Multifunctional Immunoliposomes Enhance the Immunotherapeutic Effects of PD-L1 Antibodies against Melanoma by Reprogramming Immunosuppressive Tumor Microenvironment.

The immunosuppressive tumor microenvironment (TME) can significantly limit the immunotherapeutic effects of the PD-L1 antibody (aPDL1) by inhibiting the infiltration of CD8+ cytotoxic T cells (CTLs) into the tumor tissues. However, how to reprogram the immunosuppressive TME and promote the infiltration of CTLs remains a huge challenge for aPDL1 to achieve the maximum benefits. Herein, the authors design a multifunctional immunoliposome that encapsulates the adrenergic receptor blocker carvedilol (CAR) and connects the "don't eat me" signal antibody (aCD47) and aPDL1 in series via a reactive oxygen species (ROS)-sensitive linker on the surface. In ROS-enriched immunosuppressive TME, the multifunctional immunoliposome (CAR@aCD47/aPDL1-SSL) can first release the outer aCD47 to block the "do not eat me" pathway, promote the phagocytosis of tumor cells by phagocytic cells, and activate CTLs. Then, the aPDL1 on the liposome surface is exposed to block the PD-1/PD-L1 signaling pathway, thereby inducing CTLs to kill tumor cells. CAR encapsulated in CAR@aCD47/aPDL1-SSL can block the adrenergic nerves in the tumor tissues and reduce their densities, thereby inhibiting angiogenesis in the tumor tissues and reprogramming the immunosuppressive TME. According to the results, CAR@aCD47/aPDL1-SSL holds an effective way to reprogram the immunosuppressive TME and significantly enhance immunotherapeutic efficiency of aPDL1 against the primary cancer and metastasis.

Structural genomics studies of human caries pathogen Streptococcus mutans.

Gram-positive bacterium Streptococcus mutans is the primary causative agent of human dental caries. To better understand this pathogen at the atomic structure level and to establish potential drug and vaccine targets, we have carried out structural genomics research since 2005. To achieve the goal, we have developed various in-house automation systems including novel high-throughput crystallization equipment and methods, based on which a large-scale, high-efficiency and low-cost platform has been establish in our laboratory. From a total of 1,963 annotated open reading frames, 1,391 non-membrane targets were selected prioritized by protein sequence similarities to unknown structures, and clustered by restriction sites to allow for cost-effective high-throughput conventional cloning. Selected proteins were over-expressed in different strains of Escherichia coli. Clones expressed soluble proteins were selected, expanded, and expressed proteins were purified and subjected to crystallization trials. Finally, protein crystals were subjected to X-ray analysis and structures were determined by crystallographic methods. Using the previously established procedures, we have so far obtained more than 200 kinds of protein crystals and 100 kinds of crystal structures involved in different biological pathways. In this paper we demonstrate and review a possibility of performing structural genomics studies at moderate laboratory scale. Furthermore, the techniques and methods developed in our study can be widely applied to conventional structural biology research practice.

Polyethyleneimine-coating enhances adenoviral transduction of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are non-hematopoietic cells with multi-lineage potential, which makes them attractive targets for regenerative medicine applications. Efficient gene transfer into MSCs is essential for basic research in developmental biology and for therapeutic applications involving gene-modification in regenerative medicine. Adenovirus vectors (Advs) can efficiently and transiently introduce an exogenous gene into many cell types via their primary receptors, the coxsackievirus and adenovirus receptors (CARs), but not into MSCs, which lack CAR expression. To overcome this problem, an Adv coated with cationic polymer polyethyleneimine (PEI) was developed. In this study, we demonstrated that PEI coating with an optimal ratio can enhance adenoviral transduction of MSCs without cytotoxicity. We also investigated the physicochemical properties and internalization mechanisms of the PEI-coated Adv. These results could help to evaluate the potentiality of the PEI-coated Adv as a prototype vector for efficient and safe transduction into MSCs.

[Methods of BMP immobilization and evaluation for Ti-based dental implant surface modification].

Ti-based biomaterial has been widely used as dental and bone implant material although its bioactivity still needs improvement especially for dental implant. The bone morphogenetic proteins(BMPs) bound to Ti-based materials will attract the mesenchymal stem cells to differentiate into osteoblast cells, which benefits the response to the protein-material surface, and finally leads to new bone formation. Several methods including physically mixing, coating, plasma immobilization and cross-linking were used to investigate how BMPs bind to Ti-based biomaterials. The latest research papers are focused on the structure and function of BMPs, and the methods to bind BMPs to Ti-based biomaterial and the evaluation methods after protein immobilization are reviewed in this paper.

Protein preparation, crystallization and preliminary X-ray crystallographic analysis of Smu.1475c from caries pathogen Streptococcus mutans.

The gene smu.1475c encodes a putative protein of 211 residues in Streptococcus mutans, a primary pathogen for human dental caries. In this work, smu.1475c was cloned into pET28a and expressed in good amount from the E. coli strain BL21 (DE3). Smu.1475c protein was purified to homogeneity in a two-step procedure of Ni2+ chelating and size exclusion chromatography. Crystals were obtained by hanging-drop vapor-diffusion method and diffracted to 2.7 angstroms resolution. The crystal belongs to orthorhombic space group P2(1)2(1)2(1) with cell dimension of a = 68.3 angstroms, b = 105.9 angstroms, c = 136.2 angstroms. The asymmetric unit is expected to contain four molecules with solvent content of 49.4%.

Protein preparation, crystallization and preliminary X-ray crystallographic analysis of SMU.961 protein from the caries pathogen Streptococcus mutans.

The smu.961 gene encodes a putative protein of 183 residues in Streptococcus mutans, a major pathogen in human dental caries. The gene was cloned into expression vector pET28a and expressed in a substantial quantity in Escherichia coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.961 was purified to homogeneity in a two-step procedure consisting of Ni2+-chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.9 A resolution at beamline I911-3, MAX-II-lab, Sweden. The crystal belonged to space group C2, with unit-cell parameters a = 98.62, b = 73.73, c = 184.73 A, beta = 98.82 degrees.

Crystallization and preliminary X-ray crystallographic studies of SMU.134 protein from caries pathogen Streptococcus mutans.

The smu.134 gene encodes a putative transcriptional regulator of 217 residues in Streptococcus mutans, a major pathogen for human dental caries. The gene was cloned into expression vector pET28alpha and expressed in soluble form in E. coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.134 was purified to homogeneity in a two step procedure of Ni(2+ ) chelating and size exclusion chromatography. Crystals suitable for X-ray diffraction were obtained by hanging-drop vapor diffusion method and diffracted to 2.6 A;. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a=55.03 A, b=80.84 A, c=107.96 A.

Get phases from arsenic anomalous scattering: de novo SAD phasing of two protein structures crystallized in cacodylate buffer.

The crystal structures of two proteins, a putative pyrazinamidase/nicotinamidase from the dental pathogen Streptococcus mutans (SmPncA) and the human caspase-6 (Casp6), were solved by de novo arsenic single-wavelength anomalous diffraction (As-SAD) phasing method. Arsenic (As), an uncommonly used element in SAD phasing, was covalently introduced into proteins by cacodylic acid, the buffering agent in the crystallization reservoirs. In SmPncA, the only cysteine was bound to dimethylarsinoyl, which is a pentavalent arsenic group (As (V)). This arsenic atom and a protein-bound zinc atom both generated anomalous signals. The predominant contribution, however, was from the As anomalous signals, which were sufficient to phase the SmPncA structure alone. In Casp6, four cysteines were found to bind cacodyl, a trivalent arsenic group (As (III)), in the presence of the reducing agent, dithiothreitol (DTT), and arsenic atoms were the only anomalous scatterers for SAD phasing. Analyses and discussion of these two As-SAD phasing examples and comparison of As with other traditional heavy atoms that generate anomalous signals, together with a few arsenic-based de novo phasing cases reported previously strongly suggest that As is an ideal anomalous scatterer for SAD phasing in protein crystallography.

Crystal structures of phosphotransferase system enzymes PtxB (IIB(Asc)) and PtxA (IIA(Asc)) from Streptococcus mutans.

Streptococcus mutans is the primary etiological agent of dental caries in man and other mammalian organisms. This bacterium metabolizes carbohydrates actively and thrives under anaerobic conditions by fermenting l-ascorbate (Asc) via the sga operon, which includes SgaT, PtxB, and PtxA. These three proteins are members of the Asc family of enzyme II (EII) complexes of the bacterial phosphotransferase system. Here, we report the crystal structure of PtxB, solved by single-wavelength anomalous dispersion phasing, and that of PtxA, solved by molecular replacement, from S. mutans. PtxB provides the first crystal structure of an EIIB from the Asc family, composed of a central beta sheet of parallel strands flanked by alpha helices on both sides. The structure of PtxB is similar to the structures of IIB(Mtl) (IIB subunit of mannitol PTS) and IIB(Cel) (IIB subunit of cellobiose) in Escherichia coli despite the low sequence identity. PtxA adopts a globular alpha/beta sandwich structure. The phosphorylation-site His68 is situated between beta2 and beta3, within a hydrophobic pocket. We found that the hydrogen bond on N(delta1) of the active-site histidine is a common means of ensuring that phosphate is on the correct N(varepsilon2) site in many EIIA families. Finally, a model of the PtxB-PtxA complex was constructed, and a PtxA-phospho-PtxB state is proposed. Analyses of the two structures shed light on the catalytic mechanism of the phosphotransferase system.

Bioinformatics and structural characterization of a hypothetical protein from Streptococcus mutans: implication of antibiotic resistance.

As an oral bacterial pathogen, Streptococcus mutans has been known as the aetiologic agent of human dental caries. Among a total of 1960 identified proteins within the genome of this organism, there are about 500 without any known functions. One of these proteins, SMU.440, has very few homologs in the current protein databases and it does not fall into any protein functional families. Phylogenetic studies showed that SMU.440 is related to a particular ecological niche and conserved specifically in some oral pathogens, due to lateral gene transfer. The co-occurrence of a MarR protein within the same operon among these oral pathogens suggests that SMU.440 may be associated with antibiotic resistance. The structure determination of SMU.440 revealed that it shares the same fold and a similar pocket as polyketide cyclases, which indicated that it is very likely to bind some polyketide-like molecules. From the interlinking structural and bioinformatics studies, we have concluded that SMU.440 could be involved in polyketide-like antibiotic resistance, providing a better understanding of this hypothetical protein. Besides, the combination of multiple methods in this study can be used as a general approach for functional studies of a protein with unknown function.

[Histological regeneration process of "neo-esophagus"].

BACKGROUND & OBJECTIVE: During the reconstruction process of organs or tissues, different implant materials can lead to different healing results because of different extracellular matrix interfaces and tissue biocompatibilities. This study was to observe the regeneration process of "neo-esophagus"after implanting an artificial esophagus, and investigate its healing mechanism. METHODS: Histopathologic studies on neo-esophagus in 1, 3, 6, 12, and 24 months after implantation were performed using gross-giant specimen technique and special staining methods. The processes of tissue molding and reconstruction, and regeneration of high-level cell organ in "neo-esophagus"were observed. RESULTS: The artificial esophagus temporarily replaced the defective esophagus at the early stage after implantation, and dropped off about 1 month after operation. The epithelization of neo-esophagus induced by host tissue was completed about 3-6 months after operation. The submucous muscle layer, mucous glands, nerve fiber, and capillaries were reconstructed about 12 months after operation. The narrowing of "neo-esophagus" occurred about 3-6 months after operation and was relieved 12 months after operation. CONCLUSIONS: The implanted artificial esophagus made of bio-material can replace the defective esophagus at the early stage, and induce connective tissues, including collagen and fibroblasts, to deposit and cover on it to form neo-esophagus. The neo-esophagus is developed by epithelization and reconstruction of submucous muscle layer, mucous glands, nerve fiber, and capillaries 12 months after operation. The narrowing of "neo-esophagus" is caused by overgrowth and contraction of scars.

Expression, purification and crystallization of an extended-spectrum beta-lactamase from Klebsiella oxytoca.

OXY-1a is an extended-spectrum beta-lactamase from the conditional pathogenic bacterium Klebsiella oxytoca. OXY-1a is responsible for the antibiotic resistance of this pathogen. A soluble form of OXY-1a with a His tag at its C-terminus was overexpressed in Escherichia coli. The recombinant protein was purified and crystallized at room temperature using PEG 4000 as the main precipitant. Two crystal forms were obtained from the same growth conditions. One was orthorhombic, with crystals that diffracted to better than 1.9 A, while the other was tetragonal, with crystals that only diffracted to about 3.0 A. Complete data sets were collected from both crystal forms. The orthorhombic crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 46.54, b = 73.43, c = 84.56 A, while the tetragonal crystal has unit-cell parameters a = b = 73.72, c = 96.81 A. The asymmetric unit of the orthorhombic crystal is estimated to contain one OXY-1a molecule, giving a crystal volume per protein weight (V(M)) of 2.25 A(3) Da(-1) and a solvent content of 45%.