From pixels to connections: exploring in vitro neuron reconstruction software for network graph generation.

Digital reconstruction has been instrumental in deciphering how in vitro neuron architecture shapes information flow. Emerging approaches reconstruct neural systems as networks with the aim of understanding their organization through graph theory. Computational tools dedicated to this objective build models of nodes and edges based on key cellular features such as somata, axons, and dendrites. Fully automatic implementations of these tools are readily available, but they may also be purpose-built from specialized algorithms in the form of multi-step pipelines. Here we review software tools informing the construction of network models, spanning from noise reduction and segmentation to full network reconstruction. The scope and core specifications of each tool are explicitly defined to assist bench scientists in selecting the most suitable option for their microscopy dataset. Existing tools provide a foundation for complete network reconstruction, however more progress is needed in establishing morphological bases for directed/weighted connectivity and in software validation.

Intra-cochlear Flushing Reduces Tissue Response to Cochlear Implantation.

INTRODUCTION: Intraoperative trauma leading to bleeding during cochlear implantation negatively impacts residual hearing of cochlear implant recipients. There are no clinical protocols for the removal of blood during implantation, to reduce the consequential effects such as inflammation and fibrosis which adversely affect cochlear health and residual hearing. This preclinical study investigated the implementation of an intra-cochlear flushing protocol for the removal of blood. METHODS: Three groups of guinea pigs were studied for 28 days after cochlear implantation; cochlear implant-only (control group); cochlear implant with blood injected into the cochlea (blood group); and cochlear implant, blood injection, and flushing of the blood from the cochlea intraoperatively (flush group). Auditory brainstem responses (ABRs) in addition to tissue response volumes were analyzed and compared between groups. RESULTS: After implantation, the blood group exhibited the highest ABR thresholds when compared to the control and flush group, particularly in the high frequencies. On the final day, the control and blood group had similar ABR thresholds across all frequencies tested, whereas the flush group had the lowest thresholds, significantly lower at 24 kHz than the blood and control group. Analysis of the tissue response showed the flush group had significantly lower tissue responses in the basal half of the array when compared with the blood and control group. CONCLUSIONS: Flushing intra-cochlear blood during surgery resulted in better auditory function and reduced subsequent fibrosis in the basal region of the cochlea. This finding prompts the implementation of a flushing protocol in clinical cochlear implantation. LEVEL OF EVIDENCE: N/A Laryngoscope, 134:1410-1416, 2024.

The two-component system WalKR provides an essential link between cell wall homeostasis and DNA replication in Staphylococcus aureus.

Among the 16 two-component systems in the opportunistic human pathogen Staphylococcus aureus, only WalKR is essential. Like the orthologous systems in other Bacillota, S. aureus WalKR controls autolysins involved in peptidoglycan remodeling and is therefore intimately involved in cell division. However, despite the importance of WalKR in S. aureus, the basis for its essentiality is not understood and the regulon is poorly defined. Here, we defined a consensus WalR DNA-binding motif and the direct WalKR regulon by using functional genomics, including chromatin immunoprecipitation sequencing, with a panel of isogenic walKR mutants that had a spectrum of altered activities. Consistent with prior findings, the direct regulon includes multiple autolysin genes. However, this work also revealed that WalR directly regulates at least five essential genes involved in lipoteichoic acid synthesis (ltaS): translation (rplK), DNA compaction (hup), initiation of DNA replication (dnaA, hup) and purine nucleotide metabolism (prs). Thus, WalKR in S. aureus serves as a polyfunctional regulator that contributes to fundamental control over critical cell processes by coordinately linking cell wall homeostasis with purine biosynthesis, protein biosynthesis, and DNA replication. Our findings further address the essentiality of this locus and highlight the importance of WalKR as a bona fide target for novel anti-staphylococcal therapeutics. IMPORTANCE The opportunistic human pathogen Staphylococcus aureus uses an array of protein sensing systems called two-component systems (TCS) to sense environmental signals and adapt its physiology in response by regulating different genes. This sensory network is key to S. aureus versatility and success as a pathogen. Here, we reveal for the first time the full extent of the regulatory network of WalKR, the only staphylococcal TCS that is indispensable for survival under laboratory conditions. We found that WalKR is a master regulator of cell growth, coordinating the expression of genes from multiple, fundamental S. aureus cellular processes, including those involved in maintaining cell wall metabolism, protein biosynthesis, nucleotide metabolism, and the initiation of DNA replication.

A semi-automated pipeline for quantifying drusen-like deposits in human induced pluripotent stem cell-derived retinal pigment epithelium cells.

Age-Related Macular Degeneration (AMD) is a highly prevalent form of retinal disease amongst Western communities over 50 years of age. A hallmark of AMD pathogenesis is the accumulation of drusen underneath the retinal pigment epithelium (RPE), a biological process also observable in vitro. The accumulation of drusen has been shown to predict the progression to advanced AMD, making accurate characterisation of drusen in vitro models valuable in disease modelling and drug development. More recently, deposits above the RPE in the subretinal space, called reticular pseudodrusen (RPD) have been recognized as a sub-phenotype of AMD. While in vitro imaging techniques allow for the immunostaining of drusen-like deposits, quantification of these deposits often requires slow, low throughput manual counting of images. This further lends itself to issues including sampling biases, while ignoring critical data parameters including volume and precise localization. To overcome these issues, we developed a semi-automated pipeline for quantifying the presence of drusen-like deposits in vitro, using RPE cultures derived from patient-specific induced pluripotent stem cells (iPSCs). Using high-throughput confocal microscopy, together with three-dimensional reconstruction, we developed an imaging and analysis pipeline that quantifies the number of drusen-like deposits, and accurately and reproducibly provides the location and composition of these deposits. Extending its utility, this pipeline can determine whether the drusen-like deposits locate to the apical or basal surface of RPE cells. Here, we validate the utility of this pipeline in the quantification of drusen-like deposits in six iPSCs lines derived from patients with AMD, following their differentiation into RPE cells. This pipeline provides a valuable tool for the in vitro modelling of AMD and other retinal disease, and is amenable to mid and high throughput screenings.

Quantitative Spatial Analysis of Neuroligin-3 mRNA Expression in the Enteric Nervous System Reveals a Potential Role in Neuronal-Glial Synapses and Reduced Expression in Nlgn3R451C Mice.

Mutations in the Neuroligin-3 (Nlgn3) gene are implicated in autism spectrum disorder (ASD) and gastrointestinal (GI) dysfunction, but cellular Nlgn3 expression in the enteric nervous system remains to be characterised. We combined RNAScope in situ hybridization and immunofluorescence to measure Nlgn3 mRNA expression in cholinergic and VIP-expressing submucosal neurons, nitrergic and calretinin-containing myenteric neurons and glial cells in both WT and Nlgn3R451C mutant mice. We measured Nlgn3 mRNA neuronal and glial expression via quantitative three-dimensional image analysis. To validate dual RNAScope/immunofluorescence data, we interrogated available single-cell RNA sequencing (scRNASeq) data to assess for Nlgn3, Nlgn1, Nlgn2 and their binding partners, Nrxn1-3, MGDA1 and MGDA2, in enteric neural subsets. Most submucosal and myenteric neurons expressed Nlgn3 mRNA. In contrast to other Nlgns and binding partners, Nlgn3 was strongly expressed in enteric glia, suggesting a role for neuroligin-3 in mediating enteric neuron-glia interactions. The autism-associated R451C mutation reduces Nlgn3 mRNA expression in cholinergic but not in VIPergic submucosal neurons. In the myenteric plexus, Nlgn3 mRNA levels are reduced in calretinin, nNOS-labelled neurons and S100 ß -labelled glia. We provide a comprehensive cellular profile for neuroligin-3 expression in ileal neuronal subpopulations of mice expressing the R451C autism-associated mutation in Nlgn3, which may contribute to the understanding of the pathophysiology of GI dysfunction in ASD.

A high-throughput cytotoxicity screening platform reveals agr-independent mutations in bacteraemia-associated Staphylococcus aureus that promote intracellular persistence.

Staphylococcus aureus infections are associated with high mortality rates. Often considered an extracellular pathogen, S. aureus can persist and replicate within host cells, evading immune responses, and causing host cell death. Classical methods for assessing S. aureus cytotoxicity are limited by testing culture supernatants and endpoint measurements that do not capture the phenotypic diversity of intracellular bacteria. Using a well-established epithelial cell line model, we have developed a platform called InToxSa (intracellular toxicity of S. aureus) to quantify intracellular cytotoxic S. aureus phenotypes. Studying a panel of 387 S. aureus bacteraemia isolates, and combined with comparative, statistical, and functional genomics, our platform identified mutations in S. aureus clinical isolates that reduced bacterial cytotoxicity and promoted intracellular persistence. In addition to numerous convergent mutations in the Agr quorum sensing system, our approach detected mutations in other loci that also impacted cytotoxicity and intracellular persistence. We discovered that clinical mutations in ausA, encoding the aureusimine non-ribosomal peptide synthetase, reduced S. aureus cytotoxicity, and increased intracellular persistence. InToxSa is a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevant S. aureus pathoadaptive mutations that promote intracellular residency.

Increasing muscle contractility through low-frequency stimulation alters tibial bone geometry and reduces bone strength in mdx and dko dystrophic mice.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by mutations or deletions in the dystrophin gene, for which there remains no cure. As DMD patients also develop bone fragility because of muscle weakness and immobilization, better understanding of the pathophysiological mechanisms of dystrophin deficiency will help develop therapies to improve musculoskeletal health. Since alterations in muscle phenotype can influence bone structure, we investigated whether modifying muscle contractile activity through low-frequency stimulation (LFS) could alter bone architecture in mouse models of DMD. We tested the hypothesis that increasing muscle contractile activity could influence bone mass and structure in dystrophin-deficient (mdx) and dystrophin- and utrophin-deficient (dko) dystrophic mice. Tibial bone structure in dko mice was significantly different from that in mdx and wild-type (C57BL/10) control mice. Effects of LFS on bone architecture differed between dystrophic and healthy mice, with LFS thinning cortical bone in both dystrophic models. Bone mass was maintained in LFS-treated healthy mice, with a reduced proportion of high-density bone and concomitant increase in low-density bone. LFS-treated dko mice exhibited a net deficit in cortical thickness and reduced high-density bone but no equivalent increase in low-density bone. These alterations in bone structure and mineral density reduced mechanical strength in mdx and dko mice. The findings reveal that muscle activity can regulate bone mass, structure, mineral accrual, and strength, especially in the context of dystrophin and/or utrophin deficiency. The results provide unique insights into the development of bone fragility in DMD and for devising interventions to improve musculoskeletal health.NEW & NOTEWORTHY Patients with Duchenne muscular dystrophy (DMD) develop bone fragility because of muscle weakness and immobilization. We investigated whether increasing muscle contractile activity through low-frequency stimulation (LFS) could alter bone architecture in dystrophin-deficient (mdx) or dystrophin- and utrophin-deficient (dko) mouse models of DMD. Chronic LFS reduced tibial diaphysis cross sections in mdx and dko mice, without affecting bone shape in healthy mice. LFS affected the distribution of bone mineral density across all phenotypes, with the magnitude of effect being dependent on disease severity.

Fine-scale mapping of physicochemical and microbial landscapes of the coral skeleton.

The coral skeleton harbours a diverse community of bacteria and microeukaryotes exposed to light, O2 and pH gradients, but how such physicochemical gradients affect the coral skeleton microbiome remains unclear. In this study, we employed chemical imaging of O2 and pH, hyperspectral reflectance imaging and spatially resolved taxonomic and inferred functional microbiome characterization to explore links between the skeleton microenvironment and microbiome in the reef-building corals Porites lutea and Paragoniastrea benhami. The physicochemical environment was more stable in the deep skeleton, and the diversity and evenness of the bacterial community increased with skeletal depth, suggesting that the microbiome was stratified along the physicochemical gradients. The bulk of the coral skeleton was in a low O2 habitat, whereas pH varied from pH 6-9 with depth. Physicochemical gradients of O2 and pH of the coral skeleton explained the ß-diversity of the bacterial communities, and skeletal layers that showed O2 peaks had a higher relative abundance of endolithic algae, reflecting a link between the abiotic environment and the microbiome composition. Our study links the physicochemical, microbial and functional landscapes of the coral skeleton and provides new insights into the involvement of skeletal microbes in the coral holobiont metabolism.

Quantification of Golgi Entry and Exit Kinetics of Protein Cargoes.

The Golgi apparatus is a pivotal secretory organelle in membrane trafficking, a hub responsible for posttranslational modifications, sorting, and trafficking of newly synthetized proteins received from the endoplasmic reticulum (ER). Different protein cargoes have been shown to travel through the Golgi stacks with different kinetics. Dysregulated transport and altered residency time of cargoes in the Golgi can impair their functionality. To study the anterograde trafficking of specific protein cargoes, innovative molecular methods have been developed to synchronize the traffic of selected cargoes from the ER in live cells. These methods of synchronization now provide the ability to quantify the Golgi entry and exit kinetics of defined cargo. In this chapter, we describe a quantitative, accurate, and semiautomated protocol to image and quantify the anterograde trafficking of individual cargo traversing the Golgi. This protocol, using free software, is compatible with different synchronization techniques, and can be used for a range of applications, such as comparing the Golgi kinetics of (1) different cargoes, (2) wild-type cargo vs mutated cargo, (3) the same cargo under different Golgi conditions, and (4) cargoes in drug screening platforms. The method can also be applied to study the localization and transit of a cargo through different organelles other than the Golgi apparatus.

Paraspeckle subnuclear bodies depend on dynamic heterodimerisation of DBHS RNA-binding proteins via their structured domains.

RNA-binding proteins of the DBHS (Drosophila Behavior Human Splicing) family, NONO, SFPQ, and PSPC1 have numerous roles in genome stability and transcriptional and posttranscriptional regulation. Critical to DBHS activity is their recruitment to distinct subnuclear locations, for example, paraspeckle condensates, where DBHS proteins bind to the long noncoding RNA NEAT1 in the first essential step in paraspeckle formation. To carry out their diverse roles, DBHS proteins form homodimers and heterodimers, but how this dimerization influences DBHS localization and function is unknown. Here, we present an inducible GFP-NONO stable cell line and use it for live-cell 3D-structured illumination microscopy, revealing paraspeckles with dynamic, twisted elongated structures. Using siRNA knockdowns, we show these labeled paraspeckles consist of GFP-NONO/endogenous SFPQ dimers and that GFP-NONO localization to paraspeckles depends on endogenous SFPQ. Using purified proteins, we confirm that partner swapping between NONO and SFPQ occurs readily in vitro. Crystallographic analysis of the NONO-SFPQ heterodimer reveals conformational differences to the other DBHS dimer structures, which may contribute to partner preference, RNA specificity, and subnuclear localization. Thus overall, our study suggests heterodimer partner availability is crucial for NONO subnuclear distribution and helps explain the complexity of both DBHS protein and paraspeckle dynamics through imaging and structural approaches.

Repurposing the mitotic machinery to drive cellular elongation and chromatin reorganisation in Plasmodium falciparum gametocytes.

The sexual stage gametocytes of the malaria parasite, Plasmodium falciparum, adopt a falciform (crescent) shape driven by the assembly of a network of microtubules anchored to a cisternal inner membrane complex (IMC). Using 3D electron microscopy, we show that a non-mitotic microtubule organizing center (MTOC), embedded in the parasite's nuclear membrane, orients the endoplasmic reticulum and the nascent IMC and seeds cytoplasmic microtubules. A bundle of microtubules extends into the nuclear lumen, elongating the nuclear envelope and capturing the chromatin. Classical mitotic machinery components, including centriolar plaque proteins, Pfcentrin-1 and -4, microtubule-associated protein, End-binding protein-1, kinetochore protein, PfNDC80 and centromere-associated protein, PfCENH3, are involved in the nuclear microtubule assembly/disassembly process. Depolymerisation of the microtubules using trifluralin prevents elongation and disrupts the chromatin, centromere and kinetochore organisation. We show that the unusual non-mitotic hemispindle plays a central role in chromatin organisation, IMC positioning and subpellicular microtubule formation in gametocytes.

Interacting partners of Golgi-localized small G protein Arl5b identified by a combination of in vivo proximity labelling and GFP-Trap pull down.

The small G protein Arl5b is localised on the trans-Golgi network (TGN) and regulates endosomes-to-TGN transport. Here, we combined in vivo and in vitro techniques to map the interactive partners and near neighbours of Arl5b at the TGN, using constitutively active, membrane-bound Arl5b(Q70L)-GFP in stably expressing HeLa cells, and the proximity labelling techniques BioID and APEX2 in parallel with GFP-Trap pull down. From MS analysis, 22 Golgi proteins were identified; 50% were TGN-localised Rabs, Arfs and Arls. The scaffold/tethering factors ACBD3 (GCP60) and PIST (GOPC) were also identified, and we show that Arl5b is required for TGN recruitment of ACBD3. Overall, the combination of in vivo labelling and direct pull downs indicates a highly organised complex of small G proteins on TGN membranes.

Extracellular Vesicles Secreted by Glioma Stem Cells Are Involved in Radiation Resistance and Glioma Progression.

Glioblastoma is the most aggressive brain tumour with short survival, partly due to resistance to conventional therapy. Glioma stem cells (GSC) are likely to be involved in treatment resistance, by releasing extracellular vesicles (EVs) containing specific molecular cargoes. Here, we studied the EVs secreted by glioma stem cells (GSC-EVs) and their effects on radiation resistance and glioma progression. EVs were isolated from 3 GSCs by serial centrifugation. NanoSight measurement, cryo-electron microscopy and live imaging were used to study the EVs size, morphology and uptake, respectively. The non-GSC glioma cell lines LN229 and U118 were utilised as a recipient cell model. Wound healing assays were performed to detect cell migration. Colony formation, cell viability and invadopodium assays were conducted to detect cell survival of irradiated recipient cells and cell invasion post GSC-EV treatment. NanoString miRNA global profiling was used to select for the GSC-EVs' specific miRNAs. All three GSC cell lines secreted different amounts of EVs, and all expressed consistent levels of CD9 but different level of Alix, TSG101 and CD81. EVs were taken up by both LN229 and U118 recipient cells. In the presence of GSC-EVs, these recipient cells survived radiation exposure and initiated colony formation. After GSC-EVs exposure, LN229 and U118 cells exhibited an invasive phenotype, as indicated by an increase in cell migration. We also identified 25 highly expressed miRNAs in the GSC-EVs examined, and 8 of these miRNAs can target PTEN. It is likely that GSC-EVs and their specific miRNAs induced the phenotypic changes in the recipient cells due to the activation of the PTEN/Akt pathway. This study demonstrated that GSC-EVs have the potential to induce radiation resistance and modulate the tumour microenvironment to promote glioma progression. Future therapeutic studies should be designed to interfere with these GSC-EVs and their specific miRNAs.

Segregation of the membrane cargoes, BACE1 and amyloid precursor protein (APP) throughout the Golgi apparatus.

The intracellular trafficking of ß-site amyloid precursor protein (APP) cleaving enzyme (BACE1) and APP regulates amyloid-ß production. Our previous work demonstrated that newly synthesized BACE1 and APP are segregated into distinct trafficking pathways from the trans-Golgi network (TGN), and that alterations in their trafficking lead to an increase in Aß production in non-neuronal and neuronal cells. However, it is not known whether BACE1 and APP are transported through the Golgi stacks together and sorted at the TGN or segregated prior to arrival at the TGN. To address this question, we have used high-resolution Airyscan technology followed by Huygens deconvolution to quantify the overlap of BACE1 and APP in Golgi subcompartments in HeLa cells and primary neurons. Here, we show that APP and BACE1 are segregated, on exit from the endoplasmic reticulum and in the cis-Golgi and throughout the Golgi stack. In contrast, the transferrin receptor, which exits the TGN in AP-1 mediated transport carriers as for BACE1, colocalizes with BACE1, but not APP, throughout the Golgi stack. The segregation of APP and BACE1 is independent of the Golgi ribbon structure and the cytoplasmic domain of the cargo. Overall, our findings reveal the segregation of different membrane cargoes early in the secretory pathway, a finding relevant to the regulation of APP processing events.

STAT3 Hyperactivation Due to SOCS3 Deletion in Murine Osteocytes Accentuates Responses to Exercise- and Load-Induced Bone Formation.

Cortical bone develops and changes in response to mechanical load, which is sensed by bone-embedded osteocytes. The bone formation response to load depends on STAT3 intracellular signals, which are upregulated after loading and are subject to negative feedback from Suppressor of Cytokine Signaling 3 (Socs3). Mice with Dmp1Cre-targeted knockout of Socs3 have elevated STAT3 signaling in osteocytes and display delayed cortical bone maturation characterized by impaired accrual of high-density lamellar bone. This study aimed to determine whether these mice exhibit an altered response to mechanical load. The approach used was to test both treadmill running and tibial compression in female Dmp1Cre.Socs3f/f mice. Treadmill running for 5 days per week from 6 to 11 weeks of age did not change cortical bone mass in control mice, but further delayed cortical bone maturation in Dmp1Cre.Socs3f/f mice; accrual of high-density bone was suppressed, and cortical thickness was less than in genetically-matched sedentary controls. When strain-matched anabolic tibial loading was tested, both control and Dmp1Cre.Socs3f/f mice exhibited a significantly greater cortical thickness and periosteal perimeter in loaded tibia compared with the contralateral non-loaded bone. At the site of greatest compressive strain, the loaded Dmp1Cre.Socs3f/f tibias showed a significantly greater response than controls, indicated by a greater increase in cortical thickness. This was due to a greater bone formation response on both periosteal and endocortical surfaces, including formation of abundant woven bone on the periosteum. This suggests a greater sensitivity to mechanical load in Dmp1Cre.Socs3f/f bone. In summary, mice with targeted SOCS3 deletion and immature cortical bone have an exaggerated response to both physiological and experimental mechanical loads. We conclude that there is an optimal level of osteocytic response to mechanical load required for cortical bone maturation and that load-induced bone formation may be increased by augmenting STAT3 signaling within osteocytes. © 2021 American Society for Bone and Mineral Research (ASBMR).

Bone Geometry Is Altered by Follistatin-Induced Muscle Growth in Young Adult Male Mice.

The development of the musculoskeletal system and its maintenance depends on the reciprocal relationship between muscle and bone. The size of skeletal muscles and the forces generated during muscle contraction are potent sources of mechanical stress on the developing skeleton, and they shape bone structure during growth. This is particularly evident in hypermuscular global myostatin (Mstn)-null mice, where larger muscles during development increase bone mass and alter bone shape. However, whether muscle hypertrophy can similarly influence the shape of bones after the embryonic and prepubertal period is unknown. To address this issue, bone structure was assessed after inducing muscle hypertrophy in the lower hindlimbs of young-adult C57BL/6J male mice by administering intramuscular injections of recombinant adeno-associated viral vectors expressing follistatin (FST), a potent antagonist of Mstn. Two FST isoforms were used: the full-length 315 amino acid isoform (FST-315) and a truncated 288 amino acid isoform (FST-288). In both FST-treated cohorts, muscle hypertrophy was observed, and the anterior crest of the tibia, adjacent to the tibialis anterior muscle, was lengthened. Hypertrophy of the muscles surrounding the tibia caused the adjacent cortical shell to recede inward toward the central axis: an event driven by bone resorption adjacent to the hypertrophic muscle. The findings reveal that inducing muscle hypertrophy in mice can confer changes in bone shape in early adulthood. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.