Beckman Laser Institute & Medical Clinic

Brain ischemia is a leading cause of morbidity and mortality in the aging population, yet no effective neuroprotective interventions currently exist. This therapeutic gap stems from two key challenges: the inability to deliver neuroprotective drugs before reperfusion and limited understanding of post-reperfusion events leading to delayed neurodegeneration. Zn2+ is an important contributor to ischemic brain injury, with mitochondrial Zn2+ uptake playing a central role in its neurotoxic effects. Ongoing Zn2+ accumulation occurs through the mitochondrial Ca2+ uniporter (MCU) in selectively vulnerable CA1 neurons after transient ischemia, contributing to delayed mitochondrial dysfunction. We previously demonstrated that post-ischemic administration of the non-selective MCU antagonist, ruthenium red attenuated neuronal damage in both an ex vivo oxygen glucose deprivation (OGD) mouse brain slice model and in an in vivo rat cardiac arrest (CA) model. In the present study, we test post-ischemic treatment with Ru265, a novel selective MCU blocker, in both ischemic models. In the slice model, OGD induced delayed Zn2+ accumulation in CA1 neuronal mitochondria (as previously reported), followed by late-onset persistent mitochondrial depolarization and sustained elevation of cytoplasmic Ca2+ levels, indicating loss of Ca2+ homeostasis. Application of Ru265 after OGD termination inhibited the mitochondrial Zn2+ accumulation and largely prevented subsequent mitochondrial depolarization and loss of cytosolic Ca2+ homeostasis. In the CA model, intravenous Ru265 administration during cardiopulmonary resuscitation largely mitigated mitochondrial damage, markedly reduced neuronal injury and improved behavioral outcomes 20-24 hours after ischemia. These findings suggest that post-ischemic MCU blockade may constitute a promising neuroprotective therapeutic strategy.

A complete understanding of RNA dynamics requires methods to continuously image transcripts in living systems. We recently developed a panel of bioluminescent tools for multiplexed RNA detection, comprising luciferase fragments fused to RNA-binding proteins (RNA lanterns). The RNA lanterns assembled a light-emitting luciferase upon binding unique, structured RNA tags. Different colors of emission were produced via bioluminescence resonance energy transfer to fluorescent protein fusions. In this work, we systematically optimized the RNA lantern platform for enhanced performance in live cells. A luciferase-fluorescent protein linkage was identified that enabled improved energy transfer and enhanced photon outputs. The RNA lantern was further optimized for selective recognition of structured RNA tags. The new RNA lantern-tag combination is orthogonal to existing probes and enabled the first demonstration of three-color bioluminescent RNA visualization in live cells. Overall, this work expands the suite of probes for real-time, multiplexed imaging of RNA.

In the present work, we developed a data-driven algorithm for the automated segmentation of fluorescence lifetime imaging microscopy (FLIM) images, enhancing the analysis of multifunctionalized nanoparticles (NPs) within living cancer cells. FLIM, a powerful microscopy technique, generates images that capture the fluorescence lifetime across a sample at the pixel level, revealing critical details about the molecular environment. Traditionally, FLIM image analysis has relied on manual segmentation with the phasor plot approach, a graphical representation of FLIM data in aGandScoordinate system, which is susceptible to user bias and inconsistency. Here, we present an automated and free of user-biased thresholding and segmentation algorithm that streamlines with clustering techniques to automatically identify phasor-clusters in the phasor plot space, reducing user dependency and providing a reproducible strategy under tested conditions image segmentation. We demonstrate its application in the context of FLIM images displaying a map of intensity heterogeneity where functionalized NPs affect the cellular metabolism of HeLa cells, reported by NADH, a bright and a dim fluorescent source, respectively, both of biological relevance. This algorithm provides a transparent and reproducible approach for FLIM image analysis, showing good agreement with expert-defined segmentation under sufficient contrast conditions, while presenting limitations in low-contrast or noisy regimes.

Alterations of gut microbiota are common in chronic kidney disease (CKD) and contribute to increased uremic toxins including indoxyl sulfate (IS), p-cresyl sulfate (pCS) and trimethylamine N-oxide (TMAO), which are linked to cerebrovascular disease risk. This study examined the kidney-gut-brain axis in CKD mice and in dialysis patients. Male and female mice with adenine-induced CKD were fed a high-amino-acid (HAA) diet to increase precursors of gut-derived uremic toxins. A subgroup of mice received antibiotics in drinking water to suppress gut microbiota and evaluate its role in toxin generation. Behavior tests, gut microbiome composition and brain histology for cerebral microhemorrhages were analyzed. CKD mice had higher serum levels of creatinine, cystatin C and gut-derived toxins, a 2.5-fold increase in brain microhemorrhages, and decreased locomotor activity. The HAA diet significantly increased serum TMAO but not IS and pCS, and all three toxins were reduced by antibiotic therapy. Sex differences were observed; in male animals, higher TMAO was associated with increased brain microhemorrhages, whereas in female mice, pCS was associated with brain microhemorrhage burden. The suppression of toxins with antibiotics improved working memory in male animals. Gut microbiota analysis revealed the expansion of Lactobacillus and Ileibacterium in CKD mice. The HAA diet and antibiotics altered gut microbiota composition without changing alpha diversity. The human study utilized biobanked serum samples and a retrospective review of brain imaging scans in a hemodialysis patient cohort; TMAO levels were associated with increased lobar microbleeds. Our study supports a role for bacterial-derived uremic toxins in the kidney-gut-brain axis and cerebral microhemorrhage formation in CKD.

We report the spectral broadening and post-compression of picosecond pulses using a multi-pass cell (MPC) operating in air. A 2 m-long bi-concave cavity with 41 reflections and 96.4% transmission was designed and implemented, optimized for the compression of 5.5 ps, 4.5 mJ pulses from a Yb:YAG laser operating at 1030 nm. A 19.3-fold spectral broadening was obtained, increasing the bandwidth from 0.3 nm to 5.8 nm, which enabled pulse compression down to 547 fs with a symmetric temporal profile close to the transform limit. These results demonstrate the feasibility of using open-air MPCs for high-energy picosecond pulse post-compression, without the need for sealed chambers, while preserving high mode quality and transmission. Scaling to pulse higher energy is discussed.

(Neuron, Vol. 39, 409–421; July 31, 2003) The authors wish to correct an error in Figure 1C of the above article. In the published version, the tau protein panel was inadvertently duplicated in place of the APP protein panel. As a result, the tau western blot data appear twice in the original figure, while the APP western blot data are absent. The corrected Figure 1C now shows APP (top panel) and tau (bottom panel) western blot results across the different founder lines, as originally intended. The updated quantification is shown in Figure 1D. Based on these results, we selected the B1 line as the founder line to derive and characterize further. The corrected figure accurately represents the experimental results that were obtained. This correction does not affect the conclusions or interpretation of the study. The corrected Figure 1 appears below. The authors apologize for this error and any confusion it may have caused. (Figure presented)

CKD is an increasing global health burden and is a recognized risk factor for vascular injury. Cerebral microbleeds (CMB), a radiologic marker of microvascular hemorrhage in the brain, are more prevalent in CKD and are predictive of stroke and cognitive decline. This review integrates evidence from epidemiologic studies, neuropathologic investigations, and experimental models to elucidate the mechanistic links between CKD and CMB. Key pathways include uremic toxin-induced endothelial injury, vascular calcification, and BP variability. These abnormalities weaken microvascular integrity and accelerate vascular remodeling, ultimately promoting microvascular rupture and CMB formation. Evidence indicates that CKD is an independent risk factor for CMB formation, even after adjustment for conventional vascular risk factors. Recognizing CKD-related CMB as a significant cerebrovascular complication has important implications for prevention of stroke and cognitive decline. Incorporating CMB assessment into CKD care and implementing targeted vascular protection and uremic toxin-lowering strategies may enhance brain health outcomes.

OBJECTIVES: Cryogen spray cooling (CSC) is critical for protecting the epidermis during laser dermatologic procedures. The widely used cryogen, R134a, has a high global warming potential (GWP), motivating interest in environmentally favorable alternatives such as HFO-1234ze. This study evaluated whether HFO-1234ze provides epidermal protection comparable to R134a during laser exposure and investigated the mechanistic basis underlying any differences in cooling performance. METHODS: Laser-induced epidermal injury in conjunction with two cryogens was assessed in K14-SCF Mc1r^e/e mice with pharmacologically controllable interfollicular epidermal pigmentation. Laser treatment sites were assigned fluences relative to baseline skin lightness (L*) to span subthreshold to suprathreshold injury conditions, with cryogen assignment randomized. Hypopigmentation at Day 7 served as the primary injury endpoint and was scored independently by three blinded raters. Surface cooling induced by each cryogen was measured separately using thin-film thermocouples on a skin phantom, and subsurface temperature distributions were estimated using one-dimensional transient heat-transfer modeling. RESULTS: Mean hypopigmentation scores did not differ significantly between R134a- and HFO-1234ze-cooled sites across the full range of pigmentation and fluence studied (p = 0.46). Heat-map analysis and regression modeling confirmed that hypopigmentation increased with fluence and darker pigmentation but was independent of cryogen type. Surface temperature measurements using skin phantom revealed that R134a produced more aggressive transient cooling than HFO-1234ze, reaching approximately 10°C lower minimum temperatures. However, numerical modeling of skin cooling using heat flux inferred from phantom measurement showed that this surface advantage decayed rapidly with depth, resulting in ≤ 2°C-3°C differences within the viable epidermis. CONCLUSIONS: Despite differences in surface cooling magnitude, R134a and HFO-1234ze provided equivalent epidermal protection under clinically relevant conditions. These findings support HFO-1234ze as an effective, lower-GWP alternative to R134a for CSC in laser dermatologic applications.