In the UK, the customary method of obtaining special low protein (LP) foods was by dispensing through a pharmacist (until 2010) for patients with inherited metabolic disorders (IMD) requiring LP diets. Recently, different home delivery services have been introduced to support patient access of low protein foods, but the effectiveness of these services is unclear.A prospective, longitudinal, observational study to examine the effectiveness and safety of patient home delivery services for LP foods over 12 months in IMD patients requiring a LP diet.IMD patients/caregivers had the choice of 2 home delivery services (Homeward® and Vitaflo at Home®) as well as access to primary care pharmacy services. Both home delivery services provided a limited range of LP foods. Over a 12-month period, a member of the IMD dietetic team conducted 4 home visits to IMD patients on LP diets using home delivery services for low protein foods. At each visit, caregivers completed a questionnaire consisting of 20 multiple choice and open questions about their prescription experience with special LP foods. The researchers also completed stock checks, assessed ‘use by dates’ and adequacy of home storage for LP foods.n = 55/58) of caregivers used their local pharmacy, 93% (n = 54/58) Homeward® and 78% (n = 45/58) Vitaflo at Home® to access LP foods. Two home delivery services were used by 41 (71%) caregivers and the remaining 17 (29%) only used one of the home delivery service companies. Each patient only stored a median of 6 (range 0–22) different LP foods at home. Overall, 45% (n = 26/58) of caregivers reported problems with their GP prescriptions. 30% (n = 16/53) of caregivers received at least one incorrect prescription when using their pharmacy , 6% errors (n = 3/53) with Homeward® and 2% (n = 1/48) with Vitaflo at Home®. 49% (n = 26/53) of caregivers said they experienced delayed receipt of LP foods from their pharmacy, compared with 11% (n = 6/55) from Homeward® and 8% (n = 4/48) Vitaflo at Home®.In total, 58 patients participated in this study. Over 12 months, 95% (Although home delivery services for special LP foods are associated with less errors and delay compared with pharmacies, inaccuracies and inefficiencies still occur and the overall system is complex. We suggest a new, simpler, less fragmented system whereby metabolic dietitians prescribe LP foods. This is likely to result in less burden on NHS resources and ensure a better treatment delivered to IMD patients. Generally, special LP foods are prescribed by the primary care GP and not by the metabolic centre or metabolic dietitian/clinician. They cannot be purchased over the counter in pharmacists or retail shops.Children and adults with inherited disorders of amino acid or protein metabolism such as phenylketonuria (PKU) or tyrosinaemia require low protein (LP) diets, as part of their treatment in order to prevent accumulation of offending amino acids or their metabolites . This neLP products are essential in the diets of patients with inherited metabolic disorders (IMD) as they: 1) provide a source of energy to support growth and prevent catabolism which may lead to metabolic instability; 2) provide bulk to aid satiety 3) help improve adherence by helping limit the consumption of higher protein foods; and 4) increase the variety of foods consumed. Most GP's only issue LP prescriptions monthly, meaning that the requirement for LP foods must be anticipated in advance. It is commonly a careful balance between ensuring that patients have enough LP food supply to ensure that energy requirements are met but not too much so that it is associated with product ‘stock piling,’ which could lead to wastage and misuse of NHS resources.Up until 2010 the usual method for obtaining LP products was patient prompted prescriptions that were generated by a GP and dispensed by a community pharmacist. This system was problematic as errors in both prescribing and dispensing of products as well as delays in obtaining products appeared common. Although normal timing for requesting each food prescription is monthly, delays >4 weeks occurred if pharmacies were unable to access LP food supplies. Some patients received incorrect protein substitutes or gluten-free foods instead of LP foods . These eFollowing the success of home delivery services for protein substitutes for patients with inherited metabolic disorders (IMD) , this seIt was unclear how effective and efficient the different systems were in terms of ordering patterns, dispensing errors and delays in accessing and delivery of LP foods compared with conventional pharmacy systems. Therefore, we conducted a prospective, longitudinal study, using a questionnaire to examine prescription issues over one year in a group of patients with IMD requiring a LP diet and in receipt of home delivery to access special LP products. We also checked patient's stock levels of special LP foods as well as home storage space and LP food wastage.22.1Eighty IMD patients requiring a LP diet aged between 0 and 16y were invited to participate in this study. All used the home delivery service for either protein substitute or LP foods. The inclusion criteria to participate in this study were: patients with an IMD disorder of protein metabolism, on a LP diet and using LP foods received by home delivery. Exclusion criteria included: non-use of LP foods and not using at least one home delivery service.2.2For 12 months, home visits where performed by dietitians from the IMD dietetics team as presented by use by dates’ and assessed adequacy of storage space for LP items.At each home visit, the IMD dietetic researcher also performed a stock check of all LP items stored in the home, examining ‘2.3The method of accessing LP items was different for each company. Homeward® requested patients/caregivers to complete a monthly order form listing the LP products required which was returned to the home delivery administrative team. For Vitaflo at Home® a monthly standing order for LP foods was organised for each patient with Vitaflo at Home®. Both companies contacted the GP practices for prescriptions but first established supplies by conducting a stock check with the patients/caregivers. The prescription was then ‘picked’ and verified by a qualified pharmacist and delivered directly to the patient's home. Neither of the home delivery services stocked nor delivered gluten-free foods to avoid delivery error. Any other brands of special LP foods from Promin and PK foods were supplied by the local pharmacist. Caregivers/patients requested prescriptions from the GP which were then delivered to the pharmacist to supply.2.4Data were analysed using descriptive statistics only.33.1n = 48; tyrosinaemia type I, n = 3; tyrosinaemia Type II, n = 1; non-pyridoxine responsive homocystinuria, n = 2; glutaric aciduria Type I, n = 2; maple syrup urine disease, n = 1; and 3-hydroxy-3- methyl glutaric aciduria, n = 1. Fifty seven of 58 patients (98%) were prescribed a protein substitute. All were prescribed special LP foods and already used a home delivery service for protein substitute or LP milk supplies. All patients lived in the West Midland region of the UK.In total, 58 patients participated in this study. Forty-nine patients were European Caucasian origin and 9 from Asian origin. Six were unable to speak English as their first language. The diagnosis of patients was: PKU, 3.2n = 54/58) of caregivers used Homeward®, 78% (n = 45/58) used Vitaflo at Home® whilst 95% (n = 55/58) used their local pharmacy for some LP products unavailable by either home delivery service. Two home delivery services were used by 41 (71%) caregivers and the remaining 17 (29%) only used one of the home delivery service companies. Caregivers obtained most of their LP foods (definition >5 items) from Homeward®  or the local pharmacy . Caregivers were asked about ease of access to their LP products; 83% (n = 48/58) said it was easy using Homeward®, compared with 76% (n = 44/58) for Vitaflo at Home®, and 57% (n = 33/58) their pharmacy. Eighty-one per cent (n = 47/58) preferred Homeward® overall.For the delivery of LP foods, 93% (n = 16/53), for Homeward®, 6% (n = 3/53) and for Vitaflo at Home®, 2% (n = 1/48). Prescription problems with the home delivery services were usually related to items being ‘out of stock’. Forty-nine per cent (n = 26/53) said they experienced delayed receipt of LP foods from their pharmacy, compared with 11% (n = 6/55) using Homeward® and 8% (n = 4/48) from Vitaflo at Home®.The number of caregivers reporting the receipt of at least one incorrect prescription for LP foods  was: for pharmacies, 30% (n = 26/58) reported some difficulty in obtaining prescriptions for LP items from their primary care GP service. Issues included: inability to access all LP products requested , unjustified refusal to prescribe the quantity of LP products requested , prescriptions posted to the incorrect home delivery company/pharmacy , delayed receipt of prescriptions , and caregiver need to ‘track’ delayed prescriptions (12% n = 7/58). Forty-three per cent (n = 25/58) said they needed more LP foods than they had been prescribed.Overall, 45% . Households were expected to store adequate food supplies for around 4 weeks. In households, the average amount of special LP food stock held for 4 weeks was a mean of 5 packets of LP pasta, 4 pizza bases, 6 packets LP sausage/burger mix and 3 packets of biscuits . This wan = 23/58) of caregivers were assessed to have inadequate storage space for LP foods . These households were unable to take larger stocks of LP items.Forty per cent (n = 9%) were identified as keeping higher levels of stock than necessary, with their supplies coming from both the chemist and home delivery service. However, this was usually associated with poor literacy of parents or misunderstanding and over prescription of food items by the GP prescription service.Families discarded minimal LP foods over the 12-month period because of ‘out of date’ shelf life . Only 5 4This study indicated that the number and variety of LP food items ordered and stored by patients and their caregivers is controlled and restrained. It also suggested that caregivers would prefer home delivery services to access their special LP foods although there is no single home delivery service that will supply all LP foods. Unfortunately, whilst access to LP foods is available from pharmacies and more than one home delivery company, with each company only delivering specific brands of special LP foods using different procedures, the overall service is open to error and inefficiencies. Prescriptions are sent to the wrong service and patients and health professionals are confused by the variable paperwork requirements associated with different companies. Generally, for professionals it is a time consuming and fragmented process. Since the completion of this study, another home delivery company has commenced delivery of Promin®, Taranis® and Mevalia® brands of special LP foods, adding to the complexity.Overall, home delivery services were associated with a lower rate of prescription error and less delay in receiving prescription items. This is similar to the results from a controlled study examining the delivery of amino acid supplements in IMD . In our In the UK, around 10,500 surgeries deliver primary care services to the UK population. Neither the GP nor their administrative team are likely to have received any specific training in IMD or specialist dietary products. Although IMD dietitians have expert knowledge, and calculate and monitor the complex dietary regimens, they are unable to prescribe special LP foods for their patients; their role is to advise primary care services only. Furthermore, England's NHS budget for LP foods is based within primary care rather than specialist commissioning services (which finance most of the English IMD services). This means that IMD dietitians have little control over the type and quantity of dietary products that are prescribed for their patients. For LP food access to improve, it is essential IMD dietitians are given greater autonomy and accountability for their prescription.Specialist LP foods are essential to provide adequate energy (around 50% of energy requirements) and satiety in a LP diet and patients should not be left without supplies. This study indicated that some of the patients kept very little reserve stock, suggesting that home delivery services for LP foods were not leading to stockpiling, which has been a concern of primary care services when sending prescription requests to the delivery services. However, the low stock levels of special LP food items held by patients is a concern, particularly the limited amounts of LP bread and flour held, as access to ‘regular’ food supplies using ‘usual’ commercial outlets is not an option. Many caregivers/patients were acutely aware about the cost of treatment and ordered minimal stock which led to low levels of LP food wastage. In fact, some caregivers ordered less food than they needed as they were frightened that their prescription requests may be challenged by the GP services. Others chose to order the same foods each month and not increase variety in order to avoid any criticism by GP practices.The current system of prescribing LP foods is 47 years old and it is very complex, fragmented, and time consuming . We beliSome European countries use a central distribution point to store and supply all the specialist LP products . Other cThere are limitations to this study. This was an observational study and we did not include a control group of patients receiving all their LP products via their local pharmacist only. We did not examine dietary patterns of patients or the impact special LP foods have on energy intake. We also did not examine in detail the reasons why a small number of patients had higher amounts of LP food stocks stored in their home. In addition, the questionnaire was not piloted before being used.5We conclude that the home delivery of special LP foods is associated with less prescription error and is preferred by caregivers and patients. However, prescriptions are issued and controlled by community GP teams who receive no training in rare IMD conditions which is problematic. The access to special LP foods from different sources is also fragmented, confusing and takes considerable caregiver, patient and health professional time to ensure that patients always have access to the correct LP supplies. We believe it is time to adopt a new system based on metabolic dietitians prescribing LP foods with only one UK centralized point delivering all the LP foods. This would be a much simpler, improved, and efficient system, whereby patients would receive all supplies from a single source with minimal burden for patients, caregivers and health professionals.All authors participated in data collection, critical revision of the paper and final approval of the version to be published. Anita MacDonald was involved in questionnaire development, data analysis and writing of the manuscript.This study was not funded.Anita MacDonald has received research funding and honoraria from Nutricia, Vitaflo International and Merck Serono. She is a member of the European Nutrition Expert Panel (Biomarin), member of Sapropterin Advisory Board (Biomarin), member of the Advisory Board entitled ELEMENT (Danone-Nutricia), and member of an Advisory Board for Arla and Applied Pharma Research. Alex Pinto has received an educational grant from Cambrooke Therapeutics and grants from Vitaflo, Nutricia, Merck Serono and Biomarin to attend scientific meetings. Sharon Evans is a research dietitian funded by Nutricia; financial support from Nutricia and Vitaflo to attend study days and conferences. Anne Daly has undertaken evaluation work for the nutritional companies – Vitaflo Ltd., Nutricia Ltd. and Metax.


Lhcx1 and Lhcx2 genes individually in Phaeodactylum tricornutum strain 4 and complementing the knockout lines with different Lhcx proteins, multiple mutants with varying qE capacities are obtained, ranging from zero to high values. We demonstrate that qE is entirely dependent on the concerted action of diatoxanthin and Lhcx proteins, with Lhcx1, Lhcx2 and Lhcx3 having similar functions. Moreover, we establish a clear link between Lhcx1/2/3 mediated inducible thermal energy dissipation and a reduction in the functional absorption cross-section of photosystem II. This regulation of the functional absorption cross-section can be tuned by altered Lhcx protein expression in response to environmental conditions. Our results provide a holistic understanding of the rapidly inducible thermal energy dissipation process and its mechanistic implications in diatoms.Diatoms possess an impressive capacity for rapidly inducible thermal dissipation of excess absorbed energy (qE), provided by the xanthophyll diatoxanthin and Lhcx proteins. By knocking out the  Phaeodactylum tricornutum Pt4 is Lhcx protein-dependent and correlates with a reduced functional absorption cross-section of photosystem II.Photosynthetic organisms can dissipate excess absorbed light energy as heat to avoid photodamage. Here the authors show that induced thermal dissipation in the diatom  The optimization of photochemistry is achieved when the rate of photon absorption is equal to the rate of electron transfer. Although this requires a large absorption cross-section of photosystem II under low light (LL) conditions, the same absorption cross-section would lead to an overflow of energy in the photosynthetic system under high light conditions, resulting in massive oxidative damage. Hence, in oxygenic photosynthetic organisms, photon capture can be regulated by adjusting the cross-section of photosystem II on time scales of minutes. In practice, this phenomenon leads to changes in the balance between excitons directed to reaction centers and those that are dissipated as heat.Upon absorption of a photon, the singlet excited state of a chlorophyll 3, and is characterized by a downregulation of chlorophyll fluorescence at high irradiance. Mechanistically, NPQ comprises several processes4. The most-rapid NPQ component, called energy-dependent quenching (qE), is strongly dependent on light intensity, reflecting the balance between fluorescence quenching and an increase in thermal dissipation5. qE has been shown to decrease the functional absorption cross-section of PSII (σPSII), thus reducing the flux of absorbed energy into photochemistry10. Consequently, light-enhanced thermal dissipation reduces the excitation pressure and hence the probability of photooxidative damage. In higher plants, however, the correlation between the onset of qE and the reduction of σPSII has recently been challenged11.There are a number of photoprotective mechanisms that thermally dissipate excess absorbed energy as heat; collectively these are called Non-Photochemical Quenching (NPQ). This phenomenon is present in all photosynthetic eukaryotes and in many cyanobacteria12. The initiation of qE is driven by the establishment of a pH gradient between the thylakoid lumen and plastid stroma (ΔpH) upon excess light exposure. It often also correlates with the conversion of xanthophyll pigments in the so-called xanthophyll cycle, which, for diatoms, is the conversion of diadinoxanthin (Dd) into diatoxanthin (Dt)15. In addition, in plants, qE requires the presence of PsbS, a specialized protein that belongs to the light harvesting complex protein (LHC) family16. In contrast, the green alga Chlamydomonas reinhardtii requires Lhcsr proteins for qE17. Similarly, in diatoms, the importance of the Lhcsr-related Lhcx1 proteins for qE has been proven by silencing the Lhcx1 genes in Phaeodactylum tricornutum18 and Cyclotella meneghiniana19.qE has been found in several major algal lineages as well as in mosses and land plants20. They constitute one of the most important phytoplankton groups in terms of productivity21 and biodiversity22. Their abundance is also based on their capacity to thrive in turbulent waters , where they can exploit the huge amount of available nutrients23. This goes in line with their variable qE capacity depending on the light characteristics of the respective habitats26. In contrast to C. reinhardtii, which contains only two different Lhcsr proteins, many diatoms possess multiple Lhcx proteins, e.g., Fragilariopsis cyclindrus contains 11 Lhcx27. For several diatom species, qE capacity correlates with the expression of different Lhcx proteins, which indicates the involvement of various Lhcx proteins in triggering qE under different environmental conditions35. P. tricornutum contains only four Lhcx proteins36 and therefore is a particularly good model to study the impact of individual Lhcx proteins on qE capacity. These four proteins are structurally similar, but are differentially expressed under varying environmental conditions32.Diatoms are unicellular microalgae with complex plastids, which were acquired during serial secondary endosymbiosis from a green and a red algaP. tricornutum, to examine two basic questions: First, besides Lhcx1, do other Lhcx proteins equally confer qE capacity and if so, is it correlated with the xanthophyll cycle. Second, is qE in diatoms correlated with a reduction of σPSII. Experimentally, we knockout the Lhcx1 gene in P. tricornutum that results in mutant lines devoid of qE. Then, we individually overexpress all other Lhcx proteins in this knockout background. In addition, we knockout the Lhcx2 gene and express the Lhcx2 and Lhcx3 protein in this knockout background. This experimental design allows us to quantify the influence of each Lhcx protein on qE and σPSII.Here, we use a model diatom, 37, we independently targeted two different sites of the Lhcx1 gene in P. tricornutum strain Pt4 (UTEX 646). Three Lhcx1 knockout (x1KO) lines were obtained  promoter/terminator. Out of several lines created , the x1KO + x2 and x1KO + x3-supplemented lines recovered qE capacity , particularly during the second hour  exposure, typical for P. tricornutum42. We observed a slight decline in σPSII as light intensities increased to 70–100 µmol photons m−2 s−1 , down to values of 400–450 Å2 PSII−1 (a reduction of 15–20%). Further increased light intensities, which induced a slight linear increase of NPQ capacity not related to qE (see above), did not further downregulate σPSII. When inhibiting Dt synthesis by addition of DTT, qE possessing strains showed a σPSII development similar to the x1KO and the x1KO + x4a strains where σPSII was unaffected by the addition of DTT . In order to corroborate our results, we compared the effect of three minutes supra-optimal light exposure on NPQ establishment and σPSII behavior in wild-type and x1KO strains using the DCMU method and applying the calculation and correction procedure as described by Tian et al.10. Using this method, we also observed a significant reduction in σPSII in the wild type after supra-optimal light exposure, which was absent in the x1KO strain  required to reduce σPSII. Strains possessing a higher qE capacity exhibited a substantial decrease of σPSII at much lower PSII excitation pressure. While for LL grown cultures 1-qL values of 0.6–0.8 were necessary to reduce σPSII in qE possessing strains, high light-acclimated cultures started to decrease σPSII at 1-qL values of ~ 0.2–0.3  we observed an almost linear correlation up to NPQ values of ~ 0.6  has been proposed to better visualize the effect of thermal dissipation46. In line with the rapid onset of qE  that had no capacity for qE, i.e., the rapid component of NPQ  and the conversion of Dd to Dt. Lhcx proteins or the xanthophyll cycle alone—at least under LL growth conditions - do not activate qE or cause a reduction in σPSII under supra-optimal irradiances.As a consequence of the lack of Lhcx1, the x1KO strain, as well as the x1KO + x4a strain, exhibit no decrease in σPSII under high light exposure. Instead, they show a slight increase in σPSII, as expected when PSII reaction centers become progressively closed, causing excitons to jump from one PSII unit to another, in search for an open PSII center54 and Lhcx proteins are not part of the PSII core55, the decrease in σPSII is indicative of thermal dissipation of absorbed energy in the LHC antenna complexes, as originally proposed by Genty et al.5. Accordingly, analysis of picosecond lifetime kinetics revealed that the exposure to supra-optimal irradiances leads to functional modifications in both antennae and PSII reaction centers, but the thermal dissipation occurs only in the antennae46. Our results also showed that the slow phase of NPQ, which is activated under prolonged exposure to higher light levels, has no effect on σPSII. A similar slow NPQ phase with no reduction in σPSII is observed in the mutants lacking qE (x1KO and x1KO + x4) as well as in all strains treated with DTT (which inhibits the formation of Dt). These results suggest that the slow phase of NPQ is related to photoinhibitory damage to the PSII reaction center (qI).As Dt binds to LHC antenna proteins4. Accordingly, the most rapid NPQ process (qE) can be easily identified by a pronounced reduction in σPSII upon exposure to high light. Whether this scenario holds true also for other algal taxa remains to be tested, but recent results in the green alga Ostreococcus taurii suggest a similar correlation between qE and σPSII9. Our measurements of a significantly decreased σPSII under qE conditions are fully consistent with the proposal that qE shortens the time an exciton can travel before being thermally dissipated, and thus decreases the effective excitation diffusion length for an exciton to reach an open PSII reaction center56.The combination of NPQ and σPSII measurements in a variety of mutants with different NPQ extents allowed us to better characterize multiple NPQ processes that are otherwise hardly distinguishable57, excitons that reach a PSII core may transfer back to the peripheral antennae where they can be thermally dissipated once all PSII reaction centers are closed. Such a mechanism has been identified in plants, termed “economic photoprotection”11, and later was also postulated to exist in diatoms51. This economic photoprotection, however, does not involve a decrease in σPSII upon induction of qE11, which is in sharp contrast to our results.Because the PSII reaction center is energetically a shallow trap2 at Y(NPQ) = 1 (and 115 Å2 if both parameters are measured after 1 s darkness). Using a chlorophyll a specific absorption coefficient of 9.8 m2 (g chlorophyll a)−1 for blue light58 (as used here for the σPSII measurements) and assuming a dimeric PSII core for diatoms in vivo59, containing 70 chlorophyll a molecules55, we calculated a functional PSII core cross-section of 112 Å2. This value is virtually identical to the one calculated at Y(NPQ) = 1 based on our σPSII vs. Y(NPQ) regression. In a similar approach for the centric diatom T. pseudonana, Campbell, and co-workers calculated with a monomeric PSII and concluded that at Y(NPQ) = 1 the remaining σPSII is provided by the PSII core and some LHCs that are not thermally downregulated9. If one assumes that the existing dimeric PSII59 is excitonically coupled and shares a common peripheral LHC antenna, then the regression to a Y(NPQ) of 1 provides a residual σPSII, which also in T. pseudonana corresponds to the functional absorption cross-section of the dimeric PSII core. In either cases, the results of Xu et al.9 and this study indicate that regulation of σPSII upon induction of qE does not involve the PSII core, otherwise the regression at Y(NPQ) of 1 would lead to σPSII values close to 0. The most parsimonious explanation is that Lhcx-mediated qE mechanistically leads to a disconnection of the peripheral LHC antennae—very much in line with recent results obtained by a different experimental approach51—and these functionally disconnected LHC antennae dissipate the excess absorbed light as heat. Such a mechanism has been identified as one compound of NPQ in Arabidopsis thaliana using electron microscopy60.As we obtained a linear correlation between the extent of Y(NPQ) and the decrease in σPSII driven directly by qE Fig. , we can 61. Moreover, traces of Lhcx proteins have been detected in photosystem II preparations34, indicating that Lhcx proteins might be more strongly connected to the PSII core than the peripheral LHC proteins. Both aspects could influence peripheral LHC antennae connectivity with the PSII cores in such a way that, upon induction of qE, the peripheral antennae are moved away slightly, exceeding the maximal distance for functional Förster resonance energy transfer to the PSII core. In such functionally disconnected LHC antennae, internal heat dissipation and chlorophyll fluorescence emission—the remaining energy dissipation pathways—would compete and a further synthesis of Dt under prolonged supra-optimal light would decrease fluorescence yield even more by increasing thermal dissipation, but without further affecting σPSII. These processes, together with photoinhibited PSII reaction centers46, may explain the observation of very high NPQ values, which do not affect σPSII51.It is not clear yet how this disconnection of the LHC antennae is achieved, but recent results indicate a strong role of Dt molecules in influencing thylakoid membrane rigidity63. These reports are corroborated by classical PAM analyses in centric diatoms, where one component of qE is independent of the conversion of Dd to Dt40 and clearly different antenna organizations exist64. However, the two qE-quenching sites concept is difficult to reconcile with the prime dependency of qE on the amount of Dt in the pennate P. tricornutum39 and with our results, which point to only one mechanism of qE. One reason for this discrepancy may be that in both time resolved fluorescence studies revealing the two quenching sites in P. tricornutum62 the time span of cells exposed to supra-optimal light conditions was much longer than necessary to induce full qE and hence also qI related processes likely had been recorded. Indeed, when Taddei et al.34 and colleagues measured LL grown P. tricornutum cells, they observed only one quenching site. Analyses of picosecond lifetime kinetics during the NPQ development in P. tricornutum revealed a functional modification in the PSII reaction center, in line with previous results65, which, however, is independent of the xanthophyll cycle and does not lead to any thermal dissipation46. Instead, this modification in the energy transfer in PSII centers reduces the probability of producing triplet chlorophyll46. Our results demonstrate that only Dt- and Lhcx-mediated qE provides thermal dissipation in the antennae, which likely involves uncoupling of functional antennae. This is characterized by one quenching site that corroborates the conclusions obtained by Kuzminov and Gorbunov46.There had been several reports on two potential qE quenching loci in diatoms based on fluorescence lifetime kinetics. The first is associated with an uncoupling of antenna complexes from the PSII core, which is independent of Dt formation. The second is taking place close to the PSII reaction center and is directly dependent on the xanthophyll cycle supposedlyP. tricornutum strain 4, which had been originally isolated from the Baltic sea and which has a lower NPQ capacity than other P. tricornutum strains18. Some analyses regarding two different quenching sites and mechanisms had been performed in another P. tricornutum strain (Pt2)62, which has a higher NPQ capacity than Pt4. Thus, in principle there could exist an additional quenching mechanism in Pt2. However, the fact that also in Pt2 there is a 1:1 dependency of NPQ on the amount of Dt39, together with the explanations for the apparent occurrence of a second quenching site provided above, rather argue against this assumption.We have to note that our analyses have been conducted in 34. We indeed observed no qE in the LL grown cells if Lhcx1 is knocked out, but this is rather owing to a much lower expression of Lhcx2 and Lhcx3 under these conditions32 , high light , blue light (Lhcx1-3), iron limitation (Lhcx2), or nitrogen starvation . We propose that one gene alone cannot contain all required regulatory cis-elements in order to respond to the multitude of environmental triggers. Instead, through gene duplications during evolution, several regulatory cis-elements could be integrated into several Lhcx gene promoters to modulate the expression of proteins with very similar functions and thus to tune the functional absorption cross-section depending on the environmental conditions.Then, why do diatoms have several different 68.Overall, our study reveals the molecular mechanism of how diatoms fine tune qE, allowing them to thrive in continuously changing light environments, where NPQ is one of the most important physiological processesPhaeodactylum tricornutum strain 4 . All strains were grown in batch cultures on a shaker at 20 °C in a 16 h day/8 h night cycle exposed to white light with an intensity of 40 µmol photons m−2 s−1 (onset at 8:00 a.m.) defined as low light (LL). Cells were cultured in sterile Provasoli's enriched F/2 seawater using Tropic Marin Classic artificial sea salts  with 16.6 g sea salt per 1 L medium. For all experiments, only cultures in the logarithmic growth phase were used, i.e., a chlorophyll a concentration of 1.5 mg L−1 or a cell concentration of 3.5 million cells mL−1 at maximum. Therefore, cultures were regularly diluted with fresh F/2 medium in order to avoid any nutrient limitation, which would strongly influence Lhcx expression32. Chlorophyll a concentration was determined as described in ref. 33. Cell counts were obtained using a Multisizer 3 Coulter Counter . For long-term light stress experiments, cells were exposed to white light with an intensity of ~ 400 µmol photons m−2 s−1 for 24 h. Light intensities were measured with a spherical quantum sensor .Experiments were performed in P. tricornutum by Serif et al.37. This method relies on Golden Gate reactions, where digestion with TypeIIs restriction enzymes and ligation are carried out in one step69. For constructing the specific TALENs, we assembled the respective TALEN monomers into the respective backbone vectors available at Addgene. For Lhcx1 we constructed two TALEN pairs, targeting two different sites in the Lhcx1 gene. For Lhcx2 we constructed one TALEN pair. All genomic target sites contained a thymine before the actual TALE recognition site. For each TALEN we assembled 18 monomers, each with a repeat variable di-residue (RVD), which recognizes one specific DNA base. The 19th monomer is only a half monomer and is included in the respective TALEN backbones37. Supplemental Table TALEN-knockout plasmids for Lhcx1 and Lhcx2 were generated following the procedure for creating TALEN-KO lines in Lhcx1, Lhcx2, Lhcx3, and Lhcx4 were cloned inside a modified pPha-T1 vector pPTbsr, respectively, which instead of a Bleomycin resistance cassette contains a Blasticidin-S resistance cassette70. The FcpA promoter of this vector was removed via deletion PCR and replaced by two different Lhcx promoters: the genes designated to complement the x1KO background were cloned in between the Lhcx1 promoter (starting 721 bases upstream to the Lhcx1 gene start codon) and terminator (617 bases downstream of the Lhcx1 stop codon), whereas for the genes designated to complement the x2KO strain the Lhcx2 promoter (605 bp upstream of translation start of Lhcx2) and terminator (422 bp downstream of Lhcx2 stop codon) was used. When the KO lines were complemented with their original gene , modified gene sequences using synonymous codons were used, in order to prevent the TALENs from cutting the newly introduced genes. Except for Lhcx1, all genes were cloned including their respective introns. In addition, the modified Lhcx1 gene was also cloned in between the original FcpA promoter and terminator of the pPTbsr vector.In order to complement/supplement our KO strains, Lhcx genes of Pt4 are slightly different from those of the sequenced Pt1 strain (https://genome.jgi.doe.gov/Phatr2/Phatr2.home.html) . All gene, promoter and terminator sequences were obtained by amplifying genomic DNA with specific primers using Kapa Hifi DNA Polymerase  and purification of the PCR product from agarose gels using Geneclean Turbo Kit  except for the modified Lhcx1 and Lhcx2 sequences, which were ordered from BioCat (Germany). Correct amplification and vector integration were verified by sequencing . All sequences for the genes, promoters and terminators used in this study are provided in Supplementary Table Lhcx3 complementing the x1KO and x2KO line, respectively, are depicted in Supplementary Fig. Sequences for the 71. Positive clones were selected on Zeocin  and Nourseothricin  containing solid medium plates containing 1.2 % Bacto Agar  in F/2 medium . Pre-screening of the colonies was performed using an Imaging PAM system , searching for clones with altered NPQ characteristic. Further characterization included western blot, PCR, and DNA sequencing. Confirmed KO clones were spread on individual agar plates in a suitable dilution to obtain clones from single cells. Three out of these were reanalyzed for each knockout line and one of each KO line was used for all follow-up experiments. In addition, whole-genome sequencing was done on the three created x1KO lines. All initial KO clones observed were homogeneous, i.e., clones obtained from single cells from the initial clones all showed the same knockout phenotype.Wild-type Pt4 cells were biolistically transformed with the TALEN bearing vectors according toLhcx1 gene by a second biolistic transformation using Blasticidin-S (4 µg mL−1) containing solid medium plates in low-salt F/2 medium  for selection. In addition, the native Lhcx2, Lhcx3, and Lhcx4 genes were transformed in the x1KO_1a line. The x2KO line was complemented with a modified Lhcx2 gene, and also transformed with the native Lhcx3 gene.Two obtained x1KO lines were complemented with a modified Lhcx1 gene of Pt4 does not contain allele specific differences in the TALEN-targeted region. The sequences of the two primer pairs for both alleles as well as of all following primers are specified in Supplementary Table Lhcx2 of Pt4 is depicted in Supplementary Fig. Lhcx2 promoter, Lhcx2 gene and Lhcx2 terminator in a length of ~ 1970 bp in wild-type. In the x2KO mutant, in one of the alleles a deletion of 918 bp occurred  according to the manufacturer's instructions. A cell pellet corresponding to 10 mL of culture was used as starting material. The concentration of genomic DNA was measured with a Nanodrop 2000 . In order to prove that both alleles were mutated, allele specific PCR was applied for the x2KO mutants only, as the Lhcx1 gene in the Pt4 strain, the x1KO strains and the Lhcx1 complemented lines. It served as a control. The primer pair Lhcx1_wild-type-fw and Lhcx1_wild-type-rev amplifies a 347 bp sequence only in the wild-type strain, whereas the primer pair Lhcx1_comp-fw and Lhcx1_wild-type-rev amplifies 262 bp only in the complemented x1KO lines. Note that for the two latter primer combinations only the forward primer was different, binding either to the wild-type sequence of Lhcx1 or the modified sequence of the complemented Lhcx1 gene with synonymous codon usage.To screen the x1KO mutants and the complemented lines, we used different primer combinations. The primer pair Lhcx1_all-fw and Lhcx1_all-rev amplifies 102 bp at the 3′-end of the FcpA promoter and the first base pairs of the modified Lhcx1 gene. This combination of FcpA promoter and modified Lhcx1 gene, as well as the modified TALEN-binding sites on which FcpA_Lhcx1-rev binds, are unique for the complementation construct and do not occur in wild type cells.To screen the x1KO_1a + FcpA_x1-complemented mutants, the primer pair FcpA_Lhcx1-fw and FcpA_Lhcx1-rev amplifies a 479 bp fragment, including the PCR was performed using HiDi polymerase according to the manufacturer’s instructions for 30 cycles , with either the primer pairs Lhcx1_wild-type-fw/Lhcx1_wild-type-rev and Lhcx1_all-fw/Lhcx1-all_rev or with Lhcx1_comp-fw/Lhcx1_wild-type-rev and Lhcx1_all-fw/Lhcx1-all_rev. PCR products were separated on 1% agarose gels.Lhcx1 and Lhcx2 in Pt4 and in order to verify the biallelic knockout of the Lhcx1 gene in the x1KO lines, the genomic DNA of Pt4 and the three x1KO strains were isolated using the MasterPure DNA Purification Kit  according to the manufacturer’s instructions and sequenced by Illumina 125 bp paired end sequencing by GATC Eurofins (Germany). Quality control for raw reads was done using FastQC. Low quality reads  were trimmed by FASTQ/A Trimmer (http://hannonlab.cshl.edu/fastx_toolkit/index.html). As no reference genome for Pt4 existed so far, we first produced a new Pt4 reference genome, by assembling and aligning the trimmed reads of Pt4 with Bowtie272 against the available genome of Pt1 CCAP 1055/1 deposited in Ensembl, which is an update of the previously deposited genome in JGI36. Then, the SAM files created by Bowtie2 were converted into BAM files using SAM tools73 and a new consensus sequence for Pt4 was created by aid of the BCF tools pipeline74. Finally, the quality controlled and trimmed reads of the x1KO mutants were mapped against the reads of the new Pt4 reference genome by aid of Bowtie2.In order to identify the correct DNA sequence for both alleles of a mL−1, and 4 mm NaHCO3 was added. Before starting experiments, cells were acclimated to dim light for 30–45 min. Kinetic measurements were performed by exposing the cells to 10 min of actinic light with an intensity of 1700 µmol photons m−2 s−1, consisting of similar proportions of red and blue light photons, followed by 18 min of recovery conditions with 40 µmol photons m−2 s−1. Saturating flashes  were applied every 30 s. For some experiment, DTT  was added 5 min prior to fluorescence recording. To monitor acclimation under prolonged high light, cells were exposed for 130 min to 1700 µmol photons m−2 s−1, followed by 30 min recovery conditions with 40 µmol photons m−2 s−1. NPQ was calculated as Fm/Fm’−1. Here, we set Fm to the first light step Fm’, as this value is usually higher than the Fm obtained for dark acclimated P. tricornutum cells.Pre-screening of mutants was performed with an Aqua-Pen  or an Imaging PAM . Fine fluorescence kinetics were recorded with a Dual-PAM or a Fluorescence Induction and Relaxation instrument (a mini-FIRe). For the Dual-PAM measurements, cells were concentrated to 10 µg chlorophyll 75. Fluorescence induction was achieved with blue light emitting diodes  and measured in the red light region . An 80 μs pulse, with peak optical power of 1 W/cm2, ensured that all PSII reaction centers were reduced with a single turnover of PSII. From this single turnover protocol, parameters such as σPSII, electron transfer rates, NPQ and Y(NPQ) can be calculated46. For all strains, these parameters were measured in response to increasing actinic light in 15 steps with 1 min acclimation at each step , where variable fluorescence (resulting in F’ and Fm’) was recorded at the end of each light step. In addition, after each light step, the actinic light was turned off for 1 s to allow for re-opening of reaction centers. This 1 s dark period was short enough to prevent NPQ relaxation14 but did allow us to directly measure Fo’, which is needed for the calculation of 1−qL. Electron transfer rates were calculated from E x σPSII’×(Fv’/Fm’)/(Fv/Fm)6. Y(NPQ) and 1−qL were calculated following9. σPSII’ was derived from F’→Fm’ transition induced by an 80 µs single turnover flash76. Using the F’→Fm’ transition allowed us to make more acquisitions, improving the signal-to-noise ratio, and reducing the error in the fitting procedure. A comparison of σPSII’ and σPSII’1s  showed little difference in the values or the interpretation of our results  and maximal (Fm) fluorescence, corresponding to states where all PSII reaction centers are open or closed, respectively, as well as the functional absorption cross-sections of PSII, were measured using a mini-FIRecf. Fig. .46. We measured lifetimes for Fo and Fm levels in the dark, followed by measurements of Fo’ and Fm’ lifetimes during exposure to 850 μmol photons m−2 s−1 (blue light) for 10 min to induce NPQ. The fluorescence acquisition interval was 20 s, short enough to avoid changes in the state of the photosynthetic units. Average fluorescence lifetimes were calculated by fitting the collected data to a sum of three exponential decays, which were deconvoluted from the instrument response function46.Fluorescence lifetimes were measured using a custom-built picosecond lifetime fluorometer as described in ref. 77. In short, pigments of cells filtered on a 1.2 µm isopore filters (Millipore) were extracted with a mixture of 81% methanol/9% 0.2 m ammonium acetate/10% ethyl acetate (vol/vol/vol). After centrifugation, the supernatant was injected onto a calibrated Hitachi LaChrom Elite HPLC system equipped with a 10 °C-cooled autosampler and a Nucleosil 120-5 C18 column . Pigments were separated using a linear gradient system consisting of eluent A  and eluent B .Pigment extraction and HPLC separation followed the protocol established byLhcx transcript level of light-stressed cells, the different strains were concentrated to 2 µg mL−1 chlorophyll a, treated with 0.8 mm NaHCO3 and exposed for 2 hours to ~700 µmol photons m−2 s−1 white light in a glass cylinder under constant water-cooling and stirring at 20 °C. For the qPCR analyses, the reference gene was 18S_rRNA (Ensembl Gene ID: EPrPhatr3G00000013183) as it is well-suited for both low and high-light acclimated P. tricornutum cultures33. Primer sequences for Lhcx1, Lhcx3, Lhcx4, and 18S were the same as in ref. 33 and are indicated in Supplementary Table Lhcx2 we used Lhcx2_qPCR_fw and Lhcx2_qPCR_rev . Each strain was measured in biological triplicates, and on top each gene per sample was measured three times. Cycle threshold values and gene amplification efficiencies were obtained by utilizing PCR Miner 4.078. Relative transcript levels were calculated according to79.RNA extraction was performed using a combination of Peqgold RNAPure and Peqgold Total RNA Kit S-Line with on column DNA digest using the Peqgold DNAse I Digest kit . cDNA synthesis was performed by means of the Primescript kit . RNA was extracted from LL grown cultures in the exponential growth phase. In order to assess 67, but using 14% lithium dodecyl sulfate-polyacrylamide gel electrophoresis for protein separation. Samples corresponding to an amount of 1 μg chlorophyll a were loaded on the gel. Proteins were blotted on a Bio-Trace PVDF  or an Amersham Protran nitrocellulose membrane  using the semidry blotting technique by means of a Biorad Trans-Blot Turbo system . Previously, either an anti-Lhcsr, raised against a recombinant Chlamydomonas Lhcsr protein, had been used to quantify Lhcx protein expression in P. tricornutum34 or it had been the anti-FCP6 raised against a specific C-terminal peptide of the FCP6 protein in Cyclotella cryptica35. We designed a new polyclonal anti-P. tricornutum-Lhcx  raised against the peptide MAQELVNGKGILEHL, because 14 , 13 (Lhcx2), and 12 (Lhcx4) out of these 15 amino acids are contained specifically in the C-terminus of the respective Lhcx proteins in P. tricornutum, thus making this antibody superior in detecting the different Lhcx proteins with a similar affinity. Anti-Lhcx was applied in 1:10,000 dilution overnight. Anti-Rubisco  served as a loading control. After binding of the secondary antibody for 1 h  HRP conjugated, AS09 602, Agrisera, Sweden; 1:10,000 dilution for Lhcx, 1:20,000 for Rubisco), signals were detected using Roti-Lumin Plus  in an Odyssey FC Imaging System .Protein extraction and separation followed the protocol described by ref. P value ≤ 0.05 was determined with a two-tailed unpaired Student’s t test calculated with SigmaPlot 12 on biological replicates as indicated in the respective legends. For gene expression we used the Pairwise Fixed Reallocation Randomization Test performed by REST2006 according to80 with 2000 randomizations.Significance with a Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


Blood eosinophil count (BEC) and fractional exhaled nitric oxide (FeNO) concentration are established biomarkers in asthma, associated particularly with the risk of exacerbations. We evaluated the relationship of BEC and FeNO as complementary and independent biomarkers of severe asthma exacerbations.9 cells/L; non-high: < 0.300 × 109 cells/L). Categorisation 2  was based on prior research. Reference groups included patients with neither biomarker raised.This observational study included data from the Optimum Patient Care Research Database. Asthma patients (18–80 years) with valid continuous data for 1 year before FeNO reading, ≥ 1 inhaled corticosteroid prescription, and BEC recorded ≤ 5 years before FeNO reading were separated into cohorts. Categorisation 1 was based on the American Thoracic Society criteria for elevated FeNO concentration  and BEC  had a numerically greater exacerbation rate  compared with patients in the reference group. Combination of high FeNO and high BEC (n = 27) resulted in a significantly greater exacerbation rate . Similarly, for categorisation 2, when both biomarkers were raised (n = 53), a significantly greater exacerbation rate was observed .The combination of high FeNO and high BEC was associated with significantly increased severe exacerbation rates in the year preceding FeNO reading, suggesting that combining FeNO and BEC measurements in primary care may identify asthma patients at risk of exacerbations.The online version of this article (10.1186/s13601-019-0282-7) contains supplementary material, which is available to authorized users. Asthma, a chronic inflammatory disorder of the airways affecting more than 315 million people worldwide, is associated with considerable morbidity, mortality, and loss of productivity . ApproxiBlood eosinophil counts and fractional exhaled nitric oxide (FeNO) concentrations are established biomarkers in asthma. A high blood eosinophil count, used as a marker for eosinophilic airway inflammation, correlates well with poor asthma control, an increased risk of severe exacerbations, and re-hospitalisations –14. ConvAlthough both blood eosinophil count and FeNO concentration are associated with eosinophilic airway inflammation, they demonstrate only a modest correlation, reflecting different parts of the T2-driven inflammation –26. NotaAnti-interleukin-5 treatment with mepolizumab lowered blood eosinophil counts without affecting FeNO concentrations , while bThis cross-sectional study was conducted using patient data from the Optimum Patient Care Research Database (OPCRD). The OPCRD is a primary care database containing high-quality anonymised data obtained from longitudinal medical records and patient-completed questionnaires in the UK health care system . Patient9 cells/L. Categorisation 1 included three cohorts: high FeNO (≥ 50 ppb) and high blood eosinophil count (≥ 0.300 × 109 cells/L), high FeNO alone (≥ 50 ppb) or high blood eosinophil count alone (≥ 0.300 × 109 cells/L), and non-high FeNO (< 25 ppb) and non-high blood eosinophil count (< 0.300 × 109 cells/L) (reference group). Categorisation 2 included four cohorts: high FeNO (≥ 35 ppb) and high blood eosinophil count (≥ 0.300 × 109 cells/L), high FeNO (≥ 35 ppb) and non-high blood eosinophil count (< 0.300 × 109 cells/L), non–high FeNO (< 35 ppb) and high blood eosinophil count (≥ 0.300 × 109 cells/L), and non-high FeNO (< 35 ppb) and non-high blood eosinophil count (< 0.300 × 109 cells/L) (reference group).Patients were classified based on their FeNO reading on the index date and the closest blood eosinophil count reading Fig. . Two setThe study period, during which both patient characteristics and outcomes were observed, consisted of the year prior to the latest documented FeNO reading.The study population consisted of patients who met the following criteria: age 18–80 years inclusive; a diagnostic Read code for asthma qualifying for inclusion in the asthma patient registry, which general practices in the UK maintain for the Quality Outcomes Framework ; active Patients were excluded from the study if they had a diagnosis Read code for chronic obstructive pulmonary disease or any chronic respiratory disease other than asthma; received a long-acting muscarinic antagonist or were prescribed maintenance oral corticosteroids (OCS); and had a forced expiratory volume in 1 s/forced vital capacity < 0.7.The primary outcome was the annual rate of severe asthma exacerbations, defined as the number of severe exacerbations in the study period per patient. A severe exacerbation was defined in line with the European Respiratory Society/ATS Position Statement  as an acSecondary outcomes included a description of demographics, lung function, comorbidities, respiratory medication use and ICS adherence for each of the patient groups characterised by biomarker concentrations. ICS adherence was defined using Medication Possession Ratio, calculated by dividing the total of 1 day’s supply by the total number of days evaluated (365 days in the study year), and expressed in percentage.All statistical analyses were conducted using Stata SE version 14.2 and R version 3.0.2.The sample size was calculated by accounting for multiple testing with a Bonferroni correction. With four comparisons and an alpha significance level of 0.0125, 800 patients were initially deemed necessary to demonstrate at least a 20% difference between groups, with a 90% power. However, this was later revised to detect a difference in a single outcome only, namely, a 20% difference in exacerbation rate between two groups of interest.t test was used to compare a continuous variable between two groups, with a non-parametric Mann–Whitney test used for small numbers of observations per group. Summary statistics were presented as counts and percentages. For missing data, percentages for categorical variables were provided as a percentage of the non-missing observations. A statistically significant result was defined as a p ≤ 0.05.Comparisons were initially unmatched for the purpose of exploring the main differences between patient groups and providing the steering committee with data in order to make a decision on which patient groups to compare. In addition, multivariate regression models were fitted to account for potential confounding of patient characteristics that may have varied between patient groups. Standardised mean difference was calculated to measure effect size. Characterisation and subsequent matched analyses of study outcomes were performed based on categorisations 1 and 2. Descriptive statistics of all characteristics were computed for each group of patients within the cohorts. Continuous variables were summarised using the number and percentage of non-missing observations, mean and standard deviation (SD) for normally distributed variables, and median and interquartile range (difference between the 25th and 75th percentiles) for non-normally distributed variables. Pearson’s Chi square test was used to compare percentages between different groups, with a Fisher’s test used in cases of small numbers of observations per group. Student’s The primary analysis for categorisation 1 compared the number of severe exacerbations for matched patients with a high FeNO and high blood eosinophil count with that of patients with a non-high FeNO and non-high blood eosinophil count (reference group). The rate of severe exacerbations was also compared between matched patients with a high FeNO or a high blood eosinophil count vs. the reference group. The analysis for categorisation 2 compared patients with a high FeNO and high blood eosinophil count vs. the reference group, a high blood eosinophil count alone vs. the reference group, and a high FeNO alone vs. the reference group.Conditional Poisson regression analysis was performed to estimate the rate ratio (RR) between the groups of interest, with a 95% confidence interval (CI). Unadjusted RRs were calculated based on previous knowledge of multivariable prediction models , 35.At the time of the study, the OPCRD contained more than 2.4 million available patient records from more than 560 practices across the UK  were matched 1:1 with the reference group (non-high FeNO and non-high blood eosinophil count).A total of 27 patients in the high FeNO and high blood eosinophil cohort matched with the reference group Table . OverallDemographics and clinical characteristics were generally similar between the matched groups. However, standalone ICS prescriptions were significantly fewer in the high FeNO or high blood eosinophil cohort compared with the reference group . Adherence to ICS was comparable between matched groups and was 52.3% and 63.3% in the high FeNO and high blood eosinophil cohort and high FeNO or high blood eosinophil cohort, respectively.Across the biomarker cohorts, more than 50% of patients were female, with most patients aged 35–65 years. Non-smokers represented 36–58.5% of the study sample.2, p = 0.0492; 26.9 vs. 29.3 kg/m2, p = 0.0063; and 26.8 vs. 29.0 kg/m2, p = 0.0386, respectively). All other baseline demographics were well-balanced between the matched groups    were almost four-times as likely to have had a severe exacerbation compared with patients with non-high FeNO (< 25 ppb) and non-high blood eosinophil count (< 0.300 × 109 cells/L) in the year preceding the FeNO reading. In patients with either a high FeNO reading or a high blood eosinophil count, the exacerbation RR was less pronounced and non-significant compared with the reference group. In categorisation 2, patients in the high FeNO (> 35 ppb) and high eosinophil count (≥ 0.300 × 109 cells/L) cohort were almost twice as likely to have severe exacerbations in the year prior to the FeNO reading compared with the reference group, whereas the high FeNO and non-high blood eosinophil count cohort and non-high FeNO and high blood eosinophil count cohort displayed a trend towards increased exacerbations relative to the reference group, that did not reach statistical significance. Therefore, the combination of blood eosinophil count and FeNO may be an even stronger marker of exacerbation risk compared with the individual biomarkers. Moreover, the use of the ATS criteria for high FeNO (≥ 50 ppb) resulted in a greater estimated exacerbation rate, indicating that a greater FeNO reading (≥ 50 ppb vs. ≥ 35 ppb) in the presence of a raised blood eosinophil count was associated with an even greater exacerbation rate. Notably, the exacerbation risk seemed to be independent of traditionally used prognostic variables such as predicted peak flow and short-acting β2-agonist use, which were not significantly different between cohorts.We observed that for categorisation 1, based on ATS criteria for FeNO cutoffs, patients with a high FeNO (≥ 50 ppb) and high blood eosinophil count (≥ 0.300 × 109 cells/L) was well within the range of peripheral blood eosinophils  that most accurately predicts sputum eosinophil count in patients with severe asthma [The cutoffs used in the study to define high FeNO concentration and high blood eosinophil count warrant further consideration. The cutoff chosen for high blood eosinophil count (≥ 0.300 × 10e asthma . For FeNe asthma , 23, 37.e asthma , 32. TheFew studies have evaluated the predictive value of the combination of blood eosinophil count and FeNO concentration in asthma. However, available studies have demonstrated that combining FeNO and blood eosinophil count has an additive effect in predicting wheeze, frequent exacerbations, impaired lung function, and bronchial hyper-responsiveness , 38. TheOther well-characterised risk factors for asthma exacerbations include prior exacerbations, OCS use, and underlying lung function impairment , 42. TheThis study has several limitations. The power analysis performed at the protocol stage demonstrated that more patients were required for sufficient power to demonstrate a difference between four groups than were available. Secondly, the OPCRD data set comprised information collected for clinical and routine use rather than specifically for research purposes. Although extensive quality control and validity checks were conducted at the practice level, the validity and completeness of individual patient records can be limited. Since blood eosinophil measurements and FeNO readings are not collected routinely, patients with asthma who had both blood eosinophil counts and FeNO measured may not have been representative of the overall asthma population. In addition, the time from when the blood eosinophil count reading was taken to the index date varied considerably. Although high blood eosinophil counts have been observed to be a stable phenotype, at least during a 1-year period , furtherResults of this study need to be confirmed in a prospective study in a larger patient population before high FeNO concentrations and high blood eosinophil counts can be advocated as a composite biomarker. Notably, patients with elevated FeNO concentration on a background of high blood eosinophil counts represent a potentially high-risk group of patients. Such severe asthma patients will benefit from studies conducted in larger epidemiological cohorts in primary care settings, as well as in severe asthma cohorts, such as the International Severe Asthma Registry , a globaThe combination of raised FeNO concentrations and raised blood eosinophil counts was associated with a greater exacerbation rate compared with neither biomarker raised in the year preceding the FeNO reading. FeNO concentration and blood eosinophil count are simple measurements that could, together, improve the identification of patients with asthma in primary and secondary care at risk of exacerbations, and thus, guide additional considerations in the treatment of their asthma.Additional file 1. Statistically significant differences between unmatched patient groups for categorisation 1 and 2 and frequency of exacerbations between matched biomarker groups.


Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) belong to the same peptide family and exert a variety of biological functions. Both PACAP and VIP have protective effects in several tissues. While PACAP is known to be a stronger retinoprotective peptide, VIP has very potent anti-inflammatory effects. The need for a non-invasive therapeutic approach has emerged and PACAP has been shown to be retinoprotective when administered in the form of eye drops as well. The cell penetrating peptide TAT is composed of 11 amino acids and tagging of TAT at the C-terminus of neuropeptides PACAP/VIP can enhance the traversing ability of the peptides through the biological barriers. We hypothesized that TAT-bound PACAP and VIP could be more effective in exerting retinoprotective effects when given in eye drops, by increasing the traversing efficacy and enhancing the activation of the PAC1 receptor. Rats were subjected to bilateral carotid artery occlusion (BCCAO), and retinas were processed for histological analysis 14 days later. The efficiency of the TAT-bound peptides to reach the retina was assessed as well as their cAMP increasing ability. Our present study provides evidence, for the first time, that topically administered PACAP and VIP derivatives (PACAP-TAT and VIP-TAT) attenuate ischemic retinal degeneration via the PAC1 receptor presumably due to a multifactorial protective mechanism. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) belong to the same peptide family. PACAP exists in 27 and 38 amino acid forms, and the shorter peptide shows 67% homology with VIP. They also share their receptors: VPAC1 and VPAC2 receptors bind both VIP and PACAP. However, PACAP also has a specific PAC1 receptor, which only binds PACAP. PACAP has a widespread occurrence in the body and a broad array of functions  and VIP-TAT (tagging TAT at the C-terminus of VIP) were chemically synthesized by GL Biochem Ltd. .g, 10 min) the peptide was subjected to fluorescence measurements performed with the multi-wavelength scanner Victor 3  at the excitation of 495 nm and the emission of 520 nm. Protein concentrations were determined using the K4000 Bradford Protein Quantification Kit . The labeling efficiency was calculated using the following formula: label efficiency (LE) = fluorescence value (FV)/peptide mass (mol) (PM), representing the fluorescence intensity (AU) per mol of peptide (mol).In order to trace the drugs, peptides were labeled with fluorescein isothiocyanate (FITC) using a FITC Protein Labeling Kit from ChangRu Biotech Ltd.  according to the manufacturer’s protocol. After the labeling reaction, gel filtration was used to remove the free FITC. In order to determine the amount of the residual free FITC, the peptides were submitted to ultrafiltration using Amicon Ultra – 0.5 mL  with a molecular sieve of 2000 Da. After centrifugation . Rats were randomly assigned to one of the experimental groups (7 rats per group) and subjected to eye drops with FITC labeled peptides (100 nmol/kg) and PBS as control. Rats were sacrificed by anesthesia 2 h after the eye drop administration and the retina was separated, weighed, washed three times with PBS and divided into two parts. One part was prepared on the glass slide with glycerin and subjected to fluorescence microscopic observation of FITC with 495 nm excitation/520 nm emission. All images, focused on the left upper regions of the retina, were taken with 500 ms exposure time. The other part of the retina was subjected to grinding and ultrasonication in PBS at a concentration of 100 mg weight tissue per milliliter of PBS. The supernatant was collected by centrifugation and the fluorescence intensity in the supernatant (100 uL) was determined. The valid fluorescence intensity (FI) for each sample treated with FITC labeled peptide was corrected by subtracting the fluorescence value of the sample treated with PBS, which was used as a blank background. The Efficiency of Traversing Eye to Retina (EtE) was expressed as the percentages of the FITC labeled peptide mass in the retina to the total FITC labeled peptide mass. The EtE was calculated using the following formula: EtE = tFI/LE/PW × 100% ; LE presents the label efficiency of each peptide which has been determined above; PW presents the peptide mass (mole)).6 /mL. Peptides were added to 500 uL cells suspension with the corresponding varying working concentrations of the detected factor. After incubation at 37 °C for 5-10 min cells were harvested and the lysates were subjected to cAMP quantification using the enzyme immunoassay kit for cAMP , following the manufacturer’s instructions. Protein concentration of each sample was determined using BCA assay, and the cAMP level of each sample was calculated following the formula: cAMP level (pmol/mg protein) = cAMP concentration (pmol/mL)/protein concentration (mg/mL). The cAMP level in each sample was plotted as the percentage (%) of the maximal cAMP level in cells treated with PACAP27 versus the logarithmic value of the peptide concentrations. All experiments were run with at least four parallel samples and were repeated three times.PAC1-CHO cells cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) at 37 °C were scraped off the surface with rubber policeman, washed twice with PBS and the density of the cells was adjusted to 2 × 10n = 17/ or VIP-TAT /n = 17/) eye drops (1 μg/drop). Dose and schedule of the eye drop treatments were based on our previous experiments . Under isoflurane anesthesia, common carotid arteries were exposed on both sides through a midline incision and then ligated with a 3–0 filament. A group of animals (sham group) underwent all steps of the operating procedure except ligation of the carotid arteries. Immediately following the operation, the right eye of the animals was treated with derivatives of PACAP (PACAP-TAT /n = 10 SHAM and n = 24 BCCAO) were killed with anesthetic and the eyes were processed for histology. The eyes were removed and the retinas were solved in phosphate buffered saline (PBS), fixed in 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (PB)  and embedded in Durcupan ACM resin . Retinas were cut at 2 μm and stained with toluidine blue dye . Sections were mounted in DPX medium  and photographs were taken with a digital CCD camera using the Spot program. Central retinal areas within 1 mm from the optic nerve were used (n = 5 measurements from one tissue block). The following parameters were measured: (i) cross-section of the retina from the outer limiting membrane (OLM) to the inner limiting membrane (ILM), (ii) the width of individual retinal layers , (iii) the number of cells/100 μm section length in the GCL, and the (iv) number of cells/1 μm2 in the OPL and in the IPL. Results are presented as mean ± SEM. Statistical comparisons were made using the two-way ANOVA test followed by Tukey’s post hoc analysis.Fourteen days after the operation, rats  was significantly less than in control retinas Figs.  and 4. PIn the present study we demonstrated the efficacy of TAT-bound PACAP and VIP peptides to reach the retina and exert a retinoprotective effect in a model of ischemic retinopathy in rats. The retinoprotective effects of PACAP are well-documented in models of many different retinopathies . However, in our present study, we confirm that VIP bound to TAT peptide could effectively traverse the ocular barriers and exert a neuroprotective effect in the retina. PACAP-TAT did not prove to have significantly higher retinoprotective efficacy than untagged PACAP, but VIP exerted much stronger retinoprotective effects when bound to TAT. These results were consistent with our previous report that TAT with similar structure with PACAP(28–38) endowed VIP with higher affinity for PAC1-R  dissolved in SOCB attenuate ischemic retinal degeneration via the PAC1 receptor presumably due to a multifactorial protective mechanism.


Right ventricular apical pacing with the resultant left ventricular dyssynchrony often leads to depressed systolic function and heart failure. This study aimed at investigating the relation between various septal locations guided by ECG and fluoroscopy and the intermediate term functional capacity of the patients.n = 15), group 2 “mid septum” (n = 25), and group 3 “low septum” (n = 10) using QRS vector and duration as well as fluoroscopic parameters. Their clinical status was assessed 6 months after device implementation using 6-min walk test (6MWT).Fifty patients who received a single lead pacemaker with assumed > 90% pacemaker dependency. Patients were randomized according to RV pacing site RV into group 1 “high septum” , although its combination with the paced QRS complex vector can signify the optimal pacing site and 6MWT showed a significant difference among the groups in favor of group 1; group 1 (413.3 ± 148.5), group 2 (359.8 ± 124.6), and group 3 (276.0 ± 98.5) P value 0.04.The study showed that paced QRS complex duration itself has no significant difference between the different septal pacing locations (There was a significant difference found between the three septal pacing sites concerning the patient functional capacity with superiority of high septal location. By contrast, different septal sites showed no significant difference of the paced QRS complex duration. To optimize the pacing site in the septum, assessment of the paced QRS vector in leads I and III is of a great benefit especially when combined with paced QRS complex duration assessment. Right ventricular (RV) apical pacing allows for safe and stable long-term pacing. However, it induces non-physiologic left ventricular (LV) activation with alteration of the intraventricular contraction sequence, which delays LV activation . This deTo overcome this potential limitation, other pacing sites throughout the whole septal segments have been tried . The sepThe degree of electrical dyssynchrony induced by RV pacing can be estimated by the duration of the paced QRS. Moreover, some studies have demonstrated that patients with a long paced QRS may be at high risk of adverse cardiac events including heart failure. A long paced QRS can result despite the non-apical placement because the lead might pace a non-favorable septal segment .The aim of our study is to investigate the relation between various septal locations guided by ECG and fluoroscopy and the intermediate term functional capacity of the patients through illustrating the relationship between different septal positions and paced QRS complex duration and the relationship between the paced QRS vector and duration of the paced QRS complex.This cross-sectional study conducted during the period from October 2017 to July 2019 at our cardiology department on 50 patients having a primary implant single lead VVI pacemaker following the recent guidelines for cardiac pacing with high probability of device dependency (more than 90%) excluding those with significant valve disease , recent (within 3 months) acute coronary syndrome, planned cardiac surgery , and ejection fraction of left ventricle less than 50%.A detailed informed verbal consent has been taken from all participants as it was just an observational study and this consent was approved by our ethics committee.Fifty patients were divided into three groups depending on the septal location with fluoroscopy guidance: group 1 high septum, group 2 mid septum, and group 3 low septum.All participants were subjected to a follow up visits for 6 months after implantation of the device. A detailed history taking, physical examination, ECG data (including assessment of paced QRS complex duration and vector configuration), and assessment of their functional status using 6MWT were done in our cardiology department.To facilitate lead placement in the RV septum, a 3 × 3 square grid is created for each patient using a fluoroscopic image in the right anterior oblique (RAO) 10° view using landmarks to demarcate the borders of the grid as shown in Fig.  6]Fig.Fig.6]FAll ECG records are made using an identical ECG device (Nihon Kohden Cardiofax C ECG-1150. Japan) of limb leads I, II, and III at speed 50 mm/s at a ventricular rate less than 80 bpm. The paced QRS complex duration is calculated from the end of the pacing spike to the latest deflection of the QRS complex in any limb lead. The non-paced QRS complex duration is measured from the earliest deflection to the latest deflection of the QRS complex in any limb leads. Optimization of the pacing site of the RV septum occurred by the maximal shortening of the QRS complex duration.Assessment of the paced QRS complex vector is simplified to determine the R/S voltage ratio in leads I and III. Three categories are defined positive, isoelectric, and negative. A positive vector is determined as R-S ratio > 1/2 R. An isoelectric vector is determined as R-S ratio < 1/2 R or S-R ratio < 1/2 S. A negative vector is defined as S-R ratio > 1/2 S. Then, they are divided into three subgroups: (1) negative or isoelectric in lead I plus positive in lead III. (2) Isoelectric or negative in lead III. (3) Positive in lead I plus positive in lead III .Standard descriptive statistics were used for the analysis; continuous parameters of QRS duration were described by means and standard deviation, while occurrences of categorical parameters  were described by count and percentages.Differences between parameters of QRS duration and vector according to several factors and their combination were assessed by ANOVA and also by chi-square tests. For the studied groups, statistically significant differences at the level of significance of 0.05 are stated.Post hoc tests analysis was done over the paced QRS complex vector result and also the 6MWT result to compare each group with the other group specifically.For data analysis, the SPSS Statistics 17.0 for Windows  was used and α = 0.05 was considered as the level of statistical significance in all analyses.n = 15), group 2 “mid septum” (n = 25), and group 3 “low septum” (n = 10). There are three patients who were excluded through the follow-up period and the reasons were death, ischemic cerebral stroke, and non-compliance. No other complications have been reported.Fifty patients were divided according to the RV lead pacing site into group 1 “high septum” (The demographic characteristics of the 50 patients are shown in Table . Risk fan = 19), B “isoelectric or negative in lead III” (n = 15), and C “positive in lead I and positive in lead III” (n = 16).All patients were subjected to vector configuration using leads I and III and classified into to three configurations accordingly: A “negative or isoelectric in lead I and positive in lead III”  counting the meters the patient can walk freely through the examination time and taking its all precautions in consideration.The septal position of the pacing lead in the right ventricle is a preferred alternative pacing site given the potential contribution of limiting LV dyssynchrony compared to RV apex stimulation. Historically, the decision of where in the septum the lead should be placed is individual, with the selection being corrected according to the paced QRS complex duration .n = 15), group 2 “mid septum” (n = 25), and group 3 “low septum” (n = 10), all of them were subjected to follow-up for 6 months after VVI pacemaker implantation.In our study, 50 patients were divided according to the RV lead pacing site by new documented fluoroscopic method into group 1 “high septum”  with no significant difference between the groups.According to the functional capacity assessment, there is significant difference that was found between the three septal pacing site  with significant superiority of high septal location.By contrast, different septal sites defined by the mentioned fluoroscopic method in the RAO 10° view showed no significant differences in paced QRS complex duration. But, assessment of the paced QRS vector in leads I and III is of a great benefit especially when combined with paced QRS complex duration assessment as group C of the QRS vector configuration showed significant longest QRS duration.According to the paced QRS complex duration, there is no significant difference among the groups concerning the paced QRS duration, which was in agreement with  whose RCAnd we disagree with another randomized controlled trial by  showed tn = 45) or interventricular septum (n = 34) under fluoroscopic guidance. The results showed that the QRS duration was significantly increased in both groups after pacing, but the difference between the pre- and post-pacing QRS duration was significantly higher in apical pacing group (57.1 ± 28.3 versus 32.8 ± 40.5 ms). This study concluded that the apical pacing has a higher probability of more LV dyssynchrony after PM implantation.Another multi-center prospective study comparing the ventricular dyssynchrony according to the position of right ventricular pacing electrode  where paRegarding the QRS vector configuration our study show high statistical significant difference among the groups in favor of group I high septum which goes with the result of a meta-analysis by  concerning the patient functional capacity with superiority of high septal location.By contrast, different septal sites defined by the mentioned fluoroscopic criteria in the RAO 10° view showed no significant difference of paced QRS complex duration.To optimize the pacing site in the septum, assessment of the paced QRS vector in leads I and III is of a great benefit especially when combined with paced QRS complex duration as when the vector configuration was “positive in lead I and positive in lead III,” it showed significant longest QRS duration.


Lipoprotein glomerulopathy (LPG) is a rare kidney disease with a poor prognosis that is related to mutation of the apoE gene. More than 10 variants of apoE associated with LPG have currently been identified.A male and his mother presented with proteinuria during a health examination. They went to hospital for further examination. Renal biopsy was performed, and the diagnosis was lipoprotein glomerulopathy (LPG), which is a rare, inherited renal disease. Medical histories were collected from the 2 LPG patients and their family members. The patients and family members underwent a routine urine test, and their renal function, blood lipids, and lipoprotein levels were examined. Genomic DNA was extracted from the peripheral blood of 7 family members, and exon 2, exon 3 and exon 4 of apoE were amplified by polymerase chain reaction (PCR). The purified PCR products were sequenced. Sequence analysis identified a 15 bp deletion (GCGCAAGCTGCGTAA) in exon 4 of the apoE gene that results in a novel 5 amino acid deletion in apoE (143 K-147R → 0). No mutations were found in exon 2 and exon 3 of the apoE gene.This family study suggests that a novel ApoE mutation (143 K-147R → 0) may be etiologically related to LPG, and other genetic or environmental factors may be associated with the occurrence of LPG. Lipoprotein glomerulopathy (LPG) is a kidney disease associated with abnormal lipid metabolism. It was first reported by Saito et al  in 1989 We enrolled a family with 8 members in total, 2 of which were diagnosed with LPG. The proband’s wife Fig. , II2 wasThe proband’s mother had two sisters and two older brothers diagnosed with uremia, of which the eldest brother had died, and their specific medical histories were unknown. The proband’s younger brother Fig. , II3 exhTo further study genetic mutations in the family, we sequenced exons of the APOE gene in its members  in exon 4 of the apoE gene in the proband resulting in a 5 amino acid deletion in apoE (143 K-147R → 0)  deletion in exon 4 of the apoE gene was discovered in 5 family members , resulting in a 5 amino acid deletion in ApoE (143 K-147R), that has not been reported in previous literature and databases. This mutation may be the molecular basis for the pathogenesis of lipoprotein glomerulopathy in this family.


DNA extraction is an essential procedure when investigating microbial communities in environmental samples by sequencing technologies. High clay soils can be problematic as DNA adsorbs to the clay particles and can thereby be preserved from lysed, non-viable cells for a substantial period of time. In order to accurately estimate the intact and living microbial community in the soil, extracellular DNA from dead, remnant bacterial cells needs to be removed prior to DNA extraction. One possibility is to use a sodium phosphate buffer to release both extracellular DNA and bacterial cells from the clay particles. After removing the extracellular DNA by centrifugation, the remaining viable cells can be harvested and DNA extracted. The described method is a modification of a procedure for separating extracellular DNA and bacterial cells from acidic clay soils.•The modified method increases bacterial cell yields from acidic clay soils, such as acid sulfate soil.•The modified method eliminates some steps from the original method, as only DNA from intact bacterial cells is required.•The indirect DNA extraction method increases the workload compared to standard direct extraction methods, but subsequent downstream analyses will give a more representative picture of the viable microbial community composition in the soil. Co-extraction of both intracellular DNA from living bacterial cells and extracellular DNA (eDNA) from dead cells by using standard direct DNA extraction methods can result in a considerable overestimation of the current, living microbial community in soil . When wo2HPO4 and 500 mM NaH2PO4, pH 7.2)Sodium phosphate buffer  is added to the beaker.3.The slurry is stirred for 5 min at 250 rpm.4.The beaker is chilled for 3 min at 4 °C.5.The slurry is stirred again for 5 min at 250 rpm.6.The slurry is transferred to a 50 mL sterile tube and mixed by vortex for 15 s.7.g for 15 min.The tube is centrifuged at 5008.The supernatant is carefully poured into a separate 50 mL sterile tube and stored in the dark at room temperature.9.12 mL of fresh sodium phosphate buffer is added to the pellet and the pellet is suspended by careful repeated pipetting using a 5 mL pipet. The slurry is transferred back to the beaker and the procedure described above (steps 3–8) is repeated a further two times.10.All supernatants are collected in the same 50 mL tube and the final soil pellet is discarded.11.g for 15 min to separate the cells from the suspension. Extracellular DNA is left in the supernatant.The pooled supernatant (approximately 30 mL) is divided into sterile 2.0 mL tubes and centrifuged at 10,00012.The supernatants are discarded and the cell pellets washed by pooling them into a 2.0 mL tube with 1 mL sodium phosphate buffer to remove excess eDNA.13.g for 15 min and the supernatant is discarded.The tube is centrifuged at 10,00014.If DNA is extracted using a commercial DNA isolation kit, the cell pellet is transferred to the kit́s bead tube using the bead tube buffer and the DNA is extracted according to the manufactureŕs instructions.15.The indirectly extracted DNA can now be used for suitable downstream analyses.1.The autoclaved sodium phosphate buffer is best stored in room temperature as it quickly crystallizes in lower temperatures.2.If soil samples with higher salt concentrations are used, 2% NaCl can be added to the sodium phosphate buffer to reduce the osmotic stress on the bacterial cells.3.Sometimes the final cell pellet after the washing step (points 12–13 in the protocol) can be hard and difficult to suspend in the bead tube buffer (point 14 in the protocol). To avoid losing cell material, pool the cell pellets in up to four tubes/sample instead of one tube to reduce the pellet amount per tube.4.The used commercial DNA isolation kit was the DNeasy Power Soil Kit (Qiagen).A260/280 = 1.97–2.09 and A260/230 = 0.79–1.18) while the direct extraction using solely the DNeasy PowerSoil Kit produced 1.41 ± 0.36 ng DNA/μL (A260/280 = 1.73–2.49 and A260/230 = 0.43–1.11). Purity of extracted DNA (Acidithiobacillus sp. (OTU_000931) and Ferrithrix sp. (OTU_001121) is furthermore higher in indirect extracted samples, which is logical since the samples were taken from an active acid sulfate soil. More details about the validation of the method can be found in the publication [The indirect DNA extraction protocol has successfully been used to describe the intact microbial community in clay rich acid sulfate soil . The indcted DNA  did not cted DNA . Furthercted DNA . Even wh