Measuring Intracompartmental Pressures in the Lower Leg: Assessing the Use of Unilateral Measurements in Patients with Bilateral Symptoms.

The purpose of the present study was to evaluate whether unilateral intracompartmental pressure (ICP) measurements correctly represent the contralateral ICP value in patients suspected to have bilateral chronic exertional compartment syndrome (CECS) in the anterior compartment of the leg. Methods: A retrospective cohort study was performed that included military service members who had been referred to a secondary care department for bilateral anterolateral exercise-related leg pain. The obtained ICP values were utilized to assess 2 possible measurement strategies to perform unilateral ICP measurements: the right-leg strategy (i.e., always testing the right leg) and the most-symptomatic-leg strategy (i.e., always testing the most symptomatic). The diagnostic cutoff value for CECS in this cohort was 35 mmHg in the first minute after provocation. Four outcome categories were created to describe the pressure classification of the second leg if only 1 leg would have been measured: correct (category 1: both values ≥35 mmHg; category 2: both values <35 mmHg) or incorrect (category 3: measured leg, ≥35 mmHg and contralateral leg, <35 mmHg; category 4: measured leg, <35 mmHg and contralateral leg, ≥35 mmHg). Results: A total of 442 patients (884 legs) were included. In 88% of patients, the unilateral value would have correctly diagnosed the other symptomatic leg, whereas in 12% of patients, the contralateral leg would have been diagnosed incorrectly. The right-leg strategy had a slightly smaller proportion of cases in which the contralateral leg would have been incorrectly diagnosed (7% compared with 8% for the most symptomatic leg strategy). In 89% of the 390 patients in categories 1 and 2, the ICP values deviated by >5 mmHg from the 35-mmHg cutoff value compared with 40% of the 52 patients in categories 3 and 4. Conclusions: In military service members with bilateral chronic anterolateral exertional pain, a unilateral ICP measurement seems to be justified, especially among those with pressure values >5 mmHg above or below the diagnostic cutoff value. When a unilateral pressure measurement is within 5 mmHg above or below the cutoff value, a bilateral ICP measurement may be warranted. Level of Evidence: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Clinical Consensus on Diagnosis and Treatment of Patients with Chronic Exertional Compartment Syndrome of the Leg: A Delphi Analysis.

AIM: Defining universally accepted guidelines for the diagnosis and treatment of chronic exertional compartment syndrome (CECS) is hampered by the absence of high-quality scientific research. The aim of this Delphi study was to establish consensus on practical issues guiding diagnosis and treatment of CECS of the leg in civilian and military patient populations. METHODS: An international expert group was queried using the Delphi technique with a traditional three-round electronic consultation. Results of previous rounds were anonymously disclosed in the questionnaire of rounds 2 and 3, if relevant. Consensus was defined as > 70% positive or negative agreement for a question or statement. RESULTS: The panel consisted of 27 civilian and military healthcare providers. Consensus was reached on five essential key characteristics of lower leg CECS. The panel achieved partial agreement regarding standardization of the diagnostic protocol, including muscle tissue pressure measurements. Consensus was reached on conservative and surgical treatment regimens. However, the experts did not attain consensus on their approach of postoperative rehabilitation and preferred treatment approach of recurrent or residual disease. A summary of best clinical practice for the diagnosis and management of CECS was formulated by experts working in civilian and military healthcare facilities. CONCLUSION: The Delphi panel reached consensus on key criteria for signs and symptoms of CECS and several aspects for conservative and surgical treatment. The panel did not agree on the role of ICP values in the diagnostic process, postoperative rehabilitation guidelines protocol, or the preferred treatment approach for recurrent or residual disease. These aspects serve as a first attempt to initiate simple guidelines for clinical practice.

Measuring intracompartmental pressures for the chronic exertional compartment syndrome: Challenging commercially available devices and their respective accuracy.

The aim of this study was to compare commercially available manometers and needles used for intracompartmental pressure measurements for accuracy. An experimental compartment simulation model was developed in order to compare four different terminal devices (Compass manometer, Stryker device, Meritrans transducer, and arterial line) and 22 types of needles. First, all possible device/needle combinations were introduced in rubber ports at the bottom of a water column. The water column was gradually drained three times for each device/needle combination and pressures were recorded by two researchers. This procedure was repeated after placement of a sample of homogenous porcine gluteal muscle tissue. When measuring the fluid column only, all Intraclass Correlation Coefficients were found to be ≥0.980, indicating good resemblance to the reference pressure. After addition of the muscle tissue to the experimental set up, accuracy of the Stryker device and arterial line remained the same. The accuracy of the Compass manometer and Meritrans transducer became less. Excellent reliability was found for 7 out of 22 needles when combined with the Stryker device and for 2 out of 22 needles when combined with the arterial line. The four terminal devices tested were all accurate when measuring pressure in a water column, whereas only the Stryker device and arterial line accurately represented the water column pressure in a porcine gluteal muscle model. Future research will have to verify whether these results can be repeated in human cadaver models and in vivo.

One out of four recruits drops out from elite military training due to musculoskeletal injuries in the Netherlands Armed Forces.

INTRODUCTION: Musculoskeletal injuries (MSIs) are among the main causes of dropout from military training. The main purpose of this study was to provide an overview of dropout rates and MSI incidence rates during elite military training. Second, this study aimed to explore restricted training days due to MSIs and to describe MSI-care by military physicians. METHODS: In a retrospective observational study, we collected dropout rates and injury surveillance data from the electronic patient records of two elite units of the Netherlands Armed Forces (NAF): the Royal Netherlands Marine Corps (RNLMC) and the Airmobile Brigade (AMB), from 1 January 2015 until 31 December 2017. RESULTS: In the RNLMC, total dropout rate was 53.9% and dropout due to MSIs was 23%. The most frequently affected locations were foot, knee and leg. In the AMB total dropout rate was 52.6% and dropout due to MSIs was 25%. In the AMB, the most frequently affected locations were back, knee and leg. Average restricted training days due to MSIs ranged between 8.3 and 20.8 days/injury. MSI-care by military physicians consisted mostly of the provision of injury-specific information and (self-)management options, imposing a specific activity restriction and referral to physiotherapy. CONCLUSION: Our study findings showed that one out of four recruits who dropout from elite military training in the NAF, do so due to MSIs. Redesigning training programmes with the objective to reduce MSIs should be given high priority, as this may reduce dropout substantially.

Network-driven discovery yields new insight into Shox2-dependent cardiac rhythm control.

The homeodomain transcription factor SHOX2 is involved in the development and function of the heart's primary pacemaker, the sinoatrial node (SAN), and has been associated with cardiac conduction-related diseases such as atrial fibrillation and sinus node dysfunction. To shed light on Shox2-dependent genetic processes involved in these diseases, we established a murine embryonic stem cell (ESC) cardiac differentiation model to investigate Shox2 pathways in SAN-like cardiomyocytes. Differential RNA-seq-based expression profiling of Shox2+/+ and Shox2-/- ESCs revealed 94 dysregulated transcripts in Shox2-/- ESC-derived SAN-like cells. Of these, 15 putative Shox2 target genes were selected for further validation based on comparative expression analysis with SAN- and right atria-enriched genes. Network-based analyses, integrating data from the Mouse Organogenesis Cell Atlas and the Ingenuity pathways, as well as validation in mouse and zebrafish models confirmed a regulatory role for the novel identified Shox2 target genes including Cav1, Fkbp10, Igfbp5, Mcf2l and Nr2f2. Our results indicate that genetic networks involving SHOX2 may contribute to conduction traits through the regulation of these genes.

Historic cohort: outcome of chronic exertional compartment syndrome-suspected patients.

INTRODUCTION: Exercise-related leg pain (ERLP) may be caused by chronic exertional compartment syndrome (CECS), occurring mainly in athletes and military recruits. In military populations, the effectiveness of surgical treatment in CECS is debated. The purpose of this study is to assess the outcome of surgical treatment for CECS in Alrijne Hospital (the Netherlands), a civilian hospital with supraregional referral function. METHODS: A historic cohort study was performed on patients with ERLP who were suspected for CECS and were referred for intracompartmental pressure measurement (ICPM) from 2013 to 2017 (n=160). Patient demographics, ICPM and survey response were analysed. RESULTS: The mean delay before visitation was 29.0±30.3 months. When comparing surgical-treated patients with CECS with conservative-treated patients with ERLP, surgical-treated patients were more satisfied, reported better recovery towards former level of performance (2.8±2.0 vs 3.9±1.7 and 2.5±1.6 vs 3.2±1.4 on a 7-point Likert scale, respectively) and better subjective injury status (79.3±22.6 vs 63.5±27.4 using the Single Assessment Numeric Evaluation score). Treatment satisfaction was 75.0% in surgical-treated CECS versus 51.4% in conservative-treated ERLP. CONCLUSION: Civilian patients report improved functional outcomes after fasciotomy for CECS. Future research should focus on non-invasive diagnostic options and methods to determine which treatment is the most appropriate for each individual patient.

Extracellular Vesicles Derived from Neural Progenitor Cells--a Preclinical Evaluation for Stroke Treatment in Mice.

Stem cells such as mesenchymal stem cells (MSCs) enhance neurological recovery in preclinical stroke models by secreting extracellular vesicles (EVs). Since previous reports have focused on the application of MSC-EVs only, the role of the most suitable host cell for EV enrichment and preclinical stroke treatment remains elusive. The present study aimed to evaluate the therapeutic potential of EVs derived from neural progenitor cells (NPCs) following experimental stroke. Using the PEG technique, EVs were enriched and characterized by electron microscopy, proteomics, rt-PCR, nanosight tracking analysis, and Western blotting. Different dosages of NPC-EVs displaying a characteristic profile in size, shape, cargo protein, and non-coding RNA contents were incubated in the presence of cerebral organoids exposed to oxygen-glucose deprivation (OGD), significantly reducing cell injury when compared with control organoids. Systemic administration of NPC-EVs in male C57BL6 mice following experimental ischemia enhanced neurological recovery and neuroregeneration for as long as 3 months. Interestingly, the therapeutic impact of such NPC-EVs was found to be not inferior to MSC-EVs. Flow cytometric analyses of blood and brain samples 7 days post-stroke demonstrated increased blood concentrations of B and T lymphocytes after NPC-EV delivery, without affecting cerebral cell counts. Likewise, a biodistribution analysis after systemic delivery of NPC-EVs revealed the majority of NPC-EVs to be found in extracranial organs such as the liver and the lung. This proof-of-concept study supports the idea of EVs being a general concept of stem cell-induced neuroprotection under stroke conditions, where EVs contribute to reverting the peripheral post-stroke immunosuppression.

Despite an improved aerobic endurance, still high attrition rates in initially low-fit recruits-results of a randomised controlled trial.

BACKGROUND: Low baseline fitness of recruits entering basic military training (BMT) is associated with an increased risk of musculoskeletal injuries (MSIs) and attrition from training. OBJECTIVE: To determine the effects of a pre-training conditioning program (PCP) on aerobic endurance, incidence of musculoskeletal injuries (MSIs), and attrition rates in BMT of a special infantry unit of the Netherlands Armed Forces. PARTICIPANTS: Recruits were considered eligible for this study when they were 'low-fit' at the start of BMT (time to complete a 2.7-km run ≥ 12'23″). INTERVENTIONS: 'Low-fit' recruits were deferred to a seven to twelve week-depending on the time between two consecutive training platoons-PCP consisting of functional training to improve several fitness domains. The control (CON) group started regular BMT without delay. RESULTS: Forty-nine recruits were included in this study; 26 in the PCP-group and 23 in the CON-group. Recruits who followed the PCP started BMT with better aerobic endurance than the CON-group who started BMT immediately (2.7 km run timings: PCP 11'32″, CON 13'16″). The risk of dropout was lower in the PCP-group (incidence density ratio (IDR) 0.63, 95%CI 0.32; 1.26), but dropout due to training-related MSIs was more frequent (IDR 1.23, 95%CI 0.32; 4.76 (per-protocol 0.94, 95%CI 0.24; 3.63), without statistically significant differences between the groups. CONCLUSION: Although a PCP was effective to improve aerobic endurance in low-fit recruits to return to meet pre-enlistment fitness criteria, we could not demonstrate an effect on dropout from military training. TRIAL REGISTRATION: Dutch trial register Trial NL6791 (NTR6977) https://www.trialregister.nl/trial/6791.

Reducing vertical ground reaction forces: The relative importance of three gait retraining cues.

BACKGROUND: Previous studies in our department demonstrated that gait retraining as part of a conservative treatment program for service members with exercise-related leg pain can lead to persistent changes in vertical ground reaction forces while running in shoes and boots. It is not known which gait retraining cue has the largest effect and whether a combination of cues is advantageous. METHODS: During a single gait retraining session, 12 male heel striking patients were given three cues in isolation: Cue 1. Change to a ball-of-foot strike; Cue 2. Increase cadence to 180 steps per minute; Cue 3. Stand up taller; and finally, all three cues combined. Runs were performed on an instrumented treadmill at 10 km/h, 1% incline and in running shoes. The three cues were randomly introduced. Measurements, taken during 30-second episodes, were stride length, cadence, and six force variables: maximum force (N) and maximum pressure (N/cm2) on the heel, mid-foot and fore-foot. FINDINGS: Each cue, i.e., each change in running technique, caused a different pattern of changes among the six force variables, mostly reductions. In isolation, cue 1 produced the largest reduction of force and pressure on the heel, resulting percentages 45.8 and 67.2 respectively (p = 0.00, p = 0.00). Overall, the combination of cues 1 + 2 + 3 ranked first in reducing forces for four of the six force variables. INTERPRETATION: Three commonly used gait retraining cues, when applied in isolation, all resulted in a reduction of most vertical ground reaction forces. The combination of the three cues is advantageous.

A pre-training conditioning program to increase physical fitness and reduce attrition due to injuries in Dutch Airmobile recruits: Study protocol for a randomised controlled trial.

BACKGROUND: Low baseline fitness of recruits entering basic military training is shown to be associated with an increased risk of musculoskeletal injuries (MSIs) and attrition of military training. This in turn leads to an increased demand for health care, increased health care costs and decreased personnel occupancy rate of military units. DESIGN: Study protocol for a randomised controlled trial. OBJECTIVE: To determine the effects of a pre-training conditioning program on cardiovascular endurance, incidence of overuse injuries, and attrition rates in Dutch Airmobile recruits undertaking initial military training. SETTING: Royal Netherlands Army, Air Assault Brigade military training course. PARTICIPANTS: Recruits are considered eligible for this study when they are 'low-fit' at the start of the initial military training. Time to complete a 2700 m run test in ≥12'23'' is used as selection criteria. INTERVENTIONS: We use a complex system approach to cover multiple domains of MSI prevention and optimise intervention circumstance; a pre-training conditioning program, training staff support, process-focused culture and health accountability. The pre-training conditioning program contains functional training to improve mobility, power, agility, lower and upper body strength and cardiovascular endurance. Cardiovascular endurance will be targeted both by endurance training and high intensity interval training. MAIN OUTCOME MEASUREMENTS: Outcome measures include cardiovascular endurance, injury incidence, attrition rates, lost training days due to injuries, and implementation fidelity of the PCP. TRIAL STATUS: Recruitment of participants commenced April 18, 2018 and final results are expected in August 2019. TRIAL REGISTRATION: Dutch trial register www.trialregister.nl/=trial/6791.

[Healthcare behavior of migraine and headache patients when treatment is accompanied by the digital migraine app]. / Gesundheitsverhalten von Migräne- und Kopfschmerzpatienten bei digitaler Therapiebegleitung mit der Migräne-App.

BACKGROUND: Tension-type headache and migraine are the second and third most prevalent disorders of mankind worldwide, after dental caries. The widespread implementation of smartphones enables the use of specific software applications (apps) for digital treatment accompaniment. In this study, the use of the migraine app (Migräne-App) for iOS and Android was examined in the practical treatment of migraine and headache patients in an extensive population sample. METHODS: An online survey was developed for the analysis of experiences as part of the treatment accompaniment and app usage. It contains questions concerning sociodemographic variables, the course of headache disorders and the previous treatment as well as the usage of the migraine app. The survey establishes compliance to the recommended treatment, the treatment plan, and treatment rules devised by the treating physician. The data collected were compared to traditional pen and paper documentation, prior to using the migraine app. RESULTS: A total of 1464 users participated in the standardized survey. The average age was 47.19 ± 11.37 years (87.4% female, 12.5% male). On average, users suffered from headaches for 27.28 ± 13.6 years. The majority (76.5%) were cared for by a general practitioner. Of the users 70.9% reported that they presented the aggregated data from the app to their physician on consultation, 76.4% reported that the migraine app helped them to adhere to the treatment plan designed together with their physician and the rules about headache therapy. It showed both a highly significant reduction of headache days per months prior to usage (13.30 ± 7.45 days) in comparison to at the time of conducting the survey (10.03 ± 7.30 days) as well as a highly significant reduction of intake of acute medication (before 7.61 ± 5.58 vs. ongoing 6.78 ± 4.72 days). CONCLUSION: The data show that the digital treatment control for therapy decisions made by the physician is highly relevant and established. Therapy compliance is improved and possible complications such as headache due to medication overuse are reduced. At the same time, a significant improvement of headache parameters and a marked overall improvement of treatment quality, amongst other things due to more easily available information and self-help tools can be observed.

Measurement of the magnetic moment of single Magnetospirillum gryphiswaldense cells by magnetic tweezers.

Magnetospirillum gryphiswaldense is a helix-shaped magnetotactic bacterium that synthesizes iron-oxide nanocrystals, which allow navigation along the geomagnetic field. The bacterium has already been thoroughly investigated at the molecular and cellular levels. However, the fundamental physical property enabling it to perform magnetotaxis, its magnetic moment, remains to be elucidated at the single cell level. We present a method based on magnetic tweezers; in combination with Stokesian dynamics and Boundary Integral Method calculations, this method allows the simultaneous measurement of the magnetic moments of multiple single bacteria. The method is demonstrated by quantifying the distribution of the individual magnetic moments of several hundred cells of M. gryphiswaldense. In contrast to other techniques for measuring the average magnetic moment of bacterial populations, our method accounts for the size and the helical shape of each individual cell. In addition, we determined the distribution of the saturation magnetic moments of the bacteria from electron microscopy data. Our results are in agreement with the known relative magnetization behavior of the bacteria. Our method can be combined with single cell imaging techniques and thus can address novel questions about the functions of components of the molecular magnetosome biosynthesis machinery and their correlation with the resulting magnetic moment.

Engineered Heart Repair.

There is a pressing need for the development of advanced heart failure therapeutics. Current state-of-the-art is protection from neurohumoral overstimulation, which fails to address the underlying cause of heart failure, namely loss of cardiomyocytes. Implantation of stem cell-derived cardiomyocytes via tissue-engineered myocardium is being advanced to realize the remuscularization of the failing heart. Here, we discuss pharmacological challenges pertaining to the clinical translation of tissue-engineered heart repair with a focus on engineered heart muscle (EHM).

All naturally occurring autoantibodies against the NMDA receptor subunit NR1 have pathogenic potential irrespective of epitope and immunoglobulin class.

Autoantibodies of the IgG class against N-methyl-d-aspartate-receptor subunit NR1 (NMDAR1) were first described in anti-NMDAR encephalitis and seen as disease indicators. Recent work on together over 5000 individuals challenged this exclusive view by showing age-dependently up to >20% NMDAR1-autoantibody seroprevalence with comparable immunoglobulin class and titer distribution across health and disease. The key question therefore is to understand the properties of these autoantibodies, also in healthy carriers, in order to assess secondary complications and possible contributions to neuropsychiatric disease. Here, we believe we provide for human NMDAR1-autoantibodies the first comprehensive analysis of their target epitopes and functionality. We selected sera of representative carriers, healthy or diagnosed with very diverse conditions, that is, schizophrenia, age-related disorders like hypertension and diabetes, or anti-NMDAR encephalitis. We show that all positive sera investigated, regardless of source (ill or healthy donor) and immunoglobulin class, provoked NMDAR1 internalization in human-induced pluripotent stem cell-derived neurons and reduction of glutamate-evoked currents in NR1-1b/NR2A-expressing Xenopus oocytes. They displayed frequently polyclonal/polyspecific epitope recognition in the extracellular or intracellular NMDAR1 domains and some additionally in NR2A. We conclude that all circulating NMDAR1-autoantibodies have pathogenic potential regarding the whole spectrum of neuronal NMDAR-mediated effects upon access to the brain in situations of increased blood-brain-barrier permeability.

Occurrence of Brachyspira hyodysenteriae in multiplier pig herds in Switzerland.

OBJECTIVE: This research was aimed to determine the occurrence of Brachyspira (B.) hyodysenteriae in Swiss multiplier pig herds. MATERIALS AND METHODS: In a pilot study a direct real-time polymerase chain reaction (PCR) method for B. hyodysenteriae was compared to culture followed by PCR on 106 samples from three herds. Subsequently 40 multiplier herds were epidemiologically characterized and analysed for the presence of B. hyodysenteriae using direct PCR on 1412 rectal swabs. For external validation 20 swabs obtained from two positive conventional herds were analysed. RESULTS: The comparison of direct PCR with culture followed by PCR resulted in a moderate agreement (kappa index: 0.58). In the two conventional herds, 35% of the samples (7/20) tested positive. Samples from 39 multipliers tested negative. In one multiplier herd, 25% (9/36) of the samples tested PCR positive. Risk factors in the multiplier herd may have been rodents or birds, but not pig purchase. CONCLUSION AND CLINICAL RELEVANCE: B. hyodysenteriae have been detected in a Swiss multiplier herd, which underlines the threat of potential spread by replacement pigs. Consequently, a Brachyspira monitoring programme was established for Swiss multiplier herds.

[Direct cloning of gene encoding a novel amylomaltase from soil bacterial DNA for large-ring cyclodextrin production].

The aim of this study was to isolate a novel amylomaltase gene from community DNA of soil samples collected from Ban Nong Khrok hot spring in Thailand without bacterial cultivation. Using PCR, a 1.5 kb full-length gene was amplified and ligated with pGEM-T easy vector to transform into Escherichia coli DH5 alpha for sequencing. The obtained gene encoding an amylomaltase consisted of 1.503 bp that translated into 500 amino acids. Amino acid sequence deduced from this gene was highly homologous with that of amylomaltase from Thermus thermophillus ATCC 33923. In order to express the enzyme, the cloned gene was subcloned into plasmid pET-17b and introduced into E. coli BL21 (DE3). The maximum expression was observed when the cloned cells were cultured at 37 degrees C for 6 h with 0.5 mM IPTG induction. By 10% SDS-PAGE, the relative molecular mass of the purified amylomaltase was approximately 58 kDa. This enzyme was optimally active at 70 degrees C and pH 9.0. In addition, the enzyme could hydrolyze pea starch to yield the large-ring cyclodextrins with degrees of polymerization of 23 and higher. It is noted that CD29 was the product in the largest quantity under all tested conditions.

[Individualized stem cell therapy]. / Individualisierte Therapie mit Stammzellen.

Stem cells are attributed with having a great potential in regenerative medicine. Pluripotent stem cells are particularly interesting because they can be multiplied indefinitely and also differentiated under defined conditions. Currently, cardiomyocytes can be differentiated very effectively from pluripotent stem cells, making the former an attractive starting material for cardiac disease modeling in a culture dish (patient in a dish) and cell based-therapy in heart failure. The rapid biotechnological advances made in recent years now enable these concepts to be translated into clinical applications.

PDE2-mediated cAMP hydrolysis accelerates cardiac fibroblast to myofibroblast conversion and is antagonized by exogenous activation of cGMP signaling pathways.

Recent studies suggest that the signal molecules cAMP and cGMP have antifibrotic effects by negatively regulating pathways associated with fibroblast to myofibroblast (MyoCF) conversion. The phosphodiesterase 2 (PDE2) has the unique property to be stimulated by cGMP, which leads to a remarkable increase in cAMP hydrolysis and thus mediates a negative cross-talk between both pathways. PDE2 has been recently investigated in cardiomyocytes; here we specifically addressed its role in fibroblast conversion and cardiac fibrosis. PDE2 is abundantly expressed in both neonatal rat cardiac fibroblasts (CFs) and cardiomyocytes. The overexpression of PDE2 in CFs strongly reduced basal and isoprenaline-induced cAMP synthesis, and this decrease was sufficient to induce MyoCF conversion even in the absence of exogenous profibrotic stimuli. Functional stress-strain experiments with fibroblast-derived engineered connective tissue (ECT) demonstrated higher stiffness in ECTs overexpressing PDE2. In regard to cGMP, neither basal nor atrial natriuretic peptide-induced cGMP levels were affected by PDE2, whereas the response to nitric oxide donor sodium nitroprusside was slightly but significantly reduced. Interestingly, despite persistently depressed cAMP levels, both cGMP-elevating stimuli were able to completely prevent the PDE2-induced MyoCF phenotype, arguing for a double-tracked mechanism. In conclusion, PDE2 accelerates CF to MyoCF conversion, which leads to greater stiffness in ECTs. Atrial natriuretic peptide- and sodium nitroprusside-mediated cGMP synthesis completely reverses PDE2-induced fibroblast conversion. Thus PDE2 may augment cardiac remodeling, but this effect can also be overcome by enhanced cGMP. The redundant role of cAMP and cGMP as antifibrotic meditators may be viewed as a protective mechanism in heart failure.

Molecular mutagenesis at Tyr-101 of the amylomaltase transcribed from a gene isolated from soil DNA.

The wild-type (WT) amylomaltase gene was directly isolated from soil DNA and cloned into a pET19b vector to express in E. coli BL21(DE3). The ORF of this gene consisted of 1,572 bp, encoding an enzyme of 523 amino acids. Though showing 99% sequence identity to amylomaltse from Thermus thermophilus ATCC 33923, this enzyme is unique in its alkaline optimum pH. In order to alter amylomaltase with less coupling or hydrolytic activity to enhance cycloamylose (CA) formation through cyclization reaction, site-directed mutagenesis of the second glucan binding site involving in CA production was performed at Tyr-101. The result revealed that the mutated Y101S enzyme showed a small increase in cyclization activity while significantly decreased disproportionation, coupling and hydrolytic activities. Mutation also resulted in the change in substrate specificity for disproportionation reaction. The WT enzyme preferred maltotriose, while the activity of mutated enzyme was the highest with maltopentaose substrate. Product analysis by HPAEC-PAD demonstrated that the main CAs of the WT amylomaltase were CA29-CA37. Y101S mutation did not change the product pattern, however, the amount of CAs formed by the mutated enzyme tended to increase especially at long incubation time.