Nicotinamide adenine dinucleotide treatment confers resistance to neonatal ischemia and hypoxia:effects on neurobehavioral phenotypes

Neonatal hypoxic-ischemic brain injury is the main cause of hypoxic-ischemic encephalopathy and cerebral palsy.Currently,there are few effective clinical treatments for neonatal hypoxic-ischemic brain injury.Here,we investigated the neuroprotective and molecular mechanisms of exogenous nicotinamide adenine dinucleotide,which can protect against hypoxic injury in adulthood,in a mouse model of neonatal hypoxic-ischemic brain injury.In this study,nicotinamide adenine dinucleotide(5 mg/kg)was intraperitoneally administered 30 minutes befo re surgery and every 24 hours thereafter.The results showed that nicotinamide adenine dinucleotide treatment improved body weight,brain structure,adenosine triphosphate levels,oxidative damage,neurobehavioral test outcomes,and seizure threshold in experimental mice.Tandem mass tag proteomics revealed that numerous proteins were altered after nicotinamide adenine dinucleotide treatment in hypoxic-ischemic brain injury mice.Parallel reaction monitoring and western blotting confirmed changes in the expression levels of proteins including serine(or cysteine)peptidase inhibitor,clade A,member 3N,fibronectin 1,5'-nucleotidase,cytosolic IA,microtubule associated protein 2,and complexin 2.Proteomics analyses showed that nicotinamide adenine dinucleotide ameliorated hypoxic-ischemic injury through inflammation-related signaling pathways(e.g.,nuclear factor-kappa B,mitogen-activated protein kinase,and phosphatidylinositol 3 kinase/protein kinase B).These findings suggest that nicotinamide adenine dinucleotide treatment can improve neurobehavioral phenotypes in hypoxic-ischemic brain injury mice through inflammation-related pathways.

Sorafenib Combined with Cryoablation to Treat Unresectable Hepatocellular Carcinoma

Objective：To evaluate the efficacy and tolerability of sorafenib combined with cryoablation in treating unresectable hepatocellular carcinoma （HCC）.Methods：Patients with unresectable advanced HCC received cryoablation and sorafenib at a dose of 400 mg twice daily in 4-week cycles on the same day of the cryoablation.Tumor response,median overall survival and the median time to radiological progression were calculated and the toxicity was evaluated.Results：Seventy-eight patients with unresectable HCC were involved in this study.The median age was 52 years （range,22-81 years）.The Eastern Cooperative Oncology Group （ECOG） performance status scores were 0 （39.7%）,1 （55.1%）,and 2 （5.1%）.Nine （11.5%） patients were at Barcelona clinic liver cancer （BCLC） stage A,twenty-four （30.8%） patients were at stage B and 45 （57.7%） patients were at stage C.Five （6.4%） achieved partial responses,and 34 （43.6%） achieved stable disease.The median time to progression （TTP） for all enrolled patients was 6.6 months and the median overall survival （OS） was 12.2 months.Conclusion：Cryoablation combined with sorafenib demonstrates good efficacy and acceptable tolerability in treating unresectable advanced HCC patients.

Autophagy inhibitor 3-methyladenine regulatesthe expression of LC3, Beclin-1 and ZnTs in ratcerebral cortex following recurrent neonatal seizures

BACKGROUND： Autophagy is a homeostatic process for intracellular recycling of bulk proteins and aging organelles. Increased autophagy has now been reported in experimental models of traumatic brain injury, stroke and excitotoxicity, and in patients with Alzheimer＇s disease and critical illness. The role of autophagy in developmental epilepsy, however, is unknown. The present study was to investigate the effects of recurrent neonatal seizure, in the presence and absence of autophagy inhibitor 3-methyladenine （3-MA）, on the acute phase gene expression of ZnTs, LC3 and Beclin-1 in rat cerebral cortex and the interaction among them.METHODS： Thirty-six Sprague-Dawley neonatal rats at postnatal day 6（P6） were randomly divided into three groups： a recurrent-seizures group （RS, n=12）, a 3-MA treated-seizure group （3-MA group, each rat pretreated with 3-methyladenine before seizures, 100nmol/μl/day, i.p., n=12） and a control group （n=12）. At 1.5 and 6 hours after the last seizures, the mRNA levels of ZnT1-ZnT3, microtubule-associated protein 1A/1B light chain 3 （LC3） and beclin-1 were detected using the real-time RT-PCR method. The LC3 protein level was examined by Western blotting.RESULTS： The levels of LC3, beclin-1 and ZnT-2 transcripts in the RS group elevated signi？ cantly at 1.5 and 6 hours after the last seizures compared with those in the control and 3-MA groups. At the interval of 1.5 hours, the mRNA level of ZnT-1 increased signi？ cantly after the last seizure compared with that in the control group. There was no signi？ cant difference in the transcript levels of ZnT-3 among the three groups. Linear correlation analysis showed that the expression of the ？ ve genes in the control group exhibited a signi？ cant inter-relationship. In the 3-MA group, however, the inter-relationship was only found between beclin-1 and ZnT-1. In the RS group, the inter-relationship was not observed.CONCLUSIONS： The autophagy/lysosomal pathway is immediately activated along with the elevated expression of ZnT1 and ZnT2 in the cerebral cortex after recurrent seizures. 3-MA is involved in the regulation of the autophagy/lysosomal pathway and ZnTs by down-regulating the expression of LC3 and beclin-1.

Effects of physical exercise on the developmental expression of hippocampal zinc transporter 1 and glutamate receptor subunit 2, and on cognitive function in a rat model of recurrent neonatal seizure

BACKGROUND： Developmental seizures are pathologically characterized by regenerative sprouting of hippocampal mossy fibers rich in Zn^2＋. Zn^2＋ metabolism in the mossy fiber pathway, and Zn^2＋ accumulation in presynaptic membrane vesicles, are dependent on zinc transporter 1 （ZnT1） and glutamate receptor subunit 2 （GluR2）. OBJECTIVE： To investigate the effects of long-term recurrent neonatal seizure, in the presence and absence of physical exercise, on the developmental expression of hippocampal zinc transporter 1 （ZnT1） and GluR2, and on cognitive function in rats. DESIGN, TIME AND SETTING： Based on behavioral examination and molecular biological research, a randomized, controlled animal experiment was performed at the Department of Neurobiology, Medical College of Soochow University, between January 2007 and April 2008. MATERIALS： Twenty-one 6-day-old Sprague Dawley rats of either gender were employed in this study. ZnT1 mRNA in situ hybridization kit was provided by Tianjin Haoyang Biological Manufacture Co.,Ltd., China. Rabbit anti-GluR2 was purchased from Santa Cruz Biotech, Inc, USA. METHODS： Rats were randomly divided into a recurrent seizure group （n = 11） and a control group （n = 10）. In the recurrent seizure group, 30-minute seizure was induced by flurothyl gas inhalation for a total of 6 consecutive days. Rats from the control group underwent experimental procedures similar to the recurrent seizure group, with the exception of flurothyl gas inhalation. Thirty minutes of treadmill exercise was performed daily by all rats at postnatal days 51–56. MAIN OUTCOME MEASURES： At postnatal day 82, rat hippocampal tissue was harvested for analysis of hippocampal ZnT1 and GluR2 expression by in situ hybridization and immunohistochemistry, respectively. Rat learning and memory capabilities were examined using the Y-maze test. RESULTS： In the recurrent seizure group, the gray scale value of ZnT1 in situ hybridization positive neurons in the hippocampal CA3 region was significantly greater （P 〈 0.05）, while the gray scale value of GluR2 immunoreactive neurons in the hippocampal hilus and dentate gyrus was significantly lower （P 〈 0.05）, than in the control group. At postnatal days 29–35, numbers of trials to criteria for successful learning were greater in the recurrent seizure group than in the control group （P 〈 0.05）; at postnatal days 61–67, the numbers of trials to criteria for successful learning were similar between the two groups （P 〉 0.05）. At postnatal days 29–35 and 61–67, there was no significant difference in memory capability between the recurrent seizure and control groups （P 〉 0.05）. CONCLUSION： Physical exercise likely improves the learning deficits caused by recurrent neonatal seizure in rats during brain development by modulating ZnT1 and GluR2 expression.

Zinc transporter-3 expression and long-term cognitive impairments in a rat model of neonatal concurrent seizure

BACKGROUND： Developmental seizures, which are pathologically characterized by regenerative sprouting of hippocampal mossy fibers, cause long-term damaging effects to synaptic plasticity. Zn^2＋ metabolism has been shown to contribute to the regenerative sprouting of hippocampal mossy fibers Furthermore, zinc transporter-3 （ZnT3） is responsible for Zn^2＋ transport in the hippocampal mossy fiber pathway. OBJECTIVE： To investigate the effects of long-term recurrent neonatal seizures on learning, memory formation and hippocampal ZnT3 expression in rats. DESIGN, TIME AND SETTING： Based on molecular biological research and behavioral examination a randomized, controlled, animal experiment was performed at the Laboratory Animal Center, Peking University Health Science Center, between October 2004 and July 2005. MATERIALS： Flurothyl was purchased from Aldrich Chemical Co., USA. ZnT3 mRNA in situ hybridization kits were provided by Tianjin Haoyang Biological Manufacture Co., Ltd., China. Morris water maze was produced by Shanghai Jiliang Science and Technology Co., Ltd., China. METHODS： Sixty, 6-day old, Wistar rats were randomly divided into three groups： single seizure （n = 21）, recurrent seizure （n = 21, one seizure daily for 6 consecutive days）, and control （n = 18）. Seizures were induced by flurothyl gas inhalation, in the single seizure and recurrent seizure groups. MAIN OUTCOME MEASURES： At postnatal days 12, 46 and 90, rat hippocampal ZnT3 mRNA expression was detected by RT-PCR; at postnatal days 46 and 90, ZnT3 mRNA expression was determined by in situ hybridization; and at postnatal days 41-46 and 85 90, rat spatial learning and memory formation were examined by the Morris water maze test. RESULTS： RT-PCR results revealed that at postnatal day 12, ZnT3 expression was significantly greater in the recurrent seizure group than in the control and single seizure groups, and at day 46, it was also significantly greater in the recurrent seizure group compared with the control group （P 〈 0.05）. In situ hybridization results showed that at postnatal day 46, the recurrent seizure group exhibited increased hippocampal ZnT3 expression over the control and single seizure groups （P〈0.05）. Morris water maze test results displayed that, in the first place navigation test at postnatal day 44, and the second test at days 87-88, the recurrent seizure group exhibited significantly higher value of average escape latency compared with the control group （P 〈 0.05）. In the two spatial probe tests, the search strategies were significantly inferior in the recurrent seizure group than in the control and single seizure groups （P 〈 0.05）. CONCLUSION： Neonatal concurrent seizures produce long-term damaging effects on hippocampal ZnT3 expression and cognitive function, while both of which have no parallel correlation.

Study on the Effect and Mechanism of Green Plum Citron Double Strain Fermentation Filtrate on Skin Barrier

To study the effect of Green plum citron double strain fermentation filtrate(DSF)on the proliferation rate of HaCaT keratinocytes by CCK8 assay.The effect of DSF on the mRNA transcription levels of filagolin(FLG),loricrin(LOR),and occludin 1(CLD1)in HaCaT cells in sodium dodecyl sulfate(SLS)model was investigated by qPCR.The effect of DSF on the content of TNF-αin the supernatant of HaCaT cells in the model was investigated by Elisa.The results showed that DSF had no toxicity to HaCaT cells,but it could increase the mRNA transcription levels of FLG,LOR and CLD1 in HaCaT cells in the model,and reduce the content of TNF-αin the supernatant of HaCaT cells in the model.DSF had a certain effect on skin care of strengthening skin barrier.

甜瓜靶斑病病原菌的生物学特性

甜瓜(Cucumis melo L.)是我国重要的瓜类经济作物,多主棒孢(Corynespora cassiicola)引起的甜瓜靶斑病是近年新突发病害,通常引起叶部靶斑症状,对甜瓜生产造成重要损失。为了更好地防控甜瓜靶斑病,系统研究了多主棒孢甜瓜分离物广西菌株的生物学特性,深入探讨了该菌的最适产孢,菌丝生长及孢子萌发条件。结果表明,病原菌菌丝生长的最适碳源是葡萄糖,最适氮源是KNO3,最适温度是27℃,最适pH是6~9,最适产孢培养基是寄主干叶培养基。分生孢子萌发的最适碳源是蔗糖和葡萄糖,最适pH是5~8。

甜瓜靶斑病病原菌鉴定

甜瓜(Cucumis melo L.)是我国重要的瓜类经济作物。2015年10月在我国广西武鸣甜瓜主要种植区进行病害调查时发现一种新的叶斑病。该病原菌疑似通常危害植物叶片引起叶斑病的多主棒孢菌。采用形态学观察,多基因鉴定和致病性试验的方法对引起该病的病原菌进行了鉴定。病原菌菌落中间墨绿色,边缘浅褐色,有明显的生长圈,气生菌丝茂密。分生孢子呈圆柱形或倒棍棒形,单生或串生,直立或稍微弯曲,具0～13个隔膜,大小为(3.1～6.4)μm×(14.0～138.9)μm。克隆测定该病原菌的4个保守序列ITS1,2、ITS4,5、ACT和TUB2。分离纯化的病原菌回接到甜瓜幼苗,表现出与田间相似的症状,符合柯赫氏法则,鉴定该病原菌为多主棒孢。该病原菌也可侵染西瓜(Citrullus vulgaris Schrad.)和黄瓜(Cucumis sativus L.)幼苗及离体叶片使其发病。

福建砂梨急性花枯病病原菌的分离与鉴定

【目的】明确近年在福建砂梨产区新发生的梨急性花枯病的病原菌种类。【方法】从产区采集显现急性花枯症状的‘翠冠’梨病样品进行组织分离,并利用形态学和分子生物学相结合的方法对获得的菌株进行种类鉴定和致病性验证。【结果】通过组织分离和纯化,并根据其菌落形态特征从福建砂梨急性花枯病样品中获得31个刺盘孢属(Colletotrichum)菌株。选取其中的14个代表菌株进行形态学观察和多基因(ITS、ACT、TUB、CHS-1及GAPDH)系统发育分析的结果显示,它们均为松针刺盘孢(C. fioriniae)。在一周花龄的砂梨(品种‘丰水梨’)、西洋梨(品种‘三季梨’)和秋子梨(品种‘南果梨’)的离体花序上喷雾接种代表菌株(CFZH1、CFZH6、CFZH15)分生孢子悬浮液的结果显示,均产生有急性花枯症状,平均发病率分别95.8%、63.9%和81.3%。【结论】福建砂梨产区导致梨急性花枯病的病原菌为松针刺盘孢(C. fioriniae)。本研究是福建砂梨产区松针刺盘孢引起梨花枯死的首次报道。

22种杀菌剂对甜瓜靶斑病菌的室内毒力测试

为筛选多主棒孢甜瓜分离物的高效杀菌剂,在室内采用菌丝生长速率法测定了22种杀菌剂对甜瓜多主棒孢SY和GX分离株菌丝生长的抑制能力。结果表明,50%咪鲜胺锰盐WP对多主棒孢甜瓜分离物SY和GX菌株的EC_(50)都最小,病菌对它的敏感性最高,抑菌效果最好;30%苯醚甲环唑SC、40%氟硅唑EC和80%福美双WG对多主棒孢甜瓜分离物GX分离株的EC_(50)均小于5μg·mL^(-1),抑菌效果较好,但在SY菌株上抑菌效果减弱。在试验浓度下,2个多主棒孢供试菌株对25%嘧菌酯SC、75%百菌清WP和90%多菌灵WG都非常不敏感,说明这3种杀菌剂对甜瓜靶斑病的防控不能起到有效作用。

Effect of Electroplating Waste water on Immune Function in Kunming Mice

Electroplating waste water is considered to be harmful to health of animals. In the present study, we tested the hypothesis that electroplating waste water would suppress immune functions in Kunming mice. Twenty-six mice were randomly divided into the control group (n = 13) and the experimental group (n = 13), in which the latter drank electroplating waste water. We found that body mass and most organ wet masses (heart, lungs, liver, kidneys, stomach, caecum, colon, testes, epididymis, seminal vesicals) were not influenced by electroplating waste water. However, stomach with its content, small intestine, small intestine with its contents and colon with its contents were higher in the experimental group than in the control group. As expected, phytohaemagglutinin (PHA) response indicative of cellular immunity was suppressed by electroplating waste water. White blood cells, thymus and spleen mass were all not response to electroplating waste water. Taken together, electroplating waste water had different effects on distinct components of immune system in Kunming mice.

我国梨产区引起黑斑病的链格孢种类鉴定与致病性研究

【目的】明确我国梨主产区引起黑斑病的链格孢属(Alternaria)真菌种类及其致病性,为制定有效防治措施提供理论依据。【方法】从我国梨主产区采集黑斑病病样进行组织分离,并利用形态学和分子生物学相结合的方法对获得的菌株进行种类鉴定和致病性验证。【结果】从我国14个省、自治区及直辖市的梨产区采集病样后,通过组织分离和纯化并依据其菌落形态特征,共获得405个链格孢属(Alternaria)菌株。对这些菌株的形态学观察和多基因(ITS、GAPDH、Alt a1、TEF 1、endoPG、及His 3)系统发育分析的结果显示,它们分别属于链格孢属(Alternaria)的6个种。其中属细极链格孢(Alternaria tenuissima)的有267个菌株、链格孢(A.alternata)115个菌株、乔木链格孢(A.arborescens)14个菌株、梨黑斑链格孢(A.gaisen)6个菌株、棉链格孢(A.gossypina)2个菌株、长柄链格孢(A.longipes)1个菌株。将这6个种的代表菌株在翠冠梨离体叶片上进行有伤接种的结果显示,它们均可致病,但其致病力之间存在差异。而在桃(Prunus persica)、猕猴桃(Actinidia chinensis)和柑橘(Citrus reticulate)离体叶片上进行有伤接种的结果显示,这些代表菌株均使桃和猕猴桃致病,但均不能使柑橘致病。【结论】引起我国梨黑斑病的病原菌有A.tenuissima、A.alternata、A.arborescens、A.gaisen、A.gossypina和A.longipes 6种链格孢属(Alternaria)真菌,其中细极链格孢(A.tenuissima)和链格孢(A.alternata)为优势种,分别占总分离菌株数的65.9%和28.4%。本研究是我国梨主产区引起黑斑病的链格孢属(Alternaria)真菌种类系统性鉴定的首次报道。

中国南方主要梨产区果生炭疽菌对苯醚甲环唑的敏感性

为明确苯醚甲环唑对果生炭疽菌Colletotrichum fructicola不同发育阶段的毒力,并建立中国南方主要梨产区(湖北、安徽、江西、江苏、浙江和福建)果生炭疽菌对苯醚甲环唑的敏感基线,采用孢子萌发法测定了苯醚甲环唑对6个代表性菌株孢子萌发和芽管伸长的影响,采用离体叶片接种法评价了苯醚甲环唑对该病菌的防治效果,采用菌丝生长速率法测定了120株果生炭疽菌对苯醚甲环唑的敏感性。结果表明:苯醚甲环唑对果生炭疽菌菌丝生长和芽管伸长的抑制效果较强,而对分生孢子萌发的抑制效果较弱,其平均EC50值分别为(0.33±0.03)、(2.61±0.26)和(29.87±1.31)μg/mL。160μg/mL的苯醚甲环唑对梨果生炭疽病的保护作用和治疗作用防效分别为92%和79%,而1000μg/mL的多菌灵则分别为51%和23%。来源于中国南方主要梨产区的120株果生炭疽菌对苯醚甲环唑均表现为敏感,EC50值范围0.27~1.12μg/mL,敏感性频率分布呈连续单峰曲线,符合正偏态分布,其平均EC50值(0.59±0.02)μg/mL可以作为我国南方主要梨产区果生炭疽菌对苯醚甲环唑的敏感基线。本研究结果为监测梨果生炭疽菌对苯醚甲环唑的田间抗药性发展奠定了基础,并为梨产区应用该药剂防治炭疽病提供了依据。

福建李叶斑病病原菌种类鉴定及致病性研究

【目的】鉴定明确近年在福建新发生的李叶斑病的病原菌种类。【方法】采集李叶斑病叶进行组织分离,对获得的菌株采用形态学和分子生物学相结合的方法进行种类鉴定和致病性研究。【结果】通过组织分离和纯化,并根据菌落形态特征共获得66个刺盘孢属(Colletotrichum)菌株。对这些菌株进行形态学观察和多基因(ACT、TUB2、CHS-1、GAPDH及ITS)系统发育分析的结果显示,它们分别归属于刺盘孢属的6个种,包括果生刺盘孢(C.fructicola)59个菌株、喀斯特刺盘孢(C.karstii)2个菌株、普洛柏刺盘孢(C.plurivorum)2个菌株、暹罗刺盘孢(C.siamense)1个菌株、无锡刺盘孢(C.wuxiense)1个菌株和李刺盘孢(C.pruni-salicinae)1个菌株,其中李刺盘孢(C.pruni-salicinae)为笔者鉴定出的1个新种。分离鉴定的6种刺盘孢的代表菌株,有伤接种结果显示它们均可使李叶片和果实致病,但其致病力明显不同,它们对桃、梨、柑橘和猕猴桃的致病也存在显著差异。【结论】引起福建李叶斑病的病原菌有果生刺盘孢、喀斯特刺盘孢、普洛柏刺盘孢、暹罗刺盘孢、无锡刺盘孢和李刺盘孢6种,其中果生刺盘孢(C.fructicola)为优势种,占刺分离获得的盘孢属(Colletotrichum)菌株的89.4%。不同刺盘孢菌的致病性存在明显差异。

我国砂梨主产区白纹羽病的病原鉴定及序列分析

【目的】鉴定明确我国砂梨主产区白纹羽病的病原菌种类,为该病的有效防控提供理论依据。【方法】在我国砂梨主产区调查白纹羽病的发生危害特点并采集病根样品,通过组织分离法和单菌丝纯化获得分离株,对获得的菌株采用形态学和分子生物学相结合的方法进行种类鉴定和致病性验证。【结果】从湖北、福建、贵州、四川和山东等砂梨主产区采样到48份病根样品,共分离获得128个座坚壳(Rosellinia)菌株。所有菌株的菌落初期为白色,9 d后有黑色素沉淀;菌丝均无色透明,产生有分支,部分在隔膜处呈梨形膨大。36个代表菌株多基因(ITS、TUB、RPB2和LSU)系统发育分析的结果显示,所获得的座坚壳(Rosellinia)菌株均属褐座坚壳菌(R.necatrix),但不同来源菌株的基因序列有异,在系统进化树中聚集在3个不同的亚分支。从3个亚分支中选取其品种来源均为翠冠梨的3个菌株HB1-1-15、FJ1-2-2和GZ1-2-3,于小麦粒上培养14 d后拌土接种到杜梨生根试管苗后均可产生与田间相同的症状,但3个菌株的致病力存在显著差异。【结论】我国砂梨主产区白纹羽病的病原均为褐座坚壳菌(R.necatrix),不同来源菌株的基因序列之间存在明显差异。本研究是我国梨白纹羽病病原系统鉴定的首次报道。

导致南方梨早期落叶的果生炭疽菌致病力分化分析

【目的】明确我国南方梨产区造成早期落叶的果生炭疽菌(Colletotrichum fructicola)的致病力分化状况并建立其室内快速测定方法。【方法】以梨的枝条、叶片和果实为材料,采用不同方法造成伤口后接种果生炭疽菌的强致病力菌株PAFQ32,通过比较各处理的测定效果筛选其室内快速测定方法,并对供试菌株的致病力进行观测和致病类型划分,分析不同菌株致病力分化与其地理来源之间的相关性。【结果】采用梨枝条、叶片和果实对果生炭疽菌致病力的观测结果显示,梨叶片经针刺后接种菌丝块的测定效果明显优于其他处理。供试菌株致病力的测定结果表明,来源于我国南方梨产区的111个果生炭疽菌菌株其致病力可划分为强、中、弱3个类型,其中强致病力菌株17个(15.3%);中等致病力菌株89个(80.2%);弱致病力菌株5个(4.5%)。不同地理来源的果生炭疽菌菌株,其致病类型的分布比例有异。【结论】果生炭疽菌的菌丝块针刺接种梨叶片的方法,可用于其致病力的室内快速测定。来源于我国南方梨产区导致早期落叶的果生炭疽菌存在明显的致病力分化,以中等致病力菌株为优势群体。

Fra-1 protooncogene regulates IL-6 expression in macrophages and promotes the generation of M2d macrophages

The tumor microenvironment （TME） plays a prominent role in the growth of tumor cells. As the major inflammatory component of the TME, M2d macrophages are educated by the TME such that they adopt an immunosnppressive role that promotes tumor metastasis and progression. Fra-1 forms activator protein-1 heterodimers with Jun partners and drives gene transcription. Fra-1 is thought to drastically induce tumorigenesis and progression. However, the functional role of Fra-1 in the generation of M2d macrophages is poorly understood to date. Here, we demonstrate that 4T1 mammary carcinoma cells, when co-cultured with RAW264.7 macrophage cells, skew the RAW264.7 macrophage cell differentiation into M2d macrophages. The 4T1 cells stimulate de novo overexpression of Fra-1 in RAW264.7 cells, and then Fra-1 binds to the interleukin 6 （IL-6） promoter to increase the production of the cytokine IL-6 in RAW264.7 cells. IL-6 acts in an autocrine fashion to skew RAW264.7 macrophage cell differentiation into M2d macrophages. These findings open new insights into how to reverse M2d macrophage-induced immune tolerance to improve the efficacy of immunotherapeutic approaches.

产漆酶真菌筛选、鉴定及酶促去除苯酚的条件优化

以愈创木酚为底物,从自然环境中成功筛选到一株高产漆酶的粗毛革孔菌(Coriolopsis gallica)NCULAC F1。对其漆酶诱导表达条件进行探究,结果表明,9种芳香化合物对NCULAC F1表达漆酶均无显著诱导作用(P>0.05);而硫酸铜具有显著诱导作用(P<0.05),且硫酸铜与2,6-二甲基苯酚具有显著的协同诱导作用(P<0.05);最佳协同诱导条件下,摇瓶发酵5 d漆酶活力达11.29 U·mL-1,比未添加任何诱导剂的对照组显著提高了138.69%(P<0.05)。提取纯化漆酶,进一步探究漆酶对苯酚的去除效果,结果表明,酶促去除苯酚的过程遵循Michaelis-Menten动力学模型,R2为0.9843,km为52.45 mg·L-1,Vmax为181.82 mg·L-1·h-1;最佳催化条件为温度70℃,pH 6.0,加酶量5 U·mL-1,介体2’2-联氨-双(3-乙基苯并噻唑啉-6-磺酸)二胺盐(ABTS)浓度0.6 mmol·L-1,在此条件下,10 min内苯酚去除率达100%。研究结果为粗毛革孔菌漆酶的高效生产和在酚类污染物控制方面的应用提供了理论参考。

Comparison of labeling methods and time course of traumatic brain injury-induced cell death in mice

BACKGROUND：Various molecular mechanisms of cell death following traumatic brain injury have been previously described.However,the time course of cell death remains unclear.TUNEL and Fluoro-Jade B labeling have been widely used to label apoptotic cells and neuronal degeneration.Propidium iodide （PI） functions as a biomarker of cell death in vivo.OBJECTIVE：To explore the role of PI labeling compared to TUNEL and Fluoro-Jade B staining for detecting neural cell death,and to observe time course of traumatic brain injury-induced cell death in mice.DESIGN,TIME AND SETTING：A randomized,controlled,animal experiment was performed at the Laboratory of Aging and Nervous Diseases,Soochow University from September 2007 to December 2008.MATERIALS：PI （B1221） was purchased from Sigma,USA.TUNEL kit was purchased from Roche Molecular Biochemicals,USA.Fluoro-Jade B was purchased from Chemicon,USA.METHODS：A total of 70 healthy,male,Kunming mice were randomly assigned to sham-surgery （n = 5） and model （n = 65） groups.Traumatic brain injury was established using the controlled cortical impact method.PI was intraperitoneally injected at 1 hour prior to animal sacrifice.MAIN OUTCOME MEASURES：TUNEL,Fluoro-Jade B,and Pl-positive cells were quantified using a double-labeling method to determine the time course of traumatic brain injury-induced cell death.RESULTS：PI labeled cells in an earlier phase of cell death than TUNEL and Fluoro-Jade B labeling.Pl-positive cells were observed immediately following injury,and the numbers rapidly increased in injured brain areas at 1 hour,peaked at 24-48 hours,and subsequently decreased at 3-21 days post-injury.TUNEL-labeled cells were significantly increased at 12 hours,while Fluoro-Jade B-labeled cells were increased at 6 hours after injury,with cells still visible at 6-48 hours post-injury.Moreover,a greater number of Pl-positive cells were observed compared to TUNEL- and Fluoro-Jade B-labeled cells.CONCLUSION：PI labeling is more sensitive and reliable than TUNEL and Fluoro-Jade B staining for detecting cell death following traumatic brain injury.Moreover,PI labeling can function as a reliable marker to estimate the entire time course of cell death.

Molecular and biological characterization of melon-infecting squash leaf curl China virus in China

It has been reported that squash leaf curl China virus(SLCCNV)infects some Cucurbitaceae crops except for melon(Cucumis melo L.).A new disease of melon exhibiting severe leaf curl and dwarfing was observed in Hainan Province of China.In this study,the pathogen was identified as SLCCNV through biological and molecular characterization.The isolate(SLCCNV-HN)possess a bipartite genome,DNA-A(HM566112.1)with the highest nucleotide identity(99%)to SLCCNV-Hn(MF062251.1)pumpkin and SLCCNV-Hn61(AM260205.1)squash isolates from China,whereas DNA-B(HM566113.1)with the highest nucleotide identity(99%)to SLCCNV-Hn(MF062252.1).Phylogenetic analyses based on the full-length SLCCNV-HN DNA-A and-B sequences indicated that SLCCNV-HN melon isolate is clustered with SLCCNV-Hn pumpkin,SLCCNV-Hn61 and SLCCNV-SY squash isolates from southern China,forming an independent cluster.Infectious clone of SLCCNV-HN was constructed and the melon plants were inoculated and the infection rate is 100%,the systemic symptoms in melon showed identical to those of melon plants infected in fields.Additionally,melon plants transmission of this virus by Bemisia tabaci with a transmission rate of 95%(19/20)showed leaf curl and dwarf symptoms 15 days post transmission,thereby fulfilling Koch’s postulates.Analysis of genomic organization and phylogenetic trees indicated that SLCCNV-HN melon isolate belongs to the Begomovirus genus.To the best of our knowledge,this is the first characterization of meloninfecting SLCCNV through its genome,infectious clone and transmission.