A molecular switch for neuroprotective astrocyte reactivity.

The intrinsic mechanisms that regulate neurotoxic versus neuroprotective astrocyte phenotypes and their effects on central nervous system degeneration and repair remain poorly understood. Here we show that injured white matter astrocytes differentiate into two distinct C3-positive and C3-negative reactive populations, previously simplified as neurotoxic (A1) and neuroprotective (A2)1,2, which can be further subdivided into unique subpopulations defined by proliferation and differential gene expression signatures. We find the balance of neurotoxic versus neuroprotective astrocytes is regulated by discrete pools of compartmented cyclic adenosine monophosphate derived from soluble adenylyl cyclase and show that proliferating neuroprotective astrocytes inhibit microglial activation and downstream neurotoxic astrocyte differentiation to promote retinal ganglion cell survival. Finally, we report a new, therapeutically tractable viral vector to specifically target optic nerve head astrocytes and show that raising nuclear or depleting cytoplasmic cyclic AMP in reactive astrocytes inhibits deleterious microglial or macrophage cell activation and promotes retinal ganglion cell survival after optic nerve injury. Thus, soluble adenylyl cyclase and compartmented, nuclear- and cytoplasmic-localized cyclic adenosine monophosphate in reactive astrocytes act as a molecular switch for neuroprotective astrocyte reactivity that can be targeted to inhibit microglial activation and neurotoxic astrocyte differentiation to therapeutic effect. These data expand on and define new reactive astrocyte subtypes and represent a step towards the development of gliotherapeutics for the treatment of glaucoma and other optic neuropathies.

LonP1 Drives Proneural Mesenchymal Transition in IDH1-R132H Diffuse Glioma.

Malignant astroctyoma and glioblastoma are diffuse CNS tumors that have markedly similar features, including microvascular proliferation and necrosis, and the latter presents higher grade and poorer survival. The Isocitrate dehydrogenase 1/2 (IDH) mutation further predicts improved survival and is present in oligodendroglioma and astrocytoma. The latter are more prevalent in younger populations with a median age of 37 years at diagnosis as compared to glioblastoma with a median age of 641,2. These tumors frequently have co-occurring ATRX and/or TP53 mutations (Brat et al., 2021). The IDH mutation is known to cause dysregulation of the hypoxia response broadly in CNS tumors and subsequent reduction in both tumor growth and treatment resistance. The frequency of tumor recurrence is high for diffuse CNS tumors. Understanding the mechanism and potential molecular targets enhancing treatment resistance and local invasion in IDH mutant diffuse glioma is necessary for developing new treatment strategies for better tumor control and improving overall survival. Recent evidence highlights the importance of local foci in IDH mutant glioma with an accelerated stress response as responsible for recurrence in these tumors. Here, we demonstrate that LonP1 drives NRF2 and subsequent proneural mesenchymal transition interdependent with the IDH mutation in response to stress and other tumor microenvironment cues. Our findings provide further evidence that targeting LonP1 may be a crucial strategy for improving the standard-of-care treatment in IDH mutant diffuse astrocytoma.

Spatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis.

Multiplexed mRNA profiling in the spatial context provides new information enabling basic research and clinical applications. Unfortunately, existing spatial transcriptomics methods are limited due to either low multiplexing or complexity. Here, we introduce a spatialomics technology, termed Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA), that integrates in situ labeling of mRNA and protein markers in cells or tissues with combinatorial fluorescence spectral and lifetime encoded probes, spectral and time-resolved fluorescence imaging, and machine learning-based decoding. We demonstrate MOSAICA's multiplexing scalability in detecting 10-plex targets in fixed colorectal cancer cells using combinatorial labeling of five fluorophores with facile error-detection and removal of autofluorescence. MOSAICA's analysis is strongly correlated with sequencing data (Pearson's r = 0.96) and was further benchmarked using RNAscopeTM and LGC StellarisTM. We further apply MOSAICA for multiplexed analysis of clinical melanoma Formalin-Fixed Paraffin-Embedded (FFPE) tissues. We finally demonstrate simultaneous co-detection of protein and mRNA in cancer cells.

Effect of Body Fat on Population Pharmacokinetics of High-Dose Methotrexate in Pediatric Patients With Acute Lymphoblastic Leukemia.

Nearly all international regimens for pediatric acute lymphoblastic leukemia (ALL) incorporate intravenous "high-dose" methotrexate (HDMTX, ≥1 g/m2 ) to penetrate the central nervous system. Dosing is routinely adjusted for body surface area (BSA), but limited data describe the pharmacokinetics of HDMTX, particularly in obese and/or large patients. To understand the impact of body size (BSA) and body fat percentage (BFP) on HDMTX pharmacokinetics, we performed a secondary analysis of 36 children and adolescents 10-21 years old treated for newly diagnosed ALL and who were enrolled in a prospective study examining body composition. All patients received 5 g/m2 of HDMTX infused over 24 hours. Plasma methotrexate concentrations were measured at 24, 42, and 48 hours. At 48 hours, ≥0.4 µmol/L was defined as "delayed elimination," necessitating prolonged supportive care. BFP was measured using dual-energy x-ray absorptiometry. A nonparametric population pharmacokinetic model was constructed with subsequent simulations to explore effects of BSA and BFP extremes. Despite standard BSA-adjusted dosing, we found significant intrapatient variability in mean MTX concentration (38%; range, 1.2%-86%). BSA and BFP were not linearly associated with increased area under the curve (AUC, P = 0.74 and P = 0.12), but both larger size (BSA) and greater obesity (BFP) were associated with an approximately 2-fold higher risk for delayed elimination at 48 hours. HDMTX AUC was not associated with toxicity. MTX pharmacokinetics vary among and even within patients despite BSA-adjusted dosing. Obesity and large size are identified as new risk factors for delayed elimination, requiring further investigation.

Studies towards the total synthesis of drimentine C. Preparation of the AB and CDEF ring fragments.

The drimentine family is a class of hybrid isoprenoids derived from actinomycete bacteria. Members of this family display weak antitumor and antibacterial activity. Herein we report our efforts toward the total synthesis of drimentine C using three distinct approaches incorporating palladium-catalyzed cyanoamidation, reductive cross-coupling, and photoredox-catalyzed α-alkylation of an aldehyde as key steps. Our synthetic efforts use a convergent synthesis to assemble the terpenoid and alkaloid portions of drimentine C from readily available l-tryptophan, l-proline, and (+)-sclareolide.

Integrating Metal-Catalyzed C-H and C-O Functionalization To Achieve Sterically Controlled Regioselectivity in Arene Acylation.

One major goal of organometallic chemists is the direct functionalization of the bonds most recurrent in organic molecules: C-H, C-C, C-O, and C-N. An even grander challenge is C-C bond formation when both precursors are of this category. Parallel to this is the synthetic goal of achieving reaction selectivity that contrasts with conventional methods. Electrophilic aromatic substitution (EAS) via Friedel-Crafts acylation is the most renowned method for the synthesis of aryl ketones, a common structural motif of many pharmaceuticals, agrochemicals, fragrances, dyes, and other commodity chemicals. However, an EAS synthetic strategy is only effective if the desired site for acylation is in accordance with the electronic-controlled regioselectivity of the reaction. Herein we report steric-controlled regioselective arene acylation with salicylate esters via iridium catalysis to access distinctly substituted benzophenones. Experimental and computational data indicate a unique reaction mechanism that integrates C-O activation and C-H activation with a single iridium catalyst without an exogenous oxidant or base. We disclose an extensive exploration of the synthetic scope of both the arene and the ester components, culminating in the concise synthesis of the potent anticancer agent hydroxyphenstatin.

Isoform-specific subcellular localization and function of protein kinase A identified by mosaic imaging of mouse brain.

Protein kinase A (PKA) plays critical roles in neuronal function that are mediated by different regulatory (R) subunits. Deficiency in either the RIß or the RIIß subunit results in distinct neuronal phenotypes. Although RIß contributes to synaptic plasticity, it is the least studied isoform. Using isoform-specific antibodies, we generated high-resolution large-scale immunohistochemical mosaic images of mouse brain that provided global views of several brain regions, including the hippocampus and cerebellum. The isoforms concentrate in discrete brain regions, and we were able to zoom-in to show distinct patterns of subcellular localization. RIß is enriched in dendrites and co-localizes with MAP2, whereas RIIß is concentrated in axons. Using correlated light and electron microscopy, we confirmed the mitochondrial and nuclear localization of RIß in cultured neurons. To show the functional significance of nuclear localization, we demonstrated that downregulation of RIß, but not of RIIß, decreased CREB phosphorylation. Our study reveals how PKA isoform specificity is defined by precise localization.

Novel Mechanistic Interplay between Products of Oxidative Stress and Components of the Complement System in AMD Pathogenesis.

Age-related macular degeneration (AMD) is a leading cause of vision loss affecting tens of millions of elderly worldwide. Early AMD includes soft drusen and pigmentary changes in the retinal pigment epithelium (RPE). As people age, such soft confluent drusen can progress into two forms of advanced AMD, geographic atrophy (GA, or dry AMD) or choroidal neovascularization (CNV, or wet AMD) and result in the loss of central vision. The exact mechanism for developing early AMD and progressing to advanced stage of disease is still largely unknown. However, significant evidence exists demonstrating a complex interplay of genetic and environmental factors as the cause of AMD progression. Together, complement factor H (CFH) and HTRA1/ARMS polymorphisms contribute to more than 50% of the genetic risk for AMD. Environmentally, oxidative stress from activities such as smoking has also demonstrated a powerful contribution to AMD progression. To extend our previous finding that genetic polymorphisms in CFH results in OxPLs and the risk-form of CFH (CFH Y402H) has reduced affinity for oxidized phospholipids, and subsequent diminished capacity which subsequently diminishes the capability to attenuate the inflammatory effects of these molecules, we compared the binding properties of CFH and CFH related protein 1 (CFHR1), which is also associated with disease risk, to OxPLs and their effects on modulating inflammation and lipids uptake. As both CFH-402H and CFHR1 are associated with increased risk to AMD, we hypothesized that like CFH-402H, CFHR1 contribution to AMD risk may also be due to its diminished affinity for OxPLs. Interestingly, we found that association of CFHR1 with OxPLs was not statistically different than CFH. However, binding of CFHR1 did not elicit the same protective benefits as CFH in that both inflammation and lipid uptake are unaffected by CFHR1 association with OxPLs. These findings demonstrate a novel and interesting complexity to the potential interplay between the complement system and oxidative stress byproducts, such as OxPLs, in the mechanistic contribution to AMD. Future work will aim to identify the molecular distinctions between CFH and CFHR1 which confer protection by the former, but not latter molecules. Understanding the molecular domains necessary for protection could provide interventional insights in the generation of novel therapeutics for AMD and other diseases associated with oxidative stress.

Materials in the vitreous during cataract surgery: nature and incidence, with two cases of histological confirmation.

BACKGROUND: To identify and classify materials in the vitreous observed during phacoemulsification cataract surgery (phaco). DESIGN: Prospective, consecutive, observational case series at one ophthalmic day surgery in Sydney, Australia. PARTICIPANTS: A total of 767 consecutive phaco cases. Cases were excluded if there was posterior capsule rupture or vitreous loss intraoperatively. METHODS: For each patient, age, gender, baseline corrected distance visual acuity, presence of pseudoexfoliation, nuclear sclerosis grade and phacoemulsification ultrasound time were recorded. The relationship between these variables and materials in the vitreous was evaluated with regression analysis. Two patients with materials in the vitreous developed an acute intraoperative rock-hard eye syndrome. In these two patients, pars plana needle aspiration of retrolenticular fluid was performed to re-establish normal intraocular pressure. Histology was undertaken to compare this fluid with known lens material retrieved from the Fluid Management System bags in two unrelated cases. MAIN OUTCOME MEASUREMENTS: Presence of materials in the vitreous during phaco. RESULTS: Materials in the vitreous were observed in either Berger's space or the anterior vitreous in 386 eyes (50.3% of cases); the majority was putatively lens material (46.5% of all cases). Pigment and ophthalmic viscoelastic device were seen in the anterior vitreous in 9.8% and 1.7% of cases, respectively. Logistic regression analysis demonstrated that higher nuclear sclerosis grade (P = 0.025), male gender (P = 0.003) and greater age (P = 0.016) were predictive of the presence of materials in the vitreous. Histological assessment with light microscopy and birefringence techniques identified the materials in the vitreous as lens material. CONCLUSION: Materials in the vitreous were seen in 50.3% of phaco cases. It has been histologically demonstrated that lens materials can be introduced into the anterior vitreous during phaco.

Oxidative stress, innate immunity, and age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of vision loss affecting tens of millions of elderly worldwide. Early AMD is characterized by the appearance of soft drusen, as well as pigmentary changes in the retinal pigment epithelium (RPE). These soft, confluent drusen can progress into two forms of advanced AMD: geographic atrophy (GA, or dry AMD) or choroidal neovascularization (CNV, or wet AMD). Both forms of AMD result in a similar clinical progression in terms of loss of central vision. The exact mechanism for developing early AMD, as well as triggers responsible for progressing to advanced stage of disease, is still largely unknown. However, significant evidence exists demonstrating a complex interplay of genetic and environmental factors as causes of AMD progression. Multiple genes and/or single nucleotide polymorphisms (SNPs) have been found associated with AMD, including various genes involved in the complement pathway, lipid metabolism and extracellular matrix (ECM) remodeling. Of the known genetic contributors to disease risk, the CFH Y402H and HTRA1/ARMS polymorphisms contribute to more than 50% of the genetic risk for AMD. Environmentally, oxidative stress plays a critical role in many aging diseases including cardiovascular disease, cancer, Alzheimer's disease and AMD. Due to the exposure to sunlight and high oxygen concentration, the oxidative stress burden is higher in the eye than other tissues, which can be further complicated by additional oxidative stressors such as smoking. Increasingly, evidence is accumulating suggesting that functional abnormalities of the innate immune system incurred via high risk genotypes may be contributing to the pathogenesis of AMD by altering the inflammatory homeostasis in the eye, specifically in the handling of oxidation products. As the eye in non-pathological instances maintains a low level of inflammation despite the presence of a relative abundance of potentially inflammatory molecules, we have previously hypothesized that the tight homeostatic control of inflammation via the innate immune system is likely critical for avoidance of disease progression. However, the presence of a multitude of potential triggers of inflammation results in a sensitive balance in which perturbations thereof would subsequently alter the inflammatory state of the retina, leading to a state of chronic inflammation and pathologic progression. In this review, we will highlight the background literature surrounding the known genetic and environmental contributors to AMD risk, as well as a discussion of the potential mechanistic interplay of these factors that lead to disease pathogenesis with particular emphasis on the delicate control of inflammatory homeostasis and the centrality of the innate immune system in this process.

Cardiac rehabilitation in African Americans: evidence for poorer outcomes compared with whites, especially in women and diabetic participants.

BACKGROUND: Cardiac rehabilitation (CR) improves coronary artery disease risk factors and mortality. Outcomes after CR in African Americans (AAs) compared with whites have not been studied extensively. METHODS: A total of 1,096 patients (169 AAs, 927 whites) were enrolled in a 36-session CR program for ischemic heart disease or postcardiac surgery. The program consisted of exercise, lifestyle modification, and pharmacotherapy. RESULTS: After CR, quality of life, blood pressure, and low-density lipoprotein cholesterol improved significantly in both AAs and whites, although to a lesser degree in AAs. Whites also had significant improvements in weight and triglyceride concentrations. Overall, mean peak exercise capacity, measured in metabolic equivalents (METs), improved by only 1.6 (95% CI 1.3-1.8) in AAs compared with 2.4 (2.3-2.6) in CCs (P< .001 for AAs vs CCs). African American women had the least improvement in METs, but changes were still significant (1.1 [CI 0.9-1.4]). The subgroup with the least improvement in METs was AA diabetic patients (1.4 (CI 1.1-1.7]). CONCLUSION: African Americans derive a significant benefit from CR, but not to the same degree as whites, based on changes in risk factors and in exercise capacity. Within both ethnic groups, both women and diabetic patients appeared to have markedly less improvement.

Effect of early enrollment on outcomes in cardiac rehabilitation.

Outpatient cardiac rehabilitation (CR) is most beneficial when delivered 1 to 3 weeks after the index cardiac event. The effects of delayed enrollment on subsequent outcomes are unclear. A total of 1,241 patients were enrolled in CR after recent (<1 year) treatment of cardiac events or postcardiac surgery. Risk factors and metabolic equivalent levels (METs) during aerobic exercise were calculated before and after CR. The mean CR delay time was 34 days (maximum of 327). Delay time >30 days was associated with older age, female gender, nonwhite race, being unemployed, and increased length of hospital stay before CR after index cardiac event (p <0.05 vs 0 to 15 and 16 to 30 days for all comparisons). Patients with delay time >30 days had significant improvements in all CR metrics, but peak METs and weight improvements were lesser in magnitude compared with patients with CR delay times 0 to 15 and 16 to 30 days. After multivariate adjustment, delay time >30 days remained an independent predictor of decreased MET improvement compared with delay time 0 to 15 days (ß = -0.59, p <0.001). In conclusion, time to enrollment in CR varies substantially and is independently linked to demographics and length of index hospital stay. Delayed enrollment in CR is directly related to patient outcomes. Although all patients showed improvements in key metrics regardless of delay time, CR was of greatest benefit, particularly for weight and exercise capacity, when initiated within 15 days of the index event.

Stapled aortic anastomoses: a minimally invasive, feasible alternative to videoscopic aortic suturing?

Widespread applications of totally laparoscopic aortic reconstructions have been limited by the long cross-clamp time required to suture the aortic anastomosis despite improvement in instrumentation. The authors' hypothesis was that a "one-step anastomosis concept" using an intraluminal stapler would allow shorter cross-clamp time but similar patency and imperviousness as videoscopic suturing techniques. An intraluminal stapler (Endopath-ILS, Ethicon) with a modified anvil was used to perform videoscopic-assisted thoracic aorta-to-iliac artery bypass with a 21 mm by 8 mm polytetrafluoroethylene (PTFE) graft in 22 sheep through a minimally invasive approach using a 5 cm thoracotomy. The graft-to-iliac artery anastomoses were hand sutured through a flank incision. Twelve sheep were used to establish the technique and 10 subsequent animals constituted the study group. Aortic cross-clamp time, imperviousness, and need for additional sutures were recorded and compared to previously reported data using videoscopic suturing in pigs. Patency was assessed by comparing lower limb arterial pressures. Macroscopic and microscopic examinations of the anastomoses were performed at different time-points within the first 3 months. Videoscopic-assisted stapled anastomoses were also performed on atherosclerotic aortas of 3 human cadavers. Stapled anastomoses between the thoracic aorta and PTFE graft were completed in 8 of 10 animals. Two animals were euthanized after stapler failure and anastomotic bleeding. Sutures to strengthen the anastomosis had to be used in 4 cases. Mean aortic cross-clamp time in 8 successful cases was 4.3 +/-2.9 minutes (range 2-11 minutes) and was significantly shorter than clamp time of videoscopic suturing technique (48.7 +/-9.4 minutes, p < 0.0001). Imperviousness was good or excellent in 4 animals and fair in 4 animals. All anastomoses were patent at the end of the procedure. Examination of the anastomosis of the 2 failed interventions showed medial aortic tear surrounding the anastomosis in 1 case and misfired staples in the other. No graft occlusion was noted during follow-up ranging from 0 to 12 weeks. At the time of harvest, no bleeding was noted after epinephrine and volume infusion to increase mean arterial pressure to 200 mm Hg for 15 minutes. Macroscopic examination of the anastomoses revealed adequate healing with circumferential stapling of the prosthesis to the aortic wall and no stenosis or thrombus except in 1 false aneurysm (1/7, 14%). Surface electron microscopy showed cells coverage of the anastomosis surface. When applied on human cadaver thoracic and abdominal aorta with atherosclerotic changes, clamping times of less than 5 minutes were achieved. However, imperviousness tested with saline was poor. An automatic stapling device allows performance of a graft-to-aorta anastomosis through a minimally invasive approach with shorter clamping time than a videoscopic suturing technique. However, the current technique of aortic stapling is unreliable and further improvements are needed.