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BioRxiv : the Preprint Server For... Jun 2024Bacteria find suitable locations for colonization by sensing and responding to surfaces. Complex signaling repertoires control surface colonization, and surface contact...
UNLABELLED
Bacteria find suitable locations for colonization by sensing and responding to surfaces. Complex signaling repertoires control surface colonization, and surface contact sensing by the flagellum plays a central role in activating colonization programs. adheres to surfaces using a polysaccharide adhesin called the holdfast. In , disruption of the flagellum through interactions with a surface or mutation of flagellar genes increases holdfast production. Our group previously identified several genes involved in flagellar surface sensing. One of these, called , codes for a protein with homology to the flagellar C-ring protein FliN. We show here that a fluorescently tagged FssF protein localizes to the flagellated pole of the cell and requires all components of the flagellar C-ring for proper localization, supporting the model that FssF associates with the C-ring. Deleting results in a severe motility defect that we show is due to a disruption of chemotaxis. Epistasis experiments demonstrate that promotes adhesion through a stator-dependent pathway when late-stage flagellar mutants are disrupted. Separately, we find that disruption of chemotaxis through deletion of or other chemotaxis genes results in a hyperadhesion phenotype. Key genes in the surface sensing network ( , , and ) contribute to both Δ dependent and Δ dependent hyperadhesion, but these genes affect adhesion differently in the two hyperadhesive backgrounds. Our results support a model in which the stator subunits of the flagella incorporate both mechanical and chemical signals to regulate adhesion.
IMPORTANCE
Biofilms pose a threat in clinical and industrial settings. Surface sensing is an early step in biofilm formation. Studying surface sensing can help develop strategies for combating harmful biofilms. Here, we use the freshwater bacterium to study surface sensing. We characterize a previously unstudied gene, , and find that it localizes to the cell pole in the presence of three proteins that make up a component of the flagellum called the C-ring. Additionally, we find that is required for chemotaxis but dispensable for swimming motility. Lastly, our results show that mutating and other genes required for chemotaxis causes a hyperadhesive phenotype. We propose that surface sensing requires chemotaxis for a robust response to a surface.
PubMed: 38948737
DOI: 10.1101/2024.06.20.599946 -
Sichuan Da Xue Xue Bao. Yi Xue Ban =... May 2024Infertility affects approximately one-sixth of the people of childbearing age worldwide, causing not only economic burdens of treatment for families with fertility...
OBJECTIVE
Infertility affects approximately one-sixth of the people of childbearing age worldwide, causing not only economic burdens of treatment for families with fertility problems but also psychological stress for patients and presenting challenges to societal and economic development. Premature ovarian insufficiency (POI) refers to the loss of ovarian function in women before the age of 40 due to the depletion of follicles or decreased quality of remaining follicles, constituting a significant cause of female infertility. In recent years, with the help of the rapid development in genetic sequencing technology, it has been demonstrated that genetic factors play a crucial role in the onset of POI. Among the population suffering from POI, genetic studies have revealed that genes involved in processes such as meiosis, DNA damage repair, and mitosis account for approximately 37.4% of all pathogenic and potentially pathogenic genes identified. FA complementation group M () is a group of genes involved in the damage repair of DNA interstrand crosslinks (ICLs), including -. Abnormalities in the genes are associated with female infertility and gene knockout mice also exhibit phenotypes similar to those of POI. During the genetic screening of POI patients, this study identified a suspicious variant in . This study aims to explore the pathogenic mechanisms of the genes of the FA pathway and their variants in the development of POI. We hope to help shed light on potential diagnostic and therapeutic strategies for the affected individuals.
METHODS
One POI patient was included in the study. The inclusion criteria for POI patients were as follows: women under 40 years old exhibiting two or more instances of basal serum follicle-stimulating hormone levels>25 IU/L (with a minimum interval of 4 weeks inbetween tests), alongside clinical symptoms of menstrual disorders, normal chromosomal karyotype analysis results, and exclusion of other known diseases that can lead to ovarian dysfunction. We conducted whole-exome sequencing for the POI patient and identified pathogenic genes by classifying variants according to the standards and guidelines established by the American College of Medical Genetics and Genomics (ACMG). Subsequently, the identified variants were validated through Sanger sequencing and subjected to bioinformatics analysis. Plasmids containing wild-type and mutant genes were constructed and introduced into 293T cells. The 293T cells transfected with wild-type and mutant human plasmids and pEGFP-C1 empty vector plasmids were designated as the EGFP - group, the EGFP - group, and the EGFP group, respectively. To validate the production of truncated proteins, cell proteins were extracted 48 hours post-transfection from the three groups and confirmed using GFP antibody. In order to investigate the impact on DNA damage repair, immunofluorescence experiments were conducted 48 hours post-transfection in the EGFP WT group and the EGFP -MUT group to examine whether the variant affected FANCM's ability to localize on chromatin. Mitomycin C was used to induce ICLs damage in both the EGFP - group and the EGFP - group, which was followed by verification of its effect on ICLs damage repair using γ-H2AX antibody.
RESULTS
In a POI patient from a consanguineous family, we identified a homozygous variant in the gene, c.1152-1155del:p.Leu386Valfs*10. The patient presented with primary infertility, experiencing irregular menstruation since menarche at the age of 16. Hormonal evaluation revealed an FSH level of 26.79 IU/L and an anti-Müllerian hormone (AMH) level of 0.07 ng/mL. Vaginal ultrasound indicated unsatisfactory visualization of the ovaries on both sides and uterine dysplasia. The patient's parents were a consanguineous couple, with the mother having regular menstrual cycles. The patient had two sisters, one of whom passed away due to osteosarcoma, while the other exhibited irregular menstruation, had been diagnosed with ovarian insufficiency, and remained childless. Bioinformatics analysis revealed a deletion of four nucleotides (c.1152-1155del) in the exon 6 of the patient's gene. This variant resulted in a frameshift at codon 386, introducing a premature stop codon at codon 396, which ultimately led to the production of a truncated protein consisting of 395 amino acids. experiments demonstrated that this variant led to the production of a truncated FANCM protein of approximately 43 kDa and caused a defect in its nuclear localization, with the protein being present only in the cytoplasm. Following treatment with mitomycin C, there was a significant increase in γ-H2AX levels in 293T cells transfected with the mutant plasmid (<0.01), indicating a statistically significant impairment of DNA damage repair capability caused by this variant.
CONCLUSIONS
The homozygous variant in the gene, c.1152-1155del:p.Leu386Valfs*10, results in the production of a truncated FANCM protein. This truncation leads to the loss of its interaction site with the MHF1-MHF2 complex, preventing its entry into the nucleus and the subsequent recognition of DNA damage. Consequently, the localization of the FA core complex on chromatin is disrupted, impeding the normal activation of the FA pathway and reducing the cell's ability to repair damaged ICLs. By disrupting the rapid proliferation and meiotic division processes of primordial germ cells, the reserve of oocytes is depleted, thereby triggering premature ovarian insufficiency in females.
PubMed: 38948269
DOI: 10.12182/20240560207 -
Circulation Research Jul 2024Exercise intolerance is an independent predictor of poor prognosis in diabetes. The underlying mechanism of the association between hyperglycemia and exercise...
BACKGROUND
Exercise intolerance is an independent predictor of poor prognosis in diabetes. The underlying mechanism of the association between hyperglycemia and exercise intolerance remains undefined. We recently demonstrated that the interaction between ARRDC4 (arrestin domain-containing protein 4) and GLUT1 (glucose transporter 1) regulates cardiac metabolism.
OBJECTIVE
To determine whether this mechanism broadly impacts diabetic complications, we investigated the role of ARRDC4 in the pathogenesis of diabetic cardiac and skeletal myopathy.
METHODS AND RESULTS
High glucose promoted translocation of MondoA into the nucleus, which upregulated transcriptional expression, increased lysosomal GLUT1 trafficking, and blocked glucose transport in cardiomyocytes, forming a feedback mechanism. This role of was confirmed in human muscular cells from type 2 diabetic patients. Prolonged hyperglycemia upregulated myocardial expression in multiple types of mouse models of diabetes. We then analyzed hyperglycemia-induced cardiac and skeletal muscle abnormalities in insulin-deficient mice. Hyperglycemia increased advanced glycation end-products and elicited oxidative and endoplasmic reticulum stress leading to apoptosis in the heart and peripheral muscle. However, deletion of augmented tissue glucose transport and mitochondrial respiration, protecting the heart and muscle from tissue damage. Stress hemodynamic analysis and treadmill exhaustion test uncovered that -knockout mice had greater cardiac inotropic/chronotropic reserve with higher exercise endurance than wild-type (WT) animals under diabetes. While multiple organs were involved in the mechanism, cardiac-specific overexpression (beyond levels observed during diabetes) using adenoassociated virus suggests that high levels of myocardial have the potential to contribute to exercise intolerance by interfering with cardiac metabolism through its interaction with GLUT1 in diabetes. Importantly, the mutation mouse line exhibited greater exercise tolerance, showing the potential therapeutic impact on diabetic cardiomyopathy by disrupting the interaction between ARRDC4 and GLUT1.
CONCLUSIONS
ARRDC4 serves as a regulator of hyperglycemia-induced toxicities toward cardiac and skeletal muscle, revealing a new molecular framework that connects hyperglycemia to cardiac/skeletal myopathy to exercise intolerance.
PubMed: 38946541
DOI: 10.1161/CIRCRESAHA.123.323158 -
FEBS Letters Jun 2024The human FoxP transcription factors dimerize via three-dimensional domain swapping, a unique feature among the human Fox family, as result of evolutionary sequence...
The human FoxP transcription factors dimerize via three-dimensional domain swapping, a unique feature among the human Fox family, as result of evolutionary sequence adaptations in the forkhead domain. This is the case for the conserved glycine and proline residues in the wing 1 region, which are absent in FoxP proteins but present in most of the Fox family. In this work, we engineered both glycine (G) and proline-glycine (PG) insertion mutants to evaluate the deletion events in FoxP proteins in their dimerization, stability, flexibility, and DNA-binding ability. We show that the PG insertion only increases protein stability, whereas the single glycine insertion decreases the association rate and protein stability and promotes affinity to the DNA ligand.
PubMed: 38946055
DOI: 10.1002/1873-3468.14972 -
RNA (New York, N.Y.) Jun 2024Direct methods for determining the fidelity of DNA polymerases are robust, with relatively little sample manipulation before sequencing. In contrast, methods for...
Direct methods for determining the fidelity of DNA polymerases are robust, with relatively little sample manipulation before sequencing. In contrast, methods for measuring RNA polymerase and reverse transcriptase fidelities are complicated by additional preparation steps that introduce ambiguity and error. Here, we describe a sequencing method, termed Roll-Seq, for simultaneously determining the individual fidelities of RNA polymerases and reverse transcriptases (RT) using Pacific Biosciences Single Molecule Real-Time sequencing. By employing reverse transcriptases with high rolling-circle activity, Roll-Seq generates long concatemeric cDNA from a circular RNA template. To discern the origin of a mutation, errors are recorded and determined to occur within a single concatemer (reverse transcriptase error) or all concatemers (RNA polymerase error) over the cDNA strand. We used Roll-Seq to measure the fidelities of T7 RNA polymerases, a Group II intron-encoded RT (Induro), and two LINE RTs (Fasciolopsis buski R2-RT and human LINE-1). Substitution rates for Induro and R2-RT are the same for cDNA and second strand synthesis while LINE-1 has 2.5-fold lower fidelity when performing second strand synthesis. Deletion and insertion rates increase for all RTs during second strand synthesis. In addition, we find that a structured RNA template impacts fidelity for both RNA polymerase and RT. The accuracy and precision of Roll-Seq enable this method to be applied as a complementary analysis to structural and mechanistic characterization of RNA polymerases and reverse transcriptases or as a screening method for RNAP and RT fidelity.
PubMed: 38942481
DOI: 10.1261/rna.080002.124 -
Science Advances Jun 2024Declined memory is a hallmark of Alzheimer's disease (AD). Experiments in rodents and human postmortem studies suggest that serotonin (5-hydroxytryptamine, 5-HT) plays a...
Declined memory is a hallmark of Alzheimer's disease (AD). Experiments in rodents and human postmortem studies suggest that serotonin (5-hydroxytryptamine, 5-HT) plays a role in memory, but the underlying mechanisms are unknown. Here, we investigate the role of 5-HT 2C receptor (5-HTR) in regulating memory. Transgenic mice expressing a humanized mutation exhibit impaired plasticity of hippocampal ventral CA1 (vCA1) neurons and reduced memory. Further, 5-HT neurons project to and synapse onto vCA1 neurons. Disruption of 5-HT synthesis in vCA1-projecting neurons or deletion of 5-HTRs in the vCA1 impairs neural plasticity and memory. We show that a selective 5-HTR agonist, lorcaserin, improves synaptic plasticity and memory in an AD mouse model. Cumulatively, we demonstrate that hippocampal 5-HTR signaling regulates memory, which may inform the use of 5-HTR agonists in the treatment of dementia.
Topics: Animals; Humans; Receptor, Serotonin, 5-HT2C; Memory; Mice; Mice, Transgenic; Neuronal Plasticity; Alzheimer Disease; Hippocampus; Serotonin; Disease Models, Animal; CA1 Region, Hippocampal; Neurons; Serotonin 5-HT2 Receptor Agonists
PubMed: 38941473
DOI: 10.1126/sciadv.adl2675 -
Membrane protein Bcsdr2 mediates biofilm integrity, hyphal growth and virulence of Botrytis cinerea.Applied Microbiology and Biotechnology Jun 2024Grey mould caused by Botrytis cinerea is a devastating disease responsible for large losses to agricultural production, and B. cinerea is a necrotrophic model fungal...
Grey mould caused by Botrytis cinerea is a devastating disease responsible for large losses to agricultural production, and B. cinerea is a necrotrophic model fungal plant pathogen. Membrane proteins are important targets of fungicides and hotspots in the research and development of fungicide products. Wuyiencin affects the permeability and pathogenicity of B. cinerea, parallel reaction monitoring revealed the association of membrane protein Bcsdr2, and the bacteriostatic mechanism of wuyiencin was elucidated. In the present work, we generated and characterised ΔBcsdr2 deletion and complemented mutant B. cinerea strains. The ΔBcsdr2 deletion mutants exhibited biofilm loss and dissolution, and their functional activity was illustrated by reduced necrotic colonisation on strawberry and grape fruits. Targeted deletion of Bcsdr2 also blocked several phenotypic defects in aspects of mycelial growth, conidiation and virulence. All phenotypic defects were restored by targeted gene complementation. The roles of Bcsdr2 in biofilms and pathogenicity were also supported by quantitative real-time RT-PCR results showing that phosphatidylserine decarboxylase synthesis gene Bcpsd and chitin synthase gene BcCHSV II were downregulated in the early stages of infection for the ΔBcsdr2 strain. The results suggest that Bcsdr2 plays important roles in regulating various cellular processes in B. cinerea. KEY POINTS: • The mechanism of wuyiencin inhibits B. cinerea is closely associated with membrane proteins. • Wuyiencin can downregulate the expression of the membrane protein Bcsdr2 in B. cinerea. • Bcsdr2 is involved in regulating B. cinerea virulence, growth and development.
Topics: Botrytis; Biofilms; Virulence; Hyphae; Plant Diseases; Fragaria; Fungal Proteins; Membrane Proteins; Vitis; Spores, Fungal; Gene Deletion
PubMed: 38940906
DOI: 10.1007/s00253-024-13238-8 -
Journal of Radiation Research Jun 2024The ionizing radiation with high linear energy transfer (LET), such as a heavy ion beam, induces more serious biological effects than low LET ones, such as gamma- and...
The ionizing radiation with high linear energy transfer (LET), such as a heavy ion beam, induces more serious biological effects than low LET ones, such as gamma- and X-rays. This indicates a difference in the DNA damage produced by low and high LET radiations and their biological effects. We have been studying the differences in DNA damage produced by gamma-rays and carbon ion beams. Therefore, we analyze mutations induced by both ionizing radiations to discuss the differences in their biological effects in this study. pUC19 plasmid DNA was irradiated by carbon ion beams in the solution containing 1M dimethyl sulfoxide to mimic a cellular condition. The irradiated DNA was cloned in competent cells of Escherichia coli. The clones harboring some mutations in the region of lacZα were selected, and the sequence alterations were analyzed. A one-deletion mutation is significant in the carbon-irradiated DNA, and the C:G↔T:A transition is minor. On the other hand, the gamma-irradiated DNA shows mainly G:C↔T:A transversion. These results suggest that carbon ion beams produce complex DNA damage, and gamma-rays are prone to single oxidative base damage, such as 8-oxoguanine. Carbon ion beams can also introduce oxidative base damage, and the damage species is 5-hydroxycytosine. This was consistent with our previous results of DNA damage caused by heavy ion beams. We confirmed the causal DNA damage by mass spectrometry for these mutations.
PubMed: 38940734
DOI: 10.1093/jrr/rrae050 -
Bioinformatics (Oxford, England) Jun 2024Insertions and deletions (indels) influence the genetic code in fundamentally distinct ways from substitutions, significantly impacting gene product structure and...
MOTIVATION
Insertions and deletions (indels) influence the genetic code in fundamentally distinct ways from substitutions, significantly impacting gene product structure and function. Despite their influence, the evolutionary history of indels is often neglected in phylogenetic tree inference and ancestral sequence reconstruction, hindering efforts to comprehend biological diversity determinants and engineer variants for medical and industrial applications.
RESULTS
We frame determining the optimal history of indel events as a single Mixed-Integer Programming (MIP) problem, across all branch points in a phylogenetic tree adhering to topological constraints, and all sites implied by a given set of aligned, extant sequences. By disentangling the impact on ancestral sequences at each branch point, this approach identifies the minimal indel events that jointly explain the diversity in sequences mapped to the tips of that tree. MIP can recover alternate optimal indel histories, if available. We evaluated MIP for indel inference on a dataset comprising 15 real phylogenetic trees associated with protein families ranging from 165 to 2000 extant sequences, and on 60 synthetic trees at comparable scales of data and reflecting realistic rates of mutation. Across relevant metrics, MIP outperformed alternative parsimony-based approaches and reported the fewest indel events, on par or below their occurrence in synthetic datasets. MIP offers a rational justification for indel patterns in extant sequences; importantly, it uniquely identifies global optima on complex protein data sets without making unrealistic assumptions of independence or evolutionary underpinnings, promising a deeper understanding of molecular evolution and aiding novel protein design.
AVAILABILITY AND IMPLEMENTATION
The implementation is available via GitHub at https://github.com/santule/indelmip.
Topics: Phylogeny; INDEL Mutation; Evolution, Molecular; Algorithms; Computational Biology
PubMed: 38940131
DOI: 10.1093/bioinformatics/btae254 -
Molecular Genetics & Genomic Medicine Jun 2024Spastic paraplegia 11 (SPG11) is the most prevalent form of autosomal recessive hereditary spastic paraplegia, resulting from biallelic pathogenic variants in the SPG11...
BACKGROUND
Spastic paraplegia 11 (SPG11) is the most prevalent form of autosomal recessive hereditary spastic paraplegia, resulting from biallelic pathogenic variants in the SPG11 gene (MIM *610844).
METHODS
The proband is a 36-year-old female referred for genetic evaluation due to cognitive dysfunction, gait impairment, and corpus callosum atrophy (brain MRI was normal at 25-years-old). Diagnostic approaches included CGH array, next-generation sequencing, and whole transcriptome sequencing.
RESULTS
CGH array revealed a 180 kb deletion located upstream of SPG11. Sequencing of SPG11 uncovered two rare single nucleotide variants: the novel variant c.3143C>T in exon 17 (in cis with the deletion), and the previously reported pathogenic variant c.6409C>T in exon 34 (in trans). Whole transcriptome sequencing revealed that the variant c.3143C>T caused exon 17 skipping.
CONCLUSION
We report a novel sequence variant in the SPG11 gene resulting in exon 17 skipping, which, along with a nonsense variant, causes Spastic Paraplegia 11 in our proband. In addition, a deletion upstream of SPG11 was identified in the patient, whose implication in the phenotype remains uncertain. Nonetheless, the deletion apparently affects cis-regulatory elements of the gene, suggesting a potential new pathogenic mechanism underlying the disease in a subset of undiagnosed patients. Our findings further support the hypothesis that the origin of thin corpus callosum in patients with SPG11 is of progressive nature.
Topics: Humans; Female; Adult; Spastic Paraplegia, Hereditary; Exons; Proteins; Codon, Nonsense; Corpus Callosum; Sequence Deletion; Phenotype
PubMed: 38938072
DOI: 10.1002/mgg3.2475