基因定点诱变删除及天然α型心钠素的分泌型表达

用基因定点诱变技术,删除了pO_1α ANF表达质粒中的33对碱基,使人α型心钠素结构基因直接与大肠杆菌分泌型表达质粒pIN-Ⅲ-OmPA中的信号肽酶切位点编码区相连,构成天然人α型心钠素的表达质粒pANF,在IPTG诱导下表达28肽的天然人α型心钠素。纯化后的表达产物具有天然心钠素的放免活性和很强的舒张血管的生物活性。     

Utilization of the tricarboxylic acid cycle, a reactor design criterion for the microaerobic production of 2,3-butanediol

A new parameter, the relative utilization of tricarboxylic acid (TCA) cycle beta, is introduced to quantitatively account for the involvement of fermentation pathways and TCA cycle in the utilization of oxygen under oxygen-limiting (microaerobic) conditions. With the facultative anaerobe Enterobacter aerogenes, which produces 2,3-butanediol, a method is proposed to calculate beta from measurement of metabolites and exhaust gas. In continuous culture beta was found to be small under oxygen limitation, indicating that the fermentation pathways were preferred over the TCA cycle and oxygen was almost entirely consumed through oxidation of reduced nicotinamide adenine dinucleotide (NADH(2)) released by fermentation under these conditions. The increase of beta at high oxygen supply revealed a saturation of oxygen utilization through fermentation pathways. It could be concluded that, for the optimal performance of a microaerobic culture, oxygen uptake rate must be kept at such a level that as much NADH(2) as possible from fermentation pathways is oxidized by oxygen, and at the same time the utilization of TCA cycle is kept at a minimum. As the dynamics of the microaerobic culture can be fast, a significant effect of reactor hydrodynamics, i.e., mixing, on the overall performance can be expected. This was confirmed experimentally, and the parameter beta proved to be a useful reactor design criterium for the microaerobic cultivation. (c) 1992 John Wiley & Sons, Inc.     

Structural and functional relationships of human DNA polymerases

A continuing theme of our laboraory, has been the understanding of human DNA polymerases at the structural level. We have purified DNA polymerases delta, epsilon and alpha from human placenta. Monoclonal antibodies to these polymerases were isolated and used as tools to study their immunochemical relationships. These studies have shown that while DNA polymerases delta, epsilon and alpha are discrete protiens, they must share common structural features by virtue of the ability of several of our monoclonal antibodies to exhibit cross-reactivity. A second approach we have taken is the molecular cloning of human DNA polymerase delta and epsilon. We have cloned the DNA polymerase delta cDNA, and this has allowed us to compare its primary structure to those of human polymerase alpha and other members of this polymerase family. Multiple sequence alignments have revealed that human DNA polymerase delta is also closely related to the herpes virus family of DNA polymerases. In situ hybridization has shown that the human DNA polymerase delta gene is localized to chromosome 19 q13.3–q13.4. In order to further determine the functional regions of the DNA polymerase δ structure we are currently expressing human pol δ inE. coli and baculovirus systems. Other work in our laboratory is directed toward examining the expression of DNA polymerase δ during the cell cycle.     

Molecular cloning and expression of a cDNA encoding endothelial cell nitric oxide synthase.

Endothelium-derived relaxing factor (EDRF), identified as nitric oxide (NO), is derived from a guanidino nitrogen of L-arginine via its metabolism by nitric oxide synthase (NOS). Herein, we report the molecular cloning of a cDNA encoding the constitutive calcium-calmodulin (Ca2+/CaM)-regulated nitric oxide synthase (ECNOS). A full-length ECNOS clone was isolated by screening a bovine aortic endothelial cell cDNA library using a fragment of rat brain NOS (bNOS) cDNA. This cDNA has an open reading frame of 3615 nucleotides encoding a 1205-amino acid protein. Membranes prepared from COS cells transfected with the ECNOS cDNA demonstrated NADPH- and Ca2+/CaM- dependent conversion of L-, but not D-, arginine to NO and citrulline that was inhibited by NG-nitro-L-arginine methyl ester. Comparison of the deduced amino acid sequence of ECNOS to the bNOS and macrophage NOS (Mac-NOS) sequences revealed 57 and 50% identity, respectively. In addition, ECNOS contains a unique N-myristylation consensus sequence (not shared by bNOS or Mac-NOS) that may explain its membrane localization.     

Isolation of Chinese hamster ovary cell lines producing Man3GlcNAc2 asparagine-linked glycans.

Chinese hamster ovary lines with two mutations, one causing accumulation of Man5GlcNAc2-P-P-dolichol and a second resulting in defective N-acetylglucosaminyltransferase I activity, synthesize asparagine-linked glycans with the structure Man3GlcNAc2. As a result, the asparagine-linked glycans produced by these lines are smaller and less heterogeneous than those produced by other currently available animal cell lines.     

The host inflammatory response contributes to disease severity in Crimean-Congo hemorrhagic fever virus infected mice

Crimean-Congo hemorrhagic fever virus (CCHFV) is an important human pathogen. In cell culture, CCHFV is sensed by the cytoplasmic RNA sensor retinoic acid-inducible gene I (RIG-I) molecule and its adaptor molecule mitochondrial antiviral signaling (MAVS) protein. MAVS initiates both type I interferon (IFN-I) and proinflammatory responses. Here, we studied the role MAVS plays in CCHFV infection in mice in both the presence and absence of IFN-I activity. MAVS-deficient mice were not susceptible to CCHFV infection when IFN-I signaling was active and showed no signs of disease. When IFN-I signaling was blocked by antibody, MAVS-deficient mice lost significant weight, but were uniformly protected from lethal disease, whereas all control mice succumbed to infection. Cytokine activity in the infected MAVS-deficient mice was markedly blunted. Subsequent investigation revealed that CCHFV infected mice lacking TNF-α receptor signaling (TNFA-R-deficient), but not IL-6 or IL-1 activity, had more limited liver injury and were largely protected from lethal outcomes. Treatment of mice with an anti-TNF-α neutralizing antibody also conferred partial protection in a post-virus exposure setting. Additionally, we found that a disease causing, but non-lethal strain of CCHFV produced more blunted inflammatory cytokine responses compared to a lethal strain in mice. Our work reveals that MAVS activation and cytokine production both contribute to CCHFV pathogenesis, potentially identifying new therapeutic targets to treat this disease.     

Intravital lipid droplet labeling and imaging reveals the phenotypes and functions of individual macrophages in vivo

Macrophages play pivotal roles in the maintenance of tissue homeostasis. However, the reactivation of macrophages toward proinflammatory states correlates with a plethora of inflammatory diseases, including atherosclerosis, obesity, neurodegeneration, and bone marrow (BM) failure syndromes. The lack of methods to reveal macrophage phenotype and function in vivo impedes the translational research of these diseases. Here, we found that proinflammatory macrophages accumulate intracellular lipid droplets (LDs) relative to resting or noninflammatory macrophages both in vitro and in vivo, indicating that LD accumulation serves as a structural biomarker for macrophage phenotyping. To realize the staining and imaging of macrophage LDs in vivo, we developed a fluorescent fatty acid analog-loaded poly(lactic-co-glycolic acid) nanoparticle to label macrophages in mice with high efficiency and specificity. Using these novel nanoparticles, we achieved in situ functional identification of single macrophages in BM, liver, lung, and adipose tissues under conditions of acute or chronic inflammation. Moreover, with this intravital imaging platform, we further realized in vivo phenotyping of individual macrophages in the calvarial BM of mice under systemic inflammation. In conclusion, we established an efficient in vivo LD labeling and imaging system for single macrophage phenotyping, which will aid in the development of diagnostics and therapeutic monitoring. Moreover, this method also provides new avenues for the study of lipid trafficking and dynamics in vivo.Supplementary key words: macrophage, inflammation, lipid droplet, nanoparticle delivery, in vivo imaging, fatty acid analog, bone marrow, systemic inflammation, lipid trafficking, biomarker

Macrophages, a type of immune cells, almost reside in all tissues of body, from the skin to the bone marrow (BM) (1). Macrophages have remarkable plasticity, and they can be activated into specific subtypes by modifying their physiology and functions in response to local environmental cues. Activated macrophages are commonly divided into proinflammatory killing subtype and anti-inflammatory repairing subtype. Proinflammatory macrophages responding to bacteria, IFN-γ, and lipopolysaccharide (LPS) are involved in host defense and inflammation, whereas anti-inflammatory macrophages responding to interleukin-4 (IL-4), IL-10, and IL-13 play a pivotal role in tissue homeostasis and remodeling (2). Increasing evidence indicates that the reactivation of macrophages toward proinflammatory states under diverse kinds of stress is correlated with a plethora of inflammatory diseases, such as atherosclerosis, diabetes, obesity, rheumatoid arthritis, neurodegeneration, and BM failure syndromes (3, 4). Thus, characterization of macrophage activation status and the underlying molecular mechanism in situ will help elucidate their functions in these diseases; however, in vivo analysis of the macrophage activation status in their native multicellular microenvironment is challenging.Although lipid droplets (LDs) have been initially described as intracellular fat storage organelles in adipocytes, increasing studies indicate that myeloid cells also form LDs under inflammation and stress (5, 6). Macrophages, as the effector cells of innate immunity, are found to form LDs to support their host defense when exposed to pathogens, such as parasites, bacteria, and viruses (7, 8, 9, 10, 11). However, abnormal LD accumulation in tissue-resident macrophages correlates with the pathogenesis of various inflammatory diseases. For instance, foam cells in atherosclerotic lesions can maintain the local inflammatory response by secreting proinflammatory cytokines (12, 13, 14). Moreover, LD-accumulating microglia contribute to neurodegeneration by producing high levels of reactive oxygen species (ROS) and secreting proinflammatory cytokines (15). These findings indicate that LD accumulation might be a hallmark of macrophages with proinflammatory functions.In this study, based on the typical activation of in vitro BM-derived macrophages, we find that proinflammatory M_{(LPS + IFN-γ)} macrophages are characterized by LD accumulation, whereas resting macrophages and anti-inflammatory M_(IL-4) and M_(IL-10) macrophages do not contain any LDs. These features also hold for Matrigel plug-recruited macrophages and tissue-resident macrophages in mice. These findings demonstrate that LD accumulation could serve as a morphological index to distinguish proinflammatory macrophages from others.It is feasible to distinguish LD-containing cells using imaging techniques, which has translational potential for identification of proinflammatory macrophages in vivo. However, current techniques for LD visualization are traditional in vitro staining method, and in vivo staining and imaging of LD in individual macrophages remains a challenge. Through nanocarrier screening, we selected the poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) as nanocarrier to deliver the lipophilic carbocyanine dye (DiI_C18(5) solid (1,1''-dioctadecyl-3,3,3'',3''-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt) [DiD]) and lipid staining dye (C1-BODIPY 500/510-C12) into macrophages. Using these dual fluorescence-labeled PLGA NPs, we achieved in situ and in vivo functional identification of single macrophages in various tissues under systemic or local inflammatory stress. Collectively, this study establishes an efficient in vivo labeling and imaging system of intracellular LDs for phenotyping the activation status and functions of individual macrophages in their dynamic niche, which is pivotal for disease diagnosis and preclinical research.     

DeepMHADTA: Prediction of Drug-Target Binding Affinity Using Multi-Head Self-Attention and Convolutional Neural Network

Drug-target interactions provide insight into the drug-side effects and drug repositioning. However, wet-lab biochemical experiments are time-consuming and labor-intensive, and are insufficient to meet the pressing demand for drug research and development. With the rapid advancement of deep learning, computational methods are increasingly applied to screen drug-target interactions. Many methods consider this problem as a binary classification task (binding or not), but ignore the quantitative binding affinity. In this paper, we propose a new end-to-end deep learning method called DeepMHADTA, which uses the multi-head self-attention mechanism in a deep residual network to predict drug-target binding affinity. On two benchmark datasets, our method outperformed several current state-of-the-art methods in terms of multiple performance measures, including mean square error (MSE), consistency index (CI), rm2, and PR curve area (AUPR). The results demonstrated that our method achieved better performance in predicting the drug–target binding affinity.