Clopidogrel in combination with aspirin is commonlyused for the prevention of thrombosis in patients with coronaryartery stents . Moreove–1 p.o.) and clopidogrel(75 mg d–1 p.o.), and 2 days later he developed diffusepetechiae that prompted evaluation. Physical examinationdid not reveal any signs of splenomegaly. Laboratory findingswere as follows: platelet count: 7 x 109 L–1; hemoglobin:13.4 g dL–1; leukocyte count: 4.7 x 109 L–1. Peripheralblood smear showed severe thrombocytopenia withoutmicroangiopathic changes. The lactate dehydrogenaselevel, prothrombin time, and activated partial thromboplastintime were normal. The diagnosis of clopidogrelassociatedautoimmune thrombocytopenic purpura wassuspected.A 63-year-old male presented with acute inferior myocardialinfarction, which was treated with primary stenting;heparin was administered periprocedurally. Thepatient was given aspirin (300 mg d9 L–1) and no new petechiae.According to the guidelines of The American Society ofHematology, bone marrow biopsy and aspiration were notperformed because bone marrow examination is not recommendedin cases of isolated thrombocytopenia highlysuspicious for the diagnoses of immune thrombocytopenicpurpura [9 L–1 while on oral steroid therapy Written informedconsent was obtained from the patient for publication.Clopidogrel and aspirin were discontinued at the timeof application. Intravenous methylprednisolone (1 g) wasgiven daily. A total of 2 units of platelets were transfusedin the first day of treatment. The patient responded in thethird day of high dose steroid therapy, with stabilizationof the platelet count , isolated thrombocytopenia,and autoimmune thrombocytopenia . AutoimmThe authors of this paper have no conflicts of interest,including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materialsincluded.


Streptococcus pyogenes exoproteins, many of which contribute to virulence, is regulated in response to nutrient availability. CodY is a transcriptional regulator that controls gene expression in response to amino acid availability. The purpose of this study was to identify differences in the expression of streptococcal exoproteins associated with deletion of the codY gene.The production of S. pyogenes to a codY mutant in the post-exponential phase of growth. We used both one and two-dimensional gel electrophoresis to separate exoproteins. Proteins that were significantly different in abundance upon repeated analysis were identified with tandem mass spectrometry. The production of the secreted cysteine protease SpeB, a secreted chromosomally encoded nuclease (SdaB), and a putative adhesion factor (Spy49_0549) were more abundant in supernatant fluids obtained from the codY mutant. In addition, hyaluronidase (HylA), CAMP factor (Cfa), a prophage encoded nuclease (Spd-3), and an uncharacterized extracellular protein (Spy49_0015) were less abundant in supernatant fluids obtained from the codY mutant strain. Enzymatic assays showed greater DNase activity in culture supernatants isolated in the post-exponential phase of growth from the codY mutant strain compared to the wild-type strain. Because extracellular nucleases and proteases can influence biofilm formation, we also measured the ability of the strains to form biofilms during growth with both rich medium  and chemically defined media (CDM). No difference was observed with rich media but with CDM the biofilms formed by the codY mutant strain had less biomass compared to the wild-type strain.We compared the secreted proteins produced by wild-type S. pyogenes exoproteins, including DNases, a protease, and hylauronidase, which together may alleviate starvation by promoting dissemination of the pathogen to nutrient rich environments and by hydrolysis of host macromolecules.Overall, the results indicate that CodY alters the abundance of a select group of  Streptococcus pyogenes is thought to be responsible for more than 500,000 deaths worldwide each year [S. pyogenes biofilms, which are composed of both proteins and extracellular DNA [ach year . Pathogeach year . Others ach year ; a secreach year -6 and otach year ,8. Proteach year ,10. Finaular DNA .S. pyogenes is auxotrophic for most amino acids, the pathogen's ability to respond to amino acid depletion is likely to be critical for survival within the human host. The response involves both the relA-dependent pathway mediated by accumulation of (p)ppGpp [relA-independent pathway [bsfF, which is a small RNA in B. subtilis[The regulation of exoprotein production is complex and involves a variety of transcriptional regulatory proteins, many of which are influenced by the availability of various metabolic substrates -14. Beca(p)ppGpp  and a re pathway ,17, medi pathway . CodY is pathway ,20. The  pathway  and bsfF subtilis.S. pyogenes, CodY controls the expression of genes involved in the response to nutritional stress, including genes encoding exoproteins. The transcript levels of 34 genes were previously compared between a wild-type strain of S. pyogenes and a codY mutant derivative by using quantitative reverse transcriptase PCR (qRT-PCR) [grabsagAsdaB/mf-1, and speB) was greater in the wild-type strain compared to the mutant strain, while the expression of the remaining seven was less (ngaprtSsclscpAskaslospeH). Subsequently, by using DNA microarrays, inactivation of codY in S. pyogenes was found to alter the transcription of approximately 17% of genes in the chromosome, including several that encoded exoproteins [In qRT-PCR) . Eleven proteins . Togetheproteins ,23.S. pyogenes to a codY mutant strain to identify potential differences derived either at the transcriptional or post-transcriptional level. The results confirmed, at the protein level, several differences in expression previously predicted by transcript analyses and identified additional exoproteins with altered abundance following the deletion of codY.The purpose of this study was to compare the exoproteins of a wild-type strain of codY mutant and a wild-type strain of S. pyogenes, the strains were grown to the stationary phase of growth and culture supernatant proteins (CSPs) were analysed by using SDS-PAGE gel electrophoresis. There was no difference in either the growth rate or growth yield of the two strains . In some instances proteins were differentially expressed in the representative gels shown in Figure To better resolve the exoproteins 2-DE was used and images of representative gels are shown in Figure ed Table . DifferecodY mutant strain compared to the wild-type strain , also known as mitogenic factor 1 (Mf-1), is encoded within the bacterial chromosome. The other secreted nuclease, Spd-3, is encoded within a prophage . Three SS. pyogenes are dispersed by the addition of exogenous proteases and DNases, indicating the matrix is composed of both protein and DNA [codY altered biofilm formation of S. pyogenes. Static biofilm assays showed that deletion of codY consistently reduced biofilm formation compared to the parental strain when cultured with CDM  was more abundant in supernatants from the n Figure , Table 1f growth . The pref growth . It is pormation ,30 and tmpetence -31. We ncodY mutant strain compared to the wild-type strain  were less abundant in CSPs obtained from the nscripts . Cfa is S. pyogenes exoproteins contribute substantially to interactions with the human host. Production is regulated by several, apparently redundant, transcriptional regulatory circuits working together to control expression. We used a proteomics approach to characterize the contribution of CodY to the regulation of S. pyogenes exoproteins. The purposes of this study were to clarify how previously identified differences in transcript levels between a wild-type and codY mutant strain are manifest at the protein level and to determine if codY deletion is associated with additional differences in the exoproteome due to post-transcriptional effects. The results confirmed, at the protein level, previously identified differences between the strains in the production of SpeB, Cfa, and SdaB. Moreover, additional exoproteins were discovered to be regulated by CodY, including the virulence associated secreted nuclease Spd-3, which is encoded by a prophage, a putative zinc binding transport protein AdcA, and HylA. Overall, the results contribute to defining the S. pyogenes exoproteome and the role CodY plays in determining its composition.cistrans isomerases  and a codY mutant were previously described [codY open reading frame was amplified by PCR and cloned into pFW6 such that the fragments flanked the aad9 gene, which confers resistance to spectinomycin [codY gene and substitution with the aad9 gene [codY mutant derivatives were inoculated from frozen stocks onto agar plates prepared with Todd-Hewitt supplemented with 0.2% yeast extract (THY) and incubated overnight at 37°C in 5% CO2. The bacteria were then suspended to an A600 of 0.08 in 40 ml chemically defined medium (CDM) [2.escribed . To consinomycin . After lad9 gene . Wild-tyum (CDM)  and incucodY mutant strain were separated and the gels were analyzed with PDQuest software (Biorad). The abundance of proteins isolated with 2-DE was determined by summing the values of the pixels comprising the protein spot. The mean abundance of each protein was then determined from the three biological replicates obtained for each strain. Gels were normalized based on the sum of all protein spots detected in each sample. The CSPs were analysed for the presence of protease activity by using QuantiCleave Protease Assay Kit, as described by the manufacturer . As a negative control, an NZ131 speB mutant strain was used. Standard curves were prepared with trypsin, as described by the manufacturer and purified SpeB protease was used as a positive control.Culture supernatant proteins were isolated from stationary phase cultures by trichloroacetic acid and acetone precipitation, as previously described . Protein1)-Fibrinopeptide B human with a RMS residual of 3.495 e-3 amu or 7.722 e0 ppm. Parent mass (MS) and fragment mass (MS/MS) peak ranges were 400–1500 Da and 65–1500 Da, respectively.Proteins of interest were excised from the SDS-PAGE gels with a robotic spot cutter (BioRad). The excised bands and spots were reduced with dithiothreitol , alkylated with iodoacetamide (Sigma), and digested with sequencing grade trypsin (Promega) overnight at 37°C. The tryptic peptides were extracted by using 1% formic acid/2% acetonitrile in water followed by a second extraction using 50% acetonitrile/50% water. The extracts were concentrated with a SpeedVac centrifuge (Thermo Savant), dissolved in a solution of water/acetonitrile/formic acid (97/3/0.1%), and injected into a liquid chromatography instrument . The peptides were desalted and concentrated online through an 180 μm X 20 mm, 5 μm Symmetry C18 nanoAcquity UPLC trap column (Waters) at a flow of 20 μL/min., with 99% solution A2  and 1% solution B2  for 20 min. The peptides were separated online in the second dimension through a BEH130C18 1.7 μm, 100 μm X 100 mm nanoAcquity UPLC column. The standard solvent gradient used was: 0 to 2 min, 3% B2 isocratic; 2 to 40 min, 3–85% B2 linear, at a flow rate of 400 nL/min. for 60 min. The eluted ions were analyzed by one full precursor MS scan (400–1500 m/z) followed by four MS/MS scans of the most abundant ions detected in the precursor MS scan while operating under dynamic exclusion or direct data acquisition system. Spectra were obtained in the positive ion mode with a nano ESI-Q-Tof micro mass spectrometer , deconvoluted, and analyzed using the MassLynx software 4.1 . A peak list (PKL format) was generated to identify +1 or multiple charged precursor ions from the mass spectrometry data file. The instrument was calibrated in MS/MS mode using 500 fmole of  in MS/MS ion search mode  were applied to conduct peptide matches (peptide masses and sequence tags) and protein searches against NCBInr v20110707  using taxonomy filter S. pyogenes . The following parameters were set for the search: carbamidomethyl (C) on cysteine was set as fixed; variable modifications included asparagine and glutamine deamidation and methionine oxidation. One missed cleavage was allowed; monoisotopic masses were counted; the precursor peptide mass tolerance was set at 2 Da; fragment mass tolerance was 0.3 Da. The MS/MS spectra were searched with MASCOT using a 95% confidence interval (C.I.% ) threshold (p < 0.05), with which a minimum score of 36 was used for peptide identification (identity or extensive homology). The protein redundancy that appeared at the database under different gi and accession numbers were limited to S. pyogenes with the first priority assigned to NZ131. All proteins identified were found within these domains.Mascot server v2.3.0 and Mascot Daemon Toolbox v2.3.0  and 200 μl of 0.1% (wt/vol) crystal violet was added to each well. After 30 min., the wells were washed twice with 200 μl of sterile deionized water to remove unbound crystal violet. The remaining crystal violet was dissolved in 200 μl of 95% ethanol and the absorbance was measured at 600 nm. Four wells were used for each strain and the average value determined. The experiment was repeated four times and the mean ± standard error of the mean is reported. The Student's t-test was used to determine if the mean values of biofilm formation differed between the strains.Biofilm formation on polystyrene microtiter plates  was done essentially as previously described . BrieflyEJM isolated and separated exoproteins, analyzed 2-DE gels, and drafted the manuscript. EAC identified proteins with mass spectrometry and co-authored the manuscript. HM constructed the strains and participated in the design of the study. MSC conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.Table S1. Tandem mass spectrometry results of proteins excised from SDS-PAGE gel (Figure l Figure .Click here for fileTable S2. Peptide characteristics used to identify proteins excised from SDS-PAGE gel (Figure Click here for fileTable S3. Tandem mass spectrometry results of proteins excised from 2-DE gel (Figure Click here for fileTable S4. Peptide characteristics used to identify proteins excised from 2-DE gel (Figure l Figure .Click here for file


Hyperkalemia is a commonly encountered electrolyte abnormality that can significantly alter normal cardiac conduction. Potentially lethal dysrhythmias associated with hyperkalemia include complete heart block and Mobitz Type II second-degree AV block. We report a unique case of Mobitz Type 1 second-degree atrioventricular (AV) block, known commonly as Wenckebach, due to hyperkalemia. The patient's symptoms and electrocardiogram (ECG) evidence of Wenckebach block resolved with lowering of serum potassium levels, with subsequent ECG showing first-degree AV block. This paper highlights an infrequently reported dysrhythmia associated with hyperkalemia that emergency physicians should be familiar with. Hyperkalemia is a common and potentially life-threatening electrolyte abnormality . While tDespite the range of heart blocks associated with hyperkalemia, Mobitz Type 1 second-degree AV block or Wenckebach is infrequently reported in the medical literature. Hyperkalemia-induced Wenckebach in the setting of a pacemaker, termed pacemaker exit block, has been previously reported . In the A 68-year-old man was brought in by ambulance after his wife called EMS upon finding her husband sitting on the toilet and complaining of head and neck pain. Per the paramedic report, the patient was found seated on the toilet, cool, pale, diaphoretic, and confused. He had no palpable radial pulses. He was found to have a heart rate of 30 beats per minute (bpm) on the field cardiac monitor. He received 0.5 mg of atropine intravenously (IV) in the field with some improvement in his heart rate and mental status and was then paced transcutaneously to a heart rate of 80 bpm. EMS also placed him on 15 liters of oxygen by non-rebreather (NRB) mask, documented a blood glucose level of 499 mg/dL, and initiated an intravenous (IV) bolus of 500 mL of normal saline. Repeat vital signs immediately prior to arrival in the Emergency Department (ED) were notable for a systolic blood pressure of 90 mmHg, pulse 80 bpm, respirations 18 breaths per minute, and pulse oximetry of 100% on 15 L NRB mask. His cardiac rhythm on the monitor demonstrated a paced rhythm at 80 bpm without ectopy, and he was transported without any further event. Upon arrival to the ED, his mental status had improved. He was awake and answered questions appropriately. He reported a history of diabetes mellitus type II, hypertension, hyperlipidemia, chronic renal insufficiency, and prostate cancer. He described one hospitalization several months prior, in Mexico, for hyperkalemia. He did not know the details of that hospitalization but denied ever undergoing hemodialysis. His medications were listed as atenolol, diltiazem, hydrochlorothiazide, losartan, and metformin. He denied any recent medication changes, overdoses, or chest pain. He had not taken his medications that morning. The external pacer was stopped, and he was found to have a heart rate of 45 bpm and a manual blood pressure of 90/50 mmHg. His physical examination was remarkable only for bradycardia and cool lower extremities with a delayed capillary refill. He did not have any increased work of breathing, abnormal breath sounds, irregular heart sounds, jugular venous distention, or lower extremity edema. His initial ECG showed a calculated ventricular rate of 36 bpm and a QRS duration of 76 milliseconds (ms). The providers further interpreted this ECG to show a prolonged PR interval and peaked T waves . His rhyA rapid serum electrolyte level was obtained using an i-Stat device. This showed serum potassium level of 8.0 mmol/L [3.6–5.0]. His serum chemistries were received shortly thereafter and were significant for sodium 130 mmol/L [137–145], potassium 8.3 mmol/L, chloride 102 mmol/L [98–107], CO2 21 mmol/L [22–30], BUN 28 mg/dL [7–21], creatinine 1.7 mg/dL [0.5–1.4], glucose 528 mg/dL [65–110], magnesium 1.6 mg/dl [1.6–2.3], and phosphorus 6.0 mg/dL [2.5–4.5]. The patient's hyperkalemia was treated with calcium gluconate, insulin, kayexalate, and albuterol nebulizer treatments. He was also given an IV bolus of two liters of normal saline. Four hours later, his serum potassium level was 6.0 mmol/L. The patient was admitted to the internal medicine service and nephrology was consulted. His hyperkalemia was thought to be secondary to renal tubular acidosis type 4. His cardiac enzymes were negative for ischemia. At the time of discharge his potassium level was 3.9 mmol/L and his creatinine was 1.1 mg/dL. The patient's beta-blocker and calcium-channel blocker medications were held throughout his hospital stay. His ECG immediately prior to discharge demonstrated resolution of the previously seen Wenckebach pattern, however, with a persistently prolonged PR interval of 232 ms consistent with type 1 AV block, and normal T wave morphology . His disThis case demonstrates an atypical presentation of hyperkalemia-induced Wenckebach that resolved to first-degree AV block with lowering of the serum potassium levels. Although electrophysiologic studies at the AV node and His-Purkinje system would be needed to confirm our theory that hyperkalemia induced the ECG pattern of Wenckebach in this patient, the absence of any other explanation, such as medication overdose, myopericarditis, rheumatic fever, or acute myocardial ischemia, makes it seem very unlikely that this was the case. The resolution of Wenckebach to first-degree AV block with aggressive treatment of hyperkalemia also suggests that this electrolyte disturbance produced the higher-grade AV block. The AV node is known to be susceptible to hyperkalemia, producing the classic prolonged PR interval and QRS widening seen often in the setting of hyperkalemia. The development of advanced heart blocks (second- and third-degree AV blocks) is generally found only in patients with pre-existing heart failure, conduction abnormalities, or other cardiac disease . Pre-exiIn our patient, in the absence of acute myocardial infarction, we propose that Wenckebach resulted due to the patient's AV node's susceptibility to hyperkalemia, which was likely heightened by the patient's use of both a beta-blocker and a calcium-channel-blocker (atenolol and diltiazem), as well as likely underlying AV node disease evidenced by his final ECG showing first-degree block. Hyperkalemia likely contributed to dampening the pacemaker function of the AV node and worsening underlying conduction disease, thereby producing Wenckebach. The previous documented cases of hyperkalemia-associated Wenckebach are interesting in that all cases occurred in settings of severe pre-existing cardiac disease. In the most dramatic cases, those of Wenckebach pacemaker exit block due to hyperkalemia, elevated potassium levels cause progressive latency in the generation of a QRS complex from the time of the pacemaker spike . HyperkaThe only other case of hyperkalemia-induced Wenckebach is reported in an 87-year-old female with a history of hypertension and congestive heart failure, whose baseline ECG demonstrated a right bundle branch block and a left anterior fascicular block . In the Hyperkalemia is a commonly encountered electrolyte abnormality that can produce life-threatening derangements in cardiac conduction. The ED physician should be aware of the range of dysrhythmias attributed to hyperkalemia—including Wenckebach and progressively higher grades of AV block—and should institute treatment rapidly to minimize patient morbidity and mortality.None of the authors of this paper have any financial or other conflicts of interest related to this submission.


Gokshuradi Yog (GY) is a polyherbal ayurvedic formulation used traditionally for several decades in India for the treatment of urolithiasis. The aim of the present study was to determine the underlying mechanism of GY action in the management of calcium oxalate urolithiasis. The effect of Gokshuradi polyherbal aqueous extracts (GPAEs) was studied on various biochemical parameters involved in calcium oxalate formation by employing in vitro and in vivo methods. GPAE exhibited significant antioxidant activity against 1, 1-diphenyl-2-picrylhydrazyl free radical and inhibited lipid peroxidation in the in vitro experiments. The rat model of urolithiasis induced by 0.75% ethylene glycol (EG) and 1% ammonium chloride (AC) in water caused polyuria, weight loss, impairment of renal function, and oxidative stress and decreased antioxidant enzyme activities in untreated control groups. However, GPAE-  treated groups caused diuresis accompanied by a saluretic effect and revealed significant increase in antioxidant enzyme activities along with decreased oxalate synthesizing biochemical parameters at higher doses. This study revealed the antiurolithic effect of GPAE mediated possibly through inhibiting biochemical parameters involved in calcium oxalate formation, along with its diuretic and antioxidant effects, hence supporting its use in the treatment of calcium oxalate urolithiasis. Calcium oxalate urolithiasis has become a rising problem in many countries due to geographical/genetic variations and modern lifestyles. People living in drought and arid area suffer more from intestinal hyper absorption of calcium oxalate leading to kidney stones. It is estimated that 12% of the world population is affected by kidney stones with a recurrence rate from 70 to 80% in males and from 47 to 60% in females .Gokshuradi polyherbal formulation (GPF) and is being practiced since many years in the management of kidney stones. In our preliminary clinical studies, GPF has revealed expulsion of kidney stones in naturally occurred urolithic patients which was confirmed by X-ray analysis conducted before and after four weeks of treatment [GPF consists of parts of five different medicinal plants that is, dry fruits of Tribulus terrestris L. seeds of Hygrophila spinosa T. Anders, and roots of Ricinnus communis L, Solanum anguivi Lamk. and Solanum surattense Burm. GPF decoction was prepared by mixing the fine powder of these individual parts at equal proportion as mentioned in the ayurvedic literature and then it was administered to the patients. The treatment modalities like surgery and drug therapy are practiced in the management of kidney stones but have some limitations. Surgical procedures like  ESWL have increased risk of stone recurrence and are also not affordable to poor sufferers. The armamentarium of therapeutic agents also does not have any effective drugs except for some diuretics like thiazides and furosemide. This warranted a search for new drug therapy which will be cost effective and can target multiple etiological risk factors in urolithiasis. Ayurveda, a classical Indian authentic literature, reported the traditional use of reatment . GPF conGPF are known to possess various pharmacological activities. In Ayurvedic medicine, the fruits of T. terrestris L. are recommended for the treatment of urinary disorders erectile dysfunction, and in traditional Chinese medicine, it has been used as an antihypertensive in coronary heart disease [H. spinosa T. Anders, another component of this formulation has been used in Ayurveda for various ailments like jaundice, hepatic obstruction, rheumatism, inflammation, pain, diuretic, and aphrodisiac and in the treatment of urinary disorders [R. communis stems have anticancer, antidiabetic, and antiprotozoal activity [Solanum anguivi (Lam.), also known as Solanum indicum sp. distichum (Schumach. and Thonn.), is cultivated for culinary purposes in many parts of Africa, India, and the Arab Peninsula. The fruits of this plant are used as nutritious vegetables on account of their high content of starch, calcium, vitamin A, ascorbic acid, and phosphate. Solanum xanthocarpum (SX) Schrad. & Wendl. (Family: Solanaceae) is commonly known as the Indian night shade or Yellow berried night shade found throughout India, mostly in dry places as a weed along roadsides and waste lands [Materia Medica, primarily as an expectorant and antipyretic. Various medicinal properties are attributed to it, particularly in the treatment of asthma, chronic cough, and catarrhal fever [dashamula (ten roots) of the Ayurveda [The individual component plants of  disease . It also disease , 7. H. sisorders , 9. Thisisorders , antitumisorders , haematiisorders , antinocisorders , hepatopisorders , 15, freisorders . R. commactivity . Solanumte lands . It has al fever . It is oAyurveda . In AyurAyurveda .GPF is well recognized in Indian traditional medicine as having antiurolithic effect, but no scientific data have been published supporting its underlying mechanism of action on calcium oxalate stones. Therefore this study was proposed to determine the therapeutic potential and mechanism of action of aqueous extracts from this formulation in the management of urolithiasis by employing in vitro and in vivo methods.Although, 2O2, trichloroacetic acid, 1,1,3,3,-tetraethoxypropane, and guanidine hydrochloride were obtained from Sigma Chemical Company, St. Louis, MO, USA. Reagents used for histological preparations were eosin spirit soluble, hematoxylin, and xylene from Himedia Chemical Limited, Bombay, India. Kits used in this study for the determination of calcium, magnesium, blood urea nitrogen (BUN), creatinine, superoxide dismutase, glutathione peroxidase, oxalate, and citrate were purchased from Spinreact, Germany, and Ambika diagnostics, India.All the chemicals used were of analytical grade. Ethylene glycol, thymol, reduced glutathione, 5-5′-dithiobis, 2-nitrobenzoic acid, thiobarbituric acid, furosemide, HPlants used for the formulation were collected from local forests of Nanded District, Maharashtra, India, and authenticated by experts. Voucher specimens were deposited in the herbarium of the School of Life Sciences, S.R.T.M. University, Maharashtra, India. Respective parts of each plant as mentioned previously were collected and then shade dried. Dried parts were cleansed of extraneous matter and then grounded to fine powder in a grinder.Didymocarpus pedicellata, Saxifraga ligulata, Rubia cordifolia, Cyperus scariosus, Achyranthes aspera, Onosma bracteatum, Vernonia cinerea, purified Shilajeet, and Hajrul Yahood Bhasma.Cystone polyherbal formulation from the Himalaya Drug Co., Bangalore, India was used as a standard drug in this study. The formulation contained GPAE was determined by free radical scavenging activity and lipid peroxidation inhibitory effects. 0.1 mM solution of DPPH radical in methanol was prepared, and 1 mL of this solution was added to 3 mL of GPAE at different concentrations to determine the free radical scavenging activity [Antioxidant effect of activity . SolutioLipid peroxidation inhibitory activity was determined in the isolated rat kidneys which were electrically homogenized in ice cold 50 mM phosphate buffer saline (PBS) and adjusted to pH 7.4. The homogenate was processed further, and lipid peroxidation inhibitory activity was determined by a reported method . The inhad libitum throughout the study except 24 h before and during 6 h of diuretic study, and while collecting 24 h urine samples, food was withdrawn.The animal care and handling was in accordance with the internationally accepted standard guidelines for use of animals, and the protocol was approved by institutional animal ethical committee (IAEC number P25/7-2011/GBR) at the National Institute of Nutrition, Hyderabad, India. Wistar rats (180–220 g) of either sex used for this study were sourced and housed at the National Centre for Laboratory Animal Sciences, Hyderabad, India, and kept in plastic cages (47 cm × 34 cm × 18 cm) with saw dust (renewed after every 48 h), under a controlled temperature of 23–25°C and 12 h light-dark cycle. Animals were given standard rat chow diet available at the centre. Animals had free access to food and water GPF was prepared by mixing equally the respective parts of each plant at a ratio of 1 : 1 : 1 : 1 : 1, respectively. In order to prepare the decoction for gastric gavages, 100 g of GPF and 500 mL of doubly distilled (dd) water were heated in an autoclave for 15 min at 121°C. After the boiling and sterilization procedure, the powder turned into a jelly-like paste which was dissolved in dd water to reach a final volume of 750 mL. Then, the solution was stored at 4°C for 7 days. Subsequently, the solution was poured out and centrifuged at a rate of 1,500 rpm for 10 min. Finally, the GPAE feeding solution containing 50 mg of formulation per milliliter was prepared. The same procedure was performed for Cystone to make 100 mg per milliliter of solution.Initially, GPAE was studied on Wistar rats of either sex (180–220 g) as described previously [GPAE  dissolved in saline. Subsequently, the animals were placed individually in metabolic and diuretic cages. The urine was collected, in graduated cylinders for 6 h at 1 h intervals. Total urine excreted out was collected and the volume was determined. The pH of the pooled urine from each animal was determined by using pH meter, Na+ and K+ concentrations on flame photometer, and Ca2+ concentration by using commercially available kits.The diuretic activity of eviously . AnimalsGPAE and Cystone was determined in an animal model of calcium oxalate urolithiasis as described by Atmani et al. [Antiurolithic activity of i et al. . The dosad libitum daily for 21 days .Group I rats, served as vehicle-treated control, received intraperitoneal (i.p.) injections of normal saline (2.5 mL) once in 24 h and water Group II rats received stone-inducing treatment for 21 days, comprised of 0.75% (w/v) EG with 1% (w/v) ammonium chloride for 5 days; following this the water supply was switched to 0.75% EG alone in water, along with saline treatment (positive control urolithic group).GPAE (50 mg/kg) through gastric gavages and simultaneously received stone-inducing treatment similar to the positive control daily for 21 days .Group III rats received GPAE (100 mg/kg) through gastric gavages and simultaneously received stone-inducing treatment similar to the positive control daily for 21 days .Group IV rats received Group V rats received standard drug-Cystone, (100 mg/kg) through gastric gavages and simultaneously received stone-inducing treatment similar to the positive control daily for 21 days .2+) and magnesium (Mg2+) contents were determined by using commercially available kits, while inorganic phosphate excretion was determined by reported method [Immediately before and at the end of a total 21 days of treatment, 24 h urine samples were collected in the presence of a few thymol crystals, by housing animals individually in the metabolic and diuretic cages. Water intake was also determined simultaneously. Following volume and pH determination, part of each 24 h urine sample was acidified to pH 2 with 5 M HCl. Both acidified and nonacidified urine samples were then centrifuged at 1500 ×g for 10 min to remove debris and supernatants were stored at −20°C until analyzed. In acidified urine samples, oxalate, calcium , centrifuged at 1500 ×g, and the supernatants were used to assess various antioxidant and oxalate forming enzymes activities and biochemical parameters like reduced glutathione (GSH), malondialdehyde (MDA), and protein carbonyl content.−1 cm−1. GSH was estimated as total nonprotein thiol (SH) group following the method described by Moron et al. [2O2 at 240 nm with a spectrophotometer [ε240 (molar extinction coefficient) = 0.0394 mmol−1 min−1 for H2O2 and expressed as μmoles of H2O2 decomposed per min under standard conditions at 25°C. Glycolate oxidase (GOx) activity in liver and lactate dehydrogenase (LDH) in both liver and kidney were determined by reported method [Kidney homogenates were estimated for MDA content by thiobarbituric acid reactive method . The pron et al. . Superoxotometer . Catalasd method , 32.50 value) with 95% confidence intervals. All statistical comparisons between the groups are made by means of one way analysis of variance with post hoc Student's t-test. The P  values less than 0.05 are regarded as significant. The concentration-response curves were analyzed by nonlinear regression using Graph Pad Prism .The data expressed are mean ± standard error of mean (S.E.M.) and the median inhibitory concentration  μg/mL  μg/mL and inhibited in vitro lipid peroxidation by 38.4 ± 1.22 and 94.5 ± 4.6% at 50 and 150 μg/mL, respectively.0) μg/mL  and inhiectively . The staGPAE on rat urine volume, pH, and Na+, K+, and Ca2+ excretion is given in GPAE increased urine volume at 50 mg/kg (P < 0.05) and 100 mg/kg (P < 0.01) indicating diuretic effect. At the next higher dose (200 mg/kg), urine volume did not increase above that of saline-treated group (P > 0.05). The reference diuretic drug, Furosemide at 10 mg/kg, showed statistically significant increase in the urine output (P < 0.01). In addition to an increase in the urine output, GPAE, and similarly Furosemide, increased urine excretion of Na+ and K+ compared to the saline-treated control animals at higher dose. GPAE caused significant increase in the urine pH and decrease in Ca2+ excretion (P < 0.01) at 100 mg/kg comparable to the standard drug, Furosemide (10 mg/kg).The effect of various doses of P < 0.01). Stone-inducing treatment also reduced pH of urine in the urolithic group as compared to that of the normal control group, although not to a significant extent. Simultaneous treatment with GPAE at 100 mg/kg in treatment groups reduced the increase in urine volume and water intake (P < 0.05) as compared with the control animals (P < 0.05). There was an increased oxalate and decreased Ca2+ concentration (P < 0.01) of the urine collected from the urolithic animals as compared to normal control. GPAE and Cystone groups exactly reversed this effect, that is, decreased the urinary oxalate (P < 0.01) and increased Ca2+ (P < 0.05) excretion at all the treated doses and remained significant at 100 mg/kg in GPAE-treated group. Other changes in the urine composition like decreased levels of citrate and Mg2+ and increased excretion of uric acid and inorganic phosphate in urolithic group were not statistically significant. GPAE- and Cystone-treated groups also did not show any statistically significant change in these parameters and remained comparable to that of control animals. The urolithic treatment revealed loss in body weight of the urolithic animals (P < 0.01), whereas GPAE- and Cystone-treated animals showed net gain in the body weights at all the tested doses. Kidneys of urolithic group animals were heavier than those the control animals (P < 0.01), whereas GPAE- (100 mg/kg) treated rat kidneys did not show any significant increase in weight as compared with control; however the GPAE- (50 mg/kg) and Cystone- (100 mg/kg) treated animal's kidneys were slightly heavier than those the control animals  treated groups as compared with the urolithic control groups wherein the GPAE at 100 mg/kg shown significant decrease of Ca (P < 0.01)  .P < 0.01) and decreased GSH level (P < 0.05) and activities of the antioxidant enzymes including SOD (P < 0.01), GPX (P < 0.05), and catalase (P < 0.01) in kidneys of the urolithic rats as compared to control animals. Activities of oxalate forming enzymes including GOx (P < 0.01) in liver and LDH (P < 0.01) in both liver and kidneys were found to be enhanced in urolithic group compared to control animals whereas treatment with GPAE was found to be protective against this effect and oxidative changes induced by urolithic treatment at higher doses  which is traditionally being utilized in Indian folk medicine since several decades to treat kidney stones was evaluated for its antiurolithic effects. In our earlier studies, we have clinically evaluated the efficacy of this preparation and found its potential in naturally induced urolithic patients [GPF and determine its underlying mechanism of action on calcium oxalate urolithiasis.Phytotherapy approach is attempted in this study as an alternative to primary healthcare. The patients . The preGPAE was determined by free radical scavenging and lipid peroxidation inhibitory activity to study its mechanism of action. Interestingly, GPAE revealed DPPH free radical scavenging and inhibited lipid peroxidation of rat kidney homogenate similar to a standard antioxidant chemical, BHT.Calcium oxalate stone development is a multifactorial process involving various etiological factors. Studies have shown that calcium oxalate crystal deposition leads to the cellular injury mediated by lipid peroxidation through free oxygen radical generation. Studies revealed that these cellular injuries favor the events of calcium oxalate retention in renal tubules which is significant for further stone development –35. ReceGPAE altered urine output in a dose dependant manner and maximum diuresis observed at 100 mg/kg. At next higher dose that is, 200 mg/kg, it did not lead to any further increase in the urine volume and remained comparable to the normal control group. The disappearance of the diuretic effect of these polyherbal extracts at this higher dose is not completely understood. Increase in urine volume by GPAE was also associated with the increase in Na+ and K+ electrolyte loss similar to the standard diuretic furosemide. Increase in urine volume, thus suggestive of possible loss of these electrolytes leading to this effect. GPAE-treated group is also revealed increase in urine pH and decrease in urine Ca2+ at 100 mg/kg like that of furosemide-treated groups. These hypocalciuric and diuretic effects of GPAE may help to reduce urinary super saturation of calcium salts and thus can prevent further stone formation.In the diuretic activity study, GPAE was evaluated in an animal model of urolithiasis induced by ethylene glycol and ammonium chloride in combination. This model is frequently used in calcium oxalate stone formation and therefore was selected for this study [GPAE- and Cystone-treated groups. These calcium oxalate crystal agglomerates get deposited in the renal tubules and further develop into renal stones [GPAE reduced the polyuria associated with lithogenic treatment as compared to urolithic control groups. As per the earlier reports, stone induction treatment leads an increase in oxalate and decrease in Ca2+ excretion. This is confirmed in the present study in which urolithic control animals showed the similar effect, and GPAE- and Cystone-treated groups reversed this effect [Further antiurolithic effect of is study , 40, 41.l stones . GPAE res effect , 43.GPAE revealed significant decrease in all these lithogenic markers at higher doses along with the antioxidant activity.Lithogenic treatment resulted in increased calcium oxalate deposition in the urolithic control groups along with oxidative stress determined from increased levels of markers of oxidative damage, that is, MDA and protein carbonyl content, along with decreased activity of antioxidant enzymes and GSH levels in kidneys accompanied with abnormal renal function. The renal tubules were dilated in entire kidneys of all untreated rats leading to disturbed renal flow by large crystal aggregates. However, GPAE at higher doses by the combination of antioxidant, diuretic, and hypocalciuric effects. The results of the formulation were found significant as compared with Cystone which is a standard drug available in the market used in the treatment of urolithiasis, and diuretic activity was comparable with the standard diuretic drug, Furosemide. The present investigation thus reports the significant antiurolithic potential of GPAE on calcium oxalate crystal retention in renal tubules thus can be attributed to its diuretic, hypocalciuric, antioxidant effect along with its potential to inhibit biochemical parameters involved in oxalate metabolism which rationalize its medicinal use for urinary stone disease. The present study will certainly benefit the scientific community worldwide in providing a source for isolation and designing a molecule in this research pipeline.The inhibitory effect of


Dracaena arborea in streptozotocin-induced ultra-structural spermatogenic alterations in Wistar rats.Infertility is a common complication in diabetic men and experimental animals, mainly due to loss of germ cells by apoptotic cell death. The aim of this study was to evaluate the effects of aqueous and ethanol extracts of Dracaena arborea aqueous (500 mg/kg) and ethanol (100 mg/kg) extracts for three weeks. A group of non diabetic rats received Millipore water (10 ml/kg) and served as healthy control group. Blood glucose was monitored at the beginning and the end of the study. One day after the last treatment, animals were sacrificed and the testes immediately removed were morphologically observed and prepared for electron microscopy analysis of spermatogenesis.Diabetic animals were orally treated with Millipore water (10 ml/kg), sildenafil citrate (1.44 mg/kg) or Dracaena arborea was devoid of any anti-hyperglycemic activity. In the untreated diabetic rats, hyperglycemia severely damaged the testes morphology as well as the spermatogenic process as evidenced by the: thickness of basement membrane of the seminiferous tubule; mitochondria alteration; abnormal spermatocyte cells displaying polymorphous nuclei, cytoplasmic vacuolization and necrosis; and disorganization and degeneration of sperm germ cells. Administration of sildenafil citrate and Dracaena arborea extracts to the diabetic rats improved testes morphology and reversed, although not completely, the impairment of spermatogenesis; this alleviating effect was more pronounced in animals treated with the aqueous extract (500 mg/kg) of Dracaena arborea.Our results showed that Dracaena arborea improves testes morphology and restores spermatogenesis in type 1 diabetic rats, without having major anti-hyperglycemic properties. These effects could be attributed to saponins, flavonoids, phenols and sterols revealed in this plant, which could be a useful component in the treatment of diabetes-induced testicular dysfunction. Dracaena arborea root extracts have been locally used by many people as aphrodisiac to treat sexual inadequacy and to stimulate sexual vigor. In a pilot study, we demonstrated that the aqueous (500 mg/kg) and ethanol (100 mg/kg) extracts of Dracaena arborea stimulate copulatory activity of normal and androgen-deprived (castrated) rats through dopaminergic and/or cholinergic pathways [Infertility is one of the major health problems in life, and approximately 30% of this problem is due to male factors. Male infertility is a multifactorial disease, with numerous factors contributing to both reduced spermatogenesis and production of dysfunctional sperm – the most prevalent underlying characteristics found in cases of idiopathic male infertility ,2. Infer ethanol 0 mg/kg eDracaena arborea, on the ultra-structural spermatogenic damages in streptozotocin-induced type 1 diabetic rats. Data from the literature considered as aphrodisiacs substances that enhance sex drive and/or sexual pleasure or can arouse sexual desire or libido; they are also agents that can be used to modify impaired sexual functions [On the basis of the above mentioned findings, the present study was undertaken to evaluate the effects of the aphrodisiac plant, unctions -14.Dracaena arborea was macerated with ethanol (95%)  for 72 hours to yield, after solvent evaporation under reduced pressure, 30 g of a brownish extract corresponding to an extraction yield of 3%. The two extracts used in the study were prepared at a final concentration of 100 mg/ml in Millipore water; the volume of oral administration was 1 ml/100 g of body weight.The plant material was harvested in Bagnoun, West Region of Cameroon and authenticated at the Cameroon National herbarium (CNH) under the voucher number 25361/SFR/Cam. The harvested fresh root barks were cut into small pieces, dried at room temperature and ground into a fine powder. For the aqueous extraction, eight hundred grams (800 g) of the powdered roots were dissolved in 5 l of distilled water and kept for 72 h at 25°C, and occasionally stirred. After filtration, the filtrate was oven-concentrated (40°C) to give 39.68 g of brownish residue corresponding to an extraction yield of 4.96%. In order to obtain the ethanolic extract, ground root bark (1 kg) of Dracaena arborea to determine the presence of flavonoids (test of Shinoda), sterols (Libermann Buchard test), phenols (ferric chloride test), alkaloids (Dragendorff test) and saponins (Saponification test). All these tests were performed using standard methods [Qualitative phytochemical evaluation was performed on aqueous and ethanolic extracts of  methods .Male Wistar rats weighing 200-250 g, grown in the Animal House of “Iuliu Haţieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania, were acclimatized for one-week in the Department of Cellular and Molecular Biology, of the above mentioned University. They were placed in Plexiglas cages under a 12-hour light: dark cycle with standard food and water ad libitum. All experiments were approved by the ethical committee of the University of Medicine and Pharmacy, Cluj-Napoca, Romania through letter Nr 687A/30.10.2012.®, Roche) with a drop of blood obtained by tail-vein puncture. Animals were considered diabetic if blood glucose values were higher than 200 mg/dl.Experimental diabetes was induced in overnight fasted rats by a single dose of streptozotocin (STZ) (50 mg/kg) dissolved in ice-cold 0.1 M sodium citrate buffer, pH 4.5 and administered i.p. One hour-thirty minutes after injection of STZ, all animals received an intraperitoneal injection of glucose 33% to overcome hypoglycemia  induced by STZ after the pancreatic β-cell destruction and massive release of insulin. 48 h after STZ treatment, the fasting blood glucose level was measured using reagent strips (Accu-ChekDracaena arborea (500 mg/kg); Group 5, diabetic rats treated with the ethanolic extract of Dracaena arborea (100 mg/kg). The vehicle (Millipore water) and the test solutions were orally administered once a day for 3 weeks using an endogastric canule. Doses of plant extracts were chosen on the basis of our pilot studies [Two weeks after the confirmation of the diabetic state, animals were randomly divided into 5 groups of 5 animals each and treated as follows: Group 1, healthy rats receiving 10 ml/kg of Millipore water only ; Group 2, diabetic rats receiving 10 ml/kg of Millipore water only (Diabetic control group); Group 3, diabetic rats treated with 1.44 mg/kg of sildenafil citrate; Group 4, diabetic rats treated with the aqueous extract of Streptozotocin  and sildenafil citrate (Pfizer)  were used in this study.In each treatment group, the effect of treatment duration on blood glucose level was analysed using two ways ANOVA repeated measures followed by Bonferroni all pair comparison test. However, two ways ANOVA followed by Bonferroni all pair comparison test was used to compare the treated groups to healthy and diabetic control animals respectively. P values of 0.05 or less were taken to imply statistical significance between the means. All these analysis were performed using Graphpad Prism version 5.1.Immediately after the sacrifice of rats under chloroform anesthesia, 3 small pieces from each testis were collected and prefixed with 2.7% glutaraldehyde in 0.1 M phosphate saline buffer for 2 hours. After being washed 4 times in the same buffer , samples were post-fixed with 1% osmium tetroxide in 0.15 M phosphate saline buffer for 2 hours, and washed again 2 times (30 min each) in the same buffer. The samples were next dehydrated in acetone solutions (prepared in distilled water), once in 30%, 50%, 70%, 80%, 90% and three times in 100% for 15 min respectively except that of 50% which was done for 30 min. All the steps from the beginning up to 70% acetone were performed at 4°C while the others (from 80 to 100%) were done at room temperature. After complete dehydration, the samples were infiltrated with Epon 812 in 4 steps: acetone-epon 2:1 (45 min), acetone-epon 1:1 (1 hour), acetone-epon 1:2 (1 hour), pure epon (overnight). The samples were then placed in gelatin capsules with fresh epon, and polymerized at 60°C for 72 hours. Trapezoidal ultrathin sections of 70–80 nm thickness were obtained from 3 seminiferous tubules of 4 blocks from each group with glass knives using a Bromma 8800 ULTRATOME III  and contrasted next with solutions of uranyl acetate (15 min) and lead citrate (8 min). The examination of these sections was performed on a Jeol JEM 1010 transmission electron microscope . Images were captured using a Mega VIEW III camera , and introduced in a database using a Soft Imaging System software . Ultra-structural features of the prepared seminiferous tubule section of each group were then evaluated.Dracaena arborea dried roots showed the presence of saponins, phenols, flavonoids and sterols.Qualitative phytochemical screening of both aqueous and ethanol extracts of Injection of streptozotocin to rats resulted in a severe atrophy of the testes Figure e compareDracaena arborea and sildenafil citrate (23.59% of variation). These results further revealed the lack of effect of Dracaena arborea extracts and sildenafil citrate in reducing blood glucose in 3 weeks treated-diabetic rats.Data from Table In the healthy control group, a normal aspect of the basement membrane with a normal diameter was observed. A normal space between this basement membrane and spermatogonia and spermatocyte was also observed Figure A. SpermaIn the untreated diabetic group, a thickening of the basement membrane was detected Figure A, B, couAs compared to healthy control group, no important changes concerning the aspect of basement membrane were observed in this group Figure A. Even iThe electron microscopic images of this group were comparable to those of the healthy control group, proving the effectiveness of the applied treatment. The aspect of the basement membrane joined to the appearances of Sertoli cells and spermatogonia were similar to that observed in the healthy control group Figure A. The prThe obtained results in this group can be placed under those obtained for diabetic rats treated with sildenafil citrate. The basement membrane and spermatogonia had a normal aspect Figure A, but moIn many animal species, STZ induces diabetes that resembles human hyperglycemic nonketotic diabetes mellitus and this effect has been extensively studied and appears to be mediated through a lowering of beta cell nicotinamide adenine dinucleotide (NAD+) and results in histopathologic alteration of pancreatic islet beta cells ,17. It iTesticular tissue damage caused by STZ-induced diabetes in rat as observed in this study, could then be responsible for the reduction and the death of germ cells ,4,27. InDracaena arborea extracts to diabetic rats alleviated, although not completely, these ultra-structural deficits by providing protection against apoptotic germ cell death and impairment in the spermatogenic process. Dracaena arborea could then act like sulphurous mineral water and sodium hydrosulphide on diabetic rat testes [Dracaena arborea extracts in testicular damages of STZ diabetic rats are similar to those obtained after application of hydrated C60 fullerene or enalapril in STZ-induced diabetic rats [Dracaena arborea, may include the antioxidant [Dracaena arborea. The androgenic hypothesis could be confirmed by the sexual stimulant effects of the aqueous and ethanolic extracts of this plant in the copulatory activity of androgen-deprived (castrated) rats as previously demonstrated by our research group [However, when compared to the untreated diabetic group, three weeks of oral administration of sildenafil citrate or t testes . These rtic rats ,32. Sinctic rats ,33, the ioxidant  and andrioxidant , phenolsioxidant , saponinioxidant ,38 and pioxidant  revealedch group . The regch group . Then, sch group . Also, sch group ,43.Dracaena arborea especially the aqueous extract (500 mg/kg), without having major anti-hyperglycemic property. These alleviating effects could be attributed to saponins, flavonoids and sterols present in this plant, which could be a useful treatment in preventing and treating diabetes-induced testicular dysfunction.Our results clearly demonstrate that the increased rate of testicular germ cell death by apoptosis in streptozotocin-induced diabetic rats was protected and regulated by the administration of sildenafil citrate and The authors declare that they have no competing interests.MWN, PW, SB, AF and HM conceived the project. MWN and SB did the literature search. MWN, SB and AF contributed to the laboratory work. AF did the electron micrographic images with their interpretations. HM and AK was responsible for the overall supervision. MWN and PW wrote the paper with input from all the authors. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/13/71/prepub


To assess the relationship between sleep quality and demographic variables, morning-evening type, and burnout in nurses who work shifts.We carried out a cross-sectional self-administered study with forced choice and open-ended structured questionnaires – Pittsburg Sleep Quality Index, Morningness-eveningness Questionnaire, and Maslach Burnout Inventory. The study was carried out at Gazi University Medicine Faculty Hospital of Ankara on 524 invited nurses from July to September 2008, with a response rate of 89.94% (n = 483). Descriptive and inferential statistics were applied to determine the risk factors of poor sleep quality.P < 0.001) and needlestick or sharp object injuries (P = 0.010) in the last month had poor sleep quality. The subjective sleep quality and sleep latency points of evening types within created models for the effect of burnout dimensions were high.Most socio-demographic variables did not affect sleep quality. Participants with poor sleep quality had quite high burnout levels. Most nurses who belonged to a type that is neither morning nor evening had poor sleep quality. Nurses who experienced an incident worsening their sleep patterns (Nurses working consistently either in the morning or at night had better sleep quality than those working rotating shifts. Further studies are still needed to develop interventions that improve sleep quality and decrease burnout in nurses working shifts. Nurses have to be accessible to patients on a twenty-four hour basis, which is only possible by shift work . Shift wAlso, people working rotating shifts had a variety of other health problems – gastrointestinal problems such as digestive disorders and ulcer, neck and back pain, fatigue, depression, tiredness, job stress, emotional disorders, and an increased risk of cardiovascular diseases -8. SleepNocturnal shift work alters the timing of light exposure and induces circadian rhythm disruption ,6. SleepSome studies investigated how circadian type influenced the performance of individuals . The cirBurnout is another important problem for nurses working shifts . It is aThe aim of this study was to assess the relationship between sleep quality and demographic variables such as arterial blood pressure, body mass index, heart beat rate, tea and coffee consumption, the relationship between sleep quality and morning-evening type, and the relationship between sleep quality and burnout in shift-worker nurses.A cross-sectional study was carried out between July and September 2008 by a self-administered, forced choice, and open-ended structured questionnaire at Gazi University Medicine Faculty Hospital, Ankara, Turkey. The research population comprised all 637 nurses working in the hospital with a bed capacity of 1085. We excluded the nurses diagnosed with psychotic, neurological, metabolic, and sleep disorders, as well as nurses who either worked less than one year or who were on vacation or on medical leave during July and September 2008. Therefore, 524 nurses were invited to participate, but 26 refused and 15 did not complete the questionnaires, which left 483 nurses who were interviewed, with an overall 89.94% (483/524) response rate.Nurses in the hospital worked 8-hour shifts from 08.00-16.00, 16.00-24.00, and 24.00-08.00; or 16-hour shifts from 16.00-08.00 and 08.00-24.00, working on average 40 hours or 48 hours per week.The study included 483 female nurses (mean age = 30.41 ± 5.7) with the following characteristics (mean ± standard deviation or median [range]): work experience – 8 (1 to 30) years; systolic blood pressure – 100 ± 12.33 mm Hg; diastolic blood pressure – 65.07 ± 9.21 mm Hg; heart beat rate – 82.23 ± 10.33; body mass index (BMI) – 22.67 ± 3.50; daily teacup – 1.00 (0 to 12); daily coffee cup – 3.00 (0 to 10). Most respondents were married (52.2%) and worked as an in-patient nurse on wards (48.4%). Of those nurses, 65.2% (n = 315) worked rotating shifts (morning-evening-night shift), 5% (n = 24) continuously worked night shifts (evening or night shift), and 29.8% (n = 144) continuously worked day shifts.Of the nurses, 65% (n = 314) were graduates from Nursing School with baccalaureate degree, 21.9% (n = 106) from Health Services Vocational School Nursing Program with associate degree, and 13% (n = 63) from Vocational Health High School Nursing Program. Health Services Vocational School Nursing Program provides 2 years of nursing education to students who had finished 11 years of basic education. Vocational Health High School Nursing Departments provide 4 years of professional training to students who had finished 8 years of basic education. With 2007 changes in nursing institutions, nursing education in Turkey is now provided only by the universities with baccalaureate degrees. Other nursing schools were closed down.For pilot trial, the first part of the question-form was given to 20 nurses who worked in another hospital and were not a part of the main research population. The pilot trial provided a test of comprehensibility and clarity of the questions, and based on it, self-administered, closed-ended, structured questionnaire interview was revised.Hospital ethics committee did not require an ethical approval for the study since it included no invasive practices for humans or animals. Instead, hospital management provided written approvals. The author contacted the chief nurse in each ward, explained the purpose of the study, and obtained a verbal permission. The participants signed the informed consent, after they had been informed in detail on characteristics and aim of this study.We collected data from nurses by using 4 anonymous self-administered questionnaires. Personal Information Form with forced choice and open-ended questions was designed by the authors to obtain information about demographic, socio-economic, individual, and work-related issues that were supposed to have effects on the scores of Pittsburg Sleep Quality Index (PSQI), Morningness-eveningness Questionnaire (MEQ), and Maslach Burnout Inventory (MBI) . Height,Pittsburg Sleep Quality Index. PSQI assesses quality and patterns of sleep through self-reported sleep habits over the last month. It is a global measure with seven subscales; subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbance, use of sleeping pills, and daytime dysfunction. A global sleep quality score is then obtained by summing the 7 components. Each component scores from 0 (not in the past month) to 3 points (3 or more times per week) and the global score ranges from 0 to 21. A PSQI total score ≥5 indicates poor sleep quality. A score of 5 yielded a diagnostic sensitivity of 89.6% and a specificity of 86.5%, with an internal consistency (α) of 0.83 and test-retest reliability (r) of 0.85 , version 16, for descriptive and inferential statistics. Statistical power was strengthened by defining poor sleep quality as a score of ≥5 on PSQI ,22. Compric test .P ≤ 0.05). The dependent variable was sleep quality  and DP (P < 0.001), as well as PA decreased sleep quality (P = 0.001). Neither MEQ type also decreased sleep quality (P = 0.016). The events experienced in the last month affecting the sleep pattern (P < 0.001), sleepiness during the shift (P = 0.004), sharp object and needlestick injuries (P = 0.010), and lateness or failure to wake up in time were associated with sleep quality (P < 0.001)  of nurses was ≥5. The mean ± standard deviation total sleep time was 6.95 ± 0.99 hours. Most of the basic socio-demographic variables did not affect sleep quality . EE (P << 0.001) .Emotional exhaustion, lateness to the work, disturbed sleep pattern in the last month, and having no roommate or no bed-partner were significantly associated with poor sleep quality .Excluding the effect of EE, DP, and PA on sleep quality, we aimed to determine the effect of morning-evening type on PSQI. In order to investigate this, dummy variables were formed according to MEQ and the effect of these variables on PSQI components was identified using linear regression method after having made the necessary corrections according to EE, DP, and PA. In the models created by considering the effect of EE, DP, and PA, subjective sleep quality and sleep latency points of evening types were high . In the Nurses working consistently either in the morning or at night had better sleep quality than those working rotating shifts . Nurses Most nurses in this study (79.1%) experienced poor sleep quality. This was expected since most of them (65.2%) worked rotating shifts. The finding is in accordance with previous studies -30.Nurses with poor sleep quality had higher burnout levels, experienced something that affected their sleep quality in the last month, were late for work because they could not awake up in the morning, had significantly more sharp object/needlestick injuries, and were sleepy at work. Alimoğlu and Dönmez  also fouNurses working rotating shifts experienced more sleeping problems and sleepiness at work than nurses working continuous day/night shifts. Furthermore, nurses in rotating shifts had more accidents or errors. Therefore, rotating shifts should be avoided to assure safety of nurses and patients .Sleep quality and work pattern had a low but significant correlation, but the MEQ type was not clearly associated with poor sleep quality. Admi et al showed that work pattern did not affect lateness, absence, or accidents and errors at work . On the Healthcare workers in the night shifts or those who worked 60 hours a week were found to have higher risk of sharp object or needlestick injuries ,37, and Most nurses in the study had difficulty to sleep after the night shift. Bad sleep quality led to twitching in legs, adaptation problems, and confusion during sleep. Other studies also showed that nurses working rotating or night shifts were more tired than others  and thatOur study showed that emotional exhaustion, any incident influencing sleep pattern in the last month, and having no roommate or having no (bed) partner affected sleep quality. Such a finding in individuals with no roommate or bed partner might be explained by their feeling lonely and in need of emotional support. Exhaustion, on the other hand, decreases or diminishes self-confidence and interest in work, as well as causes fatigue and weakness . An indiMorning-evening type and shift pattern of nurses did not affect sleep quality in our study. Chung et al, on the other hand, reported that chronotype affected sleep quality . These cIn this study, evening type nurses had poorer subjective sleep quality and sleep latency, which were subscales of PSQI, than nurses of other two types. The administration in the hospital under study did not, however, arrange shifts according to chronotype. Evening type participants were shown to have more negative habits than morning types . MorningPoor sleep quality due to increased burnout level in nurses working rotating shift was an interesting finding, since it might have been expected that nurses working night shifts have lower job stress level than others. Similarly, nurses working fixed day shifts had increased EE and DP scores. Adaptation of biological rhythm would be a lot easier for nurses working fixed shifts than rotating shifts. Although separate studies were available, we did not find any study that investigated MBI, PSQI, and work patterns together. Jamal and Baba , for exaA limitation of this study is that it was carried out on a given nurse population in Ankara, with a self-reported questionnaire, which might result in a biased reply from each respondent. Therefore, generalizations should be made carefully and be limited only to the investigated population. In conclusion, our findings showed that majority of nurses experienced poor sleep quality and had increased levels of burnout, especially rotating shift nurses. Most of the nurses who did not belong to either morning or evening type had poor sleep quality. Further studies are needed to plan interventions that decrease burnout and improve sleep quality for shift-work nurses. These interventions would likely improve nurses’ overall well-being and working conditions and patients’ safety. The hospital administrations should take workers’ chronotypes into account when forming the shift lists.


To investigate a possible role of Cefditoren, a recently marketed in Greece third-generation oral cephalosporin in urinary infections of outpatients.During a multicenter survey of Enterobacteriaceae causing UTIs in outpatients during 2005–2007, Cefditoren MICs were determined by agar dilution method in a randomly selected sample of uropathogens. Susceptibility against 18 other oral/parenteral antimicrobials was determined according to Clinical and Laboratory Standards Institute methodology.Escherichia coli, 142 Proteus mirabilis and 91 Klebsiella spp) was studied; MIC50/MIC90 of Cefditoren was 0.25/0.5 mg/L respectively, with 97.1% of the isolates being inhibited at 1 mg/L. All 12 strains producing ESBLs or AmpC enzymes were resistant to cefditoren. Susceptibility rates (%) for amoxicillin/clavulanic acid, cefuroxime axetil, cefotaxime, ciprofloxacin, trimethoprim/sulfamethoxazole and fosfomycin were 93.1- 94.1- 96.8-93.1-71.9 and 92.8% respectively. Cefditoren MIC was significantly higher in nalidixic/ciprofloxacin non-susceptible strains; resistance to cefditoren was not associated with resistance to mecillinam, fosfomycin nitrofurantoin and aminoglycosides. Multivariate analysis demonstrated history of urinary infection in the last two weeks or three months as risk factors for cefditoren resistance.A total of 563 isolates (330 Cefditoren exhibited enhanced in vitro activity against the most common uropathogens in the outpatient setting, representing an alternative oral treatment option in patients with risk factors for resistance to first-line antibiotics. Urinary tract infections (UTIs) represent the most frequent bacterial infection encountered in the community setting being caused in their vast majority by members of the family of Enterobacteriaceae ,2. RecenCefditoren is a third generation oral cephalosporin with a broad spectrum of activity comprising Gram-positive and Gram-negative bacterial species ,8. AfterThe present study was conducted in order to investigate a possible role of cefditoren in the treatment of UTIs treated in the outpatient setting. Given the expanded spectrum of cefditoren, strains of uncomplicated as well as complicated UTIs were included.From January 2005 to March 2007 a Greek multicentre surveillance network was formed by private or public microbiology laboratories, representative of the whole country, in order to obtain Enterobacteriaceae isolates from outpatients with UTI. Personal data were collected anonymously. The research protocol was approved by the Ethics Committees of the cooperating hospitals, namely “Attikon” University General Hospital of Athens, “Sismanoglion” General Hospital of Athens and “G. Gennimatas” General Hospital of Athens. Primary care centers and Private Institutions participating in this study do not have Ethics Committees. Isolated strains were shipped to the central laboratory . The network’s structure and methodology have been reported previously .4 CFU/ml. Bacterial isolates were identified by biochemical profiling using API systems .A single urine culture per outpatient referred for a urine sample culture to each collaborating centre was collected along with a detailed questionnaire tracking demographic and clinical information. Clinical data included: reason for giving a urine sample, symptoms, history of UTI , recent use of antibiotics (in the previous 2 weeks and 3 months), history of admission to the hospital and/or insertion of a urinary catheter during the previous year, presence of diabetes mellitus, nephrolithiasis, presence of urinary catheter and pregnancy on sampling . A cultuE. coli, or Proteus mirabilis or Klebsiella spp. As defined by the study protocol every fourth isolate per center was selected for the cefditoren study; 7 isolates were dropped out due to contamination.Isolates included in the current study were randomly selected from the total collection, which included 2446 microorganisms, 2280 of them being either Antimicrobial susceptibility testing to all antibiotics except Cefditoren was performed using the disk diffusion method, according to the Clinical Laboratory Standard Institute recommendations, CLSI 2011 [Escherichia coli ATCC 25922 was used as a quality control microorganism and was included in each run. Cefditoren standard powder was supplied by GlaxoSmithKline (Hellas). In lack of CLSI or EUCAST established breakpoints for Cefditoren, isolates with MIC ≤ 1 mg/L were considered as sensitive according to the recent literature [Susceptibility to Cefditoren was tested by agar dilution method, according to CLSI 2011 methodology. terature -14. Thisterature .Phenotypic identification of extended-spectrum β-lactamases (ESBL) production was confirmed following the CLSI 2011) guidelines  guidelin. Other mMale gender, pregnancy, history of urinary tract infection in the last two weeks, history of admission to the hospital in the last 30 days, the presence of diabetes mellitus or nephrolithiasis and the presence of a urinary catheter on sampling were considered as complicating factors .Female non pregnant patients, without complicating factors, presenting with at least one urinary symptom , and a positive urine culture were assigned to the group “Acute Uncomplicated Cystitis” (AUC) .In the univariate analysis proportions of NS (non susceptible) or R (resistant) strains between categorical variables were compared using the Pearson’s chi-square test or the Fischer’s exact test where appropriate. A p-value <0.05 was considered as statistically significant. Univariate predictors with p < 0.1 were tested for inclusion in a step-wise multivariate model. Nonsignificant variables were removed sequentially until only those significant at p < 0.1 remained. The analysis was carried out using Stata 10.0 .E. coli, 142 Proteus mirabilis and 91 Klebsiella spp-three K. oxytoca and 88 K. pneumoniae). Fully evaluable clinical information was available for 334/563 isolates (59.3%), i.e. 214/330 E. coli (64.8%), 75/142 P. mirabilis (52.8%) and 45/91 Klebsiella spp (49.5%). Women accounted for 88.3% of the patients with a mean age of 45.8 years (SD ± 18.5ys). A total of 318 cases of acute uncomplicated cystitis were available for evaluation. Demographic and clinical data are listed in detail in Table Cefditoren was studied against a total of 563 isolates , 3/142 (2.1%), and 3/91 (3.3%) respectively, while respective values for MIC50/MIC90 were 0.25/0.50, 0.125/0.25, and 0.25/0.5 mg/L . In Figure E. coli, P. mirabilis and Klebsiella spp is displayed.The in vitro activity of the antimicrobial agents tested against all the isolates in the study are listed in detail in Table E coli, 1 Klebsiella spp), while plasmid-mediated Amp-C in 3 strains (3 P. mirabilis). Production of inhibitor-resistant TEM β-lactamases and penicillinase production/hyperproduction was identified in 11 strains . All ESBL-producer strains were resistant to cefditoren (MIC range 16–128 mg/L); the same applied for all three plasmidic AmpC producers possessing MIC of cefditoren at 64 mg/L. TEM/hyperproducers displayed more variable MICs against cefditoren; in 7/11 the MIC was equal to 1 mg/L, whereas it ranged between 2–4 mg/L for the 4 remaining strains of this kind.Phenotypic identification of ESBL production was evident in 9 strains (8 E. coli isolates with different mechanisms of resistance. For ampicillin non-susceptible strains (104 strains) cefditoren MIC50/MIC90 values were 0.25/1 mg/L, whereas for amoxicillin-clavulanate and ciprofloxacin non-susceptible strains (21 and 16 strains respectively) the respective values were 0.5/128 mg/L. Finally, cefditoren MIC50/MIC90 values of cotrimoxazole non-susceptible strains (n = 78) were 0.25/4 mg/L.Cefditoren displayed good in vitro activity against E. coli isolates from AUC cases are listed in Table E coli isolates. Non-susceptibility rates for Proteus mirabilis and Klebsiella spp. isolates from AUC cases are listed in Table Non-susceptibility rates for In the total isolates yield 563 strains), cefditoren non-susceptibility was positively associated with the co-existence of non-susceptibility to other antimicrobials, with the exceptions of mecillinam, imipenem, fosfomycin, nitrofurantoin, gentamicin and netilmicin. Cefditoren MIC was significantly higher in nalidixic/ciprofloxacin non-susceptible strains. The same applied for ciprofloxacin MIC in cefditoren non-susceptible isolates  . On the hoxazole .E. coli strains [The accompanying questionnaire enabled us to classify UTIs as “complicated” or “uncomplicated” and to elucidate risk factors for resistance to cefditoren beyond the well-recognized use of an antibiotic of the same class in the previous 15 days-3 months ,19,20. T strains . Accordi strains ,17,18. A strains  has prom strains ,23. FurtA certain weakness of this study is the lack of clinical data for almost one third of the studied isolates. However the lack of significant differences in resistance rates against all antibiotics tested between the populations with and without clinical information, as well as the large and representative sample of our study argues in favor of an extended applicability of the conclusions in our community. Furthermore the addition of a pool of isolates without clinical data allowed for a simulation of real life treatment scenario of an outpatient with UTI. Another possible weakness is the lack of clinical therapeutic data, which would enable us to detect therapeutic failure in the community with currently used agents, but this was beyond the design and the scope of the current study.st line options, initially treated with a parenteral antibiotic. A strong argument for the selection of cefditoren could be the lack of cross-resistance with aminoglycosides, mecillinam, nitrofurantoin and fosfomycin.Certainly no strong argument exists for the use of cefditoren as first line option in acute uncomplicated UTIs since other treatment options mentioned above retain good activity. However many UTIs treated in the community setting are not episodes of acute uncomplicated cystitis mandating concentrations in the upper urinary tract; patients are often pretreated with other first-line classes of antibiotics; apart from recent antibiotic use patients with complicated urinary tract infections treated as outpatients frequently bear risk factors for resistance to multiple classes of antibiotics ,20. CefdIn summary, cefditoren was the most potent in vitro antibiotic available for oral use in our country, against all three major pathogens causing UTIs in the outpatient setting. A potential was demonstrated for its use as empirical treatment in outpatients with no recent history of UTI, as an alternative to first line antimicrobials or in patients with risk factors for resistance to currently indicated first treatment options. Clinical studies are warranted to clearly define its possible role in the treatment of UTIs in outpatients.All authors declared no competing interests related to this manuscript.DH carried out the in vitro procedures and input of data, and helped to draft the manuscript. GP: Participated in the design of the study, drafting and refinement of the manuscript, interpretation of data and also carried out in vitro procedures. IK: Management of the database, input and evaluation of data, statistical analysis, drafting of the manuscript. NL: Carried out laboratory procedures with determination of in vitro susceptibilities and input of data. MS: Carried out laboratory procedures and evaluation of data. ID: Carried out laboratory procedures and evaluation of data. GKN: Performed the statistical analysis. EL: Carried out laboratory work and evaluation of in vitro data. HG: Conceived the study, participated in the study design, drafting and refinement of the manuscript and interpretation of data. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/228/prepub


In addition, butein concentration-dependently repressed the phosphorylation of Akt, mTOR, and the major downstream effectors, p70S6K, 4E-BP1, and eIF4E in EPCs. Taken together, our results demonstrate for the first time that butein exhibits the antiangiogenic effect both in vitro and in vivo by targeting the translational machinery. Butein is a promising angiogenesis inhibitor with the potential for treatment of cancer and other angiogenesis-related diseases.Compelling evidence indicates that bone marrow-derived endothelial progenitor cells (EPCs) can contribute to postnatal neovascularization and tumor angiogenesis. EPCs have been shown to play a “catalytic” role in metastatic progression by mediating the angiogenic switch. Understanding the pharmacological functions and molecular targets of natural products is critical for drug development. Butein, a natural chalcone derivative, has been reported to exert potent anticancer activity. However, the antiangiogenic activity of butein has not been addressed. In this study, we found that butein inhibited serum- and vascular endothelial growth factor- (VEGF-) induced cell proliferation, migration, and tube formation of human EPCs in a concentration dependent manner without cytotoxic effect. Furthermore, butein markedly abrogated VEGF-induced vessels sprouting from aortic rings and suppressed microvessel formation in the Matrigel implant assay  Angiogenesis plays a critical role in physiological conditions such as embryonic development, reproduction, tissue repair, and bone remodeling. In contrast, angiogenesis is an important process for tumor progression and various inflammatory diseases . AngiogeCirculating endothelial progenitor cells (EPCs) have been shown to play important roles in maintaining vascular integrity and facilitating tissue repair. Circulating EPCs are mobilized from the bone marrow into the bloodstream and induce neovascularization during tissue ischemia , 6. EmerTranslational control has a crucial impact on cancer development and progression, directing both mRNA translation and protein synthesis that regulate tumor cell proliferation, transformation, angiogenesis, and metastasis . NumerouDalbergia odorifera , the stem bark of cashews (Semecarpus anacardium), and the traditional Chinese and Tibetan medicinal herbs such as Caragana jubata and Rhus verniciflua Stokes. Previous reports have demonstrated that butein has various pharmacological effects, such as antioxidant and anti-inflammatory activities -2,5-diphenyltetrazolium bromide (MTT), crystal violet, and other chemicals were purchased from Sigma . MV2 complete medium was purchased from PromoCell . Defined fetal bovine serum (FBS) and all cultured reagents were purchased from HyClone . Recombinant human VEGF was purchased from R&D Systems . Lactate dehydrogenase (LDH) assay reagents were purchased from Promega . Antibodies to phospho-Akt (Ser473), Akt, phospho-mTOR (Ser2448), and phospho-eIF4E (Ser209) were purchased from Epitomics . Antibodies to phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-p70S6K (Thr389), phospho-4E-BP-1 (Thr37/46), and GAPDH were purchased from Cell Signaling Technologies .http://dx.doi.org/10.1155/2013/943187). Experiments were conducted on EPCs between passages 8 and 12.Ethical approval was granted by the Institutional Review Board of Mackay Medical College, New Taipei City, Taiwan (reference number: P1000002). Informed consent was obtained from healthy donors before the collection of peripheral blood (80 mL). The peripheral blood mononuclear cells (PBMCs) were fractionated from other blood components by centrifugation on Ficoll-Paque plus  according to the manufacturer's instructions. CD34-positive progenitor cells were obtained from the isolated PBMCs using CD34 MicroBead Kit and MACS Cell Separation System . The isolation and maintenance of CD34-positive EPCs were performed as described previously , 33. Bri3 cells/well) were seeded onto 96-well plates. After 24 h incubation, the culture medium was removed and cells were incubated with fresh MV2 complete medium containing 10% FBS for 72 h in the absence or presence of butein. Then, cell proliferation was determined by crystal violet staining, which was performed by staining with 0.5% crystal violet in 20% methanol. The dye was subsequently eluted in the solution containing 0.1 M sodium citrate and 75% ethanol, and absorbance was measured at 540 nm with ELISA-reader.For crystal violet assay, EPCs (5 × 103 cells/well) were incubated in 96-well plates and starved with MV2 complete medium for 16 h. Then, the culture medium was removed, and cells were incubated with fresh MV2 complete medium containing VEGF (20 ng/mL) for 48 h in the absence or presence of butein. Cells were incubated with MTT (0.5 mg/mL) for 2 h. Formazan crystal was lysed by dimethyl sulfoxide (DMSO), and absorbance was measured at 550 nm with ELISA-reader.For MTT colorimetric assay, EPCs  for 48 h in the absence or presence of butein. The percentage of LDH release was calculated from the ratio of LDH activity in the medium to LDH activity in the cell lysate.EPCs were seeded onto 96-well plates in a density of 5 × 10μm pore size . EPCs (5 × 104 cells/well) were seeded onto the upper chamber with MV2 complete medium and then incubated in the bottom chamber with MV2 complete medium containing 10% FBS or VEGF (20 ng/mL) with the indicated concentrations of butein. After 16 h of treatment, cells on the upper side of the filters were mechanically removed, and those which migrated on the lower side were fixed with 4% formaldehyde then stained with 0.5% crystal violet for 10 min. Cell migration was quantified by counting the number of stained cells in 10 random fields with the inverted phase contrast microscope and photographed.Cell migration assay was performed using Transwell chambers with 8.0 4 cells) were seeded per well on the layer of polymerized Matrigel in MV2 complete medium containing 10% FBS or VEGF (20 ng/mL) with the indicated concentrations of butein, followed by incubation for 10–16 h at 37°C. Photomicrographs of capillary tube formation were taken with the inverted phase contrast microscope. Tube formation was quantified by measuring the long axis of each tube in 3 random fields per well by using Image-Pro Plus software.Matrigel , which was used to promote the differentiation of EPCs into a capillary tube-like structure, was added to 48-well plates. The Matrigel-coated 48-well plates were incubated at 37°C for 30 min to allow for polymerization. After gel formation, EPCs  with or without butein was then added to the well. The cultured medium was changed every 3 days. Sprouting endothelial cells were observed and photographed on day 8. The area of sprouting vessels was measured quantitatively by Image-Pro Plus software.Assay was performed as previously described with modification . Aortas DIVAA was performed as described previously . BrieflyCells were lysed with lysis buffer as described previously . Cell hot-test, and P values less than 0.05 were considered significant.Data are presented as the mean ± SEM for the indicated number of separate experiment. Statistical analyses of data were performed with one-way ANOVA followed by Student's μM. However, treatment of higher concentration of butein (30 μM) caused a significant LDH release /Akt and extracellular signal-related kinase 1/2 (ERK1/2) signaling pathway play important roles in cell proliferation, survival, migration, and angiogenesis , 37. Theex vivo and in vivo anti-angiogenic effect of butein, suggesting that butein may be a potential angiogenesis inhibitor  did not induce LDH release of EPCs, indicating that this antiproliferation effect of butein was not due to its cytotoxicity  . The growth inhibitory action of butein at low concentration (1–10 μM) is considered to be a more specific effect on endothelial cells that are stimulated by angiogenic growth factors. Taken together, we suggest that low concentration of butein exhibits novel anti-angiogenic activity and may contribute to suppress tumor growth and metastasis in vivo.Butein that is derived from numerous traditional herbal medicines displayed promising anticancer activities against a broad spectrum of human cancer cells via different mechanisms. Butein inhibited migration and invasion through the ERK1/2 and NF-er cells . Butein er cells . Butein er cells . In addier cells , 27, 28. Figures . Butein Importantly, our study is the first to show the effect of butein on AKT/mTOR-mediated translational pathway. Previous evidence implies that Akt and Erk signaling is the key mediator for vasculogenic functions, such as proliferation, survival, migration, and differentiation, which are activated by a variety of stimuli in endothelial cells and EPCs , 41. Sevin vitro and in vivo. We demonstrate that butein inhibits EPCs proliferation, migration, and tube formation by targeting the AKT/mTOR translation dependent signaling pathway. Since the contribution of EPCs during tumor angiogenesis is important for the initiation and promotion of tumor neovessel formation, our data provide evidence for butein that could be a potential candidate of angiogenesis inhibitor. Based on the findings herein, we suggest that butein is a promising natural product worthy of further development for the treatment of human cancer and other angiogenesis-related diseases.In conclusion, this study discovers a novel mechanism by which butein impairs angiogenesis Figure S1. Characterization of human CD34-positive endothelial progenitor cells. (A) Representative flow cytometry analysis showed that EPCs were positive for CD34, KDR, and CD31. (B) The endothelial cell property was examined by UEA-1 staining. Corresponding negative isotype controls are shown in black.5 cells/well) were treated with the indicated concentration of butein for 16 h in the presence of VEGF (20 ng/ml), and tubular morphogenesis was recorded by microscope. Tube formation was quantified by measuring the length of tubes in three random fields per well with the use of Image-Pro Plus. (B) HUVECs (5x104 cells/well) were seeded onto the upper chamber, then treated with the indicated concentrations of butein for 16 h in the presence of VEGF (20 ng/ml) as a chemoattractant in the lower chamber. Cells that migrated to the underside of the filter membrane were stained, photographed, and quantified by microscope. Data are expressed as mean ± S.E.M. of three independent experiments. ∗∗∗p < 0.001 compared with the control group.Figure S2. Effects of Butein on tube formation and migration of HUVECs. (A) HUVECs (1.2x10Click here for additional data file.


We report the clinical presentation and laboratory findings of a 69-year-old man with fragile X-associated tremor ataxia syndrome (FXTAS), a progressive neurodegenerative disorder, who was noted to have monoclonal gammopathy of undetermined significance (MGUS), a plasma cell proliferative disorder and a precursor disease of multiple myeloma. Both MGUS and FXTAS are associated with microRNA (miRNA) dysregulation. We speculate that individuals with FXTAS may be predisposed to MGUS and further studies are warranted regarding this association. FMR1) gene premutation (CGG expansion of 55–200 repeats). The clinical features of FXTAS are progressive intention tremor, cerebellar ataxia, autonomic dysfunction, peripheral neuropathy, cognitive decline, middle cerebellar peduncle signs (MCPs), and white matter disease combined with brain atrophy on MRI [Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult progressive neurodegenerative disorder that affects predominantly male carriers of the fragile X mental retardation 1  and subsequent sequestration of critical proteins necessary for normal cell function [The penetrance of FXTAS in male carriers older than age 50 is approximately 30 to 45.5% and 8 to 16.5% in females , 5, 6. Oof FXTAS . The elefunction . Mitochofunction , 11. Thifunction , 13 assocalcemia, renal insufficiency, anemia, and bone lesions) [Monoclonal gammopathy of undetermined significance (MGUS) is a plasma cell proliferative disorder characterized by a plasma cell level of less than 10% in the bone marrow, a monoclonal paraprotein band less than 30 g/L, and no end organ damage (CRAB—hyperlesions) . It is alesions) . The dialesions) . Howeverlesions) . Individlesions) –20. Racelesions) , family lesions) , and explesions)  increaseβ [The pathogenesis of MGUS is hypothesized to begin with an abnormal response to antigenic stimulation mediated by toll-like receptors (TLRs) and interleukin IL-6 receptors and IL-1β , 24. Thiβ . When itβ .Dysregulation of microRNAs (miRNAs), the small noncoding RNAs involved in posttranscriptional gene regulation, plays a role in MGUS progression to MM . MiRNAs FMR1 mRNAs that leads to global miRNA dysregulation [MiRNAs are encoded throughout the genome and are transcribed into pri-miRNA. The pri-miRNA is cleaved in the nucleus by the Drosha RNase III endonuclease. Pre-miRNAs are transported to the cytoplasm. In cytoplasm, Dicer, a ribonuclease III, cleaves the pre-miRNA into the mature miRNA which forms a complex with RNA-induced silencing complex (RISC). This complex directs the miRNA to the target mRNA leading either to degradation of target mRNA or translational repression . In FXTAgulation . We specFMR1 mRNA level of 2.33 ± 0.02 times normal. His intention tremor began at age 55 and has been progressive. Currently, it interferes with his activities of daily living (ADL) including eating and handwriting. His gait imbalance began at age 58 and he started using a walker at age 66. His short-term memory problems began at age 63. His stamina has decreased significantly over the last few years and he has some decreased sensation in his lower legs in addition to pain with walking. He has autonomic dysfunction including bladder incontinence and erectile dysfunction beginning at age 66. His past medical history includes hypertension, type 2 diabetes, hyperlipidemia, hypothyroidism, sleep apnea, and migraine headaches (A 69-year-old Caucasian man with FXTAS had a premutation of 98 to 103 CGG repeats and an eadaches .His family history includes a carrier daughter with fragile X-associated primary ovarian insufficiency (FXPOI). His brother has had severe FXTAS with two surgeries for deep brain stimulation. His mother, a carrier, has also had tremor, cognitive decline, and non-Hodgkin's lymphoma and it is likely that she also has FXTAS.On physical examination, his blood pressure was 167/62 mmHg, the heart rate was 71 bpm, the head circumference was 59.5 cm, his height was 172.7 cm, and his weight was 87.5 kg. The ears were slightly prominent. His neurological examination was abnormal with a severe intention tremor with finger to nose touching but absent at rest. There were no deep tendon reflexes but positive primitive reflexes including the snout reflex. His vibration sense was decreased in both feet. His gait was ataxic and broad based. He cannot tandem walk and he had a positive pull test. On MRI, he had significant brain atrophy and white matter disease including a positive MCP sign.As a part of a workup for his mild normocytic anemia, serum protein electrophoresis with immunofixation revealed 1 g/dL IgG kappa monoclonal paraproteinemia. His serum creatinine was mildly elevated at 1.3 mg/dL with estimated GFR of 55 mL/min. Calcium and highly sensitive C-reactive protein were within the normal reference range. The quantitative immunoglobulins revealed normal IgG and IgA levels with decreased IgM . The free kappa light chain is elevated at 7.81 mg/dL (upper reference range 1.92 mg/dL) with normal lambda light chain (1.88 mg/dL). The kappa to lambda light chain ratio was elevated at 4.15 (upper reference range 1.65). The beta-2 microglobulin is elevated at 3.38 mcg/mL (upper reference range 1.8 mcg/mL). The skeletal survey did not reveal any lytic or sclerotic lesions. The bone marrow biopsy revealed a normocellular marrow (30% cellularity) with less than 5% kappa light chain restricted plasma cells . This is the first clinical report of MGUS in an individual with FXTAS. Although MGUS is an asymptomatic condition, it is associated with peripheral neuropathy, osteoporosis, and thromboembolic events. Symptoms shared by FXTAS and MGUS include peripheral neuropathy. His risk factors for developing an MGUS included advancing age, male sex, and having a mother with non-Hodgkin's lymphoma, a hematologic cancer. We also suggest that those with FXTAS have a greater risk for MGUS through the common finding of miRNA dysregulation in both disorders.The DROSHA/DGCR8 sequestration mechanism in FXTAS leads to miRNA alteration, specifically downregulation of several miRNAs . Thus, iFMR1 transcriptional activity [It is unknown whether there is an increased risk of having cancer in those with a gene premutation, although there is one case report of a man with lung tumor and full mutation where the tumor's origin was in a cell with premutation instead of full mutation, indicating that the neoplasia development may have been facilitated by the premutation . There iactivity  and may activity .The current report of MGUS and FXTAS combined with the commonality of miRNA mechanisms in these two conditions suggests that this association may be more common than previously realized and this deserves further study. One possibility is screening for the premutation when MGUS is identified. This would be very important if FXTAS symptoms are present or if there is a family history of fragile X-associated disorders including FXPOI or fragile X syndrome or autism.