Phosphorylated transcription factor PuHB40 mediates ROS-dependent anthocyanin biosynthesis in pear exposed to high light.

Plants are increasingly vulnerable to environmental stresses because of global warming and climate change. Stress-induced reactive oxygen species (ROS) accumulation results in plant cell damage and even cell death. Anthocyanins are important antioxidants that scavenge ROS to maintain ROS homeostasis. However, the mechanism underlying ROS-induced anthocyanin accumulation is unclear. In this study, we determined that the HD-Zip I family member transcription factor PuHB40 mediates ROS-dependent anthocyanin biosynthesis under high-light stress in pear (Pyrus ussuriensis). Specifically, PuHB40 induces the PuMYB123-like-PubHLH3 transcription factor complex for anthocyanin biosynthesis. PuHB40-mediated transcriptional activation depends on its phosphorylation level, which is regulated by protein phosphatase PP2A. Elevated ROS content maintains high PuHB40 phosphorylation levels, while also enhancing PuHB40-induced PuMYB123-like transcription by decreasing PuPP2AA2 expression, ultimately leading to increased anthocyanin biosynthesis. Our study reveals a pathway regulating ROS-induced anthocyanin biosynthesis in pear, further clarifying the mechanism underlying abiotic stress-induced anthocyanin biosynthesis, which may have implications for improving plant stress tolerance.

ABRE-BINDING FACTOR3-WRKY DNA-BINDING PROTEIN44 module promotes salinity-induced malate accumulation in pear.

Malate impacts fruit acidity and plays a vital role in stress tolerance. Malate accumulation is induced by salinity in various plants as a metabolite in coping with this stress. However, the exact molecular mechanism responsible for salinity-induced malate accumulation remains unclear. Here, we determined that salinity treatment induces malate accumulation in pear (Pyrus spp.) fruit, calli, and plantlets compared to the control. Genetic and biochemical analyses established the key roles of PpWRKY44 and ABRE-BINDING FACTOR3 (PpABF3) transcription factors in promoting malate accumulation in response to salinity. We found that PpWRKY44 is involved in salinity-induced malate accumulation by directly binding to a W-box on the promoter of the malate-associated gene aluminum-activated malate transporter 9 (PpALMT9) to activate its expression. A series of in-vivo and in-vitro assays revealed that the G-box cis-element in the promoter of PpWRKY44 was targeted by PpABF3, which further enhanced salinity-induced malate accumulation. Taken together, these findings suggest that PpWRKY44 and PpABF3 play positive roles in salinity-induced malate accumulation in pears. This research provides insights into the molecular mechanism by which salinity affects malate accumulation and fruit quality.

A modified flexible GnRH antagonist protocol using antagonist early cessation and a gonadotropin step-down approach improves live birth rates in fresh cycles: a randomized controlled trial.

STUDY QUESTION: Can pregnancy outcomes following fresh elective single embryo transfer (eSET) in gonadotropin-releasing hormone (GnRH) antagonist protocols increase using a gonadotropin (Gn) step-down approach with cessation of GnRH antagonist on the day of hCG administration (hCG day) in patients with normal ovarian response? SUMMARY ANSWER: The modified GnRH antagonist protocol using the Gn step-down approach and cessation of GnRH antagonist on the hCG day is effective in improving live birth rates (LBRs) per fresh eSET cycle. WHAT IS KNOWN ALREADY: Currently, there is no consensus on optimal GnRH antagonist regimens. Studies have shown that fresh GnRH antagonist cycles result in poorer pregnancy outcomes than the long GnRH agonist (GnRHa) protocol. Endometrial receptivity is a key factor that contributes to this phenomenon. STUDY DESIGN, SIZE, DURATION: An open label randomized controlled trial (RCT) was performed between November 2021 and August 2022. There were 546 patients allocated to either the modified GnRH antagonist or the conventional antagonist protocol at a 1:1 ratio. PARTICIPANTS/MATERIALS, SETTING, METHODS: Both IVF and ICSI cycles were included, and the sperm samples used were either fresh or frozen from the partner, or from frozen donor ejaculates. The primary outcome was the LBRs per fresh SET cycle. Secondary outcomes included rates of implantation, clinical and ongoing pregnancy, miscarriage, and ovarian hyperstimulation syndrome (OHSS), as well as clinical outcomes of ovarian stimulation. MAIN RESULTS AND THE ROLE OF CHANCE: Baseline demographic features were not significantly different between the two ovarian stimulation groups. However, in the intention-to-treat (ITT) population, the LBRs in the modified antagonist group were significantly higher than in the conventional group (38.1% [104/273] vs. 27.5% [75/273], relative risk 1.39 [95% CI, 1.09-1.77], P = 0.008). Using a per-protocol (PP) analysis which included all the patients who received an embryo transfer, the LBRs in the modified antagonist group were also significantly higher than in the conventional group (48.6% [103/212] vs. 36.8% [74/201], relative risk 1.32 [95% CI, 1.05-1.66], P = 0.016). The modified antagonist group achieved significantly higher implantation rates, and clinical and ongoing pregnancy rates than the conventional group in both the ITT and PP analyses (P < 0.05). The two groups did not show significant differences between the number of oocytes retrieved or mature oocytes, two-pronuclear zygote (2PN) rates, the number of embryos obtained, blastocyst progression and good-quality embryo rates, early miscarriage rates, or OHSS incidence rates (P > 0.05). LIMITATIONS, REASONS FOR CAUTION: A limitation of our study was that the subjects were not blinded to the treatment allocation in the RCT trial. Only women under 40 years of age who had a good prognosis were included in the analysis. Therefore, use of the modified antagonist protocol in older patients with a low ovarian reserve remains to be investigated. In addition, the sample size for Day 5 elective SET was small, so larger trials will be required to strengthen these findings. WIDER IMPLICATIONS OF THE FINDINGS: The modified GnRH antagonist protocol using the Gn step-down approach and cessation of GnRH antagonist on hCG day improved the LBRs per fresh eSET cycle in normal responders. STUDY FUNDING/COMPETING INTEREST(S): This project was funded by grant 2022YFC2702503 from the National Key Research & Development Program of China and grant 2021140 from the Beijing Health Promotion Association. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: The RCT was registered in the Chinese Clinical Trial Registry; Study Number: ChiCTR2100053453. TRIAL REGISTRATION DATE: 21 November 2021. DATE OF FIRST PATIENT'S ENROLLMENT: 23 November 2021.

FBN2 pathogenic variants in congenital contractural arachnodactyly with severe cardiovascular manifestations.

PURPOSE: Congenital contractural arachnodactyly (CCA) is an extremely rare autosomal dominant connective tissue genetic disorder caused by pathogenic variants in FBN2. CCA is characterized by arachnodactyly, camptodactyly, contracture of major joints, scoliosis, pectus deformities, and crumpled ears, but rarely with lethal cardiovascular manifestations as in Marfan syndrome. It is imperative to conduct a comprehensive analysis and review of the pathogenesis of CCA resulting from pathogenic variants in FBN2 gene. MATERIALS AND METHODS: Using whole-exome sequencing and Sanger sequencing, we identified a novel pathogenic splice-altering variant (c.4472-3C>A) in intron 34 of FBN2 gene in a CCA pedigree. The transcriptional result of the splicing-altering variant was analyzed by RNA sequencing. We systematically analyzed the clinical manifestations of all reported cases of CCA caused by splicing-altering pathogenic variants and focused on all the pathogenic variants in FBN2 gene that are associated with severe cardiovascular manifestations. RESULTS: The splice-altering variant (c.4472-3C>A) in FBN2 was demonstrated to result in the exon 35 skipping and cause an in-frame deletion. Furthermore, we identified exons 31 to 35 may be a hotspot region in FBN2 gene associated with severe cardiovascular phenotype. CONCLUSIONS: This study enriched the pathogenic spectrum of CCA and identified a hotspot region in FBN2 gene associated with severe cardiovascular manifestations. We recommend that patients carrying pathogenic variants in exons 31 to 35 of FBN2 pay more attention to cardiac evaluation.

Adipocyte Rnf20 ablation increases the fast-twitch fibers of skeletal muscle via lysophosphatidylcholine 16:0.

Both adipose tissue and skeletal muscle are highly dynamic tissues and interact at the metabolic and hormonal levels in response to internal and external stress, and they coordinate in maintaining whole-body metabolic homeostasis. In our previous study, we revealed that adipocyte-specific Rnf20 knockout mice (ASKO mice) exhibited lower fat mass but higher lean mass, providing a good model for investigating the adipose-muscle crosstalk and exploring the effect of the adipocyte Rnf20 gene on the physiology and metabolism of skeletal muscle. Here, we confirmed that ASKO mice exhibited the significantly increased body weight and gastrocnemius muscle weight. Fiber-type switching in the soleus muscle of ASKO mice was observed, as evidenced by the increased number of fast-twitch fibers and decreased number of slow-twitch fibers. Serum metabolites with significant alteration in abundance were identified by metabolomic analysis and the elevated lysophosphatidylcholine 16:0 [LysoPC (16:0)] was observed in ASKO mice. In addition, lipidome analysis of gonadal white adipose tissue revealed a significant increase in LysoPCs and LysoPC (16:0) in ASKO mice. Furthermore, knockdown of Rnf20 gene in 3T3-L1 cells significantly increased the secretion of LysoPC, suggesting that LysoPC might be a critical metabolite in the adipose-muscle crosstalk of ASKO mice. Furthermore, in vitro study demonstrated that LysoPC (16:0) could induce the expression of fast-twitch muscle fibers related genes in differentiated C2C12 cells, indicating its potential role in adipose-muscle crosstalk. Taken together, these findings not only expand our understanding of the biological functions of Rnf20 gene in systemic lipid metabolism, but also provide insight into adipose tissue dysfunction-induced physiological alterations in skeletal muscle.

High-Frequency and High-Current Transmission Techniques for Multiple Earth Electrical Characteristic Measurement Systems Based on Adaptive Impedance Matching through Phase Comparison.

With the increase in groundwater exploration, underground mineral resource exploration, and non-destructive investigation of cultural relics, high-resolution earth electrical characteristic measurement has emerged as a mainstream technique owing to its advantageous non-destructive detection capability. To enhance the transmission power of the high-frequency transmitter in high-resolution multiple earth electrical characteristic measurement systems (MECS), this study proposes a high-frequency, high-current transmission technique based on adaptive impedance matching and implemented through the integration of resonant capacitors, a controllable reactor, high-frequency transformers, and corresponding control circuits. A high-current precisely controllable reactor with a 94% inductance variation range was designed and combined with resonant capacitors to reduce circuit impedance. Additionally, high-frequency transformers were employed to further increase the transmission voltage. A prototype was developed and tested, demonstrating an increase in transmission current at frequencies between 10 and 120 kHz with a peak active power of 200 W. Under the same transmission voltage, compared to the transmission circuit without impedance matching, the transmission current increased to a maximum of 16.7 times (average of 10.8 times), whereas compared to the transmission circuit using only traditional impedance matching, the transmission current increased by a maximum of 10.0 times (average of 4.2 times), effectively improving the exploration resolution.

Bone Morphogenetic Protein 2 Enhances Porcine Beige Adipogenesis via AKT/mTOR and MAPK Signaling Pathways.

Bone morphogenetic protein 2 (BMP2) has been reported to regulate adipogenesis, but its role in porcine beige adipocyte formation remains unclear. Our data reveal that BMP2 is significantly induced at the early stages of porcine beige adipocyte differentiation. Additionally, supplementing rhBMP2 during the early stages, but not the late stages of differentiation, significantly enhances porcine SVF adipogenesis, thermogenesis, and proliferation. Furthermore, compared to the empty plasmid-transfected-SVFs, BMP2-overexpressed SVFs had the enhanced lipid accumulation and thermogenesis, while knockdown of BMP2 in SVFs exhibited the opposite effect. The RNA-seq of the above three types of cells revealed the enrichment of the annotation of thermogenesis, brown cell differentiation, etc. In addition, the analysis also highlights the significant enrichment of cell adhesion, the MAPK cascade, and PPARγ signaling. Mechanistically, BMP2 positively regulates the adipogenic and thermogenic capacities of porcine beige adipocytes by activating PPARγ expression through AKT/mTOR and MAPK signaling pathways.

SnRNA-Seq of Pancreas Revealed the Dysfunction of Endocrine and Exocrine Cells in Transgenic Pigs with Prediabetes.

Diabetes poses a significant threat to human health. Exocrine pancreatic dysfunction is related to diabetes, but the exact mechanism is not fully understood. This study aimed to describe the pathological phenotype and pathological mechanisms of the pancreas of transgenic pigs (PIGinH11) that was constructed in our laboratory and to compare it with humans. We established diabetes-susceptible transgenic pigs and subjected them to high-fat and high-sucrose dietary interventions. The damage to the pancreatic endocrine and exocrine was evaluated using histopathology and the involved molecular mechanisms were analyzed using single-nucleus RNA-sequencing (SnRNA-seq). Compared to wild-type (WT) pigs, PIGinH11 pigs showed similar pathological manifestations to type 2 diabetes patients, such as insulin deficiency, fatty deposition, inflammatory infiltration, fibrosis tissue necrosis, double positive cells, endoplasmic reticulum (ER) and mitochondria damage. SnRNA-seq analysis revealed 16 clusters and cell-type-specific gene expression characterization in the pig pancreas. Notably, clusters of Ainar-M and Endocrine-U were observed at the intermediate state between the exocrine and endocrine pancreas. Beta cells of the PIGinH11 group demonstrated the dysfunction with insulin produced and secret decreased and ER stress. Moreover, like clinic patients, acinar cells expressed fewer digestive enzymes and showed organelle damage. We hypothesize that TXNIP that is upregulated by high glucose might play an important role in the dysfunction of endocrine to exocrine cells in PIGinH11 pigs.

Cold-induced phase separation for the simple and reliable extraction of sex hormones for subsequent LC-MS/MS analysis.

Sex hormones, including androgens, estrogens, and progestogens, are important biomarkers for various diseases. Quantification of sex hormones is typically conducted by LC-MS/MS. At present, most methods require liquid-liquid extraction or solid phase extraction for sample preparation. However, these pretreatments are prone to compromise LC-MS/MS throughput. To improve on the current standard practices, we investigated cold-induced phase separation for sex hormone extraction. After protein precipitation with acetonitrile and adjusting the solution constitution with water, samples were stored at -30°C for 10 min to generate two distinct phases: an acetonitrile-rich layer on top of a water-rich layer. During this process, the hydrophobic sex hormones spontaneously separate into the upper layer. This simple and reliable cold-induced phase separation-based LC-MS/MS methodology was used here to simultaneously detect estrone, estradiol, estriol, testosterone, androstenedione, dehydroepiandrosterone, progesterone, and 17-hydroxyprogesterone in serum. Validation of this method indicated satisfactory performance, including acceptable linearity, accuracy, precision, and tractability. Compared with the mainstream liquid-liquid extraction-based method, this new method exhibits significant progress in throughput, which shortens the time cost of sample preparation from 90 to 40 min. We propose that this method can be an excellent alternative for sex hormone analysis in routine clinical laboratories.

Stearoyl-CoA Desaturase is Essential for Porcine Adipocyte Differentiation.

Fat deposition, which influences pork production, meat quality and growth efficiency, is an economically important trait in pigs. Numerous studies have demonstrated that stearoyl-CoA desaturase (SCD), a key enzyme that catalyzes the conversion of saturated fatty acids into monounsaturated fatty acids, is associated with fatty acid composition in pigs. As SCD was observed to be significantly induced in 3T3-L1 preadipocytes differentiation, we hypothesized that it plays a role in porcine adipocyte differentiation and fat deposition. In this study, we revealed that SCD is highly expressed in adipose tissues from seven-day-old piglets, compared to its expression in tissues from four-month-old adult pigs. Moreover, we found that SCD and lipogenesis-related genes were induced significantly in differentiated porcine adipocytes. Using CRISPR/Cas9 technology, we generated SCD-/- porcine embryonic fibroblasts (PEFs) and found that the loss of SCD led to dramatically decreased transdifferentiation efficiency, as evidenced by the decreased expression of known lipid synthesis-related genes, lower levels of oil red O staining and significantly lower levels of triglyceride content. Our study demonstrates the critical role of SCD expression in porcine adipocyte differentiation and paves the way for identifying it as the promising candidate gene for less fat deposition in pigs.

Catalytic mechanism of UDP-glucose dehydrogenase.

UDP-glucose dehydrogenase (UGDH), an oxidoreductase, catalyzes the NAD+-dependent four-electron oxidation of UDP-glucose to UDP-glucuronic acid. The catalytic mechanism of UGDH remains controversial despite extensive investigation and is classified into two types according to whether an aldehyde intermediate is generated in the first oxidation step. The first type, which involves the presence of this putative aldehyde, is inconsistent with some experimental findings. In contrast, the second type, which indicates that the first oxidation step bypasses the aldehyde via an NAD+-dependent bimolecular nucleophilic substitution (SN2) reaction, is consistent with the experimental phenomena, including those that cannot be explained by the first type. This NAD+-dependent SN2 mechanism is thus more reasonable and likely applicable to other oxidoreductases that catalyze four-electron oxidation reactions.

FGF21 mediates the protective effect of fenofibrate against acetaminophen -induced hepatotoxicity via activating autophagy in mice.

Overdose of acetaminophen (APAP) induces acute liver injury due in part to destruction of mitochondria and resulted oxidative stress. Recently, FGF21 has been demonstrated to be an endocrine factor to protect liver from oxidative stress. The aim of present study is to explore the role of fibroblast growth factor 21 (FGF21) in the protective effect of fenofibrate, an agonist of peroxisome proliferator-activated receptor alpha (PPARα), against acetaminophen (APAP)-induced liver injury. Mice and primary cultured hepatocytes were used to test the potential hepatoprotective effect of fenofibrate against APAP-induced hepatotoxicity. FGF21 deficient mice were used to evaluate the role of FGF21 in fenofibrate against APAP-induced acute liver injury. Post-treatment with fenofibrate significantly inhibits APAP-induced hepatotoxicity, as evidenced by decreased serum ALT and AST levels and hepatic necrosis in liver tissue as well as increased the surviving rate in response to APAP overdose, whereas this protective effect of fenofibrate is largely attenuated in FGF21 KO mice. Interestingly, administration of fenofibrate efficiently increases autophagy, which was companied with alleviating hepatotoxicity in APAP-treated WT mice. However, such effect is significantly attenuated in APAP-treated FGF21 KO mice. In conclusion, our findings suggest that fenofibrate against APAP-induced hepatotoxicity is at least in part mediated by up-regulating the expression of FGF21, which in turn promotes autophagy-mediated hepatoprotective effects.

Remarkably Enhanced Room-Temperature Hydrogen Sensing of SnO2 Nanoflowers via Vacuum Annealing Treatment.

In this work, SnO2 nanoflowers synthesized by a hydrothermal method were employed as hydrogen sensing materials. The as-synthesized SnO2 nanoflowers consisted of cuboid-like SnO2 nanorods with tetragonal structures. A great increase in the relative content of surface-adsorbed oxygen was observed after the vacuum annealing treatment, and this increase could have been due to the increase in surface oxygen vacancies serving as preferential adsorption sites for oxygen species. Annealing treatment resulted in an 8% increase in the specific surface area of the samples. Moreover, the conductivity of the sensors decreased after the annealing treatment, which should be attributed to the increase in electron scattering around the defects and the compensated donor behavior of the oxygen vacancies due to the surface oxygen adsorption. The hydrogen sensors of the annealed samples, compared to those of the unannealed samples, exhibited a much higher sensitivity and faster response rate. The sensor response factor and response rate increased from 27.1% to 80.2% and 0.34%/s to 1.15%/s, respectively. This remarkable enhancement in sensing performance induced by the annealing treatment could be attributed to the larger specific surface areas and higher amount of surface-adsorbed oxygen, which provides a greater reaction space for hydrogen. Moreover, the sensors with annealed SnO2 nanoflowers also exhibited high selectivity towards hydrogen against CH4, CO, and ethanol.

Missing in metastasis B, regulated by DNMT1, functions as a putative cancer suppressor in human lung giant-cell carcinoma.

Missing in metastasis B (MIM-B) has been widely reported to inhibit cancer cell invasion and proliferation in a variety of human cancers. However, the functions of MIM-B in lung cancers are still controversial. In addition, the mechanisms and regulation of MIM-B are poorly understood. In the present study, we found that the invasion level of 95C human lung giant-cell carcinoma cells was elevated when MIM-B was knocked down, while the invasion of 95D was suppressed when MIM-B was overexpressed, proving that MIM-B suppresses human lung giant-cell carcinoma cell invasion, which is similar to its function in most cancers. Furthermore, we reported that an increase in DNA methylation density in the promoter of MIM-B by DNA methyltransferase 1 (DNMT1) is correlated with the silencing of MIM-B expression and the high metastasis of 95D human lung giant-cell carcinoma cell line. Taken together, MIM-B, which is regulated by DNMT1 through DNA methylation, is a putative cancer suppressor in human lung giant-cell carcinoma.

CXCL16 deficiency attenuates acetaminophen-induced hepatotoxicity through decreasing hepatic oxidative stress and inflammation in mice.

Chemokine C-X-C ligand 16 (CXCL16), a single-pass Type I membrane protein belonging to the CXC chemokine family, is related to the inflammatory response in liver injury. In present study, we investigated the pathophysiological role of CXCL16, a unique membrane-bound chemokine, in acetaminophen (APAP)-induced hepatotoxicity in mice. Mice were injected with APAP, and blood and tissue samples were harvested at different time points. The serum high-mobility group box 1 and CXCL16 levels were quantified by sandwich immunoassays. The liver tissue sections were stained with hematoxylin-eosin or with dihydroethidium staining. The expressions of CXCL16 and other cytokines were examined by real-time polymerase chain reaction. Ly6-B, p-jun N-terminal kinase (p-JNK), and JNK expressions were measured by western blot analysis. Intracellular glutathione, reactive oxygen species, and malondialdehyde levels were also measured. APAP overdose increased hepatic CXCL16 mRNA and serum CXCL16 protein levels. CXCL16-deficient mice exhibited significantly less liver injury and hepatic necrosis, as well as a lower mortality than wild-type (WT) mice in response to APAP-overdose treatment. APAP elevated the production of oxidative stress and decreased mitochondrial respiratory chain activation in WT mice, which was strongly reversed in CXCL16-knockout mice. In addition, CXCL16 deficiency inhibited the neutrophil infiltration and the production of proinflammatory cytokines triggered by APAP-overdose treatment. Our study revealed that CXCL16 is a critical regulator of liver immune response to APAP-induced hepatotoxicity, thus providing a potential strategy for the treatment of drug-induced acute liver failure by targeting CXCL16.

Selective killing of gastric cancer cells by a small molecule targeting ROS-mediated ER stress activation.

Gastric cancer is one of the leading causes of cancer mortality in the world. Curcumin is a natural product with multiple pharmacological activities, while its clinical application has been limited by the poor chemical stability. We have previously designed a series of curcumin derivatives with high stability and anticancer potentials. The present study aims to identify the anti-cancer effects and mechanisms of WZ26, an analog of curcumin, in gastric cancer cells. In vitro, WZ26 showed higher chemical stability and much stronger anti-proliferative effects than curcumin, accompanied by dose-dependent induction of cell cycle arrest and apoptosis in gastric cancer cells. Mechanistically, the novel compound WZ26 induced ROS production, resulting in the activation of JNK-mitochondrial and ER stress apoptotic pathways. Blockage of ROS production totally reversed WZ26-induced JNK activation, Bcl-2/Bax decrease, ER stress activation, and final cell apoptosis in SGC-7901 cells. WZ26 also exhibited potent anti-tumor effects in human gastric cancer cell xenograft models. WZ26 could be considered as a potential chemotherapeutic agent for the treatment of advanced gastric cancer. In addition, this study also demonstrated that ROS production could be act as a vital candidate pathway for inducing tumor cell apoptosis by targeting mitochondrial and ER stress-related death pathway. © 2015 Wiley Periodicals, Inc.

Wip1 knockout inhibits the proliferation and enhances the migration of bone marrow mesenchymal stem cells.

Mesenchymal stem cells (MSCs), a unique population of multipotent adult progenitor cells originally found in bone marrow (BM), are extremely useful for multifunctional therapeutic approaches. However, the growth arrest and premature senescence of MSCs in vitro prevent the in-depth characterization of these cells. In addition, the regulatory factors involved in MSCs migration remain largely unknown. Given that protein phosphorylation is associated with the processes of MSCs proliferation and migration, we focused on wild-type p53-inducible phosphatase-1 (Wip1), a well-studied modulator of phosphorylation, in this study. Our results showed that Wip1 knockout significantly inhibited MSCs proliferation and induced G2-phase cell-cycle arrest by reducing cyclinB1 expression. Compared with WT-MSCs, Wip1(-/-) MSCs displayed premature growth arrest after six passages in culture. Transwell and scratch assays revealed that Wip1(-/-) MSCs migrate more effectively than WT-MSCs. Moreover, the enhanced migratory response of Wip1(-/-) MSCs may be attributed to increases in the induction of Rac1-GTP activity, the pAKT/AKT ratio, the rearrangement of filamentous-actin (f-actin), and filopodia formation. Based on these results, we then examined the effect of treatment with a PI3K/AKT and Rac1 inhibitor, both of which impaired the migratory activity of MSCs. Therefore, we propose that the PI3K/AKT/Rac1 signaling axis mediates the Wip1 knockout-induced migration of MSCs. Our findings indicate that the principal function of Wip1 in MSCs transformation is the maintenance of proliferative capacity. Nevertheless, knocking out Wip1 increases the migratory capacity of MSCs. This dual effect of Wip1 provides the potential for purposeful routing of MSCs.

A rabbit pulmonary vein myocyte isolation method based on simultaneous heart and pulmonary vein perfusion.

Myocytes in the pulmonary veins (PV) play a pivotal role in the development of paroxysmal atrial fibrillation (AF). It is therefore important to understand physiological characteristics of these cells. Studies on these cells are, however, markedly impeded by the fact that single PV myocytes are very difficult to obtain due to lack of effective isolation methods. In this study, we described a novel PV myocyte isolation method. The key aspect of this method is to establish a combination of retrograde heart perfusion (via the aorta) and anterograde PV perfusion (via the pulmonary artery). With this simultaneous perfusion method, a better perfusion of the PV myocytes can be obtained. As results, the output and viability of single myocytes isolated by simultaneous heart and PV perfusion method were increased compared with those in conventional retrograde heart perfusion method.

Anti-inflammatory effects of novel curcumin analogs in experimental acute lung injury.

BACKGROUND: Acute lung injury (ALI) and its most severe form acute respiratory distress syndrome (ARDS) have been the leading cause of morbidity and mortality in intensive care units (ICU). Currently, there is no effective pharmacological treatment for acute lung injury. Curcumin, extracted from turmeric, exhibits broad anti-inflammatory properties through down-regulating inflammatory cytokines. However, the instability of curcumin limits its clinical application. METHODS: A series of new curcumin analogs were synthesized and screened for their inhibitory effects on the production of TNF-α and IL-6 in mouse peritoneal macrophages by ELISA. The evaluation of stability and mechanism of active compounds was determined using UV-assay and Western Blot, respectively. In vivo, SD rats were pretreatment with c26 for seven days and then intratracheally injected with LPS to induce ALI. Pulmonary edema, protein concentration in BALF, injury of lung tissue, inflammatory cytokines in serum and BALF, inflammatory cell infiltration, inflammatory cytokines mRNA expression, and MAPKs phosphorylation were analyzed. We also measured the inflammatory gene expression in human pulmonary epithelial cells. RESULTS: In the study, we synthesized 30 curcumin analogs. The bioscreeening assay showed that most compounds inhibited LPS-induced production of TNF-α and IL-6. The active compounds, a17, a18, c9 and c26, exhibited their anti-inflammatory activity in a dose-dependent manner and exhibited greater stability than curcumin in vitro. Furthermore, the active compound c26 dose-dependently inhibited ERK phosphorylation. In vivo, LPS significantly increased protein concentration and number of inflammatory cells in BALF, pulmonary edema, pathological changes of lung tissue, inflammatory cytokines in serum and BALF, macrophage infiltration, inflammatory gene expression, and MAPKs phosphorylation . However, pretreatment with c26 attenuated the LPS induced increase through ERK pathway in vivo. Meanwhile, compound c26 reduced the LPS-induced inflammatory gene expression in human pulmonary epithelial cells. CONCLUSIONS: These results suggest that the novel curcumin analog c26 has remarkable protective effects on LPS-induced ALI in rat. These effects may be related to its ability to suppress production of inflammatory cytokines through ERK pathway. Compound c26, with improved chemical stability and bioactivity, may have the potential to be further developed into an anti-inflammatory candidate for the prevention and treatment of ALI.

[Research Progress of Collagen-based Three-dimensional Porous Scaffolds Used in Skin Tissue Engineering].

Collagen is a kind of natural biomedical material and collagen based three-dimensional porous scaffolds have been widely used in skin tissue engineering. However, these scaffolds do not meet the requirements for artificial skin substitutes in terms of their poor mechanical properties, short supply, and rejection in the bodies. All of these factors limit their further application in skin tissue engineering. A variety of methods have been chosen to meliorate the situation, such as cross linking and blending other substance for improving mechanical properties. The highly biomimetic scaffolds either in structure or in function can be prepared through culturing cells and loading growth factors. To avoid the drawbacks of unsafety attributing to animals, investigators have fixed their eyes on the recombinant collagen. This paper reviews the the progress of research and application of collagen-based 3-dimensional porous scaffolds in skin tissue engineering.