The pyoverdine siderophore is produced by Pseudomonas aeruginosa to access iron. Its synthesis involves the complex coordination of four nonribosomal peptide synthetases (NRPSs), which are ...responsible for assembling the pyoverdine peptide backbone. The precise cellular organization of these NRPSs and their mechanisms of interaction remain unclear. Here, we used a combination of several single-molecule microscopy techniques to elucidate the spatial arrangement of NRPSs within pyoverdine-producing cells. Our findings reveal that PvdL differs from the three other NRPSs in terms of localization and mobility patterns. PvdL is predominantly located in the inner membrane, while the others also explore the cytoplasmic compartment. Leveraging the power of multicolor single-molecule localization, we further reveal co-localization between PvdL and the other NRPSs, suggesting a pivotal role for PvdL in orchestrating the intricate biosynthetic pathway. Our observations strongly indicates that PvdL serves as a central orchestrator in the assembly of NRPSs involved in pyoverdine biosynthesis, assuming a critical regulatory function.
Super-resolution microscopy allows biological systems to be studied at the nanoscale, but has been restricted to providing only positional information. Here, we show that it is possible to perform ...multi-dimensional super-resolution imaging to determine both the position and the environmental properties of single-molecule fluorescent emitters. The method presented here exploits the solvatochromic and fluorogenic properties of nile red to extract both the emission spectrum and the position of each dye molecule simultaneously enabling mapping of the hydrophobicity of biological structures. We validated this by studying synthetic lipid vesicles of known composition. We then applied both to super-resolve the hydrophobicity of amyloid aggregates implicated in neurodegenerative diseases, and the hydrophobic changes in mammalian cell membranes. Our technique is easily implemented by inserting a transmission diffraction grating into the optical path of a localization-based super-resolution microscope, enabling all the information to be extracted simultaneously from a single image plane.
Purpose
The two-dimensional fluoroscopic method of percutaneous pedicle screw instrumentation has been clinically described as reliable method in the caudal thoracic and lumbosacral spine. Its ...accuracy has not been clearly reported in the cranial thoracic spine. The aim of this in vitro study was to investigate percutaneous pedicle screw placement accuracy according to pedicle dimensions and vertebral levels.
Methods
Six fresh-frozen human specimens were instrumented with 216 screws from T1 to S1. Pedicle isthmus widths, heights, transversal pedicles and screws were measured on computed tomography. Pedicle cortex violation ≥ 2 mm was defined as screw malposition.
Results
The narrowest pedicles were at T3–T5. A large variability between transversal pedicle axes and percutaneous pedicle screw was present, depending on the spinal level. Screw malposition rates were 36.1% in the cranial thoracic spine (T1–T6), 16.7% in the caudal thoracic spine (T7–T12), and 6.9% in the lumbosacral spine (L1–S1). The risk for screw malposition was significantly higher at cranial thoracic levels compared to caudal thoracic (
p
= 0.006) and lumbosacral (
p
< 0.0001) levels. Cortex violation ≥ 2 mm was constantly present if the pedicle width was < 4.8 mm.
Conclusion
Percutaneous pedicle screw placement appears safe in the caudal thoracic and lumbosacral spine. The two-dimensional fluoroscopic method has a limited reliability above T7 because of smaller pedicle dimensions, difficulties in visualizing radiographic pedicle landmarks and kyphosis.
One salient feature of reverse transcription in retroviruses, notably in the human immunodeficiency virus type 1, is that it requires the homologous nucleocapsid (NC) protein acting as a chaperoning ...partner of the genomic RNA template and the reverse transcriptase, from the initiation to the completion of viral DNA synthesis. This short review on the NC protein of human immunodeficiency virus type 1 aims at briefly presenting the flexible nature of NC protein, how it interacts with nucleic acids via its invariant zinc fingers and flanking basic residues, and the possible mechanisms that account for its multiple functions in the early steps of virus replication, notably in the obligatory strand transfer reactions during viral DNA synthesis by the reverse transcriptase enzyme.
Abstract
Although the human immunodeficiency virus type 1 lipid envelope has been reported to be enriched with host cell sphingomyelin and cholesterol, the molecular mechanism of the enrichment is ...not well understood. Viral Gag protein plays a central role in virus budding. Here, we report the interaction between Gag and host cell lipids using different quantitative and super-resolution microscopy techniques in combination with specific probes that bind endogenous sphingomyelin and cholesterol. Our results indicate that Gag in the inner leaflet of the plasma membrane colocalizes with the outer leaflet sphingomyelin-rich domains and cholesterol-rich domains, enlarges sphingomyelin-rich domains, and strongly restricts the mobility of sphingomyelin-rich domains. Moreover, Gag multimerization induces sphingomyelin-rich and cholesterol-rich lipid domains to be in close proximity in a curvature-dependent manner. Our study suggests that Gag binds, coalesces, and reorganizes pre-existing lipid domains during assembly.
The nucleocapsid protein NCp7 of HIV-1 possesses nucleic acid chaperone properties that are thought to be crucial throughout the viral life cycle. These properties promote the rearrangement of ...nucleic acids into their most thermodynamically stable conformations. These NCp7 properties involve two components, namely nucleic acid destabilization and activation of the annealing of complementary sequences. Biophysical techniques have been found extremely powerful to decipher the molecular mechanisms underlying these two components. We propose here an overview of the recent reports that examine the nucleic acid chaperone properties of NCp7 by these techniques.
Bacterial outer membrane vesicles (OMVs), as well as OMV-associated small RNAs, have been demonstrated to play a role in host-pathogen interactions. The presence of larger RNA transcripts in OMVs has ...been less studied and their potential role in host-pathogen interactions remains largely unknown. Here we analyze RNA from OMVs secreted by
serovar Typhimurium (
Typhimurium) cultured under different conditions, which mimic host-pathogen interactions.
Typhimurium was grown to exponential and stationary growth phases in minimal growth control medium (phosphate-carbon-nitrogen, PCN), as well as in acidic and phosphate-depleted PCN, comparable to the macrophage environment and inducing therefore the expression of
pathogenicity island 2 (SPI-2) genes. Moreover,
pathogenicity island 1 (SPI-1), which is required for virulence during the intestinal phase of infection, was induced by culturing
Typhimurium to the stationary phase in Lysogeny Broth (LB). For each condition, we identified OMV-associated RNAs that are enriched in the extracellular environment relative to the intracellular space. All RNA classes could be observed, but a vast majority of rRNA was exported in all conditions in variable proportions with a notable decrease in LB SPI-1 inducing media. Several mRNAs and ncRNAs were specifically enriched in/on OMVs dependent on the growth conditions. Important to note is that some RNAs showed identical read coverage profiles intracellularly and extracellularly, whereas distinct coverage patterns were observed for other transcripts, suggesting a specific processing or degradation. Moreover, PCR experiments confirmed that distinct RNAs were present in or on OMVs as full-length transcripts (
;
;
;
;
;
;
;
;
;
;
), whereas others seemed to be rather present in a processed or degraded form. Finally, we show by a digestion protection assay that OMVs are able to prevent enzymatic degradation of given full-length transcripts (
,
,
, and
. In summary, we show that OMV-associated RNA is clearly different in distinct culture conditions and that at least a fraction of the extracellular RNA is associated as a full-length transcripts with OMVs, indicating that some RNAs are protected by OMVs and thereby leaving open the possibility that those might be functionally active.
Fraternal twins: Oligoureas and γ‐peptides are isosteric, quasi‐isostructural helical foldamers endowed with distinct biomolecular recognition properties. Combination of the two backbones to generate ...urea/amide hybrids (see picture) was found to give more potent yet less cytotoxic antimicrobial helical foldamers.
IntroductionSoccer is the most popular sport in the world. This contact sport carries the risk of exposure to repeated head impacts in the form of subconcussions, defined as minimal brain injuries ...following head impact, with no symptom of concussion. While it has been suggested that exposure to repetitive subconcussive events can result in long-term neurophysiological modifications, and the later development of chronic traumatic encephalopathy, the consequences of these repeated impacts remain controversial and largely unexplored in the context of soccer players.Methods and analysisThis is a prospective, single-centre, exposure/non-exposure, transverse study assessing the MRI and neuropsychological abnormalities in professional retired soccer players exposed to subconcussive impacts, compared with high-level athletes not exposed to head impacts. The primary outcome corresponds to the results of MRI by advanced MRI techniques (diffusion tensor, cerebral perfusion, functional MRI, cerebral volumetry and cortical thickness, spectroscopy, susceptibility imaging). Secondary outcomes are the results of the neuropsychological tests: number of errors and time to complete tests. We hypothesise that repeated subconcussive impacts could lead to morphological lesions and impact on soccer players’ cognitive skills in the long term.Ethics and disseminationEthics approval has been obtained and the study was approved by the Comité de Protection des Personnes (CPP) No 2021-A01169-32. Study findings will be disseminated by publication in a high-impact international journal. Results will be presented at national and international imaging meetings.Trial registration numberNCT04903015.
The HIV-1 nucleocapsid protein (NCp7) is a nucleic acid chaperone required during reverse transcription. During the first strand transfer, NCp7 is thought to destabilize cTAR, the (-)DNA copy of the ...TAR RNA hairpin, and subsequently direct the TAR/cTAR annealing through the zipping of their destabilized stem ends. To further characterize the destabilizing activity of NCp7, we locally probe the structure and dynamics of cTAR by steady-state and time resolved fluorescence spectroscopy. NC(11-55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem. This destabilization is achieved through zinc-finger-dependent binding of NC to the G(10) and G(50) residues. Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G(10) and G(50) the appropriate mobility and accessibility for specific recognition by NC. Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway.
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