Single-cell profile reveals the landscape of cardiac immunity and identifies a cardio-protective Ym-1hi neutrophil in myocardial ischemia-reperfusion injury.

Myocardial ischemia-reperfusion injury (MIRI) is a major hindrance to the success of cardiac reperfusion therapy. Although increased neutrophil infiltration is a hallmark of MIRI, the subtypes and alterations of neutrophils in this process remain unclear. Here, we performed single-cell sequencing of cardiac CD45+ cells isolated from the murine myocardium subjected to MIRI at six-time points. We identified diverse types of infiltrating immune cells and their dynamic changes during MIRI. Cardiac neutrophils showed the most immediate response and largest changes and featured with functionally heterogeneous subpopulations, including Ccl3hi Neu and Ym-1hi Neu, which were increased at 6 h and 1 d after reperfusion, respectively. Ym-1hi Neu selectively expressed genes with protective effects and was, therefore, identified as a novel specific type of cardiac cell in the injured heart. Further analysis indicated that neutrophils and their subtypes orchestrated subsequent immune responses in the cardiac tissues, especially instructing the response of macrophages. The abundance of Ym-1hi Neu was closely correlated with the therapeutic efficacy of MIRI when neutrophils were specifically targeted by anti-Lymphocyte antigen 6 complex locus G6D (Ly6G) or anti-Intercellular cell adhesion molecule-1 (ICAM-1) neutralizing antibodies. In addition, a neutrophil subtype with the same phenotype as Ym-1hi Neu was detected in clinical samples and correlated with prognosis. Ym-1 inhibition exacerbated myocardial injury, whereas Ym-1 supplementation significantly ameliorated injury in MIRI mice, which was attributed to the tilt of Ym-1 on the polarization of macrophages toward the repair phenotype in myocardial tissue. Overall, our findings reveal the anti-inflammatory phenotype of Ym-1hi Neu and highlight its critical role in myocardial protection during the early stages of MIRI.

Single-cell profiling reveals kidney CD163+ dendritic cell participation in human lupus nephritis.

OBJECTIVES: The current work aimed to provide a comprehensive single-cell landscape of lupus nephritis (LN) kidneys, including immune and non-immune cells, identify disease-associated cell populations and unravel their participation within the kidney microenvironment. METHODS: Single-cell RNA and T cell receptor sequencing were performed on renal biopsy tissues from 40 patients with LN and 6 healthy donors as controls. Matched peripheral blood samples from seven LN patients were also sequenced. Multiplex immunohistochemical analysis was performed on an independent cohort of 60 patients and validated using flow cytometric characterisation of human kidney tissues and in vitro assays. RESULTS: We uncovered a notable enrichment of CD163+ dendritic cells (DC3s) in LN kidneys, which exhibited a positive correlation with the severity of LN. In contrast to their counterparts in blood, DC3s in LN kidney displayed activated and highly proinflammatory phenotype. DC3s showed strong interactions with CD4+ T cells, contributing to intrarenal T cell clonal expansion, activation of CD4+ effector T cell and polarisation towards Th1/Th17. Injured proximal tubular epithelial cells (iPTECs) may orchestrate DC3 activation, adhesion and recruitment within the LN kidneys. In cultures, blood DC3s treated with iPTECs acquired distinct capabilities to polarise Th1/Th17 cells. Remarkably, the enumeration of kidney DC3s might be a potential biomarker for induction treatment response in LN patients. CONCLUSION: The intricate interplay involving DC3s, T cells and tubular epithelial cells within kidneys may substantially contribute to LN pathogenesis. The enumeration of renal DC3 holds potential as a valuable stratification feature for guiding LN patient treatment decisions in clinical practice.

The Impact of the Nervous System on Arteries and the Heart: The Neuroimmune Cardiovascular Circuit Hypothesis.

Three systemic biological systems, i.e., the nervous, the immune, and the cardiovascular systems, form a mutually responsive and forward-acting tissue network to regulate acute and chronic cardiovascular function in health and disease. Two sub-circuits within the cardiovascular system have been described, the artery brain circuit (ABC) and the heart brain circuit (HBC), forming a large cardiovascular brain circuit (CBC). Likewise, the nervous system consists of the peripheral nervous system and the central nervous system with their functional distinct sensory and effector arms. Moreover, the immune system with its constituents, i.e., the innate and the adaptive immune systems, interact with the CBC and the nervous system at multiple levels. As understanding the structure and inner workings of the CBC gains momentum, it becomes evident that further research into the CBC may lead to unprecedented classes of therapies to treat cardiovascular diseases as multiple new biologically active molecules are being discovered that likely affect cardiovascular disease progression. Here, we weigh the merits of integrating these recent observations in cardiovascular neurobiology into previous views of cardiovascular disease pathogeneses. These considerations lead us to propose the Neuroimmune Cardiovascular Circuit Hypothesis.

Pairing of single-cell RNA analysis and T cell antigen receptor profiling indicates breakdown of T cell tolerance checkpoints in atherosclerosis.

Atherosclerotic plaques form in the inner layer of arteries triggering heart attacks and strokes. Although T cells have been detected in atherosclerosis, tolerance dysfunction as a disease driver remains unexplored. Here we examine tolerance checkpoints in atherosclerotic plaques, artery tertiary lymphoid organs and lymph nodes in mice burdened by advanced atherosclerosis, via single-cell RNA sequencing paired with T cell antigen receptor sequencing. Complex patterns of deteriorating peripheral T cell tolerance were observed being most pronounced in plaques followed by artery tertiary lymphoid organs, lymph nodes and blood. Affected checkpoints included clonal expansion of CD4+, CD8+ and regulatory T cells; aberrant tolerance-regulating transcripts of clonally expanded T cells; T cell exhaustion; Treg-TH17 T cell conversion; and dysfunctional antigen presentation. Moreover, single-cell RNA-sequencing profiles of human plaques revealed that the CD8+ T cell tolerance dysfunction observed in mouse plaques was shared in human coronary and carotid artery plaques. Thus, our data support the concept of atherosclerosis as a bona fide T cell autoimmune disease targeting the arterial wall.

Cardiovascular Brain Circuits.

The cardiovascular system is hardwired to the brain via multilayered afferent and efferent polysynaptic axonal connections. Two major anatomically and functionally distinct though closely interacting subcircuits within the cardiovascular system have recently been defined: The artery-brain circuit and the heart-brain circuit. However, how the nervous system impacts cardiovascular disease progression remains poorly understood. Here, we review recent findings on the anatomy, structures, and inner workings of the lesser-known artery-brain circuit and the better-established heart-brain circuit. We explore the evidence that signals from arteries or the heart form a systemic and finely tuned cardiovascular brain circuit: afferent inputs originating in the arterial tree or the heart are conveyed to distinct sensory neurons in the brain. There, primary integration centers act as hubs that receive and integrate artery-brain circuit-derived and heart-brain circuit-derived signals and process them together with axonal connections and humoral cues from distant brain regions. To conclude the cardiovascular brain circuit, integration centers transmit the constantly modified signals to efferent neurons which transfer them back to the cardiovascular system. Importantly, primary integration centers are wired to and receive information from secondary brain centers that control a wide variety of brain traits encoded in engrams including immune memory, stress-regulating hormone release, pain, reward, emotions, and even motivated types of behavior. Finally, we explore the important possibility that brain effector neurons in the cardiovascular brain circuit network connect efferent signals to other peripheral organs including the immune system, the gut, the liver, and adipose tissue. The enormous recent progress vis-à-vis the cardiovascular brain circuit allows us to propose a novel neurobiology-centered cardiovascular disease hypothesis that we term the neuroimmune cardiovascular circuit hypothesis.

Effects of Human Activities on Evapotranspiration and Its Components in Arid Areas.

With the increasing impact of human activities on the environment, evapotranspiration (ET) has changed in arid areas, which further affects the water resources availability in the region. Therefore, understanding the impact of human activities on ET and its components is helpful to the management of water resources in arid areas. This study verified the accuracy of Fisher's model (PT-JPL model) for ET estimation in southern Xinjiang, China by using the evaporation complementarity theory dataset (AET dataset). The ET and the evapotranspiration components (T:E) of six land-use types were estimated in southern Xinjiang from 1982 to 2015, and the impact of human activities on ET was analyzed. In addition, the impact of four environmental factors (temperature (Temp), net radiation (Rn), relative humidity (RH), and NDVI) on ET were evaluated. The results showed that the calculated ET values of the PT-JPL model were close to the ET values of the AET dataset. The correlation coefficient (R2) was more than 0.8, and the NSE was close to 1. In grassland, water area, urban industrial and mining land, forest land, and cultivated land, the ET values were high, and in unused land types, the ET values were the lowest. The T:E values varied greatly in urban industrial and mining land, forest land, and cultivated land, which was due to the intensification of human activities, and the values were close to 1 in summer in recent years. Among the four environmental factors, temperature largely influenced the monthly ET. These findings suggest that human activities have significantly reduced soil evaporation and improved water use efficiency. The impact of human activities on environmental factors has caused changes in ET and its components, and appropriate oasis expansion is more conducive to regional sustainable development.

Neuroimmune cardiovascular interfaces in atherosclerosis.

Two pairs of biological systems acting over long distances have recently been defined as major participants in the regulation of physiological and pathological tissue reactions: i) the nervous and vascular systems form various blood-brain barriers and control axon growth and angiogenesis; and ii) the nervous and immune systems emerge as key players to direct immune responses and maintain blood vessel integrity. The two pairs have been explored by investigators in relatively independent research areas giving rise to the concepts of the rapidly expanding topics of the neurovascular link and neuroimmunology, respectively. Our recent studies on atherosclerosis led us to consider a more inclusive approach by conceptualizing and combining principles of the neurovascular link and neuroimmunology: we propose that the nervous system, the immune system and the cardiovascular system undergo complex crosstalks in tripartite rather than bipartite interactions to form neuroimmune cardiovascular interfaces (NICIs).

A Prognostic Signature for Colon Adenocarcinoma Patients Based on m6A-Related lncRNAs.

N6-methyladenosine (m6A) modification is a common epigenetic modification. It is reported that lncRNA can be regulated by m6A modification. Previous studies have shown that lncRNAs associated with m6A regulation (m6A-lncRNAs) serve as ideal prognostic biomarkers. However, whether lncRNAs are involved in m6A modification in colon adenocarcinoma (COAD) needs further exploration. The objective of this study was to construct an m6A-lncRNAs-based signature for patients with COAD. We obtained the RNA sequencing data and clinical information from The Cancer Genome Atlas (TCGA). Pearson correlation analysis was employed to recognize lncRNAs associated with m6A regulation (m6A-lncRNAs). 24 prognostic m6A-lncRNAs was identified by univariate Cox regression analysis. Gene set enrichment analysis (GSAE) was used to investigate the potential cellular pathways and biological processes. We have also explored the relationship between immune infiltrate levels and m6A-lncRNAs. Then, a predictive signature based on the expression of 13 m6A-lncRNAs was constructed by the Lasso regression algorithm, including UBA6-AS1, AC139149.1, U91328.1, AC138207.5, AC025171.4, AC008760.1, ITGB1-DT, AP001619.1, AL391422.4, AC104532.2, ZEB1-AS1, AC156455.1, and AC104819.3. ROC curves and K M survival curves have shown that the risk score has a well-predictive ability. We also set up a quantitative nomogram on the basis of risk score and prognosis-related clinical characteristics. In summary, we have identified some m6A-lncRNAs that correlated with prognosis and tumor immune microenvironment in COAD. In addition, a potential alternative signature based on the expression of m6A-lncRNAs was provided for the management of COAD patients.

Exosomal miR-625-3p secreted by cancer-associated fibroblasts in colorectal cancer promotes EMT and chemotherapeutic resistance by blocking the CELF2/WWOX pathway.

Migration, invasion, epithelial-mesenchymal transformation (EMT), and chemotherapeutic resistance are the leading causes of therapeutic failure in people with colorectal cancer (CRC). The migration of exosomal miRNA between cancer cells and the tumor microenvironment is directly associated with malignant behavior in cancer-associated fibroblasts (CAFs). In the context of earlier research, the purpose of the current study was to assess the role and potential mechanism of miR-625-3p released by CAFs in CRC cells. Exosomes were extracted and purified from CAFs conditioned medium by ultracentrifugation. Western blot, immunohistochemistry, CCK-8, transwell assay, H&E staining, Tunnel, real-time PCR, double luciferase assay, RNA-binding protein immunoprecipitation (RIP), and immunofluorescence double staining experiments were used to investigate the effects of CAFs-Exo and miR-625-3p on CRC cell invasion, migration, proliferation, EMT, chemotherapeutic resistance, and molecular mechanisms. The current results indicated that CAFs-Exo was directly internalized by CRC cells, and exosomal miR-625-3p derived from CAFs might promote migration, invasion, EMT and chemotherapeutic resistance in CRC cells by inhibiting the CELF2/WWOX pathway, providing a potential candidate for CRC prediction and treatment.

The C1q-ApoE complex: A new hallmark pathology of viral hepatitis and nonalcoholic fatty liver disease.

We recently identified a high-affinity C1q-ApoE complex in human artery atherosclerotic intima lesions and in human amyloid plaques of Alzheimer's Disease brains defining a common pathogenetic pathway of two diverse diseases, i.e. atherosclerosis and dementia. C1q is the initiating and controlling protein of the classical complement cascade (CCC), which occupies a key role in multiple acute and chronic inflammatory tissue responses. C1q is largely produced by myeloid cells including Kupffer cells (KCs) and subsequently secreted into the circulation as an inactive preprotein. Its binding partner, Apolipoprotein E (ApoE), is produced by KCs and hepatocytes and it is also secreted into the circulation, where it regulates essential steps of lipid transport. In addition to its major source, ApoE can be produced by non-liver cells including immune cells and multiple other cells depending on local tissue contexts. To initiate the CCC cascade, C1q must be activated by molecules as varied as oxidized lipids, amyloid fibrils, and immune complexes. However, ApoE is mute towards inactive C1q but binds at high-affinity to its activated form. Specifically, our studies revealed that ApoE is a CCC-specific checkpoint inhibitor via the formation of the C1q-ApoE complex. We proposed that it may arise in multiple if not all CCC-associated diseases and that its presence indicates ongoing CCC activity. Here, we turned to the liver to examine C1q-ApoE complexes in human B- and C-viral hepatitis and nonalcoholic fatty liver disease (NAFLD). In addition, we used multidrug-resistance-2 gene-knockout (Mdr2-KO) mice as a model for inflammatory liver disease and hepatocellular carcinoma (HCC) pathogenesis. In normal murine and human livers, KCs were the major C1q-producing cell type while hepatocytes were the primary ApoE-forming cell type though the C1q-ApoE complex was rare or nonexistent. However, significant numbers of C1q-ApoE complexes formed in both Mdr2-KO, human viral hepatitis, and NAFLD around portal triads where immune cells had infiltrated the liver. Additionally, high numbers of C1q-ApoE complexes emerged in human livers in areas of extracellular lipid droplets across the entire liver parenchyma in NAFLD-affected patients. Thus, the C1q-ApoE complex is a new pathological hallmark of viral hepatitis B and C and NAFLD.

Drought effects on tree growth, water use efficiency, vulnerability and canopy health of Quercus variabilis-Robinia pseudoacacia mixed plantation.

Drought-induced forest canopy die-back and tree mortality have been commonly recorded in the lithoid mountainous regions of northern China. However, the capacity of trees to regulate their carbon and water balance in response to drought remains inadequately understood. We measured tree growth, intrinsic water use efficiency (iWUE), vulnerability, and canopy health during drought events using dendrochronology, C isotope measurements, and a tree canopy health survey in a mixed plantation of Quercus variabilis and Robinia pseudoacacia. Resistance (Rt), recovery (Rc), resilience (Rs), and increased amplitude in iWUE compared to the indices 3 years before drought (iWUEr) were calculated for each species across the dominant tree (D), co-dominant tree (CD), and suppressed tree (S). Our results revealed that D and CD showed lower Rt, higher Rc, and higher iWUEr than S. After exposure to multiple sequential drought events, Q. variabilis showed an increasing trend in Rt, and R. pseudoacacia showed a decreasing trend in Rc. R. pseudoacacia exhibited a more conservative strategy towards drought, resulting in a negative SRt-iWUEr (slope of the linear model fitted to capture the trend between Rt and iWUEr) during drought events than Q. variabilis. For individual trees, lower Rc or positive SRt-iWUEr Q. variabilis and negative SRt-iWUEr R. pseudoacacia were more susceptible to canopy die-back. In conclusion, our study offers a new perspective for improved management practices in the design of silvicultural actions for forestry plantations in lithoid mountainous areas with increasing drought risk.

Six Genes Associated with Lymphatic Metastasis in Colon Adenocarcinoma Linked to Prognostic Value and Tumor Immune Cell Infiltration.

Objective: The aim of the study is to explore the relationship between lymphatic metastasis genes, prognosis, and immune cell infiltration in patients with colon cancer. Methods: Based on the Cancer Genome Atlas Program (TCGA) database, differentially expressed genes and prognostic genes related to colon adenocarcinoma (COAD) lymphatic metastasis were screened and intersected. We used lasso and univariate Cox regression analysis to screen core genes and establish a preliminary prediction model. GO and KEGG enrichment analysis was used for lymphatic metastasis-related genes, and single GSEA was used for the final screening results. Finally, we evaluated the relationship between identified genes and immune cell infiltration. Results: A total of 1727 genes were differentially expressed between COAD patients with TNM stages of N0 and N1. After further screening, six core genes (RNU4-2, ZNF556, RNVU1-15, NSA2P6, RN7SL767P, and RN7SL473P) were obtained, and a preliminary prediction model was established, in which ZNF556 was a risk factor, and the rest were protective factors. Single GSEA showed that pathways such as systemic lupus erythematosus might play an important role in the initial lymphatic metastasis of COAD. GO and KEGG enrichment analysis of 1727 genes supported this result. Immune infiltration analysis showed that six genes were significantly correlated with T cell and NK cell families. Conclusion: Six core genes may affect COAD initial lymphatic metastasis through the systemic lupus erythematosus pathway and immune cell infiltration.

Neuroimmune cardiovascular interfaces control atherosclerosis.

Atherosclerotic plaques develop in the inner intimal layer of arteries and can cause heart attacks and strokes1. As plaques lack innervation, the effects of neuronal control on atherosclerosis remain unclear. However, the immune system responds to plaques by forming leukocyte infiltrates in the outer connective tissue coat of arteries (the adventitia)2-6. Here, because the peripheral nervous system uses the adventitia as its principal conduit to reach distant targets7-9, we postulated that the peripheral nervous system may directly interact with diseased arteries. Unexpectedly, widespread neuroimmune cardiovascular interfaces (NICIs) arose in mouse and human atherosclerosis-diseased adventitia segments showed expanded axon networks, including growth cones at axon endings near immune cells and media smooth muscle cells. Mouse NICIs established a structural artery-brain circuit (ABC): abdominal adventitia nociceptive afferents10-14 entered the central nervous system through spinal cord T6-T13 dorsal root ganglia and were traced to higher brain regions, including the parabrachial and central amygdala neurons; and sympathetic efferent neurons projected from medullary and hypothalamic neurons to the adventitia through spinal intermediolateral neurons and both coeliac and sympathetic chain ganglia. Moreover, ABC peripheral nervous system components were activated: splenic sympathetic and coeliac vagus nerve activities increased in parallel to disease progression, whereas coeliac ganglionectomy led to the disintegration of adventitial NICIs, reduced disease progression and enhanced plaque stability. Thus, the peripheral nervous system uses NICIs to assemble a structural ABC, and therapeutic intervention in the ABC attenuates atherosclerosis.

Laser Capture Microdissection-Based mRNA Expression Microarrays and Single-Cell RNA Sequencing in Atherosclerosis Research.

A major goal of methodologies related to large scale gene expression analyses is to initiate comprehensive information on transcript signatures in single cells within the tissue's anatomy. Until now, this could be achieved in a stepwise experimental approach: (1) identify the majority of transcripts in a single cell (single cell transcriptome); (2) provide information on transcripts on multiple cell subtypes in a complex sample (cell heterogeneity); and (3) give information on each cell's spatial location within the tissue (zonation transcriptomics). Such genetic information will allow construction of functionally relevant gene expression maps of single cells of a given anatomically defined tissue compartment and thus pave the way for subsequent analyses, including their epigenetic modifications. Until today these aims have not been achieved in the area of cardiovascular disease research though steps toward these goals become apparent: laser capture microdissection (LCM)-based mRNA expression microarrays of atherosclerotic plaques were applied to gain information on local gene expression changes during disease progression, providing limited spatial resolution. Moreover, while LCM-derived tissue RNA extracts have been shown to be highly sensitive and covers a range of 10-16,000 genes per array/small amount of RNA, its original promise to isolate single cells from a tissue section turned out not to be practicable because of the inherent contamination of the cell's RNA of interest with RNA from neighboring cells. Many shortcomings of LCM-based analyses have been overcome using single-cell RNA sequencing (scRNA-seq) technologies though scRNA-seq also has several limitations including low numbers of transcripts/cell and the complete loss of spatial information. Here, we describe a protocol toward combining advantages of both techniques while avoiding their flaws.

Combined Single-Cell RNA and Single-Cell α/ß T Cell Receptor Sequencing of the Arterial Wall in Atherosclerosis.

Although various pro- and anti-inflammatory T cell subsets have been observed in murine and human atherosclerosis, principal issues of T cell immunity remain unanswered: Is atherosclerosis progression critically affected by aberrant T cell responses? Are tolerance checkpoints compromised during atherosclerosis progression? Answers to these questions will determine if we are at the cusp of developing T cell-dependent therapeutic strategies. Rapid advances in single cell RNA sequencing (scRNA-seq) and single cell α/ß T cell receptor (TCR) (scTCR) sequencing allows to address these issues in unprecedented ways. The majority of T cells recognize peptide antigen-MHC complexes presented by antigen-presenting cells which, in turn, trigger activation and proliferation (clonal expansion) of cognate TCR-carrying T cells. Thus, clonal expansion and their corresponding transcriptome are two similarly important sides of T cell immunity and both will-as hypothesized-affect the outcome of atherosclerosis. Here, we combined scRNA-seq and scTCR-seq in single cells. Moreover, we provide single T cell transcriptomes and TCR maps of three important tissues involved in atherosclerosis This approach is anticipated to address principal questions concerning atherosclerosis autoimmunity that are likely to pave the long sought way to T cell-dependent therapeutic approaches.

Tissue Clearing Approaches in Atherosclerosis.

Recent advances in cardiovascular research have led to a more comprehensive understanding of molecular mechanisms of atherosclerosis. It has become apparent that the disease involves three layers of the arterial wall: the intima, the media, and a connective tissue coat termed the adventitia. It is also now appreciated that arteries are surrounded by adipose and neuronal tissues. In addition, adjacent to and within the adventitia, arteries are embedded in a loose connective tissue containing blood vessels (vasa vasora) and lymph vessels, artery-draining lymph nodes and components of the peripheral nervous system, including periarterial nerves and ganglia. During atherogenesis, each of these tissues undergoes marked structural and cellular alterations. We propose that a better understanding of these cell-cell and cell-tissue interactions may considerably advance our understanding of cardiovascular disease pathogenesis. Methods to acquire subcellular optical access to the intact tissues surrounding healthy and diseased arteries are urgently needed to achieve these aims. Tissue clearing is a landmark next-generation, three-dimensional (3D) microscopy technique that allows to image large-scale hitherto inaccessible intact deep tissue compartments. It allows for detailed reconstructions of arteries by a combination of labelling, clearing, advanced microscopies and other imaging and data-analysis tools. Here, we describe two distinct tissue clearing protocols; solvent-based modified three-dimensional imaging of solvent-cleared organs (3DISCO) clearing and another using aqueous-based 2,2'-thiodiethanol (TDE) clearing, both of which complement each other.

Long-Term Effects of Climate and Competition on Radial Growth, Recovery, and Resistance in Mongolian Pines.

Understanding the response of tree growth and drought vulnerability to climate and competition is critical for managing plantation forests. We analyzed the growth of Mongolian pines in six forests planted by the Three-North Shelter Forest Program with tree-ring data and stand structures. A retroactive reconstruction method was used to depict the growth-competition relationships of Mongolian pines during the growth period and their climatic responses under different competition levels. Drought vulnerability was analyzed by measuring the basal area increment (BAI) of different competition indices (CIs). In young trees, differences in BAIs in stands with different CIs were not statistically significant. After 15-20 years, medium- and high-CI stands had significantly lower tree-ring widths (TWs) and BAIs than the low-CI stands (p < 0.05). The standardized precipitation evapotranspiration index (SPEI), precipitation, relative humidity, and vapor pressure deficit were major factors affecting tree growth. On a regional scale, climate outweighed competition in determining radial growth. The relative contribution of climatic factors increased with the gap in SPEI between plantation sites and the native range, while the reverse pattern of the competition-growth relationship was observed. Drought reduced TWs and BAIs at all sites. Stands of different CIs exhibited similar resistance, but, compared with low-CI stands, high- and medium-CI stands had significantly lower recovery, resilience, and relative resilience, indicating they were more susceptible to drought stresses. Modeled CI was significantly negatively related to resistance, resilience, and relative resilience, indicating a density-dependence of tree response to drought. After exposure to multiple sequential drought events, the relative resilience of high-CI stands decreased to almost zero; this failure to fully recover to pre-drought growth rates suggests increased mortality in the future. In contrast, low-CI stands are more likely to survive in hotter, more arid climates. These results provide a better understanding of the roles of competition and climate on the growth of Mongolian pines and offer a new perspective for investigating the density-dependent recovery and resilience of these forests.

Imaging atherosclerotic plaques by targeting Galectin-3 and activated macrophages using (89Zr)-DFO- Galectin3-F(ab')2 mAb.

Rationale: The high expression of Galectin-3 (Gal3) in macrophages of atherosclerotic plaques suggests its participation in atherosclerosis pathogenesis, and raises the possibility to use it as a target to image disease severity in vivo. Here, we explored the feasibility of tracking atherosclerosis by targeting Gal3 expression in plaques of apolipoprotein E knockout (ApoE-KO) mice via PET imaging. Methods: Targeting of Gal3 in M0-, M1- and M2 (M2a/M2c)-polarized macrophages was assessed in vitro using a Gal3-F(ab')2 mAb labeled with AlexaFluor®488 and 89Zr- desferrioxamine-thioureyl-phenyl-isothiocyanate (DFO). To visualize plaques in vivo, ApoE-KO mice were injected i.v. with 89Zr-DFO-Gal3-F(ab')2 mAb and imaged via PET/CT 48 h post injection. Whole length aortas harvested from euthanized mice were processed for Sudan-IV staining, autoradiography, and immunostaining for Gal3, CD68 and α-SMA expression. To confirm accumulation of the tracer in plaques, ApoE-KO mice were injected i.v. with Cy5.5-Gal3-F(ab')2 mAb, euthanized 48 h post injection, followed by cryosections of the body and acquisition of fluorescent images. To explore the clinical potential of this imaging modality, immunostaining for Gal3, CD68 and α-SMA expression were carried out in human plaques. Single cell RNA sequencing (scRNA-Seq) analyses were performed to measure LGALS3 (i.e. a synonym for Gal3) gene expression in each macrophage of several subtypes present in murine or human plaques. Results: Preferential binding to M2 macrophages was observed with both AlexaFluor®488-Gal3-F(ab')2 and 89Zr-DFO-Gal3-F(ab')2 mAbs. Focal and specific 89Zr-DFO-Gal3-F(ab')2 mAb uptake was detected in plaques of ApoE-KO mice by PET/CT. Autoradiography and immunohistochemical analyses of aortas confirmed the expression of Gal3 within plaques mainly in macrophages. Moreover, a specific fluorescent signal was visualized within the lesions of vascular structures burdened by plaques in mice. Gal3 expression in human plaques showed similar Gal3 expression patterns when compared to their murine counterparts. Conclusions: Our data reveal that 89Zr-DFO-Gal3-F(ab')2 mAb PET/CT is a potentially novel tool to image atherosclerotic plaques at different stages of development, allowing knowledge-based tailored individual intervention in clinically significant disease.