[5-Year-Follow-up of the MEmbeR Multicenter Study on Medical-Occupational Rehabilitation]. / 5-Jahres-Follow-Up der multizentrischen Evaluationsstudie zur medizinisch-beruflichen Rehabilitation (MEmbeR).

In Germany, medical-occupational rehabilitation represents an essential link between rehabilitation programs focusing either on medical or occupational rehabilitation. Its main objective is return to work. The current study presents the vocational integration 5 years after medical-occupational rehabilitation and determines possible prognostic factors for long-term occupational integration. To evaluate the effectiveness of medical-occupational rehabilitation, a 5-year-follow-up interview was conducted with participants (n=105) of the multicenter study on medical-occupational rehabilitation (MEmbeR). As a main result, 76% of the participants were still employed 5 years after medical-occupational rehabilitation and the return to work rate was 57%. Prognostic factors for long-term occupational integration could not be identified. However, a low degree of disability, an unrestricted capacity for teamwork as well as an unrestricted ability to judge might be beneficial factors for a successful reintegration. The high amount of participants who returned to work 5 years after medical-occupational rehabilitation, supports the concept of medical-occupational rehabilitation. However, more studies are needed to identify further factors influencing the outcome.

Extraction yields and anti-oxidant activity of proanthocyanidins from different parts of grape pomace: effect of mechanical treatments.

INTRODUCTION: White grape pomace is not subject to maceration, keeping nearly all polyphenols of grapes, so they represent important sources of bioactive compounds such as proanthocyanidins. Preparation of plant polyphenol extracts is usually performed using raw material powder. However, the fine particles make the further extraction procedure steps more difficult. OBJECTIVE: To study the effect of mechanical treatments on extraction yields and anti-oxidant activity from different parts of white grape pomace. METHODS: Skins, stems and seeds were isolated from the pomace of white winemaking process. Sequential solvent extraction of polyphenols, first using 80% methanol in water followed by 75% acetone in water, was carried out on both entire and milled (< 1 mm) grape solids; extraction on seed polyphenols was also performed in its squashed form. The phenolic content of each extract was verified by spectrometric and HPLC methods and its anti-oxidant activity was evaluated by the 1,1-diphenyl-2-picrylhydrazyl test. RESULTS: More total polyphenols can be extracted from each milled tissue than from its entire form. Seeds present the highest total phenol, oligomeric and polymeric proanthocyanidin content, and similar extraction yield was found between milled and squashed tissues. The HPLC analysis showed no difference in extraction yield of low-molecular-weight proanthocyanidins between milled and entire stems. Anti-oxidant activity showed a positive correlation with total phenol content, galloyled oligomers and polymeric proanthocyanidins. CONCLUSION: The use of entire stems and squashed seeds for solvent extraction of polyphenols makes manipulation simpler and more cost-efficient, providing similar extraction yields to using their powdery forms.

[The MEmbeR Multicenter Study on medical-occupational rehabilitation]. / Multizentrische Evaluationsstudie zur medizinisch-beruflichen Rehabilitation (MEmbeR).

INTRODUCTION: MEmbeR is a prospective multi-center study on medical-occupational rehabilitation in Germany. METHODS: 196 neurological, psychiatric, orthopaedic, and internal medicine patients from 21 rehabilitation centres all across Germany have been enrolled and followed-up for 2 years after discharge. Primary outcome parameter was defined as return to work. Further, the SF-12 and a Mini-ICF-Rating have been used. RESULTS: Mean age was 34.1 (9.9) years, length of stay 150.0 (223.5) days. Prior to occupational rehabilitation, 69.9% were unable to work, 2 years after discharge only 5.6%. Rate of participants seeking a job was reduced from 19.7% to 3.1%. In summary, 78.1% returned to work. Employed participants were younger (32.8 [9.7] vs. 38.5 [9.4] years, p=0.001) and less disabled (Degree of Disablement [GdB]: 20.0 [31.2] vs. 36.1 [33.7], p<0.05). CONCLUSION: The multicenter cohort study MEmbeR provides further knowledge about the outcome of medical-occupational rehabilitation in Germany.

[Long-term course of patients in neurological rehabilitation Phase B. Results of the 6-year follow-up in a multicenter study]. / Langzeitverlauf von Patienten der neurologischen Rehabilitation Phase B. Ergebnisse der 6-Jahres-Nachuntersuchung einer Multicenterstudie.

BACKGROUND: After conclusion of emergency care for severe neurological diseases patients in Germany are admitted at an early stage to so-called Phase B rehabilitation. No studies have been carried out on the long-term course of these patients. PATIENTS AND METHODS: In a prospective study in 2002 patients in Phase B from 9 centers were included and follow-up investigations were carried out after 5 and 6 years. Assessment instruments used were the Barthel index, the Rankin scale and the EQ-5D. Factors for the risk of a poor outcome and the chances for a good outcome were evaluated using multivariate logistic regression. RESULTS: A total of 1,280 patients were included in the study. A high age increased the risk of dying with a hazard quotient (HQ) of 1.05 (95% CI: 1.04-1.06) and high point counts in the coma remission scale (HQ 0.93; 95% CI: 0.92-0.96) and Barthel index (HQ 0.97; 95% CI: 0.97-0.98) on discharge reduced the risk of dying after 5 years. The factors swallowing impairment (OR 3.1; 95% CI: 1.7-5.5) and obligatory surveillance at the end of rehabilitation (OR 3.2; 95% CI: 1.2-8.6) increased the risk of a poor result in the Rankin scale 2-4 and the factors communication disorder (OR 5.0; 95% CI: 2.0-12.8) and PEG (percutaneous endoscopic gastrostomy) (OR 19.7; 95% CI: 2.7-144.4) on discharge increased the risk of a reduced health-related quality of life (defined as EQ-5D VAS <70) after 6 years. CONCLUSIONS: If support for bodily functions can be successfully reduced during Phase B rehabilitation, the patients will have a good outcome with respect to 5-year survival. If this is not successful the outcome is unfavorable with respect to survival and with respect to achieving self-sufficiency and health-related quality of life after 6 years.

Microbial transformation of ginsenosides Rb1, Rb3 and Rc by Fusarium sacchari.

AIMS: This study examined the transformation pathways of ginsenosides G-Rb(1) , G-Rb(3) , and G-Rc by the fungus Fusarium sacchari. METHODS AND RESULTS: Ginsenosides G-Rb(1) , G-Rb(3) and G-Rc were isolated from leaves of Radix notoginseng, and their structural identification was confirmed using NMR. Transformation of G-Rb(1) , G-Rb(3) and G-Rc by Fusarium sacchari was respectively experimented. Kinetic evolutions of G-Rb(1) , G-Rb(3) and G-Rc and their metabolites during the cell incubation were monitored by HPLC analysis. High-performance liquid chromatography (HPLC) was used for monitoring the transformation kinetics of bioactive compounds during F. sacchari metabolism. CONCLUSIONS: Ginsenoside C-K was transformed by F. sacchari from G-Rb(1) via G-Rd or via G-F(2) , or from G-Rb(1) via firstly Rd and then G-F(2) , and C-Mx was transformed by F. sacchari or directly from Rb(3) , or from Rb(3) via Gy-IX, while G-Mc was transformed by F. sacchari directly from G-Rc. Furthermore, C-K could be also formed from G-Rc via notoginsenoside Fe (N-Fe). SIGNIFICANCE AND IMPACT OF THE STUDY: The results showed an important practical application in the preparation of ginsenoside C-K. As our precious research indicated C-K possessed much more antitumor activities than C-Mx and G-Mc, so according to the transformation pathways proposed by this work, the production of antitumor compound C-K may be performed by biotransformation of G-Rb(1) previously isolated from PNLS.

A new class of anthocyanin-procyanidin condensation products detected in red wine by electrospray ionization multi-stage mass spectrometry analysis.

In our previous work, we have identified, in a model wine solution containing malvidin 3-glucoside, epicatechin and acetaldehyde, a new condensation product--hydroxylethyl-malvidin-3-glucoside-ethyl-epicatechin. The objective of this work was to verify the presence of such new condensation products in red wine. For this purpose, red wine was fractionated into various fractions by column chromatography on LiChroprep RP 18 and on Toyopearl 40 (F). The phenolic composition of each fraction was verified by HPLC-DAD and direct-infusion ESI-MS(n) analysis. In addition to the well-known anthocyanins and their acetyl and coumaroyl derivatives, and several direct and indirect anthocyanin-(epi)catechin condensation products, a new class of pigmented products, namely hydroxyethyl-anthocyanin-ethyl-flavanol compounds, have been detected in red wine. The new class of pigmented products would be expected to be the major pigments responsible for the color of aged red wine.

Enzyme replacement therapy with alglucosidase alfa in 44 patients with late-onset glycogen storage disease type 2: 12-month results of an observational clinical trial.

Late-onset glycogen storage disease type 2 (GSD2)/Pompe disease is a progressive multi-system disease evoked by a deficiency of lysosomal acid alpha-glucosidase (GAA) activity. GSD2 is characterized by respiratory and skeletal muscle weakness and atrophy, resulting in functional disability and reduced life span. Since 2006 alglucosidase alfa has been licensed as a treatment in all types of GSD2/Pompe disease. We here present an open-label, investigator-initiated observational study of alglucosidase alfa enzyme replacement therapy (ERT) in 44 late-onset GSD2 patients with various stages of disease severity. Alglucosidase alfa was given i.v. at the standard dose of 20 mg/kg every other week. Assessments included serial arm function tests (AFT), Walton Gardner Medwin scale (WGMS), timed 10-m walk tests, four-stair climb tests, modified Gowers' maneuvers, 6-min walk tests, MRC sum score, forced vital capacities (FVC), creatine kinase (CK) levels and SF-36 self-reporting questionnaires. All tests were performed at baseline and every 3 months for 12 months of ERT. We found significant changes from baseline in the modified Gowers' test, the CK levels and the 6-min walk test (341 +/- 149.49 m, median 342.25 m at baseline; 393 +/- 156.98 m; median 411.50 m at endpoint; p = 0.026), while all other tests were unchanged. ERT over 12 months revealed minor allergic reactions in 10% of the patients. No serious adverse events occurred. None of the patients died or required de novo ventilation. Our clinical outcome data imply stabilization of neuromuscular deficits over 1 year with mild functional improvement.

microRNAs and the regulation of glucose and lipid metabolism.

Over recent years, metabolic disorders such as type 2 diabetes have finally become recognized as a major challenge to global health. The attention of scientists therefore has to focus on improving our understanding of the molecular mechanisms behind these diseases and towards the design of new drug therapy strategies. The pathophysiology of diabetes is undoubtedly complex, oftentimes characterized by varying states of insulin resistance and impaired beta-cell function; however, the identification of new pathways is constantly improving our understanding of the disease. We and others have recently shown that microRNAs (miRNAs) can play a role in insulin secretion and glucose homostasis. Thus, in this review, we will discuss the potential role of miRNAs in type 2 diabetes and related metabolic diseases.

Fractionation of red wine polyphenols by solid-phase extraction and liquid chromatography.

A systematic method for separation of aged red wine polyphenols into various distinct fractions using combined techniques of solid-phase extraction and liquid chromatography was proposed. The aged red wine polyphenols were separated into various distinct fractions including phenolic acid fraction, monomer flavanol fraction, oligomer procyanidin fraction, anthocyanin and its pyruvic acid derivative fraction, free or non-colored proanthocyanidin fraction, fraction of direct condensation products between anthocyanins and proanthocyanidins and fraction of other pigmented complexes. The phenolic composition of each fraction was verified by HPLC with diode array detection (HPLC-DAD), thiolysis, vanillin assay, HPLC coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS) and multi-stage MS fragment analysis. For the first time, anthocyanins and their pyruvic derivatives were separated from other phenolic compounds, while free or non-pigmented polymer proanthocyanidins from other pigmented complexes. The fractionation method would be of particular interest in further studying the detailed composition of polymeric polyphenols in red wine.

Proanthocyanidin composition in the seed coat of lentils (Lens culinaris L.).

Lentils (Lens culinaris L.) are a popular food in many countries. However, little is known about their phenolic composition. Because polyphenols in lentils are located essentially in their seed coat, the objective of this work was to study the composition of proanthocyanidins, the major group of polyphenols, in this part of the tissue. The use of C(18) Sep-Pak cartridges permitted the fractionation of lentil seed coat extract into monomer, oligomer, and polymer proanthocyanidin fractions. Subsequent thiolysis of oligomer and polymer fractions followed by HPLC analysis allowed the mean degree of polymerization (mDP) and the structural composition of proanthocyanidins to be determined. A fractionation of lentil seed coat extracts on a polyamide column followed by HPLC and HPLC-DAD-MS analyses was used to identify the individual proanthocyanidins. The results showed that the major monomeric flavan-3-ol was (+) catechin-3-glucose, with lesser amounts of (+)-catechin and (-)-epicatechin. In the oligomer fraction, various dimer, trimer, and tetramer proanthocyanidins constituted of catechin, gallocatechin, and catechin gallate units were identified, and several procyanidins and prodelphinidins from pentamers to nonamers constitute the polymer fraction. The most abundant proanthocyanidins in the seed coat of lentils are the polymers (65-75%), with a mDP of 7-9, followed by the oligomers (20-30%), with a mDP of 4-5.

SCA17 caused by homozygous repeat expansion in TBP due to partial isodisomy 6.

An expanded polyglutamine domain in the TATA-binding protein (TBP) has been described in patients with spinocerebellar ataxia type 17 (SCA17) characterized by cerebellar ataxia associated with dementia. TBP is a general transcription initiation factor that regulates the expression of most eukaryotic genes transcribed by RNA polymerase II. SCA17, as an autosomal dominantly inherited progressive neurodegenerative disorder, is caused by heterozygous expansion of a CAG repeat coding for glutamine. Alleles with 27 to a maximum of 44 glutamine residues were found as the normal range, whereas expansions above 45 repeat units were considered pathological. Here, we present a patient with a very severe phenotype with a late onset but rapidly progressing ataxia associated with dementia and homozygous 47 glutamine residues caused by an apparent partial isodisomy 6. This extraordinary case has important implications for the insights of TBP and SCA17. The expanded polyglutamine domain in both TBP copies is not correlated with embryonic death indicating that the normal function of the protein is not disrupted by this kind of mutation but may account for the dementia seen in this patient.

Adenosine-induced expression of interleukin-6 in astrocytes through protein kinase A and NF-IL-6.

In various neurologic diseases, astrocytes express interleukin-6 (IL-6), which is an endogenous pyrogen, a neuroprotective factor, and a regulator of the blood-brain barrier. The expression of IL-6 in astrocytes is stimulated by extracellular adenosine through A(2B) receptors. To investigate the signaling cascade that induces IL-6 gene transcription further, we transfected primary mouse astrocytes with a reporter gene construct, in which luciferase expression is directed by the human IL-6 promoter. Expression of PKI, an inhibitor of protein kinase A (PKA), interfered with IL-6 transcription indicating that PKA mediates the effect of adenosine. The CAAT box of the IL-6 promoter is necessary for the stimulation by adenosine as a mutation in this element reduced the stimulation by adenosine. Indeed, the cAMP agonist forskolin increased the binding of the transcription factors NF-IL-6 and C/EBPdelta to the CAAT box of the IL-6 promoter in nuclear extracts of astrocytes. Inhibition of the de novo synthesis of NF-IL-6 by cycloheximide or an antisense oligonucleotide reduced the enhancement of NF-IL-6 binding to the CAAT box and inhibited stimulation of IL-6 transcription by forskolin. In addition, overexpression of NF-IL-6 induced IL-6 transcription. This suggests that adenosine induces the de novo synthesis of NF-IL-6 through activation of PKA and thereby stimulates transcription of IL-6 in astrocytes.

Expression of cell death-associated phospho-c-Jun and p53-activated gene 608 in hippocampal CA1 neurons following global ischemia.

Persistent activation of c-Jun N-terminal kinases (JNKs) and phosphorylation of c-Jun has been shown in various cell death paradigms. Inhibition of the JNK signal transduction pathway prevented neuronal cell death both in vitro and in vivo. In the present study, nuclear phospho-c-Jun immunoreactivity became apparent selectively in vulnerable hippocampal CA1 neurons at 24 h after transient global cerebral ischemia. A high constitutive expression of phospho-JNK1 was detected by immunoblot analysis of hippocampal extracts. Expression of JNK interacting protein-1 (JIP-1), which facilitates JNK signaling, remained unchanged in post-ischemic hippocampal neurons. By contrast, p53-activated gene 608 (PAG608), which promotes cell death in vitro, was strongly induced in post-ischemic CA1 neurons. Our data suggest that transcription factors p53 and phospho-c-Jun may contribute to programmed CA1 cell death following ischemia.

Low molecular weight organic compounds of chestnut wood (Castanea sativa L.) and corresponding aged brandies.

Oak and chestnut species have been largely used for the aging of brandies, but nowadays chestnut is rarely used. There have been no previous studies regarding the cooperage utilization of chestnut wood. This study provides, for the first time, specific information about the characterization of the northern Portuguese Castanea sativa wood and examines the influence of this wood and its heat treatment on the chemical composition of two-year-aged brandies, by the quantitative determination (HPLC) of low molecular weight phenolic compounds. The predominance of gallic acid among the analyzed extractable compounds both in chestnut wood and in the corresponding aged brandies was remarkable. The heat treatment has a very significant influence on the majority of extractable compounds analyzed. Thus, it could be responsible for the related sensorial properties of aged brandies and greatly affect their general balance.

Brain-derived neurotrophic factor improves long-term potentiation and cognitive functions after transient forebrain ischemia in the rat.

We investigated the effect of brain-derived neurotrophic factor (BDNF) on hippocampal long-term potentiation (LTP) and cognitive functions after global cerebral ischemia in the rat. After four-vessel occlusion, BDNF was administered via an osmotic minipump continuously over 14 days intracerebroventricularly. Electrophysiological experiments were performed 14 days after cerebral ischemia. Test stimuli and tetanization were delivered to the Schaffer collaterals of the hippocampus and field excitatory postsynaptic potentials (fEPSP) were recorded in the CA1 region. Cognitive impairment was analyzed repeatedly with a passive avoidance test, a hole-board test, and with an activity center on the same animal. In sham-operated animals, LTP was consistantly induced after delivering a tetanus (increase of initial slope of fEPSP to 173 +/- 12% of baseline; n = 6). After transient forebrain ischemia LTP could not be induced (117 +/- 4% of baseline; n = 7). In ischemic animals treated with BDNF, LTP could be induced (168 +/- 28% of baseline; n = 8). Transient forebrain ischemia resulted in a significant decrease in spatial discrimination performance but not of associative memory. The ratios for working memory (WM) and reference memory (RM) 15 days after ischemia were lower in the ischemic rats (n = 10) than in the sham-operated control animals (n = 10; WM: 22 +/- 6 vs 72 +/- 7; RM: 30 +/- 7 vs 72 +/- 5). Postischemic intracerebroventricular BDNF infusion increased both WM (63 +/- 4; n = 10) and RM (58 +/- 5; n = 10). The spontaneous locomotor activity did not differ significantly in the three groups. These data indicate a protective effect of BDNF for synaptic transmission and cognitive functions after transient forebrain ischemia.

Thrombolysis in acute ischemic stroke: controlled trials and clinical experience.

Thrombolytic therapy with recombinant tissue plasminogen activator (rtPA) is approved in the United States for treatment of acute ischemic stroke. Approval was granted after a large, randomized, placebo-controlled study by the National Institute of Neurological Disorders and Stroke (NINDS) showed a significant improvement in 3-month outcomes with rtPA despite a significant risk for symptomatic hemorrhage. Two other trials, the first and second European Cooperative Acute Stroke Study (ECASS I and II), have shown comparable results, but neither was statistically positive for the predefined primary end point. An analysis of the risk/benefit profile of rtPA therapy based on the results of these three trials indicates that the treatment is effective and, when administered within 3 hours of symptom onset at a dose of 0.9 mg/kg, the benefits by far outweigh the risks for eligible patients. Even with the 6-hour time window of the two ECASS trials, a combined analysis of the three studies shows the number of disabled or dead patients to be significantly reduced. Preliminary data collected on the use of rtPA outside of clinical trials in the United States and Europe suggest that, when rtPA is used according to the trial protocol, the risks and benefits are similar to those observed in clinical trials. However, even within the United States, rtPA is underutilized. The most substantial treatment barrier is the narrow time window, which may be expanded if long-term experience shows that this is possible. Most stroke patients arrive at the hospital too late to be eligible for screening and treatment. Education of the public and physicians may help to overcome this difficulty.

Bradykinin induces interleukin-6 expression in astrocytes through activation of nuclear factor-kappaB.

Bradykinin, a mediator of inflammation, is produced in the brain during trauma and stroke. It is thought to open the blood-brain barrier, although the mechanism is unclear. We have investigated, therefore, the effect of bradykinin on the expression of interleukin-6 (IL-6), a putative modulator of the blood-brain barrier, in astrocytes. IL-6 gene transcription was evaluated by transient transfection of the human IL-6 promoter linked to the luciferase gene. In murine astrocytes, bradykinin stimulated IL-6 secretion and gene transcription. The effect of bradykinin was blocked by KN-93, an inhibitor of Ca2+/calmodulin-dependent protein kinases, and by bisindolylmaleimide I, an inhibitor of protein kinase C, suggesting the involvement of these protein kinases. Mutations in the multiple response element and the binding site for nuclear factor-kappaB (NF-kappaB), but not in other known elements of the IL-6 promoter, interfered with induction of IL-6 transcription. The involvement of NF-kappaB was supported further by the finding that overexpression of nmIkappaB alpha, a stable inhibitor of NF-kappaB, inhibited the induction of IL-6 by bradykinin. Bradykinin activated NF-kappaB in primary astrocytes as shown by increased DNA binding of NF-kappaB. These data demonstrate that bradykinin stimulates IL-6 expression through activation of NF-kappaB, which may explain several inflammatory effects of bradykinin.

Inhibition of caspases prevents cell death of hippocampal CA1 neurons, but not impairment of hippocampal long-term potentiation following global ischemia.

An essential role for caspases in programmed neuronal cell death has been demonstrated in various in vitro studies, and synthetic caspase inhibitors have recently been shown to prevent neuronal cell loss in animal models of focal cerebral ischemia and traumatic brain injury, respectively. The therapeutic utility of caspase inhibitors, however, will depend on preservation of both structural and functional integrity of neurons under stressful conditions. The present study demonstrates that expression and proteolytic activity of caspase-3 is up-regulated in the rat hippocampus after transient forebrain ischemia. Continuous i.c.v. infusion of the caspase inhibitor N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone significantly attenuated caspase-3-like enzymatic activity, and blocked delayed cell loss of hippocampal CA1 neurons after ischemia. Administration of N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone, however, did not prevent impairment of induction of long-term potentiation in post-ischemic CA1 cells, suggesting that caspase inhibition alone does not preserve neuronal functional plasticity.

[Moderate hypothermia for the treatment of malignant middle cerebral artery infarct]. / Moderate Hypothermie zur Behandlung des malignen Mediainfarktes.

Moderate hypothermia was induced in 30 patients with malignant middle cerebral artery (MCA) territory infarction. Patients were kept at 33 degrees C body-core temperature for 48 to 72 h, and ICP, CPP, and brain temperature were monitored. Outcome at 4 weeks and at 3 months after the stroke as well as side effects of moderate hypothermia were analysed. Mortality of malignant MCA infarction could be reduced from 80% in historical controls, to 43% (13/30) under hypothermia. During hypothermia elevated ICP values could be significantly reduced. Herniation due to a secondary rise of ICP after rewarming was the cause of death in all 13 patients. The most frequent complication of moderate hypothermia was pneumonia in 12 of the 30 patients (40%). Other severe side effects of hypothermia could not be detected. Moderate hypothermia may improve clinical outcome in patients with malignant MCA infarction.

Familial hemiplegic migraine with cerebellar ataxia and paroxysmal psychosis.

Familial hemiplegic migraine is a rare autosomal dominant disorder associated with stereotypic neurologic aura phenomena including hemiparesis. So far two chromosomal loci have been identified. Families linked to the chromosome 19 locus display missense mutations within the CACNL1A4 gene. Here we report on a family with familial hemiplegic migraine and cerebellar ataxia with recurrent episodes of acute paranoid psychosis with anxiety and visual hallucinations associated with migraine attacks. Based on the clinical and haplotype evidence indicating linkage to chromosome 19 in this family, we hypothesize that a dysfunction of the mutated calcium channel may be involved not only in the development of hemiplegic migraine but also in the acute psychotic episodes observed in these patients.