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Efficient isolation and downstream bioinformation analysis of circulating tumor cells (CTCs) in whole blood contribute to the early diagnosis of cancer and investigation of cancer metastasis. However, the separation and release of CTCs remain a great challenge due to the extreme rarity of CTCs and severe interference from other cells in complex clinical samples. Herein, we developed a low-cost and easy-to-fabricate aptamer-functionalized wafer with a three-dimensional (3D) interconnected porous structure by grafting polydopamine (PDA), poly(ethylene glycol) (PEG), and aptamer in sequence (Ni@PDA-PEG-Apt) for the capture and release of CTCs. The Ni@PDA-PEG-Apt wafer integrated the features of Ni foam with a 3D interconnected porous structure offering enough tunnels for cells to flow through and enhancing aptamer-cell contact frequency, the spacer PEG with flexible and high hydrophilic property increasing anti-interference ability and providing the wafer with more binding sites for aptamer, which result in an enhanced capture specificity and efficiency for CTCs. Because of these advantages, the Ni@PDA-PEG-Apt wafer achieved a high capture efficiency of 78.25%. The captured cancer cells were mildly released by endonuclease with up to 61.85% efficiency and good proliferation. Furthermore, tumor cells were injected into mice and experienced circulation in vivo. In blood samples after circulation, 65% of target tumor cells can be efficiently captured by the wafer, followed by released and recultured cells with high viability. Further downstream metabolomics analysis showed that target cancer cells remained with high biological activity and can be well separated from MCF-10A cells based on metabolic profiles by the PCA analysis, indicating the great potential of our strategy for further research on the progression of cancer metastasis. Notably, not only is the wafer cheap with a cost of only 3.58 U.S. dollars and easily prepared by environmental-friendly reagents but also the process of capturing and releasing tumor cells can be completed within an hour, which is beneficial for large-scale clinical use in the future.

A novel porous graphitized carbon biomaterial (CS-900-1C) was fabricated for a highly efficient capture of N-glycans and the discovery of five specific N-glycans expressed during diabetes progression.

A novel MOFs-303-functionalized magnetic probe with strong hydrophilicity was synthesized for the highly efficient analysis of N-linked glycopeptides and the discovery of potential biomarkers with hepatocellular carcinoma.

Intestinal HDL is hepatoprotective High-density lipoprotein (HDL) is important for cholesterol metabolism and may have anti-inflammatory and antimicrobial properties. Although HDL is mainly produced by the liver, the intestine is also a source. Han et al. show in mice that intestinal HDL is not routed to the systemic circulation. Rather, in the form of HDL3, it is directly transported to the liver through the hepatic portal vein. There, it sequesters bacterial lipopolysaccharide from the gut that can trigger inflammation and liver damage. In various models of liver injury, loss of enteric HDL exacerbated pathology. By contrast, drugs elevating intestinal HDL improved disease outcomes. HDL3 is enriched in human portal venous blood, suggesting that enteric HDL may be targetable for the treatment of liver disease. Science , abe6729, this issue p. eabe6729

Cholesterol esters are a key ingredient of foamy cells in atherosclerotic lesions; their formation is catalyzed by two enzymes: acyl-CoA:cholesterol acyltransferases (ACATs; also called sterol O-acyltransferases, or SOATs) ACAT1 and ACAT2. ACAT1 is present in all body cells and is the major isoenzyme in macrophages. Whether blocking ACAT1 benefits atherosclerosis has been under debate for more than a decade. Previously, our laboratory developed a myeloid-specific Acat1 knockout (KO) mouse (Acat1−M/−M), devoid of ACAT1 only in macrophages, microglia, and neutrophils. In previous work using the ApoE KO (ApoE−/−) mouse model for early lesions, Acat1−M/−M significantly reduced lesion macrophage content and suppressed atherosclerosis progression. In advanced lesions, cholesterol crystals become a prominent feature. Here we evaluated the effects of Acat1−M/−M in the ApoE KO mouse model for more advanced lesions and found that mice lacking myeloid Acat1 had significantly reduced lesion cholesterol crystal contents. Acat1−M/−M also significantly reduced lesion size and macrophage content without increasing apoptotic cell death. Cell culture studies showed that inhibiting ACAT1 in macrophages caused cells to produce less proinflammatory responses upon cholesterol loading by acetyl low-density lipoprotein. In advanced lesions, Acat1−M/−M reduced but did not eliminate foamy cells. In advanced plaques isolated from ApoE−/− mice, immunostainings showed that both ACAT1 and ACAT2 are present. In cell culture, both enzymes are present in macrophages and smooth muscle cells and contribute to cholesterol ester biosynthesis. Overall, our results support the notion that targeting ACAT1 or targeting both ACAT1 and ACAT2 in macrophages is a novel strategy to treat advanced lesions.

Arterial inflammation is a hallmark of atherosclerosis and appropriate management of this inflammation represents a major unmet therapeutic need for cardiovascular disease (CVD) patients. Here, we review the diverse contributions of immune cells to atherosclerosis, including mechanisms of activation in this context and the cytokine circuits that underlie disease progression, and the recent application of these insights in the form immunotherapy to treat cardiovascular disease. Recent studies on the cardiovascular comorbidity that arises in autoimmunity are revealing additional roles for cytokines in atherosclerosis. We discuss these findings and highlight data pointing to interleukin (IL)-1b, tumor necrosis factor, and IL-17 as cytokines that, at least in some settings, may present effective targets to reduce cardiovascular disease progression.

Objective: vMIP-II (viral macrophage inflammatory protein 2)/vCCL2 (viral chemotactic cytokine ligand 2) binds to multiple chemokine receptors, and vMIP-II-based positron emission tomography tracer ( 64 Cu-DOTA-vMIP-II: vMIP-II tracer) accumulates at atherosclerotic lesions in mice. Given that it would be expected to react with multiple chemokine receptors on monocytes and macrophages, we wondered if its accumulation in atherosclerosis lesion-bearing mice might correlate with overall macrophage burden or, alternatively, the pace of monocyte recruitment. Approach and Results: We employed a mouse model of atherosclerosis regression involving adenoassociated virus 8 vector encoding murine Apoe (AAV-mApoE) treatment of Apoe −/− mice where the pace of monocyte recruitment slows before macrophage burden subsequently declines. Accumulation of 64 Cu-DOTA-vMIP-II at Apoe −/− plaque sites was strong but declined with AAV- mApoE -induced decline in monocyte recruitment, before macrophage burden reduced. Monocyte depletion indicated that monocytes and macrophages themselves were not the only target of the 64 Cu-DOTA-vMIP-II tracer. Using fluorescence-tagged vMIP-II tracer, competitive receptor blocking with CXCR4 antagonists, endothelial-specific Cre-mediated deletion of CXCR4, CXCR4-specific tracer 64 Cu-DOTA-FC131, and CXCR4 staining during disease progression and regression, we show endothelial cell expression of CXCR4 is a key target of 64 Cu-DOTA-vMIP-II imaging. Expression of CXCR4 was low in nonplaque areas but strongly detected on endothelium of progressing plaques, especially on proliferating endothelium, where vascular permeability was increased and monocyte recruitment was the strongest. Conclusions: Endothelial injury status of plaques is marked by CXCR4 expression and this injury correlates with the tendency of such plaques to recruit monocytes. Furthermore, our findings suggest positron emission tomography tracers that mark CXCR4 can be used translationally to monitor the state of plaque injury and monocyte recruitment.

SummaryAssembly of high density lipoprotein (HDL) requires apoA1 and the cholesterol transporter ABCA1. Although the liver generates most HDL in blood, HDL synthesis also occurs in the small intestine. However, distinct functions for intestinal HDL are unknown. Here we show that HDL in the portal vein, which connects intestine to liver, derivesd mainly from intestine and potently neutralizes lipopolysaccharide (LPS). In a mouse model of short bowel syndrome where liver inflammation and fibrosis was driven by the LPS receptor TLR4, loss of intestine-derived HDL worsened liver injury, whereas liver status was improved by therapeutics that elevated and depended upon intestinal HDL production. Thus, protection of the liver from injury in response to gut-derived signals like LPS is a major function of intestinally synthesized HDL.

We dissect metabolic variability of mononuclear phagocyte (MNP) subpopulations across different tissues through integrative analysis of three large scale datasets. Specifically, we introduce ImmGen MNP Open Source dataset that profiled 337 samples and extended previous ImmGen effort which included 202 samples of mononuclear phagocytes and their progenitors. Next, we analysed Tabula Muris Senis dataset to extract data for 51,364 myeloid cells from 18 tissues. Taken together, a compendium of data assembled in this work covers phagocytic populations found across 38 different tissues. To analyse common metabolic features, we developed novel network-based computational approach for unbiased identification of key metabolic subnetworks based on cellular transcriptional profiles in large-scale datasets. Using ImmGen MNP Open Source dataset as baseline, we define 9 metabolic subnetworks that encapsulate the metabolic differences within mononuclear phagocytes, and demonstrate that these features are robustly found across all three datasets, including lipid metabolism, cholesterol biosynthesis, glycolysis, and a set of fatty acid related metabolic pathways, as well as nucleotide and folate metabolism. We systematically define major features specific to macrophage and dendritic cell subpopulations. Among other things, we find that cholesterol synthesis appears particularly active within the migratory dendritic cells. We demonstrate that interference with this pathway through statins administration diminishes migratory capacity of the dendritic cells in vivo. This result demonstrates the power of our approach and highlights importance of metabolic diversity among mononuclear phagocytes.

Background Exhausted T cells and regulatory T cells (Tregs) comprise diverse subsets of tumor immunosuppressive microenvironment that play key roles in tumor progress. Understanding subset diversity in T cells is a critical question for developing cancer immunotherapy. Methods A total of 235 specimens from surgical resections of hepatocellular carcinoma (HCC) patients were examined for infiltration of exhausted T cell (Tex) in tumor and adjacent tissue. We conducted deep single‐cell targeted immune profiling on CD3+ cells collected from tumor tissues, adjacent normal tissues (ANTs) and peripheral blood of HCC patients. Total 10 cell clusters were identified with distinct distributions and characteristics. Results We observed transitional differentiation of exhausted CD8+T cells and Tregs increasingly enriched in tumor tissue. The accumulation and location of Tex were related to the differences in the long‐term clinical outcome of HCC. Furthermore, data of single‐cell RNA‐seq showed that (1) cells transforming from effector CD8+ T cells to exhausted CD8+ T cells simultaneously expressed upregulated effector molecules and inhibitory receptors, (2) indicated alteration of gene expression related to stress response and cell cycle at early exhaustion stage, and (3) immunosuppressive Treg had profound activation in comparison to resting Tregs. Conclusions T cell exhaustion is a progressive process, and the gene‐expression profiling displayed T cell exhaustion and anergy are different. Accordingly, it is possible that functional exhaustion is caused by the combination effects of passive defects and overactivation in stress response. The results help to understand the dynamic framework of T cells function in cancer which is important for designing rational cancer immunotherapies.

Epidemiological results revealed that there is an inverse correlation between high-density lipoprotein (HDL) cholesterol levels and risks of atherosclerotic cardiovascular disease (ASCVD). Mounting evidence supports that HDLs are atheroprotective, therefore, many therapeutic approaches have been developed to increase HDL cholesterol (HDL-C) levels. Nevertheless, HDL-raising therapies, such as cholesteryl ester transfer protein (CETP) inhibitors, failed to ameliorate cardiovascular outcomes in clinical trials, thereby casting doubt on the treatment of cardiovascular disease (CVD) by increasing HDL-C levels. Therefore, HDL-targeted interventional studies were shifted to increasing the number of HDL particles capable of promoting ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux. One such approach was the development of reconstituted HDL (rHDL) particles that promote ABCA1-mediated cholesterol efflux from lipid-enriched macrophages. Here, we explore the manipulation of rHDL nanoparticles as a strategy for the treatment of CVD. In addition, we discuss technological capabilities and the challenge of relating preclinical in vivo mice research to clinical studies. Finally, by drawing lessons from developing rHDL nanoparticles, we also incorporate the viabilities and advantages of the development of a molecular imaging probe with HDL nanoparticles when applied to ASCVD, as well as gaps in technology and knowledge required for putting the HDL-targeted therapeutics into full gear.

Cholesterol esters are a key ingredient of foamy cells in atherosclerotic lesions; their formation is catalyzed by two enzymes: acyl-CoA:cholesterol acyltransferases (ACATs; also called sterol O-acyltransferases, or SOATs) ACAT1 and ACAT2. ACAT1 is present in all body cells and is the major isoenzyme in macrophages. Whether blocking ACAT1 benefits atherosclerosis has been under debate for more than a decade. Previously, our laboratory developed a myeloid-specific Acat1 knockout (KO) mouse (Acat1(−M/−M)), devoid of ACAT1 only in macrophages, microglia, and neutrophils. In previous work using the ApoE KO (ApoE(−/−)) mouse model for early lesions, Acat1(−M/−M) significantly reduced lesion macrophage content and suppressed atherosclerosis progression. In advanced lesions, cholesterol crystals become a prominent feature. Here we evaluated the effects of Acat1(−M/−M) in the ApoE KO mouse model for more advanced lesions and found that mice lacking myeloid Acat1 had significantly reduced lesion cholesterol crystal contents. Acat1(−M/−M) also significantly reduced lesion size and macrophage content without increasing apoptotic cell death. Cell culture studies showed that inhibiting ACAT1 in macrophages caused cells to produce less proinflammatory responses upon cholesterol loading by acetyl low-density lipoprotein. In advanced lesions, Acat1(−M/−M) reduced but did not eliminate foamy cells. In advanced plaques isolated from ApoE(−/−) mice, immunostainings showed that both ACAT1 and ACAT2 are present. In cell culture, both enzymes are present in macrophages and smooth muscle cells and contribute to cholesterol ester biosynthesis. Overall, our results support the notion that targeting ACAT1 or targeting both ACAT1 and ACAT2 in macrophages is a novel strategy to treat advanced lesions.

Acyl-CoA:cholesterol acyltransferase 1 (Acat1) converts cellular cholesterol to cholesteryl esters and is considered a drug target for treating atherosclerosis. However, in mouse models for atherosclerosis, global Acat1 knockout (Acat1(-/-)) did not prevent lesion development. Acat1(-/-) increased apoptosis within lesions and led to several additional undesirable phenotypes, including hair loss, dry eye, leukocytosis, xanthomatosis, and a reduced life span. To determine the roles of Acat1 in monocytes/macrophages in atherosclerosis, we produced a myeloid-specific Acat1 knockout (Acat1(-M/-M)) mouse and showed that, in the Apoe knockout (Apoe(-/-)) mouse model for atherosclerosis, Acat1(-M/-M) decreased the plaque area and reduced lesion size without causing leukocytosis, dry eye, hair loss, or a reduced life span. Acat1(-M/-M) enhanced xanthomatosis in apoe(-/-) mice, a skin disease that is not associated with diet-induced atherosclerosis in humans. Analyses of atherosclerotic lesions showed that Acat1(-M/-M) reduced macrophage numbers and diminished the cholesterol and cholesteryl ester load without causing detectable apoptotic cell death. Leukocyte migration analysis in vivo showed that Acat1(-M/-M) caused much fewer leukocytes to appear at the activated endothelium. Studies in inflammatory (Ly6C(hi)-positive) monocytes and in cultured macrophages showed that inhibiting ACAT1 by gene knockout or by pharmacological inhibition caused a significant decrease in integrin β 1 (CD29) expression in activated monocytes/macrophages. The sparse presence of lesion macrophages without Acat1 can therefore, in part, be attributed to decreased interaction between inflammatory monocytes/macrophages lacking Acat1 and the activated endothelium. We conclude that targeting ACAT1 in a myeloid cell lineage suppresses atherosclerosis progression while avoiding many of the undesirable side effects caused by global Acat1 inhibition.

Objective— Macrophages play important roles in the pathogenesis of atherosclerosis, but their dynamics within plaques remain obscure. We aimed to quantify macrophage positional dynamics within progressing and regressing atherosclerotic plaques. Approach and Results— In a stable intravital preparation, large asymmetrical foamy macrophages in the intima of carotid artery plaques were sessile, but smaller rounded cells nearer plaque margins, possibly newly recruited monocytes, mobilized laterally along plaque borders. Thus, to test macrophage dynamics in plaques over a longer period of time in progressing and regressing disease, we quantified displacement of nondegradable phagocytic particles within macrophages for up to 6 weeks. In progressing plaques, macrophage-associated particles appeared to mobilize to deeper layers in plaque, whereas in regressing plaques, the label was persistently located near the lumen. By measuring the distance of the particles from the floor of the plaque, we discovered that particles remained at the same distance from the floor regardless of plaque progression or regression. The apparent deeper penetration of labeled cells in progressing conditions could be attributed to monocyte recruitment that generated new superficial layers of macrophages over the labeled phagocytes. Conclusions— Although there may be individual exceptions, as a population, newly differentiated macrophages fail to penetrate significantly deeper than the limited depth they reside on initial entry, regardless of plaque progression, or regression. These limited dynamics may prevent macrophages from escaping areas with unfavorable conditions (such as hypoxia) and pose a challenge for newly recruited macrophages to clear debris through efferocytosis deep within plaque.

BACKGROUND: Phosphoinositide 3-kinase (PI3K) plays an important role in cellular proliferation and tumor progression. The objective of this study is to evaluate the potential mechanism and therapeutic effects of new PI3K inhibitor SHBM1009 on various cancer cells of digestive system on proliferation. METHODS: Six human hepatocellular carcinoma cell lines including 97H, 97L, A3, F11, MHCC-1, SMMC7721, one gastric cell line SGC-7901 and three primary testicular cancer TYST,TYST1,TYST2 cells were treated by 100ng/ml epidermal growth factor with or without 1uM NVP-BEZ-235 and SHBM1009 in Cell-IQ system in 24-well plates for 48h and up to 72h. The cell bio-behaviors, especially for cell proliferation of total cell number were measured by a real-time cell monitoring system, Cell-IQ continuous cell culturing platform. Images were captured at 30 min intervals. Cell-IQ system uses machine vision technology for monitoring and recording time-lapse data, and the cell functions and morphological parameters were quantified and analyzed. RESULTS: SHBM1009 could prevent EGF-induced cancer cells proliferation. Different patterns of inhibitory effects of SHBM1009 and NVP-BEZ-235, a dual PI3K/mechanistic target of rapamycin inhibitor, on EGF-induced cancer cells proliferation were observed. CONCLUSIONS: PI3K plays a critical role in the development of cancer progress, including proliferation, differentiation and anti-apoptosis. SHBM1009, a new PI3K inhibitor, could be new therapeutic alternatives for cancer treatment.

Macrophages are phagocytes that play important roles in health and diseases. Acyl-CoA:cholesterol acyltransferase 1 (ACAT1) converts cellular cholesterol to cholesteryl esters and is expressed in many cell types. Unlike global Acat1 knockout (KO), myeloid-specific Acat1 KO ( Acat1−) does not cause overt abnormalities in mice. Here, we performed analyses in age- and sex-matched Acat1−M/−Mand wild-type mice on chow or Western diet and discovered that Acat1−M/−Mmice exhibit resistance to Western diet-induced obesity. On both chow and Western diets, Acat1−M/−Mmice display decreased adipocyte size and increased insulin sensitivity. When fed with Western diet, Acat1−M/−Mmice contain fewer infiltrating macrophages in white adipose tissue (WAT), with significantly diminished inflammatory phenotype. Without Acat1, the Ly6Chimonocytes express reduced levels of integrin-β1, which plays a key role in the interaction between monocytes and the inflamed endothelium. Adoptive transfer experiment showed that the appearance of leukocytes from Acat1−M/−Mmice to the inflamed WAT of wild-type mice is significantly diminished. Under Western diet, Acat1−M/−Mcauses suppression of multiple proinflammatory genes in WAT. Cell culture experiments show that in RAW 264.7 macrophages, inhibiting ACAT1 with a small-molecule ACAT1-specific inhibitor reduces inflammatory responses to lipopolysaccharide. We conclude that under Western diet, blocking ACAT1 in macrophages attenuates inflammation in WAT. Other results show that Acat1−M/−Mdoes not compromise antiviral immune response. Our work reveals that blocking ACAT1 suppresses diet-induced obesity in part by slowing down monocyte infiltration to WAT as well as by reducing the inflammatory responses of adipose tissue macrophages.

The lymphatic vasculature plays a key role in regulating tissue fluid homeostasis, lipid transport, and immune surveillance throughout the body. Although it has been appreciated that the heart relies on lymphatic vessels to maintain fluid balance and that such balance must be tightly maintained to allow for normal cardiac output, it has only recently come to light that the lymphatic vasculature may serve as a therapeutic target with which to promote optimal healing following myocardial ischemia and infarction. This article reviews the subject of cardiac lymphatic vessels and highlights studies that imply targeting of lymphatic vessel development or transport using vascular endothelial growth factor-C therapy may serve as a promising avenue for future clinical application in the context of ischemic injury.

HDL is cardioprotective, but plasma HDL levels do not necessarily predict cardiovascular outcomes. The major HDL-associated protein apoA-I picks up its cholesterol from cells within extravascular compartments to return it to plasma and then bile. Yet, tools are lacking to quantify the important step of HDL transit through extravascular spaces. Here, we developed recombinant photoactivatable apoA-I to quantify endogenous HDL recirculation. Using the tool, we studied HDL passage through skin in healthy mice versus those with experimental psoriasis, wherein collagen density increased in the skin in a CD4 + T cell-dependent manner. In control mice, photoactivated HDL mobilized to plasma within 2 h but was retained in collagen-enriched skin of mice with psoriasis. These data suggest that cardiovascular comorbidity in psoriasis might be linked to T cell-mediated structural changes in skin that impedes systemic recirculation of HDL. This new tool is likely to find wide application in HDL research.

Summary Lipoproteins trapped in arteries drive atherosclerosis. Extravascular low-density lipoprotein undergoes receptor uptake, whereas high-density lipoprotein (HDL) interacts with cells to acquire cholesterol and then recirculates to plasma. We developed photoactivatable apoA-I to understand how HDL passage through tissue is regulated. We focused on skin and arteries of healthy mice versus those with psoriasis, which carries cardiovascular risk in man. Our findings suggest that psoriasis-affected skin lesions program interleukin-17-producing T cells in draining lymph nodes to home to distal skin and later to arteries. There, these cells mediate thickening of the collagenous matrix, such that larger molecules including lipoproteins become entrapped. HDL transit was rescued by depleting CD4+ T cells, neutralizing interleukin-17, or inhibiting lysyl oxidase that crosslinks collagen. Experimental psoriasis also increased vascular stiffness and atherosclerosis via this common pathway. Thus, interleukin-17 can reduce lipoprotein trafficking and increase vascular stiffness by, at least in part, remodeling collagen.

A new structure of microwave field effect transistor Dielectric Resonator Oscillator (DRO) working at 8.5GHz is presented with negative resistance theory and harmonic balance theory. In order to magnify the output power and enhance the load pulling, a Buffer Amplifier (BA) is designed after the DRO. The nonlinearity and linearity of this structure are analyzed and optimized with commercial software ADS. Then the properties of this structure are simulated with commercial software HFSS. Simulation results show that DRO co-integrated with BA a high output power of 13.426dBm, at an oscillation frequency of 8.5GHz.

Dipeptidyl peptidase IV (DPP-IV) is a drug target in the treatment of human type II diabetes. It is a type II membrane protein with a single transmembrane domain (TMD) anchoring the extracellular catalytic domain to the membrane. DPP-IV is active as a dimer, with two dimer interacting surfaces located extracellularly. In this study, we demonstrate that the TM of DPP-IV promotes DPP-IV dimerization and rescues monomeric DPP-IV mutants into partial dimers, which is specific and irreplaceable by TMs of other type II membrane proteins. By bioluminescence resonance energy transfer (BRET) and peptide electrophoresis, we found that the TM domain of DPP-IV is dimerized in mammalian cells and in vitro. The TM dimer interaction is very stable, based on our results with TM site-directed mutagenesis. None of the mutations, including the introduction of two prolines, resulted in their complete disruption to monomers. However, these TM proline mutations result in a significant reduction of DPP-IV enzymatic activity, comparable to what is found with mutations near the active site. A systematic analysis of TM structures deposited in the Protein Data Bank showed that prolines in the TM generally produce much bigger kinking angles than occur in nonproline-containing TMs. Thus, the proline-dependent reduction in enzyme activity may result from propagated conformational changes from the TM to the extracellular active site. Our results demonstrate that TM dimerization and conformation contribute significantly to the structure and activity of DPP-IV. Optimal enzymatic activity of DPP-IV requires an optimal interaction of all three dimer interfaces, including its TM.

Reverse cholesterol transport (RCT) is the pathway for removal of peripheral tissue cholesterol and involves transport of cholesterol back to liver for excretion, starting from cellular cholesterol efflux facilitated by lipid-free apolipoprotein A1 (ApoA1) or other lipidated high-density lipoprotein (HDL) particles within the interstitial space. Extracellular cholesterol then is picked up and transported through the lymphatic vasculature before entering into bloodstream. There is increasing evidence supporting a role for enhanced macrophage cholesterol efflux and RCT in ameliorating atherosclerosis, and recent data suggest that these processes may serve as better diagnostic biomarkers than plasma HDL levels. Hence, it is important to better understand the processes governing ApoA1 and HDL influx into peripheral tissues from the bloodstream, modification and facilitation of cellular cholesterol removal within the interstitial space, and transport through the lymphatic vasculature. New findings will complement therapeutic strategies for the treatment of atherosclerotic vascular disease.

Viral infection results in the generation of massive numbers of activated effector CD8 + T cells that recognize viral components. Most of these are short-lived effector T cells (SLECs) that die after clearance of the virus. However, a small proportion of this population survives and forms antigen-specific memory precursor effector cells (MPECs), which ultimately develop into memory cells. These can participate in a recall response upon reexposure to antigen even at protracted times postinfection. Here, antiapoptotic myeloid cell leukemia 1 (MCL1) was found to prolong survival upon T cell stimulation, and mice expressing human MCL1 as a transgene exhibited a skewing in the proportion of CD8 + T cells, away from SLECs toward MPECs, during the acute phase of vaccinia virus infection. A higher frequency and total number of antigen-specific CD8 + T cells were observed in MCL1 transgenic mice. These findings show that MCL1 can shape the makeup of the CD8 + T cell response, promoting the formation of long-term memory. IMPORTANCE During an immune response to a virus, CD8 + T cells kill cells infected by the virus, and most die when the infection resolves. However, a small proportion of cells survives and differentiates into long-lived memory cells that confer protection from reinfection by the same virus. This report shows that transgenic expression of an MCL1 protein enhances survival of memory CD8 + T cells following infection with vaccinia virus. This is important because it shows that MCL1 expression may be an important determinant of the formation of long-term CD8 + T cell memory.

Acyl-CoA:cholesterol acyltransferase 1 (ACAT1) is a resident endoplasmic reticulum enzyme that prevents the buildup of cholesterol in membranes by converting it to cholesterol esters. Blocking ACAT1 pharmacologically or byAcat1gene knock-out (KO) decreases amyloidopathy in mouse models for Alzheimer's disease. However, the beneficial actions of ACAT1 blockage to treat Alzheimer's disease remained not well understood. Microglia play essential roles in the proteolytic clearance of amyloid β (Aβ) peptides. Here we show thatAcat1gene KO in mouse increases phagocytic uptake of oligomeric Aβ1–42 and stimulates lysosomal Aβ1–42 degradation in cultured microglia andin vivo. Additional results show thatAcat1gene KO or a specific ACAT1 inhibitor K604 stimulates autophagosome formation and transcription factor EB-mediated lysosomal proteolysis. Surprisingly, the effect of ACAT1 blockage does not alter mTOR signaling or endoplasmic reticulum stress response but can be modulated by agents that disrupt cholesterol biosynthesis. To our knowledge, our current study provides the first example that a small molecule (K604) can promote autophagy in an mTOR-independent manner to activate the coordinated lysosomal expression and regulation network. Autophagy is needed to degrade misfolded proteins/peptides. Our results implicate that blocking ACAT1 may provide a new way to benefit multiple neurodegenerative diseases.

Acyl-coenzyme A:cholesterol acyltransferases (ACATs), which are members of the membrane-bound O-acyltransferase family, catalyze the conversion of cholesterol to cholesteryl esters. Mammals have two isoenzymes: ACAT1 and ACAT2. Both enzymes are drug targets for treating human diseases. ACAT1 is present in various cell types. It contains nine transmembrane domains (TMDs), with the active site His460 located within TMD7, and the active site Asn421 located within the fourth large cytoplasmic loop. In human ACAT1, a single-nucleotide polymorphism exists for residue 526: the codon is either CAG for Gln, or CGG for Arg. Gln526/Arg526 is present within the C-terminal loop. Its biochemical significance is unknown. In addition, within the C-terminal half of ACAT1, numerous residues conserved with those of ACAT2 are present; the functions of these conserved residues are largely unknown. Here, we performed single-substitution mutagenesis experiments to investigate the roles of individual residues present in the C-terminal loop, including Gln526/Arg526, and the eight conserved Pro residues located near/in various TMDs. The results show that the enzyme activity of ACAT1 with Gln526 is less active than that of ACAT1 with Arg526 by 40%. In addition, several residues in the C-terminal loop are important for maintaining proper ACAT1 protein stability. Other results show that Pro347 plays an important role in modulating enzyme catalysis. Overall, our results imply that the CAG/CGG polymorphism can be utilized to perform ACAT1 activity/human disease susceptibility studies, and that Pro347 located near TMD5 plays an important role in modulating enzyme catalysis.

Objective— To investigate the role of acyl-CoA:cholesterol acyltransferase 1 (ACAT1) in hematopoiesis. Approach and Results— ACAT1 converts cellular cholesterol to cholesteryl esters for storage in multiple cell types and is a potential drug target for human diseases. In mouse models for atherosclerosis, global Acat1 knockout causes increased lesion size; bone marrow transplantation experiments suggest that the increased lesion size might be caused by ACAT1 deficiency in macrophages. However, bone marrow contains hematopoietic stem cells that give rise to cells in myeloid and lymphoid lineages; these cell types affect atherosclerosis at various stages. Here, we test the hypothesis that global Acat1 −/− may affect hematopoiesis, rather than affecting macrophage function only, and show that Acat1 −/− mice contain significantly higher numbers of myeloid cells and other cells than wild-type mice. Detailed analysis of bone marrow cells demonstrated that Acat1 −/− causes a higher proportion of the stem cell–enriched Lin − Sca-1 + c-Kit + population to proliferate, resulting in higher numbers of myeloid progenitor cells. In addition, we show that Acat1 −/− causes higher monocytosis in Apoe −/− mouse during atherosclerosis development. Conclusions— ACAT1 plays important roles in hematopoiesis in normal mouse and in Apoe −/− mouse during atherosclerosis development.

Video coding is a complex process, comprising a combination of spatial, temporal and statistical data reduction techniques. Of these techniques, motion estimation taking advantage of inter-frame information redundancy plays the most important role. So finding the most efficient motion estimation algorithm remains an important research problem. In this paper, a New Diamond Search (NDS) algorithm is proposed to improve the efficiency of video coding. The NDS algorithm applies Cross Search Pattern (CSP) in the very beginning search steps and switch using Large Diamond Search Pattern (LDSP) and Small Diamond Search Pattern (SDSP), so as to avoid the problem of local optimum. Experimental results show that the proposed NDS algorithm is obviously improved in search speed and search accuracy compared with the Diamond Search algorithm. The NDS algorithm can achieve very close quality compared to full search but with 18.51times speedup. In term of speed, compared with the DS algorithm, the NDS algorithm can achieve more than 125% speedup.

DPP-IV is a prolyl dipeptidase, cleaving the peptide bond after the penultimate proline residue. It is an important drug target for the treatment of type II diabetes. DPP-IV is active as a dimer, and monomeric DPP-IV has been speculated to be inactive. In this study, we have identified the C-terminal loop of DPP-IV, highly conserved among prolyl dipeptidases, as essential for dimer formation and optimal catalysis. The conserved residue His750 on the loop contributes significantly for dimer stability. We have determined the quaternary structures of the wild type, H750A, and H750E mutant enzymes by several independent methods including chemical cross-linking, gel electrophoresis, size exclusion chromatography, and analytical ultracentrifugation. Wild-type DPP-IV exists as dimers both in the intact cell and in vitro after purification from human semen or insect cells. The H750A mutation results in a mixture of DPP-IV dimer and monomer. H750A dimer has the same kinetic constants as those of the wild type, whereas the H750A monomer has a 60-fold decrease in kcat. Replacement of His750 with a negatively charged Glu (H750E) results in nearly exclusive monomers with a 300-fold decrease in catalytic activity. Interestingly, there is no dynamic equilibrium between the dimer and the monomer for all forms of DPP-IVs studied here. This is the first study of the function of the C-terminal loop as well as monomeric mutant DPP-IVs with respect to their enzymatic activities. The study has important implications for the discovery of drugs targeted to the dimer interface.

The dipeptidyl peptidase (DPP) family members, including DPP-IV, DPP8, DPP9 and others, cleave the peptide bond after the penultimate proline residue and are drug target rich. The dimerization of DPP-IV is required for its activity. A propeller loop located at the dimer interface is highly conserved within the family. Here we carried out site-directed mutagenesis on the loop of DPPIV and identified several residues important for dimer formation and enzymatic activity. Interestingly, the corresponding residues on DPP9 have a different impact whereby the mutations decrease activity without changing dimerization. Thus the propeller loop seems to play a varying role in different DPPs.Structured summary of protein interactionsDPP-IV and DPP-IVphysically interact by comigration in gel electrophoresis (View interaction: 1, 2, 3, 4)DPP9 and DPP9bind by circular dichroism (View interaction)DPP-IV and DPP-IVbind by circular dichroism (View interaction: 1, 2, 3, 4, 5)DPP-IV and DPP-IVbind by cosedimentation in solution (View interaction: 1, 2, 3, 4, 5)ADAbinds to DPP-IV by surface plasmon resonance (View interaction: 1, 2, 3, 4, 5, 6)DPP9 and DPP9bind by cosedimentation in solution (View interaction)