Spatial architecture of high-grade glioma reveals tumor heterogeneity within distinct domains.

Background: High-grade gliomas (HGGs) are aggressive primary brain cancers with poor response to standard regimens, driven by immense heterogeneity. In isocitrate dehydrogenase (IDH) wild-type HGG (glioblastoma, GBM), increased intratumoral heterogeneity is associated with more aggressive disease. Methods: Spatial technologies can dissect complex heterogeneity within the tumor ecosystem by preserving cellular organization in situ. We employed GeoMx digital spatial profiling, CosMx spatial molecular imaging, Xenium in situ mapping and Visium spatial gene expression in experimental and validation patient cohorts to interrogate the transcriptional landscape in HGG. Results: Here, we construct a high-resolution molecular map of heterogeneity in GBM and IDH-mutant patient samples to investigate the cellular communities that compose HGG. We uncovered striking diversity in the tumor landscape and degree of spatial heterogeneity within the cellular composition of the tumors. The immune distribution was diverse between samples, however, consistently correlated spatially with distinct tumor cell phenotypes, validated across tumor cohorts. Reconstructing the tumor architecture revealed two distinct niches, one composed of tumor cells that most closely resemble normal glial cells, associated with microglia, and the other niche populated by monocytes and mesenchymal tumor cells. Conclusions: This primary study reveals high levels of intratumoral heterogeneity in HGGs, associated with a diverse immune landscape within spatially localized regions.

Intracranial intraoperative radiotherapy (IORT): evaluation of electrocorticography and peri-operative seizure risk.

BACKGROUND: Intra-operative radiotherapy (IORT) for brain metastases (BMs) and primary brain tumors has emerged as an adjuvant radiation modality that allows for consolidation of care into a single anesthetic episode with surgical resection. Yet, there is a paucity of data regarding the impact that IORT may have on peri-operative and long-term seizure risk. METHODS: A retrospective analysis of patients receiving IORT during tumor resection was performed via registry including data regarding peri-operative anti-seizure medications and anesthetic agents. Intra-operative neuromonitoring was performed using electrocorticography (ECoG) captured before-, during-, and after-IORT then analyzed for evidence of seizure or significant baseline changes. Kaplan-Meir estimations were used for overall survival analysis relative to documented clinical seizure incidence post-IORT. RESULTS: Of the 24 consecutive patients treated with IORT during tumor resection included, 18 (75%) patients were diagnosed with BMs while 6 (25%) had newly-diagnosed glioblastoma. Mean and median survival times were 487 and 372 days, respectively. Clinical seizures occurred in 3 patients post-IORT, 2 BMs patients within 9 months and 1 glioblastoma patient at 14 months. IORT time represented 9.5% of anesthetic time. ECoG recordings were available for 5 patients (4 BMs; 1 glioblastoma), with mean recording durations of 13% of the total anesthetic time and no evidence of high-frequency oscillations or seizure activity. CONCLUSIONS: IORT is an option for delivery of definitive radiation in surgically resected brain tumors without increasing the peri-operative or long-term risk of seizure. ECoG data during the delivery of radiation fail to demonstrate any electrophysiological changes in response to ionizing radiation.

Defining the identity and the niches of epithelial stem cells with highly pleiotropic multilineage potency in the human thymus.

Thymus is necessary for lifelong immunological tolerance and immunity. It displays a distinctive epithelial complexity and undergoes age-dependent atrophy. Nonetheless, it also retains regenerative capacity, which, if harnessed appropriately, might permit rejuvenation of adaptive immunity. By characterizing cortical and medullary compartments in the human thymus at single-cell resolution, in this study we have defined specific epithelial populations, including those that share properties with bona fide stem cells (SCs) of lifelong regenerating epidermis. Thymic epithelial SCs display a distinctive transcriptional profile and phenotypic traits, including pleiotropic multilineage potency, to give rise to several cell types that were not previously considered to have shared origin. Using here identified SC markers, we have defined their cortical and medullary niches and shown that, in vitro, the cells display long-term clonal expansion and self-organizing capacity. These data substantively broaden our knowledge of SC biology and set a stage for tackling thymic atrophy and related disorders.

Association of Thoracic Aortic Aneurysm Size With Long-term Patient Outcomes: The KP-TAA Study.

Importance: The risk of adverse events from ascending thoracic aorta aneurysm (TAA) is poorly understood but drives clinical decision-making. Objective: To evaluate the association of TAA size with outcomes in nonsyndromic patients in a large non-referral-based health care delivery system. Design, Setting, and Participants: The Kaiser Permanente Thoracic Aortic Aneurysm (KP-TAA) cohort study was a retrospective cohort study at Kaiser Permanente Northern California, a fully integrated health care delivery system insuring and providing care for more than 4.5 million persons. Nonsyndromic patients from a regional TAA safety net tracking system were included. Imaging data including maximum TAA size were merged with electronic health record (EHR) and comprehensive death data to obtain demographic characteristics, comorbidities, medications, laboratory values, vital signs, and subsequent outcomes. Unadjusted rates were calculated and the association of TAA size with outcomes was evaluated in multivariable competing risk models that categorized TAA size as a baseline and time-updated variable and accounted for potential confounders. Data were analyzed from January 2018 to August 2021. Exposures: TAA size. Main Outcomes and Measures: Aortic dissection (AD), all-cause death, and elective aortic surgery. Results: Of 6372 patients with TAA identified between 2000 and 2016 (mean [SD] age, 68.6 [13.0] years; 2050 female individuals [32.2%] and 4322 male individuals [67.8%]), mean (SD) initial TAA size was 4.4 (0.5) cm (828 individuals [13.0% of cohort] had initial TAA size 5.0 cm or larger and 280 [4.4%] 5.5 cm or larger). Rates of AD were low across a mean (SD) 3.7 (2.5) years of follow-up (44 individuals [0.7% of cohort]; incidence 0.22 events per 100 person-years). Larger initial aortic size was associated with higher risk of AD and all-cause death in multivariable models, with an inflection point in risk at 6.0 cm. Estimated adjusted risks of AD within 5 years were 0.3% (95% CI, 0.3-0.7), 0.6% (95% CI, 0.4-1.3), 1.5% (95% CI, 1.2-3.9), 3.6% (95% CI, 1.8-12.8), and 10.5% (95% CI, 2.7-44.3) in patients with TAA size of 4.0 to 4.4 cm, 4.5 to 4.9 cm, 5.0 to 5.4 cm, 5.5 to 5.9 cm, and 6.0 cm or larger, respectively, in time-updated models. Rates of the composite outcome of AD and all-cause death were higher than for AD alone, but a similar inflection point for increased risk was observed at 6.0 cm. Conclusions and Relevance: In a large sociodemographically diverse cohort of patients with TAA, absolute risk of aortic dissection was low but increased with larger aortic sizes after adjustment for potential confounders and competing risks. Our data support current consensus guidelines recommending prophylactic surgery in nonsyndromic individuals with TAA at a 5.5-cm threshold.

GLIS1-3: Links to Primary Cilium, Reprogramming, Stem Cell Renewal, and Disease.

The GLI-Similar 1-3 (GLIS1-3) genes, in addition to encoding GLIS1-3 Krüppel-like zinc finger transcription factors, also generate circular GLIS (circGLIS) RNAs. GLIS1-3 regulate gene transcription by binding to GLIS binding sites in target genes, whereas circGLIS RNAs largely act as miRNA sponges. GLIS1-3 play a critical role in the regulation of many biological processes and have been implicated in various pathologies. GLIS protein activities appear to be regulated by primary cilium-dependent and -independent signaling pathways that via post-translational modifications may cause changes in the subcellular localization, proteolytic processing, and protein interactions. These modifications can affect the transcriptional activity of GLIS proteins and, consequently, the biological functions they regulate as well as their roles in disease. Recent studies have implicated GLIS1-3 proteins and circGLIS RNAs in the regulation of stemness, self-renewal, epithelial-mesenchymal transition (EMT), cell reprogramming, lineage determination, and differentiation. These biological processes are interconnected and play a critical role in embryonic development, tissue homeostasis, and cell plasticity. Dysregulation of these processes are part of many pathologies. This review provides an update on our current knowledge of the roles GLIS proteins and circGLIS RNAs in the control of these biological processes in relation to their regulation of normal physiological functions and disease.

Persistence of improved glucose homeostasis in Gclm null mice with age and cadmium treatment.

Antioxidant signaling/communication is among the most important cellular defense and survival pathways, and the importance of redox signaling and homeostasis in aging has been well-documented. Intracellular levels of glutathione (GSH), a very important endogenous antioxidant, both govern and are governed by the Nrf2 pathway through expression of genes involved in its biosynthesis, including the subunits of the rate-limiting enzyme (glutamate cysteine ligase, GCL) in GSH production, GCLC and GCLM. Mice homozygous null for the Gclm gene are severely deficient in GSH compared to wild-type controls, expressing approximately 10% of normal GSH levels. To compensate for GSH deficiency, Gclm null mice have upregulated redox-regulated genes, and, surprisingly, are less susceptible to certain types of oxidative damage. Furthermore, young Gclm null mice display an interesting lean phenotype, resistance to high fat diet-induced diabetes and obesity, improved insulin and glucose tolerance, and decreased expression of genes involved in lipogenesis. However, the persistence of this phenotype has not been investigated into old age, which is important in light of studies which suggest aging attenuates antioxidant signaling, particularly in response to exogenous stimuli. In this work, we addressed whether aging compromises the favorable phenotype of increased antioxidant activity and improved glucose homeostasis observed in younger Gclm null mice. We present data showing that under basal conditions and in response to cadmium exposure (2 mg/kg, dosed once via intraperitoneal injection), the phenotype previously described in young (<6 months) Gclm null mice persists into old age (24+ months). We also provide evidence that transcriptional activation of the Nrf2, AMPK, and PPARγ pathways underlie the favorable metabolic phenotype observed previously in young Gclm null mice.

GLIS1-3: emerging roles in reprogramming, stem and progenitor cell differentiation and maintenance.

Recent studies have provided evidence for a regulatory role of GLI-similar (GLIS) transcription factors in reprogramming, maintenance and differentiation of several stem and progenitor cell populations. GLIS1, in conjunction with several other reprogramming factors, was shown to markedly increase the efficiency of generating induced pluripotent stem cells (iPSC) from somatic cells. GLIS2 has been reported to contribute to the maintenance of the pluripotent state in hPSCs. In addition, GLIS2 has a function in regulating self-renewal of hematopoietic progenitors and megakaryocytic differentiation. GLIS3 plays a critical role during the development of several tissues. GLIS3 is able to promote reprogramming of human fibroblasts into retinal pigmented epithelial (RPE) cells. Moreover, GLIS3 is essential for spermatogonial stem cell renewal and spermatogonial progenitor cell differentiation. During pancreas development, GLIS3 protein is first detectable in bipotent pancreatic progenitors and pro-endocrine progenitors and plays a critical role in the generation of pancreatic beta cells. Here, we review the current status of the roles of GLIS proteins in the maintenance and differentiation of these different stem and progenitor cells.

Genetic determinants of susceptibility to silver nanoparticle-induced acute lung inflammation in mice.

Silver nanoparticles (AgNPs) are employed in a variety of consumer products; however, in vivo rodent studies indicate that AgNPs can cause lung inflammation and toxicity in a strain- and particle type-dependent manner, but mechanisms of susceptibility remain unclear. The aim of this study was to assess the variation in AgNP-induced lung inflammation and toxicity across multiple inbred mouse strains and to use genome-wide association (GWA) mapping to identify potential candidate susceptibility genes. Mice received doses of 0.25 mg/kg of either 20-nm citrate-coated AgNPs or citrate buffer using oropharyngeal aspiration. Neutrophils in bronchoalveolar lavage fluid (BALF) served as markers of inflammation. We found significant strain- and treatment-dependent variation in neutrophils in BALF. GWA mapping identified 10 significant single-nucleotide polymorphisms (false discovery rate, 15%) in 4 quantitative trait loci on mouse chromosomes 1, 4, 15, and 18, and Nedd4l (neural precursor cell expressed developmentally downregulated gene 4-like; chromosome 18), Ano6 (anocatmin 6; chromosome 15), and Rnf220 (Ring finger protein 220; chromosome 4) were considered candidate genes. Quantitative RT-PCR revealed significant inverse associations between mRNA levels of these genes and neutrophil influx. Nedd4l, Ano6, and Rnf220 are candidate susceptibility genes for AgNP-induced lung inflammation that warrant additional exploration in future studies.-Scoville, D. K., Botta, D., Galdanes, K., Schmuck, S. C., White, C. C., Stapleton, P. L., Bammler, T. K., MacDonald, J. W., Altemeier, W. A., Hernandez, M., Kleeberger, S. R., Chen, L.-C., Gordon, T., Kavanagh, T. J. Genetic determinants of susceptibility to silver nanoparticle-induced acute lung inflammation in mice.

Tricuspid leaflet repair: innovative solutions.

Tricuspid regurgitation (TR) represents a significant disease process and when severe, is associated with increased mortality. Recent guidelines support a more aggressive approach to tricuspid valve (TV) surgery, especially when encountered with left-sided valvular pathology. While annuloplasty has been the standard treatment for TR, it may not provide as effective or durable a repair compared to annuloplasty combined with TV repair techniques. Several of these approaches are discussed including bicuspidalization, anterior leaflet augmentation, edge to edge repair, neochords, leaflet resection and combined approaches. Although patient cohorts in most of the studies examining these techniques are small, the long-term durability of TV repair is significant.

Surgical Unroofing of Hemodynamically Significant Left Anterior Descending Myocardial Bridges.

BACKGROUND: Left anterior descending artery myocardial bridges (MBs) range from clinically insignificant incidental angiographic findings to a potential cause of sudden cardiac death. Within this spectrum, a group of patients with isolated, symptomatic, and hemodynamically significant MBs despite maximally tolerated medical therapy exist for whom the optimal treatment is controversial. We evaluated supraarterial myotomy, or surgical unroofing, of the left anterior descending MBs as an isolated procedure in these patients. METHODS: In 50 adult patients, we prospectively evaluated baseline clinical characteristics, risk factors, and medications for coronary artery disease, relevant diagnostic data (stress echocardiography, computed tomography angiography, stress coronary angiogram with dobutamine challenge for measurement of diastolic fractional flow reserve, and intravascular ultrasonography), and anginal symptoms using the Seattle Angina Questionnaire. These patients then underwent surgical unroofing of their left anterior descending artery MBs followed by readministration of the Seattle Angina Questionnaire at 6.6-month (range, 2 to 13) follow-up after surgery. RESULTS: Dramatic improvements were noted in physical limitation due to angina (52.0 versus 87.1, p < 0.001), anginal stability (29.6 versus 66.4, p < 0.001), anginal frequency (52.1 versus 84.7, p < 0.001), treatment satisfaction (76.1 versus 93.9, p < 0.001), and quality of life (25.0 versus 78.9, p < 0.001), all five dimensions of the Seattle Angina Questionnaire. There were no major complications or deaths. CONCLUSIONS: Surgical unroofing of carefully selected patients with MBs can be performed safely as an independent procedure with significant improvement in symptoms postoperatively. It is the optimal treatment for isolated, symptomatic, and hemodynamically significant MBs resistant to maximally tolerated medical therapy.

The pulmonary inflammatory response to multiwalled carbon nanotubes is influenced by gender and glutathione synthesis.

Inhalation of multiwalled carbon nanotubes (MWCNTs) during their manufacture or incorporation into various commercial products may cause lung inflammation, fibrosis, and oxidative stress in exposed workers. Some workers may be more susceptible to these effects because of differences in their ability to synthesize the major antioxidant and immune system modulator glutathione (GSH). Accordingly, in this study we examined the influence of GSH synthesis and gender on MWCNT-induced lung inflammation in C57BL/6 mice. GSH synthesis was impaired through genetic manipulation of Gclm, the modifier subunit of glutamate cysteine ligase, the rate-limiting enzyme in GSH synthesis. Twenty-four hours after aspirating 25µg of MWCNTs, all male mice developed neutrophilia in their lungs, regardless of Gclm genotype. However, female mice with moderate (Gclm heterozygous) and severe (Gclm null) GSH deficiencies developed significantly less neutrophilia. We found no indications of MWCNT-induced oxidative stress as reflected in the GSH content of lung tissue and epithelial lining fluid, 3-nitrotyrosine formation, or altered mRNA or protein expression of several redox-responsive enzymes. Our results indicate that GSH-deficient female mice are rendered uniquely susceptible to an attenuated neutrophil response. If the same effects occur in humans, GSH-deficient women manufacturing MWCNTs may be at greater risk for impaired neutrophil-dependent clearance of MWCNTs from the lung. In contrast, men may have effective neutrophil-dependent clearance, but may be at risk for lung neutrophilia regardless of their GSH levels.

A novel approach to ischemic mitral regurgitation (IMR).

Ischemic mitral regurgitation (IMR) is a complicated medical condition with varying degrees of coronary artery disease and mitral regurgitation (MR). The traditional surgical treatment option for those with indications for intervention is coronary artery bypass grafting (CABG) plus or minus mitral valve repair or replacement (MVR). Percutaneous coronary intervention, hybrid coronary revascularization (HCR), and conventional CABG are three techniques available to address coronary artery disease (CAD). Percutaneous edge-to-edge repair, minimally invasive, and traditional sternotomy are accepted approaches for the treatment of MR. When taken in combination, there are nine methods available to revascularize the myocardium and restore competency to the mitral valve. While most of these treatment options have not been studied in detail, they may offer novel solutions to a widely variable and complex IMR patient population. Thus, a comparative analysis including an examination of potential benefits and risks will be helpful and potentially allow for more patient-specific treatment strategies.

Susceptibility to quantum dot induced lung inflammation differs widely among the Collaborative Cross founder mouse strains.

Quantum dots (QDs) are engineered semiconductor nanoparticles with unique physicochemical properties that make them potentially useful in clinical, research and industrial settings. However, a growing body of evidence indicates that like other engineered nanomaterials, QDs have the potential to be respiratory hazards, especially in the context of the manufacture of QDs and products containing them, as well as exposures to consumers using these products. The overall goal of this study was to investigate the role of mouse strain in determining susceptibility to QD-induced pulmonary inflammation and toxicity. Male mice from 8 genetically diverse inbred strains (the Collaborative Cross founder strains) were exposed to CdSe-ZnS core-shell QDs stabilized with an amphiphilic polymer. QD treatment resulted in significant increases in the percentage of neutrophils and levels of cytokines present in bronchoalveolar lavage fluid (BALF) obtained from NOD/ShiLtJ and NZO/HlLtJ mice relative to their saline (Sal) treated controls. Cadmium measurements in lung tissue indicated strain-dependent differences in disposition of QDs in the lung. Total glutathione levels in lung tissue were significantly correlated with percent neutrophils in BALF as well as with lung tissue Cd levels. Our findings indicate that QD-induced acute lung inflammation is mouse strain dependent, that it is heritable, and that the choice of mouse strain is an important consideration in planning QD toxicity studies. These data also suggest that formal genetic analyses using additional strains or recombinant inbred strains from these mice could be useful for discovering potential QD-induced inflammation susceptibility loci.

MLL3 and MLL4 Methyltransferases Bind to the MAFA and MAFB Transcription Factors to Regulate Islet ß-Cell Function.

Insulin produced by islet ß-cells plays a critical role in glucose homeostasis, with type 1 and type 2 diabetes both resulting from inactivation and/or loss of this cell population. Islet-enriched transcription factors regulate ß-cell formation and function, yet little is known about the molecules recruited to mediate control. An unbiased in-cell biochemical and mass spectrometry strategy was used to isolate MafA transcription factor-binding proteins. Among the many coregulators identified were all of the subunits of the mixed-lineage leukemia 3 (Mll3) and 4 (Mll4) complexes, with histone 3 lysine 4 methyltransferases strongly associated with gene activation. MafA was bound to the ∼1.5 MDa Mll3 and Mll4 complexes in size-fractionated ß-cell extracts. Likewise, closely related human MAFB, which is important to ß-cell formation and coproduced with MAFA in adult human islet ß-cells, bound MLL3 and MLL4 complexes. Knockdown of NCOA6, a core subunit of these methyltransferases, reduced expression of a subset of MAFA and MAFB target genes in mouse and human ß-cell lines. In contrast, a broader effect on MafA/MafB gene activation was observed in mice lacking NCoA6 in islet ß-cells. We propose that MLL3 and MLL4 are broadly required for controlling MAFA and MAFB transactivation during development and postnatally.

In vivo approaches to assessing the toxicity of quantum dots.

The small size and heavy metal composition of quantum dots (QDs) combined with their growing consumer product and biomedical research applications have generated concern over their safety. In an occupational setting where QD-enabled products are being manufactured, inhalation is a likely route of exposure. Since current research indicates that QDs could cause inflammation and toxicity in the respiratory tract, it is important that a variety of methods be available to further characterize this potential respiratory hazard. This chapter focuses primarily on in vivo methods for modeling the inhalation and assessing the pulmonary toxicity of QDs.

Isolation and characterization of intestinal stem cells based on surface marker combinations and colony-formation assay.

BACKGROUND & AIMS: Identification of intestinal stem cells (ISCs) has relied heavily on the use of transgenic reporters in mice, but this approach is limited by mosaic expression patterns and difficult to directly apply to human tissues. We sought to identify reliable surface markers of ISCs and establish a robust functional assay to characterize ISCs from mouse and human tissues. METHODS: We used immunohistochemistry, real-time reverse-transcription polymerase chain reaction, and fluorescence-activated cell sorting (FACS) to analyze intestinal epithelial cells isolated from mouse and human intestinal tissues. We compared different combinations of surface markers among ISCs isolated based on expression of Lgr5-green fluorescent protein. We developed a culture protocol to facilitate the identification of functional ISCs from mice and then tested the assay with human intestinal crypts and putative ISCs. RESULTS: CD44(+)CD24(lo)CD166(+) cells, isolated by FACS from mouse small intestine and colon, expressed high levels of stem cell-associated genes. Transit-amplifying cells and progenitor cells were then excluded based on expression of GRP78 or c-Kit. CD44(+)CD24(lo)CD166(+) GRP78(lo/-) putative stem cells from mouse small intestine included Lgr5-GFP(hi) and Lgr5-GFP(med/lo) cells. Incubation of these cells with the GSK inhibitor CHIR99021 and the E-cadherin stabilizer Thiazovivin resulted in colony formation by 25% to 30% of single-sorted ISCs. CONCLUSIONS: We developed a culture protocol to identify putative ISCs from mouse and human tissues based on cell surface markers. CD44(+)CD24(lo)CD166(+), GRP78(lo/-), and c-Kit(-) facilitated identification of putative stem cells from the mouse small intestine and colon, respectively. CD44(+)CD24(-/lo)CD166(+) also identified putative human ISCs. These findings will facilitate functional studies of mouse and human ISCs.

Current view: intestinal stem cells and signaling.

Studies using mice have yielded significant amounts of information regarding signaling pathways, such as Wnt, bone morphogenic protein, PtdIns(3,4,5) kinase, and Notch, involved in intestinal development and homeostasis, including stem cell regulation and lineage specification and maturation. However, attempts to model signals definitively that control intestinal stem cells have been difficult because of a long-standing and recently reenergized debate surrounding their location. Although crypt-based columnar cells have been recently shown to display self-renewal and multipotential capacity, a large body of evidence supports long-term label-retaining cells, located on average at the +4 position just above the Paneth cells, as putative stem cells. Herein, we propose that both these cell types represent true intestinal stem cells maintained in different states (quiescent vs actively cycling), presumably via interactions with different microenvironments. Finally, we review current findings regarding the roles of Wnt, bone morphogenic protein, PtdIns(3,4,5) kinase, and Notch pathways within the intestine.

BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-beta-catenin signaling.

In humans, mutations in BMPR1A, SMAD4 and PTEN are responsible for juvenile polyposis syndrome, juvenile intestinal polyposis and Cowden disease, respectively. The development of polyposis is a common feature of these diseases, suggesting that there is an association between BMP and PTEN pathways. The mechanistic link between BMP and PTEN pathways and the related etiology of juvenile polyposis is unresolved. Here we show that conditional inactivation of Bmpr1a in mice disturbs homeostasis of intestinal epithelial regeneration with an expansion of the stem and progenitor cell populations, eventually leading to intestinal polyposis resembling human juvenile polyposis syndrome. We show that BMP signaling suppresses Wnt signaling to ensure a balanced control of stem cell self-renewal. Mechanistically, PTEN, through phosphatidylinosital-3 kinase-Akt, mediates the convergence of the BMP and Wnt pathways on control of beta-catenin. Thus, BMP signaling may control the duplication of intestinal stem cells, thereby preventing crypt fission and the subsequent increase in crypt number.