mSphere. 2024 Oct 31:e0055324. doi: 10.1128/msphere.00553-24. Online ahead of print.

ABSTRACT

Promiscuous biotin ligases derived from the bacterial enzyme BirA are used to identify proteins vicinal to a bait protein, thereby defining its proxisome. Despite the popularity of this approach, surprisingly little is known about its use in prokaryotes. Here, we compared the activity of four widely used promiscuous biotin ligases in the cytoplasm of Shigella flexneri, a pathogenic subgroup of Escherichia coli. Our data indicate that the kinetics of TurboID's biotinylating activity is the highest of those tested. In addition, TurboID showed reduced interaction with the natural BirA binding partners, BccP and the biotin operator, when compared to its ancestor BioID. We therefore evaluated the ability of TurboID to probe the proxisome of the type III secretion system (T3SS) chaperone IpgC and the transcriptional activator MxiE. When the T3SS is inactive (off-state), these proteins are inhibited by forming complexes with the T3SS substrates OspD1 and IpaBC, respectively. In contrast, when the T3SS is active (on-state), OspD1 and IpaBC are secreted allowing MxiE and IpgC to interact together and activate their target genes. The results obtained with the IpgC and TurboID fusions capture a good fraction of these known interactions. It also suggests that the availability of IpgC increases in the on-state, resulting in a greater number of proteins detected in its vicinity. Among these is the T3SS ATPase SpaL (also known as Spa47 or SctN), further supporting the notion that chaperones escort their substrate to the T3SS. Interestingly, a specific subset of proteins conserved in E. coli completes the IpgC proxisome in the on-state.IMPORTANCEPromiscuous biotin ligases are widely used to study protein function in eukaryotes. Strikingly, their use in prokaryotes has been rare. Indeed, the small volume and the cytoplasmic location of the biotin ligase's natural binding partners in these organisms pose unique challenges that can interfere with the study of the proxisome of proteins of interest. Here, we evaluated four of the most common promiscuous biotin ligases and found TurboID was best suited for use in the cytoplasm of Shigella flexneri. Using this method, we extended the proxisome of IpgC beyond its known direct binding partners involved in the regulation of the type III secretion system (T3SS) signaling cascade. Of particular interest for further study are transcription factors and housekeeping proteins that are enriched around IpgC when the T3SS is active. We propose a model in which the increased availability of IpgC in the on-state may allow cross-talk of the T3SS with other cellular processes.

PMID:39480076 | DOI:10.1128/msphere.00553-24

J Proteome Res. 2024 Oct 31. doi: 10.1021/acs.jproteome.4c00586. Online ahead of print.

ABSTRACT

Recent improvements in proteomics technologies have fundamentally altered our capacities to characterize human biology. There is an ever-growing interest in using these novel methods for studying the circulating proteome, as blood offers an accessible window into human health. However, every methodological innovation and analytical progress calls for reassessing our existing approaches and routines to ensure that the new data will add value to the greater biomedical research community and avoid previous errors. As representatives of HUPO's Human Plasma Proteome Project (HPPP), we present our 2024 survey of the current progress in our community, including the latest build of the Human Plasma Proteome PeptideAtlas that now comprises 4608 proteins detected in 113 data sets. We then discuss the updates of established proteomics methods, emerging technologies, and investigations of proteoforms, protein networks, extracellualr vesicles, circulating antibodies and microsamples. Finally, we provide a prospective view of using the current and emerging proteomics tools in studies of circulating proteins.

PMID:39479990 | DOI:10.1021/acs.jproteome.4c00586

FEMS Microbiol Ecol. 2024 Oct 30:fiae147. doi: 10.1093/femsec/fiae147. Online ahead of print.

ABSTRACT

Uptake hydrogenase (Hup) recycles H2 formed by nitrogenase during nitrogen fixation, thereby preserving energy. Among root nodule bacteria, most rhizobial strains examined are Hup-, while only one Hup- Frankia inoculum had been identified. Previous analyses had led to the identification of two different [NiFe] hydrogenase syntons. We analysed the distribution of different types of [NiFe] hydrogenase in the genomes of different Frankia species. Our results show that Frankia strains can contain four different [NiFe] hydrogenase syntons representing groups 1f, 1h, 2a and 3b according to Søndergaard et al. (2016); no more than three types were found in any individual genome. The phylogeny of the structural proteins of groups 1f, 1h and 2a follows Frankia phylogeny; the phylogeny of the accessory proteins does not consistently. An analysis of different [NiFe] hydrogenase types in Actinomycetia shows that under the most parsimonious assumption, all four types were present in the ancestral Frankia strain. Based on Hup activities analysed and the losses of syntons in different lineages of genome reduction, we can conclude that groups 1f and 2a are involved in recycling H2 formed by nitrogenase while group 1h and group 3b are not.

PMID:39479807 | DOI:10.1093/femsec/fiae147

J Mol Cell Biol. 2024 Oct 30:mjae048. doi: 10.1093/jmcb/mjae048. Online ahead of print.

NO ABSTRACT

PMID:39479794 | DOI:10.1093/jmcb/mjae048

Infect Drug Resist. 2024 Oct 26;17:4695-4706. doi: 10.2147/IDR.S485102. eCollection 2024.

ABSTRACT

PURPOSE: This study aimed to evaluate the clinical utility of metagenomic next-generation sequencing (mNGS) in sepsis patients who received early empirical antibiotic treatment.

PATIENTS AND METHODS: A retrospective analysis was conducted on clinical data from sepsis patients diagnosed in the Emergency Intensive Care Unit (EICU) between April 2019 and May 2023. All patients underwent standard conventional microbiological testing. Patients were categorized into either the mNGS group or the control group based on whether they underwent mNGS tests. Baseline variables were matched using propensity scores.

RESULTS: Out of 461 sepsis patients screened, 130 were included after propensity matching, with 65 patients in each group. Despite prior antibiotic treatment, 57 cases (87.69%) in the mNGS group had positive mNGS results, exceeding the culture detection rate (52.31%). Besides, a higher proportion of patients in the mNGS group experienced antibiotic adjustments compared to the control group (72.31% vs 53.85%). Mortality rates were also compared based on the duration of antibiotic exposure before mNGS sampling. Patients exposed to antibiotics for less than 24 hours had a lower mortality rate compared to those exposed for over 8 days (22.22% vs 42.86%). COX multivariate analysis identified mNGS testing, underlying diseases, lymphocyte percentage, infection site (respiratory and bloodstream) as independent risk factors for mortality in sepsis patients.

CONCLUSION: With increased antibiotic exposure time, the positive rate of culture testing significantly decreased (44.44% vs 59.52% vs 35.71%, P = 0.031), whereas the positive rate of mNGS remained stable (77.78% vs 88.10% vs 92.86%, P = 0.557). mNGS demonstrated less susceptibility to antibiotic exposure. Early mNGS detection positively impacted the prognosis of sepsis patients.

PMID:39479400 | PMC:PMC11523945 | DOI:10.2147/IDR.S485102

Research (Wash D C). 2024 Oct 30;7:0513. doi: 10.34133/research.0513. eCollection 2024.

ABSTRACT

Accurate visualization and 3-dimensional (3D) morphological profiling of small model organisms can provide quantitative phenotypes benefiting genetic analysis and modeling of human diseases in tractable organisms. However, in the highly studied nematode Caenorhabditis elegans, accurate morphological phenotyping remains challenging because of notable decrease in image resolution of distant signal under high magnification and complexity in the 3D reconstruction of microscale samples with irregular shapes. Here, we develop a robust robotic system that enables the contactless, stable, and uniform rotation of C. elegans for multi-view fluorescent imaging and 3D morphological phenotyping via the precise reconstruction of 3D models. Contactless animal rotation accommodates a variety of body shapes and sizes found at different developmental stages and in mutant strains. Through controlled rotation, high-resolution fluorescent imaging of C. elegans structures is obtained by overcoming the limitations inherent in both widefield and confocal microscopy. Combining our robotic system with machine learning, we create, for the first time, precise 3D reconstructions of C. elegans at the embryonic and adult stages, enabling 3D morphological phenotyping of mutant strains in an accurate and comprehensive fashion. Intriguingly, our morphological phenotyping discovered a genetic interaction between 2 RNA binding proteins (UNC-75/CELF and MBL-1/MBNL), which are highly conserved between C. elegans and humans and implicated in neurological and muscular disorders. Our system can thus generate quantitative morphological readouts facilitating the investigation of genetic variations and disease mechanisms. More broadly, our method will also be amenable for 3D phenotypic analysis of other biological samples, like zebrafish and Drosophila larvae.

PMID:39479356 | PMC:PMC11522223 | DOI:10.34133/research.0513

Histol Histopathol. 2024 Oct 7:18829. doi: 10.14670/HH-18-829. Online ahead of print.

ABSTRACT

BACKGROUND: The number of older organ donors is increasing due to the aging population. Aged kidneys often face problems such as delayed graft function but previous murine experiments suggested the possibilities of rejuvenation, for example, in a parabiosis setting between old and young mice. To investigate kidney-graft rejuvenation, we compared an old-to-young (O-Y) patient transplantation group and a transplantation group with donors/recipients of approx. the same age (SA) with the renal senescence marker p16 in kidney biopsy samples at baseline and one year post-transplantation.

METHODS: We retrospectively analyzed our hospital's 32 cases of living-donor ABO-compatible transplants performed between 2013-2020. Both the baseline and one-year biopsy (n=9) or only the baseline biopsy (n=32) were analyzed. We divided the nine cases into an O-Y group (donors' median age 68 yrs, recipients 41, difference -27) and an SA group (donors' median age 53 yrs, recipients 51.5, difference -3.5). p16 was stained with the clones JC8 and E6H4 to determine the precise p16-positive rate.

RESULTS: The 32 baseline biopsies' p16-positive rate was weakly related to donor age, suggesting that the p16-positive rate can help evaluate kidney senescence. The (n=5) O-Y group's p16-positive rates were at baseline 0.08 and one year 0.12; the (n=4) SA group's rate was 0.03 at both baseline and one year.

CONCLUSIONS: No kidney rejuvenation was observed, even when old donor kidneys went to young recipients.

PMID:39478629 | DOI:10.14670/HH-18-829

Nature. 2024 Oct 30. doi: 10.1038/s41586-024-08150-0. Online ahead of print.

ABSTRACT

As cancers progress, they become increasingly aggressive-metastatic tumours are less responsive to first-line therapies than primary tumours, they acquire resistance to successive therapies and eventually cause death1,2. Mutations are largely conserved between primary and metastatic tumours from the same patients, suggesting that non-genetic phenotypic plasticity has a major role in cancer progression and therapy resistance3-5. However, we lack an understanding of metastatic cell states and the mechanisms by which they transition. Here, in a cohort of biospecimen trios from same-patient normal colon, primary and metastatic colorectal cancer, we show that, although primary tumours largely adopt LGR5+ intestinal stem-like states, metastases display progressive plasticity. Cancer cells lose intestinal cell identities and reprogram into a highly conserved fetal progenitor state before undergoing non-canonical differentiation into divergent squamous and neuroendocrine-like states, a process that is exacerbated in metastasis and by chemotherapy and is associated with poor patient survival. Using matched patient-derived organoids, we demonstrate that metastatic cells exhibit greater cell-autonomous multilineage differentiation potential in response to microenvironment cues compared with their intestinal lineage-restricted primary tumour counterparts. We identify PROX1 as a repressor of non-intestinal lineage in the fetal progenitor state, and show that downregulation of PROX1 licenses non-canonical reprogramming.

PMID:39478232 | DOI:10.1038/s41586-024-08150-0

Nature. 2024 Oct;634(8036):1178-1186. doi: 10.1038/s41586-024-08087-4. Epub 2024 Oct 30.

ABSTRACT

To study the spatial interactions among cancer and non-cancer cells1, we here examined a cohort of 131 tumour sections from 78 cases across 6 cancer types by Visium spatial transcriptomics (ST). This was combined with 48 matched single-nucleus RNA sequencing samples and 22 matched co-detection by indexing (CODEX) samples. To describe tumour structures and habitats, we defined 'tumour microregions' as spatially distinct cancer cell clusters separated by stromal components. They varied in size and density among cancer types, with the largest microregions observed in metastatic samples. We further grouped microregions with shared genetic alterations into 'spatial subclones'. Thirty five tumour sections exhibited subclonal structures. Spatial subclones with distinct copy number variations and mutations displayed differential oncogenic activities. We identified increased metabolic activity at the centre and increased antigen presentation along the leading edges of microregions. We also observed variable T cell infiltrations within microregions and macrophages predominantly residing at tumour boundaries. We reconstructed 3D tumour structures by co-registering 48 serial ST sections from 16 samples, which provided insights into the spatial organization and heterogeneity of tumours. Additionally, using an unsupervised deep-learning algorithm and integrating ST and CODEX data, we identified both immune hot and cold neighbourhoods and enhanced immune exhaustion markers surrounding the 3D subclones. These findings contribute to the understanding of spatial tumour evolution through interactions with the local microenvironment in 2D and 3D space, providing valuable insights into tumour biology.

PMID:39478210 | DOI:10.1038/s41586-024-08087-4

Nat Methods. 2024 Oct 30. doi: 10.1038/s41592-024-02328-0. Online ahead of print.

ABSTRACT

Tumors are complex assemblies of cellular and acellular structures patterned on spatial scales from microns to centimeters. Study of these assemblies has advanced dramatically with the introduction of high-plex spatial profiling. Image-based profiling methods reveal the intensities and spatial distributions of 20-100 proteins at subcellular resolution in 103-107 cells per specimen. Despite extensive work on methods for extracting single-cell data from these images, all tissue images contain artifacts such as folds, debris, antibody aggregates, optical aberrations and image processing errors that arise from imperfections in specimen preparation, data acquisition, image assembly and feature extraction. Here we show that these artifacts dramatically impact single-cell data analysis, obscuring meaningful biological interpretation. We describe an interactive quality control software tool, CyLinter, that identifies and removes data associated with imaging artifacts. CyLinter greatly improves single-cell analysis, especially for archival specimens sectioned many years before data collection, such as those from clinical trials.

PMID:39478175 | DOI:10.1038/s41592-024-02328-0

Nat Microbiol. 2024 Nov;9(11):2774-2784. doi: 10.1038/s41564-024-01833-4. Epub 2024 Oct 30.

ABSTRACT

Harnessing beneficial microorganisms is seen as a promising approach to enhance sustainable agriculture production. Synthetic communities (SynComs) are increasingly being used to study relevant microbial activities and interactions with the plant host. Yet, the lack of community standards limits the efficiency and progress in this important area of research. To address this gap, we recommend three actions: (1) defining reference SynComs; (2) establishing community standards, protocols and benchmark data for constructing and using SynComs; and (3) creating an infrastructure for sharing strains and data. We also outline opportunities to develop SynCom research through technical advances, linking to field studies, and filling taxonomic blind spots to move towards fully representative SynComs.

PMID:39478084 | DOI:10.1038/s41564-024-01833-4

Nat Commun. 2024 Oct 30;15(1):9370. doi: 10.1038/s41467-024-53775-4.

NO ABSTRACT

PMID:39478018 | DOI:10.1038/s41467-024-53775-4

Nat Commun. 2024 Oct 30;15(1):9402. doi: 10.1038/s41467-024-53535-4.

ABSTRACT

Deep single-cell multi-omic profiling offers a promising approach to understand and overcome drug resistance in relapsed or refractory (rr) acute myeloid leukemia (AML). Here, we combine single-cell ex vivo drug profiling (pharmacoscopy) with single-cell and bulk DNA, RNA, and protein analyses, alongside clinical data from 21 rrAML patients. Unsupervised data integration reveals reduced ex vivo response to the Bcl-2 inhibitor venetoclax (VEN) in patients treated with both a hypomethylating agent (HMA) and VEN, compared to those pre-exposed to chemotherapy or HMA alone. Integrative analysis identifies both known and unreported mechanisms of innate and treatment-related VEN resistance and suggests alternative treatments, like targeting increased proliferation with the PLK inhibitor volasertib. Additionally, high CD36 expression in VEN-resistant blasts associates with sensitivity to CD36-targeted antibody treatment ex vivo. This study demonstrates how single-cell multi-omic profiling can uncover drug resistance mechanisms and treatment vulnerabilities, providing a valuable resource for future AML research.

PMID:39477946 | DOI:10.1038/s41467-024-53535-4

Nat Commun. 2024 Oct 30;15(1):9386. doi: 10.1038/s41467-024-53677-5.

ABSTRACT

All free-living microorganisms homeostatically maintain the fluidity of their membranes by adapting lipid composition to environmental temperatures. Here, we quantify enzymes and metabolic intermediates of the Escherichia coli fatty acid and phospholipid synthesis pathways, to describe how this organism measures temperature and restores optimal membrane fluidity within a single generation after a temperature shock. A first element of this regulatory system is a temperature-sensitive metabolic valve that allocates flux between the saturated and unsaturated fatty acid synthesis pathways via the branchpoint enzymes FabI and FabB. A second element is a transcription-based negative feedback loop that counteracts the temperature-sensitive valve. The combination of these elements accelerates membrane adaptation by causing a transient overshoot in the synthesis of saturated or unsaturated fatty acids following temperature shocks. This strategy is comparable to increasing the temperature of a water bath by adding water that is excessively hot rather than adding water at the desired temperature. These properties are captured in a mathematical model, which we use to show how hard-wired parameters calibrate the system to generate membrane compositions that maintain constant fluidity across temperatures. We hypothesize that core features of the E. coli system will prove to be ubiquitous features of homeoviscous adaptation systems.

PMID:39477942 | DOI:10.1038/s41467-024-53677-5

J Hazard Mater. 2024 Oct 28;480:136328. doi: 10.1016/j.jhazmat.2024.136328. Online ahead of print.

ABSTRACT

Antimony (Sb) pollution in soil-rice systems can affect human health by enriching of food chains. Currently, the mechanism of the negative role underlying microorganisms in plant responses to Sb stress remains clear. The results of this study showed that the presence of arbuscular mycorrhizal (AM) fungi, a common symbiotic microorganism in rhizosphere soil, significantly enhanced Sb uptake by upland rice and inhibited its growth. Furthermore, we explained the reasons for the adverse effects of AM fungi mediation on upland rice growth under Sb stress from a molecular perspective. The results also showed that AM fungi affect the biological processes of the response of upland rice to oxidative stress and the functions of its antioxidant active molecules throughout the vegetative growth phase of upland rice, and that the phenylpropanoid biosynthesis pathway is significantly downregulated. At the same time, phenylalanine/tyrosine ammonia-lyase (PTAL) in the pathway was significantly expressed in the middle and late stages of vegetative growth of upland rice. Therefore, PTAL can act as a potential reference protein to investigate the response of upland rice to Sb stress mediated by AM fungi. These findings enrich our understanding of the impact of Sb pollution on soil-plant systems in real soil environments.

PMID:39476691 | DOI:10.1016/j.jhazmat.2024.136328

J Inorg Biochem. 2024 Oct 26;262:112765. doi: 10.1016/j.jinorgbio.2024.112765. Online ahead of print.

ABSTRACT

A synthetic procedure was designed for the preparation and characterization of Ag and Ru complexes containing NHC ligands functionalized with PEG fragments. Stability studies were conducted to gain insight of the species in water and other solvents like DMSO, or with reagents like imidazole as representative group for histidine amino acid. The presence of Cl atoms instead of H in the 4,5 positions of the N-heterocyclic carbene afforded higher water stability. The complexes containing PEG units must be considered inactive as anticancer agents. To enhance the anticancer activity of PEG-containing complexes, the balance between hydrophilicity and hydrophobicity was adjusted using a silane moiety, and an anionic carbosilane dendrimer as a lipophilic carrier.

PMID:39476504 | DOI:10.1016/j.jinorgbio.2024.112765

Annu Rev Pathol. 2024 Oct 30. doi: 10.1146/annurev-pathmechdis-111523-023417. Online ahead of print.

ABSTRACT

Pathology has always been fueled by technological advances. Histology powered the study of tissue architecture at single-cell resolution and remains a cornerstone of clinical pathology today. In the last decade, next-generation sequencing has become informative for the targeted treatment of many diseases, demonstrating the importance of genome-scale molecular information for personalized medicine. Today, revolutionary developments in spatial transcriptomics technologies digitalize gene expression at subcellular resolution in intact tissue sections, enabling the computational analysis of cell types, cellular phenotypes, and cell-cell communication in routinely collected and archival clinical samples. Here we review how such molecular microscopes work, highlight their potential to identify disease mechanisms and guide personalized therapies, and provide guidance for clinical study design. Finally, we discuss remaining challenges to the swift translation of high-resolution spatial transcriptomics technologies and how integration of multimodal readouts and deep learning approaches is bringing us closer to a holistic understanding of tissue biology and pathology.

PMID:39476415 | DOI:10.1146/annurev-pathmechdis-111523-023417

Elife. 2024 Oct 30;13:RP93115. doi: 10.7554/eLife.93115.

ABSTRACT

Explaining biodiversity is a fundamental issue in ecology. A long-standing puzzle lies in the paradox of the plankton: many species of plankton feeding on a limited variety of resources coexist, apparently flouting the competitive exclusion principle (CEP), which holds that the number of predator (consumer) species cannot exceed that of the resources at a steady state. Here, we present a mechanistic model and demonstrate that intraspecific interference among the consumers enables a plethora of consumer species to coexist at constant population densities with only one or a handful of resource species. This facilitated biodiversity is resistant to stochasticity, either with the stochastic simulation algorithm or individual-based modeling. Our model naturally explains the classical experiments that invalidate the CEP, quantitatively illustrates the universal S-shaped pattern of the rank-abundance curves across a wide range of ecological communities, and can be broadly used to resolve the mystery of biodiversity in many natural ecosystems.

PMID:39476367 | DOI:10.7554/eLife.93115

Microb Genom. 2024 Oct;10(10). doi: 10.1099/mgen.0.001305.

ABSTRACT

Bacteria are fascinating research objects in many disciplines for countless reasons, and whole-genome sequencing (WGS) has become the paramount methodology to advance our microbiological understanding. Meanwhile, access to cost-effective sequencing platforms has accelerated bacterial WGS to unprecedented levels, introducing new challenges in terms of data accessibility, computational demands, heterogeneity of analysis workflows and, thus, ultimately its scientific usability. To this end, a previous study released a uniformly processed set of 661 405 bacterial genome assemblies obtained from the European Nucleotide Archive as of November 2018. Building on these accomplishments, we conducted further genome-based analyses like taxonomic classification, multilocus sequence typing and annotation of all genomes. Here, we present BakRep, a searchable large-scale web repository of these genomes enriched with consistent genome characterizations and original metadata. The platform provides a flexible search engine combining taxonomic, genomic and metadata information, as well as interactive elements to visualize genomic features. Furthermore, all results can be downloaded for offline analyses via an accompanying command line tool. The web repository is accessible via https://bakrep.computational.bio.

PMID:39475723 | DOI:10.1099/mgen.0.001305

Anal Chem. 2024 Oct 30. doi: 10.1021/acs.analchem.4c04187. Online ahead of print.

ABSTRACT

Many chiral carboxylic acids with α-amino, α-hydroxyl, and α-methyl groups are concurrently present in mammals establishing unique molecular phenotypes and multiple biological functions, especially host-microbiota symbiotic interactions. Their chirality-resolved simultaneous quantification is essential to reveal the biochemical details of physiology and pathophysiology, though challenging with their low abundances in some biological matrices and difficulty in enantiomer resolution. Here, we developed a method of the chirality-resolved metabolomics with sensitivity-enhanced quantitation via probe-promotion (Met-SeqPro) for analyzing these chiral carboxylic acids. We designed and synthesized a hydrazide-based novel chiral probe, (S)-benzoyl-proline-hydrazide (SBPH), to convert carboxylic acids into amide diastereomers to enhance their retention and chiral resolution on common C18 columns. Using the d5-SBPH-labeled enantiomers as internal standards, we then developed an optimized ultrahigh-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous quantification of 60 enantiomers of 30 chiral carboxylic acids in one run. This enantiomer-resolved method showed excellent sensitivity (LOD < 4 fmol-on-column), linearity (R2 > 0.992), precision (CV < 15%), accuracy (|RE| < 20%), and recovery (80-120%) in multiple biological matrices. With the method, we then quantified 60 chiral carboxylic acids in human urine, plasma, feces, and A549 cells to define their metabolomic phenotypes. This provides basic data for human phenomics and a promising tool for investigating the mammal-microbiome symbiotic interactions.

PMID:39475527 | DOI:10.1021/acs.analchem.4c04187