Acacia honey on sodium arsenite-induced oxidative stress in the heart, lung and kidney tissues of male Wistar rats. Male Wistar albino rats divided into four groups of five rats each were administered distilled water, Acacia honey (20%), sodium arsenite (5 mg/kg body weight), Acacia honey, and sodium arsenite daily for one week. They were sacrificed anesthetically using 60 mg/kg sodium pentothal. The tissues were used for the assessment of glutathione peroxidase, catalase, and superoxide dismutase activities, protein content and lipid peroxidation. Sodium arsenite significantly (P < 0.05) suppressed the glutathione peroxidase, catalase, superoxide dismutase activities with simultaneous induction of lipid peroxidation. Administration of Acacia honey significantly increased (P < 0.05) glutathione peroxidase, catalase, and superoxide dismutase activities with concomitant suppression of lipid peroxidation as evident by the decrease in malondialdehyde level. From the results obtained, Acacia honey mitigates sodium arsenite induced-oxidative stress in male Wistar albino rats, which suggest that it may attenuate oxidative stress implicated in chemical carcinogenesis.Cancer is a leading cause of death worldwide and its development is frequently associated with oxidative stress-induced by carcinogens such as arsenicals. Most foods are basically health-promoting or disease-preventing and a typical example of such type is honey. This study was undertaken to investigate the ameliorative effects of  Apis mellifera , five (5) free flavonoids  and abscisic acid [Honey is a viscous, supersaturated sugar solution derived from nectar gathered and modified by the honeybee, ellifera . It is cellifera –4. Acaci effects . We have effects  and lungin vitro . Contrarioxidant . Charactsic acid .Acacia honey in relation to oxidative stress in brain, liver and plasma, to the best of our knowledge no such work has been done on heart, lung and kidney tissues with sodium arsenite in the presence of Acacia honey. Therefore, this study was undertaken to investigate the effects of Acacia honey on sodium arsenite-induced oxidative stress in the heart, lung, and kidney tissues of male Wistar rats. This work will further contribute to the existing knowledge in the area of functional foods-based research.Free radicals cause oxidative damage to lipids, proteins and nucleic acids leading to many biological complications including carcinogenesis, mutagenesis, aging, and atherosclerosis. Arsenic50 was used [Acacia honey was dissolved in distilled water to prepare a 20% (v/v) honey solution and 5 mL/kg body weight was used [Sodium arsenite (5 mg/kg body weight) equivalent to two-tenth of the oral LDwas used  in all eAcacia honey was collected from Achida town, Wurno Local Government Area, Sokoto State, Nigeria, in December 2012, from Acacia flower. It was identified by pollen grain analysis and maintained at the Department of Biochemistry, Ahmadu Bello University, Zaria, Nigeria. It was kept at 4°C until analysis.The  Group 1: distilled water . Acacia honey at 5 mL/kg body weight.Group 2: 20% v/v  Group 3: 5 mg/kg body weight sodium arsenite (positive control). Acacia honey + 5 mg/kg body weight sodium arsenite.Group 4: 20% v/v Twenty (20) male albino rats of Wistar (150–195 g) were used in the present study. They were allowed to adapt for one week under standard laboratory conditions of 12-hour light-dark cycle before commencement of all experiments. The rats were maintained in the animal house of Department of Biochemistry, Ahmadu Bello University, Zaria, Nigeria with protocol for the study approved by the Institutional Animal right review committee. During acclimatization, the rats were allowed free access to NIH-07 pelletized diet and water. In addition, all rats were humanely cared for in accordance with the National Institute of Health (NIH) Guide for the care and use of Laboratory Animals. They were randomly divided into four (4) groups of five (5) rats each. The animals were treated daily for one week as shown in the experimental design as follows.Twenty four (24) hours after the administration of the last treatment, the rats were kept without food overnight and were humanely sacrificed with sodium pentothal (60 mg/kg body weight). Heart, lung, and kidney tissues were collected. The tissues were homogenized in 1 : 5 of 0.9% sodium chloride (ice-cold). The supernatant was collected after centrifugation at 3500 ×g at 4°C for 10 minutes and kept at −80°C until further analysis. Each time the supernatant was outside the freezer, it was kept in ice bags. By using Auto Analyzer Hitachi Roche 7020 , the total protein contents of the tissues were determined based on the standard manufacturer's protocol.2O2 [Lipid peroxidation was determined by measuring malondialdehyde (MDA) formed by thiobarbituric acid reaction . Catalas2O2 . The lev2O2 , while t2O2  was adopP values <0.05 were considered statistically significant for differences in mean using the least significant difference (LSD).To address the biological variability and stability of the samples, each and every experiment was repeated at least three times and the results were expressed as mean ± standard deviation. Differences between the groups were analyzed by one-way analysis of variance (ANOVA) with the aid of Statistical Package for Social Sciences (SPSS) software, SPSS Inc., Chicago, IL, USA, Standard version 19. P < 0.05) reduced level of oxidative stress as evident by the lower level of MDA compared to the group induced with sodium arsenite without treatment. On the other hand, administration of 20% (v/v) honey significantly (P < 0.05) increased SOD, catalase, and glutathione peroxidase levels when compared to the sodium arsenite induced group without treatment. There is, however, no significant (P > 0.05) difference in the total protein in all the groups.Assessment of the oxidative stress biomarkers in the heart of the animals is presented in P < 0.05) reduced oxidative stress as evident by the lower level of MDA compared to the group induced with sodium arsenite without treatment. Contrariwise, administration of honey  significantly (P < 0.05) increased SOD, catalase, and glutathione peroxidase levels when compared to the sodium arsenite induced group without treatment. Again, there is no significant (P < 0.05) difference in the total protein in all the groups.P < 0.05) showed reduced oxidative stress as evident by the lower level of MDA compared to the group induced with sodium arsenite without treatment. On the other hand, 20% honey significantly (P < 0.05) increased SOD, catalase, and glutathione peroxidase levels when compared to the sodium arsenite induced group without treatment. However, the group administered with honey alone does not have significantly (P > 0.05) higher SOD compared to sodium arsenite induced group without treatment. There is still no significant (P > 0.05) difference in the total protein in all the groups.Acacia honey and sodium arsenite administration in vivo.Antioxidants role in the maintenance of health and chemoprevention of disorders and diseases has received great attention . As a reAcacia honey. The antioxidative potentialities of honey have been reported [It has been established that free radicals cause oxidative damage to lipids, proteins, and nucleic acids leading to many biological complications including carcinogenesis, mutagenesis, aging, and atherosclerosis . Arsenitreported , 32 and reported –36. Basireported . It is ereported . This prreported –44 whichAcacia honey may ameliorate oxidative stress in the heart, lung, and kidney of Wistar rats. It also suggests that Acacia honey may mitigate the effect of arsenicals-induced oxidative stress implicated in chemical carcinogenesis.The findings presented in this research show that


SD rats received ascending aorta constriction (TAC) to establish a model of myocardial hypertrophy. The animals were treated orally for a period of six weeks. QL significantly inhibited cardiac hypertrophy due to ascending aortic constriction and improved hemodynamics. This effect was linked to the expression levels of the signaling factors in connection with upregulated energy and the regulation of glucose and lipid substrate metabolism and with a decrease in metabolic intermediate products and the protection of mitochondrial function. It is concluded that QL may regulate the glycolipid substrate metabolism by activating AMPK/PGC-1α axis and reduce the accumulation of free fatty acids and lactic acid, to protect cardiac myocytes and mitochondrial function.Qiliqiangxin capsule (QL) was developed under the guidance of TCM theory of collateral disease and had been shown to be effective and safe for the treatment of heart failure. The present study explored the role of and mechanism by which the herbal compounds QL act on energy metabolism,  Hypertension is one of the most common cardiovascular diseases and a major cause of heart failure. Increase of pressure load due to long-term hypertension will cause myocardial fibrosis and left ventricular compensatory hypertrophy. The accompaning neurohumoral factors, endocrine and metabolic abnormalities further promote ventricular remodeling; structural changes would result in the decline in coronary flow reserve, which eventually leads to heart diastolic and systolic dysfunction , 2. At pSalvia miltiorrhiza, Pepperweed Seed, Rhizoma Alismatis, Polygonatum odoratum, Ramulus Cinnamomi, Carthamus tinctorius, Cortex Periplocae, Tangerine Peel, and other herbs, see Supplementary Material available online at http://dx.doi.org/10.1155/2013/378298. The origin, harvest time, medicinal composites, and processing technology of the herbs were strictly normalized and standardized for different batches of QL capsules. The labeled compounds were verified and standardized according to the Chinese Pharmacopoeia (2005) to achieve quality control of the QL capsule. The aqueous extract of 10 different batches QL was randomly sampled and analysed for the chemical fingerprints by using ultrahigh performance liquid chromatography (UPLC). The similarity analysis of the fingerprints was performed; the similarities were found to be within the range of 0.978 to 1.000, showing that the overall quality of QL had good reproducibility  was manufactured by Changzhou Pharmaceutical Factory. Lactic acid detection kit ; free fatty acid detection kit ; adenosine triphosphate ATP (Lot no. 100111202), adenosine diphosphate ADP (Lot no. 10090203190), and rhodamine 123 (Rhodamine-123) were purchased from Sigma Chemical Co., adenosine monophosphate AMP , mitochondrial extraction kit . Trizol Reagent (Invitrogen); reverse transcriptase (M-MLV), ribonuclease inhibitor (RNasin), dNTP, Taq DNA polymerase, and random primers were purchased from Promega Corporation, USA; Hot Start Fluorescent PCR Core of Reagent Kits (SYBR Green I) was obtained from BBI.n = 15), middle-dose group , low-dose group , and the doses of administered QL powder were 1.0, 0.5, and 0.25 g/kg/d, respectively; the volume was 10 mL/kg/d; Model group ; Sham group ; Sham group and the test group were administered with an equal volume of 0.5% sodium carboxymethyl cellulose (CMC-Na), orally administered for 6 weeks.Male Sprague-Dawley rats (body weight 250–270 g), 150, were provided by the Beijing Vital River Laboratory Animal Technology Co., Ltd  2012-0001). The animals were housed at five/cage, fed with standard diet and water ad libitum, and were subject to a 12 h light and 12 h dark cycle. All animal experimental protocols were approved by Animal Care and Use Committee of Hebei Medical University and complied with laboratory animal management and use regulations. Chronic heart failure model was established by thoracic aortic coarctation (TAC) , includiAfter treatment for six weeks, the animals were anesthetized to perform the separation of the right carotid artery and cannulation. Left ventricular systolic pressure (LVSP) and left ventricular pressure (LVP) were measured by using BL-420E polygraph . After collecting blood from the abdominal aorta, the heart was rapidly removed, rinsed with cold physiological saline, and water adsorbed by filter paper. The excess tissues around the hearts were removed, and hearts were weighed on a balance and then immediately put into liquid nitrogen for storage. Heart weight index was calculated using the formula: index = heart weight (HW)/body weight (BW).Blood was stored at room temperature for 2 h and centrifuged for 10 min at 3500 rpm; the serum was then loaded into EP tube. The optical density value was measured by using the XD711 ELISA analyzer  according to the experimental method specified in the instruction of lactic acid detection kit; serum free fatty acids were measured by using Hitachi 7080 biochemical analyzer.5/mL in the culture medium. Rhodamine 123 was used as fluorescent probe . The suspension was incubated for 15 min in the dark place, and then the changes in the myocardium mitochondria MMP were measured using flow cytometer  with excitation wavelength and emission wavelength at 480 nm and 530 nm, respectively.About 0.3 g of fresh myocardial tissue was prepared and rinsed by physiological saline. Myocardial tissue was sheared and washed with PBS buffer. Cell mass was filtered with 300 mesh metal mesh and single cell suspension was collected. The supernatant was discarded after centrifugation . Single cell suspension was adjusted to 1 × 10Fresh myocardial tissue was collected at the end of the experiment and extracted with mitochondrial extraction kit to prepare mitochondrial suspension by differential centrifugation at low temperature. Protein content in myocardial mitochondrial suspension was determined by Coomassie brilliant blue. 2.5 mL of GENMED medium liquid was added into the reaction glass tank, mixed, and sealed, and the mitochondria, State IV substrate solution and State III substrate solution were added in proper sequence as instructed by the manufacturer. Mitochondrial State III and State IV respiration rate in the closed reaction system were determined using dissolved oxygen electrolytic analyzer , and mitochondrial respiratory control ratio (RCR) was calculated according to the following formula: RCR = State III respiration rate/State IV respiration rate.μL) was collected and added 25 μL 2 mol/L to adjust pH to 6.5, centrifuged again at 4000 r/min for 10 min at 4°C, and the supernatant was filtered with a 0.45 micron membrane. The supernatant preparation was loaded onto a 10 A high performance liquid chromatography detector , followed by separation and detection at wavelength of 254 nm. ATP, ADP, and AMP levels were calculated according to the elution peak area and standard concentrations. The results were expressed at μmol/g tissue. Total adenylate pool (ATP + ADP + AMP) and the energy charge [EC = (ATP + 0.5 × ADP)/(ATP + ADP + AMP)] were calculated.The cryopreserved myocardial tissues were weighed and added 0.4 mol/L precooling perchloric acid at 5 mL/g ratio, homogenized on the ice bath, and then centrifuged at 4000 r/min for 10 min at 4°C. Supernatant . PCR thermal cycling parameters were as follows: the denaturing step at 96°C for 4 min, followed by 40 cycles annealing step at 94°C 30 s, 58°C 30 s, and 72°C 30 s. Fluorescence signal was collected in each cycle of the third step 72°C 30 s. Using GAPDH gene as an internal reference and by comparing target gene expression and with Control group, the relative quantitative value  was calculated and used for statistical analysis. The primer sequences were shown in Myocardial tissue about 100 mg and 1ml precooled lysate were homogenised in ice bath. Supernatant was collected after centrifugation , and the protein concentration of supernatant, measured by Nanodrop 2000 spectrophotometer, was adjusted to the final concentration of 1%. The samples were mixed with equal volumes of 5 × SDS sample buffer, and then boiled for 5 min, centrifuged, and loaded onto 12% SDS-PAGE for electrophoresis. After being transferred onto a polyvinylidene difluoride membrane (PVDF), the membranes were blocked with 5% milk tris-buffered saline-tween 20 and probed with primary antibody (1 : 1000) and HRP conjugated secondary antibody (1 : 10000), separated by extensive washings. The membrane was treated with enhanced chemiluminescence substrate and the bands on the membrane were visualized and analyzed using UVP BioImaging System.P < 0.05.All data were presented as mean ± standard deviation, single factor analysis of variance (ANOVA) was performed with the statistical software SPSS 17.0, Dunnertt'sT3 was used for unequal variances, and the difference was statistically significant at P < 0.01). Compared with Model group, the intervention of six weeks' administration with QL reduced the LVP and LVSP levels to some extent. Model group increased significantly compared with Sham group. Each treatment group had significantly lower heart weight index (P < 0.05), especially in the QL-H and Captopril group in which the heart weight was significantly lower (P < 0.01)  .P < 0.01). Low dose and high dose groups of QL reduced serum lactate level in the pressure overload rat (P < 0.05); the high-dose group significantly lowered serum lactate level (P < 0.01). Although the positive group also reduced the lactic acid level, the difference was not significant. Compared with Model group, free fatty acid level was significantly lower in Captopril group and each dose group of QL (P < 0.05). The high dose group had a highly significant difference (P < 0.01).As shown in Figures P < 0.05) . The effects of Captopril group and QL-H group were highly significant (P < 0.01), as shown in Compared with Sham group, myocardial mitochondrial membrane potential was declined after model establishment. After drug intervention for six weeks, myocardial mitochondrial membrane potential was significantly elevated compared with Model group , showing a disturbance of myocardial energy supply occurred in postoperative rats; the ventricular reconstruction was accompanied by metabolic remodeling. Compared with Model group, the drug intervention group significantly increased the adenosine pool and energy charge value; total adenosine pool and energy charge value were higher in QL-L group than Model group . The increase of total adenylate and energy charge value in myocardial tissue was most notable in Captopril group and QL-H group ; the relative expression levels of each group indicator were increased to some extent after drug intervention. The expression levels of AMPK, PGC-1α, CPT-I, and GLUT4 mRNA were significantly different in QL-H group and Captopril group compared with the Model group   .α, CPT-I, and GLUT4 in Model group decreased significantly . Compared with Model group, the above protein expression in treatment group increased at varying degrees . Compared with Sham group, the protein expression of p-AMPK and AMPK in Model group showed no statistical difference (P > 0.05). Compared with Model group, the expression level of p-AMPK in treatment group increased significantly, in which the QL-M and QL-H group increased obviously (P < 0.01). The total amount of AMPK in each group did not show significant difference (P > 0.05)  .QL capsules are traditional Chinese medicine (TCM) formula, which was developed according to TCM theory. The QL has extracts obtained from 11 herbs. Pharmacological studies have found that QL contains a number of active substances such as ginseng saponin, astragalus saponin, flavonoid, cardenolide, and phenolic acid. There is a significant effect in the clinical treatment of chronic heart failure as shown in previous studies , 10.Failure or hypertrophic myocardium caused imbalance between demand increase and production decrease of ATP, which was the root cause of decrease in myocardial metabolic reserve and cardiac function degradation , 12. TheMyocardial remodeling can decrease oxygen level in the myocardial tissue and affects substrate oxidation, thus resulting in the reducing of aerobic metabolic efficiency and the shift of energy supply to anaerobic glycolysis. In this case, free fatty acids (FFA) and glucose aerobic oxidation are decreased, and insulin resistance may occur , 15. TheThe energy metabolism in cardiac myocytes is closely related to mitochondrial function status. Mitochondrial damage and significant reduction of oxidative phosphorylation efficiency can be caused by ischemia and hypoxia and the intermediate metabolite accumulation in failing heart. There is an internal negative and external positive transmembrane potential difference in mitochondrial inner-membrane. When the membrane potential decreases uncoupling of oxidative phosphorylation, ATP depletion and increase in oxygen radicals take place, thereby inducing cardiac myocytes into the irreversible process of apoptosis. High-energy phosphate is a direct source of energy for maintaining normal life activities of myocardial cells. The adenylate pool reflected the energy reserve state and the energy metabolic state. The results from the present study showed that, compared with Sham group, mitochondrial adenylate pool and energy charge value, RCR, and membrane potential were significantly lower in Model group, which were the same as the results of Ingwall , indicatα is a key nuclear receptor coactivator that can induce mitochondrial biogenesis, modulate number and mass of mitochondrial, fatty acid oxidation, and thermogenesis [α, compared with the Model group. Thereby, QL regulated energy metabolism by activating AMPK/PGC-1α signal factor. Specifically, QL regulated fatty acid and glucose metabolism, reduced the levels of FFA and LA through activation of AMPK. The improved mitochondrial respiratory function by QL has a relationship with its regulation of PGC-1α and the PGC-1α downstream. PGC-1α has many biological effects, for example, through the TRβ1, NRFS, and ERRS it affects mitochondrial biogenesis [α mRNA [α, a signaling pathway involved in energy metabolism, was therefore the pathway regulated by QL.AMP-activated protein kinase (AMPK) is a key modulator of lipid and glucose metabolism and energy balance. When ATP level is reduced, AMPK is rapidly activated, and becomes involved in the cell energy regulation . AMPK anogenesis . The curogenesis . Previou [α mRNA . AMPK/PGα axis and reduce the accumulation of free fatty acids and lactic acid, to protect cardiac myocytes and mitochondrial function.In summary, the improvement of cardiac energy metabolism is an important part of delaying heart failure progression. QL reduced heart weight index of rat model of TAC-induced pressure overload, improved hemodynamic parameters, and was related to the increase of myocardial high-energy phosphate content as well as the improvement of energy reserves and the metabolic state. We argue that QL may regulate the glycolipid substrate metabolism by activating AMPK/PGC-1QL is composed of a number of herbal plants. As the chemical components of this medicine are complex and varied, liquid chromatography mass spectrometry (LC-MS/MS) was adopted to study chemical component, and nearly 200 compounds were identified. Toxicological study showed the security of clinical application.Click here for additional data file.


Background: Glycoprotein IIB/IIIA inhibitors are occasionally utilized during carotid artery stenting (CAS) in the presence or absence of a visualized intra-operative thrombus.Objective: We assess the hemorrhagic and clinical outcomes associated with the use of eptifibatide during CAS.Methods: A retrospective analysis of prospectively collected data on patients with the diagnosis of carotid artery stenosis underwent CAS in a single center. We identified those who received intravenous eptifibatide intra-operatively and compared to the rest of the cohort. Hemorrhagic outcomes included intracerebral hemorrhage (ICH) or groin hematoma that occurred during the hospital stay.Results: In this analysis, 81 patients had CAS during a 3-year span; 16 of those had received 15 mg of intravenous eptifibatide intra-operatively. The mean age of the treated and untreated patients was similar . One patient (1.2%) in this series had ICH in the perioperative period that occurred in the non-eptifibatide group. Five patients (6.2%) in this series had groin hematoma; only one in the non-eptifibatide group required surgical repair. No mortality was reported and clinical outcomes including discharge modified Rankin scale, NIH stroke scale, as well as discharge destination were similar in both groups. A stratified analysis among those who underwent an urgent CAS showed no significant differences in the risks of hemorrhages or any clinical outcome (P > 0.05).Conclusion: The use of eptifibatide during CAS is safe. The risk of any hemorrhagic complication is rare in this series; however, a prospective study to validate this observation will be helpful. In-stent thrombosis is a potential problem associated with carotid artery stenting (CAS); a process attributed to intimal injury during percutaneous arterial interventions resulting in platelet activation and thrombus formation  associated with the use of GP IIb/IIIa antagonists , preoperative NIH stroke Scale (NIHSS), severity of the carotid stenosis, and whether patients were pre-treated with clopidogrel.Any patient undergoing CAS must meet the center for Medicare and Medicaid services eligibility criteria for high risk symptomatic carotid disease or have high-grade asymptomatic stenosis in the setting of high-risk clinical features Krajcer, . Two grot-test and Wilcoxon rank sum test were used to compare the mean and median of continuous variables. The primary outcome was ICH that was identified during the hospital stay or after patients were discharged from the hospital. ICH was identified by conventional or DYNA computerized tomography scans, and confirmed by MRI when appropriate. All groin hematomas were reported including the clinically significant hematoma . In addition, we identified functional outcomes based on the discharge mRS as well as the NIHSS. Lastly, we assessed the length of stay, discharge locations, and hospital mortality rates. A stratified analysis was performed for those who were hospitalized for acute stroke or TIA and in whom CAS was performed during the same hospital stay.Chi-square and Fisher’s exact tests were used to compare categorical proportions; and P = 0.13). Males were predominant in both groups . Eight patients (9.8%) were asymptomatic with the remainder of the sample having had TIA, stroke, or amaurosis fugax, 22.2, 62.9, and 3.7%, respectively. The proportion of symptomatic patients did not differ in each group (Table P = 0.07). Vascular risk factors of diabetes, hypertension, coronary, and peripheral arterial disease, hyperlipidemia, history of TIA or stroke, and smoking were similar in both groups (P > 0.05). Similarly, the mean mRS (1.18 ± 1.10 versus 0.84 ± 0.91) upon initial hospitalization as well as the stroke severity (NIHSS: 1.75 ± 2.26 versus 2.06 ± 2.70) were similar in both groups (P > 0.05).From the initial sample of 87 patients, 6 were excluded because was performed as part of an intervention for ongoing acute ischemic stroke, including those who underwent mechanical thrombectomy. Eighty one patients with the mean age of 65.5 were included in this analysis; 16 were treated with eptifibatide during CAS. The mean age of the treated and untreated population was similar . In this cohort, no mortality was reported and 56 of 81 patients (69.1%) discharged to home; and 25 (30.8%) patients discharged to rehabilitation centers. Angiographically, this cohort demonstrated severe carotid disease with the mean stenosis rate of 84.1%; which was similar in both groups  had ICH following CAS that was attributed to reperfusion syndrome  is compatible with previous reports which ranged between 0.36 and 4.1% (Cheung et al., This study is limited by the small sample of those who were treated with eptifibatide during CAS, thus may have led to a selection bias. In addition, the use of eptifibatide in our series was not uniform, i.e., eptifibatide was given for therapeutic and preventative purposes. Regardless, this study suggests the relative safety of eptifibatide use during CAS when performed electively or urgently. Risk-benefit assessment in large prospective studies or national registry would be useful.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


During a Legionnaires’ disease (LD) outbreak, combined epidemiological and environmental investigations were conducted to identify prevention recommendations for facilities where elderly residents live independently but have an increased risk of legionellosis.Legionella.Survey responses (n = 143) were used to calculate attack rates and describe transmission routes by estimating relative risk (RR) and 95% confidence intervals (95% CI). Potable water collected from five apartments of LD patients and three randomly-selected apartments of residents without LD (n = 103 samples) was cultured for L. pneumophila serogroup 1, monoclonal antibody type Knoxville , sequence type 222. An indistinguishable strain was detected in 31 (74%) of 42 potable water samples.Eight confirmed LD cases occurred among 171 residents (attack rate = 4.7%); two visitors also developed LD. One case was fatal. The average age of patients was 70 years (range: 62–77). LD risk was lower among residents who reported tub bathing instead of showering . Two respiratory cultures were characterized as Legionella prevention plan. When Legionella colonization of potable water is detected in these facilities, remediation is indicated to protect residents at higher risk. If LD occurs among residents, exposure reduction, heightened awareness, and clinical surveillance activities should be coordinated among stakeholders. For prompt diagnosis and effective treatment, clinicians should recognize the increased risk and atypical presentation of LD in older adults.Managers of elderly-housing facilities and local public health officials should consider developing a  Legionella bacteria. In the United States, ~8,000-18,000 persons with community-acquired LD are hospitalized annually [Legionnaires’ disease (LD) is a potentially-fatal pneumonia caused by inhalation of water aerosols containing annually -4 and LDannually . Immunosannually , are morannually . SwallowMost LD cases occur sporadically; relatively few patients with LD are diagnosed and reported to public health officials . TherefoLegionella colonization (if any) at the complex, and recommend interventions to prevent additional cases.On October 15, 2009, the Baltimore City Health Department and the Maryland Department of Health and Mental Hygiene contacted the Centers for Disease Control and Prevention (CDC) regarding an outbreak of LD among residents of an apartment complex for seniors in Baltimore. In the previous 5 weeks, four residents and a visitor to the complex had been hospitalized with LD. One patient had died and another was receiving intensive care. We investigated to find additional cases, identify exposures associated with disease transmission, determine the source of Epidemiological and environmental investigations were conducted at the apartment complex, which consisted of four adjacent buildings. Two buildings were built in 2004; the third and fourth buildings were built in 2006 and 2009, respectively. Occupancy of the buildings began soon after construction of each was complete. An age of 62 years or older was required for residency. Single residents occupied most (>90%) of the 340 apartment units at the complex and virtually all units were occupied by October, 2009. Common areas were available to residents . Medical services were not available onsite, but communal meals and social gatherings occurred regularly.Legionella from respiratory secretions, or a four-fold increase in serum antibody titer to L. pneumophila serogroup 1) between August 1, 2009 and December 31, 2009 among a resident of any apartment building in the complex. Possible cases were defined similarly, but had either incomplete  or no specific laboratory testing for Legionella infection performed. Respiratory illnesses in persons with negative Legionella testing were considered non-cases.For case finding, we defined confirmed cases of LD as a diagnosis of clinical or radiographic pneumonia and laboratory-confirmation of LD . The exposure period for the study was defined as August 1 through the date of survey interview. After informed consent was obtained, a standardized questionnaire was administered in person. We queried respondents about their demographic characteristics, water exposures at the complex and within the community, signs or symptoms of respiratory illness, healthcare use and diagnoses, smoking, and comorbid conditions . Complaints about municipal water service interruptions and water quality  were also recorded. We made at least two attempts on separate days to interview each resident; visitors were not included in the cohort study.Microsoft Access database  and analyzed it in SAS version 9.2 . Attack rates (AR) for specific transmission risk factors (present vs. absent) were compared to calculate relative risks (RR) with 95% confidence intervals (95% CIs) and Fisher’s exact tests. A p-value less than 0.10 was considered statistically significant because of the small number of LD cases.We maintained data in a Our environmental investigation began with a physical inspection, noting the orientation of the apartment buildings, the design of the individual apartment units, and points of water use  in each unit. Common areas where water exposures might have occurred were also noted. Diagrams of the potable water system were not available at this stage of the investigation.On October 17, we collected 103 samples from five apartments of LD patients in affected buildings (A-1 and A-2) and three randomly-selected apartments of residents who had not developed LD in A-2 and each unaffected buildings (U-1 and U-2). (A resident of a randomly-selected apartment in A-1 developed LD after the apartment was sampled.) Specific sampling locations in the apartments included showerheads, sink faucets in kitchens and bathrooms, and water heater tanks (n = 56 samples). We also collected samples from outdoor fire hydrants (n = 6), from the buildings’ water intake valves (n = 19), and the sink faucets and water heating tanks of common areas in buildings A-1 and A-2 (n = 22). We collected samples of municipal water and expansion tanks, designed to absorb excess water pressure caused by thermal expansion or “water hammers” , on October 21 and October 24, respectively. Biofilm swabs and one-liter bulk water samples were collected according to published procedures . Water tLegionella laboratory, bulk water samples were filtered through polycarbonate 0.2 μm filters. The filters were placed in 5 ml of sterile water and vortexed for 60 seconds. One hundred μl of this suspension were placed on buffered charcoal-yeast extract media with and without antibiotics. Plates were incubated at 95–98.6°F (35–37°C) in 2.5% CO2 to culture Legionella. We also forwarded two patients’ clinical isolates to CDC.At the CDC Serogrouping and determination of monoclonal antibody patterns ,14 were This study was determined an emergency public health investigation exempt from institutional review board (IRB) review; standard procedures to protect patient confidentiality were followed.The demographics of residents and LD patients were similar. Almost all of the 352 residents were either African-American 91%) or Korean-American (7%). Three-quarters (75%) of residents were female. Residents varied in age . In comparison, all eight residents who developed LD between September and December of 2009 were African-American. Six (75%) were female. Among residents, patients were younger than non-patients, but this difference was not significant (p = 0.09) (Table % or KoreMost (80%) cases were identified by urine antigen testing performed during hospitalization, with the exception of one case that was detected retrospectively ~47 days after illness onset Figure . LD occuThe eight LD cases that occurred among 171 residents of A-1 and A-2 represented an attack rate of 4.7%  or while cleaning the kitchen and bathroom using a bucket filled with water patient 10) , but temperature differences were observed in water collected from faucets at the bottom of tanks in sampled apartments (range: 70–100°F/21.1–37.8°C).st. More than half of residents (56%) reported changes in the taste, odor, or color of their water during the same period.Three-quarters (75%) of residents who were interviewed noted at least one interruption in water service since August 1L. pneumophila from patients 6 and 7 were characterized as L. pneumophila serogroup 1, monoclonal antibody type Knoxville . The outbreak strain was determined to be sequence type 222 .Clinical isolates of L. pneumophila 1  was exclusively detected in apartments within buildings A-1 and A-2 where all LD patients resided .Among the 103 pre-remediation samples collected, Legionella respiratory cultures were available from two patients and characterized as L. pneumophila serogroup 1, monoclonal antibody type Knoxville , sequence type 222. An indistinguishable strain was detected in numerous potable water sources from apartment units of two buildings. Epidemiological data from a cohort study with a high response rate indicated that all patients were primarily or exclusively exposed to colonized potable water in apartments. Taken together, the environmental and cohort studies implicated Legionella colonization of the potable water system as the source of the outbreak.A large LD outbreak occurred at an apartment complex for seniors in Baltimore between September and December, 2009. Ten cases were confirmed among eight residents and two visitors to the complex; one case was fatal. Legionella growth. Chlorine disinfection was not detected at the point of use in any of the residents’ apartments; hot water temperatures were generally within the ideal range for Legionella amplification (77°F-108°F/25°C-42.2°F). Importantly, the potable water system was colonized with a monoclonal antibody 2 positive [L. pneumophila (sequence type 222) that has been previously associated with outbreaks in the northeastern United States and Canada [The convergence of several factors led to the outbreak. Residents of the apartment complex were particularly susceptible to LD because of their advanced age, high prevalence of underlying conditions, and smoking habits. Although the size of the cohort limited statistical power, we confirmed that attack rates were highest in persons who smoked or had an immunocompromising condition. However, two younger visitors (including one without underlying risk factors) also developed LD. This finding suggested that the type, frequency, and duration of exposures may also determine disease risk. Also, the potable water system’s conditions were conducive to positive , a viruld Canada ,20. NeveIncreases in the burden of LD  may be rLegionella prevention plan in coordination with local public health officials as well as plumbers, engineers, or other environmental consultants. This plan may involve completing an environmental assessment to identify opportunities to reduce colonization risk [http://www.cdc.gov/legionella/index.html.) Hazard analyses and critical control points (HACCP), an industry method designed to improve food safety, can be applied to Legionella prevention – as outlined by forthcoming practices from the American Society of Heating, Refrigerating and Air-Conditioning Engineers, Inc. (ASHRAE) Standard Project Committee 188 (http://spc188.ashraepcs.org/). Facility managers may also consider periodic environmental monitoring of the potable water system to identify Legionella colonization in the context of a full HACCP plan. When possible, potable water systems should be designed to minimize the potential for Legionella colonization .Facility managers should consider developing a ion risk .  of LD cases in the United States [If LD cases have been detected among residents, exposure reduction, heightened awareness, and clinical surveillance  should bd States . CaregivLegionella pneumonia among residents and visitors to facilities with colonization or the occurrence of LD cases. In addition to Legionella urine antigen testing, clinicians should attempt to recover respiratory secretions for Legionella culture from any patient linked to an outbreak and any patient hospitalized with severe community-acquired pneumonia of unknown etiology because clinical Legionella isolates are critical for establishing an association with an environmental exposure and can direct appropriate antimicrobial therapy . Healthcare providers should also work closely with patients to eliminate modifiable risk factors for pneumonia (e.g. smoking) [The clinical presentation of LD pneumonia can be atypical, particularly among elderly patients ,30. Thersmoking) , optimizLegionella prevention and control strategies based on whether Legionella colonization of potable water has been detected and especially when LD cases occur. Physicians specializing in infectious diseases and other persons providing healthcare services to elderly residents should be aware of the increased risk of LD in this population; awareness is a prerequisite for the provision of quality healthcare, including prompt diagnosis and effective treatment as well as counseling on the importance of risk modification and exposure reduction to prevent additional morbidity and mortality.Managers of elderly-housing facilities, local public health officials, and other stakeholders should implement The authors declare that they have no competing interests. The findings and conclusions in this article are those of the authors and do not necessarily represent the official position of U.S. Center for Disease Control and Prevention.BJS, JLF, LMH, KJS, OF, DB, and LAH lead the investigation, including design of the environmental and cohort studies. BJS, JLF, KN, MGM, LMH, KJS, JMU, JK, and DB collected environmental and epidemiological data. EB, NAK, TT, and BSF conducted laboratory analyses. BJS, JLF, and LAH drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/291/prepub


RBC folate concentrations were measured at baseline, and after 6, 12, 29 and 40 weeks. At 40 weeks, the overall prevalence of having a RBC folate <906 nmol/L decreased to 18% and 35% in the 400 µg and 140 µg groups, respectively, while remaining relatively unchanged at 58% in the placebo group. After 40 weeks, there was no evidence of a difference in RBC folate between the two treatment groups (P = 0.340), nor was there evidence of a difference in the odds of a RBC folate <906 nmol/L (P = 0.078). In conclusion, the average daily intake of folic acid received from the proposed fortification program would increase RBC folate concentrations in reproductive age women to levels associated with a low risk of NTDs.  Primary prevention of most folate-responsive neural tube defects (NTDs) may not require 400 μg folic acid/day but may be achieved by attaining a high maternal folate status. Using RBC folate ≥906 nmol/L as a marker for NTD risk reduction, the study aimed to determine the change in blood folate concentrations in reproductive age women in response to long-term folic acid supplementation at 400 µg/day and 140 µg/day . Participants were randomly assigned to a daily folic acid supplement of 140 µg ( As a result, public health authorities worldwide have recommended consuming 400 μg folic acid per day during the periconceptional period to reduce the risk of first occurrence NTDs ,2,3,4. Tet al.  documentMandatory folic acid fortification of enriched cereal-grain products in the United States and Canada, implemented in 1998, has substantially improved the folate status of reproductive age women  and resuA sample of healthy, reproductive age women (18–40 y) were recruited in July 2008 from the staff and student population at the University of Otago, Dunedin, New Zealand, and from the local community through advertisements in the newspaper and word-of-mouth. Women were not included in the study if they were pregnant, lactating, or were planning a pregnancy in the next 12 months. Additional exclusion criteria for the study were regular use of folic acid-containing supplements in the previous 6 months; self-reported history of cardiovascular, gastrointestinal, hepatic, renal or hematologic disease; and use of medications that interfere with folate metabolism . Ethical approval was obtained from the Human Ethics Committee of the University of Otago, Dunedin, New Zealand, and all participants gave written, informed consent. This study was registered with the Australian New Zealand Clinical Trials Registry  as ACTRNThe study was a 40 week double-blinded, placebo-controlled trial conducted from August 2008 through May 2009. Participants attended an early morning clinic at baseline, and after 6, 12, 29 and 40 weeks. At the baseline visit, sociodemographic and general health data were collected using a self‑administered questionnaire; height and weight were taken according to standardized procedures  and measThe study supplements were manufactured by New Zealand Nutritionals Ltd  as hard tablets, each containing microcrystalline cellulose as a filler (placebo), and either 140 μg (317 nmol) folic acid, or 400 μg (907 nmol) folic acid. Folate contents of the tablets were analyzed by microbiological assay in our laboratory at the beginning of the study. The actual amounts in the tablets aimed to provide 140 μg and 400 μg folic acid were 133 μg and 359 μg folic acid, respectively. Participants, investigators and biostatistician were blinded to the treatment. Supplements were coded by a third-party and randomization data was kept strictly confidential in a sealed envelope until all of the data analyses had been performed. Participants were instructed to take one tablet per day and to return all unused pills. Three bottles of tablets were prepared for each participant, and distributed at baseline, and weeks 12 and 29. Compliance with respect to supplement intake was assessed by counting returned pills. Dietary intake of folate was determined via 3-day weighed food records completed at baseline. The diet records were completed over two non-consecutive weekdays and one weekend day. The total folate intakes, in µg, were tabulated using an electronic version of the 2006 New Zealand Food Composition Tables  and exprLactobacillus rhamnosus . Erythrocyte folate concentration was calculated from whole-blood values using individual packed cell volumes and after correction for plasma folate concentration. To avoid between-run variation, all study visit samples from each participant were measured in one batch. Accuracy and inter-assay variability were monitored with the use of two external quality controls: whole blood folate standard  with a certified value of 29.5 nmol/L (mean 27.3 (SD 4.6); CV 17%) and a human serum folate standard reference material  with a total folate information value as determined by microbiological assay of 44 nmol/L (mean 42.5 (SD 8.7); CV 20%). A plasma folate concentration <6.8 nmol/L or a RBC folate concentration <317 nmol/L was used to indicate folate deficiency [Blood samples were collected in EDTA-treated tubes, transported on ice and processed within one hour of collection. After measurement of the hematocrit, red blood cell hemolysates were prepared (dilution (1:10) of whole blood with 1% ascorbic acid) and incubated at 37 °C for 30 min before storage at −80 °C. The remaining whole blood was centrifuged (1500 g for 15 min at 4 °C) to separate the plasma and aliquots were stored immediately at −80 °C. Plasma and whole blood folate concentration was determined by microbiological assay  using teficiency . 2 tests for categorical variables (with Fisher’s exact test used where expected cell counts were below 5 for more than 20% of cells). The outcome variables were plasma and RBC folate concentrations and suboptimal RBC concentration (determined as <906 nmol/L). The following known or potential predictors were identified a priori: age, BMI, education, ethnicity, use of oral contraceptives and dietary folate. Education was collapsed into three categories: secondary school or less, post-secondary education (university or college), or advanced degree. Participant ethnicity was classified as New Zealand European, Maori and Pacific Peoples, Asian, or other ethnicities. Plasma and RBC folate concentrations were natural log transformed where this resolved issues with skew and/or heteroscedasticity in residuals. Linear and logistic mixed models were used to assess intervention group effects and included a random participant effect. Difference in changes between groups was assessed using treatment group-by-time interactions. Selected error covariance structures  were investigated for linear mixed models to see if they improved model fit according to the Akaike information criterion (AIC). Non-linear associations were investigated, and where appropriate, modelled using fractional polynomials. Variables with P < 0.25 in models including treatment group, time, and their interaction were included in the final regression models. All tests were 2-sided with statistical significance as determined by P < 0.05.All statistical analyses were performed by using Stata . Characteristics of participants completing the study and dropping out before the final blood sample were compared using t-tests for continuous variables (after log-transformations where appropriate) and χP ≥ 0.325). With respect to study supplement intake, 93% (134 out of 144) participants returned at least one of their three study bottles and of these, 90% (120 out of 134) had compliance of 70% or higher. Of the 144 women enrolled in the study, 119 completed the intervention trial . BaselinP < 0.001). Relative to the placebo group, plasma folate increased statistically significantly in both supplemented groups; furthermore, the mean (95% CI) increase in plasma folate of 107%  in the 400 μg group was statistically significantly greater (P = 0.007) than the group receiving 140 μg folic acid/day ). In contrast, after 40 weeks of supplementation, there were no statistically significant differences in the observed increases in RBC folate concentrations between the two treatment groups  versus 51% , P = 0.340).The geometric mean plasma and RBC folate concentrations at baseline and over the 40 week intervention period are shown in P = 0.009). There was no evidence that the odds changed among women taking 140 µg until week 29 (P < 0.001). At week 40, no statistically significant differences were detected in the odds ratio of having a RBC folate <906 nmol/L between treatment groups (P = 0.078).The decreasing prevalence of RBC folate concentration <906 nmol/L in the supplemented groups is illustrated in i.e., ≥906 nmol/L). Since 1996, New Zealand has permitted voluntary folic acid fortification of selected foods, however few foods have been fortified under this policy. It is estimated that the mandatory fortification of bread will increase average daily folic acid intakes among the target population by 136 μg. Our intervention trial was designed to evaluate changes in RBC folate at the proposed estimated daily intake level compared to the current official recommended dose of 400 μg folic acid/day for the primary prevention of NTD. After 40 weeks supplementation, blood folate status increased significantly in both treatment groups, however there was no evidence that mean RBC folate concentrations were significantly higher in the group of women taking 400 μg folic acid/day as compared to the 140 μg group. Moreover, there was no evidence of a difference in the odds of having a RBC folate <906 nmol/L between treatment groups at 40 weeks. The present study demonstrates that although the prevalence of folate deficiency is low, nearly two‑thirds of our study population had baseline RBC folate concentrations below the level thought to be protective against NTDs  [The overall prevalence of NTDs in New Zealand is difficult to determine accurately due to inadequate data collection around the number of NTD-affected pregnancies. Complete data  are only available for 1998 to 2003. These annual totals demonstrate a downward trend in the rate of NTD affected pregnancies rate from 17.4 to 11.2 per 10,000 births . In 2003et al. . On the  [et al.  and the  [et al.  likely uonsumed) . Thus, tonsumed) . Furtheronsumed) , which iMandatory folic acid fortification of bread in New Zealand has been deferred until May 2012 due to public concerns regarding cost-effectiveness and the impact on consumer choice . There a


Highly efficient gene transfer could be achieved in the cultured primary synoviocytes transfected with the combination of BL and US exposure, compared to treatment with plasmid DNA (pDNA) alone, pDNA plus BL, or pDNA plus US. When BL was injected into the knee joints of mice, and US exposure was applied transcutaneously to the injection site, highly efficient gene expression could be observed in the knee joint transfected with the combination of BL and US exposure, compared to treatment with pDNA alone, pDNA plus BL, or pDNA plus US. The localized and prolonged gene expression was also shown by an in vivo luciferase imaging system. Thus, this local gene delivery system into joint synovium using the combination of BL and US exposure may be an effective means for gene therapy in joint disorders.Recently, we have developed novel polyethylene glycol modified liposomes  entrapping an ultrasound (US) imaging gas, which can work as a gene delivery tool with US exposure. In this study, we investigated the usefulness of US-mediated gene transfer systems with BL into synoviocytes  Intra-articular gene therapy is considered a feasible technique to deliver therapeutic proteins to suppress inflammation and destruction of the joints in rheumatoid arthritis and osteoarthritis, because it could minimize extra-articular adverse effects linked to the systemic injection of drugs , 2. To ain vitro and the joint synovium in vivo. The establishment of a method to deliver genes into joints by the combination of BL and US exposure may facilitate the development of a safe and efficient gene therapy for joint disorders. In the present study, we investigated the usefulness of US-mediated gene transfer systems with BL into synoviocytes sn-glycero-3-phosphocholine (DPPC)  and 1,2-distearoyl-sn-glycero-3-phosphatidyl-ethanolamine-polyethylen eglycol (DSPE-PEG2000-OMe) (NOF Corporation) in a molar ratio of 94 : 6 were prepared by a reverse phase evaporation method. In brief, the reagents were dissolved 1 : 1 (v/v) in chloroform/diisopropyl ether. Phosphate-buffered saline was added to the lipid solution, and the mixture was sonicated and then evaporated at 47°C. The organic solvent was completely removed, and the size of the liposomes was adjusted to less than 200 nm using extruding equipment and a sizing filter (pore size: 200 nm) . The lipid concentration was measured using a Phospholipid C test Wako . BL were prepared from liposomes and perfluoropropane gas . First, 2 mL sterilized vials containing 0.8 mL liposome suspension (lipid concentration: 1 mg/mL) were filled with perfluoropropane gas, capped, and then pressurized with a further 3 mL perfluoropropane gas. The vial was placed in a bath-type sonicator   for 5 min to form BL.Bubble liposomes were prepared by the previously described methods , 23, 26.The plasmid pCMV-Luc is an expression vector encoding the firefly luciferase gene under the control of a cytomegalovirus promoter. The plasmid pDsRed-Express-N1  is an expression vector encoding the red fluorescent protein under the control of a cytomegalovirus promoter.4) were seeded in the wells of a 48-well plate . Five micrograms of pDNA and 60 μg BL were mixed together with culture medium containing 10% FBS and added to the cells. The cells were immediately exposed to US  for 10 sec through a 6-mm diameter probe placed in the well. A Sonopore 3000  was used to generate the US. The cells were washed twice with culture medium and cultured for two days. The cell lysate was prepared with lysis buffer (0.1 M Tris-HCl (pH 7.8), 0.1% Triton X-100, and 2 mM EDTA). Luciferase activity was measured using a luciferase assay system  and a luminometer . The activity is indicated as relative light units (RLU) per mg of protein.Primary synoviocytes (HFLS), which are primary fibroblast-like cells derived from the inflamed synovial tissue of rheumatoid arthritis patients, were purchased from Cell Applications, Inc. . The culture was performed according to the manufacturer's instructions. The day before transfection, cells (3 × 104) were seeded in the wells of a 48-well plate . Then, 0.25 μg pDNA  wsa diluted in Opti-MEM (GIBCO). Next, 1.25 μg Lipofectamine 2000 (LF2000)  was diluted in Opti-MEM. These solutions were mixed and added to the cells. After 4 and 24 hours, the cells were washed with PBS and cultured for two days. The experiments were performed according to the manufacturers' instructions.The day before transfection, HFLS-RA (4 × 10Cell lysate was prepared with lysis buffer (0.1 M Tris-HCl (pH 7.8), 0.1% Triton X-100, and 2 mM EDTA). Luciferase activity was measured using a luciferase assay system  and a luminometer . The activity is indicated as relative light units (RLU) per mg protein. To analyze DsRed expression, the treated cells were observed with a fluorescence microscope .n = 4 in each group). ICR mice  were anesthetized with an i.p. injection of sodium pentobarbital (80 mg/kg) throughout each procedure. A 40 μL suspension of pDNA (20 μg) and BL (30 μg) was injected into the knee joint of the ICR mice, and US exposure  was immediately applied at the injection site. Five days after the injection, the mice were euthanized and sacrificed, and the knee joint tissue in the US-exposed area was collected and homogenized. The tissue homogenates were prepared with lysis buffer (0.1 M Tris-HCl (pH 7.8), 0.1% Triton X-100, and 2 mM EDTA). Luciferase activity was measured using a luciferase assay system  and a luminometer . Activity is indicated as relative light units (RLU) per mg of protein. To determine the efficiency of gene delivery, animals were divided into five experimental groups and one control group (n = 4 in each group). ICR mice  were anesthetized with an i.p. injection of sodium pentobarbital (80 mg/kg) throughout each procedure. A 40 μL suspension of pDNA (20 μg) and BL (30 μg) was injected into the knee joint of the ICR mice, and US exposure  was immediately applied at the injection site. Several days after the injection, the mice were anaesthetized and i.p. injected with D-luciferin (150 mg/kg) . After 10 min, luciferase expression was observed with an in vivo luciferase imaging system (IVIS) (Xenogen Corporation). The image of a representative of the 4 mice was used for each treatment group in this experiment.To determine the efficiency of gene delivery, animals were divided into five experimental groups and one control group (μm thickness) were evaluated for the expression of luciferase protein by immunostaining. The sections were deparaffinized in xylene, rehydrated through graded ethanol, and equilibrated in PBS. The sections were incubated with biotin-labeled rabbit antiluciferase antibody . The following day, after three washes in PBS, immunoreactivity was detected using an antigoat IgG/HRP and diaminobenzidine (DAB). After color development, the joint sections were counterstained with hematoxylin and were then dehydrated, cleared, and mounted on slides. The gene-transfected joint tissues were preserved in 10% PFA, decalcified with EDTA, and then embedded in paraffin and sectioned. Sections . Data were considered significant when P < .05. The t-test was used to calculate statistical significance. All data are shown as the mean ± SD (It is known that microbubbles improve cell and tissue permeability by cavitation upon US exposure –15. We f in vitro could be affected by increasing the US exposure time and intensity [2) in transfection, US intensity of 2.5 W/cm2 was modestly higher than other treated groups (in vitro transfection in synoviocytes.Previously, our report demonstrated that gene transfection efficiencyntensity , 23. We,d groups . These rμL solution of pDNA and BL was injected into the knee joint of the mice, and US exposure was immediately applied at the injection site. As a result, marked gene expression could be enhanced efficiently only with the combination of BL and US exposure when compared with other treatments (in vivo luciferase imaging system during 2–24 days after transfection into a joint treated with pDNA, BL, and US exposure. The high level of gene expression persisted for 7–24 days after transfection using BL and US exposure (A local gene delivery system to the joint synovium by BL and US exposure may be easily applied, because the injected BL may be retained in the confined joint space and percutaneous US exposure may induce cavitations on the surface of the synovium. We, therefore, attempted to deliver pCMV-Luc, luciferase-expressing plasmid DNA, into the joint synovium of mice using BL and US and to determine the level of the gene expression. A 40 eatments . Exceediexposure . Gene exexposure .  Gene transfer into cartilage may be difficult because cavitation induction with BL and US cannot reach chondrocytes embedded in extracellular matrix in articular cartilage, leading to no transfection. However, successful gene transfection into chondrocytes might be achieved by BL and US exposure, because articular chondrocytes in RA or OA are exposed by degradation of the extracellular matrix in articular cartilage in this stage of the disease. It is known that cartilage degradation is the main pathological feature in OA; however, synovial factors are closely related to this progress , while sin vitro and the joint synovium in vivo. In the future, this local gene delivery method with BL and US exposure might be used in non-viral gene therapy for joint disorders, such as RA and OA.In this study, we showed that the combination of BL and US exposure could be an effective gene delivery method into synoviocytes


This study investigated the major clinical determinants of late gadolinium enhancement (LGE) at ventricular insertion points (VIPs) commonly seen in patients with pulmonary hypertension (PH).Forty-six consecutive PH patients (mean pulmonary artery pressure ≥25 mmHg at rest) and 21 matched controls were examined. Right ventricular (RV) morphology, function and LGE mass volume at VIPs were assessed by cardiac magnetic resonance (CMR). Radial motion of the left ventricular (LV) wall and interventricular septum (IVS) was assessed by speckle-tracking echocardiography. Paradoxical IVS motion index was then calculated. Univariate and multivariate regression analysis were conducted to characterize the relationship between LGE volume at VIPs and PH-related clinical indices, including the paradoxical IVS motion index.Mean pulmonary arterial pressure (MPAP) of PH patients was 38±9 mmHg. LGE at VIPs was observed in 42 of 46 PH patients, and the LGE volume was 2.02 mL (0.47–2.99 mL). Significant correlations with LGE volume at VIPs were observed for MPAP (r = 0.50) and CMR-derived parameters . In multiple regression analysis, paradoxical IVS motion index alone significantly predicted LGE volume at VIPs (p<0.001).LGE at VIPs seen in patients with PH appears to reflect altered IVS motion rather than elevated RV pressure or remodeling. Long-term studies would be of benefit to characterize the clinical relevance of LGE at VIPs. Pulmonary hypertension (PH) is defined as mean pulmonary arterial pressure (PAP) ≥25 mmHg at rest. Cardiac magnetic resonance (CMR) imaging is an established modality for the objective assessment of RV geometry and function. To date, CMR studies of PH have described increased size and impaired function of the right ventricle. Regarding this, a possible explanation is that LGE at VIPs develops as a result of elevated RV pressure and resultant RV remodeling in PH. Indeed, previous studies have shown significant associations between LGE volume at VIPs and measurements of pulmonary hemodynamics and RV morphology. The present study investigated the underlying mechanisms of LGE at IVSs in PH by evaluating the association between LGE at VIPs and clinical parameters. Particular focus was made on the possible contribution of paradoxical IVS motion assessed by speckle-tracking echocardiography to the development of LGE at IVSs.In this single-center, case-control, prospective, observational study, subjects who met the entry criteria [mean PAP of ≥25 mmHg and pulmonary capillary wedge pressure (PCWP) of ≤15 mmHg at rest] were consecutively enrolled between April 2010 and December 2012. Exclusion criteria were the presence of comorbid disease that might affect cardiac morphology and function, an unstable PH condition requiring treatment modifications, and inability to obtain or analyze electrocardiogram-gated CMR images. Patients with atrial fibrillation/flutter were excluded based on the last criterion. Age- and gender-matched subjects who did not have cardiac and/or respiratory diseases were recruited from the (para)medical staff of our institution to serve as control subjects. Subjects with systemic hypertension were considered eligible in both groups when blood pressure was well controlled and when echocardiography exhibited no structural changes of the left ventricle and atrium.Patients with PH underwentechocardiography, CMR and right heart catheterization (RHC) within a 1-week interval during which they were clinically stable. RHC was conducted according to the guidelines for the diagnosis and treatment of PH, All PH patients and control subjects gave informed written consent to participate, and the study protocol was approved by the ethics committee of the Hokkaido University Graduate School of Medicine.CMR studies were performed using a 1.5-Tesla Philips Achieva magnetic resonance imaging system  equipped with Master gradients . Imaging was performed with 10–15 sec breath-holding during expiration, using a vector-cardiographic method for electrocardiogram gating. From coronal localizing images that demonstrated the gross cardiac anatomy, an orthogonal stack of axial slices was planned to cover the heart from a level just below the diaphragm to the bronchial bifurcation, covering the heart in diastole. Axial slices were acquired using a steady-state free precession pulse sequence . A slice thickness of 10 mm was used to minimize the number of image acquisitions, and to reduce the number and duration of the breath-holding.3. Ventricular volume and mass were indexed by body surface area.Obtained images were evaluated using commercially available software . RV and left ventricular (LV) endocardial borders of contiguous axial slices were manually traced and the obtained time-volume curves allowed for calculation of RV and LV end-diastolic volume (EDV) and end-systolic volumes (ESV), and ejection fraction (EF). Epicardial ventricular borders were also manually contoured for quantification of the volume of RV and LV walls. The IVS was regarded as a part of LV wall. RV and LV masses were calculated by multiplying each wall volume by 1.05 g/cmIn patients with PH, Gd-DTPA  was intravenously administered. Ten minutes after the injection, a breath-holding, inversion-recovery (IR)-prepared, three dimensional turbo field echo pulse sequence with electrocardiogram gating was performed to obtain a delayed-enhancement image with fat saturation of spectral presaturation with inversion recovery. The imaging parameters were as follows: slice thickness = 5 mm; FOV = 400 mm; matrix size = 157×256; TR/TE = 3.8 ms/1.2 ms; flip angle = 15°; and number of signal averages = 1. For each subject, the inversion time was adjusted to null the signal from the normal myocardium; the typical inversion time was 260 to 280 ms. Hyper-enhanced regions at the anterior and posterior VIPs were manually contoured on each short-axis slice using the same software, yielding total LGE volume. This procedure was conducted by an examiner who was not aware of the measurements of echocardiographic study.Echocardiograms were obtained using Vivid q . LV end-diastolic and end-systolic dimensions were assessed from the parasternal long-axis view. The eccentricity index, an index of IVS displacement toward left ventricle, was obtained using the parasternal short-axis view at both end-systole and end-diastole. For the objective assessment of the regional motion of the LV wall and IVS, established speckle-tracking analysis was conducted using commercially available software . With this technique, LV free wall and IVS were automatically divided into six segments, and the radial motion of each segment was visualized by six lines in different colors. Intraobserver agreement for the two measurements: IVS volume and paradoxical IVS motion index was assessed by comparing the measurements of repeated analysis in five randomly chosen control subjects and in 10 randomly chosen PH patients (T.S.). Interobserver agreement of the two measurements was assessed using the same patients (n = 15) by comparing the results measured by T.S. and those obtained by a second, experienced cardiologist (I.T.). The second cardiologist was not aware of the echocardiographic measurements by the first examiner or of the CMR measurements. Bland-Altman analysis and intraclass correlations (ICC) were used to assess reproducibility.t test, or the Wilcoxon test as appropriate. LGE volume in VIPs was compared between PH patients who were on vasodilation therapy and those who were not, and among PH patients treated with different regimens of vasodilators.Continuous variables are expressed as mean ± standard deviation (SD) for those normally distributed or otherwise as medians and interquartile ranges (IQR). Departures from normality were detected with the Shapiro-Wilk statistic. Differences in measurements between the control and PH groups were assessed with the chi-square test, Student’s Correlations between LGE volume in VIPs and other clinical parameters were evaluated by Pearson’s correlation coefficient. The statistical power of our study was greater than 0.8 with a significance level of 0.05, Multiple regression analysis was also conducted to examine if any measurement predicted LGE volume at VIPs in an independent manner. In this analysis, six variables that were likely to be associated with LGE at VIPs  were chosen as explanatory variables.All statistical analyses were performed using JMP® Version 9 , and p values less than 0.05 were considered to represent statistical significance.A total of 51 Japanese patients met the entry criteria, but five patients were excluded based on pre-specified exclusion criteria. The mean duration between the initial recognition of any PH-related symptoms/signs and the diagnosis of PH was 43 months . Twenty-one age- and gender-matched healthy controls were also enrolled. When comparing the 46 PH patients  and the 21 control subjects , no significant demographic differences were apparent including in body mass index, smoking history and the prevalence of cardiovascular/respiratory diseases. 2, control 66 [52–77] mL/m2; p<0.001) and RV mass index , and a smaller RV EF  and LV EF . LGE at VIPs was found in 41 of 46 PH patients. Median LGE volume of PH patients was 2.02 [0.47–2.99] mL. LGE volume in VIPs of PH patients who were receiving vasodilators was 2.19 [0.49–3.02] mL, which was not significantly different from that of PH patients who were not on such treatment . Also, no significant difference in LGE volume at VIPs were observed when comparing among the three different treatment regimens used in PH patients (p = 0.976) ; phosphodiesterase 5 inhibitor with or without beraprost, 2.27 [0.34–2.75] mL (n = 5); endothelin receptor antagonists with or without beraprost, 2.33 [1.19–2.44] mL (n = 9).CMR images were acquired from all participants, and the image quality was sufficient for the subsequent analysis. When compared with control subjects, PH patients exhibited a greater RV EDV index  than in controls  (p<0.001 for both indices). Paradoxical IVS motion index was also significantly greater in PH patients (0.23±0.14) than in controls (0.04±0.03) (p<0.001).LGE volume at VIPs significantly correlated with mean PAP , RV EDV index , RV ESV index , RV mass index , RV EF , end-diastolic eccentricity index , end-systolic eccentricity index , and paradoxical IVS motion index , but not with cardiac index (CI) , pulmonary vascular resistance (PVR) , LV EDV index , LV ESV index , LV mass index , LV EF , and the disease duration of PH ..Bland-Altman analysis of the intraobserver variability of LGE volume at VIPs showed low mean differences and limits of agreement (0.02±0.30 ml). The ICC was 0.99. Regarding interobserver variability, Bland-Altman analysis showed similarly small mean differences and limits (0.2±6.1 ml). The ICC was 0.97.Bland-Altman analysis of the intraobserver variability of the paradoxical IVS motion index showed low mean differences and limits of agreement (0.8% ±3.5%). The ICC was 0.98. Regarding interobserver variability, Bland-Altman analysis revealed similarly small mean differences and limits (−1.0% ±4.4%). The ICC was 0.96.In the present prospective observational case-control study, 46 PH patients and 21 matched controls were examined. Results suggested the following: (1) LGE at VIPs is a common CMR finding (positive in 42 (91%) of 46 PH patients); (2) early systolic paradoxical motion of VIPs is also common; (3) the volume of LGE correlates with the pulmonary hemodynamics, and with CMR and echocardiographic parameters of the right ventricle; and (4) the paradoxical IVS motion index is the only independent explanatory variable of LGE volume at VIPs in PH.Recent studies have indicated significant associations between LGE at VIPs and various parameters of PH. For example, mean PAP, RV volume and RV mass index have been reported to positively correlate with LGE volume. Abnormal IVS motion has been documented by M-mode Contrast pooling at VIPs was suggested as the primary mechanism of LGE in PH in a recent autopsy report. Freed et al. recently reported that the presence of myocardial LGE at VIPs is a marker of poor prognosis in PH One limitation of the present study is the inclusion of PH patients with diverse etiologies. Some underlying diseases of PH are known to affect the myocardium; thus, the experimental results might have been affected by the different underlying illnesses among the patient population. Also, in the subgroup analysis, the number of PH patients on different treatment regimens was small. Thus, any confounding influence of the specific treatment on the experimental results cannot be excluded. Also, the present study analyzed patients with relatively less advanced PH, which makes it difficult to extrapolate the findings of this study to those with advanced PH. Further, IVS motion was evaluated using a methodology that is not commonly used. However, speckle-tracking echocardiography is an established method of regional analysis of IVS motion. Indeed, the calculated intra- and inter-observer reproducibility of the paradoxical IVS motion index were favorably high. Lastly, we did not conduct pathological observations in the present study. Thus, further clinicopathological studies are warranted to clarify the true mechanisms of LGE at VIPs in PH.In conclusion, the present CMR study of PH demonstrated an independent association of LGE at VIPs with early systolic paradoxical IVS motion, but not with indices of pulmonary hemodynamics and RV morphology. This suggests that LGE at VIPs is a hallmark of altered IVS motion but not of PH per se. The clinical relevance of this distinct CMR finding must be clarified in future long-term studies.


Sporisorium scitaminea, which should have massively increased the amount of data available for transcriptome profile analysis. After mapping to sugarcane EST databases in NCBI, we obtained 2015 differentially expressed genes, of which 1125 were upregulated and 890 downregulated by infection. Gene ontology (GO) analysis revealed that the differentially expressed genes involve in many cellular processes. Pathway analysis revealed that metabolic pathways and ribosome function are significantly affected, where upregulation of expression dominates over downregulation. Differential expression of three candidate genes involved in MAP kinase signaling pathway, ScBAK1 (GenBank Accession number: KC857629), ScMapkk (GenBank Accession number: KC857627), and ScGloI (GenBank Accession number: KC857628), was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Real-time quantitative PCR (qRT-PCR) analysis concluded that the expression of these genes were all up-regulated after the infection of S. scitaminea and may play a role in pathogen response in sugarcane. The present study provides insights into the molecular mechanism of sugarcane defense to S. scitaminea infection, leading to a more comprehensive understanding of sugarcane-smut interaction.To understand the molecular basis of sugarcane-smut interaction, it is important to identify sugarcane genes that respond to the pathogen attack. High-throughput tag-sequencing (tag-seq) analysis by Solexa technology was performed on sugarcane infected with  Sporisorium scitaminea, is an important sugarcane fungal disease worldwide and is also one of the most important sugarcane diseases in China [Sugarcane is the most important sugar crop and accounts for more than 90 percent of total sugar production in China . Sugarcain China . It was in China . The losin China , 4. Nowain China . Becausein China , 7.S. scitaminea. Heinze et al. [S. scitamineum infection. Borrás-Hidalgo et al. [S. scitaminea, among which 52 were upregulated and 10 were downregulated. LaO et al. [S. scitaminea. Que et al. [S. scitamineum. This is the first report of proteomic investigation of sugarcane exposed to S. scitamineum.Cultivation of resistant varieties is the most effective disease-control measure , 8. In oe et al.  conducteo et al.  obtainedO et al.  investigO et al.  obtainedO et al. , 136 trae et al.  applied As discussed above, the mechanism of sugarcane smut defense has been studied using various techniques. However, sugarcane is a highly heterozygous crop and there is no genomic resource for this genus, so more attention is needed to establish the molecular interactions between sugarcane and pathogen. Differentially expressed genes can be investigated using a multitude of methods, such as cDNA microarray, cDNA-AFLP, RDA, SAGE, and SSH –17. HoweS. scitaminea. Yacheng 05–179, a highly resistant sugarcane genotype, was used as plant material. RNA expression profile sequencing analysis of two samples, before and after sugarcane smut fungus inoculation, was then conducted by Solexa sequencing. This study aims to discover the pathogenesis-related differentially expressed genes and further understand the interaction mechanisms between sugarcane and S. scitaminea at the molecular level.The application of Solexa technology for the identification of differentially expressed genes associated with the molecular mechanism of interaction between the sugarcane-smut was therefore explored. In the present study, the Illumina Solexa sequencing technology was used to reveal the response of sugarcane to infection by S. scitaminea) was collected from Liucheng 03–182 plants and stored at 4°C.The sugarcane genotypes, Yacheng 05–179 and Liucheng 03–182, were chosen as smut-resistant and smut-susceptible plant material, respectively. These two sugarcane genotypes were provided by the Key Lab of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture . Sugarcane smut fungus . Both RNA samples of Yacheng 05–179 and Liucheng 03–182 were used for Real-time quantitative PCR at time point 12, 24, 36, 48, and 72 hours.Total RNA was isolated using the TRIzol reagent according to the manufacturer's instructions (Invitrogen). Dried RNA samples were dissolved in diethylpyrocarbonate-treated Hμg total RNA, and then the first and second strand cDNA was synthesized. The bead-bound cDNA was subsequently digested with restriction enzyme Nla III, which recognized and cut off the CATG sites. The 3′ cDNA fragments attached to the oligo (dT) beads were washed away, and then 5′ cDNA fragments were ligated to the Illumina Adaptor 1, which contained a recognition site for the endonuclease Mme I for cutting 17 bp downstream of the recognition site (CATG), producing tags with adaptor 1. After removing 3′ fragments with magnetic beads precipitation, Illumina adaptor 2 is ligated to the 3′ ends of tags, acquiring tags with different adaptors of both ends to form a tag library. After 15 cycles of linear PCR amplification, 95 bp fragments are purified by 6% TBE PAGE Gel electrophoresis. The two constructed tag libraries were fixed onto the Illumina Sequencing Chip (flowcell) for cluster generation through situ amplification and were deep-sequenced using Illumina Genome Analyzer. After image analysis, base calling, and quality calibration, the raw data was produced [The gene expression libraries were prepared using Illumina Gene Expression Sample Prep Kit according to the manufacturer's instructions. Specified Experimental Process, use magnetic oligo (dT) beads adsorption to purify mRNA from 6 produced , 26.The expressions of three candidate genes in MAP kinase signaling pathway were determined by RT-PCR and Real-time quantitative PCR (qRT-PCR). Gene-specific primers were designed according to the gene sequences using the Primer Premier 5.0 software and were synthesized commercially .μL reaction mix contained 2.5 μL 10 × PCR Buffer (plus Mg2+), 2.5 μL deoxynucleotide triphosphates (dNTPs) (2.5 mM), 1.0 μL first-strand cDNA, 1.0 μL each of forward and reverse primers (10 μM), and 0.125 μL Ex-Taq enzyme (5 U/μL). The ddH2O was added as supplement. The PCR amplification program consisted of predenaturation for 4 min at 94°C; denaturation for 1 min at 94°C, annealing for 1 min at annealing temperature, and extension for 1.5 min at 72°C for 35 cycles; and a final extension for 10 min at 72°C. Then, 5 μL of the product was electrophoresed on 1.0% agarose gel and viewed under UV light.The first-strand cDNA was synthesized from total RNA using PrimeScript 1st strand cDNA synthesis kit (Takara). The primers for RT-PCR confirmation are listed in 25S rRNA (BQ536525) gene was chosen as the internal control in the qRT-PCR analysis [μL samples were run on the ABI PRISM7500 real time PCR System using SYBR Green PCR Master Mix (Applied Biosystems). The qRT-PCR reaction conditions were held at 50°C for 2 min, predenatured at 95°C for 10 min, and then kept at 94°C for 15 s and at 60°C for 60 s for 40 cycles. Three replicas were set for each sample. When the reaction was completed and the melting curve was analyzed. The 2−△△CT method was adopted to analyze the qRT-PCR results [The analysis . The fir rRNA BQ5525 gene S. scitaminea inoculation, respectively . After comparing with sugarcane EST database, 61.36% and 57.88% of clean tags, and 21.43% and 18.81% of distinct clean tags could be matched exactly with the reference sequences in two samples, respectively. Then, there are 639,019 and 610,306 clean tags for inoculation and mock libraries are uniquely mapped to database, while the number of distinct clean tags are 72812 and 64,815 (http://dx.doi.org/10.1155/2013/298920), respectively. However, because of incomplete sequences, there are a large proportion total clean tags (about 30%) and distinct tags (about 65%) which could not be aligned to the reference sequences .Matching the tags to genes is an important step to annotate sequences and can reveal the molecular events behind the gene expression . In thisHeterogeneity and redundancy are two significant characteristics of mRNA expression . We analS. scitaminea inoculation was more than or equal to twofold (|log2Ratio| ≥ 1) and (2) if the false discovery rate value of the single sample was less than 0.001 (FDR ≤ 0.001) [2Ratio| ≥ 5, and 3 upexpressed genes in the MAP kinase signaling pathway  if the average fold change of gene expression before and after ≤ 0.001) . By usin pathway .http://www.ncbi.nlm.nih.gov/). As shown in ScBAK1, ScMapkk, and ScGloI, which belongs to MAP kinase signaling pathway, were upregulated (Gene ontology (GO) analysis was performed by mapping each differentially expressed gene on to the reference sugarcane EST database in NCBI (egulated .ScBAK1 (GenBank Accession number: KC857629), ScMapkk (GenBank Accession number: KC857627), and ScGloI (GenBank Accession number: KC857628) were obtained from sugarcane based on the bud full-length cDNA library, of which the length is 1,291 bp, 1,302 bp, and 1,091 bp, with ORF length of 1,004 bp, 1,068 bp, and 885 bp, respectively , and bioinformatics analysis of the corresponding differentially expressed genes has to only rely on sugarcane EST databases. Therefore, in the absence of corresponding sequenced genome information as a reference, many of the differentially expressed genes cannot be functionally annotated. However, the genomes of sorghum, maize and rice can act as reference information. According to sugarcane UniGenes identified and annotated by RNA-Seq and sorghum, maize and rice reference genome, we hope to establish a platform for future genetic and functional genomic research in sugarcane. In the present study, a total of 4,847,568 and 4,883,691 21 bp length clean tags that corresponded to 446,284 and 423,464 distinct tags for Yacheng 05–179 before and after  reports –34. ThisS. scitaminea and molecular changes of the host sugarcane variety makes the interactions between sugarcane, the environment, and S. scitaminea more complicated. Previous studies on sugarcane-smut interaction revealed that sugarcane genes encoding proteins homologous to chitinases as well as transcripts related to the pathways of both phenylpropanoids and flavonoids were shown to be involved in the sugarcane resistance after 7 days of S. scitamineum infection [Differential gene expression during sugarcane-smut interaction is likely to be induced following pathogen challenge, which leads to up or downregulation of gene expression , 22–24. nfection . Sugarcanfection . Two monScMapkk and ScGloI genes could be verified in further smut-resistant and smut-susceptible genotypes with different genetic backgrounds. This is important for sugarcane smut resistance breeding because the interaction of sugarcane, smut pathogen and environment leads to the instability of the resistance phenotype tested by field-inoculation. In addition, only 48 candidate genes were screened in this study, and there are still many differentially expressed genes to be tested.The study provides the potential to develop a molecular marker for smut resistance selection if the up-regulation of expression of S. scitaminea infection. When subjected to biotic and abiotic stress, the MAP kinase signaling pathway is activated [ScBAK1, ScMapkk, and ScGloI were up-regulated in both smut-resistant genotype and smut-susceptible genotype, but the expression in smut-resistant genotype was greater than that in the smut-susceptible genotype especially for ScMapkk and ScGloI genes. The expression of these three genes may play a role in defending against infection by pathogens in sugarcane. When sugarcane was infected by the smut pathogen, the expression of key enzymes were up-regulated, leading to the activation of MAP kinase signaling pathway and the triggering of the genes involved with plant defence (Plants possess integrated signaling networks that mediate the perception and responses to biotic and abiotic stresses which govern plant growth and development. MAP kinase signaling pathway is a conserved signaling pathway common in plants. The present results permit us to hypothesize about the role of the MAP kinase pathway on the sugarcane early response against the ctivated –40 and pctivated , 42. The defence , which wIn conclusion, the usefulness of the Solexa sequencing in identifying genes related to sugarcane smut defense has been successfully demonstrated in this study. However, most of the molecular mechanisms of sugarcane-smut interaction are as yet unknown. More genes related to sugarcane defense and their expression profiles in response to smut infection should be analyzed further. The present study provides a Solexa sequencing platform for gene expression research on this crop and also a reference for studying the molecular mechanism in nonmodel organisms.Additional File 1: Statistics of clean tag alignment.pdfAdditional File 2: Distribution of clean tag copy number.pdfAdditional File 3: 2015 differentially expressed genes identified in sugarcane.xlsAdditional File 4: Gene ontology analysis for differentially expressed genes.xlsAdditional File 5: Pathway annotation of 305 differently expressed genes in sugarcane.xlsAdditional File 6: Raw data of qRT-PCR for ScBAK1, ScMapkk and ScGloI genes.xlsClick here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Herbs and spices are very important and useful as therapeutic agent against many pathological infections. Increasing multidrug resistance of pathogens forces to find alternative compounds for treatment of infectious diseases.Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae, Shigella sonnei, Staphylococcusepidermidis and Salmonella typhi. The antibacterial activity was determined by disc diffusion method.In the present study the antimicrobial potency of garlic and ginger has been investigated against eight local clinical bacterial isolates. Three types of extracts of each garlic and ginger including aqueous extract, methanol extract and ethanol extract had been assayed separately against drug resistant All tested bacterial strains were most susceptible to the garlic aqueous extract and showed poor susceptibility to the ginger aqueous extract. The (minimum inhibitory concentration) MIC of different bacterial species varied from 0.05 mg/ml to 1.0 mg/ml.In the light of several socioeconomic factors of Pakistan mainly poverty and poor hygienic condition, present study encourages the use of spices as alternative or supplementary medicine to reduce the burden of high cost, side effects and progressively increasing drug resistance of pathogens. Microbial pathogenecity and other infectious diseases have been controlled by use of commercially available antimicrobial drugs since last many years. Tremendous use of antibiotics has developed multiple drug resistance (MDR) in many bacterial pathogens. The increasing drug resistance is the main hindrance in successful treatment of infectious diseases and to the control of microbial pathogenecity . SimilarThe antimicrobial activity of spices is due to specific phytochemicals or essential oils . The maiin vitro antimicrobial activity of some local spices of Pakistan, that are routinely used in food, has been investigated against clinically important bacterial pathogens.In the present study, Allium sativum) and ginger  used in the present study were purchased from the local market of Lahore, Pakistan.Garlic  under sterile conditions and placed in shaking water bath at 37°C for 16 hours. The bacterial cells were harvested at 3000 rpm for 15 minutes at 4°C, washed twice with phosphate buffer saline (pH 7.4) and resuspended in LB broth. The inoculum concentration was adjusted to 107 CFU of each test bacterial strain culture and evenly spread on entire surface of each plate. The sterile discs (5 mm diameter) were dipped aseptically in different extracts for one minute and placed over nutrient agar plates seeded with bacterial culture. The plates were left at ambient temperature for 15 minutes and then incubated at 37°C for 16 hours and observed for zone of inhibition. The diameter of inhibition zones was measured in milimeters. Antimicrobial assay was performed in triplicate with each bacterial strain.The antimicrobial assay of spices was performed by disc diffusion method as described by Kirby-Bauer . All the7 CFU of each bacterial cultures separately. Plates were placed at 37°C for 16 hours. The zone of inhibition in each case was measured as the diameter of the clearing zones and results were recorded. Each experiment was performed in triplicate.MIC of different garlic and ginger extracts was determined by the method described by Natta et al  after miValues are mean of ± SD (standard deviation) of three replicates.Use of garlic and ginger as a natural supplement is considered healthy choice for the treatment of cardiovascular diseases ,20, hypeE. coli and Shigella. Garlic methanol extract was least effective against all tested bacteria. The antimicrobial activity shown by garlic extracts in this study agrees with the findings of others [In the present study antibacterial effect of garlic and ginger was evaluated by disc diffusion method. The results indicated that different extracts of spices have broad spectrum antibacterial activity with variable degree of sensitivity of tested bacterial species toward these extracts. The controls did not show any antimicrobial activity. The data presented in Table f others ,14,29-31E. coli and Shigella were also more susceptible to the ginger extracts. E. coli showed maximum susceptibility to the ginger ethanol extracts while Shigella showed maximum susceptibility to both ginger methanol and ethanol extract. The results of antimicrobial effect of ginger in the study are in accordance with most of the reports published regarding ginger antimicrobial activity [The results given in Table activity -36. The activity . The resactivity , which eactivity  has repoE. coli, GiEE> GiME> GaAE> GiAE> GaME> GaE; 2) P. aeruginosa, GaAE> GiEE> GiME> GaEE> GiAE>GaME; 3) Bacillus subtilis, GaAE> GiEE> GaEE> GiAE>GaME> GiME; 4) Shigella, GiEE, GiME> GaEE> aAE> GiAE> GaME; 5) S.aureus, GaAE> GiME> GiEE, GiAE> GaEE> GaME; 6) K. pneumoniae, GaAE> GaEE> GiME> GiEE, GiAE, GaME; 7) S.epidermidis, GaAE> GiEE> GiAE> GiME> GaEE> GaME; 8) S.typhi, GaAE> GiME> GiEE> GiAE> GaEE> GaME.The order of antibacterial activity of different garlic and ginger extracts against tested clinical isolates of pathogenic bacteria was as follow: 1) E. coli, P. aeruginosa, B. subtilis, S. aureus, K. pneumoniae, S. epidermidis and S. typhi were 0.1 mg/ml, 0.09 mg/ml, 0.1 mg/ml, 0.2 mg/ml, 0.2 mg/ml, 0.09 mg/ml and 0.02 mg/ml respectively with garlic aqueous extract except Shigella which showed lowest MIC value (0.07 mg/ml) with garlic methanol extract. Ethanol and methanol extract of ginger had lower MIC in comparison to the ginger aqueous extract against tested bacterial strains , P. aeruginosa (0.4 mg/ml), B. subtilis (0.3 mg/ml) and S. epidermidis (0.05 mg/ml) was observed with ginger methanol extract while for S. aureus (0.3 mg/ml), K. pneumoniae (0.05 mg/ml) and S. typhi (0.08 mg/ml) was observed with ginger ethanol extract. The ginger methanol and ethanol extract showed lowest and same MIC value for Shigella (0.05 mg/ml).The minimum inhibitory concentration (MIC) was determined by making the dilutions of different extracts of garlic and ginger ranging from 100 mg/ml to 0.01 mg/ml. The MIC values of different garlic and ginger extracts are summarized in FigureS.epidermidis>S.aureus>B. subtilis>P. aeruginosa>K. pneumoniae = S.typhi>E. coli = Shigella. It was interesting to note that clinical isolates, both Gram negative and Gram positive bacteria were sensitive to all tested extracts of garlic and ginger but Gram positive bacteria were more sensitive than Gram negative bacteria. This result is in accordance with the findings of Chandarana [The decreasing susceptibility of tested pathogenic bacteria was observed in this order: andarana ; Onyeagbandarana  and de-Sandarana .It is established in the study that spices reduce and inhibit the growth of food pathogens therefore the use of spices would decrease the chances of food poisoning and increase the food shelf life. Several socioeconomic factors are major cause of miserable health condition of poor people of Pakistan which includes; poverty, unhygienic conditions, overcrowding, contamination of food /water by poor sanitary practices, limited awareness of seriousness of foodborne diseases and importance of hygiene. While living in such conditions, use of spices (garlic/ginger) in diet can reduce the risk of food contamination, protect the consumer from different foodborne diseases, improve their health status and combat with the foodborne diseases by using small quantity of spices (garlic/ginger) in diet. In this study heat effect on antimicrobial activity of garlic and ginger was not checked as it is already reported that antimicrobial activity of garlic is affected by heating at 100°C for 30–60 minutes . Therefoin vivo experiments is required to be carried for low cost treatment with few side effects and for prevention of recurrent infection.In conclusion, the results of present study have provided the justification for therapeutic potential of spices. The practice of using spices as supplementary or alternative medicine in developing countries like Pakistan will not reduce only the clinical burden of drug resistance development but also the side effects and cost of the treatment with allopathic medicine. Further clinical evaluation of spices in The authors declare that they have no competing interests.All authors equally participated in designing experiments, acquisition, analysis and interpretation of data. Prof. Amin Athar critical revise the manuscript and approved the final version of manuscript. All authors read and approved the final manuscript.