scholarly journals Mobile-CRISPRi: Enabling Genetic Analysis of Diverse Bacteria

2018 ◽  
Author(s):  
Jason M. Peters ◽  
Byoung-Mo Koo ◽  
Ramiro Patino ◽  
Gary E. Heussler ◽  
Cameron C. Hearne ◽  
...  
Keyword(s):  
Gene Networks ◽  
Essential Genes ◽  
Genetic Dissection ◽  
Human Pathogens ◽  
Major Barrier ◽  
Bacterial Gene ◽  
Bacterial Physiology ◽  
Individual Gene ◽  
Host Microbe Interactions ◽  
The Individual

Introductory paragraphThe vast majority of bacteria, including human pathogens and microbiome species, lack genetic tools needed to systematically associate genes with phenotypes. This is the major impediment to understanding the fundamental contributions of genes and gene networks to bacterial physiology and human health. CRISPRi, a versatile method of blocking gene expression using a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs (sgRNAs), has emerged as a powerful genetic tool to dissect the functions of essential and non-essential genes in species ranging from bacteria to human. However, the difficulty of establishing effective CRISPRi systems in non-model bacteria is a major barrier to its widespread use to dissect bacterial gene function. Here, we establish “Mobile-CRISPRi”, a suite of CRISPRi systems that combine modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation. Focusing predominantly on human pathogens associated with antibiotic resistance, we demonstrate the efficacy of Mobile-CRISPRi in Proteobacteria and Firmicutes at the individual gene scale by examining drug-gene synergies and at the library scale by systematically phenotyping conditionally essential genes involved in amino acid biosynthesis. Mobile-CRISPRi enables genetic dissection of non-model bacteria, facilitating analyses of microbiome function, antibiotic resistances and sensitivities, and comprehensive screens for host-microbe interactions.

scholarly journals Perturbations of Ribosomal Protein Expression Reveal Overlapping Gene Networks between Drosophila Minutes and Human Cancer

2020 ◽  
Author(s):  
Jai A Denton ◽  
Mariana Velasque ◽  
Floyd A Reed
Keyword(s):  
Ribosomal Proteins ◽  
Gene Networks ◽  
Ribosome Biogenesis ◽  
Human Cancer ◽  
Whole Body ◽  
Individual Gene ◽  
Gene Level ◽  
The Individual ◽  
Time Specific

AbstractRibosomal proteins (RPs) are critical to all cellular operations through their key roles in ribosome biogenesis and translation, as well as their extra-ribosomal functions. Although highly tissue- and time-specific in expression, little is known about the macro-level roles of RPs in shaping transcriptomes. A wealth of RP mutants exist, including the Drosophila melanogaster Minutes, with RP encoding genes that vary from greatly under-expressed to greatly over-expressed. Leveraging a subset of these mutants and using whole-body RNA sequencing, we identified the RP macro transcriptome and then sought to compare it with transcriptomes of pathologies associated with failures of ribosomal function. Gene-based analysis revealed highly variable transcriptomes of RP mutations with little overlap in genes that were differentially expressed. In contrast, weighted gene co-expression network analysis (WGCNA) revealed a highly conserved pattern across all RP mutants studied. When we compared network changes in RP mutants, we observed similarities to transcriptome alterations in human cancer, and thus confirming the oncogenic role of RPs. Therefore, what may appear stochastic at the individual gene level, forms clearly predictable patterns when viewed as a whole.

scholarly journals Single-cell RNA sequencing reveals the mesangial identity and species diversity of glomerular cell transcriptomes

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bing He ◽  
Ping Chen ◽  
Sonia Zambrano ◽  
Dina Dabaghie ◽  
Yizhou Hu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Human Kidney ◽  
Cell Types ◽  
Model Organisms ◽  
Mural Cell ◽  
Glomerular Cell ◽  
Individual Gene ◽  
The Individual

AbstractMolecular characterization of the individual cell types in human kidney as well as model organisms are critical in defining organ function and understanding translational aspects of biomedical research. Previous studies have uncovered gene expression profiles of several kidney glomerular cell types, however, important cells, including mesangial (MCs) and glomerular parietal epithelial cells (PECs), are missing or incompletely described, and a systematic comparison between mouse and human kidney is lacking. To this end, we use Smart-seq2 to profile 4332 individual glomerulus-associated cells isolated from human living donor renal biopsies and mouse kidney. The analysis reveals genetic programs for all four glomerular cell types (podocytes, glomerular endothelial cells, MCs and PECs) as well as rare glomerulus-associated macula densa cells. Importantly, we detect heterogeneity in glomerulus-associated Pdgfrb-expressing cells, including bona fide intraglomerular MCs with the functionally active phagocytic molecular machinery, as well as a unique mural cell type located in the central stalk region of the glomerulus tuft. Furthermore, we observe remarkable species differences in the individual gene expression profiles of defined glomerular cell types that highlight translational challenges in the field and provide a guide to design translational studies.

scholarly journals Pseudomonas Lipopeptide-Mediated Biocontrol: Chemotaxonomy and Biological Activity

Molecules ◽  
2022 ◽  
Vol 27 (2) ◽  
pp. 372
Author(s):  
Feyisara Eyiwumi Oni ◽  
Qassim Esmaeel ◽  
Joseph Tobias Onyeka ◽  
Rasheed Adeleke ◽  
Cedric Jacquard ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
In Vitro Assays ◽  
Bacterial Physiology ◽  
Lineage Specificity ◽  
Microbe Interactions ◽  
Host Microbe Interactions ◽  
The One ◽  
Taxonomic Groups ◽  
Plant Pathogen Interactions

Pseudomonas lipopeptides (Ps-LPs) play crucial roles in bacterial physiology, host–microbe interactions and plant disease control. Beneficial LP producers have mainly been isolated from the rhizosphere, phyllosphere and from bulk soils. Despite their wide geographic distribution and host range, emerging evidence suggests that LP-producing pseudomonads and their corresponding molecules display tight specificity and follow a phylogenetic distribution. About a decade ago, biocontrol LPs were mainly reported from the P. fluorescens group, but this has drastically advanced due to increased LP diversity research. On the one hand, the presence of a close-knit relationship between Pseudomonas taxonomy and the molecule produced may provide a startup toolbox for the delineation of unknown LPs into existing (or novel) LP groups. Furthermore, a taxonomy–molecule match may facilitate decisions regarding antimicrobial activity profiling and subsequent agricultural relevance of such LPs. In this review, we highlight and discuss the production of beneficial Ps-LPs by strains situated within unique taxonomic groups and the lineage-specificity and coevolution of this relationship. We also chronicle the antimicrobial activity demonstrated by these biomolecules in limited plant systems compared with multiple in vitro assays. Our review further stresses the need to systematically elucidate the roles of diverse Ps-LP groups in direct plant–pathogen interactions and in the enhancement of plant innate immunity.

scholarly journals Network-based pathogenicity prediction for variants of uncertain significance

2021 ◽  
Author(s):  
Mayumi Kamada ◽  
Atsuko Takagi ◽  
Ryosuke Kojima ◽  
Yoshihisa Tanaka ◽  
Masahiko Nakatsui ◽  
...  
Keyword(s):  
Computational Method ◽  
Major Barrier ◽  
Clinical Interpretation ◽  
Individual Gene ◽  
Variants Of Uncertain Significance ◽  
Uncertain Significance ◽  
Benchmark Datasets ◽  
Genomic Scale ◽  
Molecular Relationships ◽  
Potential Pathogenicity

While the number of genome sequences continues to increase, the functions of many detected gene variants remain to be identified. These variants of uncertain significance constitute a major barrier to precision medicine. Although many computational methods have been developed to predict the function of these variants, they all rely on individual gene features and do not consider complex molecular relationships. Here we develop PathoGN, a molecular network-based approach for predicting variant pathogenicity. PathoGN significantly outperforms existing methods using benchmark datasets. Moreover, PathoGN successfully predicts the pathogenicity of 3,994 variants of uncertain significance in the real-world database ClinVar and designates potential pathogenicity. This is the first computational method for the clinical interpretation of variants using biomolecular networks, and we anticipate our method to be broadly useful for the clinical interpretation of variants and for assigning biological function to unknown variants at the genomic scale.

scholarly journals Technical considerations when designing a gene expression panel for renal transplant diagnosis

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
F. Toulza ◽  
K. Dominy ◽  
T. Cook ◽  
J. Galliford ◽  
J. Beadle ◽  
...  
Keyword(s):  
Gene Expression ◽  
Renal Transplant ◽  
Biological Significance ◽  
Tissue Sampling ◽  
Individual Gene ◽  
Antibody Mediated Rejection ◽  
Gene Level ◽  
Diagnostic Panel ◽  
The Individual ◽  
Gene Panels

Abstract Gene expression analysis is emerging as a new diagnostic tool in transplant pathology, in particular for the diagnosis of antibody-mediated rejection. Diagnostic gene expression panels are defined on the basis of their pathophysiological relevance, but also need to be tested for their robustness across different preservatives and analysis platforms. The aim of this study is the investigate the effect of tissue sampling and preservation on candidate genes included in a renal transplant diagnostic panel. Using the NanoString platform, we compared the expression of 219 genes in 51 samples, split for formalin-fixation and paraffin-embedding (FFPE) and RNAlater preservation (RNAlater). We found that overall, gene expression significantly correlated between FFPE and RNAlater samples. However, at the individual gene level, 46 of the 219 genes did not correlate across the 51 matched FFPE and RNAlater samples. Comparing gene expression results using NanoString and qRT-PCR for 18 genes in the same pool of RNA (RNAlater), we found a significant correlation in 17/18 genes. Our study indicates that, in samples from the same routine diagnostic renal transplant biopsy procedure split for FFPE and RNAlater, 21% of 219 genes of potential biological significance do not correlate in expression. Whether this is due to fixatives or tissue sampling, selection of gene panels for routine diagnosis should take this information into consideration.

scholarly journals The functional ClpXP protease of Chlamydia trachomatis requires distinct clpP genes from separate genetic loci

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Stefan Pan ◽  
Imran T. Malik ◽  
Dhana Thomy ◽  
Beate Henrichfreise ◽  
Peter Sass
Keyword(s):  
Chlamydia Trachomatis ◽  
Bacterial Species ◽  
Human Pathogens ◽  
Bacterial Physiology ◽  
Sexually Transmitted ◽  
Clp Proteases ◽  
Obligate Intracellular Pathogen ◽  
Antibacterial Target ◽  
Insight Into

Abstract Clp proteases play a central role in bacterial physiology and, for some bacterial species, are even essential for survival. Also due to their conservation among bacteria including important human pathogens, Clp proteases have recently attracted considerable attention as antibiotic targets. Here, we functionally reconstituted and characterized the ClpXP protease of Chlamydia trachomatis (ctClpXP), an obligate intracellular pathogen and the causative agent of widespread sexually transmitted diseases in humans. Our in vitro data show that ctClpXP is formed by a hetero-tetradecameric proteolytic core, composed of two distinct homologs of ClpP (ctClpP1 and ctClpP2), that associates with the unfoldase ctClpX via ctClpP2 for regulated protein degradation. Antibiotics of the ADEP class interfere with protease functions by both preventing the interaction of ctClpX with ctClpP1P2 and activating the otherwise dormant proteolytic core for unregulated proteolysis. Thus, our results reveal molecular insight into ctClpXP function, validating this protease as an antibacterial target.

scholarly journals Deep transcriptomic profiling reveals the similarity between endothelial cells cultured under static and oscillatory shear stress conditions

2016 ◽  
Vol 48 (9) ◽  
pp. 660-666 ◽  
Author(s):  
Congzhen Qiao ◽  
Fan Meng ◽  
Inhwan Jang ◽  
Hanjoong Jo ◽  
Y. Eugene Chen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Function ◽  
Stress Conditions ◽  
Individual Gene ◽  
Gene Level ◽  
The Individual ◽  
Oscillatory Shear Stress ◽  
Whole Transcriptome ◽  
Transcriptome Level

Atherosclerosis is a multifactorial disease that preferentially develops in specific regions in the arterial tree. This characteristic is mainly attributed to the unique pattern of hemodynamic shear stress in vivo. High laminar shear stress (LS) found in straight lumen exerts athero-protective effects. Low or oscillatory shear stress (OS) present in regions of lesser curvature and arterial bifurcations predisposes arterial intima to atherosclerosis. Shear stress-regulated endothelial function plays an important role in the process of atherosclerosis. Most in vitro research studies focusing on the molecular mechanisms of endothelial function are performed in endothelial cells (ECs) under cultured static (ST) condition. Some findings, however, are not recapitulated in subsequent translational studies, mostly likely due to the missing biomechanical milieu. Here, we profiled the whole transcriptome of primary human coronary arterial endothelial cells (HCAECs) under different shear stress conditions with RNA sequencing. Among 16,313 well-expressed genes, we detected 8,177 that were differentially expressed in OS vs. LS conditions and 9,369 in ST vs. LS conditions. Notably, only 1,618 were differentially expressed in OS vs. ST conditions. Hierarchical clustering of ECs demonstrated a strong similarity between ECs under OS and ST conditions at the transcriptome level. Subsequent pairwise heat mapping and principal component analysis gave further weight to the similarity. At the individual gene level, expressional analysis of representative well-known genes as well as novel genes showed a comparable amount at mRNA and protein levels in ECs under ST and OS conditions. In conclusion, the present work compared the whole transcriptome of HCAECs under different shear stress conditions at the transcriptome level as well as at the individual gene level. We found that cultured ECs are significantly different from those under LS conditions. Thus using cells under ST conditions is unlikely to elucidate endothelial physiology. Given the revealed high similarities of the endothelial transcriptome under OS and ST conditions, it may be helpful to understand the underlying mechanisms of OS-induced endothelial dysfunction from static cultured endothelial studies.

Mechanisms by Which Small RNAs Affect Bacterial Activity

2019 ◽  
Vol 98 (12) ◽  
pp. 1315-1323
Author(s):  
L. Lei ◽  
Y. Yang ◽  
Y. Yang ◽  
S. Wu ◽  
X. Ma ◽  
...  
Keyword(s):  
Stress Responses ◽  
Small Rnas ◽  
Environmental Changes ◽  
Expression Profiles ◽  
Bacterial Flora ◽  
Oral Disease ◽  
Bacterial Gene ◽  
Bacterial Physiology ◽  
Cellular Processes ◽  
Regulatory Rnas

The oral cavity contains a distinct habitat that supports diverse bacterial flora. Recent observations have provided additional evidence that sRNAs are key regulators of bacterial physiology and pathogenesis. These sRNAs have been divided into 5 functional groups: cis-encoded RNAs, trans-encoded RNAs, RNA regulators of protein activity, bacterial CRISPR (clustered regularly interspaced short palindromic repeat) RNAs, and a novel category of miRNA-size small RNAs (msRNAs). In this review, we discuss a critical group of key commensal and opportunistic oral pathogens. In general, supragingival bacterial sRNAs function synergistically to fine-tune the regulation of cellular processes and stress responses in adaptation to environmental changes. Particularly in the cariogenic bacteria Streptococcus mutans, both the antisense vicR RNA and msRNA1657 can impede the metabolism of bacterial exopolysaccharides, prevent biofilm formation, and suppress its cariogenicity. In Enterococcus faecalis, selected sRNAs control the expression of proteins involved in diverse cellular processes and stress responses. In subgingival plaques, sRNAs from periodontal pathogens can function as novel bacterial signaling molecules that mediate bacterial-human interactions in periodontal homeostasis. In Porphyromonas gingivalis, the expression profiles of putative sRNA101 and sRNA42 were found to respond to hemin availability after hemin starvation. Regarding Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), a major periodontal pathogen associated with aggressive periodontitis, the predicted sRNAs interact with several virulence genes, including those encoding leukotoxin and cytolethal distending toxin. Furthermore, in clinical isolates, these associated RNAs could be explored not only as potential biomarkers for oral disease monitoring but also as alternative types of regulators for drug design. Thus, this emerging subspecialty of bacterial regulatory RNAs could reshape our understanding of bacterial gene regulation from their key roles of endogenous regulatory RNAs to their activities in pathologic processes.

Molecular Profiling and Commercial Predication Assays in Ovarian Cancer: Still Not Ready for Prime Time?

2014 ◽  
pp. 139-147 ◽  
Author(s):  
Elise C. Kohn
Keyword(s):  
Ovarian Cancer ◽  
Germ Line ◽  
Molecular Profiling ◽  
Added Value ◽  
Prime Time ◽  
Cancer Type ◽  
Brca1 And Brca2 ◽  
Major Barrier ◽  
The Individual

Short of early detection to allow curative primary intervention, the other major barrier to further success in treatment of ovarian cancers is matching the best treatment to the proper ovarian cancer type and to the individual patient. There are several decades of experience applying in vitro chemoresponse testing for solid tumors including ovarian cancer. This concept, first described in 1979, has yet to receive level one evidence supporting its application, despite the testing of numerous assays commercially as well as in academic centers and its use for tens of thousands of patients at a significant cost. The approach—rather than undergoing rigorous scientific examination—is now being muddied by the development of commercial molecular profiling assays from which treatment suggestions are provided. Molecular profiling as a research tool has added value to our understanding and treatment of patients with ovarian cancer. Morphologic and histochemical characterizations coupled now with increasing knowledge of ovarian cancer type-specific molecular patterns is improving our ability to properly diagnosis ovarian cancer type and thus guide therapy. With the exception of the role of germ-line and possibly somatic BRCA1 and BRCA2 mutations and their true predictiveness for probable response to poly(ADP-ribose) polymerase inhibition, molecular typing and profiling has yet to identify druggable molecular targets in ovarian cancer. Its use should be continued as a research and learning tool, and its results should be subjected to clinical trial validation. For very different reasons, neither chemoresponse assays nor molecular profiling are ready for prime time, yet.

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