Forward Genetic Dissection of Biofilm Development by Fusobacterium nucleatum: Novel Functions of Cell Division Proteins FtsX and EnvC.

Fusobacterium nucleatum is a key member of the human oral biofilm. It is also implicated in preterm birth and colorectal cancer. To facilitate basic studies of fusobacterial virulence, we describe here a versatile transposon mutagenesis procedure and a pilot screen for mutants defective in biofilm formation. Out of 10 independent biofilm-defective mutants isolated, the affected genes included the homologs of the Escherichia coli cell division proteins FtsX and EnvC, the electron transport protein RnfA, and four proteins with unknown functions. Next, a facile new gene deletion method demonstrated that nonpolar, in-frame deletion of ftsX or envC produces viable bacteria that are highly filamentous due to defective cell division. Transmission electron and cryo-electron microscopy revealed that the ΔftsX and ΔenvC mutant cells remain joined with apparent constriction, and scanning electron microscopy (EM) uncovered a smooth cell surface without the microfolds present in wild-type cells. FtsX and EnvC proteins interact with each other as well as a common set of interacting partners, many with unknown function. Last, biofilm development is altered when cell division is blocked by MinC overproduction; however, unlike the phenotypes of ΔftsX and ΔenvC mutants, a weakly adherent biofilm is formed, and the wild-type rugged cell surface is maintained. Therefore, FtsX and EnvC may perform novel functions in Fusobacterium cell biology. This is the first report of an unbiased approach to uncover genetic determinants of fusobacterial biofilm development. It points to an intriguing link among cytokinesis, cell surface dynamics, and biofilm formation, whose molecular underpinnings remain to be elucidated.IMPORTANCE Little is known about the virulence mechanisms and associated factors in F. nucleatum, due mainly to the lack of convenient genetic tools for this organism. We employed two efficient genetic strategies to identify F. nucleatum biofilm-defective mutants, revealing FtsX and EnvC among seven biofilm-associated factors. Electron microscopy established cell division defects of the ΔftsX and ΔenvC mutants, accompanied with a smooth cell surface, unlike the microfold, rugged appearance of wild-type bacteria. Proteomic studies demonstrated that FtsX and EnvC interact with each other as well as a set of common and unique interacting proteins, many with unknown functions. Importantly, blocking cell division by MinC overproduction led to formation of a weakly adherent biofilm, without alteration of the wild-type cell surface. Thus, this work links cell division and surface dynamics to biofilm development and lays a foundation for future genetic and biochemical investigations of basic cellular processes in this clinically significant pathogen

GPER-induced signaling is essential for the survival of breast cancer stem cells.

G protein-coupled estrogen receptor-1 (GPER), a member of the G protein-coupled receptor (GPCR) superfamily, mediates estrogen-induced proliferation of normal and malignant breast epithelial cells. However, its role in breast cancer stem cells (BCSCs) remains unclear. Here we showed greater expression of GPER in BCSCs than non-BCSCs of three patient-derived xenografts of ER- /PR+ breast cancers. GPER silencing reduced stemness features of BCSCs as reflected by reduced mammosphere forming capacity in vitro, and tumor growth in vivo with decreased BCSC populations. Comparative phosphoproteomics revealed greater GPER-mediated PKA/BAD signaling in BCSCs. Activation of GPER by its ligands, including tamoxifen (TMX), induced phosphorylation of PKA and BAD-Ser118 to sustain BCSC characteristics. Transfection with a dominant-negative mutant BAD (Ser118Ala) led to reduced cell survival. Taken together, GPER and its downstream signaling play a key role in maintaining the stemness of BCSCs, suggesting that GPER is a potential therapeutic target for eradicating BCSCs

Pioneering Astaxanthin-Tumor Cell Membrane Nanoparticles for Innovative Targeted Drug Delivery on Melanoma

BackgroundRecently, the use of the tumor or its secretions as drug carriers has gradually become popular, with the advantages of high biocompatibility and enhanced drug delivery to specific cells. Melanoma is the most malignant tumor of all skin cancers; it is the most metastatic and, therefore, the most difficult to treat. The main purpose of this study is to develop nanovesicles with tumor cell membrane secretion properties to encapsulate target substances to enhance the therapeutic effect of cancer.MethodsAstaxanthin was selected as an anticancer drug due to our previous research finding that astaxanthin has extremely high antioxidant, anti-ultraviolet damage, and anti-tumor properties. The manufacturing method of the astaxanthin nanovesicle carrier is to mix melanoma cells and astaxanthin in an appropriate ratio and then remove the genetic material and inflammatory factors of cancer cells by extrusion.ResultsIn terms of results, after the co-culture of astaxanthin nanovesicles and melanoma cancer cells, it was confirmed that the ability of astaxanthin nanovesicles to inhibit the growth and metastasis of melanoma cancer cells was significantly better than the same amount of astaxanthin alone, and it had no effect on normal Human cells are also effective. There was no apparent harm on normal cells, indicating the ability of the vesicles to be selectively transported.ConclusionOur findings illustrated the potential of astaxanthin nanovesicles as an anticancer drug

Natural orifice transluminal endoscopic surgery: A transtracheal approach for the thoracic cavity in a live canine model

BackgroundThe present study aimed to evaluate the performance of transtracheal thoracic exploration and pericardial window creation in a canine survival model.MethodsTransthoracic exploration was performed in 14 dogs. Under general anesthesia, after an incision in the right lateral wall of the middle–lower portion of the trachea was made, a 9-mm metal tube was advanced into the thoracic cavity. For thoracic cavity exploration and pericardial window creation, a flexible bronchoscope was introduced through the metal tube into the thoracic cavity. After thoracoscopy, a Dumon stent (Novatech, Grasse, France) was used to cover the tracheal incision site and facilitate healing. Animals were evaluated by endoscopy 1 and 2 weeks later. Animals were humanely killed, and necropsy was performed 2 weeks after the transtracheal natural orifice transluminal endoscopic surgery.ResultsFourteen dogs underwent transtracheal thoracic exploration lasting for an average of 110 minutes (range, 80–150), with 3 perioperative deaths. At 2 weeks after pericardial window creation, endoscopy revealed normal healing of the tracheal incision sites in all 11 surviving animals. Necropsy on the 11 animals at 2 weeks showed 9 adhesions around the pericardial window and 5 adhesions around the tracheal incision region. No mediastinitis or abscesses could be identified.ConclusionsTranstracheal thoracic exploration is technically feasible. Increasing surgical experience together with improvement in endoscopic techniques will further facilitate the development of natural orifice transluminal endoscopic surgery for thoracic diseases

Transforming growth factor-β1 induces matrix metalloproteinase-9 and cell migration in astrocytes: roles of ROS-dependent ERK- and JNK-NF-κB pathways

<p>Abstract</p> <p>Background</p> <p>Transforming growth factor-β (TGF-β) and matrix metalloproteinases (MMPs) are the multifunctional factors during diverse physiological and pathological processes including development, wound healing, proliferation, and cancer metastasis. Both TGF-β and MMPs have been shown to play crucial roles in brain pathological changes. Thus, we investigated the molecular mechanisms underlying TGF-β1-induced MMP-9 expression in brain astrocytes.</p> <p>Methods</p> <p>Rat brain astrocytes (RBA-1) were used. MMP-9 expression was analyzed by gelatin zymography and RT-PCR. The involvement of signaling molecules including MAPKs and NF-κB in the responses was investigated using pharmacological inhibitors and dominant negative mutants, determined by western blot and gene promoter assay. The functional activity of MMP-9 was evaluated by cell migration assay.</p> <p>Results</p> <p>Here we report that TGF-β1 induces MMP-9 expression and enzymatic activity via a TGF-β receptor-activated reactive oxygen species (ROS)-dependent signaling pathway. ROS production leads to activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun-N-terminal kinase (JNK) and then activation of the NF-κB transcription factor. Activated NF-κB turns on transcription of the MMP-9 gene. The rat MMP-9 promoter, containing a NF-κB <it>cis</it>-binding site, was identified as a crucial domain linking to TGF-β1 action.</p> <p>Conclusions</p> <p>Collectively, in RBA-1 cells, activation of ERK1/2- and JNK-NF-κB cascades by a ROS-dependent manner is essential for MMP-9 up-regulation/activation and cell migration induced by TGF-β1. These findings indicate a new regulatory pathway of TGF-β1 in regulating expression of MMP-9 in brain astrocytes, which is involved in physiological and pathological tissue remodeling of central nervous system.</p

Identification and pathogenicity of Aeromonas veronii isolated from sexually mature female Hypophthalmichthys molitrix

An outbreak of bacterial septicemia in female silver carp *Hypophthalmichthys molitrix*, which caused significant death of the fish in the Yantian Reservoir, was investigated. The pathogen was isolated from diseased fish and identified as *Aeromonas veronii* utilizing biochemical characteristics and molecular methods analyses. An artificial infection experiment indicated that the strain caused 100% mortality of juvenile silver carp and mature female fish with eggs but no death of the mature male fish. Silver carp challenged with *A. veronii* showed similar clinical signs with naturally infected fish. The histopathological study revealed that *A. veronii* infection caused the increment of hemosiderin granules and the vacuole formation in tissues of the spleen and livers, as well as the collapse, deformation, and disintegration of egg cells. The ACP, AKP, and CAT enzyme activities in the serum of both naturally and artificially infected silver carp were decreased significantly with the severe infection. In this study, *A. veronii* was isolated and identified as the primary bacterial pathogen causing the mass death of sexually mature female silver carp with eggs in Yantian Reservoir