Electrical stimulation of the dorsal clitoral nerve in the treatment of idiopathic defecatory urgency. A pilot study.

PURPOSE: To investigate the effect of dorsal clitoral nerve stimulation (DCNS) on bothersome urgency to defecate with or without fecal incontinence and the patient-reported discomfort or adverse effect with the method. METHODS: For dorsal clitoral nerve stimulation, a battery powered, handheld stimulator was used, set to a pulse width of 200 µs and a frequency of 20 Hz. One electrode was placed at the preputium of the clitoris and acted as cathode while an anode electrode was placed on the belly. Prior to stimulation the patients were asked to complete a bowel habit diary throughout 14 consecutive days before and during stimulation. RESULTS: Fourteen out of the 16 patients included completed the study. A decrease in the number of episodes (per day) with strong urgency declined in eight patients but increased in four cases during the stimulation period. An increase in episodes with moderate or mild urgency was observed in 11 and 6 cases, respectively, and a decrease in defecation without the feeling of urgency or passive incontinence decreased in two thirds of the patients. Two patients discontinued the study prematurely, on due to worsening in symptoms and one due to pelvic pain. CONCLUSION: Although the results may be promising, much still must be learned about the method including mode and duration of stimulation, better electrodes and more patient friendly equipment together with the development of better questionnaires to assess the patient burden of urgency.

Quantifying deformations and strains in human intervertebral discs using Digital Volume Correlation combined with MRI (DVC-MRI).

Physical disruptions to intervertebral discs (IVDs) can cause mechanical changes that lead to degeneration and to low back pain which affects 75% of us in our lifetimes. Quantifying the effects of these changes on internal IVD strains may lead to better preventative strategies and treatments. Digital Volume Correlation (DVC) is a non-invasive technique that divides volumetric images into subsets, and measures strains by tracking the internal patterns within them under load. Applying DVC to MRIs may allow non-invasive strain measurements. However, DVC-MRI for strain measurements in IVDs has not been used previously. The purpose of this study was to quantify the strain and deformation errors associated with DVC-MRI for measurements in human IVDs. Eight human lumbar IVDs were MRI scanned (9.4 T) for a 'zero-strain study' (multiple unloaded scans to quantify noise within the system), and a loaded study (2 mm axial compression). Three DVC methodologies: Fast-Fourier transform (FFT), direct correlation (DC), and a combination of both FFT and DC approaches were compared with subset sizes ranging from 8 to 88 voxels to establish the optimal DVC methodology and settings which were then used in the loaded study. FFT + DC was the optimal method and a subset size of 56 voxels (2520 µm) was found to be a good compromise between errors and spatial resolution. Displacement and strain errors did not exceed 28 µm and 3000 microstrain, respectively. These findings demonstrate that DVC-MRI can quantify internal strains within IVDs non-invasively and accurately. The method has unique potential for assessing IVD strains within patients.

Propagation phase-contrast micro-computed tomography allows laboratory-based three-dimensional imaging of articular cartilage down to the cellular level.

OBJECTIVE: High-resolution non-invasive three-dimensional (3D) imaging of chondrocytes in articular cartilage remains elusive. The aim of this study was to explore whether laboratory micro-computed tomography (micro-CT) permits imaging cells within articular cartilage. DESIGN: Bovine osteochondral plugs were prepared four ways: in phosphate-buffered saline (PBS) or 70% ethanol (EtOH), both with or without phosphotungstic acid (PTA) staining. Specimens were imaged with micro-CT following two protocols: 1) absorption contrast (AC) imaging 2) propagation phase-contrast (PPC) imaging. All samples were scanned in liquid. The contrast to noise ratio (C/N) of cellular features quantified scan quality and were statistically analysed. Cellular features resolved by micro-CT were validated by standard histology. RESULTS: The highest quality images were obtained using propagation phase-contrast imaging and PTA-staining in 70% EtOH. Cellular features were also visualised when stained in PBS and unstained in EtOH. Under all conditions PPC resulted in greater contrast than AC (p < 0.0001 to p = 0.038). Simultaneous imaging of cartilage and subchondral bone did not impede image quality. Corresponding features were located in both histology and micro-CT and followed the same distribution with similar density and roundness values. CONCLUSIONS: Three-dimensional visualisation and quantification of the chondrocyte population within articular cartilage can be achieved across a field of view of several millimetres using laboratory-based micro-CT. The ability to map chondrocytes in 3D opens possibilities for research in fields from skeletal development through to medical device design and treatment of cartilage degeneration.

Standardizing compression testing for measuring the stiffness of human bone.

OBJECTIVES: The ability to determine human bone stiffness is of clinical relevance in many fields, including bone quality assessment and orthopaedic prosthesis design. Stiffness can be measured using compression testing, an experimental technique commonly used to test bone specimens in vitro. This systematic review aims to determine how best to perform compression testing of human bone. METHODS: A keyword search of all English language articles up until December 2017 of compression testing of bone was undertaken in Medline, Embase, PubMed, and Scopus databases. Studies using bulk tissue, animal tissue, whole bone, or testing techniques other than compression testing were excluded. RESULTS: A total of 4712 abstracts were retrieved, with 177 papers included in the analysis; 20 studies directly analyzed the compression testing technique to improve the accuracy of testing. Several influencing factors should be considered when testing bone samples in compression. These include the method of data analysis, specimen storage, specimen preparation, testing configuration, and loading protocol. CONCLUSION: Compression testing is a widely used technique for measuring the stiffness of bone but there is a great deal of inter-study variation in experimental techniques across the literature. Based on best evidence from the literature, suggestions for bone compression testing are made in this review, although further studies are needed to establish standardized bone testing techniques in order to increase the comparability and reliability of bone stiffness studies.Cite this article: S. Zhao, M. Arnold, S. Ma, R. L. Abel, J. P. Cobb, U. Hansen, O. Boughton. Standardizing compression testing for measuring the stiffness of human bone. Bone Joint Res 2018;7:524-538. DOI: 10.1302/2046-3758.78.BJR-2018-0025.R1.

Site-specific ion occupation in the selectivity filter causes voltage-dependent gating in a viral K+ channel.

Many potassium channels show voltage-dependent gating without a dedicated voltage sensor domain. This is not fully understood yet, but often explained by voltage-induced changes of ion occupation in the five distinct K+ binding sites in the selectivity filter. To better understand this mechanism of filter gating we measured the single-channel current and the rate constant of sub-millisecond channel closure of the viral K+ channel KcvNTS for a wide range of voltages and symmetric and asymmetric K+ concentrations in planar lipid membranes. A model-based analysis employed a global fit of all experimental data, i.e., using a common set of parameters for current and channel closure under all conditions. Three different established models of ion permeation and various relationships between ion occupation and gating were tested. Only one of the models described the data adequately. It revealed that the most extracellular binding site (S0) in the selectivity filter functions as the voltage sensor for the rate constant of channel closure. The ion occupation outside of S0 modulates its dependence on K+ concentration. The analysis uncovers an important role of changes in protein flexibility in mediating the effect from the sensor to the gate.

The effect of long-term bisphosphonate therapy on trabecular bone strength and microcrack density.

OBJECTIVES: Bisphosphonates (BP) are the first-line treatment for preventing fragility fractures. However, concern regarding their efficacy is growing because bisphosphonate is associated with over-suppression of remodelling and accumulation of microcracks. While dual-energy X-ray absorptiometry (DXA) scanning may show a gain in bone density, the impact of this class of drug on mechanical properties remains unclear. We therefore sought to quantify the mechanical strength of bone treated with BP (oral alendronate), and correlate data with the microarchitecture and density of microcracks in comparison with untreated controls. METHODS: Trabecular bone from hip fracture patients treated with BP (n = 10) was compared with naïve fractured (n = 14) and non-fractured controls (n = 6). Trabecular cores were synchrotron scanned and micro-CT scanned for microstructural analysis, including quantification of bone volume fraction, microarchitecture and microcracks. The specimens were then mechanically tested in compression. RESULTS: BP bone was 28% lower in strength than untreated hip fracture bone, and 48% lower in strength than non-fractured control bone (4.6 MPa vs 6.4 MPa vs 8.9 MPa). BP-treated bone had 24% more microcracks than naïve fractured bone and 51% more than non-fractured control (8.12/cm2vs 6.55/cm2vs 5.25/cm2). BP and naïve fracture bone exhibited similar trabecular microarchitecture, with significantly lower bone volume fraction and connectivity than non-fractured controls. CONCLUSION: BP therapy had no detectable mechanical benefit in the specimens examined. Instead, its use was associated with substantially reduced bone strength. This low strength may be due to the greater accumulation of microcracks and a lack of any discernible improvement in bone volume or microarchitecture. This preliminary study suggests that the clinical impact of BP-induced microcrack accumulation may be significant.Cite this article: A. Jin, J. Cobb, U. Hansen, R. Bhattacharya, C. Reinhard, N. Vo, R. Atwood, J. Li, A. Karunaratne, C. Wiles, R. Abel. The effect of long-term bisphosphonate therapy on trabecular bone strength and microcrack density. Bone Joint Res 2017;6:602-609. DOI: 10.1302/2046-3758.610.BJR-2016-0321.R1.

Microindentation - a tool for measuring cortical bone stiffness? A systematic review.

OBJECTIVES: Microindentation has the potential to measure the stiffness of an individual patient's bone. Bone stiffness plays a crucial role in the press-fit stability of orthopaedic implants. Arming surgeons with accurate bone stiffness information may reduce surgical complications including periprosthetic fractures. The question addressed with this systematic review is whether microindentation can accurately measure cortical bone stiffness. METHODS: A systematic review of all English language articles using a keyword search was undertaken using Medline, Embase, PubMed, Scopus and Cochrane databases. Studies that only used nanoindentation, cancellous bone or animal tissue were excluded. RESULTS: A total of 1094 abstracts were retrieved and 32 papers were included in the analysis, 20 of which used reference point indentation, and 12 of which used traditional depth-sensing indentation. There are several factors that must be considered when using microindentation, such as tip size, depth and method of analysis. Only two studies validated microindentation against traditional mechanical testing techniques. Both studies used reference point indentation (RPI), with one showing that RPI parameters correlate well with mechanical testing, but the other suggested that they do not. CONCLUSION: Microindentation has been used in various studies to assess bone stiffness, but only two studies with conflicting results compared microindentation with traditional mechanical testing techniques. Further research, including more studies comparing microindentation with other mechanical testing methods, is needed before microindentation can be used reliably to calculate cortical bone stiffness.Cite this article: M. Arnold, S. Zhao, S. Ma, F. Giuliani, U. Hansen, J. P. Cobb, R. L. Abel, O. Boughton. Microindentation - a tool for measuring cortical bone stiffness? A systematic review. Bone Joint Res 2017;6:542-549. DOI: 10.1302/2046-3758.69.BJR-2016-0317.R2.

A comparative study of the effects of different bioactive fillers in PLGA matrix composites and their suitability as bone substitute materials: A thermo-mechanical and in vitro investigation.

Bone substitute composite materials with poly(L-lactide-co-glycolide) (PLGA) matrices and four different bioactive fillers: CaCO3, hydroxyapatite (HA), 45S5 Bioglass(®) (45S5 BG), and ICIE4 bioactive glass (a lower sodium glass than 45S5 BG) were produced via melt blending, extrusion and moulding. The viscoelastic, mechanical and thermal properties, and the molecular weight of the matrix were measured. Thermogravimetric analysis evaluated the effect of filler composition on the thermal degradation of the matrix. Bioactive glasses caused premature degradation of the matrix during processing, whereas CaCO3 or HA did not. All composites, except those with 45S5 BG, had similar mechanical strength and were stiffer than PLGA alone in compression, whilst all had a lower tensile strength. Dynamic mechanical analysis demonstrated an increased storage modulus (E') in the composites (other than the 45S5 BG filled PLGA). The effect of water uptake and early degradation was investigated by short-term in vitro aging in simulated body fluid, which indicated enhanced water uptake over the neat polymer; bioactive glass had the greatest water uptake, causing matrix plasticization. These results enable a direct comparison between bioactive filler type in poly(α-hydroxyester) composites, and have implications when selecting a composite material for eventual application in bone substitution.

Dissipation layers in Rayleigh-Bénard convection: a unifying view.

Boundary layers play an important role in controlling convective heat transfer. Their nature varies considerably between different application areas characterized by different boundary conditions, which hampers a uniform treatment. Here, we argue that, independent of boundary conditions, systematic dissipation measurements in Rayleigh-Bénard convection capture the relevant near-wall structures. By means of direct numerical simulations with varying Prandtl numbers, we demonstrate that such dissipation layers share central characteristics with classical boundary layers, but, in contrast to the latter, can be extended naturally to arbitrary boundary conditions. We validate our approach by explaining differences in scaling behavior observed for no-slip and stress-free boundaries, thus paving the way to an extension of scaling theories developed for laboratory convection to a broad class of natural systems.

Statistical analysis of global wind dynamics in vigorous Rayleigh-Bénard convection.

Experimental and numerical studies of thermal convection have shown that sufficiently vigorous convective flows exhibit a large-scale thermal wind component sweeping along small-scale thermal boundary layer instabilities. A characteristic feature of these flows is an intermittent behavior in the form of irregular reversals in the orientation of the large-scale circulation. There have been several attempts toward a better understanding and description of the phenomenon of flow reversals, but so far most of these models are based on a statistical analysis of few-point measurements or on simplified theoretical assumptions. The analysis of long-term data sets (>5×10(5) turnover times τ(t)=d/u(rms)) obtained by numerical simulations of turbulent two-dimensional Rayleigh-Bénard convection allows us to get a more comprehensive view of the spatio-temporal flow behavior. By means of a global statistical analysis of the characteristic spatial modes of the flow we extract information about the stability of dominant large-scale modes as well as the reversal paths in state subspace. We examine probability density functions and drift vector fields of two-dimensional state subspaces spanned by different large-scale spatial modes. This also provides information about the coexistence of dominant modes.

Mannan-binding lectin (MBL) and MBL-associated serine protease 2 (MASP-2) genotypes in colorectal cancer.

Mannan-binding lectin (MBL) and MBL-associated serine protease 2 (MASP-2) are key factors of the lectin pathway of complement activation. Polymorphisms of the MBL2 and MASP-2 genes affect serum levels of MBL and MASP-2. In patients with colorectal cancer (CRC), the MBL and MASP-2 serum levels are increased and high MASP-2 levels are associated with recurrence and poor survival, whereas low MBL levels predict post-operative pneumonia. It is not known whether these associations are genetically based. In this study, the MBL and MASP-2 genotypes are investigated in 593 patients with CRC and 348 healthy controls. The potential association between genetic profile and infections, recurrence and survival is evaluated. Four single-nucleotide polymorphisms (SNPs) of MBL2 were analysed using TaqMan assays, with characterization of MBL2 wildtype A, variants B, C and D and alleles H/L, Y/X and P/Q. The SNP D120G for MASP-2 was determined. Serum levels of MBL and MASP-2 were measured. The MBL2 and MASP-2 genotype distribution was similar among patients with CRC and healthy controls and MBL2 genotype significantly associated with MBL concentration in serum (P<0.0001). No significant association between MBL2/MASP-2 genotype and post-operative infectious complications (P=0.33 and 0.22), recurrent cancer or survival (P=0.74 and P=0.61 respectively) was found. Thus, the increased serum levels of MBL and MASP-2 found in patients with CRC are not explained for by genetic profiles. In contrast to what has been demonstrated for serum levels of MBL and MASP-2, the genotypes do not predict disease course of the CRC patients.

Primary stability in reversed-anatomy glenoid components.

Reversed-anatomy shoulder replacement is advocated for patients with poor rotator cuff condition, for whom an anatomical reconstruction would provide little or no stability. Modern generations of this concept appear to be performing well in the short-term to midterm clinical follow-up. These designs are almost always non-cemented, requiring a high degree of primary stability to encourage bone on-growth and so to establish long-term fixation. Six different inverse-anatomy glenoid implants, currently on the market and encompassing a broad range of geometrical differences, were compared on the basis of their ability to impart primary stability through the minimization of interface micromotions. Fixing screws were only included in the supero-inferior direction in appropriate implants and were always inclined at the steepest available angle possible during surgery (up to a maximum of 30 degrees). The extent of predicted bony on-growth was, of course, highly dependent on the threshold for interface micromotion. In some instances an additional 30 per cent of the interface was predicted to promote bone on-growth when the threshold was raised from 20 microm to 50 microm. With maximum thresholds of micromotion for bone on-growth set to 30 microm, the Zimmer Anatomical device was found to be the most stable of the series of the six designs tested herein, achieving an additional 3 per cent (by surface area) of bone on-growth above the closest peer product (Biomet Verso). When this threshold was raised to 50 microm, the Biomet Verso design was most stable (3 per cent above the second-most stable design, the Zimmer Anatomical). Peak micromotions were not a good indicator of the predicted area of bone on-growth and could lead to some misinterpretation of the implant's overall performance. All but one of the implants tested herein provided primary stability sufficient to resist motions in excess of 150 microm at the interface.

A numerical tool for the reconstruction of the physiological kinematics of the glenohumeral joint.

The aim of this study was to develop and test a robust approach to apply a joint coordinate system (JCS) to imaging data sets of the glenohumeral joint and to reconstruct the kinematics with six degrees of freedom (6DOF) in order to investigate shoulder pathologies related to instability. Visible human data were used to reconstruct bony morphology. Landmarks were used to define axes for body-fixed Cartesian coordinate frames on the humerus and scapula. These were applied to a three-cylinder open-chain JCS upon which the humeral 6DOF motions relative to the scapula were implemented. Software was written that applies 6DOF input variables to rotate and translate the nodes of the surface geometry of the humerus relative to the scapula in a global coordinate frame. The instantaneous relative position and orientation of the humerus for a given set of variables were thus reconstructed on the bone models for graphical display. This tool can be used for graphical animation of shoulder kinematics, demonstrating clinical assessments, and allowing further analysis of the function of tissues within the joint.

Collagen binding is a key factor for the hemorrhagic activity of snake venom metalloproteinases.

Snake venom metalloproteinases (SVMPs) are multifunctional enzymes involved in several symptoms following snakebite, such as severe local hemorrhage. Multidomain P-III SVMPs are strongly hemorrhagic, whereas single domain P-I SVMPs are not. This indicates that disintegrin-like and cysteine-rich domains allocate motifs that enable catalytic degradation of ECM components leading to disruption of capillary vessels. Interestingly, some P-III SVMPs are completely devoid of hemorrhagic activity despite their highly conserved disintegrin-like and cysteine-rich domains. This observation was approached in the present study by comparing the effects of jararhagin, a hemorrhagic P-III SVMP, and berythractivase, a pro-coagulant and non-hemorrhagic P-III SVMP. Both toxins inhibited collagen-induced platelet aggregation, but only jararhagin was able to bind to collagen I with high affinity. The monoclonal antibody MAJar 3, that neutralizes the hemorrhagic effect of Bothrops venoms and jararhagin binding to collagen, did not react with berythractivase. The three-dimensional structures of jararhagin and berythractivase were compared to explain the differential binding to collagen and MAJar 3. Thereby, we pinpointed a motif within the Da disintegrin subdomain located opposite to the catalytic domain. Jararhagin binds to both collagen I and IV in a triple helix-dependent manner and inhibited in vitro fibrillogenesis. The jararhagin-collagen complex retained the catalytic activity of the toxin as observed by hydrolysis of fibrin. Thus, we suggest that binding of hemorrhagic SVMPs to collagens I and IV occurs through a motif located in the Da subdomain. This allows accumulation of toxin molecules at the site of injection, close to capillary vessels, where their catalytic activity leads to a local hemorrhage. Toxins devoid of this motif would be more available for vascular internalization leading to systemic pro-coagulant effects. This reveals a novel function of the disintegrin domain in hemorrhage formation.

The palmar locking compression plate is biomechanically comparable to the dorsal pi plate for dorsally comminuted, intraarticular wrist fractures.

A clinically appropriate fracture model and testing regimen were used to test the null hypothesis that a palmarly applied locking plate was inferior to a dorsally applied Pi plate in the stabilisation of dorsally comminuted intraarticular wrist fractures. Sixteen standardised fractures of Synbone models of the radius were stabilised using either a palmar locking compression T plate (the experimental group) (n=8) or a dorsally applied Pi plate (the control group) (n=8). The constructs were tested on an Instron materials testing machine. Deformation was monitored during 500 loading cycles to 200 N. The mean permanent deformation and stiffness favoured the palmar locking compression T plate over the dorsal Pi plate (P=0.036). However, the absolute difference was only 0.5 mm. Such a small difference is unlikely to be clinically detectable and, therefore, we conclude that there is no clinically significant difference between the two types of fixation.

Developments in shoulder arthroplasty.

Indications for shoulder arthroplasty are numerous, mainly owing to glenohumeral osteoarthritis, rheumatoid arthritis, or fracture of the proximal humerus. However, the anatomy and the biomechanics of the shoulder are complex and shoulder arthroplasty has evolved significantly over the past 30 years. This paper presents the main recent evolutions in shoulder replacement, the questions not answered yet, and the main future areas of research. The review focuses firstly on the design, positioning, and fixation of the humeral component, secondly on the design, positioning, and fixation of the glenoid implant, and thirdly on other concepts of shoulder arthroplasty such as the reversed prosthesis, the cementless surface replacement arthroplasty, and the bipolar arthroplasty. This review demonstrates that more research is needed. Although, in the long term, large randomized trials are needed to settle the fundamental questions of what type of replacement and which kind of fixation should be used, biomechanical research in the laboratory should be focused primarily on the comprehension of glenoid loosening, which is a major cause of total shoulder arthroplasty failure, and the significance of radiolucent lines which are often seen but with no clear understanding about their relation with failure.

Strengths and limits of Beta distributions as a means of reconstructing the true single-channel current in patch clamp time series with fast gating.

Single-channel current seems to be one of the most obvious characteristics of ion transport. But in some cases, its determination is more complex than anticipated at first glance. Problems arise from fast gating in time series of patch-clamp current, which can lead to a reduced apparent (measured) single-channel current. Reduction is caused by undetected averaging over closed and open intervals in the anti-aliasing filter. Here it is shown that fitting the measured amplitude histograms by Beta distributions is an efficient tool of reconstructing the true current level from measured data. This approach becomes even more powerful when it is applied to amplitude distributions-per-level. Simulated time series are employed to show that the error sum is a good guideline for finding the correct current level. Furthermore, they show that a Markov model smaller than the one used for gating analysis can be used for current determination (mostly O-C, i.e., open-closed). This increases the reliability of the Beta fit. The knowledge of the true current level is not only important for the understanding of the biophysical properties of the channel. It is also a prerequisite for the correct determination of the rate constants of gating. The approach is applied to measured data. The examples reveal the limits of the analysis imposed by the signal-to-noise ratio and the shape of the amplitude distribution. One application shows that the negative slope of the I-V curve of the human MaxiK channel expressed in HEK293 cells is caused by fast gating.

A subsequent fit of time series and amplitude histogram of patch-clamp records reveals rate constants up to 1 per microsecond.

Fast gating in time series of patch-clamp current demands powerful tools to reveal the rate constants of the adequate Hidden Markov model. Here, two approaches are presented to improve the temporal resolution of the direct fit of the time series. First, the prediction algorithm is extended to include intermediate currents between the nominal levels as caused by the anti-aliasing filter. This approach can reveal rate constants that are about 4 times higher than the corner frequency of the anti-aliasing filter. However, this approach is restricted to time series with very low noise. Second, the direct fit of the time series is combined with a beta fit, i.e., a fit of the deviations of the amplitude histogram from the Gaussian distribution. Since the "theoretical" amplitude histograms for higher-order Bessel filters cannot be calculated by analytical tools, they are generated from simulated time series. In a first approach, a simultaneous fit of the time series and of the Beta fit is tested. This simultaneous fit, however, inherits the drawbacks of both approaches, not the benefits. More successful is a subsequent fit: The fit of the time series yields a set of rate constants. The subsequent Beta fit uses the slow rate constants of the fit of the time series as fixed parameters and the optimization algorithm is restricted to the fast ones. The efficiency of this approach is illustrated by means of time series obtained from simulation and from the dominant K+ channel in Chara. This shows that temporal resolution can reach the microsecond range.

Effect of CO2 supply on formation of reactive oxygen species in Arabidopsis thaliana.

Light-induced generation of reactive oxygen species (ROS) in 2-week-old leaves of Arabidopsis thaliana was studied by means of the ROS-sensitive dyes nitroblue tetrazolium (NBT) and 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (DCF-DA). Superposition of pictures of chlorophyll fluorescence and DCF fluorescence indicated that the origin of ROS was in the chloroplasts. Experiments were done with zero, 0.1, or 10 mM NaHCO3 in the infiltration medium. Energy quenching in photosystem II was higher under low CO2 concentrations as measured by chlorophyll fluorescence. DCF fluorescence showed that CO2 deficiency led to an increase of ROS generation. In contrast, the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea reduced the light-induced increase of DCF fluorescence. This indicates that ROS production does not primarily result from over-reduction of photosystem II as caused by impeding electron flow in the electron transfer chain. More likely, it is an effect of diverting electron flux normally aimed at carboxylation in the Calvin cycle to other sinks more prone to the generation of toxic radicals. There was no significant effect of salicyl hydroxamate (a blocker of the alternative oxidase), showing that the mitochondrial electron transfer chain seems to play a minor role as already indicated by the superposition of chlorophyll and DCF fluorescence.