A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...

Nanjing Medical University

Anthrax is a highly lethal infectious disease caused by the bacterium Bacillus anthracis, and the associated shock is closely related to the lethal toxin (LeTx) produced by the bacterium. The central role played by the 63 kDa protective antigen (PA63) region of LeTx in the pathophysiology of anthrax makes it an excellent therapeutic target. In the present study, a human/murine chimeric IgG mAb, hmPA6, was developed by inserting murine antibody variable regions into human constant regions using antibody engineering technology. hmPA6 expressed in 293F cells could neutralize LeTx both in vitro and in vivo. At a dose of 0.3 mg/kg, it could protect all tested rats from a lethal dose of LeTx. Even administration of 0.6 mg/kg hmPA6 48 h before LeTx challenge protected all tested rats. The results indicate that hmPA6 is a potential candidate for clinical application in anthrax treatment.

A Novel Chimeric Anti-PA Neutralizing Antibody for Postexposure Prophylaxis and Treatment of Anthrax.

To evaluate and compare the effects of nanostructured, diamondlike, carbon (DLC) coating and nitrocarburizing on the frictional properties and biocompatibility of orthodontic stainless steel archwires.

Effects of nanostructured, diamondlike, carbon coating and nitrocarburizing on the frictional properties and biocompatibility of orthodontic stainless steel wires.

Osteoclasts originate from bone marrow monocyte/macrophage lineage cells, which are important for bone health. Class A scavenger receptor (SR-A) is a multifunctional molecule that functions during differentiation of monocyte into macrophages and osteoclasts. To further characterize the role of SR-A in osteoclasts, we used the murine tooth movement model (TM) and the murine anterior cruciate ligament transection model of osteoarthritis (ACLT OA). In these two models the bones involved are of different origin and have different properties. Bone resorption was decreased in SR-A mice compared to SR-A mice. Further evaluation showed that the number of multinucleated osteoclasts in SR-A mice, compared to SR-A mice, was significantly decreased both  and . The levels of interleukin-6 (IL-6) produced by osteoclasts were reduced in SR-A mice compared to SR-A mice. In the  marrow-derived osteoclast formation assay and in both mouse models, osteoclastogenesis was restored to normal in SR-A mice by administration of recombinant murine IL-6. Moreover, neutralization of IL-6 reduced the number of osteoclasts formed in SR-A mice of TM model. Both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal protein kinase (JNK), but not p38, signaling pathways were downregulated in receptor activator of nuclear factor-κB ligand (RANKL)-stimulated SR-A osteoclasts. Importantly, when treated with either ERK or JNK inhibitor, the numbers of osteoclasts generated from RANKL-induced bone marrow derived-macrophages of SR-A mice, and their IL-6 production, were significantly decreased. This suggests that SR-A activates the ERK and JNK signaling pathways, and promotes production of IL-6 by osteoclasts to further stimulate osteoclast formation.

Class A Scavenger Receptor Exacerbates Osteoclastogenesis by an Interleukin-6-Mediated Mechanism through ERK and JNK Signaling Pathways.

Osteoblasts play a major role in bone remodeling and are regulated by transcription factors. GATA4, a zinc finger transcription factor from the GATA family, has an unclear role in osteoblast differentiation. In this study, the role of GATA4 in osteoblast differentiation was studied both in vitro and in vivo by GATA4 knockdown. GATA4 expression increased during osteoblast differentiation. GATA4 knockdown in osteoblast precursor cells reduced alkaline phosphatase activity and decreased the formation of calcified nodule in an osteogenic-induced cell culture system. In vivo, micro-CT showed that local injection of lentivirus-delivered GATA4 shRNA caused reduced new bone formation during tooth movement. Histological analyses such as total collagen and Goldner's trichrome staining confirmed these results. In vivo immunohistochemical analysis showed reduced expression of osterix (OSX), osteopontin (OPN), and osteocalcin (OCN) in the shGATA4 group (P < 0.05). Consistently, both western blotting and quantitative reverse-transcription PCR proved that expression of osteogenesis-related genes, including OSX, OPN, and OCN, was significantly repressed in the shGATA4 group in vitro (P < 0.01). For further analysis of the pathways involved in this process, we examined the MAPK signaling pathway, and found knockdown of GATA4, downregulated p38 signaling pathways (P < 0.01). Collectively, these results imply GATA4 is a regulator of osteoblastic differentiation via the p38 signaling pathways.

GATA4 regulates osteoblastic differentiation and bone remodeling via p38-mediated signaling.

Corticision is a common technique to accelerate orthodontic tooth movement; however, not much is known about the underlying mechanisms. In this study, we investigated the mechanism of alveolar tissue remodeling after corticision in a rat model of tooth movement (TM) by analyzing the differential transcriptome. A total of 36 male rats were equally divided into TM and TM with corticision (TM+C) groups. Alveolar bone response was examined using micro-computed tomography (micro-CT). Osteoclasts and osteoblasts were quantified on tartrate-resistant acid phosphatase (TRAP) and Goldner's trichrome staining. The transcriptomes of alveolus around the left maxillary first molar were determined on RNA sequencing (RNA-Seq), and the expression of selected differentially expressed genes (DEGs) validated on quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Immunohistochemical examination of alveolar tissue was performed to examine the expressions of correlative proteins of the selected signaling pathway in the TM and TM+C groups. The ratio of bone volume to total volume (BV/TV), and the trabecular number (Tb.N) were significantly decreased, while the movement distance and the trabecular separation (Tb.Sp) was significantly increased in the TM+C group. However, no significant between-group difference in trabecular thickness (Tb.Th) was observed. On histomorphometric analysis, a significant increase in the number of osteoclasts and increased bone resorption was observed in the TM+C group. A total of 399 DEGs were identified on RNA-SEq. Eleven selected genes were confirmed on qRT-PCR, which included components of the Ras signaling pathway. Four proteins of the Ras signaling pathway showed a higher expression in the TM+C group. Our findings indicate that corticision may speed up orthodontic tooth movement by accelerating osteoclastogenesis mediated via the Ras signaling pathway.

Effect of corticision on orthodontic tooth movement in a rat model as assessed by RNA sequencing.

Transcription factor GATA4 regulates cardiac and osteoblast differentiation. However, its role in tooth development is not clear. Therefore, we generated Wnt1-Cre;GATA4  mice, with conditional inactivation of the GATA4 gene in the dental papilla mesenchymal cells. Phenotypic analysis showed short root deformity along with reduced expressions of odonto/osteogenic markers. Proliferation (but not apoptosis) of cells around the apical area of the root was attenuated. In vitro, we knocked down GATA4 expression in stem cells of dental apical papilla (SCAPs). Proliferation, migration and odonto/osteogenic differentiation of SCAPs were affected in the shGATA4 group. Overexpression of GATA4 in SCAPs increased mineralization. Based on our previous iTRAQ results, guanine nucleotide binding proteins 3 (GNAI3) is one of the distinct proteins after GATA4 deletion. G protein signaling is involved in bone development, remodeling, and disease. In this study, both GATA4 deletion in the mouse root and knock-down in human SCAPs decreased the expression of GNAI3. Dual-luciferase and ChIP assay confirmed the direct binding of GATA4 to the GNAI3 promoter, both in vitro and in vivo. GNAI3 knock-down significantly decreased the odonto/osteogenic differentiation ability of SCAPs. We thus establish the role of GATA4 as a novel regulator of root development and elucidate its downstream molecular events.

Role of GATA binding protein 4 (GATA4) in the regulation of tooth development via GNAI3.

Platelet-rich plasma (PRP) is used in the clinic as an autologous blood product to stimulate bone regeneration and chondrogenesis. Numerous studies have demonstrated that PRP affects bone remodeling by accelerating osteoblast formation. With the research perspective focusing on osteoclasts, the present study established a mouse model of mandibular advancement to examine the effect of PRP on osteoclast differentiation induced by modification of the dynamics of the temporomandibular joint (TMJ). The lower incisors of the mice were trimmed by 1 mm and the resultant change in mandibular position during the process of eating induced condylar adaptation to this change. PRP significantly increased the bone mass and decreased osteoclastic activity, in vitro as well as in vivo. Mechanistically, the reduced expression of receptor activator of nuclear factor-κB ligand (RANKL)‑induced differentiation marker genes, including nuclear factor of activated T-cells, cytoplasmic 1, c-fos and tartrate-resistant acid phosphatase, and that of the resorptive activity marker genes such as cathepsin k, carbonic anhydrase 2 and matrix metalloproteinase 9, indicated that PRP suppresses RANKL-induced osteoclast differentiation. A microarray analysis revealed that several genes associated with the Wnt pathway were differentially expressed, which indicated the involvement of this pathway in osteoclast differentiation. Furthermore, the activation of the Wnt pathway was verified by reverse transcription-quantitative polymerase chain reaction and immunoblot analysis of Dickkopf-related protein 1 and β-catenin. The results of the present study indicated that PRP inhibits osteoclast differentiation through activation of the Wnt pathway.

Platelet-rich plasma inhibits RANKL-induced osteoclast differentiation through activation of Wnt pathway during bone remodeling.

The role of GATA-binding protein 4 (GATA4) in neural crest cells (NCCs) is poorly defined. Here we showed that mouse NCCs lacking GATA4 exhibited developmental defects in craniofacial bone, teeth, and heart. The defects likely occurred due to decreased cell proliferation at the developmental stage. The in vitro results were consistent with the mouse model. The isobaric tags for relative and absolute quantitation assay revealed that BARX1 is one of the differentially expressed proteins after GATA4 knockdown in NCCs. On the basis of the results of dual-luciferase, electro-mobility shift, and chromatin immunoprecipitation assays, Barx1 expression is directly regulated by GATA4 in NCCs. In zebrafish, gata4 knockdown affects the development of NCCs derivatives. However, the phenotype in zebrafish could be partly rescued by co-injection of gata4 morpholino oligomers and barx1 mRNA. This study identified new downstream targets of GATA4 in NCCs and uncovered additional evidence of the complex regulatory functions of GATA4 in NCC development.

GATA4 as a novel regulator involved in the development of the neural crest and craniofacial skeleton via Barx1.

Bacillus anthracis causes a highly lethal infectious disease primarily due to toxin-mediated injury. Antibiotics are no longer effective to treat the accumulation of anthrax toxin, thereby new strategies of antibody treatment are essential. Two anti- anthrax protective antigen (PA) antibodies, hmPA6 and PA21, have been reported by our lab previously.

Different mechanisms of two anti-anthrax protective antigen antibodies and function comparison between them.

Glioblastoma multiforme (GBM) is the most common and lethal type of primary brain tumor. More than half of GBMs contain mutation(s) of PTEN/PI3K/AKT, making inhibitors targeting the PI3K pathway very attractive for clinical investigation. However, so far, PI3K/AKT/mTOR inhibitors have not achieved satisfactory therapeutic effects in clinical trials of GBM. In this study, we aimed to develop a high-throughput screening method for high-throughput identification of potential targeted agents that synergize with PI3K inhibitors in GBM.

A high-throughput drug combination screen identifies an anti-glioma synergism between TH588 and PI3K inhibitors.

 deletion or mutation occurs in 30% to 60% of patients with glioblastoma (GBM) and is associated with poor prognosis. Efficacious therapy for this subgroup of patients is currently lacking. To identify potential target(s) to selectively suppress -deficient GBM growth, we performed a three-step synthetic lethal screen on LN18  wild-type (WT) and knockout (KO) isogeneic GBM cell lines using a library containing 606 target-selective inhibitors. A MCL1 inhibitor UMI-77 identified in the screen exhibited excellent suppression on the proliferation, colony formation, 3D spheroid, and neurosphere formation of -deficient GBM cells. Mechanistically, loss of  in GBM cells led to upregulation of MCL1 in posttranslational level via inhibition of GSK3β, and consequently confer cells resistance to apoptosis. Pharmacologic inhibition or knockdown of MCL1 blocked this PI3K-GSK3β-MCL1 axis and caused reduction of several antiapoptotic proteins, finally induced massive caspase-3 cleavage and apoptosis. In both subcutaneous and orthotopic GBM models, knockdown of MCL1 significantly impaired the  growth of -deficient xenografts. Moreover, the combination of UMI-77 and temozolomide synergistically killed -deficient GBM cells. Collectively, our work identified MCL1 as a promising target for -deficient GBM. For future clinical investigations, priority should be given to the development of a selective MCL1 inhibitor with efficient brain delivery and minimal  toxicity.

Targeting the Synthetic Vulnerability of -Deficient Glioblastoma Cells with MCL1 Inhibitors.

serotype 2 ( 2) is an important swine pathogen and also an emerging zoonotic agent. HtpsA has been reported as an immunogenic cell surface protein on the bacterium. In the present study, we constructed an isogenic mutant strain of A, namely ΔA, to study its role in the development and virulence of  2. Our results showed that the mutant strain lost its typical encapsulated structure with decreased concentrations of sialic acid. Furthermore, the survival rate in whole blood, the anti-phagocytosis by RAW264.7 murine macrophage, and the adherence ability to HEp-2 cells were all significantly affected in the ΔA. In addition, the deletion of A sharply attenuated the virulence of  2 in an infection model of mouse. RNA-seq analysis revealed that 126 genes were differentially expressed between the ΔA and the wild-type strains, including 28 upregulated and 98 downregulated genes. Among the downregulated genes, many were involved in carbohydrate metabolism and synthesis of virulence-associated factors. Taken together, A was demonstrated to play a role in the morphological development and pathogenesis of the highly virulent  2 05ZYH33 strain.

Inactivation of the A gene affects capsule development and pathogenicity of .

Previous studies have revealed that activation of the Toll‑like receptor 4 (TLR4)‑mediated proinflammatory signaling pathway plays an important role in acute inflammation, sepsis and chronic inflammatory disorders. Moreover, TLR4 significantly contributes to lipopolysaccharide (LPS)‑induced immune response. Thus, modulation of the TLR4 pathway is an important strategy to specifically target these pathologies. The aim of the present study was to develop a complete human anti‑TLR4 IgG2 antibody by screening human TLR4 Fab from a phage‑display library and integrating it with constant regions of the heavy chain of human IgG2 via antibody engineering. ELISA, a BLItz system and fluorescence‑activated cell sorting were used to assess its affinity. Furthermore, mouse‑derived peritoneal macrophages were treated with human anti‑TLR4 IgG2 and induced with LPS in vitro. Reverse transcription‑quantitative PCR and western blotting were used to determine mRNA expression levels of cytokines and phosphorylation levels of signaling pathways, respectively. It was found that human anti‑TLR4 IgG2 bound to TLR4 with a high affinity of 8.713x10‑10 M, and that preincubation with anti‑TLR4 IgG2 inhibited the LPS‑induced production of tumor necrosis factor‑α, interferon‑β and interleukin‑6 mRNA expression levels in mouse peritoneal macrophages. It was also demonstrated that human anti‑TLR4 IgG2 inhibited LPS‑induced TLR4 signaling by reducing the phosphorylation of the NF‑κB, mitogen‑activated protein kinase and interferon regulatory factor 3 signaling pathways. In addition, human anti‑TLR4 IgG2 protected mice from LPS challenge with a survival rate of 40% and also significantly increased the survival time in the cecal ligation and puncture model. Therefore, it was speculated that human anti‑TLR4 IgG2 plays a protective role against sepsis‑associated injury and is potentially applicable for the treatment of infection‑associated immune dysfunction.

Tingting Zhou

Department of Cell Biology,
School of Basic Medical Sciences,
Department of Cell Biology, School of Basic Medical Sciences

Nanjing Medical University

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

Tingting Zhou

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cell Biology,School of Basic Medical Sciences,Department of Cell Biology, School of Basic Medical Sciences

Nanjing Medical University

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

Department of Cell Biology,
School of Basic Medical Sciences,
Department of Cell Biology, School of Basic Medical Sciences