Quantitative diffusion imaging and genotype-by-sex interactions in a rat model of Alexander disease.

PURPOSE: The clinical diagnosis and classification of Alexander disease (AxD) relies in part on qualitative neuroimaging biomarkers; however, these biomarkers fail to distinguish and discriminate different subtypes of AxD, especially in the presence of overlap in clinical symptoms. To address this gap in knowledge, we applied neurite orientation dispersion and density imaging (NODDI) to an innovative CRISPR-Cas9 rat genetic model of AxD to gain quantitative insights into the neural substrates and brain microstructural changes seen in AxD and to potentially identify novel quantitative NODDI biomarkers of AxD. METHODS: Multi-shell DWI of age- and sex-matched AxD and wild-type Sprague Dawley rats (n = 6 per sex per genotype) was performed and DTI and NODDI measures calculated. A 3 × 2 × 2 analysis of variance model was used to determine the effect of genotype, biological sex, and laterality on quantitative measures of DTI and NODDI across regions of interest implicated in AxD. RESULTS: There is a significant effect of genotype in the amygdala, hippocampus, neocortex, and thalamus in measures of both DTI and NODDI brain microstructure. A genotype by biological sex interaction was identified in DTI and NODDI measures in the corpus callosum, hippocampus, and neocortex. CONCLUSION: We present the first application of NODDI to the study of AxD using a rat genetic model of AxD. Our analysis identifies alterations in NODDI and DTI measures to large white matter tracts and subcortical gray nuclei. We further identified genotype by sex interactions, suggesting a possible role for biological sex in the neuropathogenesis of AxD.

21-Gene Recurrence Score and Survival Outcomes in the Phase III Multicenter TAILORx Clinical Trial.

BACKGROUND: Recurrence score (RS) based on a 21-gene genomic assay is frequently used to estimate risk of distant recurrence for choice of adjuvant chemotherapy in breast cancer. It remains unclear whether RS is an independent prognostic factor for breast cancer-specific survival (BCSS) and overall survival (OS) in the TAILORx trial population. METHODS: We evaluated the association of RS with BCSS and OS plus recurrence-free interval (RFI) and invasive disease-free survival (DFS) using multivariable Cox proportional hazards regression analysis, adjusting for clinicopathologic measures, in 8,916 patients with hormone receptor-positive, HER2-negative, node-negative breast cancer. Likelihood ratio (LR) test was used to assess the relative amount of prognostic information provided by RS to BCSS, OS, RFI, and DFS, comparatively. RESULTS: Event rates for BCSS, OS, RFI, and DFS were 1.7%, 5.2%, 5.6%, and 12.6%, respectively, by up to 11.6 years of follow-up. Compared with low-range RS (0-10), patients with midrange (11-25) and high-range (26-100) RS had inferior BCSS (adjusted hazard ratio [aHR], 5.12 [95% CI, 2.09-16.92] and 8.03 [95% CI, 2.91-28.47], respectively) and RFI (aHR, 1.68 [95% CI, 1.23-2.36] and 3.05 [95% CI, 2.02-4.67], respectively), independent of clinicopathologic factors. High-range score was associated with an increased risk of DFS (aHR, 1.56 [95% CI, 1.20-2.04]) but not significantly associated with OS (aHR, 1.44 [95% CI, 0.95-2.18]). Midrange score was associated with neither DFS (aHR, 1.15 [95% CI, 0.96-1.38]) nor OS (HR 1.14 [95% CI, 0.87-1.52]). LR-χ2 values were 83.0 and 65.1 for RFI and BCSS, respectively, and 17.5 and 33.6 for OS and DFS, respectively (P<.0001). CONCLUSIONS: RS is an independent measure for BCSS and recurrence prognoses relative to OS in early-stage breast cancer. It carries more prognostic information for breast cancer-specific outcomes.

Histoplasty Modification of the Tumor Microenvironment in a Murine Preclinical Model of Breast Cancer.

PURPOSE: To develop a noninvasive therapeutic approach able to alter the biophysical organization and physiology of the extracellular matrix (ECM) in breast cancer. MATERIALS AND METHODS: In a 4T1 murine model of breast cancer, histoplasty treatment with a proprietary 700-kHz multielement therapy transducer using a coaxially aligned ultrasound (US) imaging probe was used to target the center of an ex vivo tumor and deliver subablative acoustic energy. Tumor collagen morphology was qualitatively evaluated before and after histoplasty with second harmonic generation. Separately, mice bearing bilateral 4T1 tumors (n = 4; total tumors = 8) were intravenously injected with liposomal doxorubicin. The right flank tumor was histoplasty-treated, and tumors were fluorescently imaged to detect doxorubicin uptake after histoplasty treatment. Next, 4T1 tumor-bearing mice were randomized into 2 treatment groups (sham vs histoplasty, n = 3 per group). Forty-eight hours after sham/histoplasty treatment, tumors were harvested and analyzed using flow cytometry. RESULTS: Histoplasty significantly increased (P = .002) liposomal doxorubicin diffusion into 4T1 tumors compared with untreated tumors (2.12- vs 1.66-fold increase over control). Flow cytometry on histoplasty-treated tumors (n = 3) demonstrated a significant increase in tumor macrophage frequency (42% of CD45 vs 33%; P = .022) and a significant decrease in myeloid-derived suppressive cell frequency (7.1% of CD45 vs 10.3%; P = .044). Histoplasty-treated tumors demonstrated increased CD8+ (5.1% of CD45 vs 3.1%; P = .117) and CD4+ (14.1% of CD45 vs 11.8%; P = .075) T-cell frequency. CONCLUSIONS: Histoplasty is a nonablative focused US approach to noninvasively modify the tumor ECM, increase chemotherapeutic uptake, and alter the tumor immune microenvironment.

Identification of MYB gene family and functional analysis of GhMYB4 in cotton (Gossypium spp.).

Myeloblastosis (MYB) transcription factors (TFs) form a large gene family involved in a variety of biological processes in plants. Little is known about their roles in the development of cotton pigment glands. In this study, 646 MYB members were identified in Gossypium hirsutum genome and phylogenetic classification was analyzed. Evolution analysis revealed assymetric evolution of GhMYBs during polyploidization and sequence divergence of MYBs in G. hirustum was preferentially happend in D sub-genome. WGCNA (weighted gene co-expression network analysis) showed that four modules had potential relationship with gland development or gossypol biosynthesis in cotton. Eight differentially expressed GhMYB genes were identified by screening transcriptome data of three pairs of glanded and glandless cotton lines. Of these, four were selected as candidate genes for cotton pigment gland formation or gossypol biosynthesis by qRT-PCR assay. Silencing of GH_A11G1361 (GhMYB4) downregulated expression of multiple genes in gossypol biosynthesis pathway, indicating it could be involved in gossypol biosynthesis. The potential protein interaction network suggests that several MYBs may have indirect interaction with GhMYC2-like, a key regulator of pigment gland formation. Our study was the systematic analysis of MYB genes in cotton pigment gland development, providing candidate genes for further study on the roles of cotton MYB genes in pigment gland formation, gossypol biosynthesis and future crop plant improvement.

The interplay between IGF-1R signaling and Hippo-YAP in breast cancer stem cells.

BACKGROUND: Both IGF-1R/PI3K/AKT/mTOR and Hippo pathways are crucial for breast cancer stem cells (BCSCs). However, their interplay remains unclear. METHODS: Four triple negative breast cancer cell lines derived from CSC of two patient-derived xenografts (PDXs), AS-B145, AS-B145-1R, AS-B244, and AS-B244-1R, were used to elucidate the role of YAP in BCSCs. YAP silenced BCSCs were analyzed by cell proliferation, aldehyde dehydrogenase (ALDH) activity, mammosphere formation, and tumorigenesis. The effects of modulating IGF-1R and IGF-1 on YAP expression and localization were evaluated. The clinical correlation of YAP and IGF-1R signaling with the overall survival (OS) of 7830 breast cancer patients was analyzed by KM plotter. RESULTS: Knockdown of YAP abates the viability and stemness of BCSCs in vitro and tumorigenicity in vivo. Depletion of IGF-1R by shRNA or specific inhibitor decreases YAP expression. In contrast, IGF-1 addition upregulates YAP and enhances its nuclear localization. YAP overexpression increased the mRNA level of IGF-1, but not IGF-1R. Data mining of clinical breast cancer specimens revealed that basal-like breast cancer patients with higher level of IGF-1 and YAP exhibit significantly shorter OS. CONCLUSIONS: YAP contributes to stemness features of breast cancer in vitro and in vivo. The expression and localization of YAP was regulated by IGF-1R and YAP expression in turns upregulates IGF-1, but not IGF-1R. Clinically, higher level of YAP and IGF-1 significantly correlated with shorter OS in basal-like breast cancer. Taken together, these findings suggest the clinical relevance of interplay between YAP and IGF-1/IGF-1R pathway in sustaining the properties of BCSCs. Video Abstract.

Increased expression of SLC25A1/CIC causes an autistic-like phenotype with altered neuron morphology.

N ε-lysine acetylation within the lumen of the endoplasmic reticulum is a recently characterized protein quality control system that positively selects properly folded glycoproteins in the early secretory pathway. Overexpression of the endoplasmic reticulum acetyl-CoA transporter AT-1 in mouse forebrain neurons results in increased dendritic branching, spine formation and an autistic-like phenotype that is attributed to altered glycoprotein flux through the secretory pathway. AT-1 overexpressing neurons maintain the cytosolic pool of acetyl-CoA by upregulation of SLC25A1, the mitochondrial citrate/malate antiporter and ATP citrate lyase, which converts cytosolic citrate into acetyl-CoA. All three genes have been associated with autism spectrum disorder, suggesting that aberrant cytosolic-to-endoplasmic reticulum flux of acetyl-CoA can be a mechanistic driver for the development of autism spectrum disorder. We therefore generated a SLC25A1 neuron transgenic mouse with overexpression specifically in the forebrain neurons. The mice displayed autistic-like behaviours with a jumping stereotypy. They exhibited increased steady-state levels of citrate and acetyl-CoA, disrupted white matter integrity with activated microglia and altered synaptic plasticity and morphology. Finally, quantitative proteomic and acetyl-proteomic analyses revealed differential adaptations in the hippocampus and cortex. Overall, our study reinforces the connection between aberrant cytosolic-to-endoplasmic reticulum acetyl-CoA flux and the development of an autistic-like phenotype.

Loss of core-fucosylation of SPARC impairs collagen binding and contributes to COPD.

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease with high morbidity and mortality worldwide. Although several mechanisms to account for deleterious immune effects were proposed, molecular description for the underlying alveolar structural alterations for COPD is lacking. Here, silencing of α1,6-fucosyltransferase (Fut8), the enzyme for core-fucosylation and highly expressed in lung stem cells, resulted in alveolar structural changes in lung organoids, recapitulating COPD. Site-specific mass spectrometry analysis demonstrated that the secreted protein acidic and rich in cysteine (SPARC), which binds collagen, contains a core-fucosylation site in its VCSNDNcfK glycopeptide. Biacore assay showed markedly reduced collagen binding of SPARC lacking core fucosylation. Molecular dynamics analysis revealed that core fucosylation of SPARC-induced dynamic conformational changes in its N-glycan, allowing terminal galactose and N-acetylglucosamine to interact with K150, P261 and H264 residues, thereby promoting collagen binding. Site-specific mutagenesis of these residues also resulted in low affinity for collagen binding. Moreover, loss of collagen and decline of core fucosylation were observed in COPD lung tissues. These findings provide a new mechanistic insight into the role of core fucosylation of SPARC in cell-matrix communication and contribution to the abnormal alveolar structures in COPD.

YULINK regulates vascular formation in zebrafish and HUVECs.

BACKGROUND: The distinct arterial and venous cell fates are dictated by a combination of various genetic factors which form diverse types of blood vessels such as arteries, veins, and capillaries. We report here that YULINK protein is involved in vasculogenesis, especially venous formation. METHODS: In this manuscript, we employed gene knockdown, yeast two-hybrid, FLIM-FRET, immunoprecipitation, and various imaging technologies to investigate the role of YULINK gene in zebrafish and human umbilical vein endothelial cells (HUVECs). RESULTS: Knockdown of YULINK during the arterial-venous developmental stage of zebrafish embryos led to the defective venous formation and abnormal vascular plexus formation. Knockdown of YULINK in HUVECs impaired their ability to undergo cell migration and differentiation into a capillary-like tube formation. In addition, the phosphorylated EPHB4 was decreased in YULINK knockdown HUVECs. Yeast two-hybrid, FLIM-FRET, immunoprecipitation, as well as imaging technologies showed that YULINK colocalized with endosome related proteins (EPS15, RAB33B or TICAM2) and markers (Clathrin and RHOB). VEGF-induced VEGFR2 internalization was also compromised in YULINK knockdown HUVECs, demonstrating to the involvement of YULINK. CONCLUSION: This study suggests that YULINK regulates vasculogenesis, possibly through endocytosis in zebrafish and HUVECs.

The Role of Cellular Immunity and Adaptive Immunity in Pathophysiology of Brain and Spinal Cord Tumors.

Major advances have been made in our understanding of CNS tumors, especially glioma, however, the survival of patients with malignant glioma remains poor. While radiation and chemotherapy have increased overall survival, glioblastoma multiforme (GBM) still has one of the worst 5-year survival rates of all human cancers. Here, in this chapter, the authors review the abrogation of the immune system in the tumor setting, revealing many plausible targets for therapy and the current immunotherapy treatment strategies employed. Notably, glioma has also been characterized as a subset of primary spinal cord tumor and current treatment recommendations are outlined here.

B3GALT5 knockout alters gycosphingolipid profile and facilitates transition to human naïve pluripotency.

Conversion of human pluripotent stem cells from primed to naïve state is accompanied by altered transcriptome and methylome, but glycosphingolipid (GSL) profiles in naïve human embryonic stem cells (hESCs) have not been systematically characterized. Here we showed a switch from globo-(SSEA-3, SSEA-4, and Globo H) and lacto-series (fucosyl-Lc4Cer) to neolacto-series GSLs (SSEA-1 and H type 2 antigen), along with marked down-regulation of ß-1,3-galactosyltransferase (B3GALT5) upon conversion to naïve state. CRISPR/Cas9-generated B3GALT5-knockout (KO) hESCs displayed an altered GSL profile, increased cloning efficiency and intracellular Ca2+, reminiscent of the naïve state, while retaining differentiation ability. The altered GSLs could be rescued through overexpression of B3GALT5. B3GALT5-KO cells cultured with 2iLAF exhibited naïve-like transcriptome, global DNA hypomethylation, and X-chromosome reactivation. In addition, B3GALT5-KO rendered hESCs more resistant to calcium chelator in blocking entry into naïve state. Thus, loss of B3GALT5 induces a distinctive state of hESCs displaying unique GSL profiling with expression of neolacto-glycans, increased Ca2+, and conducive for transition to naïve pluripotency.