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MBio Jun 2024Transposon sequencing (Tn-seq) is a powerful genome-wide technique to assess bacterial fitness under varying growth conditions. However, screening via Tn-seq is...
UNLABELLED
Transposon sequencing (Tn-seq) is a powerful genome-wide technique to assess bacterial fitness under varying growth conditions. However, screening via Tn-seq is challenging. Dose limitations and host restrictions create bottlenecks that diminish the transposon mutant pool being screened. Here, we have developed a murine model with a disruption in that renders the resulting RECON mouse resistant to high-dose infection. We leveraged this model to perform a Tn-seq screen of the human pathogen . We identified 135 genes which were required for growth in mice including novel genes not previously identified for host survival. We identified organ-specific requirements for survival and investigated the role of the folate enzyme FolD in liver pathogenesis. A mutant lacking was impaired for growth in murine livers by 2.5-log compared to wild type and failed to spread cell-to-cell in fibroblasts. In contrast, a mutant in which encodes a transcription factor that represses an operon involved in D-allose catabolism, was attenuated in both livers and spleens of mice by 4-log and 3-log, respectively, but showed modest phenotypes in models. We confirmed that dysregulation of the D-allose catabolism operon is responsible for the growth defect, as deletion of the operon in the ∆ background rescued virulence. By undertaking an unbiased, genome-wide screen in mice, we have identified novel fitness determinants for host infection, which highlights the utility of the RECON mouse model for future screening efforts.
IMPORTANCE
is the gram-positive bacterium responsible for the food-borne disease listeriosis. Although infections with are limiting in healthy hosts, vulnerable populations, including pregnant and elderly people, can experience high rates of mortality. Thus, understanding the breadth of genetic requirements for survival will present new opportunities for treatment and prevention of listeriosis. We developed a murine model of infection using a RECON mouse that is restrictive to systemic infection. We utilized this model to screen for genes required via transposon sequencing. We identified the liver-specific gene and a repressor, , that only exhibits an growth defect. AlsR controls the expression of the D-allose operon which is a marker in diagnostic techniques to identify pathogenic . A better understanding of the role of the D-allose operon in human disease may further inform diagnostic and prevention measures.
PubMed: 38940553
DOI: 10.1128/mbio.01332-24 -
MSphere Jun 2024The adaptation of gene deletion methods based on the CRISPR-Cas9 system has facilitated the genetic manipulation of the pathogenic yeast , because homozygous mutants of...
Probing gene function in wild-type strains by Cas9-facilitated one-step integration of two dominant selection markers: a systematic analysis of recombination events at the target locus.
UNLABELLED
The adaptation of gene deletion methods based on the CRISPR-Cas9 system has facilitated the genetic manipulation of the pathogenic yeast , because homozygous mutants of this diploid fungus can now be generated in a single step, allowing the rapid screening of candidate genes for their involvement in a phenotype of interest. However, the Cas9-mediated double-strand breaks at the target site may result in an undesired loss of heterozygosity (LOH) on the affected chromosome and cause phenotypic alterations that are not related to the function of the investigated gene. In our present study, we harnessed Cas9-facilitated gene deletion to probe a set of genes that are constitutively overexpressed in strains containing hyperactive forms of the transcription factor Mrr1 for a possible contribution to the fluconazole resistance of such strains. To this aim, we used gene deletion cassettes containing two different dominant selection markers, and , which confer resistance to nourseothricin and hygromycin, respectively, for simultaneous genomic integration in a single step, hypothesizing that this would minimize undesired LOH events at the target locus. We found that selection for resistance to both nourseothricin and hygromycin strongly increased the proportion of homozygous deletion mutants among the transformants compared with selection on media containing only one of the antibiotics, but it did not avoid undesired LOH events. Our results demonstrate that LOH on the target chromosome is a significant problem when using Cas9 for the generation of gene deletion mutants, which demands a thorough examination of recombination events at the target site.
IMPORTANCE
is one of the medically most important fungi and a model organism to study fungal pathogenicity. Investigating gene function in this diploid yeast has been facilitated by the adaptation of gene deletion methods based on the bacterial CRISPR-Cas9 system, because they enable the generation of homozygous mutants in a single step. We found that, in addition to increasing the efficiency of gene replacement by selection markers, the Cas9-mediated double-strand breaks also result in frequent loss of heterozygosity on the same chromosome, even when two different selection markers were independently integrated into the two alleles of the target gene. Since loss of heterozygosity for other genes can result in phenotypic alterations that are not caused by the absence of the target gene, these findings show that it is important to thoroughly analyze recombination events at the target locus when using Cas9 to generate gene deletion mutants in .
PubMed: 38940507
DOI: 10.1128/msphere.00388-24 -
Development (Cambridge, England) Jun 2024The vertebral column is a characteristic structure of vertebrates. Genetic studies in mice have shown that Hox-mediated patterning plays a key role in specifying...
The vertebral column is a characteristic structure of vertebrates. Genetic studies in mice have shown that Hox-mediated patterning plays a key role in specifying discrete anatomical regions of the vertebral column. Expression pattern analyses in several vertebrate embryos provided correlative evidence that the anterior boundaries of Hox expression coincide with distinct anatomical vertebrae. However, because functional analyses have been limited to mice, it remains unclear which Hox genes actually function in vertebral patterning in other vertebrates. In this study, various zebrafish hox mutants were generated for loss-of-function phenotypic analysis to functionally decipher the Hox code responsible for the zebrafish anterior vertebrae between the occipital and thoracic vertebrae. We found that hox genes in HoxB- and HoxC-related clusters participate in regulating the morphology of the zebrafish anterior vertebrae. In addition, medaka hoxc6a was found to be responsible for anterior vertebral identity as in zebrafish. Based on phenotypic similarities with Hoxc6 knockout mice, our results suggest that the Hox patterning system, including at least Hoxc6, may have been functionally established in the vertebral patterning of the common ancestor of ray-finned and lobe-finned fishes.
PubMed: 38940461
DOI: 10.1242/dev.202854 -
Journal of Integrative Plant Biology Jun 2024In flowering plants, sexual reproductive success depends on the production of viable pollen grains. However, the mechanisms by which QUA QUINE STARCH (QQS) regulates...
In flowering plants, sexual reproductive success depends on the production of viable pollen grains. However, the mechanisms by which QUA QUINE STARCH (QQS) regulates pollen development and how transcriptional activators facilitate the transcription of QQS in this process remain poorly understood. Here, we demonstrate that INDUCER OF CBF EXPRESSION 1 (ICE1), a basic helix-loop-helix (bHLH) transcription factor, acts as a key transcriptional activator and positively regulates QQS expression to increase pollen germination and viability in Arabidopsis thaliana by interacting with INDETERMINATE DOMAIN14 (IDD14). In our genetic and biochemical experiments, overexpression of ICE1 greatly promoted both the activation of QQS and high pollen viability mediated by QQS. IDD14 additively enhanced ICE1 function by promoting the binding of ICE1 to the QQS promoter. In addition, mutation of ICE1 significantly repressed QQS expression; the impaired function of QQS and the abnormal anther dehiscence jointly affected pollen development of the ice1-2 mutant. Our results also showed that the enhancement of pollen activity by ICE1 depends on QQS. Furthermore, QQS interacted with CUT1, the key enzyme for long-chain lipid biosynthesis. This interaction both promoted CUT1 activity and regulated pollen lipid metabolism, ultimately determining pollen hydration and fertility. Our results not only provide new insights into the key function of QQS in promoting pollen development by regulating pollen lipid metabolism, but also elucidate the mechanism that facilitates the transcription of QQS in this vital developmental process.
PubMed: 38940322
DOI: 10.1111/jipb.13725 -
Development (Cambridge, England) Jul 2024During heart development, the embryonic ventricle becomes enveloped by the epicardium, which adheres to the outer apical surface of the heart. This is concomitant with...
During heart development, the embryonic ventricle becomes enveloped by the epicardium, which adheres to the outer apical surface of the heart. This is concomitant with onset of ventricular trabeculation, where a subset of cardiomyocytes lose apicobasal polarity and delaminate basally from the ventricular wall. Llgl1 regulates the formation of apical cell junctions and apicobasal polarity, and we investigated its role in ventricular wall maturation. We found that llgl1 mutant zebrafish embryos exhibit aberrant apical extrusion of ventricular cardiomyocytes. While investigating apical cardiomyocyte extrusion, we identified a basal-to-apical shift in laminin deposition from the internal to the external ventricular wall. We find that epicardial cells express several laminin subunits as they adhere to the ventricle, and that the epicardium is required for laminin deposition on the ventricular surface. In llgl1 mutants, timely establishment of the epicardial layer is disrupted due to delayed emergence of epicardial cells, resulting in delayed apical deposition of laminin on the ventricular surface. Together, our analyses reveal an unexpected role for Llgl1 in correct timing of epicardial development, supporting integrity of the ventricular myocardial wall.
Topics: Animals; Laminin; Zebrafish; Pericardium; Heart Ventricles; Zebrafish Proteins; Myocytes, Cardiac; Cell Polarity; Mutation
PubMed: 38940292
DOI: 10.1242/dev.202482 -
Oncology Letters Aug 2024Glioblastoma multiforme (GBM) is an aggressive brain cancer that occurs more frequently than other brain tumors. The present study aimed to reveal a novel mechanism of...
Glioblastoma multiforme (GBM) is an aggressive brain cancer that occurs more frequently than other brain tumors. The present study aimed to reveal a novel mechanism of temozolomide resistance in GBM using bioinformatics and wet lab analyses, including meta-Z analysis, Kaplan-Meier survival analysis, protein-protein interaction (PPI) network establishment, cluster analysis of co-expressed gene networks, and hierarchical clustering of upregulated and downregulated genes. Next-generation sequencing and quantitative PCR analyses revealed downregulated [tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 1 (), calcium voltage-gated channel auxiliary subunit α2Δ1 (), calpain 6 () and a disintegrin and metalloproteinase with thrombospondin motifs 6 ()] and upregulated [serum amyloid (), growth differentiation factor 15 () and ubiquitin specific peptidase 26 ()] genes. Different statistical models were developed for these genes using the Z-score for P-value conversion, and Kaplan-Meier plots were constructed using several patient cohorts with brain tumors. The highest number of nodes was observed in the PPI network was for and . The PPI network model for all genes contained 35 nodes and 241 edges. Immunohistochemical staining was performed using isocitrate dehydrogenase (IDH)-wild-type or IDH-mutant GBM samples from patients and a significant upregulation of TIE1 (P<0.001) and CAPN6 (P<0.05) protein expression was demonstrated in IDH-mutant GBM in comparison with IDH-wild-type GBM. Structural analysis revealed an IDH-mutant model demonstrating the mutant residues (R132, R140 and R172). The findings of the present study will help the future development of novel biomarkers and therapeutics for brain tumors.
PubMed: 38939621
DOI: 10.3892/ol.2024.14511 -
Health Care Science Aug 2023The coronavirus disease (COVID-19), which the World Health Organization classified as the Sixth Public Health Emergency Of International Concern (PHEIC) on January 30,... (Review)
Review
The coronavirus disease (COVID-19), which the World Health Organization classified as the Sixth Public Health Emergency Of International Concern (PHEIC) on January 30, 2020, is no longer a PHEIC. Millions were affected due to unawareness. The increase in fatalities and shortage of medicine was the first outrage of COVID-19. As per the Johns Hopkins COVID-19 resource center database, it was observed that the disease has spread dynamically across 200+ nations worldwide affecting more than 600 million people from 2019 to 2023, and over thousands of people were victimized regularly at a 2% mortality rate (approx.). In the midway, the mutant variants of concern like omicron, and delta have also created havoc and caused significant impact on public health, global economy, and lifestyle. Since 2019, 3 years now passed and the dynamic disease statistics seem decelerated; moreover, the prevalence of COVID-19 is also fading. The Johns Hopkins resource center has also stopped recording the data of the global pandemic recently from March 10, 2023. Hence, based on the facts, we are presenting a concise report on the pandemic from 2019 to 2023, which includes a brief discussion of the global pandemic. We have highlighted global epidemiology, emphasizing the Indian COVID scenario, vaccination across the globe, and the psychosocial and geopolitical consequences of COVID-19 with a brief background to pathology, clinical management, and the worldwide response against triage. A lot has changed and still needs to change after three tough years of COVID-19. Even though science has progressed and advanced research in medicine is pointing toward future generations, there is no standard care supplied for COVID-19-like calamities. COVID-19 cases might have declined but its influence on the society is still stagnant. This COVID experience has taught us that, despite our bleak beginnings, there is always hope for the future and that we must act with foresight to improve things for future generations.
PubMed: 38939523
DOI: 10.1002/hcs2.57 -
Journal of Extracellular Biology Aug 2023Non-coding RNAs (ncRNAs) are important regulators of gene expression. They are expressed not only in cells, but also in cell-derived extracellular vesicles (EVs). The...
Non-coding RNAs (ncRNAs) are important regulators of gene expression. They are expressed not only in cells, but also in cell-derived extracellular vesicles (EVs). The mechanisms controlling their loading and sorting remain poorly understood. Here, we investigated the impact of mutations on the non-coding RNA content of small melanoma EVs. After purification of small EVs from six different patient-derived melanoma cell lines, we characterized them by small RNA sequencing and lncRNA microarray analysis. We found that mutations are associated with a specific micro and long non-coding RNA content in small EVs. Then, we showed that long and small non-coding RNAs enriched in mutant small EVs share a common sequence motif, highly similar to the RNA-binding motif of Sam68, a protein interacting with hnRNP proteins. This protein thus may be an interesting partner of p53, involved in the expression and loading of the ncRNAs. To conclude, our data support the existence of cellular mechanisms associate with mutations which control the ncRNA content of small EVs in melanoma.
PubMed: 38939511
DOI: 10.1002/jex2.105 -
Frontiers in Oncology 2024Field cancerization is suggested to arise from imbalanced differentiation in individual basal progenitor cells leading to clonal expansion of mutant cells that...
INTRODUCTION
Field cancerization is suggested to arise from imbalanced differentiation in individual basal progenitor cells leading to clonal expansion of mutant cells that eventually replace the epithelium, although without evidence.
METHODS
We performed deep sequencing analyses to characterize the genomic and transcriptomic landscapes of field change in two patients with synchronous aerodigestive tract tumors.
RESULTS
Our data support the emergence of numerous genetic alterations in cancer-associated genes but refutes the hypothesis that founder mutation(s) underpin this phenomenon. Mutational signature analysis identified defective homologous recombination as a common underlying mutational process unique to synchronous tumors.
DISCUSSION
Our analyses suggest a common etiologic factor defined by mutational signatures and/or transcriptomic convergence, which could provide a therapeutic opportunity.
PubMed: 38939336
DOI: 10.3389/fonc.2024.1272432 -
JACS Au Jun 2024Monoclonal antibodies (mAbs) have gradually dominated the drug markets for various diseases. Improvement of the therapeutic activities of mAbs has become a critical...
Monoclonal antibodies (mAbs) have gradually dominated the drug markets for various diseases. Improvement of the therapeutic activities of mAbs has become a critical issue in the pharmaceutical industry. A novel endo-β--acetylglucosaminidase, EndoSz, from is discovered and applied to enhance the activities of mAbs. Our studies demonstrate that the mutant EndoSz-D234M possesses an excellent transglycosylation activity to generate diverse glycoconjugates on mAbs. We prove that EndoSz-D234M can be applied to various marketed therapeutic antibodies and those in development for antibody remodeling. The remodeled homogeneous antibodies (mAb-G2S2) produced by EndoSz-D234M increase the relative ADCC activities by 3-26-fold. We further report the high-resolution crystal structures of EndoSz-D234M in the -form at 2.15 Å and the complex form with a bound G2S2-oxazoline intermediate at 2.25 Å. A novel pH-jump method was utilized to obtain the complex structure with a high resolution. The detailed interactions of EndoSz-D234M and the carried G2S2-oxazoline are hence delineated. The oxazoline sits in a hole, named the oxa-hole, which stabilizes the G2S2-oxazoline in transit and catalyzes the further transglycosylation reaction while targeting Asn-GlcNAc (+1) of Fc. In the oxa-hole, the H-bonding network involved with oxazoline dominates the transglycosylation activity. A mobile loop2 (a.a. 152-159) of EndoSz-D234M reshapes the binding grooves for the accommodation of G2S2-oxazoline upon binding, at which Trp154 forms a hydrogen bond with Man (-2). The long loop4 (a.a. 236-248) followed by helix3 is capable of dominating the substrate selectivity of EndoSz-D234M. In addition, the stepwise transglycosylation behavior of EndoSz-D234M is elucidated. Based on the high-resolution structures of the -form and the bound form with G2S2-oxazoline as well as a systematic mutagenesis study of the relative transglycosylation activity, the transglycosylation mechanism of EndoSz-D234M is revealed.
PubMed: 38938812
DOI: 10.1021/jacsau.4c00004