Platelet count was significantly lower in PVE patients caused by

Platelet count was significantly lower in PVE patients caused by S. aureus (p = 0.039). HT was more common and hemoglobin level was lower in the S. aureus group, but no statistically significant differences were identified (67% vs. 35%, p = 0.169; 11.2 mg/dL vs. 9.4 mg/dL, p = 0.050, respectively). Table 4 Comparison

between stroke patients caused by PH-797804 mw Staphylococcus aureus and other Inhibitors,research,lifescience,medical organisms Discussion The current study demonstrated that 1) embolic stroke was seen in about a quarter of patients with PVE (26/111 patients, 23%), 2) nearly half of embolic strokes in PVE patients were accompanied by HT (11/26 patients, 42%), 3) in-hospital mortality of PVE patients with embolic stroke was 15% (4/26 patients), 4) in-hospital mortality appeared higher in patients with HT compared to those without HT (27% vs. 7%), although statistically significant differences were not identified likely due to the limited Inhibitors,research,lifescience,medical number of patients identified for study, and 5) predictors for HT of ischemic stroke were not identified from the present study, even though we recruited patients through a multicenter study. Stroke remains a debilitating complication

of left-sided IE in 20-40% of patients and has been associated with poor outcomes.17) In the present study, stroke was seen in 23% of PVE patients, a proportion similar to previous reports of IE patients.9) As a result, Inhibitors,research,lifescience,medical we speculate that the risk of stroke may not be influenced by the type of infected valve (native vs. prosthetic). Not surprisingly, HT was Inhibitors,research,lifescience,medical observed in nearly half of embolic patients as prior evidence demonstrated increased frequency of HT in embolic stroke than in non-embolic stroke patients. Mortality rate for our overall data was 11% and 15% in stroke patients. Interestingly,

mortality rate of stroke patients without HT appeared much lower than stroke patients with HT (7% vs. 37%). Therefore, Inhibitors,research,lifescience,medical stroke following IE of mechanical heart valves might represent a poor prognostic factor, specifically when associated with HT. PT values were not different between stroke patients with and without HT both at the onset of IE and at stroke presentation. Although most of the patients with HT had supratherapeutic CYTH4 PT values (PT/INR > 3), HT also occurred in 2 patients with suboptimal PT levels. Therefore, other factors yet to be uncovered may also be associated with the development of HT in PVE. PT levels remained prolonged even after discontinuing anticoagulation in most of our patients – perhaps due to uncontrolled infection – suggesting that the clinical benefits of stopping anticoagulants in PVE patients with elevated PT values remain unknown. In our population, 8 of 11 stroke patients (73%) were complicated with HT at the time of stroke diagnosis, demonstrating the difficulty in preventing HT by discontinuing anticoagulation. IE caused by S. aureus has the worst prognosis and has a high rate of embolic episodes with subsequent neurologic involvement.

The synthesis of MEL is markedly increased at night in all speci

The synthesis of MEL is markedly increased at night in all species studied to date, independent of whether the animal is diurnally or nocturnally active, and the duration of the nocturnal peak is positively related to the duration of the night.5,6 In nonmammalian vertebrates, the rhythmic synthesis and secretion of MEL is the direct

output of the clock located XL184 within the pineal. Inhibitors,research,lifescience,medical In mammals, however, the pineal does not. retain clock and photoreceptive properties. The synthesis of MEL is driven through multisynaptic neural pathways7,8 by the circadian clock located in the suprachiasmatic nuclei (SCN) of the hypothalamus, and is therefore also an output of the circadian clock. MEL is synthesized Inhibitors,research,lifescience,medical from the amino acid tryptophan, which is first converted into 5-hydroxytryptophan by tryptophan hydroxylase, before being decarboxylated into serotonin (5-hydroxytryptamine, 5-HT). From 5-HT, two major enzymatic steps are involved. The first, is N-acetylation by the arylalkylamine-N-acetyltransferase (AA-NAT) to yield Af-acetylserotonin.The regulation of AA-NAT, with Inhibitors,research,lifescience,medical its sharp increase in activity at night, has received considerable attention as a major regulatory step in rhythmic MEL synthesis.9,10 The second step is

the transfer of a methyl group from 5-adenosylmethionine to the 5-hydroxy group of N-acetylserotonin catalyzed by the hydroxyindolc O-methyltransferase (HIOMT),to yield MEL.11 The rapid Inhibitors,research,lifescience,medical 6-hydroxylation of MEL in the liver means that. it. has a short half-life in the circulation and, therefore, the circulating MEL concentrations precisely reflect its pineal synthesis. MEL is produced primarily by the pineal gland. However, numerous other MEL sources have been identified. The retina

is an important source in nonmammalian vertebrates: not only is MEL rhythmically synthesized in Inhibitors,research,lifescience,medical this structure, but its release from here also contributes to the nocturnal pattern of circulating MEL. In mammals, MEL synthesis within the retina was demonstrated a long time ago.12 However, it is only since the demonstration of a true circadian release of MEL from the hamster retina in vitro (suggesting the presence of a retinal clock) too that the importance of this structure as an extrapincal source of MEL has been recognized.13 In contrast to nonmammalian vertebrates, mammalian retinal MEL does not contribute to circulating MEL. The Harderian and lachrymal glands, gastrointestinal tract, red blood cells, platelets, and mononuclear cells have also been identified as sites of MEL synthesis. MEL does not seem to be released into the general circulation from these tissues, at least, not under normal physiological conditions.14 Moreover, in these tissues the synthesis of MEL is not rhythmic. The presence of MEL is not restricted to vertebrates. MEL has been found in the head, eyes, optic lobe, and brain of various invertebrates in many taxa.

One of the most common reasons for declining HIV screening is lac

One of the most common reasons for declining HIV screening is lack

of perception of risk for HIV infection [25,28,30,32,35,39-41,50,51],[53,58,61-64]. Due to the high prevalence of reported sexual risk and alcohol misuse by ED patients, many techniques have been utilized, with mixed results, to increase uptake of HIV screening, including opt-out HIV screening [36,45,54-57,60,61,65], financial incentives [66], ED staff or clinician-initiated testing [51,54,67], oral fluid sampling for testing [53], prevention counseling [64], and video or computer-based Inhibitors,research,lifescience,medical interventions [43,68,69]. Although a number of studies have examined alcohol misuse, HIV risk, and HIV screening, there is a paucity of research on the intersection of these issues. One approach to improve HIV screening uptake may be to combine alcohol-related and HIV risk interventions in order to increase self-perceived risk and potentially Inhibitors,research,lifescience,medical increase acceptance of screening among ED patients. Before creating such interventions to improve HIV screening uptake and reduce HIV risk and Inhibitors,research,lifescience,medical alcohol misuse, the interrelationships among alcohol misuse, HIV risk and uptake of HIV screening in the absence of interventions among ED patients

need to be established. Our interests in this study were to examine the intersection of alcohol misuse and sexual risk for HIV in its relationship to HIV screening uptake among ED patients. In particular, our objectives were to determine the association between: (1) reported alcohol misuse and HIV sexual risk behaviors; (2) reported alcohol misuse and HIV screening uptake; and (3) reported sexual risk and HIV screening uptake in Inhibitors,research,lifescience,medical the absence

of any interventions. We hypothesized that those who reported greater alcohol use and sexual risk for Inhibitors,research,lifescience,medical HIV would be more inclined to accept HIV screening. Methods Study design and setting From July 2009 to August 2009, 18- to 64-year-old ED patients were randomly selected for inclusion in this study. This investigation had two components: (1) a cross-sectional study examining the prevalence of alcohol misuse and HIV sexual risk among ED patients, (2) and an examination Casein kinase 1 of opt-in HIV screening in this population. The study was conducted at two academic EDs (Rhode Island Hospital and The GSK1363089 in vitro Miriam Hospital) located in Providence, Rhode Island, that are affiliated with the Alpert Medical School of Brown University. Rhode Island Hospital is a level 1 trauma center, receiving over 100,000 annual adult patient visits, and the Miriam Hospital is a community hospital, receiving over 55,000 annual adult patient visits. The Rhode Island Hospital Institutional Review Board approved this study. Verbal consent was obtained for the cross-sectional component of the study, and written consent was obtained for the HIV testing component.

More severe forms of the disorder require progressive sleep phase

More severe forms of the disorder require progressive sleep phase delay in 3-hour steps round the clock until a satisfactory timing is achieved which then has to be fixed. Additional measures to maintain

the improved sleep schedule include early-morning exposure to bright light and firm agreement with the adolescent to maintain a new pattern of social activities and sleep. Melatonin in the evening might also help. Difficulties achieving and maintaining an improved sleep-wake schedule by these means are compounded Inhibitors,research,lifescience,medical if there is a vested interest in maintaining the abnormal sleep pattern, for example, to avoid school (“motivated sleep phase delay”). Psychological problems, including depression, may well also make successful treatment less likely. Inhibitors,research,lifescience,medical The teenager’s reluctance to go to bed earlier and to get up at the required time is often misinterpreted as “typical difficult adolescent behavior” causing trouble in the family. Otherwise the condition might be mistakenly viewed as the usual form of school non attendance, primary depression, or substance misuse. Differential diagnosis of childhood parasomnias Parasomnias are repetitive unusual behaviors or strange experiences

that occur just before, during, or arising Inhibitors,research,lifescience,medical out of sleep, or on waking. The many parasomnias (some primary sleep disorders, others secondary to medical or psychiatric conditions) now officially recognized (over 30 in ICSD-2)

indicate how commonly and in many ways (some subtle, others dramatic) sleep can be disturbed by episodic events. Confusion between the different parasomnias seems to be common. For Inhibitors,research,lifescience,medical example, in pediatric textbook accounts, sleep terrors and nightmares (two very different types of parasomnia) are mistaken for each other. Indeed, sometimes there is a tendency to call any dramatic parasomnia a nightmare. Correct diagnosis is important Inhibitors,research,lifescience,medical because different parasomnias each have their own significance and call for contrasting types of advice and treatment. The following brief account is concerned with the main features to be recognized in reaching the correct diagnosis. Fiiiphasis is placed on just some nearly of the more dramatic parasomnias (namely arousal disorders, nightmares, and sleep-related epileptic seizures) as these often cause most confusion and concern. Detailed accounts of these parasomnias and others are provided elsewhere.24 Often an accurate diagnosis can be made by means of a detailed account of the subjective and objective sequence of events from the onset of the episode to its resolution, and of the circumstances in which the episode occurred, including its duration and timing. Audiovisual recording (including by means of home recording by parents) can be very selleck inhibitor informative and often adds details that are missed in descriptions given in the clinic.

Inertial cavitation is a more violent phenomenon, in which the bu

Inertial cavitation is a more violent phenomenon, in which the bubble grows during the rarefaction phase and then rapidly PD184352 datasheet collapses which leads to its destruction. The collapse is often accompanied by the loss of bubble sphericity and formation of high velocity liquid jets. If the bubble collapse occurs next to a cell, the jets may be powerful enough to cause disruption of the cell membrane Inhibitors,research,lifescience,medical (25),(26). In blood vessels, violently collapsing bubbles can damage the lining of the vessel

wall or even disrupt the vessel altogether. One may assume that the disruption occurs due to bubble growth and corresponding distension of the vessel wall. However, it was shown that most damage occurs Inhibitors,research,lifescience,medical as the bubble rapidly collapses and the vessel wall is bent inward or invaginated, causing high amplitude shear stress (27). Stable cavitation may lead to a phenomenon called “microstreaming” (rapid movement of fluid near the bubble due to its oscillating motion). Microstreaming can produce high shear forces close to the bubble that can disrupt cell membranes and may play a role in ultrasound-enhanced drug or gene delivery when damage to the cell membrane is transient (28). Cavitation activity is the major mechanism that is utilized when mechanical damage to tissue is a goal. At its extreme, when very high rarefactional pressures (> 20 MPa) are used, a cloud of cavitating bubbles can cause

Inhibitors,research,lifescience,medical complete tissue lysis at the focus (29). In such treatments the thermal effect is usually to be avoided, therefore, short bursts of very high amplitude ultrasound of low frequency (usually below 2 MHz) are used. The time-averaged intensity remains low, and the thermal dose delivered to the tissue is not sufficient to cause thermal damage. Cavitation can also promote heating if longer HIFU pulses or Inhibitors,research,lifescience,medical continuous ultrasound is used (30)-(32). The energy of the incident ultrasound wave is transferred very efficiently into stable oscillation of resonant-size bubbles. This oscillatory motion causes microstreaming around the bubbles and that, in turn, leads to additional Inhibitors,research,lifescience,medical tissue heating through

viscous friction, which can lead to coagulative necrosis. Cediranib (AZD2171) Nonlinear ultrasound propagation effects Nonlinear effects of ultrasound propagation are observed at high acoustic intensities and manifest themselves as distortion of the pressure waveform: a sinusoidal wave initially generated by an ultrasound transducer becomes sawtooth-shaped as it propagates through water or tissue (Figure 2D). This distortion represents the conversion of energy contained in the fundamental frequency to higher harmonics that are more rapidly absorbed in tissue since ultrasound absorption coefficient increases with frequency. As a result, tissue is heated much faster than it would if nonlinear effects did not occur. Therefore, it is critical to account for nonlinear effects when estimating a thermal dose that a certain HIFU exposure would deliver.

They do it in order to support

other new patients… I woul

They do it in order to support

other new patients… I would have wished this support to continue, because most of the patients are too weak and it is going to be difficult for them to survive without this service.’ C2 facility G, age 24, patient’s brother Other ways of providing support were also reported, Inhibitors,research,lifescience,medical including kitchen gardening initiatives and cooperative income generation. One man, who was running a small shop selling vegetables he had grown, explained how the service had helped him: ‘[Service staff] advise[d] us to make a group of around 10 people and contribute some little money as members concerned, so

that Inhibitors,research,lifescience,medical when you are in need you go there then get something, or else [they] just teach us some skills–farming, bead making, hand crafts–so that when you are in need of money you either sell cabbages, tomatoes or you just sell your hand crafts as they Inhibitors,research,lifescience,medical taught you.’ P5 facility H, age 38, not on ART 3. Spiritual distress a. Description of spiritual distress Illness was often experienced by patients through the lens of their spiritual or religious beliefs: ‘After some time I started getting serious drug side effects

Inhibitors,research,lifescience,medical [interviewee breaks down in tears]. I could not overcome the drug side effects and I was tempted to stop, probably by an evil spirit. But with prayers I continued taking the drugs.’ P7 facility G, female, age 56, on ART Enzalutamide Although the supportive role of spiritual practices and beliefs was described, patients experienced existential despair and hopelessness and questioned God: ‘My biggest worry is about my future. I have no child Inhibitors,research,lifescience,medical enough and my dreams are shuttered down and it hurts lots. It is an innermost pain which I can’t explain to anybody. When you see people who have died because of this disease and yet they have been on ARVs and yet they die mysteriously… I feel weighed down and lose hope most often.’ P3 facility L, male, age 36, on ART This was reiterated by staff: ‘At times when people get sick you realise that there is that tension or tendency of saying that God is away from me because if it were true that God is alive then he would not have let me fall sick in the way that I am. So you will find that there is that kind of antagonism between him and his God.

Home care organizations engage several home care providers, defi

Home care organizations engage several home care providers, defined according to their educational level as registered (home care) nurses, certified nurse assistants and home helps. To facilitate the readability of this paper, we will use the term “nurses” for all these home care providers. Some home care organizations have teams of district selleckchem nurses specialized in palliative care and/or home care technology but in the Netherlands palliative care is part of regular health care, so most clients will be cared for by educated nurses with at least Inhibitors,research,lifescience,medical three or four years of general professional education. Often these nurses

cooperate with the family GP, because he is responsible for the medical care of his patients living at home. The general practitioner often refers patients to home care, although in the Inhibitors,research,lifescience,medical Netherlands a formal needs assessment by an independent assessment agency first has to

be conducted before home care financed via the national insurance can be delivered. In other countries terminally ill patients might be referred to specialist palliative care, but the approach of Dutch government is that palliative care should be part of regular health care, in most cases provided by generalists (GPs and nurses) [17]. In order to guarantee the provision of good palliative Inhibitors,research,lifescience,medical care, GPs and nurses can consult multidisciplinary specialist consulting teams of the Comprehensive Cancer Centres in the Netherlands. Moreover Inhibitors,research,lifescience,medical palliative care has become an important subject in the primary curriculum of GPs and at post-curricular courses for doctors and nurses organized for instance by the Comprehensive Cancer Centres. The definition of Turkish or Moroccan migrants is not dependent on their formal nationality; they might have adopted the Dutch nationality. In this paper we define Turkish or Moroccan migrants as persons who live in the Netherlands but who were born in Turkey or Morocco respectively, or whose parents (or one of their parents) were born in Turkey or Morocco. We defined a ‘terminally ill client’ as a client suffering from an incurable illness and having a probable life

expectancy of not more than six months. Fossariinae The Inhibitors,research,lifescience,medical central terms ‘home care’, ‘terminally ill clients’ and ‘Turkish and Moroccan clients’ were clearly defined in our questionnaires. Participants In order to recruit nurses and general practitioners who have experience of home care for terminally ill Turkish and Moroccan patients we first established where these migrants are mainly living. Using data from Statistics Netherlands we identified the 30 areas with the largest number of Turkish and Moroccan inhabitants. In order to find general practitioners with experience with these migrant groups, we looked for the addresses of all GP practices located in the 30 relevant areas. As their number was far too large, we searched for addresses of practices within the urban areas with indications of social and economic deprivation in the 30 relevant areas.

Strengths and limitations The present study benefited from a lar

Strengths and limitations The present study benefited from a large sample relative to the fMRI literature. It extends the literature by simultaneously examining

anxious apprehension and anxious arousal, as opposed to examining only one anxiety type or ignoring the distinction. Given that these anxiety types are correlated, inferences based on the measurement of only one anxiety type may not reflect effects specific to that Inhibitors,research,lifescience,medical anxiety type. The study also benefited from statistically controlling comorbid depression, which often confounds studies of anxiety due to shared general distress. Additionally, the present study examined habituation at the level of Syk inhibitor neural activation, which can reveal effects that may cancel out at the level of behavior. However, present findings should be interpreted in the context of some limitations. First, although the present study purposefully

chose Inhibitors,research,lifescience,medical stimuli that would not elicit an extremely strong fear response, in order to foster habituation, the stimuli may not be as relevant to pathological anxiety as stimuli that evoke a much stronger fear response (e.g., spiders for individuals with spider Inhibitors,research,lifescience,medical phobia). Second, it is unclear whether the stimuli used in the present investigation were experienced as threatening, and this may have limited the level of fear experienced by participants. Future research could explore a variety of stimuli that may engage fear

more strongly. Third, the present study interprets the observed habituation in Broca’s Inhibitors,research,lifescience,medical area as reflecting habituation in worry. However, it is possible that the activation in Broca’s area observed in the present study reflects processes other than, or in addition to, worry. For example, it is possible that Broca’s area activation better reflects Inhibitors,research,lifescience,medical engagement in processing of the specific word stimuli, rather than actual verbal rehearsal of worries. Even if true, engagement in word processing may still be driven by worry (e.g., worry about performing the task or word meaning), an inference that seems reasonable given that anxious apprehension moderated this activation. One possible method of differentiating between worry and word Oxymatrine processing specific to the stimuli would be to disentangle block-level and stimulus-specific variance, given that worry should be increased during negative blocks but not necessarily tied to specific stimuli. Although there is jitter in the presentation of words in the present study, the length of the jitter is small compared to the HRF, and the timing of the stimuli (2000 ± 225 msec) is approximately equal to the fMRI sampling rate (TR = 2 sec). In order to differentiate between these signals without aliasing, TR would have to be less than 1 sec or ITI greater than 4 sec.

These data highlight that there is still controversy with the me

These data highlight that there is still controversy with the mechanism of action of polymeric micro- and nanoparticles. PLGA micro- and nanospheres can be used for systemic or mucosal immunization [84–86]. PLGA-based systems are able to be phagocytosed by DCs, even by the oral route [87] and enhance their immunostimulatory capacity [88], leading to the upregulation of maturation markers CD40 and CD80 and release of IL-6. It has been shown that Hp91 synthetic peptide (a peptide that can induce

potent antigen-specific cytotoxic T-lymphocyte responses), both encapsulated or conjugated to the surface of PLGA nanoparticles, is able to activate both human and mouse DCs more potently than the free peptide [88]. Inhibitors,research,lifescience,medical PLGA microspheres

have been extensively studied by our research group. Different synthetic peptides have been entrapped into these microspheres, such as malarial SPf66, and have been administered by subcutaneous, intradermal [89], oral [17], or nasal [90] routes in mice. Microencapsulated SPf66 induced Inhibitors,research,lifescience,medical a superior immune response than the one obtained with the administration of the peptide adjuvanted Inhibitors,research,lifescience,medical with alum and comparable with the response obtained with FCA. In addition, these particles have been administered to Aoutus monkeys leading to high antibody levels and protection against P. falciparum challenge [91]. To our knowledge, only one clinical trial has been carried out using PLGA and synthetic peptides [92]. This phase I study evaluated the safety and immunogenicity of a synthetic HIV peptide (HIV-1 MN V3) administered intramuscularly with alum and a similar product encapsulated into PLGA microspheres administered by the oral route. However, the oral administration of this Inhibitors,research,lifescience,medical vaccine did not trigger significant humoral, cellular, or mucosal immune responses. 2.4. Liposomes Liposomes are synthetic spheres comprised by phospholipid bilayers (Figure 4). According to their structure and size, liposomes can be classified into multilamellar vesicles Inhibitors,research,lifescience,medical (MLV), small unilamellar vesicles (SUV), intermediate unilamellar vesicles (IUV), or large unilamellar vesicles (LUV) [93]. For vaccine delivery, antigens

can be encapsulated into the aqueous core, integrated in the lipid bilayer or adsorbed on the surface [4]. Figure 4 Scheme of liposomes structure. Antigens are differently incorporated based on their nature. Hydrophilic antigens can be encapsulated into the aqueous core; amphipathic antigens and are integrated into the phospholipid bilayer, and lipidic antigens are adsorbed … The mechanism of action of liposomes is not well defined. Passive targeting, derived of their particulate nature, and tendency to interact with macrophages is likely to be an important factor, particularly for nontargeted liposomes [94]. Among the different lipids available, cationic ones have a better ability to initiate and potentiate the immune response.

2000) However, it has been challenging to replicate previously i

2000). However, it has been challenging to replicate previously identified

candidate gene underlying susceptibility to depression (Lopez-Leon et al. 2008) or explain substantial variance in the phenotype. Genome-wide association studies (GWAS) have been unsuccessful in identifying significant individual genetic variants either (Sullivan et al. 2009; Lewis et al. 2010; Muglia et al. 2010; Shi et al. 2011; Shyn et al. 2011; Wray et al. 2012; Hek et al. 2013; Ripke et al. 2013). These negative findings have led to speculation that depression is particularly heterogeneous both clinically and etiologically, Inhibitors,research,lifescience,medical which could dramatically reduce statistical power to identify causal loci (Craddock et al. 2008). In addition, it is often hypothesized that for complex disorders including major depressive disorders, each individual risk allele only has low contribution, with odds ratio typically in the region of 1.05–1.2 (Mitchell 2012). Inhibitors,research,lifescience,medical Statistical power may be improved by increasing sample size, which is not always feasible; by improving precision of phenotype measurement; Inhibitors,research,lifescience,medical or by combining information from multiple loci such as creating polygenic scores (PS) or taking into account interactions between genes or loci, which may effectively increase the magnitude of the genetic effects. Measurement has proven particularly

challenging for depression-related phenotypes. Psychiatric research emphasizes distinctions between categorical diagnoses (binary phenotypes) and dimensional symptom measures (continuous phenotypes) (Maes et al. 1992; Prisciandaro and Roberts 2009). Because categorical diagnoses better distinguish individuals with true Selleckchem I BET 762 psychopathology, genetic determinants might be easier to identify when contrasting Inhibitors,research,lifescience,medical Inhibitors,research,lifescience,medical diagnosed cases to healthy controls. However, if the heritability reflects the independent influence of many genes with small effects, the phenotype is likely to be continuously distributed and more closely related to symptom-based measures. If so, using a binary outcome measure dichotomizing a continuous

phenotype reduces statistical power compared to using a dimensional quantitative measure with the same sample size (Helzer et al. 2006; Kraemer 2007), In general, diagnostic and symptom-based measures are highly but not perfectly correlated (Radloff 1977). tuclazepam A second challenge in measuring depression phenotypes is appropriately defining the time period of assessment. Genetic risks are carried throughout life, but the phenotype manifest at any given moment reflects both stable genetic contributions and fluctuating, transient contextual influences. These temporary variations reduce statistical power to detect genotype–phenotype associations (assuming transient contextual influences are independent of genetics). Depression phenotypes in genetic association studies are often assessed at a single time point when symptom measures are used.