy condition.

PMID:39152998 | DOI:10.1093/bjr/tqae164

Commun Chem. 2024 Aug 16;7(1):184. doi: 10.1038/s42004-024-01273-5.

ABSTRACT

The gut microbiota offers an extensive resource of enzymes, but many remain uncharacterized. To distinguish the activities of similar annotated proteins and mine the potentially applicable ones in the microbiome, we applied an effective Activity-Based Metaproteomics (ABMP) strategy using a specific activity-based probe (ABP) to screen the entire gut microbiome for directly discovering active enzymes and their potential applications, not for exploring host-microbiome interactions. By using an activity-based cyclophellitol aziridine probe specific to α-galactosidases (AGAL), we successfully identified and characterized several gut microbiota enzymes possessing AGAL activities. Cryo-electron microscopy analysis of a newly characterized enzyme (AGLA5) revealed the covalent binding conformations between the AGAL5 active site and the cyclophellitol aziridine ABP, which could provide insights into the enzyme's catalytic mechanism. The four newly characterized AGALs have diverse potential activities, including raffinose family oligosaccharides (RFOs) hydrolysis and enzymatic blood group transformation. Collectively, we present a ABMP platform that facilitates gut microbiota AGALs discovery, biochemical activity annotations and potential industrial or biopharmaceutical applications.

PMID:39152233 | DOI:10.1038/s42004-024-01273-5

Sci Rep. 2024 Aug 16;14(1):18987. doi: 10.1038/s41598-024-70023-3.

ABSTRACT

The role of sub-Saharan Africa in the global spread of influenza viruses remains unclear due to insufficient spatiotemporal sequence data. Here, we analyzed 222 codon-complete sequences of influenza A viruses (IAVs) sampled between 2011 and 2013 from five countries across sub-Saharan Africa (Kenya, Zambia, Mali, Gambia, and South Africa); these genomes were compared with 1209 contemporaneous global genomes using phylogeographical approaches. The spread of influenza in sub-Saharan Africa was characterized by (i) multiple introductions of IAVs into the region over consecutive influenza seasons, with viral importations originating from multiple global geographical regions, some of which persisted in circulation as intra-subtype reassortants for multiple seasons, (ii) virus transfer between sub-Saharan African countries, and (iii) virus export from sub-Saharan Africa to other geographical regions. Despite sparse data from influenza surveillance in sub-Saharan Africa, our findings support the notion that influenza viruses persist as temporally structured migrating metapopulations in which new virus strains can emerge in any geographical region, including in sub-Saharan Africa; these lineages may have been capable of dissemination to other continents through a globally migrating virus population. Further knowledge of the viral lineages that circulate within understudied sub-Saharan Africa regions is required to inform vaccination strategies in those regions.

PMID:39152215 | DOI:10.1038/s41598-024-70023-3

Nat Commun. 2024 Aug 16;15(1):7067. doi: 10.1038/s41467-024-50363-4.

ABSTRACT

RNA-binding proteins (RBPs) have pivotal functions in RNA metabolism, but current methods are limited in retrieving RBP-RNA interactions within endogenous biological contexts. Here, we develop INSCRIBE (IN situ Sensitive Capture of RNA-protein Interactions in Biological Environments), circumventing the challenges through in situ RNA labeling by precisely directing a purified APOBEC1-nanobody fusion to the RBP of interest. This method enables highly specific RNA-binding site identification across a diverse range of fixed biological samples such as HEK293T cells and mouse brain tissue and accurately identifies the canonical binding motifs of RBFOX2 (UGCAUG) and TDP-43 (UGUGUG) in native cellular environments. Applicable to any RBP with available primary antibodies, INSCRIBE enables sensitive capture of RBP-RNA interactions from ultra-low input equivalent to ~5 cells. The robust, versatile, and sensitive INSCRIBE workflow is particularly beneficial for precious tissues such as clinical samples, empowering the exploration of genuine RBP-RNA interactions in RNA-related disease contexts.

PMID:39152130 | DOI:10.1038/s41467-024-50363-4

Cells Dev. 2024 Aug 14:203964. doi: 10.1016/j.cdev.2024.203964. Online ahead of print.

ABSTRACT

The current dogma in cancer biology contends that cancer is an identity problem: mutations in a cell's DNA cause it to "go rogue" and proliferate out of control. However, this largely ignores the role of cell-cell interaction and fails to explain phenomena such as cancer reversion, the existence of epigenetic cancers, and foreign-body carcinogenesis. In this proof-of-concept paper, we draw on criminology to propose that cancer may alternatively be conceptualized as a relational problem: Although a cell's genetics is essential, the influence of its interaction with other cells is equally important in determining its phenotype. We create a simple agent-based network model of interactions among normal and cancer cells to demonstrate this idea. We find that both high mutation rates and low levels of connectivity among cells can promote oncogenesis. Viewing cancer as a breakdown in communication networks among cells in a tissue complements the gene-centric paradigm nicely and provides a novel perspective for understanding and treating cancer.

PMID:39151750 | DOI:10.1016/j.cdev.2024.203964

Stem Cell Reports. 2024 Aug 5:S2213-6711(24)00213-3. doi: 10.1016/j.stemcr.2024.07.004. Online ahead of print.

ABSTRACT

Variability between human pluripotent stem cell (hPSC) lines remains a challenge and opportunity in biomedicine. In this study, hPSC lines from multiple donors were differentiated toward neuroectoderm and mesendoderm lineages. We revealed dynamic transcriptomic patterns that delineate the emergence of these lineages, which were conserved across lines, along with individual line-specific transcriptional signatures that were invariant throughout differentiation. These transcriptomic signatures predicted an antagonism between SOX21-driven forebrain fates and retinoic acid-induced hindbrain fates. Replicate lines and paired adult tissue demonstrated the stability of these line-specific transcriptomic traits. We show that this transcriptomic variation in lineage bias had both genetic and epigenetic origins, aligned with the anterior-to-posterior structure of early mammalian development, and was present across a large collection of hPSC lines. These findings contribute to developing systematic analyses of PSCs to define the origin and consequences of variation in the early events orchestrating individual human development.

PMID:39151428 | DOI:10.1016/j.stemcr.2024.07.004

Sci Immunol. 2024 Aug 16;9(98):eade7530. doi: 10.1126/sciimmunol.ade7530. Epub 2024 Aug 16.

ABSTRACT

How group 3 innate lymphoid cells (ILC3s) regulate mucosal protection in the presence of T cells remains poorly understood. Here, we examined ILC3 function in intestinal immunity using ILC3-deficient mice that maintain endogenous T cells, T helper 17 (TH17) cells, and secondary lymphoid organs. ILC3s were dispensable for generation of TH17 and TH22 cell responses to commensal and pathogenic bacteria, and absence of ILC3s did not affect IL-22 production by CD4 T cells before or during infection. However, despite the presence of IL-22-producing T cells, ILC3s and ILC3-derived IL-22 were required for maintaining homeostatic functions of the intestinal epithelium. T cell-sufficient, ILC3-deficient mice were capable of pathogen clearance and survived infection with a low dose of Citrobacter rodentium. However, ILC3s promoted pathogen tolerance at early time points of infection by activating tissue-protective immune pathways. Consequently, ILC3s were indispensable for survival after high-dose infection. Our results demonstrate a context-dependent role for ILC3s in immune-sufficient animals and provide a blueprint for uncoupling of ILC3 and TH17 cell functions.

PMID:39151019 | DOI:10.1126/sciimmunol.ade7530

STAR Protoc. 2024 Aug 14;5(3):103246. doi: 10.1016/j.xpro.2024.103246. Online ahead of print.

ABSTRACT

Despite numerous neuropathological criteria for the evaluation of microscopic tissue slides, there are no systemic standards for sectioning postmortem human brain tissue. Here, we present a protocol for postmortem brain hybrid structured-light scanning to facilitate 3D printing of sectioning matrices. We describe steps for tissue preparation, 3D scanning and printing, and matrix-guided sectioning. This protocol can be used for streamlining tissue histopathology as well as in brain morphology volumetric analyses and applications in medical education. For complete details on the use and execution of this protocol, please refer to Barannikov et al.1.

PMID:39150849 | DOI:10.1016/j.xpro.2024.103246

mSystems. 2024 Aug 16:e0073524. doi: 10.1128/msystems.00735-24. Online ahead of print.

ABSTRACT

Targeted high-throughput sequencing (HTS) has revolutionized the way we look at bacterial communities. It can be used for the species-specific detection of bacteria as well as for the determination of the microbiome and resistome and can be applied to samples from almost any environment. However, the results of targeted HTS can be influenced by many factors, which poses a major challenge for its use in clinical diagnostics. In this study, we investigated the impact of the DNA extraction method on the determination of the bacterial microbiome and resistome by targeted HTS using principles from metrology and diagnostics such as repeatability and analytical sensitivity. Sputum samples spiked with Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa at three different concentrations (103-106 cells/mL) were used. DNA was extracted from each sample on 2 separate days in three replicates each using three different extraction methods based on cetrimonium bromide, magnetic beads, and silica membranes. All three spiked bacteria were detected in sputum, and the DNA extraction method had no significant effect on detection. However, the DNA extraction method had significant effects on the composition of the microbiome and the resistome. The sequencing results were repeatable in the majority of cases. The silica membrane-based DNA extraction kit provided the most repeatable results and the highest diversity of the microbiome and resistome. Targeted HTS has been shown to be a reliable tool for determining the microbiome and resistome; however, the method of DNA extraction should be carefully selected to minimize its impact on the results.

IMPORTANCE: High-throughput sequencing (HTS) is one of the crucial new technologies that gives us insights into previously hidden parts of microbial communities. The DNA extraction method is an important step that can have a major impact on the results, and understanding this impact is of paramount importance for their reliable interpretation. Our results are of great value for the interpretation of sputum microbiome and resistome results obtained by targeted HTS. Our findings allow for a more rational design of future microbiome studies, which would lead to higher repeatability of results and easier comparison between different laboratories. This could also facilitate the introduction of targeted HTS in clinical microbiology for reliable identification of pathogenic bacteria and testing for antimicrobial resistance (AMR). As AMR is a major threat to public health, the improved methods for determining AMR would bring great benefits to both the healthcare system and society as a whole.

PMID:39150245 | DOI:10.1128/msystems.00735-24

New Phytol. 2024 Aug 16. doi: 10.1111/nph.20065. Online ahead of print.

NO ABSTRACT

PMID:39149858 | DOI:10.1111/nph.20065