Large Scale Bacterial Colony Screening of Diversified FRET Biosensors

Biosensors based on Forster Resonance Energy Transfer (FRET) between fluorescent protein mutants have started to revolutionize physiology and biochemistry. However, many types of FRET biosensors show relatively small FRET changes, making measurements with these probes challenging when used under sub-optimal experimental conditions. Thus, a major effort in the field currently lies in designing new optimization strategies for these types of sensors. Here we describe procedures for optimizing FRET changes by large scale screening of mutant biosensor libraries in bacterial colonies. We describe optimization of biosensor expression, permeabilization of bacteria, software tools for analysis, and screening conditions. The procedures reported here may help in improving FRET changes in multiple suitable classes of biosensors

Spatial proteomics reveals secretory pathway disturbances caused by neuropathy-associated TECPR2

Hereditary sensory and autonomic neuropathy 9 (HSAN9) is a rare fatal neurological disease caused by mis- and nonsense mutations in the gene encoding for Tectonin beta-propeller repeat containing protein 2 (TECPR2). While TECPR2 is required for lysosomal consumption of autophagosomes and ER-to-Golgi transport, it remains elusive how exactly TECPR2 is involved in autophagy and secretion and what downstream sequels arise from defective TECPR2 due to its involvement in these processes. To address these questions, we determine molecular consequences of TECPR2 deficiency along the secretory pathway. By employing spatial proteomics, we describe pronounced changes with numerous proteins important for neuronal function being affected in their intracellular transport. Moreover, we provide evidence that TECPR2's interaction with the early secretory pathway is not restricted to COPII carriers. Collectively, our systematic profiling of a HSAN9 cell model points to specific trafficking and sorting defects which might precede autophagy dysfunction upon TECPR2 deficiency. Disease-associated mutations in the protein TECPR2 have so far been mainly studied with respect to autophagy. Using complementary proteomics approaches, the authors identify trafficking and sorting defects along the secretory pathway upon TECPR2 deficiency and provide evidence that TECPR2 associates with the ER-Golgi interface

Direct materials deposition: designed macro and microstructure

Solid freeform fabrication of engineering materials is now possible using the Direct Metal Deposition (DMD) technique. Closed loop optical feedback system for DMD makes realistic components with dimensional accuracy of 0.01 inch. On the other hand, close control of the process parameter can provide microstructure of choice. Such continued capability to control macro and microstructure is creating considerable interest. H13 tool steel is one of the difficult alloys for deposition due to residual stress accumulation from martensitic transformation. However, it is the material of choice for the die and tool industry. DMD offers Copper chill blocks and water cooling channels as the integral part of the tool. On the other hand ZrO 2 was co-deposited with nickel superalloys using DMD. This process thus is amenable to produce both macro and microstructure to a designed specification. This paper briefly reviews the state of the art of DMD and describes the microstructure and mechanical properties of selected engineering alloy systems deposited by DMD.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42335/1/10019-3-3-118_90030118.pd

Spatial Transcriptomics-correlated Electron Microscopy maps transcriptional and ultrastructural responses to brain injury

Understanding the complexity of cellular function within a tissue necessitates the combination of multiple phenotypic readouts. Here, we developed a method that links spatially-resolved gene expression of single cells with their ultrastructural morphology by integrating multiplexed error-robust fluorescence in situ hybridization (MERFISH) and large area volume electron microscopy (EM) on adjacent tissue sections. Using this method, we characterized in situ ultrastructural and transcriptional responses of glial cells and infiltrating T-cells after demyelinating brain injury in male mice. We identified a population of lipid-loaded foamy microglia located in the center of remyelinating lesion, as well as rare interferon-responsive microglia, oligodendrocytes, and astrocytes that co-localized with T-cells. We validated our findings using immunocytochemistry and lipid staining-coupled single-cell RNA sequencing. Finally, by integrating these datasets, we detected correlations between full-transcriptome gene expression and ultrastructural features of microglia. Our results offer an integrative view of the spatial, ultrastructural, and transcriptional reorganization of single cells after demyelinating brain injury. To understand complexity of cellular function, multiple phenotypic readouts are needed. Here, authors devised an approach integrating location, transcriptome, ultrastructure, and lipid content to characterize single-cell states after brain injury

Loss of NPC1 enhances phagocytic uptake and impairs lipid trafficking in microglia

Niemann-Pick type C disease is a rare neurodegenerative disorder mainly caused by mutations in NPC1, resulting in abnormal late endosomal/lysosomal lipid storage. Although microgliosis is a prominent pathological feature, direct consequences of NPC1 loss on microglial function remain not fully characterized. We discovered pathological proteomic signatures and phenotypes in NPC1-deficient murine models and demonstrate a cell autonomous function of NPC1 in microglia. Loss of NPC1 triggers enhanced phagocytic uptake and impaired myelin turnover in microglia that precede neuronal death. Npc1(-/-) microglia feature a striking accumulation of multivesicular bodies and impaired trafficking of lipids to lysosomes while lysosomal degradation function remains preserved. Molecular and functional defects were also detected in blood-derived macrophages of NPC patients that provide a potential tool for monitoring disease. Our study underscores an essential cell autonomous role for NPC1 in immune cells and implies microglial therapeutic potential. Niemann-Pick type C disease is a rare childhood neurodegenerative disorder predominantly caused by mutations in NPC1, resulting in abnormal late endosomal and lysosomal defects. Here the authors show that NPC1 disruption largely impairs microglial function

Pro-inflammatory activation following demyelination is required for myelin clearance and oligodendrogenesis

Remyelination requires innate immune system function, but how exactly microglia and macrophages clear myelin debris after injury and tailor a specific regenerative response is unclear. Here, we asked whether pro-inflammatory microglial/macrophage activation is required for this process. We established a novel toxin-based spinal cord model of de- and remyelination in zebrafish and showed that pro-inflammatory NF-κB-dependent activation in phagocytes occurs rapidly after myelin injury. We found that the pro-inflammatory response depends on myeloid differentiation primary response 88 (MyD88). MyD88-deficient mice and zebrafish were not only impaired in the degradation of myelin debris, but also in initiating the generation of new oligodendrocytes for myelin repair. We identified reduced generation of TNF-α in lesions of MyD88-deficient animals, a pro-inflammatory molecule that was able to induce the generation of new premyelinating oligodendrocytes. Our study shows that pro-inflammatory phagocytic signaling is required for myelin debris degradation, for inflammation resolution, and for initiating the generation of new oligodendrocytes

Apolipoprotein E aggregation in microglia initiates Alzheimer’s disease pathology by seeding β-amyloidosis

The seeded growth of pathogenic protein aggregates underlies the pathogenesis of Alzheimer’s disease (AD), but how this pathological cascade is initiated is not fully understood. Sporadic AD is linked genetically to apolipoprotein E (APOE) and other genes expressed in microglia related to immune, lipid, and endocytic functions. We generated a transgenic knockin mouse expressing HaloTag-tagged APOE and optimized experimental protocols for the biochemical purification of APOE, which enabled us to identify fibrillary aggregates of APOE in mice with amyloid-β (Aβ) amyloidosis and in human AD brain autopsies. These APOE aggregates that stained positive for β sheet-binding dyes triggered Aβ amyloidosis within the endo-lysosomal system of microglia, in a process influenced by microglial lipid metabolism and the JAK/STAT signaling pathway. Taking these observations together, we propose a model for the onset of Aβ amyloidosis in AD, suggesting that the endocytic uptake and aggregation of APOE by microglia can initiate Aβ plaque formation

Distribution of the reef manta ray Mobula alfredi and the oceanic manta ray Mobula birostris in the Philippines: A collaborative effort for conservation

This is the peer reviewed version of the following article: [Rambahiniarison, J., Agustines, A., Alexopoulos, K., Araujo, G., Armstrong, A., Arnold, S., Barruga, A., Cañete, T., Conales, S., Delijero, K., Enolva, N. P., Flam, A. L., Keane, E., Labaja, J., Legaspi, C., Murie, C., Murray, R., Oliver, S. P., Pierce, S. J., Ponzo, A., ... Barr, Y. (2023). Distribution of the reef manta ray Mobula alfredi and the oceanic manta ray Mobula birostris in the Philippines: A collaborative effort for conservation. Journal of Fish Biology, 102(2), 492-503], which has been published in final form at [https://doi.org/10.1111/jfb.15283]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited.Little is known about manta ray population size, structure, and connectivity in the Philippines. In collaboration with dive operators, non-governmental organizations, and authorities, sightings of manta rays were collated into a single national database. Using in-water photographs and videos gathered through citizen science and dedicated research efforts, this study compiled sightings between 2004 and 2020, showing 22 separate sites throughout the archipelago with manta rays present. A total of 392 individual reef manta rays (Mobula alfredi) and 107 oceanic manta rays (Mobula birostris) were identified from the collected footage. Four specific sites in the provinces of Masbate and Palawan together hosted 87% of all identified individuals and accounted for 94% of sightings, highlighting these areas are key aggregation sites. This study also reports movements of M. birostris within the Philippines, based on photo-identification of three individuals moving 150 km between Cebu and Masbate. Despite the growing number of recreational divers in Daanbantayan and San Jacinto, an 80% decline in M. birostris sightings was observed at these sites. To ensure effective future conservation, it is recommended that efforts focus on the identification and protection of manta ray hotspots and migratory corridors, the creation of a sustainable tourism framework, and most importantly, on the implementation of mitigation strategies to reduce fisheries interactions.Large Marine Vertebrates Research Institute Philippines was partially funded by Prince Bernard Foundation, the Shark Conservation Fund, The Rufford Foundation, Fondation Ensemble and PADI Foundation. AOA was partially funded by the ARC-Linkage Grant: LP150100669 Connectivity and movements of large pelagic species of ecotourism value. SJP and CAR were partially funded by Aqua-Firma and Waterlust. The Manta Trust, Philippines Project was funded by the Save Our Seas Foundation. The WWF Philippines Palawan projects were funded by Grieg Foundation and WWF Singapore

Direct Materials Deposition: Designed Macro and Microstructure

AbstractRapid Fabrication of three-dimensional shapes of engineering materials such as H 13 tool steel and Nickel super alloys are now possible using Direct Materials Deposition (DMD) technique. H 13 tool steel is one of the difficult alloys for deposition due to residual stress accumulation from martensitic transformation. However, it is the material of choice for the die and tool industry. DMD offers Copper chill blocks and water cooling channels as the integral part of the tool. On the other hand ZrO2 was co-deposited with nickel superalloys using DMD. This process thus is amenable to produce both macro and microstructure to a designed specification. This paper briefly reviews the state of the art of DMD and describes the microstructure and mechanical properties of selected engineering alloy systems deposited by DMD.</jats:p