Alleviation of cadmium-induced photoinhibition and oxidative stress by melatonin in Chlamydomonas reinhardtii.

As one of the most threatening challenges to the natural environment and human health, cadmium (Cd) pollution has seriously impacted natural organisms. Green algae, such as Chlamydomonas reinhardtii (C. reinhardtii), can provide a safer, lower cost, and more effective ecological approach to the treatment of heavy metal ions in wastewater due to their sorption properties. However, heavy metal ions affect C. reinhardtii when adsorbed. Melatonin is able to protect the plant body from damage when the plant is under biotic/abiotic stress. Therefore, we investigated the effects of melatonin on the cell morphology, chlorophyll content, chlorophyll fluorescence parameters, enzymatic activity of the antioxidant system, gene expression, and the ascorbic acid (AsA)-glutathione (GSH) cycle of C. reinhardtii under the stress of Cd (13 mg/L). Our results indicated that Cd significantly induced photoinhibition and overaccumulation of reactive oxygen species (ROS). By application with the concentration of 1.0 µM melatonin, the algal solute of C. reinhardtii under the Cd stress gradually regained its green color, the cell morphology became intact, and the photosynthetic electron transport function was retained. However, in the melatonin-silenced strain, there was a significant decrease in all of the above indicators. In addition, the use of exogenous melatonin or the expression of endogenous melatonin genes could enhance the intracellular enzyme activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR). It also upregulated the expression of active enzyme genes such as SOD1, CAT1, FSD1, GSH1, GPX5, and GSHR1. These results indicate that the presence of melatonin effectively protects the activity of photosynthetic system II in C. reinhardtii, enhances antioxidant activity, upregulates gene expression in the AsA-GSH cycle, and reduces the level of ROS, thereby alleviating the damage caused by Cd toxicity.

Exogenous melatonin improved the growth and development of naked oat seedlings under cadmium stress.

Heavy metal pollution has become a global problem, which affect more and more crop yields. Melatonin (MT) is widely used in plant stress resistance to alleviate the toxicity caused by heavy metals and other stresses. In this paper, the effects of exogenous 50 µM and 100 µM MT on the growth and development of naked oat seedlings under cadmium stress (25 mg L-1) were studied. The results showed that different concentrations of MT could promote the growth of naked oat seedlings under 25-mg L-1 cadmium stress. The application of exogenous melatonin could significantly increase the plant height, fresh weight, dry weight, chlorophyll, and proline contents of naked oats. MT could also reduce the contents of hydrogen peroxide, superoxide anion, and malondialdehyde in the cells of naked oat seedlings, and increase the activities of SOD, POD, and CAT. In addition, exogenous melatonin could affect the gene expression of LOX, POX, and Asmap1 in MAPK family and NAC and WRKY1 in TFS family in naked oat seedlings, thus promoting the growth and development of naked oat seedlings. In conclusion, this study is the first to demonstrate that MT is able to alleviate the negative effects to treat naked oat seedlings with cadmium stress. Therefore, melatonin has the potential to be applied in crops threatened by heavy metals.

Chinese multidisciplinary expert consensus: Guidelines on percutaneous transthoracic needle biopsy.

Biopsy has been used to diagnose thoracic diseases for more than a century. Percutaneous needle biopsy plays a crucial role in the diagnosis, staging, and treatment planning for tumors in the lungs, thoracic wall, hilum, and mediastinum. With the continuous improvement in imaging techniques, the range of clinical applications for percutaneous needle biopsy is also expanding. It has become important to improve Chinese professionals' and technicians' understanding of percutaneous transthoracic needle biopsy (PTNB) in order to standardize operating procedures and to strengthen perioperative management. However, there is currently no Chinese expert consensus that provides systematic standardization and guidance for PTNB in clinical practice. The Committee of Chinese Society of Interventional Oncology (CSIO) of the Chinese Anti-Cancer Association (CACA) initiated a Chinese multidisciplinary expert consensus on PTNB. The consensus includes image-guided methods, indications, contraindications, multidisciplinary team recommendations, biopsy procedures, daytime/outpatient biopsy, complications, pathological examination, and management of negative results.

Clip domain prophenoloxidase activating protease is required for Ostrinia furnacalis Guenée to defend against bacterial infection.

The prophenoloxidase (PPO) activating system in insects plays an important role in defense against microbial invasion. In this paper, we identified a PPO activating protease (designated OfPAP) containing a 1203 bp open reading frame encoding a 400-residue protein composed of two clip domains and a C-terminal serine protease domain from Ostrinia furnacalis. SignalP analysis revealed a putative signal peptide of 18 residues. The mature OfPAP was predicted to be 382 residues long with a calculated Mr of 44.8 kDa and pI of 6.66. Multiple sequence alignment and phylogenetic analysis indicated that OfPAP was orthologous to the PAPs in the other lepidopterans. A large increase of the transcript levels was observed in hemocytes at 4 h post injection (hpi) of killed Bacillus subtilis, whereas its level in integument increased continuously from 4 to 12 hpi in the challenged larvae and began to decline at 24 hpi. After OfPAP expression had been silenced, the median lethal time (LT50) of Escherichia coli-infected larvae (1.0 day) became significantly lower than that of E. coli-infected wild-type (3.0 days, p < 0.01). A 3.5-fold increase in E. coli colony forming units occurred in larval hemolymph of the OfPAP knockdown larvae, as compared with that of the control larvae not injected with dsRNA. There were notable decreases in PO and IEARase activities in hemolymph of the OfPAP knockdown larvae. In summary, we have demonstrated that OfPAP is a component of the PPO activation system, likely by functioning as a PPO activating protease in O. furnacalis larvae.

Tenascin-c renders a proangiogenic phenotype in macrophage via annexin II.

Tenascin-c is an extracellular matrix glycoprotein, the expression of which relates to the progression of atherosclerosis, myocardial infarction and heart failure. Annexin II acts as a cell surface receptor of tenascin-c. This study aimed to delineate the role of tenascin-c and annexin II in macrophages presented in atherosclerotic plaque. Animal models with atherosclerotic lesions were established using ApoE-KO mice fed with high-cholesterol diet. The expression of tenascin-c and annexin II in atherosclerotic lesions was determined by qRT-PCR, Western blot and immunohistochemistry analysis. Raw 264.7 macrophages and human primary macrophages were exposed to 5, 10 and 15 µg/ml tenascin-c for 12 hrs. Cell migration as well as the proangiogenic ability of macrophages was examined. Additionally, annexin II expression was delineated in raw 264.7 macrophages under normal condition (20% O2 ) for 12 hrs or hypoxic condition (1% O2 ) for 6-12 hrs. The expression of tenascin-c and annexin II was markedly augmented in lesion aorta. Tenascin-c positively regulated macrophage migration, which was dependent on the expression of annexin II in macrophages. VEGF release from macrophages and endothelial tube induction by macrophage were boosted by tenascin-c and attenuated by annexin II blocking. Furthermore, tenascin-c activated Akt/NF-κB and ERK signalling through annexin II. Lastly, hypoxia conditioning remarkably facilitates annexin II expression in macrophages through hypoxia-inducible factor (HIF)-1α but not HIF-2α. In conclusion, tenascin-c promoted macrophage migration and VEGF expression through annexin II, the expression of which was modulated by HIF-1α.

Cloning, expression and characterization of Ostrinia furnacalis serpin1, a regulator of the prophenoloxidase activation system.

Serine protease inhibitors of the serpin superfamily are regulators of proteases involved in a variety of physiological processes including immune responses. In this study, we have isolated a full-length serpin cDNA from Ostrinia furnacalis. The 1188 bp open reading frame encodes a 395-residue protein with a theoretical molecular mass of 43.3 kDa and an isoelectric point of 4.92. Ofserpin1 contains a putative signal peptide followed by a conserved domain including a reactive center loop (RCL) with a hinge region (E(344) to S(353)) and a predicted P1-P1' cleavage site (Leu(360)-Ser(361)). Ofserpin1 mRNA and protein were detected in all the tested tissues, particularly in hemocytes and integument. The recombinant protein inhibited chymotrypsin and trypsin in a dose-dependent manner, and were significantly cleaved by the enzyme trypsin and chymotrypsin. Ofserpin1 impeded the prophenoloxidase activation cascade by 45.6% at 16.5 µg, and affected activity of prophenoloxidase activating protease. Levels of Ofserpin1 transcripts in the integument were higher than those in hemocytes, fat body and midgut. After an immune challenge with Staphylococcus aureus and Escherichia coli, the relative mRNA levels of Ofserpin1 decreased in 2-10h post-infection (hpi) in integument and hemocytes compared to the untreated control. Our results suggested that Ofserpin1 has serine protease inhibitory activity and is likely involved in the regulation of prophenoloxidase activation system in O. furnacalis.

Induction of matrix metalloproteinase-3 (MMP-3) expression in the microglia by lipopolysaccharide (LPS) via upregulation of glycoprotein nonmetastatic melanoma B (GPNMB) expression.

Substantial evidence suggests that inflammation is an important contributor to many neurodegenerative disorders. Activated microglial cells play an important role in releasing pro-inflammatory factors, including tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) for inducing inflammation. Recently, some reports have suggested that glycoprotein nonmetastatic melanoma B (GPNMB) is highly expressed in microglia after LPS treatment. However, the role of GPNMB in activated microglia is not clearly understood. In this study, we used RT-PCR and Western blotting to detect GPNMB and matrix metalloproteinase-3 (MMP-3) expressions in activated microglia. GPNMB small interfering RNA (siRNA) or MMP-3 inhibitor was applied on microglial BV2 cells, and ELISA was performed to measure the expressions of TNF-α and IL-1ß in BV2 cells. Levels of iNOS and NO in BV2 cells were also determined. We found that the levels of GPNMB and MMP-3 were significantly increased in BV2 cells after LPS treatment. Moreover, we found that GPNMB significantly upregulated the expression of MMP-3 in BV2 cells, and high expression of MMP-3 was dependent on the level of GPNMB. Inhibition of GPNMB or MMP-3 expression by GPNMB siRNA or MMP-3 inhibitor dramatically suppressed the expressions of TNF-α, IL-1ß, iNOS, and NO in activated microglia. All of these results suggest that GPNMB is involved in the inflammatory responses of microglia.

[Gene expression of Notch1 and Jagged1 in children with acute leukemia].

OBJECTIVE: To study the gene expression of Notch1 and Jagged1 in children with acute leukemia (AL) and their possible roles in the pathogenesis of AL. METHODS: Mononuclear cells from bone marrow or peripheral blood of 47 children with AL and 20 controls (normal children or children with nonmalignant hematologic disease) were collected from February 2009 to July 2011. A two-step method to semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the gene expression of Notch1 and Jagged1. Of the 47 children with AL, there were 26 cases of B-ALL, 6 cases of T-ALL and 15 cases of AML. RESULTS: The positive expression rate of Notch1 in the ALL and AML groups was higher than in the control group (P<0.05). The expression level of Notch1 in T-ALL children was higher than in B-ALL children (P<0.01). The positive expression rate of Jagged1 in the ALL and AML groups was not significantly different from the control group, however, the expression level of Jagged1 in the ALL and AML groups was higher than in the control group (P<0.05). CONCLUSIONS: There are significant differences in the gene expression of Notch1 between children with different types of ALL, and a higher expression of Notch1 relates to T-ALL. The activation of Notch1 signal is common in children with AL. The abnormal gene expression of Notch1 in children with AML shows the role of Notch1 in AML. The gene expression of Jagged1 in children with ALL or AML is abnormal, and this needs to be confirmed by further research.

[Genetic transformation of Nicotiana tabacum L. by Agrobacterium tumefaciens carrying genes in the melatonin biosynthesis pathway and the enhancement of antioxidative capability in transgenic plants].

Arylalkylamine N-acetyltransferase (AANAT) and Hydroxyindole O-methyltransferase(HIOMT) are the key regulation enzymes in the melatonin biosynthesis pathway in mammals. The AANAT and HIOMT genes were constructed into a binary plant expression vector YXu55. Using leaf strips as the recipiences, we efficiently transformed tobacco (Nicotiana tabacum) variety qinyan 95 by the Agrobacterium mediated method. After gradient selection with gentamycin, a number of transgenic plants were regenerated. Southern blot and RT-PCR analyses showed that the AANAT-HIOMT genes were integrated into the genome of the transgenic plants and the target genes could express at the level of RNA transcription. By RP-HPLC, we measured the melatonin contents in transgenic plants. The results showed that the melatonin level in YXu55 (containing the gentamycin-resistance gene, the AANAT gene and HIOMT gene) transgenic plants were much higher than those in pZP122 (control containing only the gentamycin-resistance gene) transgenic plants and nontransgenic plants. The content of melatonin in pZP122 transgenic plants was nearly the same as that in nontransgenic plants. Physiological determination of antioxidative characteristics demonstrated that 1) the capacity of total antioxidation, 2) the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) and 3) the content of glutathione (GSH) were increased in YXu55 transgenic plants containing the AANAT-HIOMT genes as compared to the control plants (pZP122 or nontransgenic plants). At the same time, malonaldehyde (MDA) content did not appear remarkably difference between transgenic plants and nontransgenic plants. The above mentioned facts indicate enhancement of melatonin levels in YXu55 transgenic plants might help to reduce damage by oxidative stress.

Treatment of alopecia by transplantation of hair follicle stem cells and dermal papilla cells encapsulated in alginate gels.

The affected individual of hair loss demands help, because hair is viewed as a sign of youth and good health. Nowadays treatment of alopecia includes drug therapy and hair transplantation. Some drugs may promote hair growth, at least temporarily, but the treatment is effective only in milder alopecia, instead of extensive alopecia. Furthermore, the side effect of long period medication could not be avoided. Hair transplantation involves harvesting small pieces of hair-bearing scalp grafts from a donor site and relocating them to a bald area. This method does not increase the number of existing hairs, but only redistributes them. The operation is sophisticated and time-consuming, thus the patient suffers a lot during the process. The discovery of hair follicle stem cells (FSC) brings gospel to the affected individual of hair loss because of its capacity of generating new hair when they interact with mesenchymal dermal papilla cells (DPC). Besides, both FSC and DPC have strong proliferative capacity and the patient's own cells could be expanded considerably in vitro. Thus we hypothesize that the microencapsulation of the two kinds of cells in alginate gels could be implanted into the bald scalp of the patient since alginate gels is effective in cell transplantation. The strategy may provide a more convenient and valid alternative to hair loss if the hypothesis proved to be practical.