Pollen tubes require the activity of carbohydrate uptake transporters to sustain their high growth rate. The work of Rottmann and colleagues in this issue (pages 2387–2399) provides evidence that expression of a hexose transporter in pollen tubes may be regulated via the hexokinase signaling pathway.Pollen, the male gametophyte (haploid generation) of flowering plants, is responsible for delivering sperm cells to the egg sac, a process essential for sexual reproduction. Interaction between pollen and stigma (pollination) initiates secretion in the stigma and pollen germination. The pollen tube emerges and grows through the style to the ovaries where it delivers the sperm cells to the ovule to accomplish fertilization (Box 1) .Pollen tubes have been studied intensively as a model system for tip growth, a process vital in diverse cell types from fungal hyphae to cancer cells . Basic r–1 in Arabidopsis; in vitro in relatively simple media, which made the first pollen transcriptome studies possible. More recent work using a semi-in vivo method where pollen is applied to a stigma, after which the style is cut – allowing the pollen tubes to grow out onto the medium – showed that certain genes are specifically induced after growth through the pistil tissue (in vitro and causes segregation distortion in vivo (Pollen tubes can sustain very rapid growth rates (5.3 µm minSTP2, STP4, STP6, STP9 and STP11 are expressed at different stages of pollen development. STP4, STP10 and STP11 are expressed in pollen tubes and may be necessary for the uptake of hexoses from the surrounding tissue. Even though all evidence points to a role for STPs in pollen, no pollen-related loss-of-function phenotype has been described for any of the STPs.However, pollen tubes also express a cell wall-localized invertase  and are STP10 in yeast was used to show that it encodes a hexose transporter with high affinity for glucose. STP10 also transports galactose and mannose, but not fructose. While no impairment of pollen function was found in an stp10 loss-of-function mutant, possibly due to redundancy, Rottmann et al. provide intriguing insight into the regulation of STP expression in pollen. STP10 is highly induced during pollen germination, both in vitro and even more in semi-in vivo pollen tubes (in vitro germination of pollen when 50mM glucose was added to the medium; fructose and mannose did not have this effect, nor did adding 50mM glucose and 50mM fructose at the same time. Expression of STP4, another pollen tube-expressed hexose transporter, was affected in the same way. The downregulation of expression of STP10 and STP4 no longer occurred in two independent hexokinase 1 mutants. Hexokinase 1 has a dual role, both as a glucose sensor and as an enzyme that converts glucose to glucose-6-phosphate (et al. suggest that the glucose signal that leads to the decrease in STP10 and STP4 expression may be transmitted via the hexokinase pathway.The paper by hosphate . The finSTP10. In a triple myb97 myb101 myb120 mutant, the pollen tubes fail to burst upon reaching the synergid and therefore do not deliver the sperm to the ovule. Interestingly, in this mutant a number of genes are no longer induced during pollen germination, among them STP10 (STP10 may therefore be a direct or indirect target of MYB regulation. The three MYBs themselves are induced during growth through the pistil tissue.More information is available concerning transcriptional control of STP10 seem to be involved in providing specificity to the expression of STP10. A promoter–GUS fusion showed expression in many more tissues than a whole-gene–GUS fusion, indicating that a transcriptional repressor may be present in intron, exon or UTR sequences.An additional interesting finding by Future work will undoubtedly have to address the issue of redundancy, and the creation and analysis of double or higher-order multiple mutants in several hexose/sugar transporter genes. We also need to know what sugars are available in the transmitting tract, especially in Arabidopsis. Such data would provide a realistic picture of the conditions encountered by growing pollen tubes and help us understand how they get the nutrition necessary for rapid growth.


When GlglpR gene was deleted, and the rates of appearance of Glp+ mutations in the crp glpR double mutant background were measured in the absence and presence of glycerol. The numbers of Glp+ cells arising was 10-fold higher in the crp glpR double mutant than in the crp mutant when glycerol was absent. In the presence of glycerol, the loss of GlpR was without effect. Thus, deletion of glpR is equivalent to the inclusion of excess glycerol in the growth medium. High level overexpression of glpR decreased mutation rate to background levels  and the far downstream site (O4) so they no longer could bind GlpR, and compared the effects on glpFK expression using a lacZ reporter gene fusion construct vs. mutation rate to Glp+ during growth in LB medium. Mutation of O4 increased glpFK operon expression about 5-fold although mutation of O1 was almost without effect , was deleted, and as expected, IS5-mediated glpFK activation was observed at high frequency. When sub-mM concentrations of cAMP were added to the growth medium, the frequency of these mutations was drastically reduced; 1 mM exogenous cAMP essentially prevented the appearance of these mutants. When glpR was deleted in the cya genetic background, the frequencies of IS5 insertion increased by about 20-fold .We consequently undertook a systematic analysis of the cAMP dependency of IS5-mediated wt E. coli cells. Glycerol utilization in these cells is strongly inhibited by the presence of the non-metabolizable glucose analogs, 2-deoxyglucose and α-methyl glucoside, which also lower cytoplasmic cAMP levels by inhibiting adenylate cyclase activity  encoding the flagellar transcriptional master switch in E. coli, FlhDC. IS5 insertion into the upstream control region of the flhDC operon was responsible. Although the mechanism was not established, the frequency of IS5 insertion clearly increased under swarming conditions in soft agar compared to growth in liquid medium or on solid agar plates where swarming does not occur  operon may also be subject to IS5-mediated directed mutation  in front of the glpFK operon (Figure glpFK operon expression and operon activation by IS5. Our results established that binding of GlpR to O4, which overlaps the −10 promoter region, primarily controls gene expression, while binding to O1 primarily controls IS5 hopping into the specific CTAA site, between 127 and 122 base pairs upstream of the glpFK transcriptional start site, that activates the glpFK promoter. Binding of the cAMP-CRP complex to its two binding sites overlapping O2 and O3 also blocks IS5 hopping to the activating site. This dual mechanism may involve changes in DNA conformation or supercoiling. The results serve to dissociate two functions of both GlpR and CRP.Directed mutation has been defined as genetic change that is specifically induced by the stress conditions that the mutation relieves, but until recently, in no case had such a mechanism been established. We have demonstrated that mutations in the n Figure  and is dn Figure , not onlglpFK promoter activation in wild type cells provides relief from starvation when glycerol is present and a cAMP-depressing toxic substance, such as 2-deoxyglucose, is simultaneously present. Such non-metabolizable sugar derivatives are synthesized by many organisms and therefore are present in nature. This example of directed mutation could therefore have been selected for during evolution. It appears to be a genuine example of directed mutation, with mutations arising at a greater rate under conditions that allow benefit to the organism. The fact that mutation rate is influenced by the presence of glycerol in a process mediated by the glycerol repressor, and by cAMP in a process mediated by CRP, provides mechanistic explanations for IS5-mediated directed mutational control. This mechanism, illustrated in Figure glpFK operon. Our studies also provide the rationale for the evolution of this elaborate mechanism of gene activation.The mechanism of IS5-mediated The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To compare the effectiveness of bevacizumab and ranibizumab in the treatment of exudative age-related macular degeneration (AMD).Multicentre, randomized, controlled, double-masked clinical trial in 327 patients. The non-inferiority margin was 4 letters.Patients ≥ 60 years of age with primary or recurrent sub- or juxtafoveal choroidal neovascularization (CNV) secondary to AMD with a total area of CNV < 12 disc areas and a best corrected visual acuity (BCVA) score between 20 and 78 letters on an EDTRS like chart in the study eye.Monthly intravitreal injections with 1.25 mg bevacizumab or 0.5 mg ranibizumab were given during one year. Intention to treat with last observation carried forward analysis was performed.Primary outcome was the change in BCVA in the study eye from baseline to 12 months.The mean gain in BCVA was 5.1 (±14.1) letters in the bevacizumab group (n = 161) and 6.4 (±12.2) letters in the ranibizumab group (n = 166) (p = 0.37). The lower limit of the 95% confidence interval of the difference in BCVA gain was 3.72. The response to bevacizumab was more varied; 24% of patients showed a gain of ≥15 letters, 11% a loss of ≥15 letters and 65% a gain or loss < 15 letters compared to 19%, 5% and 76% respectively for ranibizumab (p = 0.038). No significant differences in absolute CRT and CRT change (p = 0.13) or in the presence of subretinal or intraretinal fluid  were observed. However, the presence of any fluid on SD-OCT  differed significantly (p = 0.020), with definite fluid on SD-OCT in 45% of the patients for bevacizumab versus 31% for ranibizumab. The occurrence of serious adverse events and adverse events was similar, with 34 SAEs and 256 AEs in the bevacizumab group and 37 SAEs and 299 AEs in the ranibizumab group .Bevacizumab was not inferior to ranibizumab. The response to bevacizumab was more varied with higher percentages of both gainers and losers and more frequently observed retinal fluid on SD-OCT at 12 months when compared to the ranibizumab group.NTR1704Trialregister.nl  Exudative age-related macular degeneration (exudative AMD) is the main cause of untreatable blindness in western countries and a major burden for the elderly population ,2. Stand®,Genentech/Hoffmann-La Roche), ranibizumab  and aflibercept . The active part of the molecule is similar in bevacizumab and ranibizumab. However, bevacizumab is the whole anti-VEGF antibody (150 kD), while ranibizumab is an antibody fragment. Bevacizumab has a longer half-life in the systemic circulation than ranibizumab while ranibizumab is believed to penetrate the retina better and has higher affinity to VEGF-A than bevacizumab. These differences could have an impact on safety and efficacy of these drugs )Bevacizumab and other anti-VEGF agents have serious side effects when used systemically for non-ocular indications, in particular in patients with an increased risk of stroke or other arterial thrombo-embolic events.  BevacizuThe follow-up time of patients in the BRAMD study is restricted to 12 months. We recognize that patients with AMD are often treated for years. It is highly conceivable however that results achieved during the first year of monthly treatment are predictive of future results. One year of intensive treatment should be sufficient to assess whether a patient will or will not continue to respond. Further, one year of treatment seemed sufficient to our public national funding agency as well to assess the non-inferiority of bevacizumab against ranibizumab.In the IVAN trial, increased atrophy was observed in patients treated monthly with ranibizumab for 2 years, ,15 The IThe number of patients and the length of follow-up are not adequate to provide a full safety report on bevacizumab versus ranibizumab. Now that our study confirms the non-inferiority by visual acuity of bevacizumab against ranibizumab and market access of bevacizumab and ranibizumab in the Netherlands have been redefined as a result of it, we need further post-marketing surveillance to keep track of the safety in the long run.We further recognize that we evaluated the efficacy of both drugs in a typically clinical trial setting, i.e. monthly injections while in every day practice most patients are treated on an as needed basis and we explicitly withhold ourselves from making statements about such treatment schedules.What might be considered an inconsistency in our protocol is the definition of treatment failure on the one hand and the indication for becoming a switcher on the other hand. Patients are considered failures or losers to therapy once they lose 15 letters or more over the year. However, already when they were still ‘stabilizers’ with a loss of between 10 to 15 letters exclusive, a cross-over to the alternative drug was indicated. We applied an ad-hoc analysis to show the percentages of patients losing or gaining more than 10 rather than 15 letters with the latter reported in the results section of this paper. There were 57 patients gaining 10 letters or more in the bevacizumab group (35%) versus 61 (37%) in the ranibizumab group. For the losers this was 20 (12%) and 13 (8%) respectively (p = 0.41). This is consistent with the results above showing more losers in the bevacizumab group.Three different SD-OCT systems were used. This could lead to some differences. In the MANTA conversion algorithms were used to deal with this problem. However we believe this is unnecessary as we analyzed the absolute differences between baseline and exit measurements, and as de Kinkelder et al. found a maximum difference in mean thickness between brands to be 7.7 micron  which is an insignificant difference .Finally, we applied the last observation carried forward at the time of switching to the alternative drug. usually, this is not done in a intention-to-treat analysis, because the gain in vision after the switch is not accounted for. However, we did not want to run the risk of demonstrating non-inferiority of bevacizumab to ranibizumab because of patients switching therapies.The BRAMD study confirms that bevacizumab is non-inferior to ranibizumab in the treatment of exudative AMD. Our study did not result in new safety signals, but definitive results should come from safety data pooled over studies. Being much cheaper it is reasonable to suggest bevacizumab as the first choice of treatment in exudative AMD. However, in our study more patients obtained suboptimal results (more than 15 letters loss) in the bevacizumab than in the ranibizumab group. Therefore, for patients with an initial poor response to bevacizumab, ranibizumab may be useful as a second line treatment option.S1 File(DOC)Click here for additional data file.S2 FileThis provides an overview of the entire study group. It also lists what their specific role was within the study.(DOC)Click here for additional data file.S3 File(DOC)Click here for additional data file.


Knowing when it is convenient to take a turn in a conversation is an important task for dialog partners. As it appears that this decision is made before the transition point has been reached, it seems to involve anticipation. There are a variety of studies in the literature that provide possible explanations for turn-end anticipation. This study particularly focuses on how turn-end anticipation relies on syntactic and/or semantic information during utterance processing, as tested with syntactically and semantically violated sentences. With a combination reaction time and EEG experiment, we used the onset latencies of the readiness potential (RP) to uncover possible differences in response preparation. Although the mean anticipation timing accuracy (ATA) values of the behavioral test were all within a similar time range , we found evidence that response preparation is indeed different for syntactically and semantically violated sentences in comparison with control sentences. Our preconscious EEG data, in the form of RP results, indicated a response preparation onset to sentence end interval of 1452 ms in normal sentences, 937 ms in sentences with syntactic violations and 944 ms in sentences with semantic violations. Compared with control sentences, these intervals resulted in a significant RP interruption for both sentence types and indicate an interruption of preconscious response preparation. However, the behavioral response to sentence types occurred at comparable time points. Figure 1 shows an example of back-channels and turn-taking in a natural dialog.Human communication usually occurs with a bi-directional information exchange, which can be performed coevally or alternately. An example of simultaneous communication would be “mimic signals”  in which communication partners are simultaneously smiling. In the visual modality, a smile can be answered with a smile while the communication partner is still smiling, i.e., the smiles can overlap without interfering with each other. However, in a more complex information exchange such as spoken language, a sequential organization of the signals is required. In contrast to non-verbal signals, it is socially restrictive not to have two interlocutors speaking at the same time because speaking and listening simultaneously is not easily possible. Indeed, in a conversation, we need to process and understand a perceived utterance before we can react by replying. Thus, in conversations between two or more interlocutors, the organization of taking turns is fundamental such that predominately only one partner speaks at a time. Turn-taking may depend on situational factors, such as the relevance and pragmatic necessity of a possible utterance, how confident the interlocutors are about the reliability of the information, and social status of the interlocutors. However, Turn-taking appears to be robust across different languages . InfantsTo test the possible influences on turn-end anticipation, Contrary to anticipation in turn-taking, a fast reaction of the listener can offer an alternative explanation for turn-taking. Turn-end anticipation is usually measured by behavioral responses, for instance, anticipation timing accuracy (ATA), which reflects conscious behavioral processes. The ATA is the accuracy of the interval from the sentence-offset to response. However, the possible similar ATA results between conditions can nevertheless fail to reflect the operation of different anticipatory performances, as compensated for by short response-preparation in late anticipation and longer response preparation in early anticipation. The current EEG experiment was conducted to test the usability of readiness potential (RP) as a neurocognitive preconscious measure during turn-end anticipation and to test the influence of syntactic and semantic violations on turn-end anticipation performance based on the RP as a preconscious measure. To investigate the RPs, our experiment used acoustically presented normal utterances (controls) and utterances containing a syntactic or a semantic violation. The forms of violations were selected as possible key factors for successful language comprehension.Even though independent sentences without context as a global semantic component might appear to be too unlike natural turn-end anticipation, To investigate turn-anticipation rather than turn-taking, we used button presses as responses to eliminate confounding variables of verbal responses, e.g., the breathing in before verbalization, the preceding higher cognitive processes for verbal response preparation and the motor complexity of verbalization. Based on previous research, two questions arise: (1) Is it possible to find response-related electrophysiological correlates providing strong evidence for early turn-end anticipation, and is the RP onset a suitable measurement that can be used to find differences in turn-end anticipation? (2) If turn-end anticipation is different for violated sentences, would syntactic incongruence, semantic incongruence or both affect turn-end anticipation?A total of 30 students (17 women and 13 men) between 19 and 35 years of age (Ø = 24.5 ± 3.5 years) were recruited from Bielefeld University. All subjects were native German speakers and right-handed with a lateralization quotient of 88.9 according to the Edinburgh Handedness Inventory . AccordiTable 1), number of words (Ø = 14.2 ± 2.4 SD), number of syllables (Ø = 22.5 ± 3.1 SD), and syntactic complexity and affectivity) along 115 non-matched filler sentences. The filler sentences consisted of a variety of syntactic structures, whereas critical sentences consisted only of a slightly deviating syntactic structure. All sentences in the experiment were spoken at a speed of 400–450 syllables per minute by a professional female speaker with natural intonation and recorded in a sound studio. The 69 critical items consisted of 23 control sentences, 23 sentences with a syntactic violation, and 23 sentences with a semantic violation. All critical sentences were pseudo-randomized among the 115 filler sentences. For this within-subject design, three groups were created in a Latin square design to ensure that the participants would not hear the same sentence from different conditions. Therefore, the participants heard at least seven sentences from each condition along with all filler sentences. The overall sentence length of all conditions varied from 3287 to 5803 ms, whereas the filler sentences varied from 1097 to 6643 ms (see Table 1). The time between the end of a critical word and the sentence end varied between 863 and 2123 ms (see Table 2). Examples from the stimuli can be seen below. A # indicates the moment at which the violations end.The stimuli in this experiment consisted of 184 sentences, of which 69 critical sentences were matched between the conditions with psycholinguistic criteria  so that the participants could not anticipate the sentence onset time after several trials. The fixation cross was continuously shown until at least 1000 ms after the sentence ended. The mean stimuli intensity ranged between 55 and 60 dB, which matches a normal face-to-face conversation. During the EEG recording, the participants listened to the sentences, and they were instructed with the following wording: “Try to push the button exactly when the sentence ends. Do not wait until the sentence has ended definitely but rather push the button at the time point you would expect its end.” After a short practice block with 20 non-critical sentences, all participants felt comfortable with the anticipation task, which was introduced in The EEG recording took place in a sound-proofed and electromagnetically shielded booth. The EEG was continuously recorded from 32 active scalp electrodes  placed at locations based on the International 10/20 system with reference at FCz. Horizontal eye movements were recorded from the left and right outer canthi (hEOG), and vertical eye movements were recorded from above and below the right eye (vEOG). The signals were sampled at 1000 Hz, amplified with a bandpass of 0.16–80 Hz and a 50 Hz notch filter by amplifiers  and recorded with BrainVision Recorder software . The impedance was kept below 5 kΩ for all channels prior to recording. A button press was recorded by a USB button-box with an internal clock.Anticipation timing accuracy data analyses were conducted using EEGLAB  under MAF-value corrected according to the formula provided by EEGLAB  under MATable 3 presents the descriptive statistics of the ATAs for the three conditions. On average, subjects were slowest when responding to sentences with semantic violations (115.6 ms) and fastest when responding to sentences with syntactic violations (92.6 ms). The control condition was in the middle, with an average response time of 108 ms. Figure 2 shows a histogram of the ATA of the three conditions to illustrate the distributions. A t-test did not reveal significant influences of the conditions on the ATA (see Table 4).Across subjects, 96.8% of the ATAs were within two standard deviations from the mean and were taken into account for statistical calculations and analyzed separately for the three conditions. Figure 3 shows that the RP in the control condition was, as expected, solid at C3, and further investigation was performed with this electrode. The RP was computed from grand average ERPs at C3 for each condition. RPs displayed a slow negative-going waveform, peaking at approximately 100 ms before response execution. Figure 4 shows the grand average RP of the three conditions at the electrode position C3.After artifact rejection, 157 epochs from the control, 158 from the syntax and 154 from the semantic condition from 25 participants formed the grand averages. Fcorrected = 9.766, p < 0.01]. Further post hoc comparisons revealed a significant RP interruption of 530 ms in the syntactic condition  compared with the control condition  and in a significant RP interruption of 500 ms in the semantic condition  compared with the control condition. The 30 ms difference between the two violation conditions was not significant. To calculate the RP onset to sentence-end interval, the ATA was subtracted from the RP onset to button-press interval. This resulted in an RP onset to sentence-end interval of 1452 ms for the control condition, 937 ms for the syntactic condition and 944 ms for the semantic condition. In addition, the condition had a significant main effect on this interval . Post hoc comparisons revealed a significant difference of 515 ms between the control condition  and syntactic condition  and a significant difference of 508 ms between the control condition and semantic condition . The difference of 7 ms between the syntactic and semantic condition was not significant. Figure 5 illustrates how the RPs provided us with a better view of the temporal processes for turn-end anticipation and response preparation, compared with behavioral ATA measurements.The RP onset started at 1560 ms before button-press in the control condition, at 1030 ms in the syntactic condition and at 1060 ms in the semantic condition. There was a significant main effect of condition on the RP onset to button-press interval [Table 4) indicate no significant influence of syntactic or semantic sentence violations on turn-end anticipation. However, similar ATAs between conditions fail to reflect the operation of different anticipatory performances, as compensated by short motor-preparation in late anticipation and longer motor-preparation in early anticipation. Although our EEG signatures display clear RP characteristics with a slow negative-going waveform with a peak at approximately 100 ms before response execution, it is possible that they contain some contingent negative variation (CNV) components. Nevertheless, this does not harm the interpretation of our observations. In our response-locked paradigm, both the RP onset and CNV after a warning are considered to be activations caused by preparation. In RPs, the latency of the onset as well as its peak can both vary differentially. Our behavioral ATA results  can be used as a preconscious measure for the time course of this anticipation. In turn anticipation for intact utterances (control), a response-related RP onset of 1452 ms before the turn end provides strong evidence for an early turn-end anticipation rather than reaction to a completed utterance. The minimal syntactic or semantic violations of sentences used in this experiment caused delayed RP onsets but similar behavioral responses. This suggests different response preparations, which are not reflected in the behavioral ATA. The possible different influence of syntactic or semantic information on the anticipation performance suggests that both syntactic and semantic incongruence likewise affect turn-end anticipation processes.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila neuro-muscular junctions, we show that Syntaxin-1A clusters are more abundant and have an increased size at active zones. A computational particle-based model of syntaxin cluster formation and dynamics is developed. The model is parametrized to reproduce Syntaxin cluster-size distributions found by STED analysis, and successfully reproduces existing FRAP results. The model shows that the neuronal membrane is adjusted in a way to strike a balance between having most syntaxins stored in large clusters, while still keeping a mobile fraction of syntaxins free or in small clusters that can efficiently search the membrane or be traded between clusters. This balance is subtle and can be shifted toward almost no clustering and almost complete clustering by modifying the syntaxin interaction energy on the order of only 1 kBT. This capability appears to be exploited at active zones. The larger active-zone syntaxin clusters are more stable and provide regions of high docking and fusion capability, whereas the smaller clusters outside may serve as flexible reserve pool or sites of spontaneous ectopic release.Synaptic vesicle fusion is mediated by SNARE proteins forming in between synaptic vesicle (v-SNARE) and plasma membrane (t-SNARE), one of which is Syntaxin-1A. Although exocytosis mainly occurs at active zones, Syntaxin-1A appears to cover the entire neuronal membrane. By using STED super-resolution light microscopy and image analysis of  For the communication between two nerve cells, a synaptic vesicle containing neurotransmitters has to fuse with the neuronal membrane at a specific fusion site, releasing its signaling molecules. The vesicle fusion is mediated by a specific family of proteins (SNAREs) that are located on the vesicle as well as on the neuronal membrane. As many other membrane proteins, SNARE proteins are not uniformly distributed over the membrane but rather exist in complexes or clusters. Syntaxin, one of the SNARE proteins, is known to form such clusters through attractive protein-protein interactions. With the help of light microscopy techniques we show that the actual size and abundance of these clusters depends on its proximity to the fusion site on the membrane. We developed a computational model of Syntaxin cluster formation that can explain the observed differences in clustering and allow us to speculate on their potential role in the process of docking and fusion of synaptic vesicles. The formation of clusters through weak protein-protein interactions allow for a highly dynamic behavior of proteins, being able to easily switch between a state with stable and almost immobile clusters and a more dynamic situation with clusters exchanging particles at high rates. Synaptic exocytosis occurs efficiently at active zones, where neurotransmitter containing synaptic vesicles (SV) are docked, primed, and released . SynaptiAlthough SNAREs catalyze exocytosis, which predominantly occurs at active zones, SNARE clusters are found all over the neuronal membrane. Previous studies have not provided evidence that SNARE proteins or microdomains would be preferentially located at active zones –29, as cDrosophila neuromuscular junctions (NMJs). Diffusion simulations [Here we use Stimulated Emission Depletion (STED) super-resolution light microscopy  to invesulations  were useFor a polyclonal Bruchpilot-C-terminal antibody, the coding sequence corresponding to the last 200 amino acids of the 190 kDa isoform  was C-term  1:1000 and mouse monoclonal anti-Syntaxin (8C3) 1:50 . Secondary antibodies for STED were used in the following concentrations: goat anti-mouse Atto590 1:100 and goat anti-rabbit star635 1:100 (Atto590 (ATTO-TEC) and star635 (Abberior) coupled to respective IgGs (Dianova)). Images from fixed samples were taken from 3rd instar larval NMJs.Dissections and immunostainings were essentially performed as described previously . But briSTED microscopy was performed as previously described in ,37. BrieImage analysis was programmed in Matlab and performed on the deconvolved data. Although the deconvolution algorithm affects the apparent cluster size, the relative size is maintained. To identify clusters of Syntaxin-1A and spots of Bruchpilot, the local maxima in the intensity profile of the respective channel of the STED images are sought. For this, a circular window of fixed diameter  is shifted over all pixels of an image (1500x1500 pixels) and it is tested whether the current pixel is the maximum value within the window. Local maximum pixels with intensities >25 a.u. are considered to be cluster centers. Thus, clusters can be distinguished when they are more than 40nm apart (for Syntaxin) and 100 nm apart (for Bruchpilot).To determine the size of the Syntaxin-1A clusters, the full-width-half-maxima area is defined as the cluster area. Starting from the cluster center, equidistant pixels are scanned until a distance is reached at which the average pixel intensity drops below half the intensity of the cluster center. All scanned pixels with an intensity above or equal to the half-maximum are considered to belong to the cluster area. The diameter of the cluster is defined as the diameter of a circular area of the same size as the cluster area. Binding of the secondary to the primary antibody may increase the distance of the fluorophore from the used epitope and therefore lead to an overestimation of the calculated absolute cluster size. However, the comparison of cluster sizes is still valid.An active zone is defined as the region surrounding a ring of Bruchpilot spots, with a 25 nm padding inside and outside of the ring structure, resulting in a closed circular area with a diameter of around 250 to 300 nm  is chosen to approximate the size of the N-terminal domain, the first particle is somewhat smaller (r1 = nm3.0 ). The ratio r2/r1 of the two sizes (here: 1.1) determines the strength of repulsion between two Syntaxin-1A molecules due to steric hindrance and thus strongly influences the average cluster size and more importantly the maximal cluster size. Ratios smaller or equal to 1.0 lead to unbounded cluster growth, while larger ratios tend to decrease cluster sizes dramatically. In our case, a ratio of 1.1 sets a practical cluster size limit at 120–140 nm as observed in STED experiments, while ratios of 1.25 and 1.5 reduce this limit to 80–90 nm and 40–45 nm, respectively The diffusion coefficient of the particles is set such that the dimer model has an effective diffusion coefficient of Dsyx = 0.2 μm2s-1, as found in single molecule experiments [Here for Syntaxin-1A a minimalistic model is chosen that permits to model the spatiotemporal behavior relevant to this study while allowing for efficient generation of long time series and thus gain statistically significant data. Each Syntaxin-1A molecule is modeled as a dimer of two connected particles. Particle 1 represents the syntaxin transmembrane helix and SNARE domain of the protein: it acts as membrane anchor and has an attractive interaction potential with other particles of the same type, thus giving rise to clustering. Particle 2 represents the N-terminal domain of syntaxin-1A and serves only to introduce the steric repulsion due to the bulky head domain. The radius of the second particle  and its strength is defined by the force constant kr which is chosen to be equal to Ea. The attractive part of the potential is active between rx,y and the interaction radius ix,y The parameter Ea defines the well depth of the attraction potential. Since the actual interaction energy is not known and may vary for different lipid compositions and protein surroundings, Ea is varied in order to find a value which can reproduce the cluster size distributions found in experiments.Here, proteins  but at tEa = K{2.8,3.2,3.6,3.8,4.0,4.2,4.4,4.8}BT we have performed twelve simulations, each consisting of 50 · 106 steps with a time step dt of 5 ns. Convergence of trajectories was tested by validating that no significant changes in the mean cluster size and cluster size distributions were observed. We then selected four values of Ea. Two values, Ea,outside = k4BT and Ea,AZ = k4.4 BT, approximately reproduce the experimental cluster size distributions found outside and at active zones, respectively. Two additional values, Ea,low = k3.6 BT and Ea,high = k4.8 BT, were chosen that exhibit more extreme cluster size distributions. For these setups we performed further simulations simply continuing from the endpoints of the convergence simulations, to analyze the equilibrium dynamics of syntaxin particles under different degrees of clustering. To study the aggregation time of syntaxin clusters at newly formed target sites such as synaptic vesicles approaching the presynaptic membrane, simulations were performed that also started from the converged configurations but with a static and highly attractive Ea = k8BT syntaxin inserted to an empty membrane site.For each tested value of rx,y + nm1  apart. In rare cases, this may lead to an overestimation of cluster sizes, since two close clusters might be joined to one larger cluster. However, we do not consider this to be a significant error. Firstly, because we average over many simulation steps and two distinct clusters will be clearly separated most of the time. Secondly, similar situation can arise for experimental data due to limited resolution making the simulated and experimental cluster size distributions comparable.Many analyses in this paper depend on the size of the syntaxin clusters. Here we define a cluster as a set of particles that are in proximity of at least one other particle in the set. Two particles are in proximity when they are not further than 1 nm apart from each other, i.e. when their centers are not further than Chemical synapses release synaptic vesicles at specialized membranes (“active zones”), whose cytoplasmic faces are covered by electron dense structures reflecting the presence of extended molecular scaffolds. These cytoplasmic active zone scaffolds are meant to facilitate synaptic vesicle release by however so far largely unknown mechanisms. Importantly, scaffold sizes of individual active zones were found to scale with their microscopic release rates –45.Drosophila neuromuscular terminals of fixed larvae. In this preparation, active zones are found evenly spaced on the surface of nearly spherical boutons, easing analysis of active zone associated regulations. Active zone scaffolds are identified via labeling the ELKS/CAST family protein Bruchpilot (BRP). BRP, a large coiled-coil domain protein serves as a direct building block of the active zone cytomatrix [We here analyzed the Syntaxin-1A cluster distribution at glutamatergic tomatrix ,34,46,47In order to visualize BRP at the AZ, we previously used the monoclonal antibody NC82, which recognizes a small epitope at the very C-terminus of BRP. As by its monoclonal nature, the MAB NC82 governs only a low labeling density and thus is limited in visualizing the true extent of the active zone scaffold being present. In order overcome this issue, we produced a fusion protein antibody against the last 200 amino acids of BRP . Higher labeling density here gets obvious by denser, more extended labeling in STED images. BRP labeled with Last200 adopts a dense ring-like structure (top view) or a bar-like structure  with a diameter of around 220-240nm . This increase in fluorescence intensity can not only be explained by the increased Syntaxin-1A cluster size, but is mainly due to the fact that there are more syntaxin-1A clusters near active zones than outside. As already reported in previous studies, –29, we pEa, was changed. Ea,outside = 4kBT and Ea,AZ = 4.4 kBT for syntaxins outside or at the active zone, respectively. Note that these different parameter choices reflect an underlying molecular mechanism. A possible explanation for the increased syntaxin interaction energy at active zones is that interactions are also modulated by the phospholipid composition of the membrane which is supposed to be different at active zones [We used a simple computational model of syntaxin-1A populated membranes and performed particle-based diffusion simulations to investigate the formation, distribution and dynamics of Syntaxin-1A clusters. Syntaxin-1A was modeled as a dimer of two particles . Particlve zones —however Ea,outside and Ea,AZ and confirms that “outside” syntaxins recover quantitatively similar to the FRAP measurements of [In order to validate the dynamics of our model, we simulated a FRAP (fluorescence recovery after photobleaching) experiment. Starting from equilibrated simulation frames, syntaxins in a disc-shaped area covering 10% of the full simulation area were marked as “bleached” while all other syntaxins were marked as “fluorescent”. The recovery of the fluorescent fraction as a result of the diffusional motion of syntaxins is then followed over time. ments of  that werEa,outside and Ea,AZ), two additional interaction energies  are considered in order to investigate the system behavior at more extreme conditions.In summary, the computational model employed here is able to reproduce the Syntaxin-1A cluster distribution and fluorescence recovery kinetics observed from experimental data. Besides the two interaction energy values chosen for active zones and outside regions  compartments and the fraction of compartments that are visited by at least one syntaxin molecule was tracked. The difference in membrane coverage between different degrees of clustering is considerable. The same area of membrane is covered up to three times faster outside of active zones than within an active zone see , e.g. 50Ea,outside, even parts of the membrane without clusters are quite homogeneously covered, and the concentration of syntaxins in clusters is less pronounced. On the other hand, for interaction energies higher than Ea,AZ, almost all syntaxins are bound to the large clusters and only a very small syntaxin population is mobile. A similar trend is apparent from the step length of particles. For low interaction energies, particle movement is similar all over the membrane, while stronger interaction energies almost abolish the movement within clusters and reduce the particle movement outside of clusters.The existing syntaxin interaction energies are right at the balance between total aggregation and free diffusion as illustrated by the density plots of syntaxin-1A particles in Thus, physiological Syntaxin-1A interaction energies seem adjusted in a way to rapidly explore the membrane and find synaptic vesicles. However, if a synaptic vesicle has been docked by a single syntaxin, will this event be able to recruit a cluster in a short time? To study this question, we simulated such a situation by introducing an attractive particle at a random position in the simulation area which is not yet occupied by syntaxin-1A. In We then turned to the physiological role of syntaxin-1A at active zones and investigated the effect of clustering on docking, priming and fusion of SVs. Docking is thought to be the initial contact formation between a synaptic vesicle and the plasma membrane, and believed to involve an interaction between syntaxin-1A/SNAP-25 with the vesicular protein synaptotagmin-1 . Thus, aIn order to quantify our simulations for vesicle docking and priming, we divided the simulation area into a 20 × 20 lattice of compartments having areas of 30 × 30nm each, roughly corresponding to the area that would be accessible to vesicles close to the membrane surface. The number of docking- and priming-competent syntaxins within each such cell was counted, and the results reported in The function of proteins, in particular membrane proteins, strongly depends on their localization and distribution in the cell and its recruitment to specific sites –59.Protein microdomains such as clusters with sizes in the range of 10 to 100 nm are smaller than the diffraction limit and can thus not be characterized by conventional light microscopy. With the advent of super-resolution microscopy techniques, more detailed investigation of protein distributions along the membrane are now possible –63. The Here we used super resolution light microscopy (STED) and image analysis to investigate the relation between Syntaxin-1A clustering and active zones. While Syntaxin-1A clusters are found all over the neuronal membrane, Syntaxin-1A clusters at active zones are significantly more abundant and on average larger in comparison to clusters outside of active zones.Protein clusters on the membrane can arise from different mechanisms. It is known that Syntxin-1As are subject to weak homophilic interactions between their SNARE domains ,6. It isEa. Here we modeled the different size distributions of syntaxin clusters inside and outside active zones by different Ea values. The molecular mechanism of this difference is still elusive. Clusters may be induced by differences in lipid composition such as lipid rafts [The actual Syntaxin-1A size distribution in our model is controlled by the magnitude of the syntaxin interaction energy id rafts ,67 or Piid rafts ,68. Thusid rafts ,15.Protein clusters may also be induced by membrane proteins or protein skeletons on the surface of membranes ,69,70. TA number of functions have been attributed to syntaxin clusters . Clusterin vivo at least three SNARE complexes [Docking of vesicles likely involves the contact formation of Syntaxin and vesicular Synaptotagmin  and precomplexes , such thomplexes –77. A hiomplexes .Here we quantified the number of docking and priming-competent Syntaxin-1A molecules that a vesicle close to the membrane would see. These numbers are greatly increased at clusters compared to regions outside clusters, or in a situation where no syntaxin clustering occurs at all. Both numbers are further increased at active zone, where syntaxin clusters are more frequent and larger. The differences in syntaxin cluster size and density are not the sole determinant of docking and fusion behavior, but combined with the fact that synaptic vesicles are tethered at active zone proteins ,78 this To identify the actual contribution of syntaxin clustering, one would have to compare the number of docked vesicles and fusion events to a system with lower clustering at active zones. The mutation of specific amino acids near the transmembrane region of syntaxin  could prIf syntaxin clusters are important for neuronal function, then the dissociation of syntaxin from clusters, the mobility of syntaxins and clusters on the membrane, and the replenishment of clusters are equally important during the cycle of vesicle exo-/endocytosis. Computational models integrating experimental data on syntaxin cluster localization and diffusion ,23,73 hakBT leads to stronger clustering at the expense of losing the free and mobile syntaxin fraction. Vice versa, slightly decreasing the syntaxin interaction energy results in losing large clusters. It thus appears that syntaxin at neuronal membranes is set up in a way to strike a balance between having most syntaxins aggregated in clusters while at the same time maintaining the ability for small fraction of free syntaxins to rapidly search the membrane surface. This balance can be shifted by subtle effects that slightly enhance or decrease the syntaxin interaction. At active zones, the system is pushed towards its upper limited of cluster aggregation while still keeping a small mobile fraction, and thus optimized for vesicle docking, priming and fusion. Outside active zones, the system is pushed towards the lower limit of cluster aggregation and optimized for mobility.Interestingly, increasing the syntaxin interaction energy only by a fraction of 1 We summarize our insights in the following model for the function of syntaxin clusters at active zones and outer regions. The large and stable clusters at the active zone represent potential sites for vesicle docking through its high local concentration of interaction partners of vesicular proteins. The periphery of these large clusters shows a high probability for SNARE complex formation, which also implies a higher fusion probability –77. TogeThe smaller and more dynamic syntaxin clusters outside the active zone might act as a flexible reserve pool of syntaxins for clusters at the active zone and might also allow for ectopic release by spontaneous aggregation and docking of vesicles approaching parts of the membrane outside of active zones. A stronger clustering at these outer regions would impair these functions. Furthermore, the difference in syntaxin interaction energies inside and outside active zones explains the much higher density of syntaxin at active zones. The more attractive clusters at the active zone act as syntaxin sinks that recruit syntaxin molecules from the outside into the active zone. Therefore, having a difference between the syntaxin interaction energies inside and outside active zones is important. A more detailed analysis of the impact of syntaxin clustering on exocytosis could be achieved by investigating interactions with other proteins involved in exocytosis, e.g. Unc-13, through colocalization studies or electrophysiology experiments. This would allow for a more sophisticated computational model including reactions leading to synaptic vesicle fusion. Such reaction models ,79 can bThe clustering of membrane proteins via weak protein-protein interactions is a fine tuned process as small changes in the protein-protein interaction can have a strong effect on the proteins dynamic behavior. Such a self-organized clustering not only can establish a specific spatial distribution of membrane proteins but can also provide an elegant way to yield location-specific functionality of these proteins and their clusters. The formation of protein clusters through weak protein-protein or protein-lipid interactions has been observed for the other SNAREs ,23, othe


This supplement has been sponsored by Bayer HealthCare. Please see acknowledgements for full disclaimer. Prescribing Information can be found in the appendices.L.GB.COM.05.2015.11280. Date of preparation: June 2015This paper provides expert recommendations on administration of aflibercept in wet age-related macular degeneration (AMD) after Year 1 (Y1), based on a roundtable discussion held in London, UK in November 2014. The goals of treatment after Y1 are to maintain visual and anatomical gains whilst minimising treatment burden and using resources effectively. The treatment decision should be made at the seventh injection visit (assuming the label has been followed) in Y1, and three approaches are proposed: (a) eyes with active disease on imaging/examination but with stable visual acuity (VA) at the end of Y1 should continue with fixed 8-weekly dosing; (b) eyes with inactive disease on imaging/examination and stable VA should be managed using a ‘treat and extend' (T&E) regimen. T&E involves treating and then extending the interval until the next treatment, by 2-week intervals, to a maximum of 12 weeks, provided the disease remains inactive. If there is new evidence of disease activity, treatment is administered and the interval to the next treatment shortened; and (c) if there has been no disease activity for ≥3 consecutive visits, a trial of monitoring without treatment may be appropriate, initiated at the end of Y1 or at any time during Y2. Where possible, VA testing, OCT imaging and injection should be performed at the same visit. The second eye should be monitored to detect fellow eye involvement. In bilateral disease, the re-treatment interval should be driven by the better-seeing eye or, if the VA is similar, the eye with the more active disease. Aflibercept is a fusion protein, combining the key binding domains of VEGF receptors 1 and 2 and the Fc portion of immunoglobulin G. Dosing for aflibercept in wet AMD is initiated with one 2 mg injection per month for three consecutive doses followed by one 2 mg injection every 2 months during the first year. Monitoring between injections is not required. After 1 year of treatment, the treatment interval may be extended based on visual and anatomical outcomes  in July 2013. Given the 90-day commissioning rule, all ophthalmology units in the UK would already be expected (as of November 2014) to have some patients who have completed 1 year of treatment with aflibercept. A national roundtable was therefore convened to discuss UK experience with aflibercept to date, and to use this experience, together with expert opinion, to develop recommendations on the practical application of aflibercept in wet AMD after Year 1. In particular, the discussion was based around maintaining the visual acuity (VA) gains from Year 1 and reducing treatment burden where possible. This paper will review the VEGF Trap-Eye: Investigation of Efficacy and Safety in Wet AMD (VIEW) study with aflibercept in wet AMD,10 and the Royal College of Ophthalmologists (RCO).11 It should be noted that the product label for ranibizumab has been changed since the RCO guidance was published, and therefore these guidelines are not presented below.Guidance on the management of wet AMD in the UK has been published by NICE9 The criteria for ranibizumab use  are as follows: best-corrected visual acuity (BCVA) between 6/12 and 6/96; no permanent damage to the central fovea; lesion size ≤12 disc areas in greatest linear dimension; and evidence of disease progression.10According to the NICE July 2013 technology appraisal (TA294), aflibercept is recommended as an option for treating wet AMD if it is used in accordance with the NICE recommendations for ranibizumab and offered at the discount agreed in the patient access scheme.12 The treatment interval should be extended by no more than 2 weeks at a time in wet AMD.12For ranibizumab, the label states that treatment should be given monthly until maximum VA is achieved and/or there are no signs of disease activity. Monitoring and treatment intervals can be extended thereafter.6As outlined above, aflibercept treatment (2 mg) is initiated with one injection per month for 3 months. Thereafter, treatment continues at 2-monthly injections for the remainder of Year 1 .68 VIEW 1 was conducted at 154 centres (n=1217) in the USA and Canada, while VIEW 2 was conducted at 172 centres (n=1240) in Europe (including the UK), the Middle East, Asia-Pacific and Latin America.7VIEW 1 and 2 were two phase III, randomised, double-blind, multicentre, head-to-head non-inferiority trials that compared aflibercept with ranibizumab in patients aged ≥50 years with wet AMD.7 From weeks 52–96, patients were monitored each month and were treated either reactively or proactively in a ‘capped pro re nata (PRN)' protocol. ‘Reactive' treatment was based on the presence of disease activity  letters from best previous score in conjunction with recurrent fluid on OCT; new-onset classic neovascularisation; new or persistent leakage on fluorescein angiography; or new macular haemorrhage). ‘Proactive' treatment meant treating irrespective of disease activity. In VIEW, proactive treatment occurred if 12 weeks had elapsed since the last injection. Reactive treatment has been shown to result in worse VA outcomes than continuous therapy,14 suggesting under-treatment in some patients, whilst it is generally accepted that proactive regimens can result in over-treatment in a proportion of patients.Patients were randomised 1:1:1:1 to aflibercept 2 mg every 4 weeks (2q4), aflibercept 2 mg every 8 weeks , aflibercept 0.5 mg every 4 weeks (0.5q4) or ranibizumab 0.5 mg every 4 weeks (Rq4). Treatment continued to week 52.8Aflibercept results are shown for the 2q8 (licensed posology) arm only. Aflibercept was non-inferior (10% margin) to ranibizumab with respect to the primary endpoint, that is the proportion of patients maintaining vision (defined as losing <15 ETDRS letters) at week 52 . These proportions remained largely similar at week 96 .post-hoc endpoint]) and change in National Eye Institute Visual Function Questionnaire-25 score.8Mean BCVA increased and was sustained over 52 weeks with both aflibercept and ranibizumab. BCVA gains from baseline to weeks 52 and 96 were similar between aflibercept and ranibizumab . In gene8 Forty-eight per cent of patients in the aflibercept arm had three or fewer injections  between weeks 52 and 96.15Between weeks 52 and 96, the mean number of injections was 4.2  and 4.7  in the aflibercept and Rq4 groups, respectively. Overall, 15.9% of patients receiving aflibercept required frequent treatment (defined here as ≥6 injections between weeks 52 and 96) and 84.1% required less frequent treatment (<6 injections between weeks 52 and 96).8Both aflibercept and ranibizumab demonstrated a favourable safety profile. Serious ocular adverse events were infrequent, occurring in 3.9% of aflibercept-treated patients and 4.4% of ranibizumab-treated patients over the 96-week treatment period. Serious systemic adverse events were typical of those seen in an elderly population receiving intravitreal treatment for wet AMD. The incidence of Antiplatelet Trialists' Collaboration events was similar between aflibercept and ranibizumab.The roundtable meeting was sponsored by Bayer HealthCare. The expert panel was made up of 10 medical retina specialists and took place in London, UK on 5 November 2014. In advance of the meeting each participant was sent a questionnaire that was used to gather information about the number of wet AMD patients treated at their unit, the treatment distribution in their practice and the aflibercept treatment approach employed at each stage of management . Participants were also asked to provide audit data on the use of aflibercept in Year 1 at their institution, and to present these to the rest of the panel at the meeting. The questionnaire and audit data were used as the basis for the discussion of aflibercept use post-Year 1.A summary of the recommendations generated by the expert panel is presented below.It should be noted that, when referring to the label, the authors refer to month- rather than week-based dosing in this paper to reflect the licensed posology for aflibercept (stated in months). However, as most clinics are set up using weekly rather than monthly schedules, practice recommendations are given using week- rather than month-based dosing. As an example, ‘8-weekly' dosing is considered to be equivalent to ‘2-monthly' dosing. The dosing schedule for aflibercept in Year 1 is shown in The goals of wet AMD treatment after Year 1 are to maintain visual and anatomical gains. These goals should be achieved by: individualising treatment regimens; minimising the treatment burden; and using resources cost-effectively. In the UK, this means delivering treatment within the local service framework and the NICE commissioning guidance. The recommendations presented here take into consideration the fact that clinic capacity limitations may have a bearing on treatment decisions; suggestions are provided for clinics where capacity is not an issue, and alternatives for clinics where capacity might be a limitation.14 Similar findings were observed in the Comparison of Age-related Macular Degeneration Treatments Trials (CATT). In Year 1, the mean number of injections for eyes receiving PRN treatment throughout Year 1 and Year 2 was 6.9 (SD: 3.0) for ranibizumab and 7.7 (SD: 3.5) for bevacizumab.16 The mean number of injections to the end of Year 2 was 12.6 (SD: 6.6) and 14.1 (SD: 7.0), respectively.13 This corresponds to approximately 5.7 injections in the ranibizumab arm and 6.4 injections in the bevacizumab arm in Year 2; the large SDs suggesting that many eyes required very little treatment. Likewise, in eyes switched to PRN treatment in Year 2, the mean number of injections was 5.0 for ranibizumab and 5.8 for bevacizumab in Year 2, but the SD values were again very wide .13As yet, there is no way to predict the amount of treatment needed with either aflibercept or other anti-VEGF therapies for an individual. This is reflected in the large range of injection frequencies found in trials. For example, in the discontinuous arm of the Inhibition of VEGF in Age-related Choroidal Neovascularisation (IVAN) study, the 25% of eyes with the lowest injection frequency required <8 injections in total to the end of Year 2, and the 25% of eyes with the highest injection frequency required >17 injections in total .15 This approach is therefore not suggested in the panel's recommendations. Three different treatment approaches are recommended for post-Year 1: (a) continuing with a fixed 8-weekly dosing regimen; (b) an individualised T&E regimen; and (c) a trial of monitoring without treatment and with extended follow-up intervals. The decision on the treatment approach after Year 1 should be made at the seventh injection in Year 1, based on the presence of active or inactive disease and taking into account the best VA and retinal anatomy achieved during the whole of Year 1. Whichever treatment approach is selected, OCT imaging should be performed and VA should be recorded at every visit post-Year 1 if possible; this should be undertaken in both eyes in order to detect fellow eye involvement early.Recommendations for the use of aflibercept in wet AMD after Year 1 are presented below and in This strategy involves continuing with proactive, fixed 8-weekly dosing. At the seventh injection visit in Year 1, the patient is re-injected and the next injection is scheduled for 8 weeks' time. OCT imaging and VA assessment are conducted at each visit if possible. Monitoring is not required between injections.Eyes with active disease (in the opinion of the treating physician) but stable VA at the end of Year 1 should continue with fixed 8-weekly dosing after Year 1. The rationale for fixed dosing at this stage is, therefore, to continue regular treatment throughout the second year of treatment in eyes with persistent disease activity at the end of Year 1.Fixed dosing should continue throughout the second year of treatment. Thereafter, individualised T&E (see below) may be considered in order to minimise treatment burden and to maximise resource utilisation.The goal of T&E (an example of a proactive treatment regimen) is to find the optimal treatment interval at which patients can achieve maximum control of disease activity and stabilisation of VA with minimum treatment burden. T&E begins with an extension of the treatment interval after the seventh injection in Year 1. Treatment should be extended by 2-week intervals, up to a maximum of 12 weeks. The goal should be to maintain a ‘stable' outcome, defined by the treating physician in consultation with each patient and based on OCT imaging and VA examination. Both imaging and examination should be carried out at every visit if possible. Monitoring is not required between injections.As an example, at the seventh injection visit in Year 1, the patient  is reviewed and re-injected. If there is no evidence of disease activity on OCT imaging and examination (in the opinion of the treating physician) and the patient has stable VA, the treatment interval is extended by 2 weeks and the next injection is scheduled for 10 weeks' time. At this time, VA is measured, an OCT scan is performed and the patient is re-injected. If the patient has stable disease (in the opinion of the treating physician), the treatment interval is extended by a further 2 weeks, and the next injection is scheduled for 12 weeks' time. Once the patient has achieved a 12-week treatment interval, treatment should continue at this frequency throughout the second year of treatment.The T&E regimen also allows the physician to shorten the treatment interval if there is new evidence of disease activity (in the opinion of the treating physician) and/or a reduction in VA. In these cases, OCT imaging is performed, VA is assessed and the patient is injected. The interval to the next treatment is shortened as appropriate. For eyes with a small increase in disease activity (e.g. those with some fluid upon OCT but stable VA), shortening the treatment interval by 2 weeks is recommended. Eyes with significant disease recurrence  should undergo more frequent monitoring, with treatment where necessary.At the end of Year 1, eyes with inactive disease (in the opinion of the treating physician) and stable VA are eligible for individualised T&E. The rationale for T&E, therefore, is to reduce the treatment burden and avoid unnecessary treatment in patients who have successfully achieved inactive disease at the end of Year 1.The T&E regimen is suitable for both a one-stop service (where OCT/VA and injection are performed on the same day) and a two-stop service (where OCT/VA and injection are performed on different days). In two-stop clinics, the use of ‘virtual clinics' can be employed to refine the patient pathway and improve capacity. Firstly, the injection visit is scheduled based on the OCT imaging and VA assessment from the previous visit. At the injection visit, OCT imaging is performed and VA is recorded prior to the injection. The OCT assessment then takes place either at the time the patient visits, or remotely  at a later date by the consultant ophthalmologist or by a non-medical member of staff under supervision. The outcome of the OCT assessment determines when the patient is asked to come back for their next injection.A trial of monitoring without treatment involves performing OCT imaging and VA assessment without giving an injection. The next planned monitoring visit should take place after an interval of between 4 and 8 weeks initially, increasing by 2-week increments, up to a maximum of 12 weeks.If active disease (in the opinion of the treating physician) develops during a trial of monitoring without treatment, the patient should go back to T&E, starting at 8 weeks and extending out to 10 and 12 weeks, once stable disease is achieved once more.At the end of Year 1 or at any time-point during Year 2, eyes that have had inactive disease and stable VA for at least three consecutive visits (including the current visit) may be considered for a trial of monitoring without treatment. A trial of monitoring without treatment avoids unnecessary treatment in patients who no longer require treatment, eliminating the treatment burden altogether.15 and data from the discontinuous treatment arms of IVAN and CATT16 reinforces the fact that a trial of monitoring without treatment may be suitable for a substantial proportion of eyes. It is unknown whether the 12-weekly injections in VIEW Year 2 were proactive (ie protocol-mandated injections because 12 weeks had elapsed since the last injection), reactive (ie injection prompted by the presence of active disease) or a combination or proactive and reactive. As a result, it is unknown exactly how little or how much treatment these patients really needed.The observation that almost half of all patients in the 2q8 arm of VIEW had three or fewer injections between weeks 52 and 96Where necessary, a trial of monitoring without treatment can be conducted purely in the ‘virtual clinic' to improve capacity. As outlined above, OCT imaging and VA assessments can be conducted in the virtual clinic, while the OCT interpretation can be done online by the consultant or by non-medical staff, under supervision.11The panel discussed the options for eyes showing a greater treatment response during the initial monthly treatment phase than during the 2-monthly treatment phase. The interval between two doses of aflibercept should not normally be shorter than 8 weeks, however the RCO guidelines for wet AMD advise that hyperactive lesions may for a short time require more intensive therapy, at the discretion of the treating physician.Eyes with significant disease reactivation during treatment in Year 2 (for example those with a significant increase in fluid and a symptomatic drop in VA) or eyes with late disease recurrence after completing initial aflibercept treatment should receive more frequent monitoring, with treatment as necessary.Given the complexity behind defining late disease recurrence after stopping treatment, however, further recommendations on managing late disease recurrence are beyond the scope of this article.Given that aflibercept has only been licensed in the UK for 2 years, very few patients have been discharged to date. As a result, the proposed discharge strategy is based on experience with only a small number of patients.Patients with stable VA who have not received any treatment for a period of 12 months or more may be considered for discharge from an active treatment service either into virtual or community clinics, or without planned further review. These patients should be seen by an ophthalmologist in person to allow for a fully informed discussion. Discharge by letter, without any direct patient contact, under these circumstances is discouraged. One should bear in mind that, in most cases, discharge will only be an option beyond the second year of treatment, that is, beyond the scope of our recommendations.17 Consequently, patients with wet AMD may re-present some time after their last injection. Follow-up at regular intervals (at least once a year), for example with a community optometrist or in virtual clinics, can be considered as alternatives to discharge, in order to check for changes in visual function in either eye. If late disease recurrence develops during this time, the patient should return to the clinic for treatment. Treatment should not be re-initiated if the patient no longer meets the criteria outlined in the NICE TA for ranibizumab.10Data from the SEVEN-UP study suggest that wet AMD is a chronic disease requiring chronic treatment.18 Thus, it is important to monitor both eyes using OCT, to ensure that fellow eye involvement is detected early. There is increasing evidence to suggest that starting treatment with vision better than 6/12 maintains good vision,19 however it should be noted that this is outside of NICE guidance and may require an individualised funding request to local commissioners.Once a patient has been diagnosed as having wet AMD in one eye, the risk of CNV in the fellow eye is high; in a study in patients with extrafoveal CNV, the 5-year incidence of CNV was reported to be 26%.If a patient is having bilateral therapy, treatment intervals should be tailored to patient visits in order to synchronise treatment of both eyes. The better-seeing eye should drive the re-treatment interval for the worse-seeing eye. However, if visual function is similar in the two eyes (difference in VA between eyes ≤5 letters), then the eye with more active disease should drive the re-treatment interval. Patients should receive the same anti-VEGF therapy in both eyes.In terms of safety considerations, the risk–benefit profile should be discussed with the patient before initiating treatment and each time the treatment regimen is altered. It should be highlighted to the patient that there is a small, added risk of ocular adverse events associated with treatment, mostly related to the injection procedure.An informed discussion with the patient is necessary to determine their treatment priorities or preferences. For example, some patients may be more willing to accept more frequent injections in order to try to achieve additional improvements in vision. Such a patient might be one who is keen to retain adequate visual acuity to continue driving. On the other hand, some patients may be unwilling to have very frequent injections and may be willing to accept some degree of disease activity. Examples include patients who have concerns about the risks associated with treatment, those who have experienced pain or an adverse event during or after injection, or those who have difficulty with regularly attending the clinic. The benefit to treatment burden may also vary according to whether the first or second eye is being treated.Recommended re-treatment criteria were developed by the panel at the roundtable meeting. The criteria were based on those used in Year 2 of the VIEW study, updated to reflect clinical experience. The definition of a ‘dry' or ‘wet' retina is highly variable between physicians, and therefore the revised criteria suggest that patients are re-treated when the treating physician considers there to be disease activity.The recommended re-treatment criteria are as follows:Patients should be re-treated if, in the opinion of the treating physician, there is new or persistent disease activity, as indicated by one or more of the following (this list provides examples but is not exhaustive):New or persistent fluid as indicated by OCT, or increase in central retinal thickness compared with the lowest previous value as measured by OCT, orLoss of vision from the best previous VA if, in the opinion of the treating physician, this is because of disease activity, orNew choroidal neovascularisation or new or persistent leakage on fluorescein angiography, orNew macular haemorrhageIt is generally accepted that the VIEW study Year 2 protocol of monthly monitoring with capped PRN treatment is not feasible for many NHS ophthalmology clinics because of capacity constraints and patient expectation, and is not mandated by the aflibercept label. The recommendations given here offer alternative treatment pathways after Year 1; the intention is to maintain visual and anatomical gains whilst minimising the treatment burden and using resources cost-effectively. Three main treatment approaches are suggested: (a) continuing with a fixed 8-weekly dosing regimen for eyes with active disease and stable VA at the end of Year 1; (b) an individualised T&E regimen for eyes with inactive disease and stable VA at the end of Year 1; and (c) a trial of monitoring without treatment for eyes with inactive disease and stable VA for three or more consecutive visits either at the end of Year 1 or at any point in Year 2.The rationale for fixed dosing at this stage is to continue regular treatment in eyes with persistent disease activity at the end of Year 1. Individualised T&E and a trial of monitoring without treatment, on the other hand, help to reduce the treatment burden and avoid unnecessary treatment in patients who have successfully achieved inactive disease by the end of Year 1. The use of virtual clinics, where the consultant ophthalmologist or a non-medical member of staff reviews OCT images remotely, can be a means of improving clinic capacity during T&E or a trial of monitoring without treatment.Whichever treatment approach is selected, an informed discussion with the patient is necessary to determine their treatment priorities and their preferences regarding disease control and treatment frequency, bearing in mind the VA gains and the response to therapy during the first year of treatment.


The aim of the present study was to determine whether aerobic exercise involving an active video game system improved asthma control, airway inflammation and exercise capacity in children with moderate to severe asthma.A randomized, controlled, single-blinded clinical trial was carried out. Thirty-six children with moderate to severe asthma were randomly allocated to either a video game group  or a treadmill group . Both groups completed an eight-week supervised program with two weekly 40-minute sessions. Pre-training and post-training evaluations involved the Asthma Control Questionnaire, exhaled nitric oxide levels (FeNO), maximum exercise testing (Bruce protocol) and lung function.No differences between the VGG and TG were found at the baseline. Improvements occurred in both groups with regard to asthma control and exercise capacity. Moreover, a significant reduction in FeNO was found in the VGG (p < 0.05). Although the mean energy expenditure at rest and during exercise training was similar for both groups, the maximum energy expenditure was higher in the VGG.The present findings strongly suggest that aerobic training promoted by an active video game had a positive impact on children with asthma in terms of clinical control, improvementin their exercise capacity and a reductionin pulmonary inflammation.NCT01438294Clinicaltrials.gov  Asthma is a chronic inflammatory disorder characterized by airway obstruction associated with recurrent episodes of wheezing, shortness of breath, chest tightness and coughing.The symptoms experienced during daily physical activities or the fear of triggering these symptoms often keep asthmatic children from engaging in physical exercise, which leads to a reduction in physical fitness ,3,4. Nevth century have raised concerns, since four fifths of children and adolescents do not follow public health guidelines regarding the recommended levels of physical activity [Changes in lifestyle in the latter half of the 20activity ,11. Althactivity , have alactivity ,14. StudThe hypothesis put forth herein is that an active video game system can be as effective as treadmill training to improve clinical control and aerobic fitness in children with asthma. Thus, the aim of the present study was to determine whether aerobic exercise involving an active video game system improved asthma control, airway inflammation and exercise capacity in children with moderate to severe asthma.This was a randomized, controlled, single-blinded clinical trial with 36 asthmatic children from a tertiary center specialized in childhood asthma, and the protocol was carried out in the University clinic . All children presenting the following inclusion criteria were invited to participate: (i) diagnosis of asthma based on the guidelines of the Global Initiative for Asthma ; (ii) meclinicaltrials.gov (NCT01438294). All caregivers gave their written informed consent prior to inclusion of the children. All evaluations were carried out by an examiner who was blinded to the allocation.The study was approved by the university ethics committee (Ethics Committee register- Nove de Julho University- 463982/2011) and registered with the Eligible children were randomly allocated to either a video game group (VGG) or treadmill group (TG). The Microsoft Excel program was used to generate a simple randomization sequence. Allocation was determined using numbered, sealed, opaque envelopes. Two envelopes were prepared for every participant. An envelope was chosen by the participant after the baseline measurements had been made. The whole randomization process was carried out by a researcher who was blinded and did not take part in the protocol.The participants were selected based on the eligibility criteria and randomly allocated to either the VGG (n = 20) or TG (n = 16). Before and after the training protocols, the participants answered questions on asthma control and were submitted to the FeNO, maximum exercise, tetrapolar bioimpedance and lung function tests. The children were submitted to all the assessments during the first week and began the training protocols the following week. The training period lasted eight weeks and involved two weekly 40-minute sessions . The game “Reflex Ridge” from Kinect Adventure  was used for training. A five minutes warm up period was carried out on a treadmill at 2 km/h prior to each session. The children then played the video game for 30 minutes  followed by a 5-minute cooling down on the treadmill again. The intensity was increased when the child successfully concluded a game level. A higher level required the child to carry out a greater number of jumps, squats, lateral movements and arm movements. Before and after each session, three measurements of the peak flow were made to detect exercise-induced bronchoconstriction (decrease ≥20% inpeakflow).A 5-minute warm up period was carried out on a treadmill at 2 km/h prior to each session, after which the exercise training was carried out for 30 minutes starting at 70% of the maximum effort determined during the maximum exercise testing. After training there was a 5-minute cooling down period. Before and after each session, three measurements of the peak flow were made. When the child maintained an exercise-intensity for two consecutive sessions with no increase in symptoms, the intensity was increased by 5% by increasing either the treadmill speed or inclination, as previously described.For both groups, the heart rate, oxygen saturation and energy expenditure were monitored during training.Maximum exercise testing was carried out on a treadmill using the Bruce protocol .The testThe fraction of exhaled nitric oxide (FeNO) was used as a marker of pulmonary inflammation, as described elsewhere. The par1) in comparison with the baseline was characterized as a positive response to the bronchodilator.Lung function was determined before and after the inhalation of 400 μg of salbutamol. The technical procedures were carried out in a climate-controlled room as recommended by the ATS. The nor1 before using the bronchodilator as a percent of the predicted value. The ACQ score is the mean of the item scores and ranges from 0 (completely controlled) to 6 (uncontrolled) obtained during a seven-day period. The cutoff point for controlled/uncontrolled asthma was 2 points. Thus the patients were classified as having their asthma controlled (< 0.75), partially controlled (0.75 to 1.5) or uncontrolled (> 1.5). The minimum clinically important difference was 0.5 on a seven-point scale [This questionnaire has seven items: five related to asthma symptoms, one on the use of short-acting ß2 agonists as a rescue drug, and one on the FEVnt scale ,25,26.All participants were evaluated individually in the afternoon to avoid the influence of circadian changes. Height, weight and abdominal circumference were determined. Tetrapolar bioimpedance was measured using the Biodynamics model 310 with electrodes on the extremities of the right upper and lower limbs.2). The waist circumference was determined with a tape measure at the navel level during expiration[Anthro plus program was used for the determination of Z scores using the standards established by the World Health Organization . BMI Z-scores were used to classify the children as obese or within the ideal weight range. Z scores between 2 and -2 were considered ideal[The body mass index (BMI) was determined as the weight divided by the height squared  and calc6. Twelve children were included to compensate for possible sample loss. Ten children (seven in the VGG and three in the TG) did not return for the follow-up evaluations even after several requests, and thus the last recorded values were used .The sample size of 24 children (12 in each group) was calculated based on the FeNO levels (primary outcome), considering a 90% power and an alpha of 5% to detect a difference of 14 ppb between the pre-training and post-training evaluations, with a standard deviation of 14.9 ppb based on previous findingsThe Kolmogorov-Smirnov test was employed to determine the data distribution. Parametric variables were expressed as the mean ± standard deviation. Non-parametric variables were expressed as the median interquartile intervals (95% CI). A two-way ANOVA with Tukey’s post hoc test was used for the comparisons between the pre-training and post-training evaluations for the parametric data, and Friedman’s test with the post hoc Dunn test for non-parametric data. The unpaired t-test was used for the analysis of energy expenditure. The statistical analysis was carried out using the Minitab 14 statistical software package. The level of significance was set at 5% (p < 0.05). The size effect was calculated using Cohen’s d and the results were interpreted based on Cohen (2008) as folloTen children (seven from the VGG and three from the TG) withdrew from the study: four due to changes in the school schedule; three abandoned the study without explanation, two dropped out due to difficulties with the parents’ schedules and one moved to another city. Thus, 26 children completed the study (13 in each group). At the baseline, both groups were similar regarding asthma control, pulmonary inflammation, lung function, body composition and anthropometric data as well as exercise capacity. All patients were treated with budesonide and long acting β2-agonists and maintained the medication dosage throughout the study.After training, a significant improvement in asthma control was found for both groups (p<0.05). However, only the children in the VGG showed a reduction in FeNO (p<0.05) ; p<0,05.The total mean energy expenditure was measured during the training protocols using a biaxial accelerometer and the data presented in metabolic equivalents (METs) and calories per minute . No difference was found between the groups regarding the mean energy expenditure, but a significantly higher maximum metabolic expenditure was found in the VGG p<0.05; . InteresBoth groups exhibited an improvement in maximum aerobic capacity after training p<0.05; ; howeverThe present study clearly demonstrated that the exercise training using an active video game system was efficient in improving clinical control and aerobic fitness and in reducing lung inflammation in children with moderate to severe asthma. Moreover, the energy expenditure during video game training was higher than that achieved with aerobic training on a treadmill. Thus, active video games seem to be an interesting exercise for asthmatic children and are probably more attractive to the pediatric population.1. The present findings support the use of aerobic training combined with medication to achieve such goals and are in agreement with data reported in previous studies [The main goal of asthma treatment is to achieve and maintain good clinical control studies ,31 regar studies  (2011) eTo the best of our knowledge, only three studies have evaluated the anti-inflammatory effects of aerobic training in asthmatic patients ,33,34. MIt has been reported that approximately 50% of preschool children do not carry out even the minimum level of physical activity recommended by the American Academy of Pediatrics (≥60 minutes/day). This inTo the best of our knowledge, aerobic exercise is the only type of physical activity that benefits asthmatic patients and suchKuys et al. (2011)  used a vThe motivation and intensity maintenance with video game training led to an improvement in the aerobic capacity of the patients. These findings are in agreement with data reported by Donovan et al. (2012)  and suggSmallwood et al. (2012) found th2 while interval training also led to such an increase but not with the same linearity [Both groups showed improvements in aerobic capacity during maximum exercise testing after the training protocols; however the TG presented better performance. Exercise on the treadmill was continuous, whereas exercise using the video game was performed at higher intensity Click here for additional data file.S1 Protocol(PDF)Click here for additional data file.