EZ spheres: A stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs

We have developed a simple method to generate and expand multipotent, self-renewing pre-rosette neural stem cells from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) without utilizing embryoid body formation, manual selection techniques, or complex combinations of small molecules. Human ESC and iPSC colonies were lifted and placed in a neural stem cell medium containing high concentrations of EGF and FGF-2. Cell aggregates (termed EZ spheres) could be expanded for long periods using a chopping method that maintained cell–cell contact. Early passage EZ spheres rapidly down-regulated OCT4 and up-regulated SOX2 and nestin expression. They retained the potential to form neural rosettes and consistently differentiated into a range of central and peripheral neural lineages. Thus, they represent a very early neural stem cell with greater differentiation flexibility than other previously described methods. As such, they will be useful for the rapidly expanding field of neurological development and disease modeling, high-content screening, and regenerative therapies based on pluripotent stem cell technology.     

1H, 13C and 15N chemical shift assignments of Ninjurin1 Extracellular N-terminal Domain

Cell adhesion molecules play a crucial role in fundamental biological processes via regulating cell–cell interactions. Nerve injury induced protein1 (Ninjurin1) is a novel adhesion protein that has no significant homology with other known cell adhesion molecules. Here we present the assignment of an 81 aa construct for human Ninjurin1 Extracellular N-Terminal (ENT) domain, which comprises the critical adhesion domain.     

20(R)-Ginsenoside Rf: A new ginsenoside from red ginseng extract

In spite of the general concept that herbal supplements are safe, there is a lack of appropriate quality control measures and regulations that often culminates in serious undesirable effects such as allergic reactions and renal and liver damage. Thus, there is a growing need to establish a suitable methodology that enables authentication and quality assurance of herbal products. The root of Panax ginseng C. A. Meyer (Araliaceae), commonly called ginseng, is traditionally recognized as a prominent herbal medicine in Far East Asia. There are two types of processed ginseng, white and red ginseng, based on processing methods, and these play a significant role in modifying ginsenosides, which are the major bioactive metabolites in these products. Herein we purify and characterize a new ginsenoside, 20(R)-ginsenoside Rf, utilizing NMR, UPLC-ESI-Q-TOF-MS and validate the metabolite is generated from its epimer, 20(S)-ginsenoside Rf during the steaming process to manufacture red ginseng. We further propose a relevant mechanism for the chemical conversion. This finding updates chemical profiling of ginseng products that can be employed in quality assurance and authentication.     

A new fused tetracyclic heterocyclic antioxidant from Serratia sp. PAMC 25557

A new fused tetracyclic heterocyclic compound, (4bR,10bR)-4b-hydroxy-10b,12-dihydrodibenzo[c,h][2,6]naphthyridine-5,11(4bH,6H)-dione (1), and a known compound, butyl 2-[(benzoyloxy)methyl]benzoate, spatozoate 2, were isolated from the broth culture of Serratia sp. PAMC 25557. The structure of 1 was determined by analyzing spectroscopic data. Compound 1 did not exhibit antimicrobial activity against Escherichia coli, Staphylococcus aureus, or Candida albicans. In addition, up to 100 μg/ml compound 1 did not show any toxicity against Artemia salina larvae. However, compound 1 showed DPPH free radical scavenging activity (IC₅₀ = 16.7 ± 0.34 μg/ml). This was the first report of spatozoate isolation from bacterial sources.     

Soluble Guanylate Cyclase α1–Deficient Mice: A Novel Murine Model for Primary Open Angle Glaucoma

Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. The molecular signaling involved in the pathogenesis of POAG remains unknown. Here, we report that mice lacking the α₁ subunit of the nitric oxide receptor soluble guanylate cyclase represent a novel and translatable animal model of POAG, characterized by thinning of the retinal nerve fiber layer and loss of optic nerve axons in the context of an open iridocorneal angle. The optic neuropathy associated with soluble guanylate cyclase α₁–deficiency was accompanied by modestly increased intraocular pressure and retinal vascular dysfunction. Moreover, data from a candidate gene association study suggests that a variant in the locus containing the genes encoding for the α₁ and β₁ subunits of soluble guanylate cyclase is associated with POAG in patients presenting with initial paracentral vision loss, a disease subtype thought to be associated with vascular dysregulation. These findings provide new insights into the pathogenesis and genetics of POAG and suggest new therapeutic strategies for POAG.     

Differential Immune Responses to Segniliparus rotundus and Segniliparus rugosus Infection and Analysis of Their Comparative Virulence Profiles

Two closely related bacterial species, Segniliparus rotundus and Segniliparus rugosus, have emerged as important human pathogens, but little is known about the immune responses they elicit or their comparative pathophysiologies. To determine the virulence and immune responses of the two species, we compared their abilities to grow in phagocytic and non-phagocytic cells. Both species maintained non-replicating states within A549 epithelial cells. S. rugosus persisted longer and multiplied more rapidly inside murine bone marrow-derived macrophages (BMDMs), induced more pro-inflammatory cytokines, and induced higher levels of macrophage necrosis. Activation of BMDMs by both species was mediated by toll-like receptor 2 (TLR2), followed by mitogen-activated protein kinases (MAPK) and nuclear factor κB (NF-κB) signaling pathways, indicating a critical role for TLR2 in Segniliparus-induced macrophage activation. S. rugosus triggered faster and stronger activation of MAPK signaling and IκB degradation, indicating that S. rugosus induces more pro-inflammatory cytokines than S. rotundus. Multifocal granulomatous inflammations in the liver and lung were observed in mice infected with S. rugosus, but S. rotundus was rapidly cleared from all organs tested within 15 days post-infection. Furthermore, S. rugosus induced faster infiltration of innate immune cells such as neutrophils and macrophages to the lung than S. rotundus. Our results suggest that S. rugosus is more virulent and induces a stronger immune response than S. rotundus.     

A Novel Balanced-Lethal Host-Vector System Based on glmS

During the last decade, an increasing number of papers have described the use of various genera of bacteria, including E. coli and S. typhimurium, in the treatment of cancer. This is primarily due to the facts that not only are these bacteria capable of accumulating in the tumor mass, but they can also be engineered to deliver specific therapeutic proteins directly to the tumor site. However, a major obstacle exists in that bacteria because the plasmid carrying the therapeutic gene is not needed for bacterial survival, these plasmids are often lost from the bacteria. Here, we report the development of a balanced-lethal host-vector system based on deletion of the glmS gene in E. coli and S. typhimurium. This system takes advantage of the phenotype of the GlmS⁻ mutant, which undergoes lysis in animal systems that lack the nutrients required for proliferation of the mutant bacteria, D-glucosamine (GlcN) or N-acetyl-D-glucosamine (GlcNAc), components necessary for peptidoglycan synthesis. We demonstrate that plasmids carrying a glmS gene (GlmS⁺p) complemented the phenotype of the GlmS⁻ mutant, and that GlmS⁺p was maintained faithfully both in vitro and in an animal system in the absence of selection pressure. This was further verified by bioluminescent signals from GlmS ⁺pLux carried in bacteria that accumulated in grafted tumor tissue in a mouse model. The signal was up to several hundred-fold stronger than that from the control plasmid, pLux, due to faithful maintenance of the plasmid. We believe this system will allow to package a therapeutic gene onto an expression plasmid for bacterial delivery to the tumor site without subsequent loss of plasmid expression as well as to quantify bioluminescent bacteria using in vivo imaging by providing a direct correlation between photon flux and bacterial number.     

Sox2 Level Is a Determinant of Cellular Reprogramming Potential

Epiblast stem cells (EpiSCs) and embryonic stem cells (ESCs) differ in their in vivo differentiation potential. While ESCs form teratomas and efficiently contribute to the development of chimeras, EpiSCs form teratomas but very rarely chimeras. In contrast to their differentiation potential, the reprogramming potential of EpiSCs has not yet been investigated. Here we demonstrate that the epiblast-derived pluripotent stem cells EpiSCs and P19 embryonal carcinoma cells (ECCs) exhibit a lower reprogramming potential than ESCs and F9 ECCs. In addition, we show that the low reprogramming ability is due to the lower levels of Sox2 in epiblast-derived stem cells. Consistent with this observation, overexpression of Sox2 enhances reprogramming efficiency. In summary, these findings suggest that a low reprogramming potential is a general feature of epiblast-derived stem cells and that the Sox2 level is a determinant of the cellular reprogramming potential.     

Comparative Genomics of 12 Strains of Erwinia amylovora Identifies a Pan-Genome with a Large Conserved Core

The plant pathogen Erwinia amylovora can be divided into two host-specific groupings; strains infecting a broad range of hosts within the Rosaceae subfamily Spiraeoideae (e.g., Malus, Pyrus, Crataegus, Sorbus) and strains infecting Rubus (raspberries and blackberries). Comparative genomic analysis of 12 strains representing distinct populations (e.g., geographic, temporal, host origin) of E. amylovora was used to describe the pan-genome of this major pathogen. The pan-genome contains 5751 coding sequences and is highly conserved relative to other phytopathogenic bacteria comprising on average 89% conserved, core genes. The chromosomes of Spiraeoideae-infecting strains were highly homogeneous, while greater genetic diversity was observed between Spiraeoideae- and Rubus-infecting strains (and among individual Rubus-infecting strains), the majority of which was attributed to variable genomic islands. Based on genomic distance scores and phylogenetic analysis, the Rubus-infecting strain ATCC BAA-2158 was genetically more closely related to the Spiraeoideae-infecting strains of E. amylovora than it was to the other Rubus-infecting strains. Analysis of the accessory genomes of Spiraeoideae- and Rubus-infecting strains has identified putative host-specific determinants including variation in the effector protein HopX1_Ea and a putative secondary metabolite pathway only present in Rubus-infecting strains.     

Fine-Needle Aspirates CYFRA 21-1 is a Useful Tumor Marker for Detecting Axillary Lymph Node Metastasis in Breast Cancer Patients

Introduction

To assess whether the value of CYFRA21-1 in the aspirates of ultrasonography-guided fine-needle aspiration biopsy (US-FNAB) can contribute to improving the performances of US-FNAB in the diagnosis of axillary lymph node (LN) metastasis in breast cancer patients.

Methods

US-FNAB was performed in 156 axillary LNs in 152 breast cancer patients (mean age: 51.4 years, range: 17–92 years). Concentrations of CYFRA21-1 were measured from washouts of the syringe used during US-FNAB. Tumor marker concentrations, US-FNAB, intraoperative sentinel node biopsy (SNB), and surgical pathology results were reviewed and analyzed. For comparison, the values of CEA and CA15-3 were also measured from washouts.

Results

Among the 156 LNs, 75 (48.1%) were benign, and 81 (51.9%) were metastases. Mean concentrations of CYFRA21-1 were significantly higher in metastasis compared to benign LNs (P<0.001). US-FNAB combined to CYFRA21-1 showed significantly higher sensitivity, NPV, and accuracy compared to US-FNAB alone (all values P<0.05). All diagnostic indices of US-FNAB combined to CYFRA21-1 were significantly higher compared to US-FNAB combined with CEA or CA15-3 (all P<0.001). Of the 28 metastatic LNs which showed metastasis on SNB, CYFRA21-1 showed higher positive rate of 75.0% (CEA or CA15-3∶60.7%, P = 0.076).

Conclusion

Measuring CYFRA 21-1 concentrations from US-FNAB aspirates improves sensitivity, NPV, and accuracy of US-FNAB alone, and may contribute to reducing up to 75.0% of unnecessary intraoperative SNB. Compared to CEA or CA15-3, CYFRA21-1 shows significantly higher performances when combined to US-FNAB in the preoperative diagnosis of LN metastasis in breast cancer patients.