Individual trabecula segmentation validation in first- and second-generation high-resolution peripheral computed tomography compared to micro-computed tomography in the distal radius and tibia.

High-resolution peripheral quantitative computed tomography (HR-pQCT) has been used for in vivo 3D visualization of trabecular microstructure. Second-generation HR-pQCT (HR-pQCT II) has been shown to have good agreement with first generation HR-pQCT (HR-pQCT I). Advanced Individual Trabecula Segmentation (ITS) decomposes the trabecula network into individual plates and rods. ITS based on HR-pQCT I showed a strong correlation to ITS based on micro-computed tomography (µCT) and identified trabecular changes in metabolic bone diseases. ITS based on HR-pQCT II has new potential because of the enhanced resolution but has yet to be validated. The objective of this study was to assess the agreement between ITS based on HR-pQCT I, HR-pQCT II, and µCT to assess the capability of ITS on HR-pQCT images as a tool for studying bone structure. Freshly frozen tibia and radius bones were scanned in the distal region using HR-pQCT I at 82 µm, HR-pQCT II at 60.7 µm, and µCT at 37 µm. Images were registered, binarized, and ITS analysis was performed. Bone volume fraction (pBV/TV, rBV/TV), number density (pTb.N, rTb.N), thickness (pTb.Th, rTb.Th), and plate-to-rod (PR) ratio (pBV/rBV) of trabecular plates and rods were obtained. Paired Student's t-tests with post hoc Bonferroni analysis were used to examine the differences. Linear regression was used to determine the correlation coefficient. The HR-pQCT I parameters were different from the µCT measurements. The HR-pQCT II parameters were different from the µCT measurements except for rTb.N, and the HR-pQCT I parameters were different from the HR-pQCT II measurements except for pTb.Th. The strong correlation between HR-pQCT II and µCT microstructural analysis (R2 = 0.55-0.94) suggests that HR-pQCT II can be used to assess changes in plate and rod microstructure and that values from HR-pQCT I can be corrected.

Directed differentiation of human iPSCs to functional ovarian granulosa-like cells via transcription factor overexpression.

An in vitro model of human ovarian follicles would greatly benefit the study of female reproduction. Ovarian development requires the combination of germ cells and several types of somatic cells. Among these, granulosa cells play a key role in follicle formation and support for oogenesis. Whereas efficient protocols exist for generating human primordial germ cell-like cells (hPGCLCs) from human induced pluripotent stem cells (hiPSCs), a method of generating granulosa cells has been elusive. Here, we report that simultaneous overexpression of two transcription factors (TFs) can direct the differentiation of hiPSCs to granulosa-like cells. We elucidate the regulatory effects of several granulosa-related TFs and establish that overexpression of NR5A1 and either RUNX1 or RUNX2 is sufficient to generate granulosa-like cells. Our granulosa-like cells have transcriptomes similar to human fetal ovarian cells and recapitulate key ovarian phenotypes including follicle formation and steroidogenesis. When aggregated with hPGCLCs, our cells form ovary-like organoids (ovaroids) and support hPGCLC development from the premigratory to the gonadal stage as measured by induction of DAZL expression. This model system will provide unique opportunities for studying human ovarian biology and may enable the development of therapies for female reproductive health.

Ovaries are responsible for forming the eggs humans and other mammals need to reproduce. Once mature, the egg cell is released into the fallopian tube where it can be potentially fertilized by a sperm. Despite their crucial role, how eggs are made in the ovary is poorly understood. This is because ovaries are hard to access, making it difficult to conduct experiments on them. To overcome this, researchers have built artificial ovaries in the laboratory using stem cells from the embryos of mice which can develop into all cell types in the adult body. By culturing these embryonic stem cells under special conditions, researchers can convert them in to the two main cell types of the developing ovary: germ cells which go on to form eggs, and granulosa cells which help eggs grow and mature. The resulting lab-grown ovary can make eggs that produce live mice when fertilized. This approach has also been applied to human induced pluripotent stem cells (iPSCs), adult human cells which have been reprogrammed to a stem-like state. While this has produced human germ cells, generating human granulosa cells has been more challenging. Here, Pierson Smela, Kramme et al. show that activating a specific set of transcription factors (proteins that switch genes on or off) in iPSCs can make them transition to granulosa cells. First, the team tested random combinations of 35 transcription factors which, based on previous literature and genetic data, were likely to play a role in the formation of granulosa cells. This led to the identification of a small number of factors that caused the human iPSCs to develop features and carry out roles seen in mature granulosa cells; this includes producing an important reproductive hormone and supporting the maturation of germ cells. Pierson Smela, Kramme et al. found that growing these granulosa-like cells together with germ cells (also generated via iPSCs) resulted in structures similar to ovarian follicles which help eggs develop. These findings could help researchers build stable systems for studying how granulosa cells behave in human ovaries. This could lead to new insights about reproductive health.

Intron-Retention Neoantigen Load Predicts Favorable Prognosis in Pancreatic Cancer.

PURPOSE: High tumor mutation burden (TMB) in many cancer types is associated with the production of tumor-specific neoantigens, a favorable outcome and response to immune checkpoint blockade (ICB) therapy. Besides mutation-derived neoantigens, aberrant intron retention also produces tumor neopeptides that could trigger an immune response. The relationship between intron-retention-derived tumor neoantigens (IR-neoAg) and clinical outcomes in pancreatic cancer remains uncertain. Here, we quantify IR-neoAg in pancreatic cancer and evaluate whether IR-neoAg load might serve as a biomarker for selecting patients who may benefit from ICB therapy. METHODS: We developed a computational approach to estimate patient-specific IR-neoAg load from transcriptome data available in The Cancer Genome Atlas pancreatic cancer cohort. Associations between IR-neoAg load and patient overall survival were evaluated using Kaplan-Meier estimates and Cox regression. Differential expression of immune checkpoint and HLA-I genes was evaluated in tumors with high IR-neoAg load. RESULTS: High IR-neoAg load predicted better overall survival in pancreatic cancer, although no association was found for TMB. IR-neoAg load remained a significant prognostic factor after adjusting for patient age, sex, tumor stage and grade, and TMB. Moreover, pancreatic tumors with both high IR-neoAg load and high HLA-I gene expression had similar gene expression profiles as other tumor types that showed response to anti-programmed cell death protein 1 therapy. CONCLUSION: IR-neoAg load is associated with favorable survival in pancreatic cancer. These findings provide strong evidence for considering IR-neoAgs when selecting patients who might benefit from ICB therapy.

MAL2 drives immune evasion in breast cancer by suppressing tumor antigen presentation.

Immune evasion is a pivotal event in tumor progression. To eliminate human cancer cells, current immune checkpoint therapy is set to boost CD8+ T cell-mediated cytotoxicity. However, this action is eventually dependent on the efficient recognition of tumor-specific antigens via T cell receptors. One primary mechanism by which tumor cells evade immune surveillance is to downregulate their antigen presentation. Little progress has been made toward harnessing potential therapeutic targets for enhancing antigen presentation on the tumor cell. Here, we identified MAL2 as a key player that determines the turnover of the antigen-loaded MHC-I complex and reduces the antigen presentation on tumor cells. MAL2 promotes the endocytosis of tumor antigens via direct interaction with the MHC-I complex and endosome-associated RAB proteins. In preclinical models, depletion of MAL2 in breast tumor cells profoundly enhanced the cytotoxicity of tumor-infiltrating CD8+ T cells and suppressed breast tumor growth, suggesting that MAL2 is a potential therapeutic target for breast cancer immunotherapy.

Long-term Bone Loss and Deterioration of Microarchitecture After Gastric Bypass in African American and Latina Women.

CONTEXT: The prevalence of obesity is burgeoning among African American and Latina women; however, few studies investigating the skeletal effects of bariatric surgery have focused on these groups. OBJECTIVE: To investigate long-term skeletal changes following Roux-en-Y gastric bypass (RYGB) in African American and Latina women. DESIGN: Four-year prospective cohort study. PATIENTS: African American and Latina women presenting for RYGB (n = 17, mean age 44, body mass index 44 kg/m2) were followed annually for 4 years postoperatively. MAIN OUTCOME MEASURES: Dual-energy x-ray absorptiometry (DXA) measured areal bone mineral density (aBMD) at the spine, hip, and forearm, and body composition. High-resolution peripheral quantitative computed tomography measured volumetric bone mineral density (vBMD) and microarchitecture. Individual trabecula segmentation-based morphological analysis assessed trabecular morphology and connectivity. RESULTS: Baseline DXA Z-Scores were normal. Weight decreased ~30% at Year 1, then stabilized. Parathyroid hormone (PTH) increased by 50% and 25-hydroxyvitamin D was stable. By Year 4, aBMD had declined at all sites, most substantially in the hip. There was significant, progressive loss of cortical and trabecular vBMD, deterioration of microarchitecture, and increased cortical porosity at both the radius and tibia over 4 years. There was loss of trabecular plates, loss of axially aligned trabeculae, and decreased trabecular connectivity. Whole bone stiffness and failure load declined. Risk factors for bone loss included greater weight loss, rise in PTH, and older age. CONCLUSIONS: African American and Latina women had substantial and progressive bone loss, deterioration of microarchitecture, and trabecular morphology following RYGB. Further studies are critical to understand the long-term skeletal consequences of bariatric surgery in this population.

Sesn3 deficiency promotes carcinogen-induced hepatocellular carcinoma via regulation of the hedgehog pathway.

Sestrin 3 (Sesn3) belongs to a small protein family that has been implicated in multiple biological processes including anti-oxidative stress, anti-aging, cell signaling, and metabolic homeostasis. However, the role of Sesn3 in hepatocellular carcinoma (HCC) remains unclear. Here we generated a Sesn3 knockout mouse model and induced HCC development by a combination of a single dose of diethylnitrosamine and chronic feeding of a choline deficient-high fat diet. After 6 months of the dietary treatment, Sesn3 knockout mice developed more severe HCC with higher levels of alpha-fetoprotein, arginase 1, and cytokeratin 19, but also higher metastatic rates than wild-type mice. Histological analysis revealed elevated extracellular matrix and cancer stem cell markers including Acta2, Cd44, and Cd133. Signaling analysis showed activated IL6-Stat3 and Akt pathways. Biochemical and microscopic analyses uncovered a novel inhibitory regulation of Gli2, a downstream transcription factor of the hedgehog signaling, by Sesn3. Two of the Gli2-regulated genes - Pdgfrb and Cd44 were upregulated in the Sesn3-deficient liver tissue. In conclusion, our data suggest that Sesn3 plays a critical tumor suppressor role in the liver partly through the inhibition of the hedgehog signaling.

Trabecular bone score (TBS)--a novel method to evaluate bone microarchitectural texture in patients with primary hyperparathyroidism.

CONTEXT: In the milder form of primary hyperparathyroidism (PHPT), cancellous bone, represented by areal bone mineral density at the lumbar spine by dual-energy x-ray absorptiometry (DXA), is preserved. This finding is in contrast to high-resolution peripheral quantitative computed tomography (HRpQCT) results of abnormal trabecular microstructure and epidemiological evidence for increased overall fracture risk in PHPT. Because DXA does not directly measure trabecular bone and HRpQCT is not widely available, we used trabecular bone score (TBS), a novel gray-level textural analysis applied to spine DXA images, to estimate indirectly trabecular microarchitecture. OBJECTIVE: The purpose of this study was to assess TBS from spine DXA images in relation to HRpQCT indices and bone stiffness in radius and tibia in PHPT. DESIGN AND SETTING: This was a cross-sectional study conducted in a referral center. PATIENTS: Participants were 22 postmenopausal women with PHPT. MAIN OUTCOME MEASURES: Outcomes measured were areal bone mineral density by DXA, TBS indices derived from DXA images, HRpQCT standard measures, and bone stiffness assessed by finite element analysis at distal radius and tibia. RESULTS: TBS in PHPT was low at 1.24, representing abnormal trabecular microstructure (normal ≥1.35). TBS was correlated with whole bone stiffness and all HRpQCT indices, except for trabecular thickness and trabecular stiffness at the radius. At the tibia, correlations were observed between TBS and volumetric densities, cortical thickness, trabecular bone volume, and whole bone stiffness. TBS correlated with all indices of trabecular microarchitecture, except trabecular thickness, after adjustment for body weight. CONCLUSION: TBS, a measurement technology readily available by DXA, shows promise in the clinical assessment of trabecular microstructure in PHPT.