The aim of this review was to identify the imaging methods at our disposal to optimally manage posterior uveitis at the present time. The focus was put on methods that have become available since the 1990s, some 30 years after fluorescein angiography had revolutionised imaging of posterior uveitis in particular imaging of the retinal vascular structures in the 1960s. We have focussed our review on precise imaging methods that have been standardised and validated and can be used universally thanks to commercially produced and available instruments for the diagnosis and follow-up of posterior uveitis. The second part of this imaging review will deal with invasive imaging methods and in particular ocular angiography. As indicated in the first part of the review on imaging of posterior uveitis, the huge progress made in imaging techniques in the last two to three decades transformed the appraisal of posterior uveitis into an exact clinical science. The problem however lies in the reluctance to embrace such a new precise and comprehensive approach, perpetuating traditional mostly obsolete approaches promoted by “consensus” conferences and adopted by official health agencies, failing to recommend precise and objective investigations, so not only hampering the diagnosis and management of posterior uveitis worldwide but also hampering the proper design of clinical trials. It is hard to believe that the standard outcome measure for studies on posterior uveitis recommended by the FDA (Food and Drug Administration of the USA) is vitreous haze based on a system proposed more than 40 years ago  and takeHLA-A29 birdshot retinochoroiditis (BRC) is one example of an unsuitable approach because of the publication of inadequate diagnostic criteria . CliniciAnother example is Vogt–Koyanagi–Harada (VKH) disease. Again a consensus workshop failed to include in their report on “the revised diagnostic criteria of VKH” the single most important imaging investigational method, ICGA, for a disease originating exclusively and involving principally the choroid . It is vThe approach of these diseases was biased because the most adequate imaging methods for choroiditis were not taken into account. Today we dispose of an array of precise imaging methods for posterior uveitis, the problem and the enigma being that so many centres often use them with reluctance .The aim of this review will concentrate on the progress made in the precision with which posterior uveitis can be investigated nowadays thanks to new or revisited techniques that have become available for routine use, such as laser flare photometry, optical coherence tomography (OCT) and fundus autofluorescence, presented in the first part of the review on imaging in posterior uveitis, and fluorescein angiography (FA), ICGA and dual FA/ICGA scoring presented in this second part of the review , 10.FA has been practised since more than 50 years. When it was developed by Novotny and Alvis in the early 1960s it represented a major progress in imaging of the posterior segment . Since tWe will not go into the details of this method used and extensively described in innumerable, articles and books during its more than 50 years of use –16.Although the periphery of the retina was always reachable by taking panorama pictures, recently developed wide-field angiographs have facilitated the access to the periphery of retina and have again attracted attention to the angiographic analysis of these areas .We will briefly review its main uses for inflammatory diseases. Because of the physical–chemical properties of the fluorescein sodium molecule (low molecular weight), there is easy extrusion of fluorescein out of inflamed retinal vessels into tissues from which it is however also quickly washed out.The main structures explored by FA and the information asked to FA are exposed hereunder.1. Optic disc: the degree of papillitis and the presence of subclinical disc inflammation, as well as the detection of pre-papillary neovascularisation are advantageously analysed by the type and degree of FA hyperfluorescence.2. Retinal vessels: are best analysed by FA that gives 1), very sensitive information on retinal vasculitis even when it is minimal, (2) displays occlusive vasculopathy of retinal arteries or arterioles  Fig. .Fig. 4FA5. The retinal pigment epithelium (RPE): lesions are well highlighted by FA. Loss of RPE will appear as hyperfluorescent areas as through such “window-defects” the choroidal fluorescein will become visible. In VKH, exudative retinal detachments damage the RPE producing loss of RPE cells on one side and pigment clumps on the other hand, , which will produce an irregular hyperfluorescent (window-defect) and hypofluorescent (masking effect) giving a mottled aspect of the RPE Fig. .6. Choriocapillaris: in the early FA angiographic phase (60″), the amount of fluorescein in the choriocapillaris is such that choriocapillaris perfusion inhomogeneities can be detected which occur in primary choriocapilaritis entities such as APMPPE. Fig. , top In 7. Chorioretinal atrophy: total chorioretinal atrophy is hyperfluorescent on FA, as the small fluorescein sodium molecule is staining the bare sclera, whereas, on ICGA full-thickness atrophy is hypofluorescent as the large protein-bound ICG molecular complex is not diffusing to the non-perfused atrophic areas Fig. .8. Choroidal inflammatory neovascularisation (CNV): can be detected and analysed by FA and represents a further clinicopathological situation usefully investigated by FA.9. Subretinal fibrosis: as can be found in chronic or end-stage VKH disease or in idiopathic multifocal choroiditis causes FA tissue staining, delineating the fibrosis .Ocular angiography comprising Fig.  FA and IBecause fluorescein sodium fluoresces in the wavelengths of the visible light, mainly gives information on the superficial structures of the fundus as indicated in the previous chapter and thus, often only emphasises signs the clinician has already identified, as, chronologically, OCT is usually already available when FA is performed.At the beginning of the 1990-decade ICGA was developed and complemented FA. The dye used, indocyanine green (ICG) has the particularity to fluoresce in the infrared wavelengths and therefore gives images of the choroid to which there was poor imaging access before ICGA. In contrast to FA, ICGA often gives additional information, undetected by clinical examination or FA or OCT. In consequence for a thorough assessment of inflammatory involvement in posterior uveitis, ICGA in most instances appears as unavoidable, as it gives information that would otherwise be lost. It is not rare that ICGA shows occult choroidal lesions and therefore has often a diagnostic value, which is rarely the case for FA. Therefore, in most cases where angiographic work-up is deemed necessary and choroidal involvement cannot be excluded a priori, dual FA and ICGA should be undertaken.ICG fluoresces at 830 nm and therefore is giving access to the choroidal vascular structures through the RPE. The molecular weight difference between ICG  Fig.  and fluoICGA is the method of choice to investigate and follow inflammatory lesions of the choroid. Early frames taken until 2 min (early phase) show the early choroidal circulation Fig. . Two angA standard ICGA protocol to analyse choroiditis has been designed –22. The When analysing ICGA in posterior inflammatory disorders, crucial differences with FA interpretation have to be borne in mind to correctly analyse the images obtained. During initial circulation, ICG is comparable to fluorescein showing the passage through arteriovenous compartments except that it shows superimposed retinal and choroidal circulations. The difference occurs during recirculation time when ICG is progressively leaking out from the fenestrated choriocapillaris, gradually and physiologically impregnating the whole choroidal thickness.This process can be altered in two ways that can be associated in the same disease, either there is a decreased fluorescence or an increased fluorescence .hypofluorescence) either (1) by a decrease of the physiological extrusion of the ICG from the choriocapillaris   Figs.  or (2) bt) Figs. . The lathyperfluorescence) by increased leakage from the larger choroidal vessels which adds to the physiological background fluorescence. The vessels appear fuzzy in the intermediate time frames and extrusion of the dye from large vessels causes late diffuse hyperfluorescence Multiple Evanescent White Dot Syndrome (MEWDS)/Acute Idiopathic BlindSpot Enlargement (AIBSE); Acute Posterior Multifocal Placoid Pigment Epitheliopathy (APMPPE)/AcuteMultifocal Ischaemic Choriocapillaropathy (AMIC) , 31, 32Idiopathic Multifocal Choroiditis  Serpiginous ChoroiditisRare entities: , Acute Zonal Occult Outer Retinopathy (AZOOR)1.2 Secondary Inflammatory ChoriocapillaropathiesAcute Syphilitic Posterior Placoid Chorioretinitis (ASPPC)2.Stromal inflammation  further subdivided into two categories2.1 Primary obligatory stromal choroiditiisVKH disease , 30Sympathetic Ophthalmia Birdshot HLA-A29 retinochoroiditis , 362.2. Stromal choroiditis as a random location of a systemic diseaseSarcoidosis Tuberculosis SyphilisOther infectious choroiditis entitiesTwo main ICGA derived lesion mechanisms determine the classification of choroiditis:This first group of diseases, formerly mostly included in the inadequate term of “white dot syndromes” results from inflammation at the level of the choriocapillaris causing areas of choriocapillaris non-perfusion and its ischaemic consequences both at the level of the choroid but also at the level of the outer retina that depends on the choriocapillaris for oxygen and nutrients . APMPPE Angiographic signs in inflammatory choriocapillaropathies [opathies , 31–33: The hallmark sign of inflammatory choriocapillaropathy is patchy or geographic ICGA hypofluorescent areas of variable sizes present in the early, intermediate and late angiographic phases but usually more clearly visible on the late frames, indicating choriocapillaris non-perfusion or hypoperfusion Figs. ,  17a, bComplete or partial regression of the ICGA hypofluorescence or absence of regression in the convalescent phase. The areas remaining hypofluorescent in the convalescent phase represent chorioretinal atrophy and correspond to areas of window effect and masking effect on FA.In diseases with a progressing course such as serpiginous choroiditis, ICGA can show diffuse choroidal hyperfluorescence at the edges of the progressing lesions in areas having no translation on fundoscopy or FA Fig. .Fig. 18DThe following ICGA signs have to be looked for:On FA there is likewise early hypofluorescence showing the choriocapillaris non-perfusion identified on ICGA.Depending on the severity of the choriocapillaris non-perfusion seen on ICGA, late FA frames either show no hyperfluorescence (for instance in mild MEWDS), discrete patchy late hyperfluorescence seen in MEWDS, or extensive late hyperfluorescence seen in APMPPE Fig. .To understand the genesis of the FA signs in choriocapillaritis it is important to be aware that late FA fluorescence is coming from retinal vessels overlying areas of ischaemic outer retina that present increased permeability in response to the ischemia due to choriocapillaris non-perfusion.In the convalescent phase there is a delayed regression of FA signs (hyperfluorescence and staining) as compared with the regression of ICGA signs.Whereas the ICGA signs are quite uniform, the corresponding FA angiographic signs depend on the severity and extension of the choriocapillaris non-perfusion and on the outer retinal damage.Any severe inflammation in an adjacent structure to the choriocapillaris  can cause inflammation at the level of the choriocapillaris and produce similar angiographic signs. In that situation we speak of secondary inflammatory choriocapillaropathy. The latter term is also used when the triggering agent is known such Treponama Pallidum in acute syphilitic posterior placoid chorioretinitis Fig. .In the second group of diseases, the primary mechanism is the development of inflammatory foci, mostly granulomatous at the level of the stroma appearing hypofluorescent on ICGA, usually associated with inflammation of larger non-fenestrated stromal vessels appearing on ICGA as fuzzy vessels in the intermediate phase followed by diffuse late choroidal hyperfluorescence. VKH disease or HLA-A29 BRC are typical illustrations of this type of choroidal inflammation. For the latter two conditions the lesion process is starting electively within the choroidal stroma and they are therefore called primary stromal choroiditis. In other cases such as sarcoidosis chorioretinitis the choroidal stroma is just one chance localisation of a systemic process (an innocent bystander) and are therefore called secondary stromal choroiditis entities. In contrast to primary stromal choroiditis where the distribution of hypofluorescent dark dots (HDDs) is uniform and their size is regular, in secondary stromal choroiditis HDDs have a random distribution Fig. . AlthougApart from the signs that allow to explore choroidal inflammation, whether at the level of the choriocapillaris or at the level of the choroidal stroma, additional ICGA signs have been reported.The optic disc is usually non fluorescent in ICGA. In case of severe inflammatory disease disc hyperfluorescence is a characteristic finding usually present in all types of hyperacute diseases such as initial attacks of Behçet’s uveitis and VKH disease, representing also a parameter to monitor the effect of therapeutic intervention. In a group of VKH patients, it was shown to regress within 1 month after introduction of systemic therapy .The choroid is the starting point of the inflammation in many diseases including MEWDS, APMPPE, multifocal choroiditis, serpiginous choroiditis, VKH, sympathetic ophthalmia and HLA-A29 BRC and is participating in the inflammatory process in many other diseases such as sarcoidosis, tuberculosis, syphilis, toxoplasmosis, posterior scleritis and many more which have extensively been analysed by ICGA –40. A reIn some diseases such as MEWDS only choroidal signs can be present and in others the clinically apparent part of the disease such as in multifocal choroiditis is only the peak of the iceberg (jellyfish effect) and the preponderant part of the disease can only be followed by ICGA Fig. . In diseUnlike for FA, it is not rare that ICGA gives the essential contribution that leads to the diagnosis. This can be the case for patients with MEWDS that have minimal translation in fundus signs or FA signs. Early HLA-A29 BRC can only be diagnosed using ICGA as the early lesions are HDDs indicating subclinical granulomas that can only be detected by ICGA. These ICGA lesions often precede the typical oval shaped depigmented “birdshot” lesions by several months or even years. Fig.  The sameAs choroidal lesions can only be detected by ICGA, it is obviously also the recommended modality to monitor the evolution and to evaluate the impact of treatment on choroidal inflammatory processes. In case of VKH disease, clinical disease, meaning inflammation involving extrachoroidal structures accessible to fundus observation, to OCT and FA, can be followed by classical means. However once clinical disease is under control, it has been shown that subclinical disease is ongoing resulting in almost 100% of cases in sunset-glow-fundus (SGF) the witness of ongoing disease destroying choroidal pigment. It was recently shown that ICGA guided treatment of VKH could avoid evolution towards SGF when treating also subclinical disease shown by ICGA Fig. .FA is performed routinely in inflammatory ocular diseases, which is probably justified in order to make a good evaluation of inflammation and to use it as a follow-up parameter for superficial fundus structures. Except for the evaluation of retinal vessels it mostly does not add essential information to clinical examination and OCT. In contrast, ICGA often furnishes additional information otherwise undetected on the choroidal compartment. Several choroidal diseases such as PICCPs  present with the “jellyfish effect or constellation” meaning presence of lesions of the choriocapillaris below the RPE with no or minimal signs seen on funduscopy or FA with nevertheless extensive choroidal involvement Fig. . SimilarTherefore, except for conditions such as pars planitis or Behçet’s disease where choroiditis is absent or insignificant, dual FA and ICGA should be performed for the assessment of posterior uveitis if angiographic analysis is deemed necessary as choroiditis cannot be excluded “a priory”. A dual FA/ICGA angiographic scoring method has recently been proposed for that very purpose.Uveitis can be considered to have developed into an exact “clinical science”, as investigational procedures allow increasingly precise appraisal of lesions in most ocular compartments. ICGA is the method of choice to explore choroidal inflammation. It is giving essential information no other method can furnish to assess and follow choroiditis. It is the most sensitive and reactive means to monitor therapeutic intervention and in that regard is not matched by new investigations such as enhanced depth imaging (EDI-OCT) measurement of choroidal thickness. Therefore, management of posterior uveitis where choroid is involved cannot be meaningfully performed without ICGA.A semiquantitative angiographic scoring system was developed by the Angiography Scoring for Uveitis Working Group (ASUWOG) in 2010  and has Angiography is based on the deviation from what is considered as normal fluorescence towards hyper or hypofluorescence. This is the case for both retinal and choroidal delta-fluorescence. The interpretation of inflammatory signs in angiography is based on this principle of hyper and/or hypofluorescence.Based on this classical phenomenon, the ASUWOG group defined inflammation score points for both retinal and choroidal delta-fluorescence. There are twice as many structures that can cause delta-fluorescence in the retina when compared with the choroid, which has less capacity to express inflammation score points based on delta-fluorescence. Therefore, its capacity to express inflammation score points is going to be artificially lower and had to be adjusted so that the inflammation score points generated by hyper/hypofluorescence could be compared between the retina and the choroid. The advantage of such a system is that choroidal and retinal inflammation can be comparatively evaluated, as inflammation points based on delta-fluorescence were the common denominator, a phenomenon occurring similarly in both structures . These pThe dual FA/ICGA scoring system has been used for comparison of retinal and choroidal inflammation , 43–46 aThe FA scoring system has also been used separately in studies of Behçet uveitis which primarily involves the retinal vasculature –54.Although, in present times, there is a continued trend towards non-invasive imaging techniques, ocular angiography still represents the mainstay of routine investigation of posterior uveitis when it is severe enough to warrant energic investigation. OCT to investigate the retina, or the choroid by EDI-OCT, have been valuable non-invasive additions, among others, for the appraisal of posterior uveitis. Their advantage is that examinations can be easily repeated and are especially useful for follow-up purposes. At present, however, to detect inflammatory macular oedema or retinal vasculitis, FA is still more sensitive than OCT . Similar


Solute transport across the chloroplast envelope is controlled by metabolite concentration gradients and transporter abundance. Overexpression of an envelope dicarboxylate transporter causes a pronounced metabolic phenotype, indicating that transporter abundance directly affects metabolism. 4 photosynthesis: OAA resulting from phosphoenolpyruvate carboxylation is imported into the chloroplast, reduced to malate by plastidic NADP-malate dehydrogenase, and then exported for transport to bundle sheath cells. Both transport steps are catalyzed by OMT1, at the rate of net carbon assimilation. To engineer C4 photosynthesis into C3 crops, OMT1 must be expressed in high amounts on top of core C4 metabolic enzymes. We report here high-level expression of ZmOMT1 from maize in rice (Oryza sativa ssp. indica IR64). Increased activity of the transporter in transgenic rice was confirmed by reconstitution of transporter activity into proteoliposomes. Unexpectedly, overexpression of ZmOMT1 in rice negatively affected growth, CO2 assimilation rate, total free amino acid content, tricarboxylic acid cycle metabolites, as well as sucrose and starch contents. Accumulation of high amounts of aspartate and the impaired growth phenotype of OMT1 rice lines could be suppressed by simultaneous overexpression of ZmDiT2. Implications for engineering C4 rice are discussed.The chloroplastic 2-oxaloacetate (OAA)/malate transporter (OMT1 or DiT1) takes part in the malate valve that protects chloroplasts from excessive redox poise through export of malate and import of OAA. Together with the glutamate/malate transporter (DCT1 or DiT2), it connects carbon with nitrogen assimilation, by providing 2-oxoglutarate for the GS/GOGAT (glutamine synthetase/glutamate synthase) reaction and exporting glutamate to the cytoplasm. OMT1 further plays a prominent role in C Oryza sativa L.) is a C3 grass and one of the top three staple crops in the world. Its highest consumption is in Asia  surrounding the vascular tissue, respectively. This spatial separation concentrates CO2 around the enzyme Rubisco, thereby reducing Rubisco oxygenase activity and the subsequent loss of energy and previously fixed CO2 during photorespiration  pathway  into mesophyll cell chloroplasts where it is reduced to malate by NADP-MDH. OMT1 also facilitates the export of malate to the cytosol. These transport steps occur at the same rate as CO2 assimilation and thus, for engineering C4 photosynthesis into C3 crops such as rice, high expression and activity of OMT1 are required. In this study, as part of the effort to engineer C4 rice, we introduced the ZmOMT1 gene from C4 maize into C3 rice to achieve sufficiently highly transport capacity for OAA and malate across the chloroplast envelope. Additionally, since C4 photosynthesis requires a complex array of biochemical and anatomical components, we investigate whether ZmOMT1 expression triggers anatomical features found in C4 plants, which could further aid continued C4 engineering efforts.OMT1 also plays an important role in CZmOMT1 in rice mesophyll cells, we transferred the pSC110:ZmOMT1:AcV5 construct into Oryza sativa spp. indica cultivar IR64 (ZmOMT1 gene (GRMZM2G383088) from maize (Zea mays var. B73) with a C-terminal AcV5 epitope tag, driven by the maize B73 mesophyll-specific ZmPEPC promoter  from the pSC110 vector (JXB online). The forward and reverse primers given in ZmOMT1 coding sequence (CDS) for later detection of expressed protein using commercially available AcV5 antibody. The final construct was transferred into freshly harvested immature embryos 8–12 d after anthesis using an Agrobacterium-mediated transformation protocol as described in –1 hygromycin B. The transgenic plants generated from hygromycin-resistant calli were transferred to Yoshida hydroponic solution . After 2 weeks, plants were transplanted to 7 liter soil pots. Plants were grown at Heinrich-Heine University (HHU) Düsseldorf, Germany under semi-controlled greenhouse conditions (16 h day/8 h night and 25 °C). Assessment of leaf gas exchange, as well as metabolite, C:N ratio, total free amino acids, and transporter activity measurements were performed at HHU.To express maize var IR64 . The conZmOMT1 in a 10 µl total volume employing the gene-specific primers given in Homozygous transgenic lines were screened by performing genomic PCR using the KAPA 3G plant PCR kit . Leaves were harvested 7 d after transplanting plants into soil; scraped leaves were directly used as the template for PCR amplification of OsOMT1 and ZmOMT1 transcript expression levels in 8-week-old plants. Leaf RNA was extracted using a QIAGEN RNeasy Mini Kit from three biological replicates. Then one-step cDNA synthesis using LunaScript™ RT SuperMix Kit  was followed by total 20 µl volume master mix preparation . PCRs were performed in a 7500 Fast Real-Time PCR System , and guided using the primer pairs given in OseEF-1a (Os03g0177500)  and treated with DNase . Using 1 µg of RNA, cDNA was synthesized by a first-stand cDNA synthesis kit , normalized to 100 ng µl−1, and used for PCR analysis in a 10 µl reaction with gene-specific primers (see To detect the mers see . OseEF-1The upper fully expanded leaves at mid-tillering stage (50–60 d old) were used to determine leaf chlorophyll content using a SPAD chlorophyll meter . The plant height and tiller number were measured at the booting stage from soil level to the base of the flag leaf on the main tiller.ZmOMT1 was checked by fractionating the isolated protein on a 12% SDS–PAGE gel, followed by western blot analysis. Primary mouse anti-AcV5 tag 1:2000  and peroxidase-conjugated secondary  antibodies were used for the detection of the AcV5 tag. The stained protein on nitrocellulose membranes was visualized by a LAS-4000 Mini luminescence image analyzer  using the ECL Western Blotting Detection Reagents .The presence of the AcV5-tagged ZmOMT1 protein in leaf membrane extracts of HindIII restriction endonuclease , separated by electrophoresis on a 0.8% agarose gel, then transferred onto Hybond N+ membrane . Blots were hybridized with a digoxigenin (DIG)-labeled ZmPEPC promoter-specific probe synthesized using the primers given in Genomic DNA was extracted from the mature leaves using the potassium acetate method as described by The seventh leaf at the mid-tillering stage was fixed and prepared for immunolocalization analysis as described in g (10 min) and 100 000 g (45 min) were then performed, using a benchtop centrifuge and ultracentrifuge, respectively. Ultimately, the isolated membrane was resuspended in 50 mM HEPES , 5 mM EDTA, 2 mM DTT, together with protease inhibitors , 25 mM EDTA, 30% (w/v) sucrose, 5 mM DTT, and appropriate protease inhibitors. Two subsequent centrifugation steps at 10 000 In vitro analysis of transporter activity was carried out using a freeze–thaw–sonication reconstitution procedure in concert with forward exchange of the substrate , and stopped at each of the above-mentioned time points by loading 150 µl of the reaction mixture to an anion exchange resin column . The resin column was previously equilibrated five times using 150 mM sodium acetate (pH 7.5). Unincorporated [14C]malate was replaced by acetate in the resin column and the incorporated label was washed through a scintillation vial containing 10 ml of Rotiszint® eco plus scintillation cocktail . Finally, the uptake of radiolabeled substrate was measured as counts per minute (CPM) by scintillation counting. To correct for background and false positives, the entire experiment was repeated using proteoliposomes without pre-loading of the substrate of interest .Uptake of radiolabeled substrates in counterexchange with non-labeled substrates was carried out at six different time points . The reaction was started by adding 950 µl of proteoliposomes into 50 µl of [2 assimilation and dark respiration rates during the tillering were measured on the middle portion of fully expanded leaves  in the standard leaf chamber of a portable photosynthesis system  between 08.00 h and 13.00 h at a constant airflow rate of 400 µmol s−1, leaf temperature of 30 °C, and a leaf to air vapor pressure deficit of between 1.0 kPa and 1.5 kPa. Leaves were acclimated in the chamber for 30 min before measurements. The response curves of the net rate of CO2 assimilation  to changing intercellular CO2 concentration  were acquired by decreasing Ca (CO2 concentration in the cuvette) from 2000 µmol CO2 mol−1 to 20 µmol CO2 mol−1 at a photosynthetic photon flux density (PPFD) of 2000 µmol photons m−2 s−1. The 2 % oxygen entering the cuvette was set by mixing nitrogen and oxygen in the CO2 free airstream through two mass flow controllers  at a flow rate of 1.5 ml min−1. Maximum Rubisco activity (Vcmax) and maximum electron transport activity (J max) were determined using the PsFit Model , CO2 compensation point , and quantum yield  were calculated as described by Rd, µmol CO2 m−2 s−1) was measured on leaves in darkness following an acclimation at a PPFD of 1000 µmol photons m−2 s−1 for 10 min at a Ca of 400 µmol CO2 mol−1, and calculated for 1100–1200 s in the dark.Leaf photosynthetic COit Model . The ligA and collect the samples for metabolite analysis under steady-state conditions, a custom gas exchange chamber was interfaced with a LI-COR 6400XT portable photosynthesis system  were randomized and performed only during the peak photosynthetic activity of the rice plants between 09.00 h and to 15.00 h.To normalize metabolite pool sizes by photosynthetic flux, two sets of rice plants of different ages  were analyzed. To measure s system  encasingThe GC/MS-based metabolite measurements were performed as described by 2O, 2.5:1 ratio), and then heated to 95 °C for 20 min. The solution was then transferred to a microwell plate after a short centrifugation of 10 s at 10 000 rpm. The total amino acid content was then measured in a Synergy HT plate reader  at a wavelength of 550 nm. Data were adjusted based on the l-leucine standard curve and related dilution factor.FAA contents were measured using the ninhydrin colorimetric method as described by g for 10 min at 4 °C to separate the soluble and insoluble fractions. The insoluble fraction containing the starch was further washed five times with 1 ml of 80% (v/v) ethanol. After centrifugation, the supernatant was discarded, the pellet was air-dried and resuspended in 500 µl of water, then gelatinized by boiling for 4 h and hydrolyzed overnight at 37 °C with 0.5 U of amyloglucosidase and 5 U of α-amylase. The starch content was measured as described in g for 10 min at 4 °C, the supernatant was assayed for sucrose content by enzymatic determination as described by The youngest fully expanded leaf during the tillering stage was harvested at 10.00 h, frozen immediately, and ground in liquid nitrogen using a mortar and pestle. A 50 mg aliquot of homogenized leaf powder was then extracted in 500 µl of ice-cold 0.7 M perchloric acid and centrifuged at 21 100 13C were analyzed based on leaf dry weight (mg) of 30-day-old and 50-day-old transgenic plants using the ISOTOPE cube elemental analyzer connected to an Isoprime 100 isotope ratio mass spectrometer . The δ 13C ratio is expressed as parts per thousand (‰) using the international standard of the Vienna Pee Dee Belemnite (VPDB).The ratio of carbon to nitrogen as well as δ −2 s−1. The middle section of the most recently fully expanded leaf was dissected into 2 mm pieces, prepared in Araldite 502 epoxy resin, and sectioned for TEM as described by Rice seeds were germinated in Petri dishes in distilled water for 4 d and then placed on a floating net in distilled water in a 19 liter bin in greenhouses at the University of Toronto. Seedlings were fertilized with 1/3 strength hydroponic media at day three after transfer and then with full-strength media every 4 d . Plants ZmOMT1 and ZmDiT2, homozygous ZmOMT1 single transgenic T2 lines  were crossed with homozygous ZmDiT2 single transgenic T2 lines  (1 progeny were selfed to produce segregating F2 populations. The pSC110:ZmDiT2:AcV5 construct used for generating DiT2 lines contained the coding sequence of ZmDiT2 (GRMZM2G40933) from Zea mays of the B73 variety and included an AcV5 epitope tag at the C-terminal end of the coding sequence. ZmDiT2 was cloned using the primers shown in ZmDiT2 lines were selected by PCR analysis, and protein accumulation was determined on western blots (To generate transgenic rice plants co-expressing DiT2-44) . The F1 rn blots .0 plants were generated, of which 87 were positive for ZmOMT1 as determined by PCR analysis of genomic DNA, and 40 of which carried a single copy of the ZmOMT1 transgene as determined by DNA gel blot analysis. Three single insertion lines  by immunoblotting  displayed perturbed phenotypes at the whole-plant level. The OMT1-79 and OMT1-87 lines were shorter ; Table 12 assimilation rate (A) in response to PPFD was measured at ambient CO2 conditions (400 µmol CO2 mol−1). The transgenic lines with highest ZmOMT1 expression (OMT1-79 and OMT1-87) had slightly lower CO2 assimilation rates than the wild type, whereas OMT1-80 had a similar rate  from the initial slope of light response curves (PPFD <100 µmol photons m−2 s−1) to the wild type (2 in rice plants. The dark respiration rates were twice as high in OMT1-79 and OMT1-87 lines compared with OMT1-80 and the wild type (2 assimilation rate (A) in response to intercellular CO2 concentration (Ci) under non-photorespiratory (2% O2) versus photorespiratory (21% O2) conditions was measured under saturating light intensity of 2000 µmol photons m−2 s−1. At 21% O2, lower photosynthetic rates were observed in OMT1-79 and OMT1-87 lines compared with the wild type and the OMT1-80 line (2 compensation points (Γ) (2), the wild type, OMT1-80, and OMT1-87 had similar photosynthetic rates at ~40 µmol CO2 m2 s−1, and similar Γ  and the maximum rate of electron transport (Jmax) were reduced in OMT1-79 and OMT1-87 lines under high photorespiratory conditions (2) and that when ZmOMT1 is expressed, ribulose 1,5-bisphosphate (RuBP) regeneration is limited at high CO2 concentrations. Together, these data indicate that ZmOMT1 overexpression leads to higher rates of photorespiration, a suggestion supported by the observation that transgenic lines have a higher Γ than the wild type at 21% but not at 2% O2.To examine the effect of overexpressing ZmOMT1 on photosynthesis in response to changing light conditions, the COlar rate . At 2000ild type , suggestild type , suggest-80 line . OMT1-79ints (Γ) . Under limilar Γ . Above anditions . TogetheThe macroscopic and physiological phenotypes of OMT1 lines were accompanied by ultrastructural changes in mesophyll cell chloroplasts. In contrast to wild-type plants, mesophyll cell chloroplasts of the OMT1 lines developed a peripheral reticulum PR;  which isZmOMT1 transgenic plants. However, in older plants, the photosynthetic rate was significantly lower in ZmOMT1 transgenic lines under ambient CO2 concentration (400 ppm) in each of the transgenic lines (2 concentration (1000 ppm) for older plants and only one line had a significantly lower photosynthetic rate as compared with the wild type under 200 ppm CO2 (The photosynthetic rate of the older ZmOMT1 transgenic plants (50–55 d old) measured in our custom-built gas exchange cuvette  was affeic lines . The pho ppm CO2 .ZmOMT1 transgenic rice lines and the wild type under different CO2 conditions was examined using GC-MS analysis. Large differences were observed among the measured metabolites of the mitochondrial tricarboxylic acid (TCA) cycle between the transgenic lines and the wild type. Malic acid, fumaric acid, iso-citric acid, succinic acid, and 2-oxoglutarate were significantly lower in all ZmOMT1 transgenic rice lines than in the wild type under different CO2 concentrations  under all CO2 concentrations but were significantly decreased under ambient CO2 (400 ppm) compared with wild-type rice , and glutamate and aspartate (transported by DiT2). If this assumption was true, then the phenotypes of ZmOMT1 single transgenic lines should be rescued by simultaneous overexpression of ZmDiT2. We hence generated double transgenic lines in which both ZmOMT1 and ZmDiT2 were expressed  leads to lower photosynthetic rates than the wild type, suggesting that OMT1 transporter activity must be precisely regulated to maintain optimal photosynthetic performance. The reduced photosynthetic rates in ZmOMT1 transgenic rice plants reveal possible relationships between photosynthesis, photorespiration, and cellular redox status. Differences in photosynthesis were significant in the plants measured in the Philippines and in older plants grown in Düsseldorf, Germany (Rd (ZmOMT1 transgenic lines were not rescued by elevated CO2 or reduced O2 when measured under growth conditions in the Philippines (2 (−2 s−1 for the A–Ci curves versus 500 μmol m−2 s−1 for the metabolite assays), meaning that phenotypic rescue may only occur under subsaturating light intensities. As photorespiratory rates increase, the increased demand for ATP relative to NAD(P)H pushes the redox status of the NADP+/NADPH pools to be more reduced unless processes either decrease plastidic NADPH  or increase ATP production (cyclic electron flux around PSI). The oxidation of NADPH, which could be increased with increased export of malate, must be finely balanced with metabolic demand so as not to directly compete with NADPH pools needed to supply the Calvin–Benson cycle or photorespiration. Under subsaturating light, there are numerous lines of evidence suggesting that the malate valve regulates this balance, particularly under photorespiratory conditions  carry a single copy of the ZmOMT1 CDS and are homozygous at the T3 generation.Fig. S4. Relative amount of some individual amino acids in OMT1 lines  and wild-type (WT) plants under different CO2 concentrations .Fig. S5. Western blot analysis of ZmOMT1 and ZmDiT2 protein expression in rice cross lines of OMT1/DiT2 .Fig. S6. RT-PCR of ZmOMT1 and ZmDiT2 mRNA expression in OMT1/DiT2 double transgenic lines together with the wild type (WT).Table S1. Primers used in this study.Table S2. V cmax and Jmax based on A/Ci data at 21% or 2% O2 using the PsFit Model.eraa343_suppl_Supplementary_Figures_TablesClick here for additional data file.


Besides, compound 10h could inhibit self-induced Aβ1–42 aggregation and AChE-induced Aβ aggregation with 32.6% and 29.4% inhibition values, respectively. The Lineweaver–Burk plot and molecular modeling study showed that compound 10h targeted both the catalytic active site (CAS) and peripheral anionic site (PAS) of BuChE. It should be noted that compound 10h was able to chelate biometals. Thus, the designed scaffold could be considered as multifunctional agents in AD drug discovery developments. To discover multifunctional agents for the treatment of Alzheimer's disease (AD), a new series of 1,2,3-triazole-chromenone derivatives were designed and synthesized based on the multi target-directed ligands approach. The in vitro biological activities included acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibition as well as anti-Aβ aggregation, neuroprotective effects, and metal-chelating properties. The results indicated a highly selective BuChE inhibitory activity with an IC Dementia is one of the noteworthy problems in the public health management as over 80% of dementia cases are suffering from Alzheimer’s disease (AD). Currently, available therapies provide temporary symptomatic relief but do not target the distractive neuropathology. Therefore, a new treatment to delay or halt disease progression has remained as an urgent medical need.The pathophysiological processes in AD have still remained unclear to this day. However, alongside its complexity, several neurodegenerative processes could be identified which include (I) aggregation of insoluble amyloid beta (Aβ) plaques mostly trigger from sequential cleavage of amyloid precursor protein (APP) by the aspartyl protease β-site APP cleaving enzyme-1 (BACE1) and γ-secretase, (II) neurofibrillary tangles (NFTs) form through hyperphosphorylation of tau proteins, (III) biometals dysfunction, and (IV) oxidative stress which in return results in synapse loss and death of neuronal cells in the brain –4.Based on the approved theory for AD, the loss of cholinergic neurons causes reduction of ACh. As a result, inhibition of the acetylcholinesterase (AChE) raises the level of ACh and improves cognitive performance at the early stage of AD. The critical point is that the AChE level decreases with the progression of AD, subsequently, AChE inhibition seems to be ineffective during the progression of AD , 6. InteBuChE has 65% amino acid sequence similarity to AChE along with mostly similar functions –14. AChEMoreover, produced Aβ peptides can aggregate into Aβ plaques which initiate pathogenic cascade leading to neuronal loss and dementia. Inhibition of the accumulation of Aβ peptide in the brain could be another therapeutic strategy against the development of AD . The metA depicted that coumarin moiety interacted with Trp231 and Phe329 residues of CAS pocket and benzyl pyridinium moiety interacted with Trp82 of BuChE PAS pocket  at variable concentrations of butyrylthiocholine as the substrate . The inhibition constant Ki was achieved by the plot of slopes versus the corresponding concentrations of compound 10h [The kinetic study for the inhibition of BuChE by compound ound 10h , 44.42 aggregation inhibition, a reported method, based on the fluorescence emission of ThT was followed. Briefly, the mixtures of Aβ1-40 peptide  and AChE , in the presence or absence of the test inhibitor were incubated for 24 h at room temperature. The final concentrations of Aβ , AChE , and the test compound are 200 µM, 2 µM and 100 µM, respectively. After co-incubation, 20 µL of the mixed solution was diluted to a final volume of 2 mL with ThT  and the absorbance was measured with a multi-mode plate reader at the excitation and emission wavelength of λex = 450 nm and λem = 485 nm, respectively [Inhibition of Aβ1-42 self-aggregation was measured by ThT fluorescence assay. The details of the method were reported in our previous study . To stud10h on neuronal PC12 cell damage induced by Aβ25-35. The cells were grown in monolayer culture on collagen-coated plates at 37 °C in a humidified atmosphere of 5% CO2. Neuronal PC12 cells were plated at a density of 5 × 105 cells/well on 96-well plates. The cells were pre-incubated with compound 10h for 3 h before human Aβ 25–35  was added. After 24 h, 90 μL the medium was taken out and 20 μL of MTT (0.5 mg/ml dissolved in RPMI containing phenol red) was added and incubated for an additional 2 h at 37 °C. The absorbance (A570 nm) was measured using a Bio-Rad microplate reader . The details were reported in our previous work [MTT reduction assay was used to evaluate the neuroprotective effect of compound ous work , 46.10h and Fe2+, Cu2+, and Zn2+ ions  were prepared in methanol. The mixture of compound 10h (1 mL) and the test ion solutions (1 mL) with the same final concentration of 20 µM in a 1 cm quartz cuvette was incubated at room temperature for 30 min. Then, the absorption spectra were recorded in the range of 200–600 nm.To study the metal chelating ability, the solutions of compound 10h-Cu2+ was also studied using the molar ratio method [10h was 20 μM and the final concentration of Cu2+ ranged from 0–44 μM with 4 μM intervals at 303.5 nm. The plot was obtained by the corresponding absorption versus mole fraction of Cu2+ to ligand 10h. All experiments were performed in triplicate.The stoichiometry of complex o method , 48. The+ [The molecular docking studies of the most potential ligand was performed on BuChE (PDB code: 4BDS)  to obser+ . The com+ .https://lmmd.ecust.edu.cn:8000/predict/ and https://preadmet.bmdrc.kr.ADME-Tox properties of the synthesized compounds were performed by using online servers especially 1-42 aggregation inhibition, metal-chelating properties, and neuroprotective effects against Aβ25-35-induced PC12 cell injury. Our results showed that these compounds had a high inhibitory potency and selectivity towards BuChE with an IC50 value of 21.71 μM for 10h as the most potent BuChE inhibitor. The inhibition kinetic analysis revealed a mixed-type inhibition pattern for this compound. The molecular modeling study of the most potent compound 10h with BuChE indicated that it bound to both CAS and PAS of the BuChE. Moreover, this compound had a significant anti-Aβ aggregation capacity and served as a metal chelator. These results indicated that this hybridization approach could be a successful strategy for the further developments of potential multifunctional candidates against AD.In summary, a new series of 1,2,3-triazole-dimethylaminoacryloyl-chromenone derivatives were designed and synthesized as multifunctional anti-Alzheimer’s agents. All the target compounds were synthesized and screened as AChE/BuChE inhibitors. The most active compound was further evaluated by the multiple biological activities including AβAdditional file 1. The supplementary file include copies of NMR spectra and elemental analysis report.


Balaena mysticetus) were hunted to near extinction in the world's first commercial whaling enterprise; this population clearly remains threatened, but nothing is known about its distribution, making assessment unfeasible. In this study, we document range, movement patterns and habitat preferences of this population, based on tagging done from an icebreaker-based helicopter. Despite their reduced abundance, Spitsbergen's bowhead whales occupy much of their historical range, stretching across the northern Barents Region from East Greenland eastward to Franz Josef Land. Unlike larger bowhead populations to the west, they do not migrate in a classical sense, but rather disperse from wintering grounds in the northernmost parts of their range during spring, returning northward again in autumn, a pattern opposite in terms of directionality compared to other Arctic bowhead whale populations. The extreme affiliation of this population with cold, ice-filled waters is a concern given ongoing climate warming and concomitant rapid sea ice habitat loss.Spitsbergen's bowhead whales ( In the early 1990s, this population was estimated to number in the few tens [The Spitsbergen stock of bowhead whales were deployed using an ARTS  [Because Spitsbergen bowhead whales are highly ice affiliated, the novel use of a helicopter tagging platform (based on a ship) was the only logistically functional option. We used an Écureuil Eurocoper AS350 to search for and approach the whales in the drifting sea ice of the western Fram Strait in late May–early June 2017. Sixteen Spot 5 satellite transmitters ( System)  air gun 3.a). During the study period, the animals were exposed to highly variable environmental conditions ranging from shallow, coastal areas to deep (max. 5018 m) offshore (max. 411 km) areas. The whales traversed areas with sea surface temperatures ranging from −1.8°C to 4.34°C (mean = 0.29 ± 1.52°C), with sea ice concentrations ranging from open water to 100% coverage. We tagged all of the animals in a restricted area in central Fram Strait , but they did not migrate directionally in any classical sense; they simply dispersed north and south, east and west , at relatively high swimming speeds (1–2 km h−1) over the summer period (c). Eleven of the whales stayed west of Svalbard, while the other two moved eastward to Franz Josef Land and somewhat beyond . During the autumn, movements were more homogeneously northward, and speed of travel was reduced . All of the whales spent the winter in relatively small areas in waters off Northeast Greenland or Franz Josef Land . Toward the end of winter and during the spring, the whales started moving more quickly once again, generally in a southward direction .Thirteen of the tagged bowhead whales provided location information; mean record length was 181 ± 199 days (range 4–709 days). Following our application of a speed, distance, angle filter and selection of a one-year study period , we retained 12 201 locations . Subsequent to creating 1 h interpolations within the identified track segments , 22 489 location estimates were obtained, 22 268 of which were retained for habitat assessment because they could be associated with oceanographic parameters a. Duringr period c. Elevenb and 2d–f; electronic supplementary material figure S7). In autumn, the whales moved into shallower areas, closer to coastlines and spent more time in lighter ice concentrations or open water areas, but SSTs were progressively colder, reaching below zero by October . As winter approached the animals returned to deeper, offshore areas where they occupied areas with high sea ice concentrations, often exceeding 90%, with SSTs below zero . In spring, few tags were still providing locations, but the whales for which we have data occupied areas with progressively declining ice concentrations and progressively higher SST values (temperatures became positive), although they remained offshore in deep, relatively cold areas . In all seasons, the whales spent most of their time inside the margins of the drifting ice, though during late summer and early autumn they made forays into open water (b).We explored movement patterns and habitat use throughout the year using GAMMs . The GAMMs showed that during summer, the whales occupied the marginal ice zone  in deep, offshore areas over the continental slope, where relatively high ice concentrations prevailed, with sea surface temperatures (SST) averaging 2°C . Proximity to coastlines also appeared to be an important variable influencing TSA; the whales were closest to the coast in autumn and furthest from the coast in winter . TSA was impacted by distance to the ice edge, particularly in the winter when the whales spent most of their time deep inside the ice, far from the edges . SST was also an important variable with regard to TSA; the whales spent most of their time in cold water, even during summer (SST < –1°C), and maintained this general cold water preference through autumn and winter, when they stayed longer periods of time in water with SSTs between −1°C and +1°C . TSA models' output summaries can be found in electronic supplementary material, table S4.4.In all seasons, the bowheads spent most of their time in cold water and in close association with sea ice. This species is the only baleen whale that resides year-round in the Arctic, and through evolutionary time they have become the most specialized baleen whale species. Not surprisingly, most of their unique characteristics are associated with their high latitude, cold water, ice-affiliated lifestyle . This inThe history of human exploitation of this population might play a part in the habitat preference we see in Spitsbergen's bowhead whales in modern times. Coastal and pelagically inclined bowhead whales in the Spitsbergen population were likely extirpated, leaving only individuals that tended to reside in ice protected refugia . Mammal Bowhead whales are shallow divers that feed on a variety of Arctic crustaceans, primarily in the top 200 m, although they can dive deeper than 500 m . AlthougBoth the Bering–Chukchi–Beaufort and the Eastern Canada West Greenland bowhead populations are recovering from historical overexploitation, and there are a growing number of promising signs for the Spitsbergen population ,13,21–23


We report a 50-year-old woman who presented with infected delayed union after ulnar shortening osteotomy. She was a chronic smoker. Implants were removed and infected tissue was debrided. Sufficient bony union was obtained after 5 months of treatment with weekly teriparatide and low-intensity pulsed ultrasound during the infection-controlled waiting period. As a treatment for ulnar abutment syndrome, ulnar shortening osteotomy with a plate is widely used, and its postoperative results are satisfactory. However, the possibility of non-union after plate fixation in ulnar shortening osteotomy is small but significant . Chan etThis report described a case of infectious delayed union with a loosened screw after ulnar shortening osteotomy that was successfully treated using teriparatide (once-weekly administration) and low-intensity pulsed ultrasound (LIPUS) during the infection-controlled waiting period after implant removal. Bony union was achieved without an additional surgical intervention.A 50-year-old woman had persistent ulnar wrist pain for over 4 months despite conservative treatment with bracing, physical therapy, and pain medication. The ulnocarpal stress test was positive without distal radioulnar joint instability and tenderness of the extensor carpi ulnaris tendon and pisiform. In addition, there was a positive ulnar variance of 2 mm on the radiograph. She had no trauma or medical history of diabetes mellitus. She had a 30-year history of smoking 20 cigarettes/day. She was diagnosed with ulnar abutment syndrome. Ulnar shortening transverse osteotomy was performed, and a seven-hole locking plate  was used for fixation . After tFive months after the operation, the patient complained of swelling and pain at the surgical site and loosening of the screw and radiolucency at the osteotomy site were observed on X-ray . White bOne month after implant removal and debridement, the infection was still under control. However, bony absorption was observed at the osteotomy site on computed tomography (CT) . For theCallus formation at the osteotomy site was observed on CT after 3 months after implant removal . Five moUsing ulnar shortening osteotomy plating systems that exert compression force on the osteotomy site, the rates of non-union can be decreased to 4%–7% . HoweverFew studies have described infectious non-union after internal fixation surgery ,5,7,8. ISeveral clinical studies have reported that LIPUS enhances the healing of fresh fractures, delayed unions, and non-unions . A systeWe presented a smoker of infectious delayed union after ulnar shortening osteotomy that was successfully treated using once-weekly teriparatide and LIPUS during the infection-controlled waiting period after implant removal without an additional surgical intervention. This combined therapy might be considered in the patients’ group with infectious delayed union after adequate infection control by debridement and long-term antibiotics and/or the patients’ group with high risk of developing non-union.


In ice-free summer, Antarctic penguins enjoy easy access to food and increase body weight and breeding success. Population trends and breeding success variability of Adélie penguins, a bioindicator of Antarctic environments, have been attributed to changing sea-ice extents; however, causative mechanisms remain unclear. By electronically tagging 175 penguins in four seasons with contrasting sea-ice conditions, we show that ice-free environments enhance, not deteriorate, foraging efficiencies and breeding success. In an ice-free season, penguins traveled by swimming rather than walking, leading to larger foraging areas, shorter trip durations, and lower energy expenditure than three ice-covered seasons. Freed from the need to find cracks for breathing, dive durations decreased, and more krill were captured per unit dive time, which may also be associated with phytoplankton blooms and increased krill density in the sunlit ice-free water. Consequently, adult body mass, chick growth rates, and breeding success increased. Our findings explain the regional population trends and demonstrate a key link among sea ice, foraging behavior, and reproductive success in this iconic species. In polar regions, different seascapes can appear from year to year due to interannual variabilities in sea-ice extents. During the last few decades, sea-ice extents have markedly decreased in the Arctic, whereas they have, counterintuitively, increased in Antarctica . These modern devices allowed us to track foraging trips, categorize behavior   loggers, accelerometers, and video cameras in four breeding seasons (resting) . HoweverN = 87 birds), part of which also had accelerometer records (N = 37 birds), showed different penguin behaviors among seasons. In the ice-covered seasons , penguins walked (and sometimes tobogganed) on the sea ice or along the coast after leaving their nests, with occasional resting behavior that lasted for minutes to hours, and then found cracks in the ice where they dived repeatedly (2(3) = 23.3, P < 0.0001] and distance of foraging trips , with the ice-free season having, on average, 3.2- to 7.9-hour (21 to 40%) shorter duration and 4.5 to 4.8 km (188 to 229%) longer distance compared with the three ice-covered seasons  than walking . The effect of locomotory modes  was also evident in the relationship between trip distance and trip duration (fig. S2), which showed that the ice-free season had by far the shortest duration for a given distance. To examine the energetic consequence of this change, a subset of GPS tracking data that also had accelerometer records (N = 37 birds) was analyzed. The proportions of time spent in walking, diving, resting at the sea surface, and resting on land or ice during the trips were highly variable among individuals, except that penguins spent little time walking in the ice-free season (fig. S3). Because of this variability, the total energy expenditure during trips, estimated from the time spent in each activity and activity-specific metabolic rates previously reported , was largely determined by trip duration alone regardless of the presence or absence of sea ice (fig. S4). Therefore, foraging trips of shorter duration observed in the ice-free season were energetically efficient.GPS tracking data  showed that, in the ice-covered seasons, penguins often slowed ascending speeds down at the end of dives, indicating that they searched for and moved to cracks for breathing (2(3) = 20.7, P < 0.0005], with the ice-free season having, on average, 15- to 27-s (22 to 33%) shorter duration (as assessed by the intercept of dive duration as a function of dive depth) compared with the three ice-covered seasons (R2 = 0.60) compared with the ice-covered seasons (R2 = 0.20 to 0.57). The difference in diving behavior was even more evident when each dive was separated into descent, bottom, and ascent phases . Descent phases had similar duration with similar variabilities for a given depth for all seasons, whereas ascent phases had shorter and less variable duration in the ice-free season compared with the ice-covered seasons (fig. S5). Thus, shorter and less variable dive durations observed in the ice-free season are primarily due to the difference in ascent phases, during which penguins were freed from the need to find cracks in the ice.The analyses of foraging dives based on accelerometer records (reathing . In the reathing . As a re seasons . Dive duN = 44 birds), estimated from penguin head accelerations relative to body accelerations (2(3) = 3.6, P = 0.31]. Water was blue in the ice-covered seasons but green in the ice-free season, as seen in the penguin-borne video footage (fig. S6). This observation agreed with unusually high chlorophyll a concentrations  measured in the ice-free season for the surface water sampled by a ship 17 to 21 km off the penguin colony.The number of krill captured in a dive (standardized for a 100-s dive)  and the growth rates of chicks , with the ice-free season having, on average, 0.2 to 0.6 kg (5 to 16%) larger female body mass, 0.3 to 0.7 kg (7 to 17%) larger male body mass, and 26 to 35 g day−1 (34 to 52%) higher chick growth rate compared with the three ice-covered seasons ,  seasons . Breedin seasons , althoug seasons .N = 39 birds) showed that Antarctic krill Euphausia superba was the single dominant prey (94% by mass) in the ice-free season. By contrast, two krill species  and fish (mostly Pagothenia borchgrevinki) were found in different proportions in the three ice-covered seasons , which is four times lower for swimming  than walking  in penguins. Moreover, penguins did not need to find cracks in the ice in the ice-free season but instead dived anywhere they wanted. This advantage likely helped penguins to further save time and to mitigate intraspecific competition for prey. In colonial breeding seabirds, intraspecific competition for prey is so severe that it can cause local prey depletion . At the foraging trip scale, the most important change was that penguins traveled by swimming rather than walking. Because swimming is four times faster than walking, penguins searched larger areas for prey in shorter durations in the ice-free season . This chAt the individual dive scale, the ice-free condition freed penguins from the need to find cracks for breathing at the end of each dive, leading to shorter and less variable ascent duration (fig. S5) and total dive duration . For exa−3) recorded off the penguin colony. In the Antarctic coastal waters with seasonal ice cover, phytoplankton growth is limited by sunlight and the loss of sea ice is followed by intensive phytoplankton blooms  prefers high-latitude, coastal habitats with heavy ice cover (E. superba (reaching 54 mm in length) is much larger than E. crystallorophias (reaching 36 mm in length) (fig. S7)  and high chlorophyll a concentrations  could deteriorate their energy balance. The penguins tagged in this study rested for extended periods exclusively on land or ice, rather than at the sea surface, during foraging trips. They never stayed at the sea surface for >5 min in the ice-covered seasons (in the 613-hour record composed of 30 trips) and did so only rarely (10 times in the 197-hour record composed of 14 trips) even in the ice-free season. To understand the case where sea ice is normally sparse, detailed biologging studies on foraging behavior and activity time budget, as demonstrated in this study, are warranted for penguin populations in the maritime Antarctic region.Our finding explains the previous analyses on the Adélie penguin populations in the continental Antarctic region (including our study site), where breeding success and population growth rates are negatively affected by increasing sea-ice extents  also play a role. In a broader context, our results show that simple physical effects of sea-ice changes on animal behavior  can have substantial fitness consequences. Biological effects that occur at lower trophic levels  are also important but are more complex and difficult to predict. Therefore, examining the physical effects of sea-ice changes on behavior for a range of polar predators  using animal-tracking tools is a promising avenue for better predicting future changes in polar ecosystems.Fieldwork was conducted at the Hukuro Cove colony  in Lützow-Holm Bay, Antarctica, from late December to early February in four seasons . All the experimental procedures were approved by the Ministry of the Environment, Japan. One hundred seventy-five Adélie penguins, rearing one or two chicks, were captured using a dip net when leaving for a foraging trip, weighed and measured, and then instrumented (see below) before they were released. The instruments were recovered by recapturing the birds when they returned to their nests. Sex of the instrumented birds was inferred from an index based on bill length, bill depth, and flipper width  showed a similar trend to the breeding success of the whole colony  and weighed every 5 days until late January or early February . Growth rate (grams per day) was calculated as the slope of least-squares regression line for body mass plotted against calendar date. Some chicks died of starvation or predation by south polar skuas e colony . Breedine colony .In the ice-free season (2016/17), chlorophyll a concentrations of surface water were measured by a ship at four locations 17 to 21 km off the penguin colony. Water samples were taken from the water pumped through the hull (from approximately 9 m depth) and processed following (−1 (−1 (−1 (−1 based on our GPS tracking data), 8.4 W kg−1, and 4.8 W kg−1 . Foraging trips were extracted from the GPS data as any return excursions from the nest with the maximum distance reached of >100 m and a duration of >1 hour. A subset of GPS tracking data that also had accelerometer records  was used to estimate penguin energy expenditure during foraging trips. To do so, penguin activity was categorized into (i) diving (when depth was >1 m), (ii) walking , (iii) resting at the sea surface , and (iv) resting on land or ice  (fig. S3). Metabolic rates during diving, walking, resting at the sea surface, and resting on land or ice were set at 14.0 W kgE. superba and E. crystallorophias) is the most important prey in our study site based on the previous . Diving bouts composed of krill-feeding dives, which are readily detected by the characteristic depth profile with repeated up-and-down movements . To charprevious  stomach 10 transformed due to their positively skewed distributions. When modeling dive duration, dive depth was included as a predictor variable. Statistical significance was tested by comparing the full models and the model without the season variable using the likelihood ratio test. Adult body mass and chick growth rates, where a single value was recorded for each individual, were compared among seasons using ANOVA.The records of penguin behavior  were compared among seasons by using linear mixed-effect models with season as a predictor variable and penguin ID as a random factor, using the software R with the extension lme4 (


Staphylococcus aureus is a facultative pathogen that can encode numerous antibiotic resistance and immune evasion genes and can cause severe infections. Reduced susceptibility to last resort antibiotics such as vancomycin and daptomycin is often associated with mutations in walRK, an essential two-component regulatory system (TCS). This study focuses on the WalK accessory membrane proteins YycH and YycI and their influence on WalRK phosphorylation. Depletion of YycH and YycI by antisense RNA caused an impaired autolysis, indicating a positive regulatory function on WalK as has been previously described. Phosphorylation assays with full-length recombinant proteins in phospholipid liposomes showed that YycH and YycI stimulate WalK activity and that both regulatory proteins are needed for full activation of the WalK kinase. This was validated in vivo through examining the phosphorylation status of WalR using Phos-tag SDS-PAGE with a yycHI deletion mutant exhibiting reduced levels of phosphorylated WalR. In the yycHI knockdown strain, muropeptide composition of the cell wall was not affected, however, the wall teichoic acid content was increased. In conclusion, a direct modulation of WalRK phosphorylation activity by the accessory proteins YycH and YycI is reported both in vitro and in vivo. Taken together, our results show that YycH and YycI are important in the direct regulation of WalRK-dependent cell wall metabolism. Staphylococcus aureus, a commensal pathogen that is infamous for causing severe nosocomial and community acquired infections, harbors 16 sets of TCS, of which 15 are not essential for bacterial growth -ATP in phosphorylation buffer  glycerol, pH 8) and incubated at 15 °C for 30 min. This time interval was chosen, because it was needed for a visible phosphorylation of the control and enabled the observation of inhibitory or activating effects. The reaction was stopped with 2x Laemmli SDS sample buffer and loaded onto a pre-cast SDS-PAGE gel . Electrophoresis was performed in accordance to the manufacturer’s instructions. The radioactive bands were visualized using a storage phosphor screen  with digitalization and quantification using the Storm Scanner System and ImageQuant TL software.Phosphorylation assays of the liposomes containing the reconstituted proteins were performed as previously described . ProteolS. aureus HG003 was grown in TSB at 37 °C until OD600 = 1.5 and cooled on ice immediately. Cells were harvested by centrifugation at 5800× g for 15 min at 4 °C and resuspended in extraction buffer . After 30 min incubation on ice, the suspension was centrifuged at 15,000× g and 4 °C for 5 min and the supernatant was used as an autolysin extract immediately. For lysis, WTA-free peptidoglycan from S. aureus HG003 was purified as described in the methods section for muropeptide analysis. Then, 30 µL of the peptidoglycan suspension were diluted in 1 mL PBS and 50 µL of the autolysin extract as well as 200 ng/mL lysostaphin were added. Lysostaphin was added to replace LytM, which was shown to be inactive in in vitro experiments [® 3000 MWCO, without the addition of lysostaphin. Lysis was observed as the reduction of OD600 over time. After 120 min of lysis, the suspension was spun down and the supernatant was used for the phosphorylation assays with WalK and WalR. Phosphorylation assays with detergent-micelles were performed using the Phos-tag SDS-PAGE method for histidine kinases as previously described [d-γ-Glu-Lys-d-Ala-D-Ala), 1 mM d-alanine , 1.5 to 6 µL of the lysed peptidoglycan, or 2 µL of purified WTA as described in the WTA extraction and quantification section. Phos-tag SDS-PAGE of the samples was conducted as described above. The Phos-tag gels were silver stained and the phosphorylation of WalR in presence of the supplement was compared to a sample without supplement.For extraction of autolysins, eriments . PGN wasescribed . Prior tS. aureus, we created antisense knockdowns of the yycH and yycI genes in the laboratory methicillin-sensitive S. aureus strain HG003 using the xylose-inducible pEPSA5 vector [S. aureus HG003  are cell wall components that should be liberated after lysis of the peptidoglycan polymer by the glucosaminidase AtlA, transglycosylase IsaA, muramidase SceD, amidases AtlA and SsaA, the endopeptidase LytM, and other enzymes that are all regulated by the WalRK TCS [S. aureus with and without the addition of lysostaphin. The resulting cell wall fragments did not show any effect on phosphorylation of WalR either , an excess of the accessory proteins had to be used for full activation. However, the high concentrations may have been necessary to saturate complex formation in the liposomes, as the direction of the insertion into the membrane must be correct for all three proteins and therefore might be limiting in the liposome system.In  studies ,22. Prev studies . The tra. aureus ,21. In tyycH or yycI yielded almost identical transcriptomes, which differed from the wild-type and caused the transcription of the autolysins Sle1, AtlA, SsaA, and IsaA to be downregulated [yycH, yycI, and yycHI mutants with no significant differences between the strains, stating that both proteins are required for their regulating effect on WalRK. Furthermore, bacterial two-hybrid assays revealed repeatedly that WalK only interacts with a YycHI complex and not with the single proteins [An activating effect of YycH and YycI has been reported in a recent study with transcriptional profiling of deletion strains. Deletion of either egulated . Vancomyproteins ,20. Our S. aureus cells. The absence of YycH and YycI in S. aureus NRS384 walR-FLAG ΔyycHI caused WalR to be less phosphorylated throughout all growth stages, which indicates the importance of YycH and YycI. WalR phosphorylation peaked at the end of the log phase, which is consistent with work conducted in B. subtilis [S. aureus [By using Phos-Tag SDS-PAGE in combination with immunoblotting, we were able to visualize the phosphorylation state of the FLAG-tagged WalR response regulator in lysates of growing subtilis ,41 and i. aureus .S. aureus HG003. Immunoblotting of growing S. aureus cells with anti-YycH and anti-YycI antibodies confirmed the reduced abundance of the proteins in cell lysates. Due to the effect of the antisense RNA on the polycistronic transcript, knockdown of YycH or YycI alone also caused a double knockdown phenotype to some extent. It has been shown before that antisense RNA targeting single genes in a cistron also can affect the adjacent genes [yycHI strain, however, was not affected. As WalRK is essential, the strain would not be able to grow under these conditions.We used an antisense RNA-based approach to knock down the production of YycH and YycI in nt genes . The expyycHI strains, Triton X-100 induced autolysis was reduced and autolysin extracts showed less lysis of M. luteus cells in zymographic analysis. Some cells appeared enlarged and might have had problems to initiate cell septation. Similar defects (irregular septation and failure to initiate cell division) have been described in cells starved for WalRK [B. subtilis, a positive regulation of WTA biosynthesis genes by WalRK was reported [S. aureus, to the best of our knowledge, the regulation of WTA biosynthesis by WalRK has not been observed. However, the expression of WalRK controlled genes is increased together with WTA biosynthesis genes during the early phase of nasal colonization [yycHI antisense strains. Further knockdown experiments employing a ΔtagO mutant of S. aureus, which is not able to produce teichoic acids [yycHI antisense strains. Alternatively, a comparison of the lysis of isolated cell walls of yycHI antisense strains with a control in the presence of externally added autolysins might help to decide whether the decreased autolysis is caused by the higher WTA content in the cell wall of the yycHI antisense strains or lower transcription of autolysins as expected in these strains [In the antisense or WalRK . Furtherreported . Furtherreported ,43. In Snization . Interesnization  and theric acids , for kno strains  or a comyycHI knockdown caused reduced autolysis, we analyzed if the cell wall muropeptide profile of this strain was affected. However, significant differences in the spectra of the muropeptide analysis could not be observed  may be


Botrytis cinerea were investigated for the first time. AJ was rich in lactic acid (4.46 g/L), acetic acid (1.52 g/L), Lactobacillus (72.45%) and Acetobacter (15.23%), which was a microbial ecosystem consisting of acid-based substances (AS) and beneficial microorganisms (BM). The results of the antifungal assays suggested that B. cinerea was effectively inhibited by AJ, with the half-maximal inhibitory concentration (IC50) of 9.24%. AJ showed the strongest and most-lasting inhibitory effect compared to cell-free supernatant and microbial solution of AJ, indicating that AS and BM and their synergistic effect contributed to the antifungal activity of AJ. Two-step inhibition’ is an antifungal mode of AJ. Firstly, AS not only inhibited the pathogen directly but also provided a dominant niche for BM of AJ. Then, BM in AJ, especially Acetobacter, proliferated and metabolized acetic acid continuously. Thus, AJ achieved high-efficiency and long-acting inhibition. AJ is a promising biological agent considering its features of an eco-friendly, low-cost and easy-to-operate biological agent in rural areas.Synthetic fungicides are eco-unfriendly to agriculture and the environment. Agricultural Jiaosu (AJ), which originates from organic wastes, has the potential to be a substitute for synthetic fungicides. In this study, the characteristics of AJ and its antifungal activity against  B. cinerea was studied for the first time.The antifungal activity of AJ against AJ was an ecosystem and achieved an effective and lasting antifungal activity.AS, MB and their synergistic effect contributed to higher inhibitory rate.Botrytis cinerea is a common and extremely harmful soil-borne pathogen in agricultural production, which can attack the leaves, flowers and fruits of crops  attracts widespread attention and large-scale application as a biological agent in agricultural practice in China. AJ has many advantages, such as sustainability, stabilisation, handleability, low-cost and use of simple equipment. This novel biotechnology can break the bottleneck of fungicides. In general, the AJ is diluted 1:200-500 times and applied to farmland once every 3–7 days. The dosage is about 2–3 L/mB. cinerea were investigated for the first time. The research was divided into five aspects: (1) physicochemical properties and microbial characteristics of AJ, (2) changes in the antifungal activity of AJ with dosage, (3) dynamic changes in the inhibitory effect of AJ with cultivation time, (4) main inhibitory factors of AJ and (5) antifungal mode by the synergistic effect between AS and BM of AJ.There is no relevant literature on the antifungal potential of AJ on phytopathogens. In this study, the antifungal activity and mechanism of AJ against Brown sugar (400 g) and jujube wastes (1200 g) were mixed with 4 L distilled water in air-tight containers for anaerobic fermentation at 30 °C for 3 months and stirred once a week. A large number of jujube wastes were produced when farmers harvest at an organic orchards in Jishan County, Shanxi Province, China. Then these wastes were collected to fermentation. The composition of jujube wastes was described by Guo . AJ was Botrytis cinerea (ACCC 36028) was purchased from the Agricultural Culture Collection of China . The pathogen was cultured on potato dextrose agar (PDA) medium at 25 °C before the experiments.The pH values of AJ were determined by a micro-pH metre . LA and AA were measured using high-performance liquid chromatography with a diode array detector . The samples were mixed with acetonitrile at a 1:1 ratio for 10 min and then filtered through a 0.22 μm filter. The details of the chromatographic procedure and conditions were described by Cai et al. , b.3, 0.015 g bromocresol purple, 15 g agar, pH 6.8 ± 0.2). High-throughput sequencing was performed on an MiSeq platform . The V3–V4 hypervariable region of the bacteria was amplified using the following universal primer set: 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The community structure was analysed by Silva database at the phylum and gene levels , which is indicative of antifungal activity, and Dck and Di represent the colony diameter of the pathogen of CK and treatments (mm), respectively.Blank control (CK) was prepared without the addition of AJ, whereas 5, 15, 25 and 50 mL AJ were added into 95, 85, 75 and 50 mL autoclaved PDA medium to create the dosage of 5%, 15%, 25% and 50%, respectively, and then shaken gently and poured into the dish. After agar gelling, the mycelial plugs (circle disks) of 50) refers to the concentration required for a drug to inhibit cell growth and virus replication by 50%, which is one of the important parameters commonly used in toxicology. Taking the 72 h colony diameter as the y-value and the dosage of AJ as the x-value, quadratic regression analysis was performed to obtain the fitting curve and polynomial equation. Half of the colony diameter of CK was taken as the y-value and entered it into the formula to get the corresponding x-value, which is the IC50 of AJ against B. cinerea  and T3, the precipitate of T2 was washed with sterile water and centrifuged at 12,000 rpm for 10 min, and the supernatant was removed. This operation was repeated three times. Finally, the pellet was mixed with 3 mL sterile water. The same amount of sterile water was used as control (CK). Under the premise of 5% dosage, 19 mL sterile PDA medium was poured into each culture dish. After the medium has cooled and solidified, 1 mL AJ with different treatments was spread onto the surface of fresh PDA medium until drying and then the 5 mm plug was placed at the centre . PCR consisted of 10 µL using SYBR® Green PCR SuperMix-UDG Kit , DNA template (1.0 µL) and double-distilled water to a total volume of 20 µL. The reported units were copies/mL. The PCR primers and the amplification protocol are given in Additional file The gene concentrations of P < 0.05 using Statistix 8.1.1 software . Excel 2016  was used to compute the mean ± standard deviation (SD) and graphical presentation.Statistical analyses were evaluated based on completely randomised variance at To evaluate the antifungal activity of AJ, its characteristics were determined. The main parameters are shown in Table Firmicutes and Proteobacteria dominated the entire community, with relative abundances of 72.80% and 15.10%, respectively. At the genus level, Lactobacillus (72.45%) and Acetobacter (15.23%) occupied the dominant niche.High-throughput analyses were used to detect the bacterial diversity of AJ. Figure Lactobacillus and Acetobacter). It was speculated that AJ had antifungal potential based on its characteristics.AJ was an acidic ecosystem consisting of AS  and BM , which indicated that AJ had a negative impact on the growth of B. cinerea. The pathogen was almost completely suppressed when the dosage of AJ was more than 15% of the whole medium. Also, the inhibitory rate was positively correlated with dosage and cultivation time. At 48 h, the value of this parameter at dosages of 5%, 15%, 25% and 50% was 10.95%, 58.79%, 74.89% and 73.17%, respectively, but there was no significant difference between the latter two treatments (P > 0.05). Interestingly, when the time was prolonged to 72 h, the inhibitory rate climbed to 42.36%, 74.54%, 84.27% and 85.98%, respectively. The inhibition rates of the latter two treatments (25% and 50%) were significantly higher than those of the former two treatments , but there was no significant difference between them (P > 0.05). At this moment, the antifungal activity was 2.87, 0.27, 0.13 and 0.18 times higher than those at 48 h.Dosage is a key factor that affects the antifungal activity in toxicological studies. The effect of the dosage of AJ on its inhibitory effect against ted Fig. . CK show50 was calculated according to the above experimental data using Excel 2016. With the colony diameter on the y-axis and the dosage on the x-axis, the fitting formula was obtained as follows: Y = 0.0382x2 − 2.7089x + 48.021, R2 = 0.9519. Half of the colony diameter of CK at 72 h (26.245 mm) was introduced into the formula as the y-value, and then IC50 was taken as the corresponding x-value. The IC50 of AJ against B. cinerea was 9.24%  but significantly larger than that of T1 and T2 (P < 0.05). At 48 h, the colony diameter of T1 was significantly smaller than that of the other three treatments (P < 0.05), but there was no significant difference between T3 and CK (P > 0.05). The colony diameter at 72 h of CK, T1, T2 and T3 were 49.27 ± 0.24, 35.78 ± 0.49, 40.29 ± 0.56 and 47.30 ± 0.41 mm, respectively. The difference in treatments was significant (P < 0.05). Visually, it was obvious that T1 showed the smallest colony diameter as compared with the other.To determine the main inhibitory factors, AJ was divided into two fractions and the antifungal assay was performed Table . T2 and P < 0.05). The inhibitory rate of T2 was between T1 and T3 and the value increased at the beginning and then decreased (P < 0.05). The peak appeared at 48 h, with 22.11% . The concentration of LA showed a decline with cultivation time from 0.52 to 0.31 g/L. As for AA, there was a trend of increase first and then gradually decreased to 0.29 g/L. Finally, the concentration of AA was only 15.18% of T1. The main component of cell-free supernatant was AS, so the pH value was only 5.09 at the beginning. Subsequently, the value dropped temporarily and then increased. The lowest pH value was observed at 48 h (4.21). Microbial solution consisted of BM of AJ, such as Lactobacillus and Acetobacter. Compared to T1, LA and AA in T3 increased slightly. The concentration of AA was only 0.36 g/L at 72 h, which was 18.85% that of T1. The pH value dropped from 6.66 to 5.08, which was equivalent to the initial value of T1.To further reveal the antifungal mechanism of AJ, the concentrations of LA and AA and the pH were dynamically measured during the inhibition process Fig. . OrganicIn summary, the pH value and the concentration of AA displayed an apparent fluctuation during the inhibition process. AJ showed the lowest pH value and the strongest acid production capacity, especially AA.B. cinerea and the main strains of AJ (Lactobacillus and Acetobacter) in different periods were quantitated by qPCR  from 1.68E+08 copies/mL at 0 h. Cell-free supernatant (T2) showed an inhibitory effect at the early stage. The gene concentration of the pathogen decreased to 2.89E+03 copies/mL within 48 h and then recovered to 3.16E + 04 copies/mL. Microbial solution (T3) had a weak influence on the growth of B. cinerea, with the highest gene concentration of 3.27E + 04 copies/mL at 72 h.PCR Fig. . B. cineAcetobacter rapidly proliferated and occupied the dominant niche, thus inhibiting the growth of the pathogen by metabolising a large amount of AA. This conclusion was consistent with the results of Fig. In summary, AJ could inhibit the pathogen effectively and persistently. B. cinerea.In Fig. When the inhibitory rates of T2 and T3 were added, the sum was less than the inhibitory rate of T1. This difference was considered as a synergistic effect of AS and BM. Figure In summary, AS were the main inhibitory factor at the early stage. BM gradually played a vital role at the late stage, which led to an increased proportion of the synergistic.B. cinerea were investigated. The following points were discussed based on the comprehensive analysis of the results.AJ not only has a strong antifungal activity but also provides a high-value-added treatment strategy of organic wastes, which achieves the dual role of resource utilisation and safety production. This product has the advantages of green, low-cost and easy operation and has been widely applied in rural China in recent years  and BM (Lactobacillus and Acetobacter). When AS and BM of AJ were separated, the two fractions still showed an inhibitory effect. These results suggested that AS and BM are the key inhibitory factors. Acetobacter was the antagonistic microorganism that played a major role during the late stage of the inhibition process . When AJ was added to the medium dominated by phytopathogens, AS inhibited the growth of the pathogen directly at the early stage (48 h) and provided a dominant niche for BM in AJ simultaneously. With the consumption or transformation of AS, the inhibitory effect gradually weakened. However, BM in AJ, especially Acetobacter, rapidly propagated and became the dominant bacteria under this condition. Then, a large amount of AA was metabolised continuously, which is a recognized as a good antibacterial substance. Therefore, AJ achieved high-efficiency and long-acting antifungal activity through this synergistic effect between AS and BM.Based on the above analysis, an antifungal mode called ‘two-step inhibition’ of AJ was proposed, as shown in Fig. Additional file 1.:Table S1. Summary of qPCR primers. Table S2. Cycling protocols of qPCR. Fig. S1. IC50 of AJ against B. cinerea. Broken, horizontal and vertical lines represent the fitting line, half of the colony diameter of CK and the corresponding dosages, respectively. Black and red circles represent the observed value and the IC50, respectively. Fig. S2. Increase part of the inhibitory rate of AJ. Blue and green bar represents the inhibitory rate of AS(T2) and BM (T3) respectively. And the red bar represents the higher part of inhibitory rate of AJ compared to the AS and BM.