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BioRxiv : the Preprint Server For... Jun 2024Cochlear hair cell stereocilia bundles are key organelles required for normal hearing. Often, deafness mutations cause aberrant stereocilia heights or morphology that...
Cochlear hair cell stereocilia bundles are key organelles required for normal hearing. Often, deafness mutations cause aberrant stereocilia heights or morphology that are visually apparent but challenging to quantify. Actin-based structures, stereocilia are easily and most often labeled with phalloidin then imaged with 3D confocal microscopy. Unfortunately, phalloidin non-specifically labels all the actin in the tissue and cells and therefore results in a challenging segmentation task wherein the stereocilia phalloidin signal must be separated from the rest of the tissue. This can require many hours of manual human effort for each 3D confocal image stack. Currently, there are no existing software pipelines that provide an end-to-end automated solution for 3D stereocilia bundle instance segmentation. Here we introduce VASCilia, a Napari plugin designed to automatically generate 3D instance segmentation and analysis of 3D confocal images of cochlear hair cell stereocilia bundles stained with phalloidin. This plugin combines user-friendly manual controls with advanced deep learning-based features to streamline analyses. With VASCilia, users can begin their analysis by loading image stacks. The software automatically preprocesses these samples and displays them in Napari. At this stage, users can select their desired range of z-slices, adjust their orientation, and initiate 3D instance segmentation. After segmentation, users can remove any undesired regions and obtain measurements including volume, centroids, and surface area. VASCilia introduces unique features that measures bundle heights, determines their orientation with respect to planar polarity axis, and quantifies the fluorescence intensity within each bundle. The plugin is also equipped with trained deep learning models that differentiate between inner hair cells and outer hair cells and predicts their tonotopic position within the cochlea spiral. Additionally, the plugin includes a training section that allows other laboratories to fine-tune our model with their own data, provides responsive mechanisms for manual corrections through event-handlers that check user actions, and allows users to share their analyses by uploading a pickle file containing all intermediate results. We believe this software will become a valuable resource for the cochlea research community, which has traditionally lacked specialized deep learning-based tools for obtaining high-throughput image quantitation. Furthermore, we plan to release our code along with a manually annotated dataset that includes approximately 55 3D stacks featuring instance segmentation. This dataset comprises a total of 1,870 instances of hair cells, distributed between 410 inner hair cells and 1,460 outer hair cells, all annotated in 3D. As the first open-source dataset of its kind, we aim to establish a foundational resource for constructing a comprehensive atlas of cochlea hair cell images. Together, this open-source tool will greatly accelerate the analysis of stereocilia bundles and demonstrates the power of deep learning-based algorithms for challenging segmentation tasks in biological imaging research. Ultimately, this initiative will support the development of foundational models adaptable to various species, markers, and imaging scales to advance and accelerate research within the cochlea research community.
PubMed: 38948743
DOI: 10.1101/2024.06.17.599381 -
BioRxiv : the Preprint Server For... Jun 2024Vertebrate radial glia progenitors (RGPs), the principal neural stem cells, balance self-renewal and differentiation through asymmetric cell division (ACD), during which...
Vertebrate radial glia progenitors (RGPs), the principal neural stem cells, balance self-renewal and differentiation through asymmetric cell division (ACD), during which unequal inheritance of centrosomes is observed. Mechanistically, how centrosome asymmetry leads to distinct daughter cell fate remains largely unknown. Here we find that the centrosome protein Pericentriolar Material 1 (Pcm1), asymmetrically distributed at the centrosomes, regulates polarized endosome dynamics and RGP fate. time-lapse imaging and nanoscale-resolution expansion microscopy of zebrafish embryonic RGPs detect Pcm1 on Notch ligand-containing endosomes, in a complex with the polarity regulator Par-3 and dynein motor. Loss of disrupts endosome dynamics, with clonal analysis uncovering increased neuronal production at the expense of progenitors. Pcm1 facilitates an exchange of Rab5b (early) for Rab11a (recycling) endosome markers and promotes the formation of Par-3 and dynein macromolecular complexes on recycling endosomes. Finally, in human-induced pluripotent stem cell-derived brain organoids, PCM1 shows asymmetry and co-localization with PARD3 and RAB11A in mitotic neural progenitors. Our data reveal a new mechanism by which centrosome asymmetry is conveyed by Pcm1 to polarize endosome dynamics and Notch signaling in regulating ACD and progenitor fate.
PubMed: 38948739
DOI: 10.1101/2024.06.17.599416 -
BioRxiv : the Preprint Server For... Jun 2024Protocadherin-15 is a core protein component of inner-ear hair-cell tip links pulling on transduction channels essential for hearing and balance. Protocadherin-15...
Protocadherin-15 is a core protein component of inner-ear hair-cell tip links pulling on transduction channels essential for hearing and balance. Protocadherin-15 defects can result in non-syndromic deafness or Usher syndrome type 1F (USH1F) with hearing loss, balance deficits, and progressive blindness. Three rationally engineered shortened versions of protocadherin-15 (mini-PCDH15s) amenable for gene therapy have been used to rescue function in USH1F mouse models. Two can successfully or partially rescue hearing, while another one fails. Here we show that despite varying levels of hearing rescue, all three mini-PCDH15 versions can rescue hair-cell mechanotransduction. Negative-stain electron microscopy shows that all three versions form dimers like the wild-type protein, while crystal structures of some engineered fragments show that these can properly fold and bind calcium ions essential for function. In contrast, simulations predict distinct elasticities and nano differential scanning fluorimetry shows differences in melting temperature measurements. Our data suggest that elasticity and thermal stability are key determinants of sustained hearing rescue by mini-PCDH15s.
PubMed: 38948700
DOI: 10.1101/2024.06.16.599132 -
BioRxiv : the Preprint Server For... Jun 2024Bridge-like lipid transport proteins (BLTPs) are an evolutionarily conserved family of proteins that localize to membrane contact sites and are thought to mediate the...
Bridge-like lipid transport proteins (BLTPs) are an evolutionarily conserved family of proteins that localize to membrane contact sites and are thought to mediate the bulk transfer of lipids from a donor membrane, typically the endoplasmic reticulum (ER), to an acceptor membrane, such as a that of the cell or an organelle . Despite the fundamental importance of BLTPs for cellular function, the architecture, composition, and lipid transfer mechanisms remain poorly characterized. Here, we present the subunit composition and the cryo-electron microscopy structure of the native LPD-3 BLTP complex isolated from transgenic . LPD-3 folds into an elongated, rod-shaped tunnel whose interior is filled with ordered lipid molecules that are coordinated by a track of ionizable residues that line one side of the tunnel. LPD-3 forms a complex with two previously uncharacterized proteins, here named "Intake" and "Spigot", both of which interact with the N-terminal end of LPD-3 where lipids enter the tunnel. Intake has three transmembrane helices, one of which borders the entrance to the tunnel; Spigot has one transmembrane helix and extends 80 Å along the cytosolic surface of LPD-3. Experiments in multiple model systems indicate that Spigot plays a conserved role in ER-PM contact site formation. Our LPD-3 complex structural data, together with molecular dynamics simulations of the transmembrane region in a lipid bilayer, reveal protein-lipid interactions that suggest a model for how the native LPD-3-complex mediates bulk lipid transport and provide a foundation for mechanistic studies of BLTPs.
PubMed: 38948693
DOI: 10.1101/2024.06.21.600134 -
Frontiers in Veterinary Science 2024(), also known as group B (GBS), is a highly infectious pathogen. Prolonged antibiotic usage leads to significant issues of antibiotic residue and resistance....
(), also known as group B (GBS), is a highly infectious pathogen. Prolonged antibiotic usage leads to significant issues of antibiotic residue and resistance. Chelerythrine (CHE) is a naturally occurring benzophenidine alkaloid and chelerythrine chloride (CHEC) is its hydrochloride form with diverse biological and pharmacological activities. However, the antibacterial mechanism of CHEC against GBS remains unclear. Thus, this study aims to investigate the antibacterial activity of CHEC on GBS and elucidate its underlying mechanism. The antibacterial effect of CHEC on GBS was assessed using inhibitory zone, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) assays, as well as by constructing a time-kill curve. The antibacterial mechanism of CHEC was investigated through techniques such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM), measurement of alkaline phosphatase (AKP) activity, determination of Na K, Ca Mg-adenosine triphosphate (ATP) activity, observation of membrane permeability, and analysis of intracellular reactive oxygen species (ROS) and mRNA expression levels of key virulence genes. The results demonstrated that the inhibition zone diameters of CHEC against GBS were 14.32 mm, 12.67 mm, and 10.76 mm at concentrations of 2 mg/mL, 1 mg/mL, and 0.5 mg/mL, respectively. The MIC and MBC values were determined as 256 μg/mL and 512 μg/mL correspondingly. In the time-kill curve, 8 × MIC, 4 × MIC and 2 × MIC CHEC could completely kill GBS within 24 h. SEM and TEM analyses revealed significant morphological alterations in GBS cells treated with CHEC including shrinkage, collapse, and leakage of cellular fluids. Furthermore, the antibacterial mechanism underlying CHEC's efficacy against GBS was attributed to its disruption of cell wall integrity as well as membrane permeability resulting in extracellular release of intracellular ATP, AKP, Na K, Ca Mg. Additionally CHEC could increase the ROS production leading to oxidative damage and downregulating mRNA expression levels of key virulence genes in GBS cells. In conclusion, CHEC holds potential as an antimicrobial agent against GBS and further investigations are necessary to elucidate additional molecular mechanisms.
PubMed: 38948675
DOI: 10.3389/fvets.2024.1408376 -
ImmunoTargets and Therapy 2024Medulloblastoma (MB) is the most prevalent paediatric brain tumour. Despite improvements in patient survival with current treatment strategies, the quality of life of...
PURPOSE
Medulloblastoma (MB) is the most prevalent paediatric brain tumour. Despite improvements in patient survival with current treatment strategies, the quality of life of these patients remains poor owing to the sequelae and relapse risk. An alternative, or, in addition to the current standard treatment, could be considered immunotherapy, such as Natural Killer cells (NK). NK cells are cytotoxic innate lymphoid cells that play a major role in cancer immunosurveillance. To date, the mechanism of cytotoxicity of NK cells, especially regarding the steps of adhesion, conjugation, cytotoxic granule polarisation in the cell contact area, perforin and granzyme release in two and three dimensions, and therapeutic efficacy in vivo have not been precisely described.
MATERIALS AND METHODS
Each step of NK cytotoxicity against the three MB cell lines was explored using confocal microscopy for conjugation, Elispot for degranulation, flow cytometry, and luminescence assays for target cell necrosis and lysis and mediators released by cytokine array, and then confirmed in a 3D spheroid model. Medulloblastoma-xenografted mice were treated with NK cells. Their persistence was evaluated by flow cytometry, and their efficacy in tumour growth and survival was determined. In addition, their effects on the tumour transcriptome were evaluated.
RESULTS
NK cells showed variable affinities for conjugation with MB target cells depending on their subgroup and cytokine activation. Chemokines secreted during NK and MB cell co-culture are mainly associated with angiogenesis and immune cell recruitment. NK cell cytotoxicity induces MB cell death in both 2D and 3D co-culture models. NK cells initiated an inflammatory response in a human MB murine model by modulating the MB cell transcriptome.
CONCLUSION
Our study confirmed that NK cells possess both in vitro and in vivo cytotoxic activity against MB cells and are of interest for the development of immunotherapy.
PubMed: 38948503
DOI: 10.2147/ITT.S458278 -
Biotechnology Reports (Amsterdam,... Jun 2024Despite powerful DNA repair systems, oxidative damage/modification to DNA is an inevitable side effect of metabolism, ionizing radiation, lifestyle habits, inflammatory...
Despite powerful DNA repair systems, oxidative damage/modification to DNA is an inevitable side effect of metabolism, ionizing radiation, lifestyle habits, inflammatory pathologies such as type-2 diabetes or metabolic syndrome, cancer and natural aging. One of the most common oxidative DNA modifications is 8-OHdG (8‑hydroxy-2'-deoxyguanosine), which is the most widely used marker in research and clinical diagnostics. 8-OHdG is easily and specifically detectable in various samples such as urine, plasma, cells and tissues via a large variety of methods like ELISA, HPLC, chromatographic methods, and immunochemistry. Formed by oxidation of guanine and being representative for the degree of DNA damage, 8-OHdG can be also used as biomarker for risk assessment of various cancers as well as degenerative diseases. Here, we present a highly specific, self-developed 8-OHdG antibody in successful comparison to a commercially one, tested in cells (FF95, HCT116, and HT22) and intestinal tissue, focusing on automatized evaluation via fluorescence/confocal microscopy.
PubMed: 38948353
DOI: 10.1016/j.btre.2024.e00833 -
Biotechnology Reports (Amsterdam,... Jun 2024The environmental and economic impact of an oil spill can be significant. Biotechnologies applied during a marine oil spill involve bioaugmentation with immobilised or...
The environmental and economic impact of an oil spill can be significant. Biotechnologies applied during a marine oil spill involve bioaugmentation with immobilised or encapsulated indigenous hydrocarbonoclastic species selected under laboratory conditions to improve degradation rates. The environmental factors that act as stressors and impact the effectiveness of hydrocarbon removal are one of the challenges associated with these applications. Understanding how native microbes react to environmental stresses is necessary for effective bioaugmentation. Herein, isolated from a marine oil spill mooring system showed hydrocarbonoclastic activity on Maya crude oil in a short time by means of total petroleum hydrocarbons (TPH) at 144 h: up to 98.79 % and 97.77 % removal. The assessment of biofilms at different temperature (30 °C and 50 °C), pH (5, 6, 7, 8, 9), salinity (30, 50, 60, 70, 80 g/L), and crude oil concentration (1, 5, 15, 25, 35 %) showed different response to the stressors depending on the strain. According to response surface analysis, the main effect was temperature > salinity > hydrocarbon concentration. The hydrocarbonoclastic biofilm architecture was characterised using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Subtle but significant differences were observed: pili in by SEM and the topographical differences measured by AFM Power Spectral Density (PSD) analysis, roughness was higher in than in In all three domains of life, the Universal Stress Protein (Usp) is crucial for stress adaptation. Herein, the A gene expression was analysed in biofilm under environmental stressors. The A expression increased up to 2.5-fold in biofilms at 30 °C, and 1.3-fold at 50 °C. The highest A expression was recorded in M. biofilms at 50 °C with 2.5 and 3-fold with salinities of 50, 60, and 80 g/L at hydrocarbon concentrations of 15, 25, and 35 %. biofilms showed greater resilience than biofilms when exposed to harsh environmental stressors. biofilms were thicker than biofilms. Both biofilm responses to environmental stressors through A gene expression were consistent with the behaviours observed in the response surface analyses. The gene is a suitable biomarker for assessing environmental stressors of potential microorganisms for bioremediation of marine oil spills and for biosensing the ecophysiological status of native microbiota in a marine petroleum environment.
PubMed: 38948351
DOI: 10.1016/j.btre.2024.e00834 -
Bio-protocol Jun 2024All aerial organs in plants originate from the shoot apical meristem, a specialized tissue at the tip of a plant, enclosing a few stem cells. Understanding developmental...
All aerial organs in plants originate from the shoot apical meristem, a specialized tissue at the tip of a plant, enclosing a few stem cells. Understanding developmental dynamics within this tissue in relation to internal and external stimuli is of crucial importance. Imaging the meristem at the cellular level beyond very early stages requires the apex to be detached from the plant body, a procedure that does not allow studies in living, intact plants over longer periods. This protocol describes a new confocal microscopy method with the potential to image the shoot apical meristem of an intact, soil-grown, flowering Arabidopsis plant over several days. The setup opens new avenues to study apical stem cells, their interconnection with the whole plant, and their responses to environmental stimuli. Key features • Novel dissection and imaging method of the shoot apical meristem of . • Procedure performed with intact, soil-grown, flowering plants. • Possibility of long-term live imaging of the shoot apical meristem. • Protocol can be adapted to different plant species.
PubMed: 38948259
DOI: 10.21769/BioProtoc.5015 -
PeerJ 2024species are among the most common pathogens in agriculture and forestry, and their control is urgently needed.
BACKGROUND
species are among the most common pathogens in agriculture and forestry, and their control is urgently needed.
METHODS
In this study, a total of 68 strains of biocontrol bacteria were isolated and identified from × rhizosphere soil.
RESULTS
The isolates were identified as by 16S rRNA. The inhibitory effect of TR-4 on was confirmed by an antagonistic experiment. The inhibitory effect of TR-4 was 98% at a concentration of 10 µl/ml bacterial solution, protection of the plant and inhibition of was evident. Moreover, the secretion of cellulase and chitosan enzymes in the TR-4 fermentation liquid cultured for three days was 9.07 mol/L and 2.15 µl/mol, respectively. Scanning electron microscopy and transmission electron microscopy confirmed that TR-4 destroyed the cell wall of , resulting in leakage of the cell contents, thus weakening the pathogenicity of the bacteria.
Topics: Brevibacillus; Plant Diseases; Soil Microbiology; Colletotrichum; RNA, Ribosomal, 16S; Plant Leaves; Rhizosphere; Microscopy, Electron, Scanning
PubMed: 38948232
DOI: 10.7717/peerj.17568