In this study, we advance our understanding of the spatial relationship between the purinosome, a liquid condensate consisting of six enzymes involved in de novo purine biosynthesis, and mitochondria. Previous research has shown that purinosomes move along tubulin toward mitochondria, suggesting a direct uptake of glycine from mitochondria. Here, we propose that the purinosome is located proximally to the mitochondrial transporters SLC25A13 and SLC25A38, facilitating the uptake of glycine, aspartate, and glutamate, essential factors for purine synthesis. We utilized the proximity ligation assay and APEX proximity labeling to investigate the association between purinosome proteins and mitochondrial transporters. Our results indicate that purinosome assembly occurs close to the mitochondrial membrane under purine-deficient conditions, with the transporters migrating to be adjacent to the purinosome. Furthermore, both targeted and non-targeted analyses suggest that the SLC25A13-APEX2-V5 probe accurately reflects endogenous cellular status. These findings provide insights into the spatial organization of purine biosynthesis and lay the groundwork for further investigations into additional proteins involved in this pathway.

BACKGROUND: Previous studies have shown that surgical technique errors especially the wrong bone tunnel position are the primary reason for the failure of anterior cruciate ligament (ACL) reconstruction. In this study, we aimed to compare the femoral tunnel position and impact on knee function during the ACL reconstruction using measuring combined with fluoroscopy method and bony marker method for femoral tunnel localization.
METHODS: A retrospective cohort study of patients undergoing ACL reconstruction using the bony marker method or measuring combined with fluoroscopy for femoral tunnel localization was conducted between January 2015 and January 2020. A second arthroscopic exploration was performed more than 1 year after surgery. Data regarding patient demographics, the femoral tunnel position, results of the Lysholm score, the International Knee Documentation Committee (IKDC) score, KT-1000 side-to-side difference, pivot shift grade, and Lachman grade of the knee were collected.
RESULTS: A total of 119 patients were included in the final cohort. Of these, 42 cases were in the traditional method group, and 77 cases were in the measuring method group. The good tunnel position rate was 26.2% in the traditional method group and 81.8% in the measuring method group (p < 0.001). At the final follow-up, the Lysholm and IKDC scores were significantly greater in the measuring method group than the traditional method group (IKDC: 84.9 ± 8.4 vs. 79.6 ± 6.4, p = 0.0005; Lysholm: 88.8 ± 6.4 vs. 81.6 ± 6.4, p < 0.001). Lachman and pivot shift grades were significantly greater in the measuring method group (p = 0.01, p = 0008). The results of KT-1000 side-to-side differences were significantly better in the measuring method group compared with those in the traditional method group (p < 0.001).
CONCLUSIONS: The combination of the measuring method and intraoperative fluoroscopy resulted in a concentrated tunnel position on the femoral side, a high rate of functional success, improved knee stability, and a low risk of tunnel deviation. This approach is particularly suitable for surgeons new to ACL reconstructive surgery.

Largely due to its simplicity, while being more like human cells compared to other experimental models, Dictyostelium continues to be of great use to discover basic molecular mechanisms and signaling pathways underlying evolutionarily conserved biological processes. However, the identification of new protein interactions implicated in signaling pathways can be particularly challenging in Dictyostelium due to its extremely fast signaling kinetics coupled with the dynamic nature of signaling protein interactions. Recently, the proximity labeling method using engineered ascorbic acid peroxidase 2 (APEX2) in mammalian cells was shown to allow the detection of weak and/or transient protein interactions and also to obtain spatial and temporal resolution. Here, we describe a protocol for successfully using the APEX2-proximity labeling method in Dictyostelium. Coupled with the identification of the labeled proteins by mass spectrometry, this method expands Dictyostelium\'s proteomics toolbox and should be widely useful for identifying interacting partners involved in a variety of biological processes in Dictyostelium.

OBJECTIVE: This study aimed to provide a method for determining the apical vertebra for pedicle subtraction osteotomy (PSO) in corrective surgery for patients with ankylosing spondylitis (AS) with thoracolumbar kyphosis (TLK).
METHODS: The medical records of AS patients with TLK who underwent PSO between May 2009 and August 2022 were retrospectively reviewed, and 235 patients were included in the study. Using the proposed method, choosing the vertebra based on Kim\'s apex (KA), which is defined as the farthest vertebra from a line drawn from the center of the T10 vertebral body to the midpoint of the S1 upper endplate, the authors analyzed 229 patients with apices at T12, L1, or L2 (excluding L3 because of the small sample size, n = 6). They divided all patients into two groups. Group A (n = 144) underwent PSO at the KA vertebra, while group B (n = 85) underwent PSO at a different level. Demographic and radiological data, including sagittal spinopelvic parameters of the entire spine, were collected. An additional analysis was performed on patients with the same KA vertebra.
RESULTS: The vertebra distributions of patients based on KA were T12 (28 [12.2%]), L1 (119 [52.0%]), and L2 (82 [35.8%]). The corrections of sagittal vertical axis (SVA; 101.0 ± 48.5 mm vs 82.0 ± 53.8 mm, p = 0.010), global kyphosis (GK; 31.6° ± 10.0° vs 26.4° ± 10.5°, p = 0.005), and TLK (29.4° ± 10.2° vs 24.2° ± 12.9°, p = 0.012) in group A were significantly greater than those in group B, and there was no difference in the corrections of thoracic kyphosis (TK), lumbar lordosis, and pelvic incidence between the two groups. On further analysis, group A showed greater correction in TK (26.2° ± 13.7° vs 0.1° ± 8.1°, p = 0.013) for patients with T12 as the KA; greater improvements in SVA (101.5 ± 44.2 mm vs 73.4 ± 48.7 mm, p = 0.020), GK (30.6° ± 11.0° vs 25.0° ± 10.4°, p = 0.046), and TLK (32.6° ± 7.8° vs 26.7° ± 9.9°, p = 0.012) for those with L1 as the KA; and significant correction in TLK (30.0° ± 6.3° vs 4.3° ± 19.5°, p = 0.008) for patients with L2 as the KA, compared with group B.
CONCLUSIONS: PSO at the apical vertebra provides a greater degree of correction of sagittal imbalance. The proposed method, selecting the vertebra based on KA, is easily reproducible for determining the apex level in AS patients with TLK.

TRPV2 voltage-insensitive, calcium-permeable ion channels play important roles in cancer progression, immune response, and neuronal development. Despite TRPV2\'s physiological impact, underlying endogenous proteins mediating TRPV2 responses and affected signaling pathways remain elusive. Using quantitative peroxidase-catalyzed (APEX2) proximity proteomics we uncover dynamic changes in the TRPV2-proximal proteome and identify calcium signaling and cell adhesion factors recruited to the molecular channel neighborhood in response to activation. Quantitative TRPV2 proximity proteomics further revealed activation-induced enrichment of protein clusters with biological functions in neural and cellular projection. We demonstrate a functional connection between TRPV2 and the neural immunoglobulin cell adhesion molecules NCAM and L1CAM. NCAM and L1CAM stimulation robustly induces TRPV2 [Ca2+]I flux in neuronal PC12 cells and this TRPV2-specific [Ca2+]I flux requires activation of the protein kinase PKCα. TRPV2 expression directly impacts neurite lengths that are modulated by NCAM or L1CAM stimulation. Hence, TRPV2\'s calcium signaling plays a previously undescribed, yet vital role in cell adhesion, and TRPV2 calcium flux and neurite development are intricately linked via NCAM and L1CAM cell adhesion proteins.

Synapses play a pivotal role in forming neural circuits, with critical implications for brain functions such as learning, memory, and emotions. Several advances in synaptic research have demonstrated the diversity of synaptic structure and function, which can form thousands of connections depending on the neuronal cell types. Moreover, synapses not only interconnect neurons but also establish connections with glial cells such as astrocytes, which play a key role in the architecture and function of neuronal circuits in the brain. Emerging evidence suggests that dysfunction of synaptic proteins contributes to a variety of neurological and psychiatric disorders. Therefore, it is crucial to determine the molecular networks within synapses in various neuronal cell types to gain a deeper understanding of how the nervous system regulates brain function. Recent advances in synaptic proteome approaches, such as fluorescence-activated synaptosome sorting (FASS) and proximity labeling, have allowed for a detailed and spatial analysis of many cell-type-specific synaptic molecules in vivo. In this brief review, we highlight these novel spatial proteomic approaches and discuss the regulation of synaptic formation and function in the brain. This knowledge of molecular networks provides new insight into the understanding of many neurological and psychiatric disorders.

Fluorescent tagging of biomolecules enables their sensitive detection during separation and determining their subcellular location. In this context, peroxidase-based reactions are actively utilized for signal amplification. To harness this potential, we developed a genetically encodable enzymatic fluorescence signal amplification method using APEX (FLEX). We synthesized a fluorescent probe, Jenfluor triazole (JFT1), which effectively amplifies and restricts fluorescence signals under fixed conditions, enabling fluorescence-based detection of subcellularly localized electron-rich metabolites. Moreover, JFT1 exhibited stable fluorescence signals even under osmium-treated and polymer-embedded conditions, which supported findings from correlative light and electron microscopy (CLEM) using APEX. Using various APEX-conjugated proteins of interest (POIs) targeted to different organelles, we successfully visualized their localization through FLEX imaging while effectively preserving organelle ultrastructures. FLEX provides insights into dynamic lysosome-mitochondria interactions upon exposure to chemical stressors. Overall, FLEX holds significant promise as a sensitive and versatile system for fluorescently detecting APEX2-POIs in multiscale biological samples.

Borrowing some quotes from Harper Lee\'s novel \"To Kill A Mockingbird\" to help frame our manuscript, we discuss methods to profile local proteomes. We initially focus on chemical biology regimens that function in live organisms and use reactive biotin species for this purpose. We then consider ways to add new dimensions to these experimental regimens, principally by releasing less reactive (i. e., more selective) (preter)natural electrophiles. Although electrophile release methods may have lower resolution and label fewer proteins than biotinylation methods, their ability to probe simultaneously protein function and locale raises new and interesting possibilities for the field.

The mammalian cell is a complex entity, with membrane-bound and membrane-less organelles playing vital roles in regulating cellular homeostasis. Organellar protein niches drive discrete biological processes and cell functions, thus maintaining cell equilibrium. Cellular processes such as signaling, growth, proliferation, motility, and programmed cell death require dynamic protein movements between cell compartments. Aberrant protein localization is associated with a wide range of diseases. Therefore, analyzing the subcellular proteome of the cell can provide a comprehensive overview of cellular biology. With recent advancements in mass spectrometry, imaging technology, computational tools, and deep machine learning algorithms, studies pertaining to subcellular protein localization and their dynamic distributions are gaining momentum. These studies reveal changing interaction networks because of \"moonlighting proteins\" and serve as a discovery tool for disease network mechanisms. Consequently, this review aims to provide a comprehensive repository for recent advancements in subcellular proteomics subcontexting methods, challenges, and future perspectives for method developers. In summary, subcellular proteomics is crucial to the understanding of the fundamental cellular mechanisms and the associated diseases.

High salinity mobilization and movement from salt-laden deposits in semi-arid landscapes impair soils and water resources worldwide. Semi-arid regions worldwide are expected to experience rising temperatures and lower precipitation, impacting water supply and spatio-temporal patterns of salinity loads and affecting downstream water quality. This study quantifies the impact of future climate on hydrologic fluxes and salt loads in the Gunnison River Watershed (GRW) (14,608 km2), Colorado, using the APEX-MODFLOW-Salt hydro-chemical watershed model and three different CMIP5 climate models projection downscaled by Multivariate Adaptive Constructed Analogs (MACA) for the period 2020-2099. The APEX-MODFLOW-Salt model accounts for the reactive transport of major salt ions (SO42-, Cl-, CO32-, HCO3-, Ca2+, Na+, Mg2+, and K+) to streams via surface runoff, rainfall erosional runoff, soil lateral flow, quick return flow and groundwater-stream exchange. Model results are analyzed for spatial and temporal trends in water yield and salt loading pathways. Although streamflow is primarily derived from surface runoff (65%), the predominant source of salt loads is the aquifer (73%) due to elevated concentrations of groundwater salt. Annual salt loading from the watershed is 582 Mkg, approximately 10% of the salt load in the Colorado River measured at Lee\'s Ferry, AZ. For future climate scenarios, annual salt loads from the watershed increased between 4.1% and 9.6% from the historical period due to increased salt loading from groundwater and quick return flow. From the results, applying the APEX-MODFLOW-Salt model with downscaled future climate forcings can be a helpful modeling framework for investigating hydrology and salt mobilization, transport, and export in historical and predictive settings for salt-affected watersheds.