Photosynthetic starch reserves that accumulate in Arabidopsis leaves during the day decrease approximately linearly with time at night to support metabolism and growth. We find that the rate of decrease is adjusted to accommodate variation in the time of onset of darkness and starch content, such that reserves last almost precisely until dawn. Generation of these dynamics therefore requires an arithmetic division computation between the starch content and expected time to dawn. We introduce two novel chemical kinetic models capable of implementing analog arithmetic division. Predictions from the models are successfully tested in plants perturbed by a night-time light period or by mutations in starch degradation pathways. Our experiments indicate which components of the starch degradation apparatus may be important for appropriate arithmetic division. Our results are potentially relevant for any biological system dependent on a food reserve for survival over a predictable time period.DOI:http://dx.doi.org/10.7554/eLife.00669.001 Many organisms build up reserves of food when it is available so that metabolism and growth can continue when food is no longer available. Plants, for example, use energy from the sun to produce starch during the day, which is then used as a source of energy during the night. In some plants the amount of starch increases linearly with time during the day, and declines linearly with time during the night, so that the plant contains very little starch when the sun rises the following day. Although a plant is likely to starve if it cannot store or consume starch effectively, very little is known about the mechanisms that plants use to ensure that they store enough starch and do not use it up too quickly.Plants are able to track the time to dawn using their internal circadian clock so, as Scialdone et al. point out, if they can also track how much starch they have stored, they might somehow be dividing the amount of starch by the time to dawn to work out the rate at which starch can be consumed so that it lasts until sunrise. But could plants actually perform such calculations?S for starch and T for time). In one model, they propose that the S molecules stimulate starch consumption, and the T molecules prevent this from happening, with the rate of starch consumption being equal to the concentration of S molecules divided by the concentration of T molecules. These models were able to reproduce the results of previous experiments, including experiments in which dawn arrived unexpectedly early or unexpectedly late.To gain some insight into this puzzle, Scialdone et al. constructed mathematical models in which information about the size of the starch store and the time until dawn was encoded in the concentrations of two kinds of molecules (called Scialdone et al. then performed experiments which confirmed predictions of the models for the pattern of starch consumption in plants lacking relevant enzymes. These experiments also revealed that an enzyme called PWD may be the point at which results of the division computation are integrated into the starch consumption pathway. More generally, this work shows that sophisticated arithmetic computations can be important in biology. Moreover, whereas computers rely on digital logic, Scialdone et al. show that arithmetic computations can also be performed by exploiting the analogue dynamics that take place between molecules.DOI:http://dx.doi.org/10.7554/eLife.00669.002 Arabidopsis thaliana this phenomenon is essential for productivity: mutants with defects in either the accumulation or the degradation of starch have reduced productivity and exhibit symptoms of starvation  or an unexpectedly early night (8 hr after dawn) was such that starch reserves were almost depleted by dawn  and denominator (expected time to dawn). Previously, we showed that the rate of starch degradation is appropriately adjusted in response to an unexpectedly early night (imposition of darkness 8 hr after dawn on plants grown in 12-hr light/12-hr dark cycles) . We founf <24 hr . In 12-hf <24 hr .When subf <24 hr . Approprf <24 hr . Approprf <24 hr . To inve by dawn .10.7554/Overall, these results demonstrate that the control of starch degradation at night to achieve almost complete consumption at the expected time of dawn can accommodate unexpected variation in the time of onset of darkness, starch content at the start of the night, and patterns of starch accumulation during the preceding day. Although the rate of degradation is different in a circadian clock mutant with an altered period from that in the wild-type, the capacity to adjust starch degradation in response to an unexpectedly early night is not compromised. It is also likely that the mechanism underlying this control is present in evolutionarily-distant species of plants.Robust computation of the appropriate starch degradation rate in response to perturbations of both the expected time to dawn and starch content is clearly consistent with the implementation of arithmetic division. Since it is conceptually unclear how such a computation might be performed, we turned to mathematical modeling to generate possible mechanisms.S whose concentration is proportional to the amount of starch in a granule. Since plants are able to adjust the rate of starch degradation according to variations in two independent quantities (the expected time to dawn and the amount of starch present), two separate species of molecule are clearly required. Therefore, we further assume the existence of a molecule T whose concentration encodes information about the expected time to dawn.The fact that starch exists as large polymers that are mostly inaccessible within granule matrices means that a separate measure is likely required to provide information about the total amount of starch present at any given time. Hence, we assume the existence of a soluble molecule T molecule concentration is proportional to the expected time to dawn, except during a period after dawn when its value must be reset . In In our first model the be reset . To compT molecule concentration increases as the expected time of dawn approaches, before being reset. If this increase is such that the T molecule concentration is approximately proportional to 1/(expected time to dawn)   then theS molecules) occur only in region(s) of granule surface of overall fixed area as each granule shrinks. This would be the case if one or more of the additional components required for starch degradation  can be implemented simply using analog post-translational chemical kinetics. Furthermore, output from both models fits our experimental data well. We note that there is some variation in the fitted parameters for both models see , which aLHY . The latter rates are those that would have ensured the complete depletion of the starch content measured at 12 hr at the expected time of dawn (24 hr). We obtained the same result by changing the duration and the starting time of the night-time light period . If instead this factor is <1, so that the starch is degraded too slowly, then rather than complete degradation the models predict that only a certain percentage of the starch will be consumed during the night regardless of the starch content at the end of the preceding light period . Accordingly, we predict that perturbations to parts of the arithmetic division or degradation apparatus might result in degradation of only a fraction of starch during the night. Interestingly, several mutants defective in proteins involved in, or related to, starch degradation exhibit approximately linearly decreasing starch content with time during the night, but have an elevated starch content at the end of the night. These include beta-amylase mutants bam3 and bam4, the debranching enzyme mutant isa3, and mutants lacking phosphoglucan water dikinase , a glucan phosphate phosphatase (sex4) and a glucan phosphate phosphatase-like protein (lsf1) , regardless of the starch content at the end of the respective preceding light period. This resulted in a progressive increase in the end-of-night starch content to an approximately constant value over 3–4 days after return to normal light/dark cycles  . We showdarkness . We re-ak cycles . Thus thS and T) and the starch degradation apparatus. If this idea is correct, we expect that mutants lacking components of the degradation apparatus involved in these interactions may also lack the ability to adjust the rate of starch degradation in response to variation in starch content or time of onset of darkness.We next attempted to gain experimental insight into how the arithmetic division mechanism modulates flux through the starch degradation pathway. In our models the computation of the starch degradation rate is the result of interactions between the molecules encoding the information about starch content and time to dawn . All six mutants show approximately linear decreases in starch content with time during the night, but they have higher end-of-night starch contents than wild-type plants. This mutant collection covers the majority of currently-known components of the chloroplastic starch degradation apparatus. For five of the six mutants, the rates of starch degradation (as determined from linear fits) were lower during an unexpectedly early night (starting 8 hr after dawn) than in a normal night (starting 12 hr after dawn), as is the case in wild-type plants  during the normal and the early night. For wild-type plants, the expected value of R is 16/12 ≈ 1.33, that is, the ratio between the expected lengths of the early and the normal night. In R for five of the six mutants are consistent with the wild-type value.To look for a mutant in this category, we imposed an unexpectedly early night on six mutants, each lacking a protein involved in starch degradation . This finding indicates that PWD—phosphoglucan, water dikinase—may be a node at which information about expected time to dawn and starch content is integrated to set an appropriate rate of starch degradation.However for the sixth mutant, ly night , as we fPWD contributes to a cycle of phosphorylation and dephosphorylation of glucosyl residues within starch polymers that is essential for normal starch degradation. Two enzymes—glucan water dikinase (GWD) and PWD—add phosphate groups to starch polymers at the granule surface, and two further enzymes SEX4 and LSF2—remove them. All four enzymes are necessary for normal rates of starch degradation, and loss of GWD almost completely prevents degradation at night . The phoS molecule may be a modulator of activities of enzymes of the phosphorylation/dephosphorylation cycle, thus generating a daily pattern of change in the accessibility of the granule surface to hydrolytic enzymes that approximately tracks starch content.Because the phosphorylation/dephosphorylation cycle modifies the granule surface, it is a potential candidate not only for flux modulation but also for the storage of information about starch content. To discover whether phosphate groups may provide information about starch content, we measured the amount of granule-bound phosphate  over the light/dark cycle. If phosphorylation simply tracks starch polymer synthesis during the day, phosphate levels per unit mass of starch will be constant and will thus contain no information about starch content. Surprisingly, however, we found a large increase/decrease in the level of phosphate per unit mass of starch over the light/dark cycle . This diOverall, our work provides a new framework and perspective for understanding the control of reserve utilization in plants at night. Our experiments provide strong support for an implementation of arithmetic division in night-time starch degradation. We used mathematical modeling to generate two simple mechanisms capable of analog implementation of such an operation. Predictions from the models were then verified and a potential point identified at which the division computation could be integrated into the starch degradation pathway. We also showed that the phosphorylation state of the starch granule surface could provide information about starch content through the day.Calidris minuta) arriving at their Arctic summer breeding grounds after a 5000-km journey have sufficient remaining lipid reserves for an average of only 0.6 days (Aptenodytes forsteri), lipid reserves are used such that they reach a critical depletion level at approximately the point at which the females are expected to return. Unexpected extension of the fast period leads to catabolism of protein and abandonment of offspring in favor of hunting for food  and Brachypodium distachyon (Bd21) plants were grown as in −2 s−1 illumination at a constant temperature of 20°C for 21 days.Plants were harvested and extracted in dilute perchloric acid for analysis of starch, which was then quantified enzymatically as previously described .. Oligonucleotide primer sequences were as follows:RNA was extracted from plant material and qPCR was performed as in Graf et al. (2010)Starch granules were prepared as in S whose number Stot in a chloroplast at the end of a light period:Starch is laid down as semi-crystalline granules in chloroplasts in the light. Granule surfaces are then subject to degradation during periods of darkness. We assume here that the computation of the degradation rate is performed autonomously inside each chloroplast. In order to correctly compute the appropriate starch degradation rate, knowledge of the total chloroplast starch content is required. We assume that there exists a molecule S molecule can exchange rapidly between the granule surfaces and the surrounding chloroplast compartment. Moreover, we propose the existence of a molecule T that encodes information about the expected time to dawn. The T molecule dynamics will presumably be controlled by the circadian clock and are assumed to be independent of the starch degradation process.We also assume that the T concentration in the compartment surrounding the granules, [TC], is proportional to the expected time to dawn Δt = tday − t during most of the light/dark cycle. Note, however, that this cannot be true at all times, as around the time of expected dawn, after [TC] has dropped to low levels, its value must be reset. So, if dawn occurs at t = 0, we assume that:β is a constant, tday is the period of the light/dark cycle and tr is the reset-time after which [TC] starts tracking the time to expected dawn . Again, this cannot be true around dawn, where [TC ] must be reset. For the sake of simplicity, we assume that during the reset period [TC] has a linear profile with time:β is a constant, and tr1 and tr2 define the time interval in which [TC] is proportional to 1/Δt  = β/Δt as required, we then find [TC](tr1) = β/(tday − tr1) and η = 1/β.For [Δt behavior for [TC] is a plausible assumption.Of course, more complex schemes could also generate a similar behavior; we simply wish to point out here that a 1/We now describe in full mathematical detail the models introduced in the main text.S concentration by the T concentration, where the T molecule concentration is proportional to Δt for most of the light/dark cycle. Such a division can be implemented by the following set of reactions:C’ refers to molecules in the compartment surrounding the granules. The reversible rS reaction describes exchange of the S molecules between the granule surface and surrounding compartment, with forward reaction parameter fS and backward rate bS. The T molecules can be recruited by the S molecules on the granule surface  and the resulting complex can then dissociate leading to the detachment of S and T from the granule surface . The S molecule on the granule can be degraded  or it can permit starch degradation .In this model, division is implemented by dividing the bS ≫ fD1 and that the dynamics of the S and T molecules can be taken to be in quasi-steady-state. As described in the main text, we assume that the degradation reactions (for both the starch and the S molecule) can only occur in a region of overall fixed area Ad as each granule shrinks. We also assume that all granules in a given chloroplast are approximately equal in area and volume. The differential equations describing the dynamics of the total number of S molecules, ΔStot, are:mS is the amount of starch degraded by an S molecule in a single degradation reaction, n is the number of granules in the chloroplast, μ is the starch degradation rate for an individual granule and μtot is the total starch degradation rate.In all the calculations in this section, we assume that S molecules continues to hold true throughout the degradation process. The total number of S molecules is V is the compartmental volume surrounding the granules, and A is the surface area of a granule. Using the above quasi-steady state assumption and the law of mass action, we obtain:kS= fS/bS, kST1= fST1/bS. Assuming that nkSA ≪ V, bS/fST2 ≲1, then n[S] A, n[ST]A ≪ [SC]V, and hence:n is proportional to the chloroplast volume V . S can recruit T molecules from the surrounding compartment and form the complex ST . As the ST complex dissociates, T is released back in the stroma, while S remains attached to the granule . The ST complex permits S degradation, with T released from the granule surface , as well as starch degradation . Similar to our assumptions for Model 1, we assume here that bST ≫ fD1, with a fixed degradation area Ad for the starch as well as for the S molecule as each granule shrinks. We also again assume that the dynamics of S and T can be taken to be in quasi-steady-state and that all granules in a given chloroplast have approximately equal areas and volumes.Reversible reaction ΔStot, and of the total number of S molecules, The differential equations describing the dynamics of the total amount of starch in a chloroplast, S molecules is V is the compartmental volume surrounding the granules, and where A is the surface area of a granule. Using the above quasi-steady state assumption and the law of mass action, we obtain:kS= fS/bS and kST= fST/bST. We assume that nkSA ≪ V, kST[TC] ≲ 1, giving n[S]A, n[ST]A ≪ [SC]V, and hence:n is proportional to the volume V. For the T molecules, we have  ν, (ΔStot/n) being the amount of starch in a single granule in mg and ν the number of starch granules g−1 FW. This leads toTo convert this equation into one for starch content TC], the above differential equation can be exactly solved analytically. If we define the functions:t* is the time at which degradation starts, with ρS(t*) = ρ0 the corresponding starch content, then, for Given βkST1)−1 sufficiently small, starch contents are almost completely depleted by the time of expected dawn, and for γ = 1, starch contents decrease linearly with time for most of the dark period, except around the time of expected dawn, when [TC] is being reset  = ρ0, and Similarly, for Model 2, we have:For tr2 close to tday starch contents are almost completely depleted by the time of expected dawn, and for γ = 1, starch contents decrease linearly with time for TC] is being reset −1, ρ0, tr; and for Model 2: γ, ρ0, tr1 and tr2.For Model 1, the values of the following parameters are required: γ,  hr after the previous dawn, instead of the normal 24 hr. We found this phenotype in cca1/lhy plants, which run out of starch earlier than 24 hr after the previous dawn. Hence, to reproduce the phenotype of cca1/lhy in our models, t0 was also used as a fitting parameter. A more extensive discussion about cca1/lhy can be found below in the ‘Mutant phenotypes’ section.The phase shift of the zero see  and whiceset see . TherefoA full list of the system parameters is shown in N mean values of starch contents measured at times ti. ti with parameter values . The set of parameters L corresponds to the maximum-likelihood estimates of the parameters under the assumption that the experimental measurements are normally distributed around the theoretical values with a constant relative error.The best fit to a given dataset was found by minimizing the function:cca1/lhy mutant plants for the early and the normal night, we did not consider the two data points closest in time to 24 hr from the datasets, as they are characterized by very low values of starch content and, therefore, are likely to be affected by higher relative errors compared to the other data points. For the same reason, for the linear fits shown in t = 24 hr in the normal night datasets and the data points at t = 22 hr and t = 24 hr in the early night datasets.Note that, in the fits of the data from the A simulated annealing algorithm was used for the minimization of By combining our results from the night-time light period experiments shown in i-th experiment was measured as:The starch degradation rate before the night-time light period in the i-th experiment was:Similarly, the starch degradation rate after the night-time light period for the N = 3 was the number of experiments. The error on this quantity was estimated by propagating standard errors of the mean. We found μA− μB= 0.43 ± 0.14 (mg g−1 FW)/hr which is significantly greater than 0 .These rates were averaged over the three experiments, and the difference between the two averages was calculated:We also calculated the average difference between −5).This value is again significantly greater than 0  hr < 24 hr, as discussed above in the ‘Parameters’ section. For the sake of simplicity, we fitted the cca1/lhy data by assuming that the latter perturbation takes place, and accordingly we used t0 as a fitting parameter.The models we discussed previously can straightforwardly explain such a phenotype by assuming that in this mutant, the time of expected dawn is shifted to a time lsf1 and sex4 mutant plants were kept in the dark for 132 hr after the end of the previous light period, then exposed to normal 12-hr light/12-hr dark cycles and the starch content measured. As tearly = 8 hr, tnormal = 12 hr the times of onset of darkness respectively for the unexpected early and normal night. From these fits, we calculated the ratio R between the starch degradation rates in the normal and the unexpected early night, normalized by the respective starch content at the time of onset of darkness:Interestingly, the same mutants could also adjust their starch degradation rate in response to an unexpected early night. In order to quantitatively verify this observation, we performed linear fits of the data from the unexpected early and normal nights:f of the initial starch content should be degraded by the time of expected dawn. Therefore, R found for lsf1 and sex4 are compatible with 1.33 (dashed line), therefore supporting the hypothesis that these mutants are able to appropriately adjust their starch degradation rate in response to an unexpected early night. We also found the same type of rate adjustment in bam3, bam4 and isa3 mutants .The first condition ensures that the number of fD2, which is the starch degradation reaction parameter. This reasoning fits well with the known functions of the above genes, all of which play roles, directly or indirectly, in starch degradation.One way to perturb only the second of the two normalization conditions shown above (and not the first), would be to alter pwd mutant lacks the glucan water dikinase responsible for the addition of the phosphate groups to the 3-position of glucose moieties in starch. This mutation generated an approximately linear decrease of starch content with time during the night, but with a rate which was too low to ensure the complete utilization of the starch reserve by the time of expected dawn. Yet, as opposed to the previously discussed mutations, this mutant did not have the ability to adjust the starch degradation rate in response to an unexpected early night .The ight see . Indeed,S and T molecules.This finding indicates that PWD is potentially a key node where information about starch content and expected time to dawn from the circadian clock are integrated to control starch degradation dynamics. This makes PWD an obvious target for future experiments that aim to elucidate the identities of the S and T molecule concentrations are divided, with the T molecule concentration tracking the time to expected dawn. In Model 2, the S and T concentrations are multiplied, with the T molecule concentration tracking the reciprocal of the time to expected dawn. The precise implementation of these two methods of performing arithmetic division could, however, vary, with slightly different reaction schemes but the same underlying principles. To illustrate this point, we briefly outline other models related to Model 1 above, where the TC molecule again has the behavior given in The models detailed above use two distinctly different methods of implementing the division operation. In one case, exemplified by Model 1, the S and T molecules can directly and reversibly associate to the granule surface (reactions rS and rT). Once bound to the surface, S and T can form a complex ST (reaction rST) that permits S molecule and starch degradation (through reactions rD1 and rD2 respectively). The T molecule can also hinder starch degradation by forming dimers , recruit the S and T molecules from the surrounding compartment (reactions rSand rT), to form the SM and TM complexes, which can then dissociate from the surface through the reactions rSMand rTM. The SM complex can permit S molecule and starch degradation (reactions rD1 and rD2). Since the T molecule hinders starch degradation by binding to M molecules and preventing them from binding to S, it can be shown that this model can also implement arithmetic division between the S and T molecule concentrations.T molecule. To discover whether our experimental conditions gave rise to such problems, we investigated the behavior of transcript levels of LHY, a central clock gene, by qPCR analysis. For the experiment shown in LHY transcript levels were slightly elevated during the night-time light period, then peaked about 2 hr later than in plants that were not exposed to the light period (LHY expression observed with the night-time light period would not be expected to occur at around 24 hr after the previous dawn.Exposure of Arabidopsis plants to light during the normal night could potentially lead to the early, light-induced expression of clock genes, and under some circumstances might re-entrain the clock. Such re-entrainment might affect the level of the t period . This re


Objectives:Acinetobacter spp. has emerged as an important opportunistic pathogen responsible for nosocomial infections in many health-care settings worldwide. The study describes the clinico-epidemiology and antimicrobial susceptibility of Acinetobacter spp. in a tertiary health-care institution.Methodology: Acinetobacter spp. were isolated from 141 specimens of the patients who reported to Universiti Kebangsaan Medical Centre (UKMMC). The sources of specimens were wound, skin and soft tissue, respiratory and urinary tract from patients in various wards. Clinio-epidemiological features of patients infected with Acinetobacter spp. were recorded. Standard bacteriological techniques with API 20NE kits and disk diffusion method were followed for identification and antibiotic sensitivity of the organisms. Results: One hundred and forty one patients with positive culture for Acinetobacter spp. were identified. Soft tissue/wound and respiratory tract were among the commonest sites of Acinetobacter spp. isolation. The isolates were most frequently obtained from ICU. All isolates were multi-drug resistant and had a resistance rate of more than 70% to most antibiotics, except polymyxin B.Conclusion: High prevalence of multi-drug resistance Acinetobacter spp. provides essential information on judicious antibiotic selection for empirical therapy in our health-care institution. Acinetobacter spp. have become a serious concern in many health-care institutions worldwide. Acinetobacter calcoaceticus–baumannii complex has been recognized as one the most common species responsible for nosocomial bacteremia, meningitis, respiratory tract and urinary tract infections.1Infections caused byAcinetobacter spp. has been reported in Malaysian hospitals.2,3 However, the result of the studies might not represent our institution.The prevalence and antimicrobial susceptibility profiling of Acinetobacter spp. The antimicrobial resistance patterns of Acinetobacter strains will be assessed.The aim of this study was to determine, the demographic and clinical profile of Setting: This is a cross-sectional retrospective observational study conducted in a tertiary healthcare facility with 830 beds.Bacterial isolates: From October 2010 to April 2011, non-duplicate isolates of Acinetobacter spp. grown from all clinical specimens of hospitalized patients were analyzed. The sources of isolates included blood, sputum, tracheal aspirate, bronchoalveolar lavage, pus, sterile body fluid and urine. In this study, nosocomial isolate was defined as isolate grown from specimen that was sampled after 48 hours of hospitalization. Non-nosocomial isolate was defined as isolate grown from specimen sampled within 48 hours of hospitalization. Colonizer was defined as isolate that had microscopy smear showing 0 to 1 pus cell/high power field. Isolate showing more than 1 pus cell/high power field was regarded as significant isolate.Patient data: Medical and demographic data of hospitalized patients with culture-positive Acinetobacter spp. were retrieved from patients’ medical records. Data that were recorded include age, gender, ward location, date of hospitalization, transfer and discharge, date of specimen sampling, specimen site, ICU stay and antibiotic usage.Laboratory identification: Microbiological data were obtained from laboratory records. Bacterial colonies grown on MacConkey plates were identified by its colonial morphology, Gram-staining and oxidase test. Genus identification was performed using conventional biochemical tests. For blood and sterile body fluid specimens, speciation was performed using API 20NE system, based on manufacturer’s instruction .Antimicrobial susceptibility test: The antimicrobial susceptibility testing was assessed by disk diffusion method, according to the guidelines of Clinical and Laboratory Standards Institute (CLSI).4 Antibiotic disks were obtained from Oxoid Ltd. (Basingstoke UK). Tigecycline susceptibility testing was assessed by disk diffusion method, according to the guidelines of The Asia Pacific Clinical Microbiology Working Group Laboratory Manual 2009. Antibiotic disks were obtained from Becton Dickinson (USA). Minimum inhibitory concentrations (MIC) for polymyxin B were determined by E-tests, based on manufacturer’s instructions . Quality control was performed with the following strains as recommended by the CLSI: Escherichia coli ATCC 25922, Escherichia coli ATCC 35218 and Pseudomonas aeruginosa ATCC 27853. Isolates were tested for susceptibility to sixteen relevant drugs. Multiple-drug resistant (MDR) Acinetobacter spp. was defined as resistance to 3 or more classes of antibiotic used to treat Acinetobacter infections.Statistical analysis: Statistical analysis was performed by using Statistical Package for the Social Science for Windows . Parametric variables were assessed using chi-squared test, as appropriate. A difference was considered statistically significant if the p-value < 0.05.Ethical considerations: The institution’s medical research and ethical committee had approved this study. Acinetobacter spp. grown from all clinical specimens were included in this study. Distribution for the 141 isolates was as shown in A total of 141 non-duplicate isolates ofAcinetobacter spp. are shown in Acinetobacter spp. infection/colonization varied from 3 days to 78 days, median duration was 13 days (IQR 7-23).The demographic characteristics and clinical epidemiology profile of hospitalized patients with culture-positive Acinetobacter spp. Thirty-seven of them (39.8%) had one course of antibiotic therapy. Thirty out of ninety-three (32.3%) patients had 2 courses of antibiotic therapy and 26/93 (28.0%) patients had three or more courses of antibiotic therapy prior to Acinetobacter spp. isolation. The most common antibiotic used was meropenem (22.5%), followed by ceftriaxone (17.6%), piperacillin-tazobactam (13.4%) and amoxicillin-clavulanate (12.0%).In this study, 93/141 patients (66.0%) received antibiotics before the isolation of Acinetobacter spp. isolates is shown in Acinetobacter isolates had more than 70% resistance to most antibiotics tested. The rates of more than 70% resistance for antibiotics were ampicillin (95.0%), cefuroxime-parenteral (80.8%), cefotaxime (78.0%), amoxicillin-clavulanate (75.9%), ciprofloxacin (73.8%), ceftazidime (73.0%), meropenem (73.0%), imipenem (72.3%), piperacillin-tazobactam (72.7%), cefepime (73.1%), ampicillin-sulbactam (70.9%) and gentamicin (70.2%). Out of 141 isolates, 79.4% were MDR Acinetobacter spp. Twenty-eight isolates were tested for polymyxin B and were 100% sensitive. The MIC90 was 2 mcg/mL. Comparison of the rate of antibiotic resistance between ICU and non-ICU isolates did not show any significant difference (p>0.05).The susceptibility profile of 141 Acinetobacter spp. have been considered as important opportunistic pathogens responsible for nosocomial infections, especially among patients in intensive care units (ICUs).5 The data in this study showed that most of these isolates were obtained from soft tissue and wound followed by respiratory tract. Most of them were isolated from the intensive care unit (ICU), which suggests that seriously ill patients in ICUs have a greater chance of becoming colonized or infected by Acinetobacter spp. especially through the soft tissue/wound and respiratory tract. Similarly Falagas et al6 reported that infections caused by Acinetobacter spp. are more common in the ICUs in Asian and European hospitals and are lower in the United States hospitals, also Carlet et al7 and Peleg et al8 mentioned the prevalence of hospital-acquired infections could be as high as 25% in an ICU and there is a problem with nosocomial infection which is only one third of hospital-acquired infections that are avoidable.In the past two decades, Acinetobacter spp. occurs in elderly patients. One study documented the distribution of MDR-Acinetobacter was greatest in the >65 age group and long term care facilities.9 Also, duration of stay in hospital effected to acquire Acinetobacter infections, as in our data point out that this duration is from 3 days to 78 days. In several studies which examined nosocomial, blood stream, and burn infections explained antibiotic-resistance Acinetobacter infections are associated with longer hospital stays.10,11Our data show that the median age of patients is 54 (31-66) which indicates the infection by Acinetobacter infection after 48 hours during hospitalization are considered as nosocomial patients. Reports of Chang et al12 and Enoch et al13 mentioned that Acinetobacter spp. emerged as a crucial pathogen in health care - associated and nosocomial infections with high mortality, and was difficult to treat efficiently. Munoz-Price5 also reported that hospital acquired Acinetobacter is often multidrug resistance and widespread.The present study showed that most patients (75.2%) exposed to Acinetobacter spp. colonization of the hospital environmental may lead to infection because they survive on both moist and dry surfaces for long periods in the hospital environment.14 This feature of Acinetobacter is helpful to survive in hospital environments and cause infection and eliminating Acinetobacter spp. from clinical materials is difficult.15In the present study, the isolates were predominantly colonizers (53.2%). Thus, Acinetobacter spp. is 66.0%. The most common antibiotics that patients received before the diagnosis of Acinetobacter spp. were meropenem (22.5%), followed by ceftriaxone (17.6%), piperacillin-tazobactam (13.4%). Other studies16,17 have reported that prior exposure to antibiotics as one of the risk factors for acquisition of Acinetobacter infections. Besides prior exposure to antibiotics18, other factors include long stay in hospital19, ICU admission, using tubes and catheter18 and furthermore, transmission between colonized or infected patients directly from hospital equipment or through the hands of health care workers, were also reported as risk factors for acquisition of Acinetobacter spp.20In this study, the frequency of patients who received antibiotics before the isolation of Acinetobacter infections are limited and there are not any controlled trials to show therapeutic choices. Also, carbapenems and colistin are the options of choice for the most drug-resistant infections.1 In view of increasing resistance of Acinetobacter to carbapenem, polymyxins have been considered an option for the treatment of multidrug resistant Acinetobacter spp. infections.6In general, treatment options for Acinetobacter isolates. Therefore, this new glycylcycline agent has bacteriostatic activity against multidrug Acinetobacter spp. to is the appropriate agent for skin and soft tissue infection caused by MDR Acinetobacter spp.23 However; there are some reports of high level resistance to tigecycline among MDR Acinetobacter spp.23 In our study, except for polymyxin B, tigecycline had high sensitivity rates to Acinetobacter isolates. Therefore, this new glycylcycline agent has bacteriostatic activity against multidrug Acinetobacter spp.23 However; there are some reports of high level resistance to tigecycline among MDR Acinetobacter spp.24In our study, except for polymyxin B, tigecycline had high sensitivity rates to Acinetobacter infections based on their clinic-epidemiological features. It also showed that there were high resistant rates of Acinetobacter isolates to common antibiotics except for polymyxin B which becomes emerging problem in combating nosocomial infections in Malaysia. The current findings might be helpful to strategize infection control measures and guidance for prudent use of antibiotic against Acinetobacter infections. In conclusion, the present study provided some information about the patients that are prone to


Poiesis & Praxis in 2001—“A new journal is launched”— this notification came along with high expectations of the addressees with respect to meaning and quality of the content of the newborn journal. The initiative for an “International Journal of Ethics of Science and Technology Assessment” followed the supposed demand for a periodic forum for the rational reflection of the consequences of scientific and technological advance for the individual and social life of the human and its environment—also beyond national perspectives. In the meantime, this broad spectrum was indeed covered with mostly professional articles, which reflected upon the quite different methods of technology assessment and scientific policy advice.When Carl Friedrich Gethmann announced Poiesis & Praxis, changes of the journal’s publication strategy toward an open access model were successfully acknowledged by both the readers and the authors.Eleven years, nine volumes, and 36 issues later, the world has changed and with it the journal as well: It came up with new topics and challenges of the scientific-technological civilization but also with new perception modes and reading habits of its addressees. Especially, the rapid development in the field of the neurosciences and hopes for clinical applications were covered by several “Focus” sections accompanied by high public attention. The same held true with respect to the special issues reflecting methodological problems of appropriate science and technology assessment, which had and have to be adjusted to current challenges regarding the relation between technology and society. Finally, during the last few years of Poiesis & Praxis in order to improve its output and impact. By inviting further coeditors to join the journal’s Editorial Board, they want to broaden its perspective—also with respect to its international entitlement. Several renowned representatives of technology assessment in Europe have already expressed their interest. Thus, the issue following the one at hand will present itself with a new appearance. Therefore, the editors of the journal would like to thank the authors and the readers for their interest in Poiesis & Praxis so far—by publishing and/or by reading. They also thank the Scientific Advisory Board for its various inputs and critics.However, the editors are now considering a relaunch of Everyone is welcome to contribute to the journal’s new appearance upcoming in the course of the next year.Stephan Lingner and Katharina Mader, November 2012


To elucidate the role of prenatal, neonatal and early postnatal variables in influencing the achievement of full enteral feeding (FEF) in very low birth weight (VLBW) infants and to determine whether neonatal intensive care units (NICUs) differ in this outcome.Population-based retrospective cohort study using data on 1,864 VLBW infants drawn from the “Emilia-Romagna Perinatal Network” Registry from 2004 to 2009. The outcome of interest was time to FEF achievement. Eleven prenatal, neonatal and early postnatal variables and the study NICUs were selected as potential predictors of time to FEF. Parametric survival analysis was used to model time to FEF as a function of the predictors. Marginal effects were used to obtain adjusted estimates of median time to FEF for specific subgroups of infants.Lower gestational age, exclusive formula feeding, higher CRIB II score, maternal hypertension, cesarean delivery, SGA and PDA predicted delayed FEF. NICUs proved to be heterogeneous in terms of FEF achievement. Newborns with PDA had a 4.2 days longer predicted median time to FEF compared to those without PDA; newborns exclusively formula-fed had a 1.4 days longer time to FEF compared to those fed human milk.The results of our study suggest that time to FEF is influenced by clinical variables and NICU-specific practices. Knowledge of the variables associated with delayed/earlier FEF achievement could help in improving specific aspects of routine clinical management of VLBW infants and to reduce practice variability. The advances in perinatal care over the last two decades have led to an improvement of neonatal survival worldwide Optimal care provided during the neonatal period has the potential to reduce the risk of short and long term adverse outcome The need to attain FEF rapidly often conflicts with the physiologic immaturity of the GI function of preterm infants and also with the occurrence of various comorbidities in the neonatal period. In this perspective, it is extremely important to investigate risk factors occurring in the early neonatal period which influence the achievement of FEF, in order to optimize the nutritional management of VLBW infants and reduce the potential for several complications. Consideration of the clinical variables associated with delayed/earlier FEF achievement could help in ameliorating specific aspects of routine clinical management of VLBW infants.The aim of the present study is to elucidate the role that some prenatal, neonatal and early postnatal variables play in favoring or delaying the achievement of FEF in VLBW infants and to determine whether neonatal intensive care units (NICUs) differ in this outcome, using data from a Regional Italian Registry.The study was carried out in conformity with the Italian law on privacy and the regulations on data management of the Regional Health Authority. As anonymized administrative data are used routinely for health-care management, the study was exempt from notification to the Ethics Committee and no specific written informed consent was required to use patients' information.Data were drawn from the “Emilia-Romagna Perinatal Network” Registry Starting from 2002, data on live-born infants were collected prospectively from admission until discharge or death; data collection was continued for infants who were transferred to other hospitals. The database was designed according to the format of the Vermont Oxford Network Registry (VON) The study population included 2,441 VLBW infants born from January 2004 to December 2009 . Newborns were excluded if they had congenital malformations, died on the first day of hospitalization or died subsequently but never received enteral feeding because of the severity of their clinical conditions. Records with missing or wrong data on all the predictors, as well as extreme outliers on hospital stay (>365 days) and time to FEF (>160 days), were also excluded.The outcome of interest was time to FEF achievement, measured as the difference in days between the date when FEF was established and the birth date. FEF was defined as enteral tolerance of at least 150 ml/kg/day of milk.For the purpose of the present study, on the basis of their clinical relevance and evidence from the literature, eleven prenatal, neonatal and early postnatal variables were selected for the analysis. NICUs were also included to examine the possible effect of different feeding practices on outcome. Because our aim was to identify early life predictors of time to FEF achievement, variables related to complications occurring later during hospitalization  were not considered.The variables and the rationale for their inclusion in the analysis are detailed below:Maternal pregnancy-induced hypertension, defined as pre-eclampsia and eclampsia: women with pre-eclampsia have higher risk of having children born preterm, with low birth weight or small for gestational age (SGA) Chorioamnionitis, which has been linked to several neonatal outcomes, including respiratory distress syndrome, for which chorioamnionitis seems to be protective, whereas the effect on intraventricular hemorrhage (IVH) and BPD appears to be detrimental Antenatal steroids prophylaxis, defined as complete (two doses) or incomplete Mode of delivery, defined as vaginal delivery (VD), which included spontaneous, induced and instrumental delivery, and delivery by cesarean section (CS). Infants born by CS have significantly different physiology at birth compared to those born by VD, including altered feeding and metabolism Gestational age (GA), as reported by the mother. If the difference between self-reported GA and GA calculated from early prenatal ultrasound examination was >1 week, the latter was used.ad hoc for the Italian population of preterm infants Weight for GA. Infants were defined as appropriate, small and large for GA  according to neonatal growth charts developed Sex. It has been demonstrated that, even with the advances in neonatal care, male preterm infants have higher mortality and poorer short- and long-term outcome than female counterparts Clinical Risk Index for Babies (CRIB) II: CRIB II consists in a five-item score including GA, BW, sex, temperature on admission, and base excess; it provides a valid and simple method of risk-adjustment for neonatal intensive care Early onset sepsis (EOS), diagnosed with a positive blood culture in the first seven days of life.ductus arteriosus (PDA), assessed by ECHO in the first week of life. The presence of PDA and its treatment can influence tissue oxygenation, especially in the mesenteric region Patent Feeding, defined as HM or formula-feeding during hospital stay. Specifically, HM feeding was defined as the use any own mother's or donor milk during hospital stay.A questionnaire was administered to the heads of the nine NICUs to collect more detailed information on feeding practices in different GA/BW groups and in relation to the newborns' clinical conditions, in order to give a qualitative description of the possible sources of variability of the outcome among the NICUs. Practices regarding PDA management were also collected.Newborns' characteristics were compared among the study NICUs using ANOVA or chi-square test as appropriate. Following significant test, post-hoc pairwise comparisons were carried out using Bonferroni correction to the probability level.The study outcome was time (in days) to FEF achievement. A parametric survival analysis was used to estimate the relationship of prenatal, neonatal and early postnatal variables with time to FEF. The main advantages of this analytical approach are the use of information provided by each individual in the cohort  and the use of an appropriate distribution for time to FEF estimation, reflecting the observed temporal pattern of occurrence of this outcome.In this study we chose a log-logistic hazard function The nine participating NICUs were included in the survival model as eight dummy variables in order to take into account the possible effect of practice differences on time to FEF. NICU ‘B’ was chosen as the reference unit because it had the highest number of newborns.a priori on a clinical basis. A backward elimination criterion was used to obtain the final model, removing one at a time those predictors whose effect on time to FEF was not significant (p>0.05).The initial model included all the predictors identified Marginal effects were obtained for each variable using the estimates of time to FEF from the final model Statistical analyses were carried out using Stata version 13.0 . Streg procedure was used for parametric survival analysis and margins procedure to estimate marginal effects.Of the 2,441 newborns, 219 (9.0%) were excluded because they had congenital malformations, 58 (2.4%) because they died on their first day of hospitalization, and 59 (2.4%) because they never received enteral feeding. Among the 2,105 subjects eligible for the study, 234 (11.1%) were excluded due to missing or wrong data and 7 because they were extreme outliers (0.3%). Thus, 1,864 newborns were included in the survival analysis. Of these subjects, 1,775 (95.2%) achieved FEF; 109 (5.9%) died during hospitalization and, of these, 20 (18.4%) died after having reached FEF. Compared with newborns included in the survival analysis, those who were not included were significantly more likely to have been born by vaginal delivery at a lower GA, and to have a higher CRIB II score and a higher incidence of IVH. Moreover, they were less likely to have received antenatal steroids, to have been oxygen dependent at 36 weeks of corrected age, to have had periventricular leukomalacia and LOS, and have required UVC for fewer days.The clinical characteristics of the sample are provided in The median time for achieving FEF was 13 days (IQR 7-24 days) and the mean time 18.25 days (SD = 17.56). The nine NICUs differed significantly on time to FEF achievement  and on the frequency of several prenatal and postnatal variables . Post-hoStarting from an initial survival model including all predictors, a final model was obtained including NICUs and seven predictors significantly associated with time to FEF achievement . The hazCorrelations among predictors of the final model higher than 0.30  were found for GA with SGA, CRIB II score and PDA, and for CRIB II score with PDA. None of these correlations exceeded 0.63, suggesting that the predictors were not redundant in the model.Marginal effects were higher for the variables which had a stronger effect on outcome : infantsRemarkable differences among NICUs were found: for example, after adjusting for the other variables in the model, newborns in NICU D achieved FEF 14.7 days later than in the reference NICU, those from NICU A 9.1 days later, and those from NICU H 10 days earlier.The secondary analysis carried out to examine the effect of PDA treatment showed that observed median time to FEF was 15.1 days in infants without PDA, and increased to 17.8 days in infants with PDA who did not receive any treatment, to 19.3 days in those receiving a pharmacological treatment, and to up to 21.7 days in those who underwent surgery in combination with a pharmacological treatment. These findings were confirmed by survival analysis: the hazard functions estimated for these subgroups  showed tThe survey, conducted to explore the different attitudes towards feeding VLBW infants, showed that the large majority of the NICUS (7 out of 9) had a standardized protocol for enteral feeding.However, feeding practices were quite heterogeneous among NICUs. Furthermore, feeding protocols in each NICU were modulated according to different cut-offs .In smaller/younger infants, minimal enteral feeding (MEF) was used in all the NICUs, starting at 0–2 days in six NICUs, and at 3–5 days in the other three NICUs; the daily increase of enteral intake was less than 15 ml/kg/day in the majority of the NICUs (7/9). Reasons for feeding withdrawal were also heterogeneous, including bilious aspirates, red blood cells transfusions, and presence and treatment of PDA.Clinical practices regarding PDA management were also examined: none of the NICUs used prophylactic PDA treatment, and all of them treated PDA only when hemodynamically significant. The first therapeutical approach to PDA was pharmacological for all the NICUs: 7 used exclusively Ibuprofen, 1 Ibuprofen for newborns less than 30 weeks GA and Indomethacin for newborns>30 weeks GA, and 1 used exclusively Indomethacin. Surgical treatment was a second-line treatment in all the NICUs, after the failure of two consecutive pharmacological courses.The present study aimed to evaluate the effect of prenatal, neonatal and early postnatal conditions on time to FEF achievement in VLBW infants.GA and PDA proved to be the strongest predictors of time to FEF. As expected, higher GA predicted an earlier achievement of FEF: specifically, each one-week increase in GA reduced time to FEF by 15.9% (estimated reduction of more than 3 days). To our knowledge, evidence linking GA to FEF achievement in VLBW infants is scanty In the present study, PDA increased time to FEF achievement by 27.6% (estimated delay of more than 4 days). The presence of a clinically significant PDA, especially when associated with sepsis, has been recognized as a risk factor for a delayed start of enteral feeding and FEF achievement Infants fed HM reached FEF slightly earlier than exclusively formula-fed infants, after adjusting for all the other newborns' characteristics. The estimated reduction of the time to FEF in HM-fed infants was 1.4 days. This reduction could be clinically significant, as results from cohort studies in extremely low birth weight newborns fed HM indicate that very early establishment of FEF with HM is associated with lower occurrence of complications such as LOS In our study population, SGA increased time to FEF achievement by 16.6%. Evidence from the literature suggests that IUGR infants have increased risk for several adverse neonatal outcomes Our results on the association between FEF, GA, SGA and PDA are consistent with a recent study based on a retrospective cohort of preterm infants An increase in CRIB II score was associated with a delay in FEF achievement. To the best of our knowledge, no previous studies assessed the role of this predictor on time to FEF in preterm infants. CRIB II score, which provides a recalibrated and simplified scoring system that avoids the potential problems of early treatment bias in utero may persist in the splanchnic circulation for several days after birth, compromising feeding tolerance and FEF achievement Maternal hypertension increased time to FEF achievement by 11.2%. Previous studies have reported a similar association Infants born by CS reached FEF later than infants born by VD. We can speculate that this delay could be attributed to the alterations in gut microbiota which occur following CS audit activities, aiming to identify “best clinical practices” and promote quality improvement in neonatal care.In addition, our results show that, after adjusting for the effect of other variables, NICUs proved to be still heterogeneous in time to FEF. This indicates that time to FEF was significantly accounted for by unmeasured NICUs characteristics, including differences in clinical practice or in the organizational set-up. The survey which was conducted in collaboration with the heads of the nine NICUs showed that most of the NICUs had a formal protocol for the nutrition of preterm infants, but these protocols were quite heterogeneous as well. The differences in clinical and nutritional practices may at least partially explain the significant variation in time to FEF among NICUs . The identification of differences in clinical practices among NICUs could be the starting point for the development of clinical One of the methodological strengths of the study is its design: the advantage of population-based studies over RCTs is that the former include data collected systematically from unselected populations. In particular, data obtained from large population-based registries, such as the VON It has to be noted that, although our population included a slightly higher proportion of very preterm newborns compared to VON data (GA<27 weeks 18.0% vs. 16.1%), mortality was lower (14.9% vs. 16.6%), as well as the incidence of neonatal complications such as NEC (4.6% vs. 6.7%) In conclusion, the present study identifies some clinical predictors of the time to FEF achievement in VLBW infants. Knowledge of risk factors associated with FEF achievement could help in ameliorating specific aspects of routine clinical management of VLBW infants. Efforts are warranted to optimize nutritional management of VLBW infants and reduce NICU variability in feeding practices in order to avoid potential complications of a delayed achievement of FEF.


Chlamydomonas reinhardtii. This screen relies on starch synthesis accumulation during nitrogen starvation followed by the supply of nitrogen and the switch to darkness. Hence multiple regulatory networks including those of nutrient starvation, cell cycle control and light to dark transitions are likely to impact the recovery of mutant candidates. In this paper we monitor the specificity of this mutant screen by characterizing the nature of the genes disrupted in the selected mutants. We show that one third of the mutants consisted of strains mutated in genes previously reported to be of paramount importance in starch catabolism such as those encoding β-amylases, the maltose export protein, and branching enzyme I. The other mutants were defective for previously uncharacterized functions some of which are likely to define novel proteins affecting starch mobilization in green algae.A screen was recently developed to study the mobilization of starch in the unicellular green alga  Starch represents the most widespread storage polysaccharide found in plants and algae. This insoluble polymer of glucose not only defines an essential source of calories for human and animal nutrition but is also used in many non-food applications including but not restricted to biofuel and hydrogen production. Starch is made of two distinct subfractions: the minor amylose fraction is synthesized by a starch granule bound isoform of starch synthase called GBSS (Granule Bound Starch Synthase). Amylopectin, the major fraction, confers to starch most of its physical properties and in particular its insoluble semi-crystalline nature. Even if amylopectin and glycogen share the same primary structure , the synthesis of these two storage polysaccharides requires a completely different set of enzymes. While bacterial glycogen is synthesized and degraded through the use in most cases of no more than 5–6 activities The starch biosynthetic process has been extensively studied thanks to both reverse and forward genetics approaches. The high number of enzymatic activities involved and the interaction found between them in the same protein complexes Arabidopsis thaliana have yielded a working model for the recurring starch degradation occurring in leaves of this model organism Chlamydomonas reinhardtii which has been by the past intensively used to study starch synthesis Significant progress in our understanding of starch mobilisation has been made mostly in the last decade. It now defines a very active domain of research that essentially concerns diurnal Arabidopsis leaf starch mobilisation , an activity producing a pink band on denaturing starch containing zymograms is lacking . This phIn order to confirm the validity of our screening procedure, the mutants displaying a possible defect in starch mobilization (i.e. chiefly the class 2 and 3 mutants) were analyzed through molecular techniques in order to identify the genomic sequences of those regions flanking the insertions. Different strategies including plasmid rescue, inverse PCR and TAIL-PCR were attempted. Even if our efforts resulted in limited success, we were still able to determine in a few of our mutants the identity of the genes disrupted by the exogenous DNA. A summary of the genes identified and of the techniques used for the identification is listed in In this work, we decided to focus on the CAT16 mutant which is the only one in our bank for which we detected a clear enzymatic defect through zymograms.BE2a and BE2b structural genes in both 137C and CAT16 while no amplification was obtained with the mutant with the primers designed to amplify a part of the BE1 gene  starch structure From 16,000 paromomycin resistant colonies obtained after insertional mutagenesis  we were able to isolate 38 independent strains which harbor a clear defect for starch mobilization with the two-step iodine screening procedure. Through kinetics of starch mobilization, we were able to show that 12 of these 38 mutants do degrade starch with the same efficiency as the wild-type. Those strains may be either false positive or strains in which the efficiency of starch degradation is not the same on solid and liquid medium. The 26 other mutants were all less effective than the wild-type with respect to starch mobilization and define two different phenotypic classes. The so-called class 2 mutants display a mild phenotype but with a significant starch degradation impairment as these 13 mutants still contain between 35 to 49% of their initial polysaccharide amounts after 1 day of degradation compared to the 28% recorded for the isogenic wild-type. The most defective strains were found in the class 3 for which 13 mutants still contain from 56 to 97% of the initial polysaccharide amounts assayed before degradation. It is worth noting that starch over accumulation and impairment in starch degradation were not linearly linked as overproducers are found in the mutants of class 1 (the one degrading starch with good efficiency) while even in class 3 mutants containing the mutants the most dramatically impaired for starch degradation, some of the strains accumulated nearly wild-type amounts of polysaccharide. These results may reflect the high complexity of the process involved in the regulation of starch deposition and catabolism in Chlamydomonas.For the 26 strains for which a real defect for starch degradation was confirmed thanks to the kinetics of polysaccharide mobilization in liquid cultures, we used several strategies including the starch structure determination, visualization of the starch hydrolytic activities through the use of zymograms and molecular techniques in order to identify the missing function responsible for the catabolic phenotype.Even if a defect in starch catabolism is not expected to lead to a modification of starch structure, two of our mutants displayed modification either in the amylopectin/amylose ratio or in the amylopectin structure. The CAT31 mutant contains a high amount of amylose as this starch subfraction represents 41% of the total polysaccharide for only 23% in the wild-type. This increase in amylose is not accompanied by a strong modification of amylopectin which displays the classical λmax value around 550 nm. At the opposite, the CAT17 mutant produces a modified amylopectin with a high λmax at 567 nm but no amylose increase in this polysaccharide can be recorded. As for now, it is difficult to explain these modifications and the link between the modified structure and the defect in starch degradation cannot be easily explained. The identification of the disrupted functions in these strains will possibly yield some understanding on the reasons for these observations.The molecular experiments including plasmid rescue, inverse PCR and TAIL PCR allowed us to identify several candidates which still need to be validated as responsible of the starch degradation defects. Nevertheless, the detection of a copy of the mutagenesis plasmid in the structural genes encoding the maltose transporter Mex (CAT3) or an isoform of β-amylase (CAT4), both functions already demonstrated to impact efficient starch mobilization, are encouraging with respect to the specificity of our mutant screen. Several disrupted functions which may be related to polysaccharide metabolism were also identified as a starch binding domain containing protein in CAT 33 or an oligosaccharyl transferase in CAT 37. Such oligosaccharyl transferase are known to be involved in protein N-glycosylation in the ER lumen sbe1a mutant was more resistant to α-amylase digestion. During kernel germination, shorter coleoptile lengths and higher residual starch were recorded leading to the hypothesis that less efficient starch utilization was taking place in this mutant. However, at this stage a role of branching enzyme 1 in starch catabolism in Chlamydomonas can only be suggested as this and the other mutants reported in this work have to go through a more detailed and comprehensive characterization. Nevertheless, the recovery of such a “mild” defect in starch catabolism suggests that the iodine screening procedure in Chlamydomonas is quite sensitive and effective in this respect.One single mutation was identified through the use of zymogram techniques allowing visualization of hydrolytic activities. The missing activity in the CAT 16 mutant was identified as a branching enzyme activity. The molecular characterizations allowed us to prove that this strain was defective for branching enzyme 1, an enzyme for which no function in starch metabolism has been reported so far. A recent study performed in maize revealed a slight modification of starch structure in this homologous mutant The discovery of mutants for Mex or β-amylase which are already known as important starch degradation enzymes reveals the two step iodine phenotypic screen as an efficient tool to isolate novel starch catabolic mutants. We believe that the sensitivity of this mutant screening procedure and the physiological plasticity of the Chlamydomonas model will enable this system to bring further light into the complex machinery responsible for starch mobilization in green plants and algae.C. reinhardtii strains used in this study were 137C (mt- nit1 nit2) and 37 (mt+ ac14 pab2). All experiments were carried out in continuous light (40 mE m−2 s−1) in the presence of acetate at 24°C in liquid cultures that were shaken without air or CO2 bubbling. Late-log phase cultures were inoculated at 105 cells mL−1 and harvested at 2–3.106 cells mL−1. Nitrogen-starved cultures were inoculated at 5.105 cells mL−1 and were harvested after 5 days at a final density of 1 to 2.106 cells mL−1. Recipes for media can be found in The wild-type reference A full account of amyloglucosidase assays, starch purification on Percoll gradient, separation of amylose and amylopectin on CL-2B gel permeation chromatography and λmax  measures can be found in RI linearized pSL18 vector AphVIII gene (aminoglycoside 3′-phosphotransferase from Streptomyces rimosus) conferring resistance to paramomycin, or with a 1,9 kb PCR product of this plasmid corresponding to the resistance cassette in order to produce transformants according to the protocol described in 5′-ACCATGATTACGCCAAGCGCGCAA-3′) and ParoR (5′-CTCGACATGCGTTCACTTCCTGTC-3′) using Dynazyme Ext  following the manufacturer recommendation with an annealing temperature at 55°C and an extension time of 2 minutes for 30 cycles.The wild-type Chlamydomonas reference strain 137C was transformed with either EcoAfter transformation, cells were plated on paromomycin supplemented TAP (10 mg/l) plates for the selection of transformant lines.The putative catabolic mutants were screened on the basis of their iodine/starch interaction which reflects the amount of polysaccharide in the cells. Cellular patches (20 µL) of the wild-type reference strain 137C, the already characterized mutants BafJ4 2; 0.1% KI) and 700 µL of distilled water. The absorbance was measured every nanometer between 400 and 700 nm to determine the wavelength of the maximal absorbance of the iodine-polysaccharide complex (λmax) which reflects the polysaccharide structure.Zymograms in starch containing gels allowing the detection of most starch hydrolases and branching enzymes have been described for both undenatured samples 5′-GAGAGTCACGACCAGGCTCTG-3′) and BE1RT3 (5′-GAAGCCGTAGTGGTTGTCGAG-3′) allow the amplification of a 357 bp fragment of BE1 mRNA while PhoBRT5 (5′-GCATGTTCCGCCAGACCA-3′) and PhoBRT3 (5′-TGCAGGAAGCGCCAGTTGA-3′) primers are used to amplify a 736 bp fragment of the PhoB mRNA used as an internal control. PCR products Total RNA was extracted from Chlamydomonas according to Merendino et al., 2003Total DNA from wild-type and mutant strains was extracted as previously described °C and an extension time of 1 minute for 30 cycles. A 650 bp genomic DNA fragment corresponding to BE1 (XM_001695339) was amplified in the reference wild-type using the BE1 FOR5 (5′-ATGGCTGCGAGGCCGCTTCAG-3′) and BE1 REV3 (5′-ACGACCGCCTACACGCCCTG-3′) primers, while BE2aFor5 (5′-CAATGGCACACCTCCTCCAC-3′) and BE2aRev3 (5′-GTTGAACTGGATCTCGTTCCAG-3′) primers were used to amplify a 516 bp DNA fragment and BE2bFor5 (5′-CTTACTCGCACCAGCAAGCTG-3′) and BE2bRev3 (5′-CTTGAAGGTGTACTGCTTGTC-3′) a 615 bp fragment corresponding respectively to the BE2a (XM_001696177) and BE2b (XM_001690270) genes.The integrity of branching enzymes genes was checked by amplifying parts of the corresponding DNA with Dynazyme Ext  following the manufacturer recommendation with an annealing temperature of 60http://www.ncbi.nlm.nih.gov/BLAST; http://www.phytozome.net/search.php?show=blast&method=Org_Creinhardtii). For TAIL-PCR the primers used were the degenerated primer AD2 (5′-NGTCGASWGANAWGAA-3′) and 3 specific primers, TAIL1 (5′-GTGCTCGTTTGTCGCTGAAAGT-3′), TAIL2 (5′-CAAATCAGTCCTGTAGCTTCA-3′), and TAIL3 (5′-ACATACGCACCAATCATGTCA-3′) used respectively for the primary, secondary and tertiary reactions. For inverse PCR, the P1 (5′-CATGTTTGCCCGAACTCGGAG-3′) and P2 (5′-CATTTGCCTGCCTTCACGCATC-3′) or the T1 (5′-TGCATGTAATGGCCAGGCCATG -3′) and T2 (5′-ACTGGCTCACGCACACGCTAAC-3′) primers allowed the amplification of the genomic DNA flanking each side of the unique EcoRI site used to linearize the pSL18 plasmid. The cycling parameters for all reactions of TAIL-PCR and inverse PCR are described in Thermal asymmetric interlaced PCR and inverse-PCR were used to obtain the DNA flanking the paramomycin cassette or pSL18 insertion sites  Homology searches were performed using the BLAST server  are indicated with a star.(TIF)Click here for additional data file.Figure S2Partial purification of branching enzyme 1 activity from the CAT16 mutant crude extract. The enzymatic defect in the CAT 16 mutant can be observed through the lack of a pink or a red band (enlighted by arrows) on native  and denaturing  starch zymograms respectively. This activity cannot be detected in the first elution fractions (E1 to E3) of the amylose column chromatography as it was the case with the wild-type crude extract as reveled by analysis on starch denaturing zymogram (b). (c) Interaction of polysaccharides with iodine. Samples 1 to 6 correspond respectively to the iodine alone (1), the interaction of the latter with the unmodified amylose in the absence (2) or in the presence of the MOS in elution buffer (3). The iodine interaction of the polysaccharide incubated with the 3 elution fractions (E1 to E3) are displayed in 4, 5 and 6. The values of the λmax of each complex is indicated on the figure in nanometers.(TIF)Click here for additional data file.


Treating acute ischaemic stroke patients with iv-rtPA is of overall benefit. An associated increase in the risk of symptomatic intracranial haemorrhage (SICH) may cause considerable harm. We investigated (i) whether novel or existing prediction models could predict SICH or poor functional outcome in rtPA treated patients and (ii) whether ischemic stroke patients at either a high predicted risk of SICH or poor functional outcome experienced less benefit from rtPA.We used the IST-3 trial data, an international, multicentre, open treatment randomised trial of rtPA versus control in 3035 acute ischemic stroke patients. We developed and internally evaluated a multivariate logistic regression model for SICH following rtPA including variables identified as important in a systematic review. We compared the discrimination (area under the receiver operating characteristic curve (AUROCC)) of our model with those existing in the medical literature. We calculated the absolute risk reduction of death or dependency with rtPA in patients at a low, medium or high predicted risk of SICH or poor functional outcome with each model.Our model had similar discrimination for SICH (AUROCC 0.68 95% CI: 0.63-0.74) to nine previously developed models . There was no evidence that patients at high predicted risk of SICH or poor functional outcome after stroke derived less benefit from rtPA.We found no evidence to support a stratified approach in administering rtPA to acute ischaemic stroke patients at a high predicted risk of intracranial haemorrhage or poor functional outcome.


Implementation Science, your support.Implementation Science, like all journals, needs to continue to develop. There will always be changes we need to make as next steps in improving the Journal for readers and improving how it runs. However, we now have our first change in Editors in Chief. We are fortunate to have been able to recruit two experienced academics who are also experienced editors—Professor Michel Wensing and Dr Anne Sales. I hope you will join me in welcoming them and give them, and continue to give  Implementation Science first accepted submissions, and February 2006 when it published its first issue, the Journal has grown from 100 submissions in 2006 to 354 in 2011. Similarly, the number of articles published has also increased from 29 in 2006 to 133 in 2011.Since November 2005, when As well as providing a high-quality, peer-reviewed outlet for traditional research articles, we have encouraged the publication of a range of articles that were previously difficult (if not impossible) to publish despite the widely held view that they were important contributions to the field—study protocols, detailed descriptions of barriers and facilitators to high-quality care, detailed descriptions of the development processes behind interventions, evaluations of the processes by which interventions achieved their effects—as well as providing space for conceptual and methodological articles.th out of 62 journals in the ‘Health Care Sciences & Services’ and 9th out of 76 journals in the ‘Health Policy & Services' categories of the Journal Citation Report. With our clear focus on implementation science, we are now the leading international journal in this field.The strong response to the Journal launch, and its year-on-year increase in submissions and publications, speak to the importance of the Journal to the emerging field of implementation science. Listed in all the major electronic databases, we attracted our first impact factor in 2010 and this has steadily risen to our current figure of 3.1. We are now ranked 4As we have grown, we have made changes to continue the efficient running of the Journal. We have recruited an increasing number of Associate Editors. We have gone from starting with just the two Co-Editors in Chief to now having one Deputy Editor in Chief and ten Associate Editors. This has been part of a steady, incremental process, and the Editors in Chief are enormously grateful to the Associate editors for the work that they contribute to running the Journal. We have re-configured our Editorial Board to form two bodies—a Senior Advisory Board to provide the Editor(s) in Chief with strategic advice when it is required and a refreshed and enlarged editorial board. We have a loyal pool of editorial board members who review for us on a regular basis, and we are very grateful for all of the time they commit to Journal activities.And so to our next transition. Last year, Brian Mittman became Editor in Chief Emeritus, leaving me as sole Editor in Chief—a role that I will step down from during the next twelve months when I will have been Editor in Chief for over seven years. I am delighted to announce that, as of 1 September 2012, two of our current editors—Professor Michel Wensing and Dr Anne Sales—are taking on the roles of Co-Editors in Chief. They and I will share the Co-Editor in Chief roles until I permanently stand down in 2013.Implementation Science, like all journals, needs to continue to develop. There are a number of changes we need to make as the next steps in improving the Journal for readers and improving how it runs. However, this change in Editors in Chief is key to the continuing development of the Journal. We are fortunate to be able to recruit two experienced academics who are also experienced editors, and it is good to be planning the transition with two such experienced pairs of hands. I hope you will join me in welcoming them and give them, and continue to give Implementation Science, your support.


South Africa not infrequently hosts high-level cardiology meetings outside the auspices of South African professional medical societies and associations. Well-known cardiologists, particularly from the USA and Europe are invited to make presentations, which bring the best and most-recent advances in the field to this country. This is of course of great value to the selected group of delegates who are invited to attend. Large sums of money are involved because all the presenters and many of those attending receive full financial support for their participation.A recent meeting in Cape Town, Cardiology and Diabetes at the Limits, is a case in point. It was arranged through the Hatter Institute in London and, typically, had as many as 30 invited cardiologists. In this instance, organisers raised funds very successfully from international pharmaceutical companies and also from their local business units. It was held outside the orbit of accountable professional medical organisations in either South Africa or the United Kingdom. These companies are hard pressed to say no, and might well wish to spend budgeted money on other forms of continuing medical education or research support, which would reach a wider audience, but are being energetically lobbied into funding such meetings.The question arises whether South Africa is being used to sidestep the ethical principles as set out for the promotion of pharmaceutical products by the International Federation of Pharmaceutical Manufacturers and Associations (IFPMA) and the affiliated Pharmaceutical Industry Association of South Africa (PIASA).et al.The recent editorial article by DJ Rothman,


Visco-elastic properties of the (neuro-)musculoskeletal system play a fundamental role in the control of posture and movement. Often, these properties are described and identified using stiffness-damping-inertia (KBI) models. In such an approach, perturbations are applied to the (neuro-)musculoskeletal system and subsequently KBI-model parameters are optimized to obtain a best fit between simulated and experimentally observed responses. Problems with this approach may arise because a KBI-model neglects critical aspects of the real musculoskeletal system.The purpose of this study was to analyze the relation between the musculoskeletal properties and the stiffness and damping estimated using a KBI-model, to analyze how this relation is affected by the nature of the perturbation and to assess the sensitivity of the estimated stiffness and damping to measurement errors. Our analyses show that the estimated stiffness and damping using KBI-models do not resemble any of the dynamical parameters of the underlying system, not even when the responses are very accurately fitted by the KBI-model. Furthermore, the stiffness and damping depend non-linearly on all the dynamical parameters of the underlying system, influenced by the nature of the perturbation and the time interval over which the KBI-model is optimized. Moreover, our analyses predict a very high sensitivity of estimated parameters to measurement errors.The results of this study suggest that the usage of stiffness-damping-inertia models to investigate the dynamical properties of the musculoskeletal system under control by the CNS should be reconsidered. It is widely acknowledged that visco-elastic properties of the (neuro-)musculoskeletal system play a fundamental role in the control of posture and movement. Visco-elastic properties arise from reflexive pathways and intrinsic properties of the muscle-tendon complex. Reflexive pathways influence dynamics through feedback from, for example, muscle spindles and Golgi-tendon organs, the gain of which is regulated by the central nervous system (CNS) K), a damping (B) element, and an inertia (I) element. Stiffness and damping are typically identified experimentally by perturbing the human musculoskeletal system and optimizing parameter values of the KBI-model to obtain a best fit between model responses and responses observed experimentally  models; for example that of the ankle joint e.g.  elbow joally e.g.. Using sWithin the musculoskeletal system, the skeleton interacts with an elastic tendon that is in turn connected in series with a visco-elastic CE. In a KBI-model, such an elastic tendon is not present see . Becausein vivo. Our analyses show that the estimated stiffness and damping will not resemble any of the dynamical parameters of the underlying system, not even when the responses are adequately captured by the KBI-model. The analyses furthermore show that the estimated stiffness and damping will be a non-linear mixture of all the dynamical parameters of the MSM, influenced by the nature of the perturbation and the time window over which the parameters are estimated. Finally, our analyses predict a very high sensitivity of estimated parameters to measurement errors. These results suggest that the usage of stiffness-damping-inertia models to experimentally investigate the dynamical properties of the musculoskeletal system that are under the control of the CNS should be reconsidered.The purpose of the present study was threefold: i) analyzing the relation between the musculoskeletal properties and the estimated stiffness and damping, ii) analyzing how this relation is affected by the nature of the perturbation and iii) assessing the sensitivity of the estimated stiffness and damping to measurement errors. Since the real dynamical properties are not known exactly and are difficult to control in an experiment, a musculoskeletal model (MSM) was used to assess the validity of using a KBI- approach to estimate stiffness and damping in vivo. As mentioned in the Since the real dynamical properties are not known exactly and are difficult to control in an experiment, a musculoskeletal model (MSM) was used to assess the validity of using a KBI- approach to estimate stiffness and damping In essence, we used the KBI-approach to try and estimate stiffness and damping of a MSM for which the dynamical parameters are known a-priori. To do so, we followed the approach identical to those described in the literature: we perturb the system (our MSM model) and subsequently optimize the parameters of the KBI-model to obtain a best fit between the responses of the KBI-model and the MSM. This was repeated for a realistic range of values of the dynamical parameters of the MSM, for different response times and perturbations see . In addiMSM and ZKBI respectively, in the Laplace domain (2)MSMp = [CE BCE KSE IMSMK]T  for the MSM, and KBIp = [K B I]T  for the KBI-model. KBIp was optimized by minimizing the following error criterion function that is only sensitive to difference in the shape of the MSM (MSMϕ) and KBI-model response (KBIϕ):For convenience, we take the parameters of each of the models in a parameter vector and define E obtained with KBIp* will be indicated as *E and corresponds to the  value of the error criterion function for a given set of MSMp. T is the time window over which the response was fitted.The value of MSMp  = [32 3.2 100 .1] to a torque impulse perturbation for a time window T of 50 ms. As can be appreciated from the figure, for this MSMp and response time, the KBI-model was very well capable of describing the dynamic behavior, leading to an error E of practically zero. The estimated stiffness K (55 Nm/rad) was somewhere between the CE (32 Nm/rad) and SE (100 Nm/rad) stiffness and the estimated damping B (0.36 Nms/rad) was about ten times lower than the CE damping (3.2 Nms/rad). MSMp set to [32 10 320 .1]) that could not be captured well by the KBI-model (E = 0.005).E, which, as stated before, captures the difference between the response of the MSM and that of the optimized KBI-model (see Eq. 3), was plotted as a function CEK and CEB. For graphical purposes, and based on the rationale that in the real musculoskeletal system CEK and SEK cannot be chosen independently (SE is a passive non-linear spring and hence its stiffness depends on the CE force), SEK was set to a fixed value of CEK . Clearly, the estimated K does not have a simple relation with the dynamic parameters of the MSM, not even in the cases in which the responses of the MSM and KBI-model were virtually identical . For all other  combinations of MSM parameters, estimated stiffness for a fixed perturbation depended non-linearly on all parameters of the MSM. For example, for a fixed CEK of the MSM, estimated stiffness could range from 1 to 6 times CEK, depending on the damping of the CE.B as a function of the MSM parameters. The resemblance between CE damping and estimated damping is in general very non-linear. More importantly, the damping estimated depends greatly on the stiffness of the CE and SE. By changing CE stiffness, but not its damping, estimated damping can change 100-fold! Furthermore, the estimated stiffness and damping in general do not resemble any of the parameters of the musculoskeletal model, not even in those cases for which the responses of the KBI-model were virtually identical to those of the MSM. Above that, estimated stiffness and damping greatly depended on the time interval over which the response were optimized and on the type of perturbation applied.MSMp and KBIp analytically for low and high frequencies (see LF BLFK]) and high frequency approximation ([HF BHFK]).As can be appreciated from cies see . Table 1LFK is obtained by the well-known expression for the stiffness of two springs connected in series. As tendon stiffness in reality never reaches infinity, the identified stiffness is always lower than the CE stiffness.  In the expression for the estimated damping LFB, the same factor SEK /( CEK+SEK) occurs to the power of two. This means that SE stiffness reduces the estimated damping even more than the estimated stiffness.Under low-frequency conditions, the identified stiffness HFB is quite remarkable: the effective damping constant increases with SE stiffness and decreases with CE damping! The higher CE damping, the less it will change its length for a given perturbation force. As a result, relatively more of the rate of change in muscle length is taken up by SE (which has zero damping), and hence estimated damping will be less. Conversely, a high SE stiffness enforces CE stretching and hence will increase the estimated damping. In general, in vivo estimated stiffness and damping will be somewhere between the low and high frequency approximation depending on the parameters values of the musculoskeletal system and on the type of perturbation applied.Under high-frequency (HF) conditions, we found a very different set of parameters for the KBI-model. The effective stiffness corresponds to the SE stiffness. This can be readily understood as for very fast perturbations CE damping prevents elongation of the CE. In that case, all muscle length changes is due to changes in tendon length and hence stiffness estimated is the stiffness of the tendon. The expression for the estimated damping constant E. In this sensitivity analysis, we calculated the maximal change in parameter values of the KBI-model that led to an arbitrarily small ΔE of 0.001, and hence assessed the sensitivity of parameters that can be expected on the basis of measurement errors. In In an experiment, inherent errors occur in both the applied perturbation and the measured response. To directly explore the sensitivity of estimated stiffness and damping to such errors, a sensitivity analysis was carried out. Any given difference between actual and measured joint angle time histories can be expressed by a corresponding value of the error criterion pKBI* plus the ΔpKBI leading to a small increase in matching error ΔE of 0.001. Evidently, the small additional matching error of 0.001 changed almost unnoticeably the responses of the KBI-model. In an experimental study, these responses would clearly have been marked as being excellent fits of those observed experimentally. Yet, such a small difference in response can correspond to very large differences in estimated stiffness and damping. As an example  as a function of CEK and CEB . As with the four examples depicted in K (ΔK/K) was 65% (±13%) when the time interval was set to 50 ms, and 12% (±7%) and 7% (±1%) when the time interval was set to 100 and 200 ms, respectively.E = 0.001) as a function of CEK and CEB . As with the sensitivity of the estimated stiffness, the general sensitivity of the estimated damping was considerable and tended to increase with decreasing time intervals over which the responses were optimized. The mean relative change of B (ΔB/B) was 880% (±1350%) when the time interval was set to 50 ms, and 65% (±99%) and 4% (±2%) when the time interval was set to 100 and 200 ms, respectively.Most of the results shown were obtained using torque impulse perturbations  within the standard error bounds. In accordance with our findings, their sensitivity analysis indicated a large within-subject variability of estimated stiffness and an even larger sensitivity of estimated damping.Our finding that the estimated dynamical parameters depend greatly on i) the nature of the perturbation, ii) the time interval over which the response is being fitted and iii) are very sensitive to measurement errors is supported by the very wide range of joint stiffness reported in the literature. For example, elbow joint stiffness values reaches from about 14 Nmrad-1 to 126 Nmrad-1 in ‘do resist’ paradigms e.g. . Based odamping. CE damping however changes not only with muscle activation, but also with contraction velocity. Therefore, changes in estimated stiffness do not necessarily reflect either changes in CE/SE stiffness due to changed motor commands per se. In addition, CE stiffness is not only influenced by activation level, but also by CE length (and hence tendon length) and activation/contraction history mechanical variables controlled by the CNS. However, such a point of view raises fundamental problems. For example, as can be appreciated from the see e.g.  and therThere is ample evidence that the dynamics of the musculoskeletal system positively contribute to stabilize posture and movement control and that the dynamics are influenced by changes in motor commands e.g. . HoweverMSM and ZKBI respectively, in the Laplace domain MSMp = [CE BCE KSE IMSMK]T for the MSM, and KBIp = [K B I]T for the KBI-model.For convenience, we take the parameters of each of the models in a parameter vector and define SEK estimated was based on a non-linear quadratic spring and its stiffness can be written as:In essence, the MSM presented in this study acts like a lumped muscle representing all muscles crossing the elbow joint that work basically in series. Parameters of muscles crossing the joint were obtained from the literature k the tendon stiffness, ΔSEl the tendon elongation, SEM the torque due to tendon force and arm the moment arm of the muscle. k was calculated such that at maximal isometric CE force, SE elongates 4%. At zero CE force (ΔSEl = 0), SEK is zero. Using the parameters of the individual muscles crossing the elbow joint, total SEK at maximal isometric CE force was estimated to be 1800 Nm/rad.With CEK is linearly dependent on CEl:The CE force-length relationship mainly originates from the changes in myofilament overlap and  is reasonably well described by a parabola. In this case w a parameter defining the active and passive slack length of a muscle, CE_rell the CE length relative to its optimum CE length (CE_optl) and CE_lM the torque due to the CE force-length relationship. Based on the sliding filament theory, this parameter was set to 0.56, yielding a maximal CE_rell range of 1±w. Maximal CEK (at active slack) for a lumped elbow muscle is about 200 Nm/rad. Obviously, at optimum CE length no stiffness originates from changes in myofilament overlap (the derivative of force-length relationship = 0) and should be seen as an upper limit. In reality, CE stiffness increases due to short-range stiffness 2+] sensitivity With CEB on considerations of the classic Hill-type (concentric) force-velocity relationship:Damping parameters of the joint are not readily obtained from the literature, partially because these parameters are often based on the KBI approach and partially because damping of individual muscle is not often reported. We based CE_nF is the normalized CE force; a and b are the Hill parameters and were set to 0.41 and 5.2 respectively ; ce_relv is the normalized contraction velocity . Taking the derivative of CE_nF with respect to CE_relv, rewritten in terms of joint damping and scaled with the maximal CE force, CEB is expressed as:CEB) on the basis of a Hill-type muscle was about 18 Nms/rad. The eccentric force-velocity slope at CEv = 0 is estimated to be about twice as high as that of the concentric part CEB of 36 Nms/rad.Estimated maximal concentric (lumped muscles) elbow joint damping  and hence SE stiffness is much greater that CE stiffness. At a relative CE length of .8 times optimum length, CE stiffness of a lumped elbow muscle maximal stimulation is about 80 Nm/rad. This value increases in reality due to LDCS and short range stiffness and hence maximal CE stiffness was set to an upper limit of 100 Nm/rad (it can be even higher at shorter CE lengths). At this CE length and at maximal activation, SE stiffness is about 10 times higher. At the same CE length but with an activation yielding a CEK of about 30 Nm/rad, SEK is about 4 times that of the CE. At a CE stiffness of 10 Nm/rad, SEK equals that of the CE. This all taken into account, we used the following parameter set:For graphical purposes and based on the rationale that MSMI = 0.10 kgm2 (the inertia of the forearm relative to the elbow joint)CEK = 10−200 Nm/radSEK = 2, 5 and 10×CEKCEB = 2−32 Nms/radMSMϕ ) using values of MSMp. For each of the combinations of MSMp, optimal values for KBIp* = [K*B*I* ] were identified that minimized the difference between MSMϕ and response of the KBI-model (KBIϕ). Optimal values were identified for three different time intervals over which the responses were fitted . These intervals were based on the range reported in the literature  of the MSM  and for high-frequency  perturbations. The low-frequency approximation that results from Taylor expansion of the impedance of the MSM (s = 0) is:To gain insight in the relation between parameters of the MSM and those of the optimized KBI-model, analytical approximations of the impedance of the MSM were derived for low-frequency  was based on the rationale that at high frequency perturbations, CE force is dominated by CE damping and as such CE stiffness was removed from the impedance of the MSM:Not surprisingly, at high perturbation frequencies inertia dominates the dynamic behavior. At CEsB will be large with respect to SEK. Ignoring SEK from Eq. 10 yields:Second, as high frequency conditions are characterized by large magnitudes of the Laplace variable s, the term This approximation is identical to the formal expansion for high frequency perturbations with an additional damping term and it was found that this approximation markedly improved the description of the impedance at high frequency conditions. Below, these analytical expressions will be used to analyze the relationship between the parameters of the MSM and KBI-model under low and high frequency conditions.E. In this sensitivity analysis, we calculated the maximal change in parameter values of the KBI-model that led to an arbitrarily small ΔE, and hence assessed the sensitivity of parameters that can be expected on the basis of measurement errors.In an experiment, inherent errors occur in both the applied perturbation and the measured response. To directly explore the sensitivity of estimated stiffness and damping to such errors, a sensitivity analysis was carried out. Any given difference between actual and measured joint angle time histories can be expressed by a corresponding value of the error criterion KBIp* at the minimum of the error function (*E), the first derivative matrix (or Jacobian) of E to the optimal parameter set KBIp* is zero by definition. Therefore, the second derivative  of E with respect to KBIp determines the change of E as a function of small changes in KBIp. Defining small changes from KBIp, ΔKBIp = [ΔK ΔB ΔI], ΔE can be approximated by a second-order Taylor expansion of E:  >For a given optimally estimated KBIp is calculated for which E changes the least. Thus, in other words, this ΔKBIp indicates the combination of parameter values that can be changed the most before the difference between the response of the MSM and KBI reaches an error ΔE  corresponding to the smallest eigenvalue (λ) of the H:Using this Hessian, the Δr ΔE see . Since Hn indicates the norm of ΔKBIp. Because v is an eigenvector of H we have Hv = λv, and therefore:E is proportional to the smallest eigenvalue of H and to the squared norm of ΔKBIp. Using Eq. 11, ΔE can be expressed in terms of ΔKBIp:So the error increase ΔE is given by:The largest simultaneous change in parameters as a function of Δv depends on all parameters of the MSM and thus was calculated for every MSMp.The sensitivity of stiffness and damping to a difference in measured/optimized response can be readily obtained using Equation 15. Note that KBIp*. The Hessians for every MSMp was estimated by computing a finite-difference approximation.All calculations were performed in Matlab (R14). A Nelder–Mead simplex search method Supporting Information S1(DOCX)Click here for additional data file.