7-15%) Further, the dechlorination potentialities of ‘Dehalococc

7-15%). Further, the dechlorination potentialities of ‘Dehalococcoides’ species living in the aquifer were evaluated by analyzing the abundance and the expression of 16S rRNA genes and reductive dehalogenase (RDase) encoding functional genes by qPCR and Reverse Transcription qPCR (RT-qPCR). ‘Dehalococcoides’ tceA gene, known to be associated to strains capable of reducing chlorinated solvents beyond cis-DCE, was found and

expressed in the field. Overall, this study proved the existence of a well-established dechlorinating microbial community able to use contaminants as substrates for their metabolic activity PD-0332991 in vitro and indicated the occurrence of reductive dechlorination at the site.”
“We examine the conditions for the transition from antagonism to mutualism between plants and their specialists nursery pollinators in a reference case which is the Trollius europaeus-Chiastocheta interaction. The mechanistic model we developed shows that a specialization of T. europaeus on Chiastocheta could be the result of an attempt to escape

over-exploitation by closing its flower. The pressure for such an escape increases with the parasite’s frequency and its pollination efficiency but decreases in the presence of alternative pollinators. The resulting specialization is a priori an unstable one, leading either to strong evolutionary oscillations, or to evolutionary suicide due to over-exploitation of the plants. It becomes stable if the plants develop a defense mechanism to regulate their parasite’s Quisinostat mw population size and limit seed-exploitation. The development of a counter-measure by the latter can destabilize the mutualism depending on the costs linked to such a trait. On the other hand, we find that a specialization on a purely

mutualistic basis would require a preexisting high diversity of flower-opening within the population. (C) 2011 Elsevier Ltd. All rights reserved.”
“Bioelectrochemical systems (BES) are increasingly being considered for bioremediation applications, such as the reductive transformation of chlorinated hydrocarbons in subsurface environments. These systems typically rely on a polarized solid-state electrode (i.e. a cathode) serving as electron donor for the microbially catalyzed reductive dechlorination Adenosine of chlorinated contaminants. The microorganisms involved in dechlorinating biocathodes are not still identified. Particularly, it is not clear whether the same microorganisms responsible for the reductive dechlorination in ‘conventional’ bioremediation systems (i.e. those based on the supply of soluble substrates as electron donors) also play a role in BES. Here, we analyzed by CARD-FISH, the microbial composition of a dechlorinating biocathode operated at different set potential, in the range from -250 mV to -750 mV (vs. the standard hydrogen electrode, SHE). The rate and extent of TCE dechlorination, as well as of competing metabolisms (i.e.

Patients were followed postoperatively for evidence and duration<

Patients were followed postoperatively for evidence and duration

of success, and treatment related complications.

Results: From January 2006 to June 2007, 18 females and 3 males with a mean age of 81.2 years (range 75 to 92) in whom Selleck Foretinib detrusor overactivity was confirmed on urodynamics and who were refractory to or intolerant of antimuscarinics were treated with intravesical botulinum toxin A. Preoperatively the mean +/- SD number of daily voids was 11.4 +/- 1.67 and the mean number of pads per day was 4.0 +/- 0.89. One month after treatment 16 of the 21 patients (76%) reported greater than 50% improvement in symptoms after 1 injection. Specifically there was a significant improvement in the mean number of voids per day (5.19 +/- 0.83, p<0.001) and in the number of pads used daily (1.3 +/- 0.60, p<0.001). Two of the remaining 5 patients demonstrated greater than 50% improvement following repeat injection, while 3 did not show improvement selleck inhibitor after 2 injections. Mean time to deterioration was 7.12 months. There were no treatment related complications.

Conclusions: Intravesical botulinum toxin A for detrusor overactivity in the elderly population appears to be efficacious and durable. Given its low incidence of adverse events, it should be considered a viable treatment

option in this population.”
“Ligands for the epidermal growth factor receptor ErbB1, such as epidermal growth factor (EGF) and Branched chain aminotransferase transforming growth factor alpha (TGF alpha), negatively regulate synaptic maturation of GABAergic neurons in the developing neocortex. Here, we evaluated the effects of these ligands in vivo on developing inhibitory

neurons in the dentate gyrus. Hippocampal slices were prepared from postnatal mice repeatedly challenged with EGF or from transgenic mice overexpressing TGF alpha. We monitored paired pulse depression of field population spikes evoked by perforant path stimulation to estimate the strength of local inhibition. Administration of EGF increased the paired pulse ratio, suggesting a reduction of inhibitory strength. A similar reduction was observed in TGF alpha transgenic mice. Monitoring miniature and evoked synaptic currents, we estimated EGF effects on synaptic input and output of GABAergic neurons. EGF treatment diminished the amplitude of excitatory postsynaptic currents (EPSCs) in the GABAergic neurons without affecting their miniature EPSCs. EGF also affected output strength of the GABAergic neurons: The frequency of miniature inhibitory postsynaptic currents (IPSCs) and the evoked IPSC/evoked EPSC ratio were decreased in granule cells. In parallel, EGF down-regulated the protein level of vesicular GABA transporter. Thus, ErbB1 ligands influence GABAergic inhibitory synaptic transmission in the developing dentate gyrus. (C) 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Our results show that while these mutations do not drastically af

Our results show that while these mutations do not drastically affect the structure of the Efb-C/C3d recognition complex, selleck compound they have significant adverse effects on both the thermodynamic and kinetic profiles of the resulting complexes. We also characterized other key interactions along the Efb-C/C3d binding interface and found an intricate network of salt bridges and

hydrogen bonds that anchor Efb-C to C3d, resulting in its potent complement inhibitory properties.”
“Objectives: The purpose of the present study was to determine the prevalence and risk factor associations for subclavian artery calcification.

Background: Arterial calcification is a marker of atherosclerosis, and its presence portends an adverse prognostic risk. The prevalence click here and associated risk factors

for aortic arch, carotid, renal, and coronary calcification have been well described. Fewer data are available for subclavian artery calcification.

Methods: Electron-beam computed tomography was used to evaluate the extent of vascular calcification in multiple arterial beds in 1387 consecutive individuals who presented for preventive medicine services at a university-affiliated disease prevention center. Laboratory values for blood pressure, lipids, anthropomorphic data, and self-reported medical history were obtained.

Results: Subclavian artery calcification was present in 439 of 1387 individuals (31.7%). Those with subclavian artery calcification were significantly older, had a smaller body mass index, and were more likely to also have calcification of nonsubclavian vascular beds. When adjusted for

cardiovascular disease risk factors, the presence of subclavian artery calcification was significantly associated with age Dimethyl sulfoxide (prevalence ratio [PR], 1.04; P < .001), hypertension (PR, 1.20; P = .01), history of smoking (PR, 1.21; P = .01), and calcification in nonsubclavian vascular beds (PR, 1.58; P = .01). Subclavian artery calcification was also associated with an increased pulse pressure (beta-coefficient = 2.2, P = .008).

Conclusions: Subclavian artery calcification is relatively common and is significantly associated with age, smoking, hypertension, and nonsubclavian vascular calcification. There may be a relationship between vascular stiffness, as manifested by a widened pulse pressure, and the presence of subclavian artery calcification. (J Vase Surg 2011;54:1408-13.)”
“Animal experimentation in the Parkinson’s disease (PD) field is a classic example of how the use of animal models to study diseases can have a significant impact on human health. Among the different neurotoxin-based animal models of PD that are presently available, the 6-hydroxydopamine (6-OHDA) and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) models have been established and validated as useful models for the development of therapeutic strategies aimed to treat motor symptoms and to study alterations of the basal ganglia that occur in this disease.

Furthermore, these unexpected and newly identified transcripts le

Furthermore, these unexpected and newly identified transcripts lead to the synthesis

of viral proteins termed HBZ (HTLV-1 basic leucine zipper) and APH-2 (antisense protein of HTLV-2), respectively. As potential open reading frames are present on the antisense strand of HTLV-3 and HTLV-4, we tested whether in vitro antisense transcription occurred in these viruses and whether these transcripts had a coding potential. Using HTLV-3 and HTLV-4 proviral DNA constructs, antisense transcripts were detected by reverse transcriptase PCR. These transcripts are spliced and polyadenylated and initiate at multiple sites from the 3′ long terminal repeat (LTR). The resulting proteins, termed APH-3 and APH-4, are devoid of a typical basic leucine zipper domain but contain basic amino acid-rich regions. Confocal microscopy and Western blotting experiments demonstrated a nucleus-restricted pattern for APH-4, DMXAA research buy while APH-3 was localized both in the cytoplasm and in the nucleus. Both proteins showed partial colocalization with nucleoli and HBZ-associated structures. Finally, Lonafarnib research buy both proteins inhibited Tax1-

and Tax3-mediated HTLV-1 and HTLV-3 LTR activation. These results further demonstrate that retroviral antisense transcription is not exclusive to HTLV-1 and HTLV-2 and that APH-3 and APH-4 could impact HTLV-3 and HTLV-4 replication.”
“Mutations of the voltage gated sodium channel gene (SCN4A) are responsible for non-dystrophic myotonia including hyperkalemic periodic paralysis, paramyotonia congenita, and sodium channel myotonia, as well as congenital myasthenic syndrome. In vitro functional analyses have demonstrated the nondystrophic mutants to show a gain-of-function defect of the channel; a disruption of fast inactivation, an enhancement of activation, or both, while the myasthenic mutation presents a loss-of function defect. This report

Inositol monophosphatase 1 presents a case of non-dystrophic myotonia that is incidentally accompanied with acquired myasthenia. The patient presented a marked warm-up phenomenon of myotonia but the repeated short exercise test suggested mutations of the sodium channel. The genetic analysis identified a novel mutation, G1292D, of SCN4A. A functional study of the mutant channel revealed marked enhancement of activation and slight impairment of fast inactivation, which should induce muscle hyperexcitability. The effects of the alteration of channel function to the myasthenic symptoms were explored by using stimulation of repetitive depolarization pulses. A use-dependent channel inactivation was reduced in the mutant in comparison to normal channel, thus suggesting an opposing effect to myasthenia. (C) 2012 Elsevier Ireland Ltd. All rights reserved.”
“High levels of homocysteine (Hcy) were suggested to contribute to the pathogenesis of schizophrenia.

“Although most anti-epileptic drugs are considered to have

“Although most anti-epileptic drugs are considered to have a primary molecular target, it is clear that their actions are unlikely to be limited to effects on a single aspect of inhibitory synaptic transmission, excitatory transmission or voltage-gated

ion channels. Systemically administered drugs can obviously selleck simultaneously access all possible targets, so we have attempted to determine the overall effect of diverse agents on the balance between GABAergic inhibition, glutamatergic excitation and cellular excitability in neurones of the rat entorhinal cortex in vitro. We used an approach developed for estimating global background synaptic excitation and inhibition from fluctuations in membrane potential obtained by intracellular recordings. We have previously validated www.selleckchem.com/products/lazertinib-yh25448-gns-1480.html this approach in entorhinal cortical neurones [Greenhill and Jones (2007a) Neuroscience 147:884-892]. Using this approach, we found that, despite their differing pharmacology, the drugs tested (phenytoin, lamotrigine, valproate, gabapentin, felbamate, tiagabine) were unified in their ability to increase the ratio of background GABAergic inhibition to glutamatergic excitation. This could occur as a result of decreased excitation concurrent with increased inhibition (phenytoin, lamotrigine, valproate), a decrease in excitation alone

(gabapentin, felbamate), or even with a differential increase in both (tiagabine). Additionally, we found that the effects on global synaptic conductances agreed well with whole cell patch recordings of spontaneous

glutamate and GABA release (our previous studies and further data presented here). The consistency with which the synaptic inhibition:excitation ratio was increased by the antiepileptic drugs tested was matched by an ability of all drugs to concurrently reduce intrinsic neuronal excitability. Thus, it seems possible that specific molecular targets among antiepileptic drugs are less important than the ability to increase the inhibition:excitation ratio and reduce overall neuronal and network excitability. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.”
“Accurate Diflunisal endoleak classification is essential following fenestrated endovascular aneurysm repair (f-EVAR). Both endoleak type and exact source of endoleak have implications upon the urgency and complexity of future management strategies. Herein we report on a patient with a documented endoleak post-f-EVAR, in which the source of blood flow into the aneurysm sac could not be determined using conventional computed tomographic angiography. Consequently, dynamic volumetric computed tomographic angiography (DV-CTA) was employed, which clearly illustrated the site of origin of the endoleak.

(C) 2012 Elsevier Inc All rights reserved “
“pGEX vectors a

(C) 2012 Elsevier Inc. All rights reserved.”
“pGEX vectors are widely used for GST-fusion protein expression in Escherichia selleck chemicals llc coli under the control of a strong IPTG inducible tac promoter. While using pGEX-4T-2 vector in heterologous protein expression we noticed that the GST or GST-fusion protein were expressed at a very low levels. Interestingly, we found a spontaneous deletion of 701 bp DNA fragment harbouring the tac promoter in both, native and recombinant pGEX-4T-2 vectors. This deletion took place

between two direct repeats of 43 bases and led to the loss of a 701 bp DNA fragment. This explained the decrease in GST or GST-fused protein level since the tac promoter, that directs transcription was deleted. The lacZ promoter, located upstream of the deleted fragment, replaced tac promoter but was less efficient. The deleted DNA also specifies part of the lacZ gene coding for the N-terminal end of the beta-galactosidase (the alpha-peptide), which is slightly functional. Consequently, bacterial cells transformed with the original pGEX are of a faint blue Caspase Inhibitor VI colour while those bearing the deleted ones are white,

when plated on X-Gal containing medium. The deletion, did not affect neither the sequence nor the molecular weight of GST and fusion protein since it took place just before the GST start codon. It occurred in E. coli TOP10 cells which are deficient in RecA protein, suggesting that the deletion did not require the RecA recombination system. (c) 2008 Elsevier Inc. All rights Carnitine palmitoyltransferase II reserved.”
“The ankyrin repeat (ANK) is the most common protein-protein interaction motif in nature, and is predominantly found in eukaryotic proteins. Genome sequencing

of various pathogenic or symbiotic bacteria and eukaryotic viruses has identified numerous genes encoding ANK-containing proteins that are proposed to have been acquired from eukaryotes by horizontal gene transfer. However, the recent discovery of additional ANK-containing proteins encoded in the genomes of archaea and free-living bacteria suggests either a more ancient origin of the ANK motif or multiple convergent evolution events. Many bacterial pathogens employ various types of secretion systems to deliver ANK-containing proteins into eukaryotic cells, where they mimic or manipulate various host functions. Studying the molecular and biochemical functions of this family of proteins will enhance our understanding of important host-microbe interactions.”
“Introduction: Pulmonary Langerhans cell histiocytosis (PLCH) is a rare cause of interstitial lung disease characterized by formation of nodules in the active phase of the disease that evolve into nonactive cystic lesions later on.

As pCREB is required for neuronal plasticity, partly because of i

As pCREB is required for neuronal plasticity, partly because of its regulation of immediate early-gene expression, the present findings reinforce the concept of an ‘extended hippocampal system’ in which hippocampal function

is dependent on distal sites such as the anterior thalamic nuclei. (C) 2012 IBRO. Published by Elsevier TSA HDAC manufacturer Ltd. All rights reserved.”
“Gerontological research suggests that depressive symptoms show antecedent and consequent relations with late-life disability. Less is known, however, about how depressive symptoms change with the progression of disability-related processes and what factors moderate such changes.

We applied multiphase growth models to longitudinal data pooled across 4 Swedish studies of very old age (N = 779, M age = 86 years at disability

onset, 64% women) to describe change in depressive symptoms prior to disability onset, at or around disability onset (the measurement wave at which assistance in personal activities of daily living was first recorded), and postdisability onset.

Results indicate that, on average, depressive symptoms Navitoclax slightly increase with approaching disability, increase at onset, and decline in the postdisability phase. Age, study membership, being a woman, and multimorbidity were related to depressive symptoms, but social support emerged as the most powerful predictor of level and change in depressive symptoms.

Our Phospholipase D1 findings are consistent with conceptual notions

implicating disability-related factors as key contributors to late-life change and suggest that contextual and psychosocial factors play a pivotal role for how well people adapt to late-life challenges.”
“A set of face stimuli called the NimStim Set of Facial Expressions is described. The goal in creating this set was to provide facial expressions that untrained individuals, characteristic of research participants, would recognize. This set is large in number, multiracial, and available to the scientific community online. The results of psychometric evaluations of these stimuli are presented. The results lend empirical support for the validity and reliability of this set of facial expressions as determined by accurate identification of expressions and high intra-participant agreement across two testing sessions, respectively. (C) 2008 Elsevier Ireland Ltd. All rights reserved.”
“After trauma brain injury, a large number of cells die, releasing neurotoxic chemicals into the extracellular medium, decreasing cellular glutathione levels and increasing reactive oxygen species that affect cell survival and provoke an enlargement of the initial lesion. Alpha-lipoic acid is a potent antioxidant commonly used as a treatment of many degenerative diseases such as multiple sclerosis or diabetic neuropathy.

Conclusions: Current data suggest that ACP and mild systemic hypo

Conclusions: Current data suggest that ACP and mild systemic hypothermic circulatory arrest can be safely applied to complex aortic arch surgery even in a subgroup of patients with up to 90 minutes of ACP. Unilateral ACP offers at least equal brain and visceral organ protection as bilateral ACP and might be advantageous in that it reduces the incidence of embolism arising from surgical manipulation on the arch vessels. (J Vistusertib mouse Thorac Cardiovasc Surg 2012;144:1042-50)”
“The aim of this study is to assess the relationship between the venous angioarchitectural

features and the clinical course of intracranial dural arteriovenous shunt (DAVS) with cortical venous reflux (CVR).

With institutional review board approval, 41 patients (M:F = 24:17; median age, 52 years (range, 1-72 years), median follow-up; 1.5 years; partial treatment, n = 36) with persistent CVR were included. We evaluated the initial presentation and the incidence of annual morbidity (hemorrhage or new/worsened nonhemorrhagic neurological deficit (NHND)) according to

Ricolinostat mw the venous angiographic patterns-isolated venous sinus, occlusion of the draining sinus, direct pial venous drainage, pseudophlebitic pattern, venous ectasia, brisk venous drainage, and length of pial vein reflux-on digital subtraction angiography. Cox regression was performed to identify independent factors for clinical course.

During 111.9 patient-years

of follow-up, the overall annual morbidity rate was 11.6 % (mortality; n = 3, rate; 2.6 %/year). Hemorrhage occurred in five patients (12.2 %, rate; 4.5 %/year) and new/worsened NHND occurred in eight patients (19.5 %, rate; 7.2 %/year). Patients with isolated venous sinus, direct pial venous drainage, and pseudophlebitic pattern were associated Etomidate with initial aggressive presentation. Venous ectasia was associated with initial hemorrhagic presentation. Brisk venous drainage was associated with initial benign presentation. Patients with isolated venous sinus showed a poor clinical course with a higher annual incidence of hemorrhage or new/worsened NHND (91.2 %/year vs 9.2 %/year; hazard ratio, 6.681; p = 0.027).

Venous angioarchitectural features may be predictive of the clinical course of DAVSs. DAVS patients with isolated venous sinus may be especially at high risk for future aggressive clinical course.”
“Proteomics holds great promise in personalized medicine for cancer in the post-genomic era. In the past decade, clinical proteomics has significantly evolved in terms of technology development, optimization and standardization, as well as in advanced bioinformatics data integration and analysis. Great strides have been made for characterizing a large number of proteins qualitatively and quantitatively in a proteome, including the use of sample fractionation, protein microarrays and MS.

HO-1 expression, its activity, the ratio of Bax/Bcl-2 protein, an

HO-1 expression, its activity, the ratio of Bax/Bcl-2 protein, and active caspase-3 fragments were all significantly higher in isolated glomeruli of diabetic rats and in high glucose-treated podocytes. These increases were inhibited by zinc protoporphyrin treatment of the rats or by HO-1 siRNA treatment of the podocytes in culture. The number of apoptotic cells was also significantly increased in the glomeruli of diabetic rats and in high glucose-treated podocytes. Inhibition of HO-1 accentuated the increase in apoptotic cells both in vivo and in vitro. Our findings suggest that HO-1 expression protects against podocyte apoptosis

under diabetic conditions. Kidney International (2009) Luminespib ic50 76, 838-848; doi:10.1038/ki.2009.286; published online 5 August 2009″
“Metabolites of arachidonic acid influence sodium chloride (NaCl) transport in the thick ascending limb. Because Acadesine price a 10 pS Cl channel is the

major type of chloride channel in the basolateral membrane of this nephron segment, we explored the effect of arachidonic acid on this channel in cell-attached patches. Addition of 5 mu mol arachidonic acid significantly decreased channel activity (a product of channel number and open probability) while linoleic acid had no effect. To determine if this was mediated by acachidonic acid per se or by its metabolites, we measured channel activity in the presence of the cyclooxygenase inhibitor indomethacin, the selective lipoxygenase inhibitor nordihydroguaiaretic acid, and the cytochrome P-450 (CYP)-omega-hydroxylation inhibitor 17-octadecynoic acid. Neither cyclooxygenase nor lipoxygenase inhibition had an effect on basal chloride channel activity; further they failed to abolish the inhibitory effect of arachidonate on the 10 pS channel. However, inhibition of CYP-omega-hydroxylation completely abolished the effect

of arachidonic acid. The similarity of the effects of 20-hydroxyeicosatetraenoic acid (20-HETE) and arachidonic acid suggests that the effect of arachidonic acid was mediated by CYP-omega-hydroxylation-dependent Galeterone metabolites. We conclude that arachidonic acid inhibits the 10 pS chloride channel in the basolateral membrane of the medullary thick ascending limb, an effect mediated by the CYP-omega-hydroxylation-dependent metabolite 20-HETE. Kidney International (2009) 76, 849-856; doi:10.1038/ki.2009.287; published online 29 July 2009″
“Transforming growth factor-beta 2 (TGF-beta 2) stimulates the expression of pro-fibrotic connective tissue growth factor (CTGF) during the course of renal disease. Because sphingosine kinase-1 (SK-1) activity is also upregulated by TGF-beta, we studied its effect on CTGF expression and on the development of renal fibrosis.

70 and 0 68, respectively Unlike serum prostate specific antigen

70 and 0.68, respectively. Unlike serum prostate specific antigen the PCA3 score did not increase

with prostate volume. PCA3 assay sensitivity and specificity were equivalent at serum prostate specific antigen less than 4, 4 to 10 and more than 10 ng/ml. A logistic regression algorithm using PCA3, serum prostate specific antigen, prostate volume and digital rectal examination result increased the AUC from 0.69 for PCA3 alone to Mdivi1 chemical structure 0.75 (p = 0.0002).

Conclusions: PCA3 is independent of prostate volume, serum prostate specific antigen level and the number of prior biopsies. The quantitative PCA3 score correlated with the probability of positive biopsy. Logistic regression results suggest that the PCA3 score could be incorporated into a nomogram for improved prediction of biopsy outcome. The results of this study provide further evidence that PCA3 is a useful adjunct to current methods for prostate cancer diagnosis.”
“Purpose: Blood levels of transforming growth factor-beta 1, interleukin-6 and interleukin-6 soluble receptor have been associated with aggressive primary and metastatic prostate cancer. We hypothesized that patients with increased plasma levels of transforming growth factor-beta 1, interleukin-6 Vemurafenib nmr and/or interleukin-6 soluble receptor after

radical prostatectomy would be more likely to harbor occult metastases, leading to disease progression despite effective local control of disease.

Materials Racecadotril and Methods: Plasma transforming growth factor-beta 1, interleukin-6 and interleukin-6 soluble receptor were measured 6 to 8 weeks after surgery in 291 consecutive

patients treated with radical prostatectomy for clinically localized disease. Discrimination and validation of multivariate Cox regression models targeting time to biochemical progression were used to quantify the added value of these markers to predictive accuracy (concordance index) after internal validation with 200 bootstrap resamples.

Results: On multivariate analysis adjusting for standard postoperative features postoperative plasma transforming growth factor-beta 1 was the only biomarker independently associated with biochemical progression (p<0.001). The addition of postoperative transforming growth factor-beta 1 improved the accuracy of the standard postoperative model from 78.4% to 84.1%, representing a 5.7% gain (p<0.001). Of patients who experienced biochemical progression postoperative transforming growth factor-beta 1 was significantly higher in those with features of aggressive disease progression, ie development of metastasis, prostate specific antigen doubling time less than 10 months and/or failure to respond to local salvage radiation therapy (p<0.001).

Conclusions: Postoperative interleukin-6 and interleukin-6 soluble receptor have limited clinical usefulness in prostate cancer.