Manganese promotes α-synuclein amyloid aggregation through the induction of protein phase transition.

α-Synuclein (α-Syn) is the major protein component of Lewy bodies, a key pathological feature of Parkinson's disease (PD). The manganese ion Mn2+ has been identified as an environmental risk factor of PD. However, it remains unclear how Mn2+ regulates α-Syn aggregation. Here, we discovered that Mn2+accelerates α-Syn amyloid aggregation through the regulation of protein phase separation. We found that Mn2+ not only promotes α-Syn liquid-to-solid phase transition but also directly induces soluble α-Syn monomers to form solid-like condensates. Interestingly, the lipid membrane is integrated into condensates during Mn2+-induced α-Syn phase transition; however, the preformed Mn2+/α-syn condensates can only recruit lipids to the surface of condensates. In addition, this phase transition can largely facilitate α-Syn amyloid aggregation. Although the Mn2+-induced condensates do not fuse, our results demonstrated that they could recruit soluble α-Syn monomers into the existing condensates. Furthermore, we observed that a manganese chelator reverses Mn2+-induced α-Syn aggregation during the phase transition stage. However, after maturation, α-Syn aggregation becomes irreversible. These findings demonstrate that Mn2+ facilitates α-Syn phase transition to accelerate the formation of α-Syn aggregates and provide new insights for targeting α-Syn phase separation in PD treatment.

A Radical Addition/Cyclization and Se-Group Transfer Strategy for the Facile Synthesis of Se-Containing Cyclopentenes under Metal-Free and Peroxide-Free Conditions.

A novel intermolecular radical addition/cyclization and Se-group transfer reaction of terminal alkynes and unsaturated alkyl selenide is presented which offers a straightforward and facile approach for the synthesis of valuable Se-containing cyclopentenes. Remarkable features of this strategy include easily accessible starting materials, metal-free and peroxide-free conditions, high atom economy, simple operation and broad substrate scope. More importantly, the reaction is easy to scale up and can be extended to the construction of six-membered carbon ring.

Amidation-Ketonization-Selenation of Terminal Alkynes Using TEMPO and Elemental Selenium.

Herein, we present a novel silver- or copper-mediated direct amidation-ketonization-selenation of terminal alkynes for the synthesis of α-oxo-selenoamides. The reaction utilized easily accessible elemental selenium as the source of selenium. In addition, the 18O labeling experiment revealed that TEMPO is the oxygen source of the carbonyl group. The reaction takes advantage of an unsaturated C≡C bond to construct new C═O, C═Se, and C-N bonds in one step.

Growth differentiation factor 11 promotes differentiation of MSCs into endothelial-like cells for angiogenesis.

Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor-ß super family. It has multiple effects on development, physiology and diseases. However, the role of GDF11 in the development of mesenchymal stem cells (MSCs) is not clear. To explore the effects of GDF11 on the differentiation and pro-angiogenic activities of MSCs, mouse bone marrow-derived MSCs were engineered to overexpress GDF11 (MSCGDF11 ) and their capacity for differentiation and paracrine actions were examined both in vitro and in vivo. Expression of endothelial markers CD31 and VEGFR2 at the levels of both mRNA and protein was significantly higher in MSCGDF11 than control MSCs (MSCVector ) during differentiation. More tube formation was observed in MSCGDF11 as compared with controls. In an in vivo angiogenesis assay with Matrigel plug, MSCGDF11 showed more differentiation into CD31+ endothelial-like cells and better pro-angiogenic activity as compared with MSCVector . Mechanistically, the enhanced differentiation by GDF11 involved activation of extracellular-signal-related kinase (ERK) and eukaryotic translation initiation factor 4E (EIF4E). Inhibition of either TGF-ß receptor or ERK diminished the effect of GDF11 on MSC differentiation. In summary, our study unveils the function of GDF11 in the pro-angiogenic activities of MSCs by enhancing endothelial differentiation via the TGFß-R/ERK/EIF4E pathway.

Synthesis of organoselenyl isoquinolinium imides via iron(III) chloride-mediated tandem cyclization/selenation of N'-(2-alkynylbenzylidene)hydrazides and diselenides.

This report describes the synthesis of organoselenyl isoquinolinium imides through a tandem cyclization between N'-(2-alkynylbenzylidene)hydrazides and diselenides. The reaction was carried out at room temperature under an ambient atmosphere using cheap iron(iii) chloride as the metallic source. The strategy shows good tolerance to a broad range of N'-(2-alkynylbenzylidene)hydrazides and diselenides, and forms C-N and C-Se bonds in one step. The obtained product is further transformed into a bioactive H-pyrazolo[5,1-a]isoquinoline skeleton easily via a silver catalyzed [3 + 2] cycloaddition.

Microvesicles facilitate the differentiation of mesenchymal stem cells into pancreatic beta-like cells via miR-181a-5p/150-5p.

Transplantation of pancreatic islet cells is a promising strategy for the long-term treatment of type 1 diabetes (T1D). The stem cell-derived beta cells showed great potential as substitute sources of transplanted pancreatic islet cells. However, the current efficiency of stem cell differentiation still cannot match the requirements for clinical transplantation. Here, we report that microvesicles (MVs) from insulin-producing INS-1 cells could induce mesenchymal stem cell (MSC) differentiation into pancreatic beta-like cells. The combination of MVs with small molecules, nicotinamide and insulin-transferrin-selenium (ITS), dramatically improved the efficiency of MSC differentiation. Notably, the function of MVs in MSC differentiation requires their entry into MSCs through giant pinocytosis. The MVs-treated or MVs combined with small molecules-treated MSCs show pancreatic beta-like cell morphology and response to glucose stimulation in insulin secretion. Using high throughput small RNA-sequencing, we found that MVs induced MSC differentiation into the beta-like cells through miR-181a-5p/150-5p. Together, our findings reveal the role of MVs or the MV-enriched miR-181a-5p/150-5p as a class of biocompatible reagents to differentiate MSCs into functional beta-like cells and demonstrate that the combined usage of MVs or miR-181a-5p/150-5p with small molecules can potentially be used in making pancreatic islet cells for future clinical purposes.

Wnt4 is crucial for cardiac repair by regulating mesenchymal-endothelial transition via the phospho-JNK/JNK.

Rational: Wnt4 plays a critical role in development and is reactivated during fibrotic injury; however, the role of Wnt4 in cardiac repair remains unclear. In this study, our aim was to clarify the pathophysiological role and mechanisms of Wnt4 following acute cardiac ischemic reperfusion injury. Methods and results: We investigated the spatio-temporal expression of Wnt4 following acute cardiac ischemic reperfusion injury and found that Wnt4 was upregulated as an early injury response gene in cardiac fibroblasts near the injury border zone and associated with mesenchymal-endothelial transition (MEndoT), a beneficial process for revascularizing the damaged myocardium in cardiac repair. Using ChIP assay and in vitro and in vivo loss- and gain-of-function, we demonstrated that Wnt4 served as a crucial downstream target gene of p53 during MEndoT. Wnt4 knockdown in cardiac fibroblasts led to decreased MEndoT and worsened cardiac function. Conversely, Wnt4 overexpression in cardiac fibroblasts induced MEndoT in these cells via the phospho-JNK/JNK signaling pathway; however, both the p53 and Wnt4 protein levels were dependent on the ß-catenin signaling pathway. JNK activation plays a critical role in the induction of MEndoT and is crucial for Wnt4 regulated MEndoT. Moreover, Wnt4 overexpression specifically in cardiac fibroblasts rescued the cardiac function worsening due to genetic p53 deletion by decreasing fibrosis and increasing MEndoT and vascular density. Conclusion: Our study revealed that Wnt4 plays a pivotal role in cardiac repair with involvement of phospho-JNK mediated MEndoT and is a crucial gene for cardiac fibroblast-targeted therapy in heart disease.

Divergent synthesis of α-functionalized amides through selective N-O/C-C or N-O/C-C/C-N cleavage of aza-cyclobutanone oxime esters.

Herein, a novel sequential ring opening reaction of aza-cyclobutanone oxime esters with isocyanides is described. The reaction proceeded smoothly under redox-neutral and mild conditions, leading to a divergent synthesis of α-cyanomethylaminoamides, α-acyloxyamides and α-acylaminoamides. In these transformations, a selective N-O/C-C or N-O/C-C/C-N cleavage was achieved only by changing the iron-catalyst system. Among them, a rare sequential N-O/C-C/C-N cleavage process with a classical Passerini or Ugi multicomponent reaction can be executed in a single step. To the best of our knowledge, this work creates a novel reaction mode of cycloketone oximes and provides new opportunities for reaction design.

Mesenchymal-endothelial transition-derived cells as a potential new regulatory target for cardiac hypertrophy.

The role of Mesenchymal-endothelial transition (MEndoT) in cardiac hypertrophy is unclear. To determine the difference between MEndoT-derived and coronary endothelial cells is essential for understanding the revascularizing strategy in cardiac repair. Using lineage tracing we demonstrated that MEndoT-derived cells exhibit highly heterogeneous which were characterized with highly expression of endothelial markers such as vascular endothelial cadherin(VECAD) and occludin but low expression of Tek receptor tyrosine kinase(Tek), isolectin B4, endothelial nitric oxide synthase(eNOS), von Willebrand factor(vWF), and CD31 after cardiac hypertrophy. RNA-sequencing showed altered expression of fibroblast lineage commitment genes in fibroblasts undergoing MEndoT. Compared with fibroblasts, the expression of p53 and most endothelial lineage commitment genes were upregulated in MEndoT-derived cells; however, the further analysis indicated that MEndoT-derived cells may represent an endothelial-like cell sub-population. Loss and gain function study demonstrated that MEndoT-derived cells are substantial sources of neovascularization, which can be manipulated to attenuate cardiac hypertrophy and preserve cardiac function by improving the expression of endothelial markers in MEndoT-derived cells. Moreover, fibroblasts undergoing MEndoT showed significantly upregulated anti-hypertrophic factors and downregulated pro-hypertrophic factors. Therefore MEndoT-derived cells are an endothelial-like cell population that can be regulated to treat cardiac hypertrophy by improving neovascularization and altering the paracrine effect of fibroblasts.

PBX homeobox 1 enhances hair follicle mesenchymal stem cell proliferation and reprogramming through activation of the AKT/glycogen synthase kinase signaling pathway and suppression of apoptosis.

BACKGROUND: PBX homeobox 1 (PBX1) is involved in the maintenance of the pluripotency of human embryonic and hematopoietic stem cells; however, the effects of PBX1 in the self-renewal and reprogramming of hair follicle mesenchymal stem cells (HF-MSCs) are unclear. The AKT/glycogen synthase kinase (GSK) 3ß pathway regulates cell metabolism, proliferation, apoptosis, and reprogramming, and p16 and p21, which act downstream of this pathway, regulate cell proliferation, cell cycle, and apoptosis induced by reprogramming. Here, we aimed to elucidate the roles of PBX1 in regulating the proliferation and reprogramming of HF-MSCs. METHODS: A lentiviral vector designed to carry the PBX1 sequence or PBX1 short hairpin RNA sequence was used to overexpress or knock down PBX1. The roles of PBX1 in proliferation and apoptosis were investigated by flow cytometry. Real-time polymerase chain reaction was performed to evaluate pluripotent gene expression. Dual-luciferase reporter assays were performed to examine the transcriptional activity of the NANOG promoter. Western blotting was performed to identify the molecules downstream of PBX1 involved in proliferation and reprogramming. Caspase3 activity was detected to assess HF-MSC reprogramming. The phosphatidylinositol 3-kinase/AKT inhibitor LY294002 was used to inhibit the phosphorylation and activity of AKT. RESULTS: Overexpression of PBX1 in HF-MSCs increased the phosphorylation of AKT and nuclear translocation of ß-catenin, resulting in the progression of the cell cycle from G0/G1 to S phase. Moreover, transfection with a combination of five transcription factors (SOMKP) in HF-MSCs enhanced the formation of alkaline phosphatase-stained colonies compared with that in HF-MSCs transfected with a combination of four transcription factors (SOMK). PBX1 upregulated Nanog transcription by activating the promoter and promoted the expression of endogenous SOX2 and OCT4. Furthermore, PBX1 expression activated the AKT/glycogen synthase kinase (GSK) 3ß pathway and reduced apoptosis during the early stages of reprogramming. Inhibition of phospho-AKT or knockdown of PBX1 promoted mitochondrion-mediated apoptosis and reduced reprogramming efficiency. CONCLUSIONS: PBX1 enhanced HF-MSC proliferation, and HF-MSCs induced pluripotent stem cells (iPSC) generation by activating the AKT/GSK3ß signaling pathway. During the reprogramming of HF-MSCs into HF-iPSCs, PBX1 activated the NANOG promoter, upregulated NANOG, and inhibited mitochondrion-mediated apoptosis via the AKT/GSK3ß pathway during the early stages of reprogramming.

Biochemical and enzymatic properties of a novel marine fibrinolytic enzyme from Urechis unicinctus.

A novel potent protease, Urechis unicinctus fibrinolytic enzyme (UFE), was first discovered by our laboratory. In this study, we further investigated the enzymatic properties and dynamic parameters of UFE. As a low molecular weight protein, UFE appeared to be very stable to heat and pH. When the temperature was <50 degrees C, the remnant enzyme activity remained almost unchanged, but when the temperature was raised to 60 degrees C the remnant enzyme activity began to decrease rapidly. UFE was quite stable in a pH range of 3.0-12.0, especially at slightly alkaline pH values. Mn(2+), Cu(2+), and Fe(2+) ions were activators of UFE, whereas Fe(3+) and Ag(+) ions were inhibitors. Fe(2+) ion along with Fe(3+) ion might regulate UFE activity in vivo. The optimum pH and temperature of UFE were about 8.0 and 50 degrees C, respectively. When using casein as substrate and a substrate concentration <0.1% casein (w/v), the reaction velocity was increased with substrate concentration. Also when using casein as substrate, the determined K(m) and V(max) of UFE were 0.5298 mg/mL and 3.0845 mol of L-tyrosine equivalent, respectively. Our systematic research results are significant when UFE is applied for medical and industrial purposes.

The bioreactor: a powerful tool for large-scale culture of animal cells.

Bioreactors play a key role in the field of biologics, where they are used for the production of recombinant therapeutic proteins by large-scale cultivation of animal cells. There are several types of bioreactors, including stirred-tank, airlift, hollow-fiber, and Rotary Cell Culture System (RCCS) designs. The stirred-tank bioreactor is one of the most commonly used types, and is used both for industrial applications and laboratory research. The RCCS, invented by NASA, is increasingly used in the area of tissue engineering for medical purposes. Important improvements have been made in the design of traditional bioreactors, and new types of bioreactor are also being developed such as Couette-Taylor bioreactor, multifunctional-membrane bioreactor, and shaking bioreactor. Work is also progressing on techniques to improve the performance of bioreactors, including perfusion culture, the use of microcarriers, and methods of suppressing apoptosis and of monitoring cell growth in real time. Given the demand for the production by animal cells for use in the growing number of clinical applications, further advances in bioreactor technology can be expected during the next few years. Two main goals will be pursued: firstly, to increase output by high density cultivation of animal cells to produce high value protein pharmaceutics or viral vectors for clinical gene therapy; and secondly, to create a three-dimension space similar to that of an in vivo environment to regenerate tissue or organ and to reproduce valuable cells that are hard to culture in the traditional culture system.

Associations between CYP1A1 and CYP2E1 polymorphisms and susceptibility to esophageal cancer in Chaoshan and Taihang areas of China.

PURPOSE: To study the causes of esophageal cancer in Chaoshan and Taihang areas. METHODS: By using gel-based DNA microarray genotyping method, four cancer-related polymorphisms including CYP1A1 m2, CYP1A1 m4, CYP2E1 Pst I and CYP2E1 Rsa I were studied with 565 (CYP1A1) or 482 (CYP2E1) cases and 468 (CYP1A1) or 466 (CYP2E1) controls. RESULTS: For CYP1A1 m2, the mutant allele frequencies were 21.3% (Chaoshan) and 19.6% (Taihang), and OR for AG versus AA genotype (Chaoshan) was 1.855 (95% CI [1.227-2.805]). For CYP1A1 m4, no mutant allele was detectable. For CYP2E1 Pst I, the mutant allele frequencies were 27.3% (Chaoshan) and 29.4% (Taihang), and OR for GG versus CC genotype (Taihang) was 3.263 (95% CI [1.059-10.052]). For CYP2E1 Rsa I, the mutant allele frequencies were 27.3% (Chaoshan) and 29.6% (Taihang), and OR for CC versus TT genotype (Taihang) was 3.167 (95% CI [1.026-9.776]). CONCLUSION: The results suggest that AG genotype of CYP1A1 in Chaoshan area and GG (CC) genotype of CYP2E1 in Taihang area are significantly associated with esophageal cancer susceptibility.

[Differentiation of human amniotic mesenchymal stem cells into insulin-secreting cells induced by regenerating pancreatic extract].

In this study, the natural biological inducer, rat regenerating pancreatic extract (RPE), was used to induce human amniotic mesenchymal stem cells (hAMSCs) into insulin-secreting cells. We excised 60% of rat pancreas in order to stimulate pancreatic regeneration. RPE was extracted and used to induce hAMSCs at a final concentration of 20 microg/mL. The experiment methods used were as follows: morphological-identification, dithizone staining, immumofluorescence analysis, reverse transcription-PCR (RT-PCR) and insulin secretion stimulated by high glucose. The results show that the cell morphology of passge3 hAMSCs changed significantly after the induction of RPE, resulting in cluster shape after induction for 15 days. Dithizone staining showed that there were scarlet cell masses in RPE-treated culture. Immumofluorescence analysis indicated that induced cells were insulin-positive expression. RT-PCR showed the positive expression of human islet-related genes Pdx1 and insulin in the induced cells. The result of insulin secretion stimulated by high glucose indicated that insulin increasingly secreted and then kept stable with prolongation of high glucose stimulation. In conclusion, hAMSCs had the potential to differentiate into insulin-secreting cells induced by RPE in vitro.