Cyclic multiplex fluorescent immunohistochemistry and machine learning reveal distinct states of astrocytes and microglia in normal aging and Alzheimer’s disease

Background Astrocytes and microglia react to Aβ plaques, neurofibrillary tangles, and neurodegeneration in the Alzheimer’s disease (AD) brain. Single-nuclei and single-cell RNA-seq have revealed multiple states or subpopulations of these glial cells but lack spatial information. We have developed a methodology of cyclic multiplex fluorescent immunohistochemistry on human postmortem brains and image analysis that enables a comprehensive morphological quantitative characterization of astrocytes and microglia in the context of their spatial relationships with plaques and tangles. Methods Single FFPE sections from the temporal association cortex of control and AD subjects were subjected to 8 cycles of multiplex fluorescent immunohistochemistry, including 7 astroglial, 6 microglial, 1 neuronal, Aβ, and phospho-tau markers. Our analysis pipeline consisted of: (1) image alignment across cycles; (2) background subtraction; (3) manual annotation of 5172 ALDH1L1+ astrocytic and 6226 IBA1+ microglial profiles; (4) local thresholding and segmentation of profiles; (5) machine learning on marker intensity data; and (6) deep learning on image features. Results Spectral clustering identified three phenotypes of astrocytes and microglia, which we termed “homeostatic,” “intermediate,” and “reactive.” Reactive and, to a lesser extent, intermediate astrocytes and microglia were closely associated with AD pathology (≤ 50 µm). Compared to homeostatic, reactive astrocytes contained substantially higher GFAP and YKL-40, modestly elevated vimentin and TSPO as well as EAAT1, and reduced GS. Intermediate astrocytes had markedly increased EAAT2, moderately increased GS, and intermediate GFAP and YKL-40 levels. Relative to homeostatic, reactive microglia showed increased expression of all markers (CD68, ferritin, MHC2, TMEM119, TSPO), whereas intermediate microglia exhibited increased ferritin and TMEM119 as well as intermediate CD68 levels. Machine learning models applied on either high-plex signal intensity data (gradient boosting machines) or directly on image features (convolutional neural networks) accurately discriminated control vs. AD diagnoses at the single-cell level. Conclusions Cyclic multiplex fluorescent immunohistochemistry combined with machine learning models holds promise to advance our understanding of the complexity and heterogeneity of glial responses as well as inform transcriptomics studies. Three distinct phenotypes emerged with our combination of markers, thus expanding the classic binary “homeostatic vs. reactive” classification to a third state, which could represent “transitional” or “resilient” glia.España Ministry of Science, Innovation, and Universities FPU fellowship to CM-CMassachusetts Alzheimer’s Disease Research Center grant P30AG062421 to BTH, and 1R56AG061196 to BTHAlzheimer’s Association (AACF17-524184 and AACF-17-524184-RAPID to AS-P

Regulatory Network and Prognostic Effect Investigation of PIP4K2A in Leukemia and Solid Cancers

Germline variants of PIP4K2A impact susceptibility of acute lymphoblastic leukemia (ALL) through inducing its overexpression. Although limited reports suggested the oncogenic role of PIP4K2A in cancers, regulatory network and prognostic effect of this gene remains poorly understood in tumorigenesis and leukemogenesis. In this study, we conducted genome-wide gene expression association analyses in pediatric B-ALL cohorts to discover expression associated genes and pathways, which is followed by the bioinformatics analyses to investigate the prognostic role of PIP4K2A and its related genes in multiple cancer types. 214 candidates were identified to be significantly associated with PIP4K2A expression in ALL patients, with known cancer-related genes rankings the top (e.g., RAC2, RBL2, and TFDP1). These candidates do not only tend to be clustered in the same types of leukemia, but can also separate the patients into novel molecular subtypes. PIP4K2A is noticed to be frequently overexpressed in multiple other types of leukemia and solid cancers from cancer cohorts including TCGA, and associated with its candidates in subtype-specific and cancer-specific manners. Interestingly, the association status varied in tumors compared to their matched normal tissues. Moreover, PIP4K2A and its related candidates exhibit stage-independent prognostic effects in multiple cancers, mostly with its lower expression significantly associated with longer overall survival (p < 0.05). Our findings reveal the transcriptional regulatory network of PIP4K2A in leukemia, and suggest its potentially important role on molecular subtypes of multiple cancers and subsequent treatment outcomes

Session 3H: LUBAC-Dependent Epithelial Signaling Regulates TRAIL Expression During Influenza A Virus Infection

The linear ubiquitin chain assembly complex (LUBAC), consists of three proteins; HOIP, the catalytic unit whose E3-ligase activity is necessary for the formation of “head-to-tail” linear ubiquitin chains, and SHARPIN and HOIL, which provide stability to the complex (Tokunaga et al. 2009). Downstream of inflammatory stimuli, LUBAC is required for the robust activation of NF-κB and subsequent upregulation of NF-kB-regulated inflammatory cytokines (Tokunaga and Iwai, 2012). During Influenza A Virus (IAV) infection, alveolar epithelial cells (AEC) are targeted for viral replication and orchestrate the inflammatory response by producing cytokines, which recruit inflammatory cells (Short et al. 2014). While infiltrating immune cells are necessary for viral clearance, they contribute to the excessive production of cytokines, which promotes AEC death, damages the alveolar epithelial barrier, and impairs proper lung function (Short et al. 2013). One of the cytokines produced, TNF-related apoptosis-inducing ligand (TRAIL), is expressed by macrophages in response to IAV-induced epithelial signaling (Herold et al. 2008). TRAIL interacts with its receptor, Death Receptor 5 (DR5), on AEC to induce death and enhance morbidity and mortality during influenza infection (Herold et al. 2008). To investigate whether TRAIL expression is driving influenza-induced mortality in mice with an alveolar epithelial specific loss of HOIP (HOIP-KO), IAV-infected wild-type (WT) and HOIP-KO mice were subjected to bronchoalveolar lavage (BAL) at 5 days post infection to obtain a cell pellet of inflammatory cells from which we then isolated RNA using a specialized RNA-binding silica membrane. Eluted purified RNA was then converted into cDNA using a reverse transcriptase and used in an qRT-PCR reaction to quantify TRAIL expression in BAL. We found that the absence of HOIP correlated with increased TRAIL expression, which was detected at nearly sixfold expression levels in cells from HOIP-KO mice as compared to those from WT mice. These results suggest the LUBAC plays a critical role in regulating the total levels of TRAIL expression in the mouse lung during IAV infection

Understanding Hydrothermal Dechlorination of PVC by Focusing on the Operating Conditions and Hydrochar Characteristics

To remove chlorine from chlorinated wastes efficiently, the hydrothermal treatment (HT) of PVC was investigated with a lower alkaline dosage in this work. Some typical operating conditions were investigated to find out the most important factor affecting the dechlorination efficiency (DE). The FTIR technique was employed to detect the functional groups in PVC and hydrochars generated to reveal the possible pathways for chlorine removal. The results show that the HT temperature was a key parameter to control the dechlorination reaction rate. At a HT temperature of 240 °C, about 94.3% of chlorine could be removed from the PVC with 1% NaOH. The usage of NaOH was helpful for chlorine removal, while a higher dosage might also hinder this process because of the surface poisoning and coverage of free sites. To some extent, the DE was increased with the residence time. At a residence time of 30 min, the DE reached a maximum of 76.74%. A longer residence time could promote the generation of pores in hydrochar which is responsible for the reduction in DE because of the re-absorption of water-soluble chlorine. According to the FTIR results, the peak intensities of both C=CH and C=C stretching vibrations in hydrochar were increased, while the peak at around 3300 cm−1 representing the –OH group was not obvious, indicating that the dehydrochlorination (elimination reaction) was a main route for chlorine removal under these conditions studied in this work

Effect of lncRNA XIST on acute myeloid leukemia cells via miR-142-5p-PFKP axis

ABSTRACTAcute myeloid leukemia (AML) is the common blood cancer in hematopoietic system-related diseases and has a poor prognosis. Studies have shown that long non-coding RNAs (lncRNAs) are closely related to the pathogenesis of a variety of diseases, including AML. However, the specific molecular mechanism remains unclear. Hence, the objective of this study was to investigate the effect and mechanism of lncRNA X inactive specific transcript (lncRNA XIST) on AML. To achieve our objective, some tests were performed. Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to detect the expression of lncRNA XIST, miR-142-5p and the platelet isoform of phosphofructokinase (PFKP). The targeting relationship between miR-142-5p and lncRNA XIST and PFKP was verified by Pearson correlation analysis, dual-luciferase reporter assay, and pull-down assay. Functional experiments were used to analyze the effect and mechanism of action of knocking down lncRNA XIST on THP-1 and U937 cells. Compared with bone marrow cells, lncRNA XIST and PFKP expression levels were up-regulated and miR-142-5p expression levels were down-regulated in AML. Further analysis revealed that lncRNA XIST targeted and bound to miR-142-5p, and PFKP was a target gene of miR-142-5p. Knockdown of lncRNA XIST significantly promoted miR-142-5p expression to down-regulate PFKP in THP-1 and U937 cells, while the cell proliferation, cell viability, and cell cycle arrest were inhibited and apoptosis was increased. Knockdown of miR-142-5p reversed the functional impact of lncRNA XIST knockdown on AML cells. In conclusion, down-regulation of lncRNA XIST can affect the progression of AML by regulating miR-142-5p

Life Cycle Assessment of Advanced Circulating Fluidized Bed Municipal Solid Waste Incineration System from an Environmental and Exergetic Perspective

The production of clean and efficient energy from municipal solid waste (MSW) is extremely urgent matter due to an increasing energy demand and environmental concerns. In this study, a high steam parameter (520 °C, 7.9 MPa) circulating fluidized bed (CFB) MSW incineration system, equipped with a mechanical, biological treatment and external heat exchanger systems, was introduced and a comparative study with a typical mechanical grate (450 °C, 5.3 MPa) incineration system and conventional CFB (485 °C, 5.3 MPa) incineration system was carried out from a life-cycle, environmental and exergetic perspective which could assess different energy and material outputs based on real operating data. Moreover, the potential system optimization of this advanced CFB system was proposed. The results showed that the advanced CFB system was more environmentally friendly and resource-efficient than conventional MSW incineration systems. The recovery of material should be given priority over energy recovery. According to the assessment of the environment, and energy and material recovery, a process improvement with an incinerated refuse-derived fuel and a semi-compost produced by MBT as a soil conditioner was highly recommended