“In Arabidopsis (Arabidopsis thaliana), jasmonate is a key signal required for stamen and pollen maturation and thus for male fertility. Using transcriptional profiling, we have previously identified a set of 13 transcription factors that are proposed to be involved in controlling jasmonate responses in stamens. This finding suggests that a transcriptional cascade regulates the many developmental and biochemical pathways required to ensure fertility; however, the organization of this cascade is currently not understood. Here, we provide a genetic characterization of the role of MYB108 and map its relationship
to MYB21 and MYB24, two other transcription factors involved in
the jasmonate response in Arabidopsis stamens. Transcriptional profiling and analysis of plants expressing a MYB108:GUS fusion protein demonstrated that MYB108 expression Adavosertib in vivo is largely confined to sporophytic tissues of the stamen. Three allelic myb108 mutants exhibited reduced male fertility that was Cytoskeletal Signaling inhibitor associated with delayed anther dehiscence, reduced pollen viability, and decreased fecundity relative to wild type. These phenotypes were all found to be exacerbated in myb108 myb24 double mutants, which also had shorter stamen filaments. Measurements of MYB108 transcript levels in wild-type and mutant flowers showed that expression of this gene is strongly dependent
on MYB21. Taken together, our results indicate that MYB108 and MYB24 have overlapping functions and act downstream of MYB21 in a transcriptional cascade that mediates learn more stamen and pollen maturation in response to jasmonate.”
“Purpose: To describe the efficacy and safety of using a single corneal graft for 2 different ocular surgeries, Descemet stripping automated endothelial keratoplasty in one patient and coverage of a glaucoma drainage device tube in another patient.\n\nMaterials and Methods: The records of 12 consecutive patients who underwent Ahmed glaucoma valve implantation using the anterior lamella of a donor cornea that had been previously used for Descemet stripping automated endothelial keratoplasty were reviewed.\n\nResults: Nine eyes (75%) had superotemporal Ahmed valve implantation and 3 eyes (25%) had inferotemporal implantation. No intraoperative complications were encountered in any of these cases. During postoperative follow-up there were no graft rejections, wound dehiscences, tube exposures, or any glaucoma drainage device-related complications. In 1 patient, graft thinning was noticed 12 months after surgery, but without erosion of the conjunctiva or exposure of the tube. Mean intraocular pressure (IOP) before surgery was 32.8 +/- 9.3 mm Hg. The mean postoperative IOPs were 13.
These can be easily registered after small changes in the hardware of the CPR compression pads.”
“Insulin stimulation of glucose uptake is achieved by redistribution of insulin-responsive glucose transporters, GLUT4, from intracellular storage compartment(s) to the plasma membrane in adipocytes and muscle cells. Although GLUT4 translocation has been investigated using various approaches, GLUT4 trafficking
properties within the cell are largely Selleck PLX3397 unknown. Our novel method allows direct analysis of intracellular GLUT4 dynamics at the single molecule level by using Quantum dot technology, quantitatively establishing the behavioral nature of GLUT4. Our data demonstrate the predominant mechanism CAL-101 in vivo for intracellular GLUT4 sequestration in the basal state to be “static retention” in fully differentiated 3T3L1 adipocytes. We also directly defined three distinct insulin-stimulated GLUT4 trafficking processes: 1) release from the putative GLUT4 anchoring system in storage compartment(s), 2) the speed at which transport GLUT4-containing vesicles move, and 3) the tethering/docking steps at the plasma
membrane. Intriguingly, insulin-induced GLUT4 liberation from its static state appeared to be abolished by either pretreatment with an inhibitor of phosphatidylinositol 3-kinase or overexpression of a dominant-interfering AS160 mutant (AS160/T642A). In addition, our novel approach revealed the possibility that, in certain insulin-resistant states, derangements in GLUT4 CYT387 mw behavior can impair insulin-responsive GLUT4 translocation.”
“The tachykinin, neurokinin 3 receptor (NK3R) is a g-protein coupled receptor that is broadly distributed in the nervous system and exerts its diverse physiological actions through multiple signaling pathways. Despite the role of the receptor system in a range of biological functions, the effects of NOR activation on chromatin dynamics and gene expression have received limited attention. The present work determined the effects of senktide, a selective NK3R agonist, on chromatin organization, acetylation,
and gene expression, using qRT-PCR, in a hypothalamic cell line (CLU 209) that expresses the NK3R Senktide (1 nM, 10 nM) caused a relaxation of chromatin, an increase in global acetylation of histone H3 and H4, and an increase in the expression of a common set of genes involved in cell signaling, cell growth, and synaptic plasticity. Pretreatment with histone acetyltransferase (HAT) inhibitor (garcinol and 2-methylene y-butylactone), that inhibits p300, p300/CREB binding protein (CBP) associated factor (PCAF), and GCN 5, prevented the senktide-induced increase in expression of most, but not all, of the genes upregulated in response to 1 nM and 10 nM senktide. Treatment with 100 nM had the opposite effect: a reduction in chromatin relaxation and decreased acetylation.
Basidiobolus ranarum usually causes subcutaneous infection, and gastrointestinal manifestations in immunocompetent patients have rarely been reported. It is difficult to diagnose gastrointestinal basidiobolomycosis because of the non-specific CDK inhibitor clinical presentation and the absence of a definite risk factor. This study identified 14 cases of gastrointestinal basidiobolomycosis, all of which were diagnosed after surgery by characteristic histopathological findings. Diagnosis of this disease requires a high index of suspicion in patients presenting with abdominal symptoms, fever, gastrointestinal
mass and eosinophilia accompanied by a high erythrocyte sedimentation rate.”
“The asialoglycoprotein receptor (ASGPR) is a high-capacity galactose-binding receptor expressed on hepatocytes that
binds its native substrates with low affinity. More potent ligands are of interest for hepatic delivery of therapeutic agents. We report several classes of galactosyl analogues with varied substitution at the anomeric, C2-, C5-, and C6-positions. Significant increases in binding affinity were noted for several trifluoromethyl-acetamide derivatives without covalent attachment to the protein. A variety of new ligands were obtained with affinity for ASGPR as ABT-737 inhibitor good as or better than that of the parent N-acetylgalactosamine, showing that modification on either side of the key C3,C4-diol moiety is well tolerated, consistent with previous models of a shallow
binding pocket. The galactosyl pyranose motif therefore offers many opportunities for the attachment of other functional units or payloads while retaining low-micromolar or better affinity for the ASGPR.”
“A Phase II study was started in Japan to evaluate the efficacy and safety of concurrent chemoradiotherapy with S-1 plus cisplatin in patients with unresectable locally Epigenetics inhibitor advanced squamous cell carcinoma of the head and neck. This study began in July 2008, and a total of 45 patients will be accrued from 13 institutions within 2 years. The primary endpoint is the clinical complete remission rate. The secondary endpoints are local progression-free survival, overall survival, progression-free survival, time to treatment failure, proportion of patients who achieve nutritional support-free survival and adverse events.”
“Bile duct injury is a serious complication of laparoscopic cholecystectomy. We report a case of spontaneous hepaticoduodenal fistula following bile duct injury. Initially, Roux-en-Y hepatico-jejunostomy had been planned for the patient, but as the patient did not show any symptoms or findings of biliary obstruction, we preferred a non-operative management. The fistula allowed adequate biliary drainage, and the patient has been followed regularly by the outpatient clinic with good clinical results for approximately five years.
In clinical practice, monoplace or multiplace hyperbaric chambers are used to achieve this. Treatment is usually given as daily 90- to 120-min-long HBO sessions at pressures between 2.0 and 2.5 absolute atmosphere, aiming for 3040 treatment sessions.\n\nThe use of HBO as treatment of diabetic JNK-IN-8 foot ulcers has been founded on weak scientific ground, although
the outcomes from previous studies are in concert with the conclusions from preclinical studies and supports the theoretical framework of HBO reversing hypoxia-induced pathology. Two well-designed randomized double-blind trials have put HBO on firmer ground and may justify adjunctive HBO treatment to a selected group of patients with nonhealing diabetic foot ulcers.\n\nSome health economic studies suggest
potential cost effectiveness, but these studies are limited by deficient primary clinical data and should be interpreted with caution. Several issues remain to be addressed, such as developing robust criteria to improve treatment protocols, determining which patients are likely to benefit, and when to start and stop treatment. Copyright (C) selleckchem 2012 John Wiley & Sons, Ltd.”
“Our postmortem study aimed to determine the impact of suicide on the number of noradrenergic neurons of the locus coeruleus (LC) in suicidal depressive patients. Noradrenergic neurons were shown by immunostaining tyrosine hydroxylase in the LC of 22 non-elderly patients with mood disorders compared to 21 age- and sex-matched normal
controls. Eleven patients were suicide victims and the other eleven died of natural causes. Seven violent suicide victims revealed SBE-β-CD inhibitor an increased number of tyrosine hydroxylase immunoreactive (TH-ir) neurons compared with non-violent suicide victims and controls. No difference was found between the number of TH-ir neurons in all suicidal patients and controls and between non-suicidal patients and controls. The differences of TH-immunoreactivity could neither be attributed to medication nor to the polarity of depressive disorder (unipolar/bipolar). The numbers of TH-ir neurons in suicidal patients correlated negatively with the mean doses of antidepressants. The study suggested a presynaptic noradrenergic dysregulation in the LC related to the level of self-aggression. Traditional antidepressants may, therefore, regulate noradrenergic activity of the LC in suicide patients, however, without demonstrating the suicide-preventing effect.”
“The antiapoptotic proteins of the Bcl-2 family are expressed at high levels in many types of cancer. However, the mechanism by which Bcl-2 family proteins regulate apoptosis is not fully understood. Here, we demonstrate the interaction of Bcl-2 with the outer mitochondrial membrane protein, voltage-dependent anion channel 1 (VDAC1). A direct interaction of Bcl-2 with bilayer-reconstituted purified VDAC was demonstrated, with Bcl-2 decreasing channel conductance.
This response was driven by a p38MAPK-dependent mechanism and was affected by radiation quality and dose. This stress response and elevation of ROS affected genomic instability by distinct pathways. Through interference with p38MAPK activity, we show that radiation-induced stress phenotypes promote genomic instability. In contrast, exposure to physiologically relevant doses of hydrogen peroxide or increasing endogenous
ROS levels with a catalase inhibitor reduced the level of KPT-8602 datasheet genomic instability. Our results implicate persistently elevated ROS following exposure to radiation as a factor contributing to genome stabilization.”
“The stereoselective degradation of the racemic diclofop-methyl in Chinese cabbage and several agricultural soils had been investigated. Both the enantiomers were analyzed by organic solvent extraction and validated by chiral high-performance liquid chromatography-diode array detector (DAD). Cellolose-tris-(3,5-dimethylphenylcarbamate)-based chiral column was used for the chiral separation of the two enantiomers applying a mixture of n-hexane and 2-propanol FK228 research buy (90:10) as mobile phase at a flow rate of 1.0 mL/min.
The elution order of the two enantiomers was distinguished by a JASCO 2000 HPLC-circular dichroism (CD) system and the first elution was (-)-enantiomer while the other was (+)-enantiomer. Racemic diclofop-methyl was fortified into 10 types of agricultural soils and foliar applied to Chinese cabbage. The assay method was linear over a range of concentrations (0.5-250 mg L-1) and the mean recovery was more than 80% for both
enantiomers. The limit of detection for both enantiomers Fosbretabulin in vivo was 0.1 mu g g(-1) in soil and plant samples. The results of the stereoselectivity, degradation of diclofop-methyl enantiomers in soil showed that the (-)-enantiomer was dissipated faster than the (+)-enantiomer in two typical soils and this enantioselectivity may be caused by microorganism, but no stereoselectivity was observed in other soils. In Chinese cabbage plant, (+)-enantiomer accumulated and eliminated faster than its antipode. (C) 2008 Elsevier Inc. All rights reserved.”
“Variable efficacies have been reported for glucocorticoid drugs as anti-inflammatory treatment after stroke. We applied an alternative drug delivery strategy, by injection of dexamethasone phosphate-containing liposomes in combination with recombinant tissue plasminogen activator (rtPA), in an experimental stroke model, and tested the hypothesis that this approach improves behavioral recovery and reduces lesion growth. Rats were subjected to right middle cerebral artery occlusion with a blood clot. After 2 h, animals were intravenously injected with rtPA plus empty long-circulating liposomes (LCL), free dexamethasone phosphate (DXP), or DXP-containing LCL (LCL-DXP).
2072ngml(-1)h, 90% CI = 126.2, 147.5) and peak exposure (C-max) by
5% (247 vs. 236ngml(-1)). Multiple doses of rifampicin increase apremilast clearance approximate to 3.6-fold and decrease apremilast mean AUC(0,) by approximate to 72% (3120 vs. 869ngml(-1)h, 90% CI = 25.7, 30.4) and C-max (from 290 vs. 166ngml(-1)) relative to that of apremilast given alone. A 30min intravenous infusion of rifampicin 600mg had negligible effects on the overall exposure (AUC(0,)) of apremilast (2980 vs. 3120ngml(-1)h, 90% CI = 88.0, 104.1). ConclusionKetoconazole slightly decreased apremilast clearance, resulting in a small increase in AUC which is probably not meaningful clinically. However, the effect of CYP3A4 induction by rifampicin on apremilast clearance is much more pronounced than that of CYP3A4 inhibition by ketoconazole. Strong CYP3A4 inducers may result in a loss of efficacy of apremilast because of decreased drug exposure.”
“Bone www.selleckchem.com/products/netarsudil-ar-13324.html regeneration in the alveolar process and dental implant are widely used and there are a lot of different products. The aim of this research was to know the bone
reparation associated to bone substitute with calcium sulfate and a biological membrane in created defects in rabbit tibiae. Were selected 12 rabbit between 3 and 6 month to be operated; using previous anesthesia protocols was do it a surgical defect in the right and left tibiae with 2.6 mm diameter; four groups were created:
group I with blood clot fill, group II with blood clot fill plus biological membrane, group Nepicastat nmr III with calcium sulfate and group IV with calcium sulfate plus biological membrane. The euthanasia was made in 21 and 42 days before surgery and was obtained histological plate using hematoxillin and eosin. The histomorphometry was made and statistical analysis using ANOVA and Turkey test with p smaller than 0.05 to obtain statistical differences. Were observed in the all created defects a regular bone reparation; the group I and II, with blood clot, showed a minor bone reparation than group III and IV with calcium sulfate, but these last one show an important inflammatory process; the group IV presented the better results at Transmembrane Transporters inhibitor 21 and 42 euthanasia days in term of bone formation. It’s conclude that calcium sulfate can be used in bone reparation of minor defects and the biological membrane can be used in guide bone regeneration with success.”
“Glucose transporter-1 (GLUT-1) is one of the major isoforms of the family of glucose transporter proteins that facilitates the import of glucose in human cells to fuel anaerobic metabolism. The present study was meant to determine the extent of the anaerobic/hypoxic state of the intratumoral microenvironment by staining for GLUT-1 in intracranial non-embolized typical (WHO grade I; n=40), brain invasive and atypical (each WHO grade II; n=38) and anaplastic meningiomas (WHO grade III, n=6).
(C) 2011 American Institute of Physics. [doi: 10.1063/1.3660283]“
“We present an application of a novel DNA separation matrix, cholesterol-bearing pullulan (CHP) nanogels, for microchip electrophoresis. The solution of the CHP showed a unique phase transition around
30 mg/ml and formed gel phase over this critical concentration. This gel phase consists of the weak buy PF-562271 hydrophobic interactions between the cholesterols could be easily deformed by external forces, and thus, loading process of the CHP nanogels into microchannels became easier. The high concentration of the CHP nanogels provided excellent resolutions especially for small DNA fragments from 100 to 1500 bp. The separation mechanism was discussed based on Ogston and Reptation models which had developed in gels or polymer solutions. The result of a single molecule imaging gave us an insight of the separation mechanism and the nanogel structures as well. (C) 2010 American buy Cilengitide Institute of Physics. [doi:10.1063/1.3479997]“
“Recent studies reveal the role of the ankle retinacula in proprioception and functional stability
of the ankle, but there is no clear evidence of their role in the outcomes of ankle sprain. 25 patients with outcomes of ankle sprain were evaluated by MRI to analyze possible damage to the ankle retinacula. Patients with damage were subdivided into two groups: group A comprised cases with ankle retinacula damage only, and Dibutyryl-cAMP in vitro group B those also with anterior talofibular ligament rupture or bone marrow edema. Both groups were examined by VAS, CRTA and static posturography and underwent three treatments of deep connective tissue massage (Fascial Manipulation technique). All evaluations were repeated after the end of treatment and at 1, 3 and
6 months. At MRI, alteration of at least one of the ankle retinacula was evident in 21 subjects, and a further lesion was also identified in 7 subjects. After treatment, VAS and CRTA evaluations showed a statistically significant decrease in values with respect to those before treatment (p < 0.0001). There were also significant improvements (p < 0.05) in stabilometric platform results. No significant difference was found between groups A and B. The initial benefit was generally maintained at follow-up. The alteration of retinacula at MRI clearly corresponds to the proprioceptive damage revealed by static posturography and clinical examination. Treatment focused on the retinacula may improve clinical outcomes and stabilometric data.”
“Antivector immunity may limit the immunogenicity of adenovirus vector vaccines. We tested sera from individuals immunized with adenovirus type 4 and 7 (Ad4 and Ad7, respectively) vaccine or naturally infected with Ad4 for their ability to neutralize a panel of E1-deleted human and chimpanzee adenoviruses (ChAd).
Group C reported significantly improved
scores at final follow-up in all SRS domains and ODI. The group with NIDDM reported improvement in all domains except for the mental health and pain domains. However, there were no significant differences between the group with NIDDM and group C in terms of SRS and ODI scores preoperatively and postoperatively.\n\nConclusion. Contrary to traditional thinking, properly selected NIDDM was not a significant risk factor for perioperative complications or additional surgeries in adult patients with spinal deformities.”
“Many of the skills and resources associated with botanic gardens and arboreta, including plant taxonomy, horticulture, and seed bank management, are fundamental to ecological restoration efforts, yet few of the world’s botanic gardens are involved in the science or practice of restoration. Thus, we examined the potential role of botanic STI571 concentration gardens in these emerging fields. We believe a reorientation of certain existing institutional strengths, such as plant-based research and knowledge transfer,
would enable many more botanic gardens worldwide to provide effective science-based support to restoration efforts. We recommend botanic gardens widen research to include ecosystems as well as species, increase involvement in practical restoration projects and training practitioners, and serve as information hubs Pitavastatin Metabolism inhibitor for data archiving and exchange.”
“Posterior expectation is widely used as a Bayesian point estimator. In this note we extend it from parametric models
selleck products to nonparametric models using empirical likelihood, and develop a nonparametric analogue of James Stein estimation. We use the Laplace method to establish asymptotic approximations to our proposed posterior expectations, and show by simulation that they are often more efficient than the corresponding classical nonparametric procedures, especially when the underlying data are skewed.”
“Background: Prenatal termination of pregnancy may underestimate risks or cause bias in epidemiological studies of birth defects if such studies measure only defects diagnosed postnatally. We aimed to estimate the proportion of all fetuses with birth defects terminated in the second trimester of pregnancy-overall and for specific defects. Methods: The study comprised all pregnancies ending in a singleton birth, miscarriage, or termination of pregnancy for which health care services were sought, as recorded in Danish medical registries between 1 January 2007 and 31 December 2011. Results: Of the 420,090 pregnancies, 307,637 fetuses survived until gestational week 12 or beyond; of these, 296,373 (96%) ended in a live birth or stillbirth and 11,264 (4%) ended in a second-trimester termination. The prevalence of birth defects among live births and stillbirths was 3% (9,790/296,373); the corresponding prevalence among second-trimester-terminated pregnancies was 14% (1,563/11,264).
The moderate decline in in-patient treatments for penile warts in men probably reflects herd immunity.”
“A method was developed for determination of amitraz and its metabolites, N-[2,4-(dimethylphenyl)N'-methylformamidine (DMPF), 2,4-dimethylformamidine
(DMF), 2,4-dimethylaniline (DMA) in whole blood. The analytes were extracted by solid-phase extraction (SPE) using dichloromethane, acetonitrile and methanol (2:1:1) mixture as elute solution. Analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the positive ion mode using multiple reaction monitoring (MRM) technique. Collision-induced dissociation (CID) of amitraz at the electrospray source in MS/MS was observed in the analytic conditions. The method was
validated in human whole blood spiked at three concentration levels. The low limit of detection (LOD) and the low limit of quantification (LOQ) Selleck LOXO-101 for all the analytes were below 0.5 mu g/L and 2 mu g/L, respectively. Recoveries were between 90.2% and 104.5%, Bias and relative standard deviation (RSD) were below 15% (n = 6). The good linear Z-IETD-FMK purchase relationships were obtained in certain concentration ranges of amitraz and its metabolites. The results demonstrated the method is exclusive, sensitive and accurate, and can be applied in forensic toxicology. (C) 2014 Elsevier B.V. All rights reserved."
"The crystal structure of the title compound, Ce[B4O6(OH)(2)](-) Wnt assay Cl, is built from polyborate sheets parallel to the (001) plane. These sheets stack along the  direction and are linked by Ce atoms exhibiting
an CeO8Cl2 coordination sphere. O-H center dot center dot center dot O and O-H center dot center dot center dot Cl hydrogen bonds additionally stabilize the structural set-up. The polyborate sheet is made up of zigzag borate chains running along the [(1) over bar 10] direction. These zigzag chains are interconnected by shared O-vertices, resulting in a two-dimensional layer with nine-membered rings. All B and O atoms (except for the terminal OH atoms) lie in the nearly planar sheets of polyborates, leading to their isotropic atomic displacement parameters being significantly smaller than usual. This may be attributed to the fact that the atomic displacement parameters correlate not only with their atomic masses but with their coordination environments also.”
“The effect of alkali and alkaline earth metal ions on the spermine induced condensates of high molecular weight DNA was studied. The spermine induced DNA precipitation was facilitated by the alkali and alkaline earth metal ions, suggesting that the metal ions instead of getting into competition with spermine in DNA binding, had acted in concert to precipitate DNA from solution.
These dips appear when the separations between nanoclusters are properly adjusted. Such a device may be used to transfer directionally the electromagnetic radiation. In the limit of equal atomic nanoclusters we reproduce the monoatomic chain results (C) 2010
Elsevier Ltd. All rights reserved”
“The inner membrane ring of the bacterial type III secretion system (TTSS) is composed of two proteins. In Chlamydia trachomatis this ring is formed by CdsD (gene name CT_664) and CdsJ (gene name CTA_0609). CdsD consists of 829 amino acids. The last 400 amino acids at its C-terminal end relate it to the type III secretion system YscD/HrpQ protein family. The C-terminal domain, consisting of amino acids 558-771, of C. trachomatis www.selleckchem.com/products/dorsomorphin-2hcl.html CdsD was overexpressed in Escherichia coli and purified using
immobilized metal-affinity chromatography (IMAC) and size-exclusion Ricolinostat concentration chromatography. The protein was crystallized using the vapour-diffusion method. A data set was collected to 2.26 angstrom resolution. The crystals have the symmetry of space group C2, with unit-cell parameters a = 106.60, b = 23.91, c = 118.65 angstrom, beta = 104.95 degrees. According to the data analysis there is expected to be one molecule in the asymmetric unit, with a Matthews coefficient of 3.0 angstrom(3) Da(-1).”
“This work combines the analytical capability of GC x GC/TOFMS, the use of retention indices and adequate software tools for the study of essential oils of Piper regnellii (Miq.) C. DC. (pariparoba) growing wild in “cerrado” landscape, Central-West region, Brazil. The leaves, stems and flowers of P. regnellii generated essential oils with 163, 119 and 110 compounds tentatively identified, respectively. The major compounds
in each essential oil were approximately selleck chemical the same, except dill apiole, which was concentrated more highly in the stems. The major compounds were: myrcene, anethole E and bicyclogermacrene (22%, 19% and 5%, respectively) in leaves, anethole E, dill apiole and myrcene (20%, 19% and 16%, respectively) in stems and anethole E, myrcene and bicyclogermacrene (24%, 18% and 9%, respectively) in flowers. This is the first time that this plant was analyzed by GC x GC/TOFMS and this technique allows identification of a higher number of compounds when compared to traditional one-dimensional chromatography. (C) 2014 Elsevier B.V. All rights reserved.”
“Background. Information has been very limited on the pharmacokinetics of the selective alpha(2)-adrenoceptor agonist dexmedetomidine in children, particularly in children < 2 yr of age.\n\nMethods. Eight children aged between 28 days and 23 months and eight children aged between 2 and 11 yr undergoing either elective bronchoscopy or nuclear magnetic resonance imaging were included in the study. Dexmedetomidine 1 mu g kg(-1) was infused i.v. over 5 min.