搜索结果：microLife

Screening for, detecting, and managing pregnancy hypertension is a core function of antenatal care. To reduce both training requirements and the risks of measurement error in blood pressure (BP) values, automated and semiautomated BP devices have been validated in pregnant women with normal BP and pregnant women with hypertension and introduced for serial antenatal measurement of BP. The study aimed to (1) determine whether or not repeated BP measurements reduced the presence of terminal digit preference and (2) discern whether or not there was evidence of threshold avoidance in the Community-Level Interventions for Preeclampsia (CLIP) trials compared with the purely observational Pregnancy Care Integrating Translational Science, Everywhere (PRECISE) cohorts. The BP 3AS1-2 and CRADLE Vital Signs Alert low-cost Microlife BP devices were used by trained research staff in the CLIP trials conducted in India, Mozambique, Nigeria (pilot trial only), and Pakistan and the PRECISE cohorts of unselected pregnant women and nonpregnant women of reproductive age recruited in the Gambia, Kenya, and Mozambique. Both devices algorithmically calculate systolic blood pressure and diastolic blood pressure values displayed on digital read-outs. All BP readings were entered manually into a digital platform, which averaged them as the BP for that visit; the first and second readings were averaged unless they were more than 10 mm Hg different, which triggered a third reading, and the second and third readings were averaged. A total of 51,875 participants had their BP measured 438,404 times. Using raw BP values, there was terminal digit preference (129,539/911,500, 14.21% vs 10%; P<.001 values ended in zero). A total of 28,929 out of 437,446 (6.61%) dBP values were 62 mm Hg, compared with 9310 of 195,349 (4.77%) from the averaged values (P<.001); errors were obviated by averaging BP values. There was evidence of both threshold preference and avoidance in the CLIP trials and the PRECISE cohort. Given the excess of 62 mm Hg values, there is a shared inherent algorithmic error in the calculation of dBP in the BP 3AS1-2 and CRADLE Vital Signs Alert devices. Averaged BP measurements are important to reduce the impact of user errors in manually recording BP values. We recommend that automated and semiautomated BP devices should be connected wirelessly to automatically transfer readings to digital health records to further optimize care.

Fluorescence microscopy has become an indispensable tool in biological research, offering powerful approaches to study protein dynamics and cellular processes in vivo. Among archaea, Haloferax volcanii has emerged as a particularly well-suited model organism for imaging studies, with a growing toolkit of established fluorescent markers, plasmids, and promoter systems. Recent advances in single-molecule imaging techniques have created new opportunities through WR806, a carotenoid-free H. volcanii strain providing reduced autofluorescence background. However, existing plasmid-based expression systems in WR806 show critical limitations in protein expression control and challenges with protein aggregation. To address these limitations, we developed pUE001, a novel plasmid system specifically designed for WR806. This system achieves precise expression control by decoupling selection and induction through strategic implementation of the trpA selection marker. Through comprehensive characterization, we demonstrate that pUE001 provides superior control over protein expression compared to the previously established pTA962 system. It enables linear, titratable expression of diverse proteins-from the highly regulated CRISPR-Cas component Cas1 to the abundant structural protein FtsZ1-while preventing protein aggregation that could compromise native cellular functions. Additionally, we performed a comprehensive analysis of WR806 to show that carotenoid depletion does not affect native cellular physiology. Finally, to demonstrate the system's utility, we investigated the role of Cas1 in UV-induced DNA repair using single-particle tracking photoactivated localization microscopy (sptPALM). Our findings reveal Cas1 colocalizing with DNA-dense cellular regions and significant, dose-dependent changes in Cas1 mobility following UV-light-induced damage, providing evidence for its possible involvement in DNA damage response processes and offering new insights into the expanding roles of CRISPR-Cas systems beyond adaptive immunity.

Microproteins (&#x2264;70 amino acids) have important and often essential roles in all kingdoms of life, influencing cell motility, regulation of membrane transport and as transcription factors. In the halophilic archaeon and model system Haloferax volcanii a significant number of &#xb5;-proteins were predicted to be zinc finger proteins. Here we used mass spectrometry-based proteomics to systematically investigate the impact of single gene deletions of 19 zinc finger &#xb5;-proteins on the proteome of H. volcanii grown in synthetic medium with glucose as sole carbon and energy source. We employed a state-of-the-art dia-PASEF acquisition strategy, detecting over 3400 proteins across the 19 deletion strains and the wild type. The comprehensive proteome coverage enabled a systematic analysis of proteome remodeling. We found that in 11 out of the 19 mutants the proteome remodeling involved proteins annotated to play a role in cell motility, matching swarming and growth rate phenotypes we observed for these strains. Taken together, our data provide the most comprehensive proteome coverage of H. volcanii to date, and the effect of 19 different zinc-finger &#xb5;-proteins deletion strains on the proteome of this organism. The combined data (available via ProteomeXchange with identifier PXD066008) provide a valuable resource for future research in the field.

Fungi, and particularly fungal pathogens, are having an increasing impact on human health and economy. At the same time, modern high-throughput technologies offer insights deep into the molecular level and thus mechanisms, giving scientists new opportunities to identify fungal biomarkers and essential components for survival and virulence. This wealth of data, however, is most often only analyzed in the context of a specific scientific question, while many more projects may benefit from a multifaceted view on e.g. fungal gene expression under various conditions. The prime challenge is the limited access to readily pre-processed data and circumventing technological biases introduced by different sequencing platforms and software tools across different projects. We here present FungiNetDB, a web platform comprising 139 fungal pathogenicity datasets and statistical analysis of more than 2000 different pairwise gene expression comparisons. FungiNetDB thus resembles a most comprehensive fungal transcriptomics resource, which can be explored without any programming knowledge, allows highly customizable filtering and cross-project comparisons and download of all offered data tables and visualizations.

Climate change is increasingly recognized as a key driver in the emergence and re-emergence of infectious diseases, including leptospirosis, a globally distributed bacterial zoonosis. The bacterial agents of leptospirosis infect humans through contact with soil or water contaminated by the urine of animal reservoirs. As a primarily waterborne disease, leptospirosis is strongly influenced by climatic conditions, including rainfall, flooding, and extreme events such as hurricanes and cyclones. Here, we present current knowledge on the links between climate change and leptospirosis, as well as perspectives on effective strategies to combat this disease, which disproportionately affects the poorest populations.

Intracellular Gram-negative pathogens employ either type IVA or type IVB secretion systems (T4SSs) to translocate effector proteins into host cells, where they modulate cellular processes to facilitate infection and promote intracellular survival. Roughly one-third of these effectors harbor hydrophobic transmembrane domains and are thus destined for integration into host cell membranes during infection. Many of these transmembrane domain-containing effectors (TMEs) localize to the membrane of the pathogen-containing vacuole, thereby contributing to its formation and remodeling. Despite the biological relevance of TMEs, the detailed molecular mechanisms governing their translocation via T4SSs and subsequent membrane integration in the host cell remain insufficiently understood. In this review, the biophysical characteristics of T4SS-secreted TMEs are systematically examined, including predictions of membrane topology and hydrophobicity. These analyses are then contextualized through comparison with recent structural analysis of both T4ASS and T4BSS machineries, as well as with mechanistic principles of eukaryotic membrane protein biogenesis. This integrative approach enables the conceptual reconstruction of the potential pathways by which TMEs are translocated through the T4SS and subsequently targeted and inserted into host membranes, offering new mechanistic insights into the poorly understood handling of bacterial TMEs from both the pathogen and host perspectives.

Quantitative information on protein abundance is crucial to understand biological processes and is therefore frequently gathered in proteomic studies. However, the quality of a quantitative proteomic dataset is greatly affected by the number of missing values, which need to be minimized to produce robust and meaningful data. In this context, small proteins (&#x2264;100 amino acids) pose specific analytical challenges, which hinder their efficient identification and quantitative characterization in complex proteomes. In this study, methods for sample preparation and MS-data processing are systematically evaluated for their contribution to identification and quantification of small proteins of Clostridioides difficile 630 &#x394;erm. Results show that small protein enrichment can enhance the number of identified and quantified proteins also for low abundant small proteins. Through application of spectral libraries for identification of MS spectra the number of robustly quantified proteins is increased and a lower limit of their detection is reached. Additionally, the dataset presented here is currently the most comprehensive protein repository for C. difficile covering 84.7% of the predicted proteome and 61.4% of all predicted small proteins of this important pathogen.

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&#x3ba;B) represents a family of important transcription factors in innate immunity. We have previously reported that the gastric pathogen Helicobacter pylori needs the actin-binding protein cortactin for efficient interleukin-8 (IL-8) secretion, which requires NF-&#x3ba;B activation. However, it remained unknown, which exact cortactin signaling mechanism contributes to IL-8 release. In fact, H. pylori profoundly activates NF-&#x3ba;B in wild-type AGS gastric epithelial cells by the effector molecule adenosine diphosphate (ADP)-&#x3b2;-d-manno-heptose (ADPH) in a type IV secretion system-dependent manner. However, the injected CagA protein might contribute to NF-&#x3ba;B activation. The ADPH-stimulated canonical NF-&#x3ba;B cascade involves alpha-kinase 1 and adapter protein TRAF-interacting protein with forkhead-associated domain (TIFA) to activate inhibitor of kappa B (I&#x3ba;B) kinases (IKKs), followed by phosphorylation-dependent degradation of I&#x3ba;B&#x3b1; and subsequent nuclear translocation of p65 NF-&#x3ba;B and IL-8 release. Here, we show that infection of cortactin knockout cells leads to reduced activation of focal adhesion kinase (FAK) and c-Sarcoma (Src) kinase resulting in diminished phosphorylation of IKK&#x3b2;&#xa0;at tyrosine residue 199 and subsequently phosphorylation of p65 at serine residue 536, both of which are associated with downregulated NF-&#x3ba;B activity. Our results were further supported using FAK and TIFA knockout cells and treatments with purified ADPH and overexpression of CagA, showing cumulative effects in wild-type, but not in knockout cells. These data demonstrate that ADPH-dependent NF-&#x3ba;B activation and IL-8 secretion are enhanced by CagA. Together, we present here a novel CagA>cortactin>FAK>Src>IKK&#x3b2;&#xa0;signaling cascade, contributing to proinflammatory responses by H. pylori.

CRISPR and their associated Cas proteins provide adaptive immunity in prokaryotes, protecting against invading genetic elements. These systems are categorized into types and are highly diverse. Genomes often harbor multiple CRISPR arrays varying in length and distance from Cas loci. However, the ecological roles of multiple CRISPR arrays and their interactions with multiple Cas loci remain poorly understood. We present a comprehensive analysis of CRISPR systems that uncovers variation between diverse Cas types regarding the occurrence of multiple arrays, the distribution of their lengths and positions relative to Cas loci, and the diversity of their repeat sequences. Some types tend to occur as the sole Cas locus present in the genome, but typically have two or more associated arrays, especially for types I-E and I-F. Multiple Cas types are also common, with some systems showing a preference for specific co-occurrence. Distinct array distributions and orientations around Cas loci indicate substantial differences in functionality and transcriptional behavior among Cas types. Our analysis suggests that arrays with identical repeats in the same genome acquire new spacers at comparable rates, irrespective of their proximity to the Cas locus. Furthermore, repeat similarities indicate that arrays of systems that often co-occur with other systems tend to have more diverse repeats than those mostly appearing alongside solitary systems. Our results indicate that co-occurring Cas-type pairs might not only collaborate in spacer acquisition but also maintain independent and complementary functions and that CRISPR systems distribute their defensive spacer repertoire equally across multiple CRISPR arrays.

In heterogeneous environments, the hyphae of filamentous fungi and oomycetes can facilitate the dispersal of other microorganisms. The use of these "fungal highways" (FH) is regulated by both physical and biological factors with their interplay resulting in variable capabilities of different microbes to establish FH. Several devices have been developed to test the movement of bacteria across mycelium. However, these methods are usually time-consuming and cannot be applied at a large scale. In this study, we developed 3D-printed experimental devices that physically separate two environments while allowing hyphal networks to act as bridges for bacterial movement. The final design allows for the simultaneous testing of up to 10 pairs and the inclusion of any culturing media. With these devices, we investigated how fungal-bacterial pairing, nutrient conditions, and inoculation strategies influence FH formation. Bacterial transport was limited in nutrient-rich media but increased under poorer nutrient conditions, consistent with enhanced exploratory growth of the mycelium. Both cis- and trans-inoculation supported FH formation, although bacterial arrival was delayed in the absence of co-inoculation. The devices were used to demonstrate that transport of bacteria by FH was relevant for the colonization of a natural substrate. Finally, we established a novel in planta assay to evaluate FH formation during host colonization. This assay demonstrated that Fusarium graminearum can transport bacteria during wheat spike colonization. Together, these results provide accessible, scalable tools to study hyphal-mediated bacterial dispersal and highlight the combined role of biological specificity and nutrient context in the establishment of FH.

暂无摘要（点击查看详情）

Fluorescent labeling is a powerful tool in microbiology allowing live cell imaging and providing insights into dynamic cellular processes, quantification of gene expression, and protein subcellular localization. Although multicolor imaging is widely used in Streptococcus pneumoniae and S.&#xa0;mutans, this is less common in other streptococcal species. To address this gap in the streptococcal molecular toolbox, we benchmarked five different fluorescent proteins. They were fused to the C-terminus of S.&#xa0;pneumoniae HlpA, a small non-specific DNA binding histone-like protein. These reporters, combined with four different antibiotic resistance genes, were engineered with various expression systems (inducible or constitutive) to form versatile cassettes. We provide methods to transfer these cassettes to different streptococcal species including S.&#xa0;salivarius, S.&#xa0;thermophilus, and S.&#xa0;pyogenes. As a proof of concept, we generated a triple labeled S. salivarius strain in which HlpA, FtsZ, and DivIVA were fused to three spectrally distinct compatible fluorescent proteins. Multiple fluorescent labeling has broad applications for deciphering a wide range of scientific problems, from cellular processes to infectious disease mechanisms. The availability of these cassettes should allow for a wider use of single-cell labeling strategies in the streptococcus clade and other closely related bacteria.

Protists, which comprise the majority of eukaryotic diversity, frequently engage in endosymbiotic relationships with other unicellular eukaryotes or prokaryotes. These interactions have profoundly shaped eukaryotic evolution, not only through the origin of endosymbiotic organelles and the subsequent diversification of eukaryotes, but also via less studied endosymbioses that have influenced the evolution of diverse eukaryotic lineages. Endosymbioses often alter host metabolic capabilities, enabling the colonisation of new ecological niches and significantly contributing to ecosystem functioning. In recent years, interest in these interactions has increased, driven by methodological innovations and new discoveries that reveal the diversity, mechanisms, and ecological roles of protist endosymbioses. Despite these advances, key questions remain: How widespread and ecologically impactful are protist endosymbioses? What functions do symbionts provide, and how do associations form, persist, or break down? Addressing these questions requires systematic studies of protists in their natural environments, combining microscopy and sequencing using both high-throughput and single-cell approaches, along with experimental manipulations of host-symbiont interactions. Here, we review current knowledge, highlight recent breakthroughs, and discuss ongoing challenges in the study of protist endosymbioses.

Microbiota play crucial roles in host health, including protection against pathogens through competitive interactions between commensal and pathogenic bacteria that are mediated by direct contact or secreted factors. We previously demonstrated that Chryseobacterium massiliae, a zebrafish commensal, protects larvae against infection by Flavobacterium covae (formerly F. columnare). Here, we investigated whether interference interactions contribute to this protective effect. We found that C. massiliae culture supernatant inhibits F. covae growth and a transposon mutagenesis screen identified mutants lacking this activity. All identified mutants carried insertions in a gene encoding a protein homologous to Bacteroidales BSAP pore-forming toxins, characterized by a Membrane Attack Complex/Perforin (MACPF) domain. We showed that this protein, which we named CSAP-1 (for Chryseobacterium Secreted Antimicrobial Protein) displays bactericidal, pore-forming activity that lyses F. covae cells. Unlike BSAP proteins from Bacteroides spp., CSAP-1 displays broader antagonistic activity, targeting multiple species across the Flavobacterium and Chryseobacterium genera-thus mediating interspecies and intergenus inhibition within Bacteroidetes. Although CSAP-1 is not essential for the in vivo protective effect of C. massiliae, administration of purified CSAP-1 alone confers significant protection to zebrafish larvae against sensitive F. covae infection. This study therefore identifies CSAP-1 as the first MACPF protein from C. massiliae with broad-spectrum inhibitory activity against members of the order Flavobacteriales. These findings highlight CSAP-1 as a promising candidate for the development of novel antimicrobial strategies and warrant further mechanistic investigation.

Type IV secretion systems (T4SS) are found in both monoderm and diderm bacteria. The broad-host-range conjugative plasmid pIP501 from Enterococcus faecalis harbors a T4SS encoding 15 tra genes responsible for the spread of antimicrobial resistance genes among diverse G+ pathogens. Eight Tra proteins (TraB, TraCB3, TraF, TraHB8, TraI, TraK, TraLB6, and TraMB8) are postulated to form the mating pair formation (MPF) complex representing the central DNA translocation pore. One of these proteins is TraF, a 52.8&#xa0;kDa transmembrane protein, which lacks any homologs in other well described T4SSs. In this study, TraF was proven to be an essential conjugative transfer protein. The TraF pulldown co-eluted all Tra proteins except TraGB1 and TraN. Bacterial-two-hybrid assay showed a strong interaction between TraF and TraMB8. We present a 1.25&#xa0;&#xc5; resolution crystal structure of the N-terminal domain of TraF, which adopts a pseudokinase fold. AlphaFold predictions of full-length TraF with membrane mimetics show a transmembrane protein with two distinct soluble domains. FoldSeek revealed a strong similarity to YukC (EssB), a transmembrane pseudokinase from type VII secretion system (T7SS). YukC was shown to function as an interaction hub by mediating contacts between its pseudokinase domain and other T7SS proteins as part of the central membrane core complex. We postulate that TraF might play an important role in T4SS complex formation.

Chlamydia trachomatis is a major human pathogen responsible for the most prevalent bacterial sexually transmitted infections. Given its high prevalence, potential for serious complications and high economic burden, understanding the pathogenesis of C. trachomatis infections is crucial for developing effective prevention and treatment strategies. Mucosal epithelial cells serve as the primary replicative niche and play a central role in infection. Historically, research has relied heavily on cancer-derived cell line models to study the interactions of&#xa0;C. trachomatis with host cells. Although these models have provided important insights, they fail to fully recapitulate key aspects of human physiology, including apical-basal polarity, a fundamental feature that shapes epithelial functions. Limited studies using polarized in vitro epithelial systems have uncovered unique mechanisms by which C. trachomatis interacts with host cells. In this review, we summarize the current, albeit limited, knowledge on the role of epithelial polarity during C. trachomatis infection and highlight this understudied aspect of chlamydial biology.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated genes) systems provide adaptive immunity in bacteria and archaea against mobile genetic elements, but the role they play in gene exchange and speciation remains unclear. Here, we investigated how CRISPR-Cas targeting affects mating and gene exchange in the halophilic archaeon Haloferax volcanii. Surprisingly, we found that CRISPR-Cas targeting significantly increased mating efficiency between members of the same species, in contrast to its previously documented role in reducing interspecies mating. This enhanced mating efficiency was dependent on the Cas3 nuclease/helicase and extended beyond the targeted genomic regions. Further analysis revealed that CRISPR-Cas targeting promoted biased recombination in favor of the targeting strain (the strain containing the CRISPR-Cas system) during mating, resulting in an increased proportion of recombinant progeny that are positive for CRISPR-Cas. To test whether an increase in recombination is sufficient to increase mating efficiency, we tested whether strains lacking the Mre11-Rad50 complex, which are known to have elevated recombination activity, also exhibited higher mating success. Indeed, these strains showed higher mating, as did cells that were exposed to DNA damage using methyl methanesulfonate. These findings suggest that CRISPR-Cas systems in archaea play roles beyond their canonical immune function. They may contribute to speciation by facilitating within-species gene exchange while limiting between-species genetic transfer, thereby maintaining species boundaries.