Asymmetric segregation of maternal mRNAs and germline-related determinants in Cephalochordate embryos: implications for the evolution of early patterning events in chordates.

How animal embryos determine their early cell fates is an important question in developmental biology. In various model animals asymmetrically localized maternal transcripts play important roles in axial patterning and cell fate specification. Cephalochordates (amphioxus), which have three living genera (Asymmetron, Epigonichthys, Branchiostoma), are an early branching chordate lineage and thus occupy a key phylogenetic position for understanding the evolution of chordate developmental mechanisms. It has been shown that in the zygote of Brachiostoma amphioxus, which possess bilateral gonads flanking both sides of their trunk region, maternal transcripts of germline determinants form a compact granule. During early embryogenesis this granule is inherited by a single blastomere that subsequently gives rise to a cluster of cells displaying typical characteristics of primordial germ cells (PGC). These PGCs then come to lie in the tailbud region and proliferate during posterior elongation of the larva to join in the gonad anlagen at the ventral tip of the developing myomeres in amphioxus larvae. However, in Asymmetron and Epigonichthys amphioxus, whose gonads are present only on the right side of their body, nothing is known about their PGC development or the cellular/morphogenetic processes resulting in the asymmetric distribution of gonads. Using conserved germline determinants as markers, we show that similarly to Brachiostoma amphioxus, Asymmetron also employ a preformation mechanism to specify their PGCs, suggesting that this mechanism represents an ancient trait dating back to the common ancestor of Cephalochordates. Surprisingly, we found that Asymmetron PGCs are initially deposited on both sides of the body during early larval development; however, the left side PGCs cease to exist in young juveniles, suggesting that PGCs are eliminated from the left body side during larval development or following metamorphosis. This is reminiscent of the PGC development in the sea urchin embryo, and we discuss the implications of this observation for the evolution of developmental mechanisms.

Handheld common-path swept-source optical coherence tomography angiography.

This study develops a handheld optical coherence tomography angiography (OCTA) system that uses a high-speed (200 kHz) swept laser with a dual-reference common-path configuration for stable and fast imaging. The common-path design automatically avoids polarization and dispersion mismatches by using one circulator as the primary system element, ensuring a cost-effective and compact design for handheld probe use. With its stable envelope (i.e., sub-µm shifts) and phase variation (corresponding to nm changes in axial displacement), the minimum detectable flow velocity is ∼ 0.08 mm/s in our experiment, which gives the common-path setup a high potential for application in a handheld OCTA system for clinical skin screening. In vivo skin structures and microvasculature networks on the dorsum of the hand and cheek of a healthy human are imaged successfully.

The Image-Histology Correlation of Subcutaneous mPEG-poly(Ala) Hydrogel-Embedded MIN6 Cell Grafts in Nude Mice.

Previously, we have successfully used noninvasive magnetic resonance (MR) and bioluminescence imaging to detect and monitor mPEG-poly(Ala) hydrogel-embedded MIN6 cells at the subcutaneous space for up to 64 days. In this study, we further explored the histological evolution of MIN6 cell grafts and correlated it with image findings. MIN6 cells were incubated overnight with chitosan-coated superparamagnetic iron oxide (CSPIO) and then 5 × 106 cells in the 100 µL hydrogel solution were injected subcutaneously into each nude mouse. Grafts were removed and examined the vascularization, cell growth and proliferation with anti-CD31, SMA, insulin and ki67 antibodies, respectively, at 8, 14, 21, 29 and 36 days after transplantation. All grafts were well-vascularized with prominent CD31 and SMA staining at all time points. Interestingly, insulin-positive cells and iron-positive cells were scattered in the graft at 8 and 14 days; while clusters of insulin-positive cells without iron-positive cells appeared in the grafts at 21 days and persisted thereafter, indicating neogrowth of MIN6 cells. Moreover, proliferating MIN6 cells with strong ki67 staining was observed in 21-, 29- and 36-day grafts. Our results indicate that the originally transplanted MIN6 cells proliferated from 21 days that presented distinctive bioluminescence and MR images.

Magnetic Resonance Imaging of Transplanted Porcine Neonatal Pancreatic Cell Clusters Labeled with Exendin-4-Conjugated Manganese Magnetism-Engineered Iron Oxide Nanoparticles.

Recently, we have shown that manganese magnetism-engineered iron oxide nanoparticles (MnMEIO NPs) conjugated with exendin-4 (Ex4) act as a contrast agent that directly trace implanted mouse islet ß-cells by magnetic resonance imaging (MRI). Here we further advanced this technology to track implanted porcine neonatal pancreatic cell clusters (NPCCs) containing ducts, endocrine, and exocrine cells. NPCCs from one-day-old neonatal pigs were isolated, cultured for three days, and then incubated overnight with MnMEIO-Ex4 NPs. Binding of NPCCs and MnMEIO-Ex4 NPs was confirmed with Prussian blue staining in vitro prior to the transplantation of 2000 MnMEIO-Ex4 NP-labeled NPCCs beneath the left renal capsule of six nondiabetic nude mice. The 7.0 T MRI on recipients revealed persistent hypointense areas at implantation sites for up to 54 days. The MR signal intensity of the graft on left kidney reduced 62-88% compared to the mirror areas on the contralateral kidney. Histological studies showed colocalization of insulin/iron and SOX9/iron staining in NPCC grafts, indicating that MnMEIO-Ex4 NPs were taken up by mature ß-cells and pancreatic progenitors. We conclude that MnMEIO-Ex4 NPs are excellent contrast agents for detecting and long-term monitoring implanted NPCCs by MRI.

Exendin-4-Conjugated Manganese Magnetism-Engineered Iron Oxide Nanoparticles as a Potential Magnetic Resonance Imaging Contrast Agent for Tracking Transplanted ß-Cells.

To specifically detect and trace transplanted islet ß-cells by magnetic resonance imaging (MRI), we conjugated manganese magnetism-engineered iron oxide nanoparticles (MnMEIO NPs) with exendin-4 (Ex4) which specifically binds glucagon-like peptide-1 receptors on the surface of ß-cells. The size distribution of MnMEIO and MnMEIO-Ex4 NPs were 67.8 ± 1.3 and 70.2 ± 2.3 nm and zeta potential 33.3 ± 0.5 and 0.6 ± 0.1 mV, respectively. MnMEIO and MnMEIO-Ex4 NPs with iron content ≤ 40 µg/mL did not affect MIN6 ß-cell viability and insulin secretion. Positive iron staining was found in MIN6 ß-cells loaded with MnMEIO-Ex4 NPs but not in those with MnMEIO NPs. A transmission electron microscope confirmed MnMEIO-Ex4 NPs were distributed in the cytoplasm of MIN6. In vitro MR images revealed a loss of signal intensity in MIN6 ß-cells labeled with MnMEIO-Ex4 NPs but not with MnMEIO NPs. After transplantation of islets labeled with MnMEIO-Ex4, the graft under kidney capsule could be visualized on MRI as persistent hypointense areas up to 17 weeks. Moreover, histology of the islet graft showed positive staining for insulin, glucagon and iron. Our results indicate MnMEIO-Ex4 NPs are safe and effective for the detection and long-term monitoring of transplanted ß-cells by MRI.

Noninvasive Tracking of mPEG-poly(Ala) Hydrogel-Embedded MIN6 Cells after Subcutaneous Transplantation in Mice.

Recently, we demonstrated the feasibility of subcutaneous transplantation of MIN6 cells embedded in a scaffold with poly(ethylene glycol) methyl ether (mPEG)-poly(Ala) hydrogels. In this study, we further tracked these grafts using magnetic resonance (MR) and bioluminescence imaging. After being incubated overnight with chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles and then mixed with mPEG-poly(Ala) hydrogels, MIN6 cells appeared as dark spots on MR scans. For in vivo experiments, we transfected MIN6 cells with luciferase and/or incubated them overnight with CSPIO overnight; 5 × 106 MIN6 cells embedded in mPEG-poly(Ala) hydrogels were transplanted into the subcutaneous space of each nude mouse. The graft of CSPIO-labeled MIN6 cells was visualized as a distinct hypointense area on MR images located at the implantation site before day 21. However, this area became hyperintense on MR scans for up to 64 days. In addition, positive bioluminescence images were also observed for up to 64 days after transplantation. The histology of removed grafts showed positive insulin and iron staining. These results indicate mPEG-poly(Ala) is a suitable scaffold for ß-cell encapsulation and transplantation. Moreover, MR and bioluminescence imaging are useful noninvasive tools for detecting and monitoring mPEG-poly(Ala) hydrogel-embedded MIN6 cells at a subcutaneous site.

Magnetic Resonance Imaging of Transplanted Porcine Neonatal Pancreatic Cell Clusters Labeled with Chitosan-Coated Superparamagnetic Iron Oxide Nanoparticles in Mice.

Neonatal pancreatic cell clusters (NPCCs) are potential tissues for the treatment of diabetes. Different from adult cells, they continuously proliferate and differentiate after transplantation. In this study, we utilized magnetic resonance imaging (MRI) to detect and monitor implanted NPCCs. NPCCs were isolated from one-day-old neonatal pigs, cultured for three days, and then incubated overnight with the contrast agent chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles. In vitro, Prussian blue staining and MR scans of CSPIO-labeled NPCCs were performed. In vivo, we transplanted 2000 CSPIO-labeled NPCCs under the kidney capsule of nondiabetic nude mice. Recipients were scanned with 7.0T MRI. Grafts were removed for histology with insulin and Prussian blue staining. After being incubated overnight with CSPIO, NPCCs showed positive iron staining and appeared as dark spots on MR scans. After transplantation of CSPIO-labeled NPCCs, persistent hypointense areas were observed at recipients' implant sites for up to 54 days. Moreover, histology showed colocalization of the insulin and iron staining in 15-, 51- and 55-day NPCC grafts. Our results indicate that transplanted NPCCs survived and differentiated to ß cells after transplantation, and that MRI is a useful tool for the detection and monitoring of CSPIO-labeled NPCC grafts.

Implanted islet mass influences the effects of dipeptidyl peptidase-IV inhibitor LAF237 on transplantation outcomes in diabetic mice.

BACKGROUND: Previous studies showed inconsistent Results of the effects of dipeptidyl peptidase (DPP)-IV inhibitors on syngeneic mouse islet transplantation. We hypothesized that the implanted islet numbers are critical for the effects of DPP-IV inhibitors on the outcomes of transplantation. METHODS: One hundred and fifty or three hundred islets were syngeneically transplanted under the renal capsule of each streptozocin-diabetic C57BL/6 mouse and recipients were then treated without or with LAF237 (10 mg/kg/day, po) for 6 weeks. After transplantation, recipients' blood glucose, body weight and intraperitoneal glucose tolerance test (IPGTT) were followed-up periodically. The graft was removed for the measurement of ß-cell mass at 6 weeks. RESULTS: In recipients with 150 islets, it was not significantly different between the LAF237- treated group (n = 14) and control group (n = 14) in terms of the blood glucose, body weight, glucose tolerance at 2, 4 and 6 weeks or the graft ß-cell mass at 6 weeks. In contrast, in recipients with 300 islets, the LAF237-treated group (n = 24) did have a lower area under the curve of the IPGTT at 4 weeks (p = 0.0237) and 6 weeks (p = 0.0113) as well as more graft ß-cell mass at 6 weeks (0.655 ± 0.008 mg vs. 0.435 ± 0.006 mg, p = 0.0463) than controls (n = 24). CONCLUSIONS: Our findings revealed 6-week treatment of LAF237 improves glucose tolerance and increases graft ß-cell mass in diabetic mice transplanted with a sufficient number but not a marginal number of islets. These indicate that the effects of DPP-IV inhibitors are influenced by the implanted islet mass.

Porcine Neonatal Pancreatic Cell Clusters Maintain Their Multipotency in Culture and After Transplantation.

Ductal epithelium is primarily detected in porcine neonatal pancreatic cell clusters (NPCCs) bearing grafts, suggesting that transplants might exhibit progenitor-like phenotypes. Here we found that soon after NPCC isolation, PDX1+/insulin- and SOX9+ pancreatic progenitor-like cells dramatically increased while dual-hormonal progenitor-like cells were routinely observed in NPCC culture. After transplantation (Tx), insulin+ cells increased and PDX1+ and SOX9+ cells gradually decreased in both non-diabetic (NDM) and streptozotocin-induced diabetic (DM) grafts over 2 months. Strikingly, a significantly higher percentage of insulin+ cells were detected in 9-day and 16-day, but not in 23-day, 30-day and 60-day grafts implying that hyperglycemia could only facilitate NPCC-derived ß cells early post-Tx. A higher percentage of NPCC-derived ß cells in early DM grafts was determined via an enhanced neogenic differentiation based on the detection of insulin+ cells budding out from PDX1+/SOX9+ epithelium. Interestingly, a drop in SOX9+ progenitor-like cells was detected 16 days post-Tx in DM grafts whilst PDX1+ cells do not show a significant difference until 60 days post-Tx between DM and NDM grafts, demonstrating that distinct progenitor-like populations fuel new ß cells post-Tx. In conclusion, PDX1+/SOX9+ cells could be quickly activated after NPCC isolation, maintain their multipotency in culture and differentiate into new ß cell post-Tx.

Preparation and in vitro evaluation of artemisinin loaded long-circulating liposomes / 药学学报

The therapeutic application of artemisinin (ART) is restricted in application due to its poor water solubility and stability. In this study, the long-circulating liposomes (L-Lip) were constructed to improve the solubility and stability of ART. The preparation method, physicochemical properties, serum stability, in vitro release profile and cytotoxicity of the ART loaded long-circulating liposomes were investigated. Using the particle size and entrapment efficiency (EE) as the evaluation index, the preparation procedure was optimized by the Box-Behnken response surface design based on the single factor screening method. The ART loaded long-circulating liposomes were prepared by filming rehydration method, and evaluated with particle size and entrapment efficiency. The optimal formulation was as follows:lipid-cholesterol=5.22:1 (mass ratio), drug-lipid=1:23.15 (mass ratio), lipid concentration=14.35 mg·mL-1, and molar percentage of mPEG=2%. The morphology of L-Lip was uniformly spherical shape according to optimal formulation. The mean size and polydispersity index (PDI) were about (113.3 ±4.7) nm and 0.227 ±0.022 respectively, the zeta potential was (-12.9 ±2.6) mV, and the entrapment efficiency (EE) of ART was (95.88 ±4.8)%. The L-Lip had good stability at 4℃ for 15 days and the particle sizes did not exhibit significant variations in 50% rat plasma over 24 h at 37℃. The in vitro release study of formulation showed a sustained release. Moreover, the cytotoxicity exhibited that blank liposomes were of great safety. Compared with the free ART, the liposome formulation achieved lower cytotoxicity at the high concentration. The L-Lip successfully prepared by a simple filming-rehydration method exhibited ideal physicochemical properties and were enhanced safety, which may sever as a promising nanoplatform for clinical application.

Phenotypic transition maps of 3D breast acini obtained by imaging-guided agent-based modeling.

We introduce an agent-based model of epithelial cell morphogenesis to explore the complex interplay between apoptosis, proliferation, and polarization. By varying the activity levels of these mechanisms we derived phenotypic transition maps of normal and aberrant morphogenesis. These maps identify homeostatic ranges and morphologic stability conditions. The agent-based model was parameterized and validated using novel high-content image analysis of mammary acini morphogenesis in vitro with focus on time-dependent cell densities, proliferation and death rates, as well as acini morphologies. Model simulations reveal apoptosis being necessary and sufficient for initiating lumen formation, but cell polarization being the pivotal mechanism for maintaining physiological epithelium morphology and acini sphericity. Furthermore, simulations highlight that acinus growth arrest in normal acini can be achieved by controlling the fraction of proliferating cells. Interestingly, our simulations reveal a synergism between polarization and apoptosis in enhancing growth arrest. After validating the model with experimental data from a normal human breast line (MCF10A), the system was challenged to predict the growth of MCF10A where AKT-1 was overexpressed, leading to reduced apoptosis. As previously reported, this led to non growth-arrested acini, with very large sizes and partially filled lumen. However, surprisingly, image analysis revealed a much lower nuclear density than observed for normal acini. The growth kinetics indicates that these acini grew faster than the cells comprising it. The in silico model could not replicate this behavior, contradicting the classic paradigm that ductal carcinoma in situ is only the result of high proliferation and low apoptosis. Our simulations suggest that overexpression of AKT-1 must also perturb cell-cell and cell-ECM communication, reminding us that extracellular context can dictate cellular behavior.