Synthesis of poly(3-hydroxyalkanoates) by mutant and recombinant Pseudomonas strains

We have studied the accumulation kinetics and physical characteristics of the poly(3-hydroxyalkanoates) (PHAs) formed by several Pseudomonas strains, mutants and recombinants. Although PHA synthesis generally begins only after an essential nutrient such as N, P, S or Mg becomes limiting, we have identified at least one strain (P. putida KT2442) that begins producing PHA during the exponential growth phase. This PHA is chemically and physically identical to that produced by P. oleovorans GPol, the strain in which we first identified PHA. Analysis of the PHA formed by a mutant strain defective in PHA degradation (P. oleovorans GPo500) revealed that the molecular mass (M_w), the monomer composition and thermal characteristics were similar to that of the PHA of the wild-type parent strain P. oleovorans GPo1. The pha locus of P. oleovorans encodes enzymes that are involved in PHA biosynthesis and degradation. It has been subcloned to study the two PHA polymerases separately in a PHA^– mutant (GPp104) derived from P. putida KT2442. The recombinant strains accumulated lower PHA levels than the wild-type strains, and the M_w of these polymers were lower than those produced by the wild-type P. oleovorans and parent strain. The monomer composition of the two PHAs formed by the two PHA polymerases differed, indicating that the PHA polymerases have different substrate specificities for the incorporation of 3-hydroxyoctanoate and 3-hydroxyhexanoate monomers into PHA. Despite these differences, the PHAs formed were essentially indistinguishable from wild-type PHAs with respect to their thermal characteristics.Correspondence to: B. Witholt     

Engagement of SIRPα Inhibits Growth and Induces Programmed Cell Death in Acute Myeloid Leukemia Cells

Background

Recent studies show the importance of interactions between CD47 expressed on acute myeloid leukemia (AML) cells and the inhibitory immunoreceptor, signal regulatory protein-alpha (SIRPα) on macrophages. Although AML cells express SIRPα, its function has not been investigated in these cells. In this study we aimed to determine the role of the SIRPα in acute myeloid leukemia.

Design and Methods

We analyzed the expression of SIRPα, both on mRNA and protein level in AML patients and we further investigated whether the expression of SIRPα on two low SIRPα expressing AML cell lines could be upregulated upon differentiation of the cells. We determined the effect of chimeric SIRPα expression on tumor cell growth and programmed cell death by its triggering with an agonistic antibody in these cells. Moreover, we examined the efficacy of agonistic antibody in combination with established antileukemic drugs.

Results

By microarray analysis of an extensive cohort of primary AML samples, we demonstrated that SIRPα is differentially expressed in AML subgroups and its expression level is dependent on differentiation stage, with high levels in FAB M4/M5 AML and low levels in FAB M0–M3. Interestingly, AML patients with high SIRPα expression had a poor prognosis. Our results also showed that SIRPα is upregulated upon differentiation of NB4 and Kasumi cells. In addition, triggering of SIRPα with an agonistic antibody in the cells stably expressing chimeric SIRPα, led to inhibition of growth and induction of programmed cell death. Finally, the SIRPα-derived signaling synergized with the activity of established antileukemic drugs.

Conclusions

Our data indicate that triggering of SIRPα has antileukemic effect and may function as a potential therapeutic target in AML.     

Clinical Evaluation of Rega 8: An Updated Genotypic Interpretation System That Significantly Predicts HIV-Therapy Response

Introduction

Clinically evaluating genotypic interpretation systems is essential to provide optimal guidance in designing potent individualized HIV-regimens. This study aimed at investigating the ability of the latest Rega algorithm to predict virological response on a short and longer period.

Materials & Methods

9231 treatment changes episodes were extracted from an integrated patient database. The virological response after 8, 24 and 48 weeks was dichotomized to success and failure. Success was defined as a viral load below 50 copies/ml or alternatively, a 2 log decrease from the baseline viral load at 8 weeks. The predictive ability of Rega version 8 was analysed in comparison with that of previous evaluated version Rega 5 and two other algorithms (ANRS v2011.05 and Stanford HIVdb v6.0.11). A logistic model based on the genotypic susceptibility score was used to predict virological response, and additionally, confounding factors were added to the model. Performance of the models was compared using the area under the ROC curve (AUC) and a Wilcoxon signed-rank test.

Results

Per unit increase of the GSS reported by Rega 8, the odds on having a successful therapy response on week 8 increased significantly by 81% (OR = 1.81, CI = [1.76–1.86]), on week 24 by 73% (OR = 1.73, CI = [1.69–1.78]) and on week 48 by 85% (OR = 1.85, CI = [1.80–1.91]). No significant differences in AUC were found between the performance of Rega 8 and Rega 5, ANRS v2011.05 and Stanford HIVdb v6.0.11, however Rega 8 had the highest sensitivity: 76.9%, 76.5% and 77.2% on 8, 24 and 48 weeks respectively. Inclusion of additional factors increased the performance significantly.

Conclusion

Rega 8 is a significant predictor for virological response with a better sensitivity than previously, and with rules for recently approved drugs. Additional variables should be taken into account to ensure an effective regimen.     

A Meta-Analysis of Retinoblastoma Copy Numbers Refines the List of Possible Driver Genes Involved in Tumor Progression

BackgroundWhile RB1 loss initiates retinoblastoma development, additional somatic copy number alterations (SCNAs) can drive tumor progression. Although SCNAs have been identified with good concordance between studies at a cytoband resolution, accurate identification of single genes for all recurrent SCNAs is still challenging. This study presents a comprehensive meta-analysis of genome-wide SCNAs integrated with gene expression profiling data, narrowing down the list of plausible retinoblastoma driver genes.MethodsWe performed SCNA profiling of 45 primary retinoblastoma samples and eight retinoblastoma cell lines by high-resolution microarrays. We combined our data with genomic, clinical and histopathological data of ten published genome-wide SCNA studies, which strongly enhanced the power of our analyses (N = 310).ResultsComprehensive recurrence analysis of SCNAs in all studies integrated with gene expression data allowed us to reduce candidate gene lists for 1q, 2p, 6p, 7q and 13q to a limited gene set. Besides the well-established driver genes RB1 (13q-loss) and MYCN (2p-gain) we identified CRB1 and NEK7 (1q-gain), SOX4 (6p-gain) and NUP205 (7q-gain) as novel retinoblastoma driver candidates. Depending on the sample subset and algorithms used, alternative candidates were identified including MIR181 (1q-gain) and DEK (6p gain). Remarkably, our study showed that copy number gains rarely exceeded change of one copy, even in pure tumor samples with 100% homozygosity at the RB1 locus (N = 34), which is indicative for intra-tumor heterogeneity. In addition, profound between-tumor variability was observed that was associated with age at diagnosis and differentiation grades.InterpretationSince focal alterations at commonly altered chromosome regions were rare except for 2p24.3 (MYCN), further functional validation of the oncogenic potential of the described candidate genes is now required. For further investigations, our study provides a refined and revised set of candidate retinoblastoma driver genes.     

Muscle contraction history: modified Hill versus an exponential decay model

In recent years, it has been recognised that improvements to classic models of muscle mechanical behaviour are often necessary for properly modelling co-ordinated multi-joint actions. In this respect, the purpose of the present study was to improve on modelling stretch-induced force enhancement and shortening-induced force depression of muscle contraction. For this purpose, two models were used: a modified Hill model and a model based loosely on mechano-chemistry of the cross-bridge cycle (exponential decay model). The models were compared with a classic Hill model and experimental data. Parameter values were based, as much as possible, on experimental findings in the literature, and tested with new experiments on the gastrocnemius of the rat. Both models describe many features of slow-ramp movements well during short contractions (300–500 ms), but long-duration behaviour is described only partly. The exponential decay model does not incorporate a force–velocity curve. Therefore, its good performance indicates that the status of the classic force–velocity characteristic may have to be reconsidered. Like movement-induced force depression and enhancement, it seems a particular manifestation of time-dependent force behaviour of muscle, rather than a fundamental property of muscle (like the length–tension curve). It is argued that a combination of the exponential decay model (or other models based on the mechano-chemistry of contraction) and structurally based models may be fruitful in explaining this time-dependent contraction behaviour. Furthermore, not in the least because of its relative simplicity, the exponential decay model may prove more suitable for modelling multi-joint movements than the Hill model. Received: 19 March 1999 / Accepted in revised form: 9 June 2000     

Stretch-shorten cycle compared with isometric preload: contributions to enhanced muscular performance

Walshe, Andrew D., Greg J. Wilson, and Gertjan J. C. Ettema.Stretch-shorten cycle compared with isometric preload: contributions to enhanced muscular performance. J. Appl. Physiol. 84(1): 97-106, 1998.To isolateany difference muscular contraction history may have on concentric workoutput, 40 trained male subjects performed three separate isokineticconcentric squats that involved differing contraction histories:1) a concentric-only (CO) squat, 2) a concentric squat preceded by anisometric preload (IS), and 3) astretch-shorten cycle (SSC) squat. Over the first 300 ms of theconcentric movement, work output for both the SSC and IS conditions wassignificantly greater (154.8 ± 39.8 and 147.9 ± 34.7 J, respectively; P < 0.001) comparedwith the CO squat (129.7 ± 34.4 J). In addition, work output afterthe SSC test over the first 300 ms was also significantly larger thanthat for the corresponding period after the IS protocol(P < 0.05). There was no difference in normalized, integrated electromyogram among any of the conditions. It was concluded that concentric performance enhancement derived from apreceding stretch of the muscle-tendon complex was largely due to theattainment of a higher active muscle state before the start of theconcentric movement. However, it was also hypothesized that contractileelement potentiation was a significant contributor to stretch-inducedmuscular performance under these conditions.

     

Energy Expenditure and Metabolic Changes of Free-Flying Migrating Northern Bald Ibis

Many migrating birds undertake extraordinary long flights. How birds are able to perform such endurance flights of over 100-hour durations is still poorly understood. We examined energy expenditure and physiological changes in Northern Bald Ibis Geronticus eremite during natural flights using birds trained to follow an ultra-light aircraft. Because these birds were tame, with foster parents, we were able to bleed them immediately prior to and after each flight. Flight duration was experimentally designed ranging between one and almost four hours continuous flights. Energy expenditure during flight was estimated using doubly-labelled-water while physiological properties were assessed through blood chemistry including plasma metabolites, enzymes, electrolytes, blood gases, and reactive oxygen compounds. Instantaneous energy expenditure decreased with flight duration, and the birds appeared to balance aerobic and anaerobic metabolism, using fat, carbohydrate and protein as fuel. This made flight both economic and tolerable. The observed effects resemble classical exercise adaptations that can limit duration of exercise while reducing energetic output. There were also in-flight benefits that enable power output variation from cruising to manoeuvring. These adaptations share characteristics with physiological processes that have facilitated other athletic feats in nature and might enable the extraordinary long flights of migratory birds as well.     

A comparison of kinematics between overarm throwing with 20% underweight, regular, and 20% overweight balls

The aim of this study was to compare the kinematics in throwing with a regular weighted handball with 20% lighter and heavier balls in female experienced handball players. In total, eight joint movements during the throw were analyzed. The analysis consisted of maximal angles, angles at ball release, and maximal angular velocities of the joint movements and their timings during the throw. Results on 24 experienced female team handball players (mean age 18.2 ± 2.1 years) showed that the difference in ball weight affected the maximal ball velocity. The difference in ball release velocity was probably a result of the significant differences in kinematics of the major contributors to overarm throwing: elbow extension and internal rotation of the shoulder. These were altered when changing the ball weight, which resulted in differences in ball release velocity.     

Dynamics of the in-run in ski jumping: a simulation study

A ski jumper tries to maintain an aerodynamic position in the in-run during changing environmental forces. The purpose of this study was to analyze the mechanical demands on a ski jumper taking the in-run in a static position. We simulated the in-run in ski jumping with a 4-segment forward dynamic model (foot, leg, thigh, and upper body). The curved path of the in-run was used as kinematic constraint, and drag, lift, and snow friction were incorporated. Drag and snow friction created a forward rotating moment that had to be counteracted by a plantar flexion moment and caused the line of action of the normal force to pass anteriorly to the center of mass continuously. The normal force increased from 0.88 G on the first straight to 1.65 G in the curve. The required knee joint moment increased more because of an altered center of pressure. During the transition from the straight to the curve there was a rapid forward shift of the center of pressure under the foot, reflecting a short but high angular acceleration. Because unrealistically high rates of change of moment are required, an athlete cannot do this without changing body configuration which reduces the required rate of moment changes.     

Evaluation of the flow-dialysis technique for analysis of protein-ligand interactions: an experimental and a monte carlo study

Flow dialysis has found widespread use in determining the dissociation constant (KD) of a protein-ligand interaction or the amount of available binding sites (E0). This method has the potency to measure both these parameters in a single experiment and in this article a method to measure simultaneously the KD and E0 is presented, together with an extensive error analysis of the method. The flow-dialysis technique is experimentally simple to perform. However, a number of practical aspects of this method can have a large impact on the outcome of KD and E0. We have investigated all sources of significant systematic and random errors, using the interaction between mannitol and its transporter from Escherichia coli as a model. Monte Carlo simulations were found to be an excellent tool to assess the impact of these errors on the binding parameters and to define the experimental conditions that allow their most accurate estimation.