We made a molecular study of Zungaro populations in an attempt to

We made a molecular study of Zungaro populations in an attempt to solve taxonomical inconsistencies and to analyze genetic diversity in natural populations of this genus. We analyzed two regions of the mitochondrial DNA (the control region and the ATPase 6 gene region) of individuals sampled from the Parana-Paraguay MLN8237 concentration River and Amazon River basins. Analyses based on p-distances and maximum likelihood phylogenetic models showed a genetic difference between populations corresponding to different species. Genetic differentiation between Zungaro populations was at the same level as that observed between other Siluriformes species, using the same DNA sequences. We conclude

that Zungaro species of the Parana-Paraguay River basin do not belong to the same species found in the Amazon basin. This finding has a significant implication for conservation of this fish, given that populations are disappearing at a high rate in the Parana-Paraguay River basin, mainly due to impoundments.”
“Iron I-BET-762 chlorosis is one of the major abiotic stresses affecting fruit trees and other crops in calcareous soils and leads to a reduction in growth and yield. Usual remediation strategies consist of amending iron to soil, which is an expensive practice, or using tolerant

cultivars, which are difficult to develop when not available. To understand the mechanisms underlying the associated physiopathy better, and thus develop new strategies to overcome the problems resulting from iron deficiency, the differential gene expression induced by iron deficiency in the susceptible citrus rootstock Poncirus trifoliata (L.) Raf. have been examined. The genes identified are putatively involved in cell CH5183284 clinical trial wall modification, in determining photosynthesis rate and chlorophyll content, and reducing oxidative stress. Additional studies on cell wall morphology, photosynthesis, and chlorophyll content, as well as peroxidase and catalase activities, support their possible functions in the response to iron deficiency in a susceptible

genotype, and the results are discussed.”
“The thermoelectric properties of Magneli phase titanium oxides Ti(n)O(2n-1) (n=2,3,…) have been investigated, paying special attention to how the thermoelectric performance can be altered by changing the microstructure. Dense polycrystalline specimens with nominal composition of TiO(2-x) (x=0.05, 0.10, 0.15, and 0.20) prepared by conventional hot-pressing are all identified to be one of the Magneli phases, in which crystallographic shear planes are regularly introduced according to the oxygen deficiency. Electrical conduction is n-type for all specimens and the carrier concentration increases with the increase in the oxygen deficiency. The values of lattice thermal conductivity, on the other hand, decrease with the increase in the oxygen deficiency, which can be attributed to phonon scattering at the crystallographic shear plane.

Women working 1-19h/week at baseline also had higher functional w

Women working 1-19h/week at baseline also had higher functional well being compared with women who were not working. There was a significant, positive association between hours worked per week and physical and social well being. At the 6-month follow-up, women working at least selective HDAC inhibitors 20h/week had higher physical and functional well being than those who were not working. Lower scores for physical and functional well being were observed among women who stopped working during the 6-month follow-up period. Conclusions Continuing to work after a breast cancer diagnosis may be beneficial to multiple areas of QOL. Strategies to help women continue working through treatment should be explored. Copyright (c) 2012

John Wiley & Sons, Ltd.”
“Neuromuscular disease or peripheral nerve damage can interrupt muscle contraction, but tissue engineered LY3039478 in vivo constructs can be created to combat this problem. Electrospinning provides a way to create a degradable nonwoven mesh that can be used to culture cells and tissues. Conductive polymers can be blended with other polymers to provide an electrical current to increase cell attachment, proliferation, and migration. We electrospun several polyaniline and poly(D,L-lactide) (PANi/PDLA) mixtures at different weight percents including the following PANi-PDLA solutions (w/v): 24% (83% PDLA/17% PANi), 24% (80% PDLA/20% PANi), 22% (75%PDLA/25% PANi), 29% (83% PDLA/17%

PANi), and 29% (80% PDLA/20% PANi). Only the 75/25 electrospun scaffold was able to conduct a current of 5 mA. The calculated

electrical conductivity for this scaffold was 0.0437 S/cm. Primary rat muscle cells were cultured on all three of the scaffolds and on tissue culture polystyrene as a positive control. Although the scaffolds degraded during this process, cells were still able to attach and proliferate on each of the different scaffolds. The cellular proliferation measurements showed no significant difference between the four groups measured. The conductivity and cellular behavior demonstrate the feasibility of fabricating a biocompatible, biodegradable, and electrically conductive PDLA/PANi scaffold. (C) 2009 Wiley Periodicals, Inc. J Appl Polym Sci 115: 1566-1572, 2010″
“Seed coat colour in common bean (Phaseolus vulgaris eFT-508 solubility dmso L.) is determined by activity of the flavonoid biosynthetic pathway resulting in the presence or absence of specific anthocyanins, tannins and glycosidic flavanols. These secondary metabolites have anti-oxidant properties in the case of anthocyanins and glycosidic flavonols and strongly influence dietary mineral bioavailability in the case of tannins. The modification of tannin content is a goal of biofortification breeding programs, while almost all bean improvement considers seed colour in selection priorities as this effects consumer preference and food quality.

Intervention: The surgical approaches used included preauricular

Intervention: The surgical approaches used included preauricular infratemporal fossa, postauricular infratemporal fossa, and pterional infratemporal fossa.

Main Outcome Measures: The most critical assessment selleck compound measures were postoperative patient morbidity and evidence of disease at follow-up visits.

Results: The surgical approach was tailored according to the radiographic mapping of the size and location of the tumor. Trismus and jaw discomfort were the most common postoperative complications. Conductive hearing loss occurred

only in patients who underwent oversewing of the cartilaginous ear canal.

Conclusion: The presenting signs and symptoms of extremely rare https://www.selleckchem.com/products/gsk-j4-hcl.html primary TMJ tumors are those often seen by otolaryngologists. Contemporary neurotologic skull base surgical techniques can optimize the successful extirpation of these lesions.”
“Short-lasting unilateral neuralgiform headache attacks with conjunctival injection and tearing (SUNCT) is a syndrome of intermittent, brief, unilateral, severe paroxysms of orbital-temporal pain recurring multiple times per day. The pain modulation is often very difficult. The reported SUNCT patient is the first who responded to a combination treatment of

oxcarbazepine and gabapentin.”
“Childhood acute leukaemia (AL) accounts for a third of childhood cancers. Analysing the spatial distribution of the incidence of AL and its lymphoblastic Ganetespib and myeloblastic subtypes may contribute to the identification of risk factors. This national registry-based study aimed to evaluate global spatial heterogeneity in the incidence rates of AL and to detect

clusters in France over the period 1990-2006 on the ‘living-zone’ scale. Between 1990 and 2006, 7675 cases of AL were registered in the National Registry of Childhood Haematopoietic malignancies. Their spatial distribution in the 1895 ‘living zone’ was first evaluated with two tests for global spatial heterogeneity (Potthoff-Witthinghill and Rogerson’s tests) and then with the SaTScan and FleXScan methods, which aim to locate spatial and space-time clusters. Over 1990-2006, no spatial heterogeneity of AL or its subtypes was evidenced. In addition, none of the most likely clusters identified with SaTScan and FleXScan over the whole period was significant, and the systematic search for space-time clusters yielded nonsignificant results. However, when three subperiods were considered, five statistically significant nonoverlapping spatial clusters were identified. This study did not find evidence of any global spatial heterogeneity of AL incidence rates in France over the period 1990-2006. Although no significant spatial cluster was detected over the whole period, the study identified a few significant spatial clusters in specific periods.

“We previously reported that low-dose X-irradiation allevi

“We previously reported that low-dose X-irradiation alleviates ischemia-reperfusion injury such as mouse paw edema. In this study, we examined active changes in the biological function of mouse liver grafts in cold storage after low-dose X-irradiation. Mouse livers were sham-irradiated or were irradiated with 0.25, 0.5, 1.0, or 5.0 Gy of X-ray and stored for 4, 8, 24, or 48 h in preservation

or saline solution. The results show that storage for 24 h in saline solution after 0.5 Gy irradiation significantly increased 17DMAG clinical trial the activity of superoxide dismutase (SOD) and catalase. Following storage for 4, 8, or 48 h in preservation solution, lipid peroxide levels of the 0.5 Gy irradiated group were significantly lower than those of the sham irradiated group. Following storage for 24 h in preservation solution, the activity of SOD and catalase of the 1.0 Gy irradiated group were significantly higher than those of the sham irradiated group. Hepatocytes stored in saline solution were vacuolated. However, no vacuole formation was observed in hepatocytes stored in preservation solution. These findings suggest that low-dose irradiation significantly activates antioxidative functions of liver grafts. Moreover, the dose at which enhancement of antioxidative function occurs in livers stored in preservation solution, which

contains glutathione, is significantly higher than that in saline solution.”
“Case Description-An 8-month-old crossbred dog was evaluated because of an oronasal fistula following 2 previous attempts to surgically correct a secondary palate defect.

Clinical Findings-Three months after initial evaluation DNA Damage inhibitor and successful closure of the soft palate section of the secondary palate defect CX-6258 JAK/STAT inhibitor via 2 surgeries, an oronasal fistula was present. The oronasal fistula was predominantly right sided, involving the rostral third of the hard palate and, at the widest aspect, spanning the transverse palate at the level of the maxillary canine teeth.

Treatment and Outcome-Following CT of the

rostral aspect of the skull, rapid prototyping technology was used to create a stereolithographic model of the skull, allowing fabrication of a customized titanium plate for intranasal stenting. The titanium plate was inserted via a rostral nasal approach and secured rostrally with 1.5-mm screws and caudally with 2.4-mm screws from the nasal bridge. An oronasal fistula formed laterally at the edge of the plate, but bonding of a dental glass ionomer product resulted in successful resolution of the fistula.

Clinical Relevance-Results suggested that rapid prototyping and creation of customized implants may provide an option for the management of large or nonreconstructible oronasal defects in dogs. (J Am Vet Med Assoc 2012;240:1316-1322)”
“The neurodegenerative disorder Alzheimer’s disease (AD) is the most common form of dementia. It is characterized by progressive impairment of cognitive functions and behavior.

After the characterization studies, the in vitro dissolution stud

After the characterization studies, the in vitro dissolution studies of these tablets in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were carried out. To evaluate the in vivo bioavailability, the tablets selleck chemical were administered orally to rabbits and drug levels in serum were determined by HPLC. Tablets containing the cyclodextrin complex showed a higher in vitro dissolution rate and bioavailability compared to the tablets containing KET alone.</.”
“We conducted this population-based cohort study by linking several databases to explore the role of socioeconomic position for accessing and keeping antihypertensive drug therapy. A total of 71 469 patients, residents

in the city of Milan (Italy) aged 40-80 Z-IETD-FMK chemical structure years, who received an antihypertensive drug during 1999 2002 were followed for 1 year starting from the first dispensation. Socioeconomic position and drug prescriptions were respectively obtained from tax registry and outpatient prescription database. The effect of socioeconomic characteristics on standardized incidence rate (SIR) of new users of antihypertensive agents, odds ratio (OR) of using combined

antihypertensive agents and non-antihypertensive drugs and hazard ratio (HR) of discontinuing antihypertensive therapy were estimated after adjustment for potential confounders. SIRs were 3.7 and 4.2 per 1000 person-months among persons at the lowest and intermediate income, respectively, and Wnt activation 2.4 and 3.0 among immigrants and Italians, respectively. Compared to persons

at the highest income, those at the lowest income had increased chances of starting with combined antihypertensive drugs (OR: 1.1; 95% confidence intervals (CIs): 1.0, 1.2), and of using drugs for heart failure (OR: 1.5; CIs: 1.3, 1.6) and diabetes (OR: 1.7; CIs: 1.6, 1.9). Compared with Italians, non-western immigrants had increased chances of starting with combined antihypertensive agents (OR: 1.2; CIs: 1.0, 1.3), of using drugs for heart failure (OR: 1.2; CIs: 1.0, 1.4) and for diabetes (OR: 1.8; CIs: 1.6, 2.1), and of interrupting antihypertensive therapy (HR: 1.1; 95% CIs: 1.0, 1.2). Despite the universal health coverage of the Italian National Health Service (NHS), social disparities affect accessing and keeping antihypertensive therapy.”
“BACKGROUND The use of ablative laser treatments is steadily increasing. A side-effect is plume, which can potentially transmit infectious material. Insufficient plume removal may lead to transmission of infectious diseases.

OBJECTIVE To introduce a newly developed circular suction technique for ablative interventions.

MATERIALS AND METHODS The new plume removal system consists of a circular master tube and four smaller suction tubes. This design guarantees plume removal around the whole treatment area. This system can be connected in principle to any common aspiration device.

Two were interventional, four were longitudinal, and the rest wer

Two were interventional, four were longitudinal, and the rest were cross-sectional studies. Four studies were qualitative while the rest used quantitative measures. Variance Vorinostat clinical trial in research designs and coping instruments led to inconsistent results. The most common theme was that emotion-focused

coping was associated with worse psychological outcomes, while the effect of problem-focused coping was less consistently associated with better psychological outcomes.

Conclusions: More longitudinal and interventional studies are needed to causally link coping strategies with psychological outcomes in IBD patients (C) 2012 European Crohn’s and Colitis Organisation. Published by Elsevier B.V. All rights reserved.”
“Glucagon-like peptide 1 (GLP-1) is a hormone secreted predominantly by the distal small

intestine and colon and released in response to enteral nutrient exposure. GLP-1-based therapies are now used widely in the management of type 2 diabetes and have the potential to be effective antiobesity agents. Although widely known as an incretin hormone, there is a growing body of evidence that GLP-1 also acts as an enterogastrone, with profound effects on the gastrointestinal Selleckchem Quisinostat motor system. Moreover, the effects of GLP-1 on gastrointestinal motility appear to be pivotal to its effect of reducing postprandial glycaemic excursions and may, potentially, represent the dominant mechanism. This review summarizes current knowledge of the enterogastrone properties of GLP-1, focusing on its effects on gut motility at physiological and pharmacological concentrations, and the motor actions of incretin-based therapies. While of potential importance, the inhibitory action of GLP-1 on gastric acid secretion is beyond the scope of this paper.”
“Arabidopsis IRX10 and IRX10-LIKE (IRX10-L) Selleckchem LY3023414 proteins are closely related members of the GT47 glycosyltransferase family. Single gene knock-outs of IRX10 or IRX10-L

result in plants with either a weak or no mutant phenotype. However irx10 irx10-L double mutants are severely affected in their development, with a reduced rosette size and infrequent formation of a small infertile inflorescence. Plants homozygous for irx10 and heterozygous for irx10-L have an intermediate phenotype exhibiting a short inflorescence compared with the wild type, and an almost complete loss of fertility. Stem sections of the irx10 homozygous irx10-L heterozygous or irx10 irx10-L double mutants show decreased secondary cell-wall formation. NMR analysis shows that signals derived from the reducing end structure of glucuronoxylan were detected in the irx10 single mutant, and in the irx10 homozygous irx10-L heterozygous combination, but that the degree of polymerization of the xylan backbone was reduced compared with the wild type.

Conclusions Comparison of eGFR calculated by MDRD and CKD-EPI eq

Conclusions. Comparison of eGFR calculated by MDRD and CKD-EPI equations demonstrated no clinically relevant difference. In addition, CKD classification by both equations agreed highly. As both equations performed equally well, the simpler and more established MDRD equation should be preferred in patients with or at high risk of CKD managed in primary selleck chemicals care. Patients’ characteristics seem to account for previously reported differences in the performance

of CKD-EPI and MDRD equations.”
“We have developed new high efficiency particulate absorbing filter materials by bonding the fiber web with the help of high pressure water jets emerging from micron sized nozzles and subsequently coating the filters with a chemical binder. Two different types of

nonwoven filters are produced by varying the water jet pressure during the bonding process. The performance characteristics of the filter materials are evaluated in terms of filtration parameters, such as filtration efficiency, dust holding capacity, and pressure drop. Filtration efficiency SNX-5422 depends on the pore characteristics, namely pore size and their distribution in the filters. The developed filter materials have shown promising performance characteristics by capturing higher amount of dust particles with a relatively low pressure drop during use. These filter materials can be used for a wide range of industrial applications, where high filtration efficiency is required at low energy consumption. A fluid flow simulation is carried out by computational fluid dynamics (CFD) to understand flow pattern during the bonding process. The CFD is also used to predict the pressure drop in the nonwoven filter

materials during filtration process. (C) 2009 Wiley Periodicals, Inc. J Appl Polym Sci 114: 275-280, 2009″
“Osteopetrorickets is a rare autosomal recessive disorder of osteoclast function characterized by abnormally dense bone and failure of resorption of calcified cartilage. Rickets is a paradoxical complication of osteopetrosis, resulting from the inability of the osteoclasts to maintain a normal calcium-phosphorus balance in the extracellular fluid. We report a patient with an unusual case of infantile PD98059 mw osteopetrorickets who was admitted with anterior fontanel bulging and was treated with haploidentical bone marrow transplantation.”
“Objective. Few studies have linked changes in bone mineral density (BMD) in peritoneal dialysis (PD) patients to the calcium concentration in peritoneal dialysate. Material and methods. In total, 236 incident patients who underwent an annual BMD determination for 2 years were enrolled. The patients were divided into two groups: the standard calcium dialysate (SCD) group (n = 190; 1.75 mmol/l) and the low-calcium dialysate (LCD) group (n = 46; 1.25 mmol/l). Results.

MeasurementsChange in self-reported

ADHD symptoms, relaps

MeasurementsChange in self-reported

ADHD symptoms, relapse to any drug use (amphetamine and other drugs) measured by urine toxicology, retention to treatment, craving and time to relapse.

FindingsThe MPH-treated group reduced their ADHD symptoms during the trial (P=0.011) and had a significantly higher proportion of drug-negative urines compared with the placebo group (P=0.047), including more amphetamine-negative APR-246 urines (P=0.019) and better retention to treatment (P=0.032).

ConclusionsMethylphenidate treatment reduces attention deficit hyperactivity disorder symptoms and the risk for relapse to substance use in criminal offenders with attention deficit hyperactivity disorder and substance dependence.”
“Objective: To formulate and evaluate solid-reversed-micellar-solution (SRMS)-based solid lipid microparticles (SLMs) for intramuscular administration of gentamicin.

Methods: SRMS formulated with Phospholipon (R) 90G and Softisan (R) 154 were used to prepare gentamicin-loaded SLMs. Characterizations based on size and morphology, stability and encapsulation efficiency (EE%) were carried out on the SLMs. In vitro release of gentamicin from the SLMs was performed in phosphate

buffer while in vivo release studies were conducted in rats.

Results: Maximum EE% of 90.0, 91.6 and 83.0% were obtained for SLMs formed with SRMS 1: 1, 1: 2 and 2: 1, respectively. Stable, PLX4032 purchase spherical and smooth SLMs of size range 9.80 +/- 1.46 mu m to 33.30 +/- 6.42 mu m were produced. The release of gentamicin in phosphate buffer varied widely with the lipid contents. Moreover, significant (p < 0.05) amount of gentamicin was released in vivo from the SLMs.

Conclusion: SRMS-based SLMs would likely offer a reliable means of delivering gentamicin intramuscularly.”
“The accurate and reliable estimation of fiber orientation distributions,

based on diffusion-sensitized magnetic resonance images is a major prerequisite for tractography algorithms or any other derived statistical analysis. In this work, we formulate the principle of fiber continuity C188-9 (FC), which is based on the simple observation that the imaging of fibrous tissue implies certain expectations for the measured images. From this principle we derive a prior for the estimation of fiber orientation distributions based on high angular resolution diffusion imaging (HARDI). We demonstrate on simulated, phantom, and in vivo data the superiority of the proposed approach. Further, we propose another application of the FC principle, named FC flow, a method to resolve complex crossing regions solely based on diffusion tensor imaging (DTI). The idea is to infer directional information in crossing regions from adjacent anisotropic areas.”
“Objectives: Correlate adenoid size as determined by lateral neck radiographs and intra-operative mirror exam. Determine if a radiologist’s assessment of the lateral neck X-ray correlates with adenoid size.

Past-month binge prevalence of 18 18% was considerably lower than

Past-month binge prevalence of 18.18% was considerably lower than the past-year prevalence of 33.51%, but change from past-month to past-year binge threshold had no significant effect on the demographic composition of binge drinkers. The standard AUDIT-C threshold over-estimates hazardous drinking IWR-1-endo research buy prevalence in older adults by up to 33%, but even the most conservative rates in this study are cause

for concern regarding the level of drinking by older people in New Zealand.

Conclusion: Older hazardous drinkers are predominantly younger, wealthier, white, partnered males, whichever threshold is used, but binge drinkers are more likely to be rural. Maori, and lack tertiary education. Further efforts are needed to determine factors underpinning hazardous drinking, especially in older Maori. (C) 2011 Elsevier Ireland Ltd. All rights reserved.”
“Andrographolide (AGP), the major phytoconstituent isolated from Andrographis paniculata was found to exhibit growth inhibition and cytotoxicity against the hormone-independent (PC-3 and DU-145) and hormone-dependent (LNCaP) prostate cancer cell lines via the microculture tetrazolium (MTT) assay. Due to its greater cytotoxic potency and selectivity towards PC-3 cells, flow cytometry was used to analyze the cell cycle distribution of control and treated PC-3 cells whereas Annexin V-FITC/PI see more flow cytometry analysis was carried out to confirm apoptosis induced by AGP in

PC-3 cells. Cell cycle and apoptotic regulatory proteins were determined by western blot analysis. AGP was found to induce G2/M cell cycle arrest which led to predominantly apoptotic mode of cell death. Mechanistically, AGP was found to downregulate CDK1 without affecting the levels of CDK4 and cyclin D1. Induction of AGP was associated with an increase CDK activity in activation and expression of caspase 8 which then is believed to have induced cleavage of Bid into tBid. In addition, activation and enhancement of executioner

caspase 9 and Bax proteins without affecting Bcl-2 protein levels were observed.”
“Whether antiplatelet therapy is associated with better outcomes among patients with infective endocarditis (IE) remains controversial. A retrospective study was conducted concerning all patients with IE, treated in a tertiary-care centre of Canada between 1991 and 2006, who satisfied the modified Duke criteria for a definite or possible IE. The primary outcome was all-cause mortality within 90 days of diagnosis. A secondary outcome was the development of major systemic embolism. In total, 241 patients satisfied the inclusion criteria, 75 of whom had been on chronic antiplatelet therapy prior to developing endocarditis. Seventy-one (29.5%) patients died. According to multivariate analysis, age, a high Charlson score, aortic valve involvement, myocardial infarction and presence of a perivalvular abscess were strongly associated with mortality. Undergoing valvular replacement (adjusted OR (AOR) 0.28, 95% CI 0.09-0.

There were no between-group differences in segment areas or circu

There were no between-group differences in segment areas or circumferences. Reaction time correlated with midbody circumference for boys with ADHD and isthmus circumference for girls with ADHD. For the entire cohort, rostral body circumference correlated with intrasubject variability. Impaired response control in ADHD is associated with anomalies in frontal interhemispheric connections.

Future studies examining callosal shape will illuminate the anatomic basis of correlations between callosal segment circumference and response control.”
“The topic of drug safety has received great attention see more in recent years. Pharmacoepidemiology is the study of the use and effects of medicines in large populations using epidemiological methods. Pharmacoepidemiologic JNK activity research can fill the knowledge gaps due to the limitations of existing pharmacovigilance systems that rely on randomised controlled trials and voluntary reporting. This review discusses the present state of pharmacoepidemiologic research in Australia. In Australia, linking administrative

data on use of medications and medical services is possible to a certain extent. Data from patient registries with respect to rheumatology are also available. These data are valuable for better understanding of the beneficial and adverse effects of medicines. Opportunities and challenges of using these data sources to address issues from clinical pharmacology learn more are also highlighted. Australia is well-placed internationally to make major contributions to the knowledge base of outcomes of medicines in the real-world setting. Developments in pharmacoepidemiology are critical

to clinicians treating patients with rheumatic and other conditions.”
“This paper studies an ultrasonic wave dispersion characteristics of a nanorod. Nonlocal strain gradient models (both second and fourth order) are introduced to analyze the ultrasonic wave behavior in nanorod. Explicit expressions are derived for wave numbers and the wave speeds of the nanorod. The analysis shows that the fourth order strain gradient model gives approximate results over the second order strain gradient model for dynamic analysis. The second order strain gradient model gives a critical wave number at certain wave frequency, where the wave speeds are zero. A relation among the number of waves along the nanorod, the nonlocal scaling parameter (e(0)a), and the length of the nanorod is obtained from the nonlocal second order strain gradient model. The ultrasonic wave characteristics of the nanorod obtained from the nonlocal strain gradient models are compared with the classical continuum model. The dynamic response behavior of nanorods is explained from both the strain gradient models. The effect of e(0)a on the ultrasonic wave behavior of the nanorods is also observed.