Two-Color Spatial Cumulant Analysis Detects Heteromeric Interactions between Membrane Proteins.

Fluorescence fluctuation spectroscopy can be used to measure the aggregation of fluorescently labeled molecules and is typically performed using time series data. Spatial intensity distribution analysis and fluorescence moment image analysis are established tools for measuring molecular brightnesses from single-color images collected with laser scanning microscopes. We have extended these tools for analysis of two-color images to resolve heteromeric interactions between molecules labeled with spectrally distinct chromophores. We call these new methods two-color spatial intensity distribution analysis and two-color spatial cumulant analysis (2c-SpCA). To implement these techniques on a hyperspectral imaging system, we developed a spectral shift filtering technique to remove artifacts due to intrinsic cross talk between detector bins. We determined that 2c-SpCA provides better resolution from samples containing multiple fluorescent species; hence, this technique was carried forward to study images of living cells. We used fluorescent heterodimers labeled with enhanced green fluorescent protein and mApple to quantify the effects of resonance energy transfer and incomplete maturation of mApple on brightness measurements. We show that 2c-SpCA can detect the interaction between two components of trimeric G-protein complexes. Thus, 2c-SpCA presents a robust and computationally expedient means of measuring heteromeric interactions in cellular environments.

TCAF1 promotes TRPV2-mediated Ca2+ release in response to cytosolic DNA to protect stressed replication forks.

The protection of the replication fork structure under stress conditions is essential for genome maintenance and cancer prevention. A key signaling pathway for fork protection involves TRPV2-mediated Ca2+ release from the ER, which is triggered after the generation of cytosolic DNA and the activation of cGAS/STING. This results in CaMKK2/AMPK activation and subsequent Exo1 phosphorylation, which prevent aberrant fork processing, thereby ensuring genome stability. However, it remains poorly understood how the TRPV2 channel is activated by the presence of cytosolic DNA. Here, through a genome-wide CRISPR-based screen, we identify TRPM8 channel-associated factor 1 (TCAF1) as a key factor promoting TRPV2-mediated Ca2+ release under replication stress or other conditions that activate cGAS/STING. Mechanistically, TCAF1 assists Ca2+ release by facilitating the dissociation of STING from TRPV2, thereby relieving TRPV2 repression. Consistent with this function, TCAF1 is required for fork protection, chromosomal stability, and cell survival after replication stress.

The role of imaging in patient selection, preoperative planning, and postoperative monitoring in human upper extremity allotransplantation.

Objective. To describe the role of imaging in vascular composite allotransplantation based on one institution's experience with upper extremity allotransplant patients. Methods. The institutional review board approved this review of HIPAA-compliant patient data without the need for individual consent. A retrospective review was performed of imaging from 2008 to 2011 on individuals undergoing upper extremity transplantation. This demonstrated that, of the 19 patients initially considered, 5 patients with a mean age of 37 underwent transplantation. Reports were correlated clinically to delineate which preoperative factors lead to patient selection versus disqualification and what concerns dictated postoperative imaging. Findings were subdivided into musculoskeletal and vascular imaging criterion. Results. Within the screening phase, musculoskeletal exclusion criterion included severe shoulder arthropathy, poor native bone integrity, and marked muscular atrophy. Vascular exclusion criterion included loss of sufficient arterial or venous supply and significant distortion of the native vascular architecture. Postoperative imaging was used to document healing and hardware integrity. Postsurgical angiography and ultrasound were used to monitor for endothelial proliferation or thrombosis as signs of rejection and vascular complication. Conclusion. Multimodality imaging is an integral component of vascular composite allotransplantation surgical planning and surveillance to maximize returning form and functionality while minimizing possible complications.

Helping hands: caring for the upper extremity transplant patient.

Caring for upper extremity transplant recipients can offer challenges and opportunities to nursing staff in combining new patient procedures, new technologies, and complex patient care needs including unique physical care, monitoring and observation, rehabilitation expectations, and psychiatric/psychosocial support. Medical professionals continue to be apprehensive about the risks of immunosuppressive therapy and the possibility of acute and chronic rejection. The sustained development and research into reliable, reduced-dose immunosuppression or immunomodulatory strategies could expand the life-enhancing benefits of reconstructive transplantation.