Compartmentalized metabolism supports midgestation mammalian development.

Mammalian embryogenesis requires rapid growth and proper metabolic regulation1. Midgestation features increasing oxygen and nutrient availability concomitant with fetal organ development2,3. Understanding how metabolism supports development requires approaches to observe metabolism directly in model organisms in utero. Here we used isotope tracing and metabolomics to identify evolving metabolic programmes in the placenta and embryo during midgestation in mice. These tissues differ metabolically throughout midgestation, but we pinpointed gestational days (GD) 10.5-11.5 as a transition period for both placenta and embryo. Isotope tracing revealed differences in carbohydrate metabolism between the tissues and rapid glucose-dependent purine synthesis, especially in the embryo. Glucose's contribution to the tricarboxylic acid (TCA) cycle rises throughout midgestation in the embryo but not in the placenta. By GD12.5, compartmentalized metabolic programmes are apparent within the embryo, including different nutrient contributions to the TCA cycle in different organs. To contextualize developmental anomalies associated with Mendelian metabolic defects, we analysed mice deficient in LIPT1, the enzyme that activates 2-ketoacid dehydrogenases related to the TCA cycle4,5. LIPT1 deficiency suppresses TCA cycle metabolism during the GD10.5-GD11.5 transition, perturbs brain, heart and erythrocyte development and leads to embryonic demise by GD11.5. These data document individualized metabolic programmes in developing organs in utero.

Mutant IDH1 regulates the tumor-associated immune system in gliomas.

Gliomas harboring mutations in isocitrate dehydrogenase 1/2 (IDH1/2) have the CpG island methylator phenotype (CIMP) and significantly longer patient survival time than wild-type IDH1/2 (wtIDH1/2) tumors. Although there are many factors underlying the differences in survival between these two tumor types, immune-related differences in cell content are potentially important contributors. In order to investigate the role of IDH mutations in immune response, we created a syngeneic pair mouse model for mutant IDH1 (muIDH1) and wtIDH1 gliomas and demonstrated that muIDH1 mice showed many molecular and clinical similarities to muIDH1 human gliomas, including a 100-fold higher concentration of 2-hydroxygluratate (2-HG), longer survival time, and higher CpG methylation compared with wtIDH1. Also, we showed that IDH1 mutations caused down-regulation of leukocyte chemotaxis, resulting in repression of the tumor-associated immune system. Given that significant infiltration of immune cells such as macrophages, microglia, monocytes, and neutrophils is linked to poor prognosis in many cancer types, these reduced immune infiltrates in muIDH1 glioma tumors may contribute in part to the differences in aggressiveness of the two glioma types.

A Feedback Loop between TGF-ß1 and ATG5 Mediated by miR-122-5p Regulates Fibrosis and EMT in Human Trabecular Meshwork Cells.

Autophagy is a cell's evolutionary conserved process for degrading and recycling cellular proteins and removing damaged organelles. There has been an increasing interest in identifying the basic cellular mechanism of autophagy and its implications in health and illness during the last decade. Many proteinopathies such as Alzheimer's and Huntington's disease are reported to be associated with impaired autophagy. The functional significance of autophagy in exfoliation syndrome/exfoliation glaucoma (XFS/XFG), remains unknown though it is presumed to be impaired autophagy to be responsible for the aggregopathy characteristic of this disease. In the current study we have shown that autophagy or ATG5 is enhanced in response to TGF-ß1 in human trabecular meshwork (HTM) cells and TGF-ß1 induced autophagy is necessary for increased expression of profibrotic proteins and epithelial to mesenchymal (EMT) through Smad3 that lead to aggregopathy. Inhibition of ATG5 by siRNA mediated knockdown reduced profibrotic and EMT markers and increased protein aggregates in the presence of TGF-ß1 stimulation. The miR-122-5p, which was increased upon TGF exposure, was also reduced upon ATG5 inhibition. We thus conclude that TGF-ß1 induces autophagy in primary HTM cells and a positive feedback loop exists between TGF-ß1 and ATG5 that regulated TGF downstream effects mainly mediated by Smad3 signaling with miR-122-5p also playing a role.

Development of a Medical Complexity Score for Pediatric Aerodigestive Patients.

OBJECTIVE: To develop a complexity scoring system to characterize the diverse population served in pediatric aerodigestive clinics and help predict their treatment outcomes. STUDY DESIGN: A 7-point medical complexity score was developed through an iterative group consensus of relative stakeholders to capture the spectrum of comorbidities among the aerodigestive population. One point was assigned for each comorbid diagnosis in the following categories: airway anomaly, neurologic, cardiac, respiratory, gastrointestinal, genetic diagnoses, and prematurity. A retrospective chart review was conducted of patients seen in the aerodigestive clinic who had ≥2 visits between 2017 and 2021. The predictive value of the complexity score for the selected outcome of feeding progression among children with dysphagia was analyzed with univariate and multivariable logistic regression. RESULTS: We analyzed 234 patients with complexity scores assigned, showing a normal distribution (Shapiro Wilk P = .406) of the scores 1-7 (median, 4; mean, 3.50 ± 1.47). In children with dysphagia, there was waning success in the improvement of oral feeding with increasing complexity scores (OR, 0.66; 95% CI, 0.51-0.84; P = .001). Tube-fed children with higher complexity scores were incrementally less likely to achieve full oral diet (OR, 0.60; 95% CI, 0.40-0.89; P = .01). On multivariable analysis, neurologic comorbidity (OR, 0.26; P < .001) and airway malformation (OR, 0.35; P = .01) were associated with a decreased likelihood to improve in oral feeding. CONCLUSIONS: We propose a novel complexity score for the pediatric aerodigestive population that is easy to use, successfully stratifies diverse presentations, and shows promise as a predictive tool to assist in counseling and resource use.

Non-linguistic auditory speech processing.

OBJECTIVES: A method for testing auditory processing of non-linguistic speech-like stimuli was developed and evaluated. DESIGN: Monosyllabic words were temporally reversed and distorted. Stimuli were matched for spectrum and level. Listeners discriminated between distorted and undistorted stimuli. STUDY SAMPLE: Three groups were tested. The Normal group was comprised of 12 normal-hearing participants. The Senior group was comprised of 12 seniors. The Hearing Loss group was comprised of 12 participants with thresholds of at least 35 dB HL at one or more frequencies. RESULTS: The Senior group scored lower than the Normal group, and the Hearing Loss group scored lower than the Senior group. Scores for forward compressed speech were slightly higher than backward compressed speech but the difference was not statistically significant. Retest scores were slightly higher than scores on the first test, but the difference was not statistically significant. CONCLUSIONS: Large differences in discrimination of distorted speech were observed among the three groups. Age and hearing loss separately affected performance. The depressed performance of the Senior group may be a result of "hidden hearing loss" that is attributed to cochlear synaptopathy. The backward-distorted speech task may be a useful non-linguistic test of speech processing that is language independent.

Alternate Causes for Pathogenesis of Exfoliation Glaucoma, a Multifactorial Elastotic Disorder: A Literature Review.

Exfoliation glaucoma (XFG) is the most recognizable form of secondary open-angle glaucoma associated with a high risk of blindness. This disease is characterized by white flaky granular deposits in the anterior chamber that leads to the elevation of intraocular pressure (IOP) and subsequent glaucomatous optic nerve damage. Conventionally, XFG is known to respond poorly to medical therapy, and surgical intervention is the only management option in most cases. Various genetic and nongenetic factors are known to be linked to the development of XFG. Despite decades of research on the genetic factors in exfoliation syndrome (XFS) by study groups and global consortia involving different ethnic populations, the pathogenesis of XFS and the mechanism of onset of glaucoma still remains an unsolved mystery. The key lies in understanding how the function of a gene (or set of genes) is altered by environmental triggers, along with other molecular events that underlie the key disease attributes, namely, oxidative stress and the disruption of the blood-aqueous barrier (BAB). It remains a challenge to evolve a theory encompassing all factions of molecular events occurring independently or parallelly that determine the disease manifestation (phenotype) or the stage of the disease in the eye (or in any tissue) in exfoliation. Our enhanced understanding of the underlying molecular pathophysiology of XFG, beyond the known genes or polymorphisms involved in the disease, will lead to improved diagnosis and management and the ability to recognize how the environment influences these key events that lead to the disease phenotype or disease progression. This review summarizes the recent observations and discoveries of four key factors that may hold the answers to the non-lysyl oxidase-like 1 (LOXL1) mechanisms behind XFG pathogenesis, namely, the epigenetic factor miRNA, disordered autophagy along with the potential involvement of mitochondrial mutations, and a compromised aqueous-blood barrier.

Regulation of PRMT5-MDM4 axis is critical in the response to CDK4/6 inhibitors in melanoma.

Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are an established treatment in estrogen receptor-positive breast cancer and are currently in clinical development in melanoma, a tumor that exhibits high rates of CDK4 activation. We analyzed melanoma cells with acquired resistance to the CDK4/6 inhibitor palbociclib and demonstrate that the activity of PRMT5, a protein arginine methyltransferase and indirect target of CDK4, is essential for CDK4/6 inhibitor sensitivity. By indirectly suppressing PRMT5 activity, palbociclib alters the pre-mRNA splicing of MDM4, a negative regulator of p53, leading to decreased MDM4 protein expression and subsequent p53 activation. In turn, p53 induces p21, leading to inhibition of CDK2, the main kinase substituting for CDK4/6 and a key driver of resistance to palbociclib. Loss of the ability of palbociclib to regulate the PRMT5-MDM4 axis leads to resistance. Importantly, combining palbociclib with the PRMT5 inhibitor GSK3326595 enhances the efficacy of palbociclib in treating naive and resistant models and also delays the emergence of resistance. Our studies have uncovered a mechanism of action of CDK4/6 inhibitors in regulating the MDM4 oncogene and the tumor suppressor, p53. Furthermore, we have established that palbociclib inhibition of the PRMT5-MDM4 axis is essential for robust melanoma cell sensitivity and provide preclinical evidence that coinhibition of CDK4/6 and PRMT5 is an effective and well-tolerated therapeutic strategy. Overall, our data provide a strong rationale for further investigation of novel combinations of CDK4/6 and PRMT5 inhibitors, not only in melanoma but other tumor types, including breast, pancreatic, and esophageal carcinoma.

In vivo efficacy of decitabine as a natural killer cell-mediated immunotherapy against isocitrate dehydrogenase mutant gliomas.

OBJECTIVE: Isocitrate dehydrogenase (IDH) mutations are found in more than 80% of low-grade gliomas and in the majority of secondary glioblastomas. IDH mutation (IDHmut) leads to aberrant production of an oncogenic metabolite that promotes epigenetic dysregulation by inducing hypermethylation to suppress transcription of various tumor suppressor genes. Hypermethylation in IDHmut gliomas leads to transcriptional repression of NKG2D ligands, especially UL16-binding protein (ULBP)-1 and ULBP-3, and subsequent evasion of natural killer (NK) cell-mediated lysis. The demethylating agent 5-aza-2'deoxycytodine (decitabine [DAC]) is a DNA methyltransferase 1 inhibitor that prevents hypermethylation and is capable of restoring NKG2D ligand expression in IDHmut gliomas to resensitize them to NK cells. Given its capacity for sustained epigenetic reprogramming, the authors hypothesized that DCA would be an effective immunotherapeutic agent in treating IDHmut gliomas in an NK cell-dependent manner by upregulating epigenetically repressed activating NKG2D ligands in IDHmut tumors. In this study, the authors sought to use a glioma stem cell, preclinical animal model to determine the efficacy of DAC in IDHmut and IDH wild-type (IDHwt) tumors, and to characterize whether the activity of DAC in gliomas is dependent on NK cell function. METHODS: Xenograft models of IDHwt and IDHmut gliomas were established in athymic-nude mice. When tumors were grossly visible and palpable, mice were treated with either DCA or dimethylsulfoxide intraperitoneally every 7 days. Tumor sizes were measured every 2 to 3 days. After the animals were euthanized, xenografts were harvested and analyzed for the following: tumor expression of NKG2D ligands, tumor susceptibility to human and murine NK cells, immunohistochemistry for NK infiltration, and tumor-infiltrating lymphocyte characterization. RESULTS: DAC significantly inhibited the growth of IDHmut xenografts in the athymic nude mice. This effect was abrogated with NK cell depletion. Ex vivo analysis of tumor cells from harvested xenografts confirmed that DAC increased NKG2D ligand ULBP-1 and ULBP-3 expressions, and enhanced susceptibility to lysis of both human and murine IDHmut glial cells with corresponding NK cells. Immunohistochemical analysis of the xenografts indicated that DCA-treated IDHmut gliomas had a greater level of NK infiltration into the tumor compared with the negative control. Finally, DCA radically altered the tumor-infiltrating lymphocyte landscape of IDHmut glioma xenografts by increasing NK cells, dendritic cells, and M1 macrophages, while decreasing suppressive monocyte infiltration. CONCLUSIONS: DCA displayed novel immunotherapeutic functions in IDHmut gliomas. This effect was critically dependent on NK cells. Additionally, DCA significantly altered the tumor immune landscape in IDHmut gliomas from suppressive to proinflammatory.

Adenosine receptors regulate exosome production.

Exosomes, small-sized extracellular vesicles, carry components of the purinergic pathway. The production by cells of exosomes carrying this pathway remains poorly understood. Here, we asked whether type 1, 2A, or 2B adenosine receptors (A1Rs, A2ARs, and A2BRs, respectively) expressed by producer cells are involved in regulating exosome production. Preglomerular vascular smooth muscle cells (PGVSMCs) were isolated from wildtype, A1R-/-, A2AR-/-, and A2BR-/- rats, and exosome production was quantified under normal or metabolic stress conditions. Exosome production was also measured in various cancer cells treated with pharmacologic agonists/antagonists of A1Rs, A2ARs, and A2BRs in the presence or absence of metabolic stress or cisplatin. Functional activity of exosomes was determined in Jurkat cell apoptosis assays. In PGVSMCs, A1R and A2AR constrained exosome production under normal conditions (p = 0.0297; p = 0.0409, respectively), and A1R, A2AR, and A2BR constrained exosome production under metabolic stress conditions. Exosome production from cancer cells was reduced (p = 0.0028) by the selective A2AR agonist CGS 21680. These exosomes induced higher levels of Jurkat apoptosis than exosomes from untreated cells or cells treated with A1R and A2BR agonists (p = 0.0474). The selective A2AR antagonist SCH 442416 stimulated exosome production under metabolic stress or cisplatin treatment, whereas the selective A2BR antagonist MRS 1754 reduced exosome production. Our findings indicate that A2ARs suppress exosome release in all cell types examined, whereas effects of A1Rs and A2BRs are dependent on cell type and conditions. Pharmacologic targeting of cancer with A2AR antagonists may inadvertently increase exosome production from tumor cells.