[Effects of canagliflozin on amino acid metabolism in atherosclerotic mice].

Objective: To explore the possible anti-atherosclerotic mechanisms of glucose co-transporter-2 inhibitor canagliflozin. Methods: ApoE-/-mice fed on Western diet were randomly assigned into the model group (n=10) and the canagliflozin group (n=10). C57BL/6J mice fed on normal diet were chosen as the control group (n=10). Mice in the canagliflozin group were gavaged with canagliflozin for 14 weeks. The presence and severity of atherosclerosis were evaluated with HE and oil red O stainings in aortic root section slices. PCR assay was performed to determine the mRNA expression levels of nitric oxide synthase. Hepatic transcriptome analysis and hepatic amino acid detection were conducted using RNA-seq and targeted LC-MS, respectively. Results: HE staining and oil red O staining of the aortic root showed that AS models were successfully established in ApoE-/-mice fed on Western diet for 14 weeks. Canagliflozin alleviated the severity of atherosclerosis in pathology. Hepatic transcriptome analysis indicated that canagliflozin impacted on amino acid metabolism, especially arginine synthesis in ApoE-/-mice. Targeted metabolomics analysis of amino acids showed that canagliflozin reduced hepatic levels of L-serine, L-aspartic acid, tyrosine, L-hydroxyproline, and L-citrulline, but raised the hepatic level of L-arginine. Compared to the model group, the canagliflozin group exhibited higher serum arginine and nitric oxide levels as well as elevated nitric oxide mRNA expression in aortic tissues (P<0.05). Conclusion: Canagliflozin regulated the amino acid metabolism, reduced the levels of glucogenic amino acids,and promoted the synthesis of arginine in atherosclerotic mice.

[Cone-beam CT analysis of vertical control of mandible and changes of temporomandibular joint in adult patients with skeletal class â ¡ malocclusion with high angle].

Objective: To investigate the shape and position changes of temporomandibular joint (TMJ) in adult skeletal class Ⅱ malocclusion with high angle patients after vertical mandibular control, and the correlation between vertical mandibular changes and condylar position changes. Methods: Twenty adult skeletal class Ⅱ malocclusion with high angle patients [6 males and 14 females, aged (21.4±2.4) years] who underwent extraction treatment and active vertical control in the Department of Orthodontics, Lanzhou University Stomatological Hospital from October 2017 to November 2020 were selected. Cone-beam CT data of the patient before and after treatment were imported into Invivo Dental 5.0 software for three-dimensional reconstruction and correction, and the vertical index of mandible in reconstructed lateral cephalogram (mandibular plane angle, posterior anterior height ratio, mandibular true rotation angle) were measured. Incisal angle and variables of condyle shape, position and articular fossa shape were measured. Paired t test was performed on the results before and after treatment, and the correlation between mandibular vertical changes and condylar position changes was determined by Pearson correlation coefficient calculation. Results: After treatment, the overbite and overjet were within normal range, and the vertical height of the molars was controlled. Compared with the measurement before treatment, mandibular plane angle and mandibular true rotation angle were decreased by 2.05°±1.22° (t=7.60, P<0.001) and 1.42°±1.92° (t=3.54, P=0.002), respectively. The posterior anterior height ratio was increased by (1.89±3.32)% (t=2.56, P=0.019). After treatment, the mediolateral diameter of condyle, the anteroposterior diameter of condyle, the maximum cross-sectional area of condyle, the height of condyle head, the width of articular fossa, the depth of articular fossa and the articular nodular angle were increased by (0.55±0.76) mm (t=-2.73, P=0.015), (0.27±3.51) mm (t=-3.23, P=0.006), (6.01±7.36) mm2 (t=-2.80, P=0.013), (0.33±0.72) mm (t=-2.14, P=0.046), (0.56±0.93) mm (t=-2.37, P=0.032), 0.33 (0.14, 0.51) mm (Z=-2.76, P=0.006) and 1.50°±2.40° (t=-2.44, P=0.028), respectively. The internal condylar space and the external condylar space were decreased by (0.33±0.49) mm (t=2.31, P=0.035) and (0.20±0.23) mm (t=3.58, P=0.003), respectively. Before orthodontic treatment, 6 patients were with anterior displacement of the condyle, 7 patients with central position of the condyle, and 7 patients with posterior displacement of the condyle. After correction, patients who were with central position of the condyle have not changed much. The posterior displaced condyle in 2 patients and anterior displaced condyle in 3 patients became in central position after treatment. The joint space index was closer to the central position in 3 patients with anterior displacement and 3 patients with posterior displacement. The position of condyle in 1 patient with posterior displacement and 1 patient with anterior displacement remained basically unchanged. There was a significant negative correlation between the change of the posterior-anterior height ratio and the change of the internal condylar space in patients (r=-0.52, P=0.019), and a low correlation with the contral condylar space and the external condylar space(r=-0.48, P=0.031; r=-0.47, P=0.035). Conclusions: Skeletal class Ⅱ malocclusion with high angle adult patients achieved normal overbite and overjet and remodeling of condyle and articular fossa occurred after orthodontic treatmnet and vertical control. There was a certain negative correlation between the change of posterior-anterior height ratio and the change of condylar position.

The effect of dexmedetomidine on myocardial ischemia/reperfusion injury in patients undergoing cardiac surgery with cardiopulmonary bypass: a meta-analysis.

OBJECTIVE: The purpose of this study was to evaluate the effect of dexmedetomidine administration on myocardial ischemia/reperfusion (I/R) injury in patients undergoing cardiac surgery with cardiopulmonary bypass (CPB). MATERIALS AND METHODS: Online databases including PubMed, the Cochrane Library, Web of Science, Medline, and EMBASE were searched for clinical trials that investigated the application of dexmedetomidine in CPB patients prior to May 2021. A total of 17 studies involving 866 patients were included in this study. RESULTS: The result of the meta-analysis showed that there was a significant difference in serum creatinine-kinase-MB (CK-MB) between the dexmedetomidine group and the control group at the end of the operation and 24 h after the operation. Compared to the control group, cardiac troponin I (cTn-I) concentration in the dexmedetomidine group was significantly decreased at the end of the operation, 24 h after the operation, and 48 h after the operation. There was also a significant difference between the dexmedetomidine group and the control group in the length of a patient's ICU stay. CONCLUSIONS: Dexmedetomidine can reduce CK-MB and cTn-I concentrations and shorten the length of ICU stays for patients undergoing cardiac surgery with CPB. It can also provide myocardial protection from I/R injury.

[Current practice patterns of preoperative bowel preparation in elective colorectal surgery: a nation-wide survey of Chinese surgeons].

Objective: To understand the current practice of preoperative bowel preparation in elective colorectal surgery in China. Methods: A cross-sectional questionnaire survey was conducted through wechat. The content of the questionnaire survey included professional title of the participants, the hospital class, dietary preparation and protocol, oral laxatives and specific types, oral antibiotics, gastric intubation, and mechanical enema before elective colorectal surgery. A stratified analysis based on hospital class was conducted to understand their current practice of preoperative bowel preparation in elective colorectal surgery. Result: A total of 600 questionnaires were issued, and 516 (86.00%) questionnaires of participants from different hospitals, engaged in colorectal surgery or general surgeons were recovered, of which 366 were from tertiary hospitals (70.93%) and 150 from secondary hospitals (29.07%). For diet preparation, the proportions of right hemicolic, left hemicolic and rectal surgery were 81.59% (421/516), 84.88% (438/516) and 84.88% (438/516) respectively. The average time of preoperative dietary preparation was 2.03 days. The study showed that 85.85% (443/516) of surgeons chose oral laxatives for bowel preparation in all colorectal surgery, while only 4.26% (22/516) of surgeons did not choose oral laxatives. For mechanical enema, the proportions of right hemicolic, left hemicolic and rectal surgery were 19.19% (99/516), 30.04% (155/516) and 32.75% (169/516) respectively. Preoperative oral antibiotics was used by 34.69% (179/516) of the respondents. 94.38% (487/516) of participants were satisfied with bowel preparation, and 55.43% (286/516) of participants believed that preoperative bowel preparation was well tolerated. In terms of preoperative oral laxatives, there was no statistically significant difference between different levels of hospitals [secondary hospitals vs. tertiary hospitals: 90.00% (135/150) vs. 84.15% (308/366), χ(2)=2.995, P=0.084]. Compared with the tertiary hospitals, the surgeons in the secondary hospitals accounted for higher proportions in diet preparation [87.33% (131/150) vs. 76.78% (281/366), χ(2)=7.369, P=0.007], gastric intubation [54.00% (81/150) vs. 36.33% (133/366), χ(2)=13.672, P<0.001], preoperative oral antibiotics [58.67% (88/150) vs. 24.86% (91/366), χ(2)=12.259, P<0.001] and enema [28.67% (43/150) vs. 15.30% (56/366), χ(2)=53.661, P<0.001]. Conclusion: Although the preoperative bowel preparation practice in elective colorectal surgery for most of surgeons in China is basically the same as the current international protocol, the proportions of mechanical enema and gastric intubation before surgery are still relatively high.

[Establishment and evaluation of an animal model of female pelvic nerve injury].

Objective: To build a stable animal model simulating pelvic nerve injury in female pelvic floor dysfunction. Methods: A total of 55 10-week-old female SD rats weighing (220±15) g were randomly divided into 3 groups: 5 for normal group, 25 for sham operation group (SO), 25 for bilateral pudendal nerve block group (BPNB). Samples of rat anterior vaginal wall were obtained in 3 days, 1 week, 1 month and 3 months after the operation. The number of nerve fibers was counted per high power field under microscope, with UCHL immunohistochemical staining of nerve fibers. RNA was extracted and the expression of RNA related to nerve tissue was tested. Results: The numbers of nerve fibers had no significant difference between the normal group and the sham operation group. The numbers of nerve fibers in anterior vaginal wall of rats in BPNB group, was obviously decreased in 3 days after the operation, reached a minimum value at 1 weeks, and lasting till 3 months. QRT-PCR indicated that the expression of UCHL mRNA in the BPNB group was significantly decreased after 1 week, 1 month and 3 months, while the expression of Nestin was significantly decreased 1 month and 3 months after the operation. Conclusions: Bilateral pudendal nerve block could be used to make rat models of anterior vaginal nerve injury for further exploratory research on pelvic nerve injury theory of pelvic floor dysfunction.

Neutrophil elastase inhibitor suppresses oxidative stress in obese asthmatic rats by activating Keap1/Nrf2 signaling pathway.

OBJECTIVE: The aim of this study was to detect the oxidative stress response in the rat model of obesity, asthma and obese asthma. Meanwhile, we aimed to investigate the inhibitory effect of neutrophil elastase inhibitor (NEI) on cellular oxidative stress in the body and whether it exerted an effect on the oxidative stress response in obese asthma through the Kelch-like ECH-associated protein 1/nuclear factor E2-related factor 2 (Keap1/Nrf2) pathway. MATERIALS AND METHODS: The obesity and asthma models were established using a total of 70 Sprague-Dawley (SD) rats. All rats were randomly divided into 7 groups. The rats with normal weight were divided into the control (CTR) group (n=10), asthma (ATM) group (n=10) and ATM+NEI group (n=10). Meanwhile, the obese rats were divided into the obesity (OBS) group (n=10), the OBS+NEI group (n=10), the OBS+ATM group (n=10) and the OBS+ATM+NEI group (n=10). After modeling, rats in NEI intervention groups were injected with Sivelestat (5 mg/kg) via the caudal vein twice a day for 1 week. The tests of cough sensitivity to capsaicin and bronchial responsiveness were performed 24 h after the last administration. Lung tissues of rats were collected for hematoxylin-eosin (HE) staining. Meanwhile, the levels of reactive oxygen species (ROS) in heart, lung and kidney tissues were detected via 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The activities of reduced glutathione (GSH), glutathione peroxidase (GSH-Px), H2O2 and total superoxide dismutase (T-SOD) in the heart, lung and kidney tissues were detected using the colorimetric method. The mRNA and protein expressions of Keap1 and Nrf2 messenger ribonucleic acid expressions in the heart, lung and kidney tissues were measured via Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Western blotting, respectively. RESULTS: NEI significantly improved the symptoms and lung pathology in rats with asthma. The level of ROS in the heart, lung and kidney tissues of the OBS group, ATM group and OBS+ATM group was significantly increased. However, NEI markedly inhibited the level of ROS in rats with asthma. The activities of antioxidant stress-related enzymes (reduced GSH, GSH-Px, H2O2 and SOD) in the heart, lung and kidney tissues of the OBS group, ATM group and OBS+ATM group were significantly decreased. However, NEI markedly promoted the activities of the related antioxidant enzymes in oxidative stress response in asthma rats. Besides, the Keap1/Nrf2 signaling pathway in the heart, lung and kidney tissues of the OBS group, ATM group and OBS+ATM group was significantly inhibited, while NEI activated the Keap1/Nrf2 signaling pathway in rats with asthma. CONCLUSIONS: NEI promotes the release of a variety of antioxidant factors, enhances the activity of antioxidant enzymes and improves the symptoms of rats with obese asthma. The possible underlying mechanism may be the activation of the Keap1/Nrf2 signaling pathway.

Molecular cloning and expression analysis of tyr and tyrp1 genes in normal and albino yellow catfish Tachysurus fulvidraco.

The full-length complementary DNA of two genes related to vertebrate albinism, the tyrosinase gene tyr and tyrosinase-related protein 1 gene tyrp1, were cloned and analysed from normal and albino yellow catfish Tachysurus fulvidraco. The open reading frames (ORF) of tyr and tyrp1 encode putative peptides of 533 and 526 amino acids (amino-acid), both of which possess two conserved copper binding sites. The homologous identities of deduced amino-acid sequences showed that both Tyr and Tyrp1 of T. fulvidraco share considerable similarity with that of channel catfish Ictalurus punctatus. Both tyr and tyrp1 were expressed in a wide range of adult tissues. Tyr gene had the highest expression level in the brain of both normal and albino T. fulvidraco. Tyrp1 had the highest expression level in the skin of normal groups, and the fin of albino groups. The messenger (m)RNA expressions of tyr and tyrp1 were detectable at different early developmental stages and varied with embryonic and larval growth. Tyr and tyrp1 mRNA have obvious tissue specificity both in normal and albino T. fulvidraco and higher expression levels were detected in the normal group revealing that tyr and tyrp1 may have an important role in pigmentation. These results will provide useful data for understanding the molecular mechanism of melanin formation and the occurrence of albinism in T. fulvidraco.

Snail-induced epithelial-mesenchymal transition in gastric carcinoma cells and generation of cancer stem cell characteristics.

Biological changes in Snail-overexpressed SGC7901 cells were studied by establishing a pEGFP-C1-Snail carrier. The significance of Snail in epithelial-mesenchymal transition (EMT) as well as the invasion and metastatic capacity of gastric cancer cells was also discussed; moreover, we attempted to verify the probable cancer stem cell characteristics of Snail-overexpressed cells. A pEGFP-C1-Snail eukaryotic expression plasmid was constructed and pEGFP-C1(-) and pEGFP-C1-Snail plasmids were extracted and transfected into SGC7901 cells using Lipofectamine 2000. Stably expressed SGC7901-N [control group containing pEGFP-C1(-)] and SGC7901-S (test group containing pEGFP-C1-Snail) cells were screened using a G418 resistance medium. Snail, E-cadherin, b-catenin, vimentin, and fibronectin gene and protein expressions were detected by real-time quantitative PCR, western blot, and immunofluorescence. Cell invasion and metastasis were tested by scratch test, invasion assay, and an adhesion experiment. The positive rate of aldehyde dehydrogenase-1 (ALDH-1) expression was analyzed by flow cytometry. The results indicated the occurrence of EMT, accompanied by morphological changes in the cells and a weakening of the cell adhesion capacity. We also observed a decrease in the expression of epithelial markers E-cadherin and b-catenin and an increase in mesenchymal (Snail and vimentin) marker expression. Moreover, the cells showed increased invasiveness and metastatic capacity, and decreased proliferative ability. Moreover, the Snail-treated SGC7901 cells moved towards the scratch and produced fewer clones compared to the control cells. Owing to its capacity for self-renewal, SGC7901-S cells produced new clones and expressed ALDH-1. Therefore, we concluded that Snail overexpression induced EMT and endowed cells with tumor stem cell characteristics.

Isolation and characterization of eighteen polymorphic microsatellite loci in Schizopygopsis younghusbandi Regan and cross-amplification in three other Schizothoracinae species.

Eighteen polymorphic microsatellite loci were isolated from Schizopygopsis younghusbandi Regan and the characterization of these loci was assessed in 46 individuals collected from the Yarlung Tsangpo River in Tibet, China. The numberof alleles per locus ranged from 2 to 14. The expected heterozygosity and Shannon-Wiener diversity index ranged from 0.022 to 0.879 and from 0.059 to 2.313, respectively. The cross-species amplification and applicability of these loci were tested in three other Schizothoracinae species belonging to Schizothorax and Oxygymnocypris. These loci will be useful for the evaluation of genetic diversity and population genetic structure in S. younghusbandi and other related species.

Ontogeny expression of ghrelin, neuropeptide Y and cholecystokinin in blunt snout bream, Megalobrama amblycephala.

Ghrelin, neuropeptide Y (NPY) and cholecystokinin (CCK) all have important roles in the regulation of feeding in fish and mammals. To better understand the role of the three peptides in appetite regulation in the early developmental stages of blunt snout bream (Megalobrama amblycephala), partial cDNA sequences of ghrelin, NPY and CCK genes were cloned. And then, real-time quantitative PCR and RT-PCR were used to detect and quantify the mRNA expressions of these genes from zygotes to larvae of 50 days after hatching (DAH). Ghrelin, NPY and CCK were all expressed throughout the embryonic and larval development stages, and the expression levels were higher in larval stages than in embryonic stages. Ghrelin and NPY mRNA expressions were upregulated at 1, 3, 5 DAH, while CCK mRNA expression was reduced significantly at 3 DAH. The mRNA expression levels of three genes in larvae varied significantly until 30 DAH. In adult fish, all three peptides were detected to be expressed in brain and several peripheral tissues. Ghrelin mRNA was mainly expressed in the intestine, whereas NPY and CCK mRNAs were mainly expressed in the brain. Taken together, these results indicate that ghrelin, NPY and CCK may have roles in early development and participate in the regulation of feeding of larvae in blunt snout bream and will be helpful for further investigation into feed intake regulation in adults of this species.

First Report of the Soybean Frogeye Leaf Spot Fungus (Cercospora sojina) Resistant to Quinone Outside Inhibitor Fungicides in North America.

Quinone outside inhibitor (QoI; also known as strobilurin) fungicides sometimes are applied to soybean (Glycine max) fields to help manage frogeye leaf spot of soybean (caused by Cercospora sojina) in the United States. In August 2010, soybean leaflets exhibiting severe frogeye leaf spot symptoms were collected from a field in Lauderdale County, TN that had been treated twice with pyraclostrobin during that growing season. The field had been planted into soybean annually since at least 2008, and a QoI fungicide had been applied to the field in each of those years. Fifteen single-spore isolates of C. sojina were recovered from the affected soybean leaflets. These isolates were identified as C. sojina based on the observed symptoms on the soybean leaflets and the morphology and size of conidiophores and conidia (3). In addition, DNA was extracted from the cultures, PCR amplification of the small subunit rDNA and internal transcribed spacer (ITS) region was conducted (2), and the resulting PCR product was sequenced at the Keck Biotechnology Center at the University of Illinois, Urbana. The resulting nucleotide sequences were compared with sequences deposited in the nucleotide database ( http://www.ncbi.nlm.nih.gov ) and showed highest homology to sequences of C. sojina. The isolates were tested for their sensitivity to technical-grade formulations of the QoI fungicides azoxystrobin, pyraclostrobin, and trifloxystrobin with an in vitro conidial germination assay with fungicide + salicylhydroxamic acid (SHAM)-amended potato dextrose agar as described by Bradley and Pedersen (1). The effective concentration at which 50% conidial germination was inhibited (EC50) was determined for all 15 C. sojina isolates, with mean values of 3.1644 (2.7826 to 4.5409), 0.3297 (0.2818 to 0.6404), and 0.8573 (0.3665 to 2.5119) µg/ml for azoxystrobin, pyraclostrobin, and trifloxystrobin, respectively. When compared with previously established mean EC50 values of C. sojina baseline isolates (4), EC50 values of the C. sojina isolates collected from the Lauderdale County, TN soybean field were approximately 249- to 7,144-fold greater than the EC50 values of the baseline isolates. These results indicate that all isolates recovered from the Lauderdale County, TN soybean field were highly resistant to QoI fungicides. To our knowledge, this is the first report of QoI fungicide resistance occurring in C. sojina, and surveys for additional QoI fungicide-resistant C. sojina isolates are needed to determine their prevalence and geographic distribution. In light of these findings, soybean growers in Tennessee and adjacent states should consider utilizing alternative frogeye leaf spot management practices such as planting resistant cultivars, rotating to nonhost crops, and tilling affected soybean residue (3). References: (1) C. A. Bradley and D. K. Pedersen. Plant Dis. 95:189, 2011. (2) N. S. Lord et al. FEMS Microbiol. Ecol. 42:327, 2002. (3) D. V. Phillips. Page 20 in: Compendium of Soybean Diseases. 4th ed. G. L. Hartman et al., eds. The American Phytopathological Society, St. Paul, MN, 1999. (4) G. Zhang et al. Phytopathology (Abstr.) 100(suppl.):S145, 2010.

The first vitellogenin receptor from a Lepidopteran insect: molecular characterization, expression patterns and RNA interference analysis.

The vitellogenin receptor (VgR) belongs to the low-density lipoprotein receptor (LDLR) superfamily, and is an important carrier for the uptake of vitellogenin (Vg) into developing oocytes of all oviparous species. The first full-length message for a VgR from a Lepidopteran insect was cloned and sequenced from the ovary of Spodoptera litura Fabricius (GenBank accession no. GU983858). The coding region consisted of 5370 bp flanked by a 49 bp 5'-untranslated region (UTR) and a 177 bp 3'-UTR, which encoded a 1798-residue protein with a predicted molecular weight (MW) of 201.69 kDa. S. litura VgR (SlVgR)comprised two ligand binding sites with four LDLR class A repeats in the first domain and seven in the second domain, an epidermal growth factor-like domain containing an LDLR class B repeat and a YWXD motif, a transmembrane domain and a cytoplasmic domain. A phylogenetic relationship placed SlVgR as a separate group from the other insects. SlVgR messenger RNA (mRNA) was specifically expressed in the ovarian tissues. The developmental expression patterns showed that VgR mRNA was first transcribed in 6(th) day female pupae and the maximum level of VgR mRNA appeared in 36-h-old adults. Immunoblot analysis detected an ovary-specific VgR protein with a MW of ∼200 kDa, whose development profiles were consistent with VgR mRNA expression patterns. RNA inteference (RNAi) specifically disrupted the VgR gene by injection of 3 or 5 µg VgR double-stranded RNA per insect in 4(th) or 6(th) day pupae. RNAi of SlVgR led to a phenotype characterized by high Vg accumulation in the haemolymph, low Vg deposition in the ovary and the failure of insect spawning. These results mean that VgR is critical for binding Vg and transporting it into the oocytes of the insect ovary, thus playing an important role in insect reproduction.

First Report of Charcoal Rot Caused by Macrophomina phaseolina on Sunflower in Illinois.

In September 2009, sunflower (Helianthus annuus L.) plants (cv. Mycogen 8C451) from a University of Illinois field research trial in Fayette County, Illinois exhibited silvery gray girdling lesions on the lower stems and premature death. When lower stems and roots were split open, the pith tissue was compressed into layers. Black microsclerotia (90 to 180 µm) were present on the outside of the lower stem tissue and in the stem vascular tissue. Five pieces (approximately 1 cm long) of symptomatic stem tissue from five different affected plants (25 pieces total) were soaked in a 0.5% solution of NaOCl for 30 s, rinsed with sterile distilled water, and placed on potato dextrose agar (PDA; Becton, Dickinson, and Company, Franklin Lakes, NJ). Gray hyphae grew from all of the stem pieces, which subsequently turned black and formed black microsclerotia (75 to 175 µm). On the basis of plant symptoms and size and color of the microsclerotia, the disease was diagnosed as charcoal rot caused by Macrophomina phaseolina (Tassi) Goid (2). To confirm that the isolated fungus was M. phaseolina, DNA was extracted from the pure culture, and PCR amplification of a subunit rDNA and internal transcribed spacer (ITS) region with primers EF3RCNL and ITS4 was performed (3). The Keck Biotechnology Center at the University of Illinois, Urbana sequenced the PCR product. The resulting nucleotide sequence shared the highest homology (99%) with sequences of M. phaseolina when compared with the subunit rDNA and ITS sequences in the nucleotide database ( http://www.ncbi.nlm.nih.gov ). A greenhouse experiment was conducted to confirm pathogenicity; the greenhouse temperature was approximately 27°C and sunflower plants (cv. Cargill 270) were grown in pots and watered daily to maintain adequate soil moisture for growth. Sterile toothpicks were infested with M. phaseolina and placed through the stems (10 cm above the soil surface) of five 40-day-old sunflower plants that were approximately at growth stage R4 (1,4). Five sterile, noninfested toothpicks were similarly placed through sunflower plants to act as controls. Parafilm was used to hold the toothpick in the stem and seal the stem injury. Thirty-five days after inoculation, the mean lesion length on stems inoculated with M. phaseolina was 595 mm and no lesions developed on the control plants. M. phaseolina-inoculated plants also began to wilt and die. Cultures identical to the original M. phaseolina isolate were reisolated from stem lesions of the M. phaseolina-inoculated plants. This is the first report of charcoal rot on sunflower in Illinois to our knowledge. Sunflower is currently not a major crop grown in Illinois, but on-going research is focused on evaluating sunflower as a potential late-planted crop to follow winter wheat. If sunflower production increases in Illinois, growers may need to take precautions to manage charcoal rot. References: (1) L. K. Edmunds. Phytopathology 54:514, 1964. (2) T. Gulya et al. Page 263 in: Sunflower Technology and Production. American Society of Agronomy, Madison, WI, 1997. (3) N. S. Lord et al. FEMS Microbiol. Ecol. 42:327, 2002. (4) A. A. Schneiter and J. F. Miller. Crop Sci. 21:901, 1981.

Occurrences of Diplodia Leaf Streak Caused by Stenocarpella macrospora on Corn (Zea mays) in Illinois.

In August 2008, long and narrow lesions were observed on leaves of corn (Zea mays L.) growing in a field in Pope County, Illinois. Lesions were 10 to 35 × 50 to 250 mm and were cream to tan. Dark pycnidia inside the lesions were immersed and approximately 350 µm in diameter. Affected leaves were collected and placed into a moist chamber to encourage the development of conidia. Conidia developed in cirri and were dark, one septate, and 7 to 11 × 59 to 87 µm. Cirri were streaked onto potato dextrose agar (PDA; Becton, Dickinson, and Company, Franklin Lakes, NJ) and cultures arising from single conidia were transferred and maintained. On the basis of the corn leaf symptoms and the morphological characteristics of the pycnidia and conidia, the fungus was tentatively identified as Stenocarpella macrospora (Earle) Sutton (1). To complete Koch's postulates, 'Garst 84H80-3000GT' corn was inoculated in the greenhouse. Conidia were produced by placing a S. macrospora isolate from Pope County, IL onto water agar containing autoclaved corn leaves and incubating at room temperature until pycnidia and conidia were produced (approximately 3 weeks). A conidial suspension was used to inoculate the leaf whorls of corn plants (approximately at the V4 growth stage). Control plants were mock inoculated with sterile water. The experiment was repeated once over time. Twenty days after inoculation, all plants inoculated with S. macrospora conidia developed lesions similar to those observed in the field, and mock-inoculated plants remained symptomless. The fungus was reisolated on PDA from the symptomatic leaves. In August 2009, symptomatic leaves similar to those observed in Pope County, IL in 2008 were observed and collected from corn fields in Gallatin and Vermillion counties. Pycnidia and conidia from these lesions were similar to those described above, and isolates from single conidia were obtained from these samples. To confirm the identity of all isolates collected, PCR amplification of the small subunit rDNA and internal transcribed spacer (ITS) region with primers EF3RCNL and ITS4 was conducted (3). The PCR product was sequenced with these primers at the Keck Biotechnology Center at the University of Illinois, Urbana. The resulting nucleotide sequence was compared with small subunit rDNA and ITS sequences deposited in the GenBank nucleotide database, which revealed 99% homology to sequences of S. macrospora. In total, six of our S. macrospora isolates from Gallatin, Pope, and Vermillion counties were submitted to the United States Department of Agriculture-Agriculture Research Service Culture Collection in Peoria, IL, where they have received NRRL Accession Nos. 54190-54195. To our knowledge, this is the first report of S. macrospora affecting corn in Illinois. Although not observed in the Illinois corn fields described above, S. macrospora has been reported to infect stalks and ears (2). Because of the large leaf lesions caused by S. macrospora and its reported aggressiveness in causing disease on leaves, ears, and stalks, this pathogen has the potential to cause severe yield and quality losses to corn in the United States (2). References: (1) M. L. Carson. Diseases of minor importance or limited occurrence. Page 23 in: Compendium of Corn Diseases. 3rd ed. The American Phytopathological Society, St. Paul, MN, 1999. (2) F. M. Latterell and A. E. Rossi. Plant Dis. 67:725, 1983. (3) N. S. Lord et al. FEMS Microbiol. Ecol. 42:327, 2002.

Age-related expression of calcium/calmodulin-dependent protein kinase II A in the hippocampus and cerebral cortex of senescence accelerated mouse prone/8 mice is modulated by anti-Alzheimer's disease drugs.

Senescence-accelerated mouse (SAM) prone/8 (SAMP8) is a good animal model to investigate the fundamental mechanisms of age-related learning and memory deficits such as Alzheimer's disease (AD) at the gene and protein levels, and SAM resistant/1 (SAMR1) is its normal control. Calcium/calmodulin-dependent protein kinase II-alpha (CaMKIIalpha) is one of the most abundant subunits of calcium/calmodulin-dependent protein kinase II in cerebral cortex and hippocampus, and is closely linked to AD. In this study, we used real time fluorescence quantitative PCR (RT-PCR) and Western blot techniques to examine the expression of CaMKIIalpha mRNA and protein in the cerebral cortex and hippocampus of SAMP8 both with aging and following treatment with anti-AD drugs (for example, natural product huperzine A (HupA) and traditional Chinese medicinal prescription Liu-Wei-Di-Huang decoction (LW), Ba-Wei-Di-Huang decoction (BW), Huang-Lian-Jie-Du decoction (HL), Dang-Gui-Shao-Yao-San (DSS) and Tiao-Xin-Fang decoction (TXF)). The results showed that the levels of both CaMKIIalpha mRNA and protein decreased significantly in the cerebral cortex of SAMR1 with aging, but increased significantly in the cerebral cortex of SAMP8. Compared with age-matched SAMR1, the expression of mRNA and protein of CaMKIIalpha significantly increased in the cerebral cortex and hippocampus of SAMP8 after 10 months of age. After SAMP8 was treated with the previously mentioned drugs, the abnormally high expression of CaMKIIalpha was relatively down-regulated. These results indicated that the expression of CaMKIIalpha in the brain of SAMP8 was abnormal and that this abnormality could be reversed with anti-AD drugs. These data suggest that CaMKIIalpha may play an important role in the age-related cognitive deterioration in AD, and may be a potential targets for anti-AD drugs.

First Report of Phomopsis Stem Canker of Sunflower in Illinois Caused by Phomopsis helianthi.

Stem cankers were observed on confection sunflower (Helianthus annuus) plants growing in a field in Champaign County, Illinois in August 2008. Lesions were brown to reddish brown, elongated (approximately 10 to 15 cm long), and centered over the area where leaf petioles connected to the stems. Stem tissues underneath the lesions were degraded. Lesions from diseased stems were cut into 5- to 7-mm pieces and immersed in a 0.5% NaOCl solution for 1 min, rinsed with sterilized distilled water, and placed into petri dishes containing acidified potato dextrose agar (APDA; 4 ml of 25% lactic acid per liter). Fungal colonies that grew from the stem lesion pieces on APDA were white, floccose, and dense with dark colored substrate mycelia. On the basis of the symptoms on sunflower plants and the growth characteristics on APDA, the fungus was tentatively identified as Phomopsis helianthi (1). To confirm the identity of the fungus, PCR amplification of the small subunit rDNA and internal transcribed spacer (ITS) region with primers EF3RCNL and ITS4 was done (2). The PCR product was sequenced with these primers at the Keck Biotechnology Center at the University of Illinois, Urbana. The resulting nucleotide sequence was compared with small subunit rDNA and ITS sequences deposited in the nucleotide database ( http://www.ncbi.nlm.nih.gov ) and showed highest homology to sequences of Diaporthe helianthi, teleomorph of P. helianthi. To confirm pathogenicity of the fungus, sunflower plants (cv. Cargill 270) were grown in the greenhouse and inoculated with the isolated fungus. The stems of sunflower plants between the V2 and V4 growth stages (3) were excised just below the uppermost node. Mycelia plugs of the fungus were placed into the large end of disposable micropipette tips (200 µl). The micropipette tip containing the fungus was subsequently placed over a cut sunflower stem. The fungal isolate was used to inoculate five stems. To serve as controls, five cut sunflower stems were inoculated with micropipette tips containing plugs of noninfested PDA and five cut stems were not inoculated. Mean lesion length on the stem was measured from the inoculated tip toward the soil line 7 days after inoculation. The experiment was replicated over time. Mean lesion length over both replications averaged 24 mm on the fungus-inoculated plants, 2 mm on the noninfested PDA-inoculated control plants, and no lesions were present on the noninoculated control plants. The fungus was reisolated on PDA from the inoculated plants in the greenhouse. To our knowledge, this is the first report of P. helianthi causing a stem canker of sunflower in Illinois. Although commercial sunflower production in Illinois is currently limited, it is being evaluated as a potential crop to follow winter wheat in portions of the state. If sunflower production were to increase in the state, growers may have to monitor for and manage Phomopsis stem canker. References: (1) T. Gulya et al. Sunflower diseases. Page 263 in: Sunflower Technology and Production. American Society of Agronomy, Madison, WI, 1997. (2) N. S. Lord et al. FEMS Microbiol. Ecol. 42:327, 2002. (3) A. A. Schneiter and J. F. Miller, Crop Sci. 21:901, 1981.

Fetal liver-conditioned medium induces hepatic specification from mouse bone marrow mesenchymal stromal cells: a novel strategy for hepatic transdifferentiation.

BACKGROUND: Although different strategies have been established for hepatic differentiation of mesenchymal stromal cells (MSC), further studies are required to define an efficient strategy to produce hepatocytes from stem cells and uncover the mechanisms of hepatic differentiation. METHODS: Bone marrow mesenchymal stromal cells (BMMSC), isolated from ICR mice, were induced by fetal liver-conditioned medium from different developmental stages, embryonic days (ED) 9.5, 11.5 and 13.5 and newborn (1 day). Differentiated cells were characterized by morphologic changes, liver-specific gene expression at mRNA and/or protein levels and in vitro functional features. RESULTS: BMMSC morphologically became epithelioid and binucleated after 7 days' exposure to fetal liver-conditioned medium from ED13.5, expressed liver-specific genes (AFP, HNF-3beta, TTR, CK18, ALB and CK19) at mRNA and/or protein levels and acquired in vitro functions characteristic of liver cells, including glycogen storage, urea production and albumin secretion. Conditioned medium derived from fetal liver at ED13.5 was most efficient on hepatic differentiation of BMMSC compared from the other three developmental stages. DISCUSSION: The present study not only provides a high-performance strategy for hepatic differentiation from BMMSC, but also implies liver at different developmental stages might secrete different types of cytokines that have diverse effects on hepatic differentiation, which could support further investigation to provide insight into fundamental processes that govern development and regeneration of the liver.

Cartilage polysaccharide induces apoptosis in human leukemia K562 cells.

In this study, we extracted a polysaccharide (short-chain polysaccharide [PS]) from porcine cartilage and examined its function in chronic myeloid leukaemia by using human K562 cells and mouse L1210 cells. Results of cell proliferation assay indicated that PS inhibited cancer cell growth at different concentrations, while it had little effect on normal cells. The presence of morphological aspects of apoptosis, such as nuclear shrinkage, was shown in H&E stained sections. The occurrence of PS-induced apoptosis was confirmed by TUNEL assay and cell cycle analysis. The results of immunofluorescent staining indicated the molecular mechanism underlying. Through interfering with the cell cycle of tumor cells, PS may induce apoptosis by downregulating the expression level of cyclin D1 and upregulating the level of p21 protein. Correlation analysis of apoptosis and MAPK suggested that inactivation of ERK was crucial for PS induced apoptosis, while JNK phosphorylation had a small effect and p38 was not involved. In vivo assay showed that PS inhibited L1210 cell growth in vivo and prolonged the life span of L1210-bearing mice. We conclude that PS is a polysaccharide with anticancer effects and induced apoptosis in human K562 cells.