Methods for studying microbial acid stress responses: from molecules to populations.

The study of how micro-organisms detect and respond to different stresses has a long history of producing fundamental biological insights while being simultaneously of significance in many applied microbiological fields including infection, food and drink manufacture, and industrial and environmental biotechnology. This is well illustrated by the large body of work on acid stress. Numerous different methods have been used to understand the impacts of low pH on growth and survival of micro-organisms, ranging from studies of single cells to large and heterogeneous populations, from the molecular or biophysical to the computational, and from well-understood model organisms to poorly defined and complex microbial consortia. Much is to be gained from an increased general awareness of these methods, and so the present review looks at examples of the different methods that have been used to study acid resistance, acid tolerance, and acid stress responses, and the insights they can lead to, as well as some of the problems involved in using them. We hope this will be of interest both within and well beyond the acid stress research community.

Long-term results after primary augmented repair of proximal tears of the anterior cruciate ligament with the Kennedy-LAD - Does it work?

BACKGROUND: The purpose of this study was to objectively evaluate the clinical functionality of the knee joint 30 years after surgical augmentation of the ACL with the "Kennedy Ligament Augmentation Device ®" (Kennedy LAD®). METHODS: The patient collective consisted of 41 patients with an average age of 59.51 years (±10.18 standard deviation). Included were all patients treated operatively with a Kennedy LAD® augmented reattachment of the ACL at the Department of Trauma Surgery between 1983 and 1985. The state of the knee joint was evaluated with the following measures: Knee injury and Osteoarthritis Outcome Score, Lysholm Score, Short Form (36) Health Survey, International Knee Documentation Committee Score (IKDC, objective + subjective form) and Tegner Activity Scale. RESULTS: Seven patients (17%) sustained a re-rupture of the Kennedy LAD® augmented ACL after a mean time of 16.28 years. Five of them underwent revision surgery. Another four patients (9.76%) showed an ACL insufficiency in clinical examination. The average IKDC Score was 74.14 ±â€¯16.62, the average Lysholm Score was 86.83 ±â€¯14.10, the average Tegner Activity Scale was 4.34 ±â€¯1.11, and the average Knee injury and Osteoarthritis Outcome Score was 86.25 ±â€¯11.64 at final follow-up. The mean Kellgren Lawrence Score of the operated knee was 2 ±â€¯0.71. CONCLUSION: An overall good outcome 30 years after primary ACL augmented repair with the Kennedy LAD® with an implant survival rate of 73% could be reached. These results therefore support the trend of ACL augmentation in selected cases. LEVEL OF EVIDENCE: Retrospective study, Level IV.

An 11-year retrospective experience of antibodies against the voltage-gated potassium channel (VGKC) complex from a tertiary neurological centre.

Acquired diseases classically associated with VGKC-complex antibodies include peripheral nerve hyperexcitability (PNH), Morvan's syndrome, limbic encephalitis (LE), and epilepsy. However, not all such patients have VGKC-complex antibodies and antibodies have been reported in patients without a defined immune-mediated syndrome. To analyse the clinical relevance of positive VGKC-complex antibodies requested on the basis of initial clinical suspicion. We retrospectively analysed patients with positive VGKC-complex antibodies (>100 pM) referred to our institution between 2001 and 2011. 1,614 VGKC-complex assays were performed in 1,298 patients. Titres >100 pM were detected in 57/1,298 (4 %) patients. A classic VGKC-complex channelopathy (60 %) was associated with VGKC-complex antibody titres >400 pM (p = 0.0004). LGI1 or CASPR2 antibodies were only detected in classic VGKC-complex channelopathies (LE; n = 3/4 and PNH; n = 1/5). VGKC-complex antibody titres <400 pM were seen with PNH (n = 15/22; 68 %) but also a heterogeneous range of central and/or peripheral nervous system disorders. Electromyography was supportive of PNH in 65 % of cases and symptomatic treatment was beneficial in 46 % of patients. Irrespective of titre, the rate of malignancy in patients with VGKC-complex antibodies was higher than the age-matched national incidence of malignancy (OR 19.9, 95 % CI 8.97-44.0 p<0.0001). Clinical phenotyping and antibody titres >400 pM can help determine VGKC-complex antibody relevance. Antibody titres <400 pM are associated with PNH but also a more heterogeneous clinical spectrum. The antibody association in the latter is of doubtful clinical relevance. The rate of malignancy was significantly higher than the national incidence irrespective of titre.

Measurement uncertainty of isotopologue fractions in fluxomics determined via mass spectrometry.

Metabolic flux analysis implies mass isotopomer distribution analysis and determination of mass isotopologue fractions (IFs) of proteinogenic amino acids of cell cultures. In this work, for the first time, this type of analysis is comprehensively investigated in terms of measurement uncertainty by calculating and comparing budgets for different mass spectrometric techniques. The calculations addressed amino acids of Pichia pastoris grown on 10% uniformly (13)C labeled glucose. Typically, such experiments revealed an enrichment of (13)C by at least one order of magnitude in all proteinogenic amino acids. Liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) analyses were performed. The samples were diluted to fit the linear dynamic range of the mass spectrometers used (10 µM amino acid concentration). The total combined uncertainties of IFs as well as the major uncertainty contributions affecting the IFs were determined for phenylalanine, which was selected as exemplary model compound. A bottom-up uncertainty propagation was performed according to Quantifying Uncertainty in Analytical Measurement and using the Monte Carlo method by considering all factors leading to an IF, i.e., the process of measurement and the addition of (13)C-glucose. Excellent relative expanded uncertainties (k = 1) of 0.32, 0.75, and 0.96% were obtained for an IF value of 0.7 by LC-MS/MS, GC-MS, and LC-TOFMS, respectively. The major source of uncertainty, with a relative contribution of 20-80% of the total uncertainty, was attributed to the signal intensity (absolute counts) uncertainty calculated according to Poisson counting statistics, regardless which of the mass spectrometry platforms was used. Uncertainty due to measurement repeatability was of importance in LC-MS/MS, showing a relative contribution up to 47% of the total uncertainty, whereas for GC-MS and LC-TOFMS the average contribution was lower (30 and 15%, respectively). Moreover, the IF actually present also depends on the isotopic purity of the carbon sources. Therefore, in the uncertainty calculation a carbon source purity factor was introduced and a minor contribution to the total uncertainty was observed. The results obtained by uncertainty calculation performed according to the Monte Carlo method were in agreement with the uncertainty value of the Kragten approach and showed a Gaussian distribution.

[Acute effects of fast dynamic stretching on rate of force development in ice hockey players: a pilot study]. / Akute Effekte des schnellen dynamischen Dehnens auf die Schnellkraft von Eishockeyspielern: eine Pilotstudie.

BACKGROUND: Fast dynamic stretching has a positive effect on rate of force development in ice hockey players. The aim of this pilot study was to evaluate the influence of fast dynamic stretching (FDS) of the gluteus maximus muscle on rate of force development. SUBJECTS/METHODS: This study included 12 healthy ice hockey players. The intervention was based on a fast dynamic stretching of the gluteus maximus muscle. For quantification, measurements were performed before and after the intervention. The rate of force development was determined by using the drop jump (DJ) for ground contact time and jump height, and sprint time was measured on ice over 20 meters. RESULTS: The intervention showed significant extension of the ground reaction time. The jump height and the sprint time showed no significant differences after FDS intervention. CONCLUSION: The data in this present randomised controlled pilot study showed that FDS interventions on the gluteus maximus muscle have no influence on rate of force development. For future studies, it is recommended that the study protocol should be modified with regard to determining the stretched muscle groups. Furthermore, the FDS intervention on the gluteus maximus muscle showed no positive influence on DJ and sprint.

Constant dopaminergic stimulation by transdermal delivery of dopaminergic drugs: a new treatment paradigm in Parkinson's disease.

Current dopaminergic therapies for the treatment of Parkinson's disease are associated with the development of long-term motor complications. Abnormal pulsatile stimulation of dopamine receptors is thought to underlie the development of motor complications. There is thus a need for therapies that mimic the normal physiological state more closely by resulting in constant dopaminergic stimulation (CDS). Several studies support the hypothesis that CDS can reverse levodopa-induced motor complications. Other potential benefits of CDS include alleviating nocturnal disturbances, minimizing daytime sleepiness, avoiding priming for motor fluctuations and dyskinesia, preventing the development of gastrointestinal dysfunction and reducing the risk of developing psychosis or behavioural disturbances. Continuous infusion of dopaminergic therapies is impractical for the routine treatment of large numbers of patients. Although catechol-O-methyltransferase inhibitors or sustained-release preparations of levodopa may be beneficial, they do not entirely eliminate pulsatile stimulation of dopamine receptors. A new dopamine agonist (rotigotine), delivered over 24 h by a once-daily transdermal patch, has been investigated in several clinical trials. Continuous delivery of rotigotine has been shown to provide 'true' CDS in animal models. The potential of true CDS therapy to prevent or reduce long-term motor and non-motor complications requires investigation in appropriately designed clinical trials.

Deep brain stimulation of the subthalamic nucleus: effectiveness in advanced Parkinson's disease patients previously reliant on apomorphine.

OBJECTIVES: To assess the efficacy of bilateral subthalamic nucleus (STN) deep brain stimulation (DBS) in patients with advanced Parkinson's disease previously reliant on apomorphine as their main antiparkinsonian medication. METHODS: Seven patients with motor fluctuations despite optimal medical treatment given as predominantly apomorphine infusion (n=6), or intermittent apomorphine injections (n=1) underwent bilateral STN DBS using frameless stereotactic surgery. Standard assessments of parkinsonism and motor fluctuations, using Unified Parkinson's Disease Rating Scale (UPDRS) were performed before and six months after surgery. Assessments were performed both on and off medication, and postoperative with the stimulators switched on and off. RESULTS: Bilateral STN DBS improved motor scores (UPDRS III) by 61% when off medication (p<0.05). Clinical fluctuations (UPDRS IV items 36-39) were reduced by 46.2% (p<0.05). Total daily apomorphine dose was reduced by 68.9% (p<0.05) and apomorphine infusion via a pump was no longer required in four patients. There were no operative complications. Two patients required treatment for hallucinations postoperatively but there was no significant change in mini-mental state examination. CONCLUSIONS: In patients with advanced Parkinson's disease, previously reliant on apomorphine, bilateral STN DBS is an effective treatment to reduce motor fluctuations and enable a reduction in apomorphine use.

Variability in position of the subthalamic nucleus targeted by magnetic resonance imaging and microelectrode recordings as compared to atlas co-ordinates.

BACKGROUND: Traditional methods for localisation of target nuclei for deep brain stimulation (DBS) have used brain atlas co-ordinates for initial targeting. It is now possible to visualise the subthalamic nucleus (STN) on magnetic resonance imaging (MRI) and determine the individual variability of its position. METHODS: The present study was performed in patients undergoing STN DBS for Parkinson's disease. The STN was directly targeted from axially obtained MRI and verified with microelectrode recordings. Postoperatively, the most effective contact was identified for each patient, and its position was calculated. RESULTS: Fifty electrodes were inserted in 25 patients. The target position varied considerably in relation to the mid-commissural point. The mean effective contact position lies just dorsal to the location of the STN in a standard brain atlas. CONCLUSION: The STN varies in position, and can be accurately targeted from MRI alone.

Use of the NeuroMate stereotactic robot in a frameless mode for movement disorder surgery.

BACKGROUND/AIMS: To evaluate the use of the NeuroMate stereotactic robot with a novel ultrasound registration system for movement disorder surgery (MDS). METHODS: Using the robot in a frameless mode, 51 patients underwent MDS. Surgical planning was carried out using MRI data obtained more than 24 h before surgery. RESULTS: 37 out of 50 targets in the subthalamic nucleus were satisfactorily identified with a single microelectrode trajectory and the final electrode positions were at a mean distance of 1.7 mm from the calculated target. There was a significant improvement in motor scores of the Unified Parkinson's Disease Rating Scale III (off medication) at 6 (43%) and 18 months (51.7%) compared to pre-operative scores (p < 0.05). CONCLUSIONS: The frameless robot using only MRI data can be used for MDS. The temporal separation of imaging from the surgical procedure provides additional time for detailed image analysis and planning.

Substrate recognition by a yeast 2'-phosphotransferase involved in tRNA splicing and by its Escherichia coli homolog.

The final step of tRNA splicing in Saccharomyces cerevisiae requires 2'-phosphotransferase (Tpt1) to transfer the 2'-phosphate from ligated tRNA to NAD, producing mature tRNA and ADP ribose-1' '-2' '-cyclic phosphate. To address how Tpt1 protein recognizes substrate RNAs, we measured the steady-state kinetic parameters of Tpt1 protein with 2'-phosphorylated ligated tRNA and a variety of related substrates. Tpt1 protein has a high apparent affinity for ligated tRNA (K(m,RNA), 0.35 nM) and a low turnover rate (k(cat), 0.3 min(-1)). Tpt1 protein recognizes both tRNA and the internal 2'-phosphate of RNAs. Steady-state kinetic analysis reveals that as RNAs lose structure and length, K(m,RNA) and k(cat) both increase commensurately. For a 2'-phosphorylated octadecamer derived from the anticodon stem-loop of ligated tRNA, K(m,RNA) and k(cat) are 5- and 8-fold higher, respectively, than for ligated tRNA, whereas for a simple substrate like pApA(p)pA, K(m,RNA) and k(cat) are 430- and 150-fold higher, respectively. Tpt1 is not detectably active on a trimer with a terminal 5'- or 3'-phosphate and is very inefficient at removal of a terminal 2'-phosphate unless there is an adjacent 3'-phosphate or phosphodiester. The K(m,NAD) for Tpt1 is substrate dependent: K(m,NAD) is 10 microM with ligated tRNA, 200 microM with pApA(p)pA, and 600 microM with pApApA(p). Preliminary analysis of KptA, a functional Tpt1 protein homologue from Escherichia coli, reveals that KptA protein is strikingly similar to yeast Tpt1 in its kinetic parameters, although E. coli is not known to have a 2'-phosphorylated RNA substrate.

New twists in understanding the fate of antisense oligodeoxynucleotide mRNA targets.

In this issue of Molecular Cell, Thoma et al. show that after a target mRNA is cleaved, upon treatment with an antisense oligodeoxynucleotide, the 3' cleavage product persists and is translated to produce an N-terminally truncated version of the protein encoded by the target mRNA.

The chemical synthesis of oligoribonucleotides with selectively placed 2'-O-phosphates.

A phosphoramidite, solid support method for the chemical synthesis of oligoribonucleotides containing 2'-O-phosphate at a selected position is presented. Synthesis of these oligoribonucleotides is based on uridine- and adenosine-(2'-O-phosphate)-3'-phosphoramidites, and a new condition for removal of 2'-O-phosphate protecting groups, which does not cleave internucleotide bonds. The structure of oligoribonucleotides with 2'-O-phosphate has been proven by enzymatic digestions and dephosphorylation by yeast 2'-phosphotransferase.

Effect of a prolonged low-dose lipopolysaccharide infusion on feed intake and metabolism in heifers.

Prolonged infusions of bacterial lipopolysaccharides (LPS) are known to model gram-negative bacterial infections, but the basic mechanisms of the LPS effects on feed intake and metabolism and their potential interdependence are largely unknown. The aim of the present study was to distinguish and to better characterize the feeding suppressive and metabolic effects of LPS. Six heifers were infused intravenously for 100 min with either 1) LPS (2 microg/kg BW) with free access to feed, 2) saline with free access to feed, or 3) saline with feeding restricted to the amount of feed consumed after LPS infusion. Feed intake, body temperature, plasma concentrations of various metabolites and hormones, and the respiratory quotient and heat production were measured. The LPS reduced feed intake and induced pronounced changes in metabolic energy turnover and fat and carbohydrate metabolism that were largely independent of the concomitant feed intake reduction. Some of the metabolic changes were biphasic; the first phase resembled a stress response with increases in plasma glucose and cortisol, and the second phase reflected a beginning energy deficit with low plasma glucose and enhanced lipolysis. The coincidence of a short-term surge of plasma insulin with marked transient decreases in plasma FFA, glycerol, and beta-hydroxybutyrate as well as with the transition from hyper- to hypoglycemia indicates that insulin plays a role in some of the metabolic responses to LPS. The failure of LPS to clearly increase energy expenditure despite the increase in body temperature suggests that anaerobic mechanisms of heat production and, perhaps, a reduced peripheral blood flow contributed to the fever. Many of the initial metabolic responses occurred before and, therefore, independent of, an increase in circulating tumor necrosis factor-alpha.

Deterioration in parkinsonism with low-dose pergolide.

The administration of dopamine agonists can have a role early in the course of Parkinson's disease, in an attempt to reduce the frequency of long-term motor complications associated with the use of levodopa. After treatment with dopamine agonists has begun, gradual dose escalation is recommended to reduce the incidence of side effects; at low doses, parkinsonian symptoms significantly decline in some patients, only to improve as the dose increases. We report a number of such patients and discuss the possible pathogenesis of this motor deterioration.

Hyperekplexia and trismus due to brainstem encephalopathy.

The brainstem is said to be the generator of pathological startle responses due to reticular reflex myoclonus or hyperekplexia. A patient with facial weakness, nystagmus, and pyramidal tract signs had generalised reflex spasms in response to auditory, visual and tactile stimuli which clinically and neurophysiologically resembled hyperekplexia. The case is unusual because as well as hyperekplexia, the patient's initial presentation was with an equally rare manifestation of brainstem pathology-brainstem mediated trismus. The causes of brainstem trismus and exaggerated startle responses are discussed with respect to their underlying mechanisms.

Disordered axial movement in Parkinson's disease.

Axial motor impairments are a common cause of disability in patients with Parkinson's disease, become more prominent with longer disease duration, and have been said to be less responsive to levodopa replacement therapy. The ability to turn in bed while lying supine before and after dopaminergic stimulation was studied in a group of 36 patients with Parkinson's disease; 23 were in Hoehn and Yahr stages 3-5 when "off", and 13 were in stages 1-2. Turning was also compared with postural stability and gait before ("off") and after ("on") dopaminergic stimulation. Failure to turn in bed was noted in 19 of the 36 patients in the "off" state, with significant associations between disturbances of gait, postural stability, rising from a chair, whole body bradykinesia, and axial rigidity. Gait, postural stability, rising from a chair, whole body bradykinesia, and axial rigidity were significantly correlated in the "off" state. Disorder of axial movement, gait, and postural stability were not dependent on age at onset of Parkinson's disease, but did relate to duration of disease. After a levodopa challenge, turning in bed returned to normal in all but one patient, and gait, postural stability, rising from a chair, whole body bradykinesia, and axial rigidity also improved in nearly all. It is concluded that in the later stages of Parkinson's disease at least some aspects of axial motor control can remain dopamine responsive.