Arabidopsis thaliana, where the genetic control of inflorescence development is best known, has a simple inflorescence, where the primary inflorescence meristem directly produces the flowers, which are thus borne in the main inflorescence axis. In contrast, legumes represent a more complex inflorescence type, the compound inflorescence, where flowers are not directly borne in the main inflorescence axis but, instead, they are formed by secondary or higher order inflorescence meristems. Studies in model legumes such as pea (Pisum sativum) or Medicago truncatula have led to a rather good knowledge of the genetic control of the development of the legume compound inflorescence. In addition, the increasing availability of genetic and genomic tools for legumes is allowing to rapidly extending this knowledge to other grain legume crops. This review aims to describe the current knowledge of the genetic network controlling inflorescence development in legumes. It also discusses how the combination of this knowledge with the use of emerging genomic tools and resources may allow rapid advances in the breeding of grain legume crops.The architecture of the inflorescence, the shoot system that bears the flowers, is a main component of the huge diversity of forms found in flowering plants. Inflorescence architecture has also a strong impact on the production of fruits and seeds, and on crop management, two highly relevant agronomical traits. Elucidating the genetic networks that control inflorescence development, and how they vary between different species, is essential to understanding the evolution of plant form and to being able to breed key architectural traits in crop species. Inflorescence architecture depends on the identity and activity of the meristems in the inflorescence apex, which determines when flowers are formed, how many are produced and their relative position in the inflorescence axis.  One of the most interesting features of plant development is the fact that all aerial parts of the plant body are generated from the activity of the shoot apical meristem (SAM). The SAM is located at the tip of the plant shoot and contains a central pool of stem cells that are able to self-maintain together with peripheral dividing cells required for organ initiation . During determinate inflorescences are those where, after floral transition, the SAM acquires the identity of a floral meristem, which forms a terminal flower . Compound inflorescences are typical, for instance, of grasses and legumes  are produced, and their position in the plant, has a profound impact on key agronomical aspects such as crop management, yield and yield stability. For instance, in crops such as tomato and grain legumes, determinate varieties have been traditionally selected because they show favorable traits for an efficient cultivation and harvest. Determinate varieties often display a shorter flowering time and earlier maturation and they are usually more compact, facilitating large-scale harvesting . Taken iIn this review, we describe the current knowledge on the genetic control of inflorescence architecture in grain legumes, and discuss the biotechnological potential of this knowledge for the development and selection of more productive and sustainable legume crop varieties.A main factor that shapes inflorescence architecture is the identity of the meristems produced in the inflorescence apex, which determines the relative position where flowers are formed.Arabidopsis thaliana is one of the best-known examples of simple indeterminate inflorescences. In Arabidopsis, upon floral transition, the vegetative meristem becomes an inflorescence meristem, which produces floral meristems laterally . The development of the Arabidopsis inflorescence can be mostly explained by the function and mutual regulation of three genes: TERMINAL FLOWER 1 (TFL1), LEAFY (LFY), and APETALA 1 (AP1) ( 1 (AP1) . These t 1 (AP1) .LFY and AP1 genes are essential for the specification of floral meristem identity in Arabidopsis. LFY codes for a plant-specific transcription factor that is expressed at very early stages in the flanks of the inflorescence meristem, at the floral “anlagen”  directing these incipient primordia to a floral meristem fate  gene, a paralogue of AP1 that is also expressed in early floral meristems as a result of LFY direct activation  and it is expressed in a subset of cells of the SAM at low level during vegetative stage , which in turn will generate floral meristems (F). In pea, the I2 usually produces 1-2 floral meristems before it ceases growing, forming a residual organ or stub . Therefore, the appearance of the I2 meristem supposes an additional level of complexity in the legume inflorescence, as compared to Arabidopsis, and different genes have been coopted to orchestrate the development of the compound inflorescence in legumes.As mentioned above, legumes are characterized by a compound indeterminate inflorescence . The ontAmong legumes, pea is the species where genetics of inflorescence development is best understood. In this section, we will review the current knowledge on the genes controlling floral meristem identity, as well as primary and secondary inflorescence meristem identity, primarily in this species and then extending to what is known for other legume species.LFY and AP1 genes from Arabidopsis. The PROLIFERATING INFLORESCENCE MERISTEM (PIM/PEAM4) and UNIFOLIATA (UNI) genes have been characterized as homologs to AP1 and LFY, respectively  have also been described and both produce flowers with a very similar phenotype to those of uni . Pea mutants in the DETERMINATE (DET) gene have a determinate inflorescence that produces 1-2 normal lateral I2s and an apparent TFL, resembling the Arabidopsis tfl1 mutant  flower but, instead, it develops as a stem that produces a flower in a lateral position and terminates into a stub, like the I2s. This shows that in pea det mutants, rather than the conversion of the inflorescence meristem into floral meristem observed in Arabidopsis tfl1 mutants, what really takes place is the conversion of the I1 meristem into an I2 meristem  was due to mutations in another TFL1-like gene, PsTFL1c, a paralogue of DET/PsTFL1a. Interestingly, the pea det lf double mutant plants are early flowering and determinate, which strongly resembles the phenotype of Arabidopsis tfl1 mutants. This has lead to the attractive idea that the TFL1 function, which in Arabidopsis controls both the vegetative and the inflorescence phases  meristem, as the formation of these meristems is crucial for the development of higher order inflorescences and hence for the formation of the characteristic legume compound inflorescences. Secondary inflorescence (I2) meristems do not form in simple inflorescences, indicating that new genetic functions must have appeared in evolution to direct the acquisition of I2 meristem identity. The identification of these novel genetic functions, and the characterization of their conservation across legumes, is important for a better understanding of compound inflorescence development.VEGETATIVE1 (VEG1) locus. veg1 mutant plants present a extreme non-flowering phenotype: no flowers or floral organs are produced in veg1 plants under any growing condition  pea, VEG1 is required to confer I2 identity to these lateral meristems and that to achieve that, VEG1 directly or indirectly represses DET expression in these meristems  conditions. Similar to veg1, gigas mutants show apparently normal vegetative development, and later in development, the induction of inflorescence markers, such as upregulation of DET and bud outgrowth  gene in Arabidopsis .A genetic model for the specification of meristem identity in the pea compound inflorescences has been proposed based on the genetic studies described above and the existing knowledge on DET (TFL1 homolog) and VEG1, respectively. DET and VEG1 repress each other expression, ensuring the balance between the indeterminate development of the apical primary inflorescence (I1) and the formation of secondary inflorescence (I2) meristems at its flanks. The identity of the floral meristems produced by the I2 is controlled by PIM, the homolog of AP1. PIM expression in the newly formed floral meristems represses VEG1 in this tissue, allowing floral development to proceed.In this model, primary and secondary inflorescence meristem identity is regulated by VEG1 in the inflorescence apex of det mutant plants would cause the apical meristem to acquire I2 identity and to develop as a terminal I2. Conversely, in a veg1 mutant, DET is ectopically expressed in the lateral meristems produced by the I1 and these lateral meristems then fail to acquire I2 identity, developing as I1 inflorescences. Finally, as it was also observed that loss-of-function of PIM leads to a missexpression of VEG1 in the newly formed “floral” meristems, this ectopic VEG1 expression would explain why these meristems do not acquire floral fate but instead develop as I2 meristems. This simple model based on the DET/VEG1/PIM regulatory module  is a more complex version of the TFL1/LFY/AP1 model  that explains the development of the Arabidopsis simple inflorescence. With the introduction of a novel genetic function, represented by VEG1, which is placed in between the mutual antagonistic activities of DET and PIM, the pea model explains elegantly how the transient I2 meristem appears and, hence, the development of the compound inflorescence in legumes.Consistent with this model, the missexpression of DET/TFL1-homologs in other grain legumes indicates that DET function is conserved in these species  produced by the primary inflorescence (I1), and the number of flowers produced by the secondary inflorescences, depends on for how long the I1 and I2 meristems, respectively, remain active.Mellilotus officinalis (yellow sweet clover) usually have 20–30 flowers.The number of flowers produced in each I2, as well as the number of I2s produced by the primary inflorescence, is characteristic of each species or cultivar . Thus, fIn spite of its possible influence on seed yield of crops, there are relatively few studies about the genetic control of flower number per I2 in legumes, which depends on the activity of the I2 meristem.Fn and Fna, whose single recessive mutations cause an increase in the number of flowers per I2, being higher in the double recessive genotypes, the so-called multipod phenotype (NEPTUNE (NEP), a gene represented by two recessive alleles, also causes a multipod phenotype  and STERILE NODES (SN), involved in photoperiod response, also strongly influence this trait, with the number of flowers being decreased by recessive sn alleles and increased by dominant HR alleles  is the grain legume where genetics of number of flowers per I2 is possibly better understood. Most chickpea genotypes have only one flower per I2, and it has been proposed that this solitary flower could be a reduction of a multi-flowered ancestor  nodes has been found to limit yield in legume crops , which iInflorescence traits amenable to improvement in legumes could be divided into two categories: (1) traits related to the identity of the meristems in the inflorescence apex, and (2) traits related to the activity of the inflorescence meristems. In this section, we discuss genetic and/or biotechnological strategies to modify inflorescence traits that might be applicable in breeding programs either to synchronize maturity facilitating mechanical harvesting or to improve and stabilize yields.Figure 4).At least two main meristem-identity related traits might be amenable to improvement: determinate growth habit and inhibition of flowering  inflorescence meristem continues growing and producing lateral (secondary) inflorescences until its senescence, in many legume crop species determinate varieties exist, in which the growth of the primary inflorescence meristem is interrupted, soon after onset of flowering, by the production of a terminal inflorescence  than determinate ones and, at the same time; they are less prone to lodging because they are shorter than indeterminate cultivars . In soybene VEG1 .TFL1 homologs should be the most direct way to obtain determinate varieties of grain legume crop species. In contrast, it is not clear that a strategy to obtain semideterminate varieties through mutation will be possible because the causative mutation(s) of semideterminacy in soybean has not been fully elucidated yet, and also because they are gain-of-function mutations, most likely in regulatory regions of the Dt2 gene, which may be difficult to translate to other legume species . The genetic control of flowering time in legumes is discussed in another review in this number  is inhibited, as these structures are transformed into vegetative branches (I1s)  and, consistent with that, pea plants where VEG1 was transiently silenced through virus-induced-silencing (VIGS) produced more vegetative nodes than the WT control  and inhibit the formation of flowers . The possibility of increasing the number of pods appears an attractive option to increase yield in grain legumes. In this sense, in the case of chickpea it has been reported that the double-flower trait has the potential to increase yield  is an inflorescence trait related to the activity of the inflorescence meristem that might be amenable to improvement in grain legumes  gene, which codes for a homeobox-type transcription factor that induces stem-cell identity and is essential to maintain the population of stem cells in the meristems. WUS expression in the center of the meristems is directly correlated with an active state of stem cells within the meristem, and when WUS expression disappears meristems arrest. In wus loss-of-function mutants very few flowers are formed and these have a reduced number of floral organs , therefore facilitating the identification of suitable alleles to breed inflorescence traits in the legume crop of interest.Access to the genome sequence of crop legumes will allow easy identification and cloning of the homologs of inflorescence architecture regulators previously identified in model grain legumes such as pea or Next generation sequencing technologies should also greatly ease the identification of candidate genes for architectural traits whose genetic basis remains unknown. The recently developed mapping-by-sequencing approaches are now starting to be routinely used to rapidly identify candidates for causal mutations in model organisms and, as the NGS technologies become more powerful and cheaper, their use is extending to crop species in spite of their usually larger genomes . These mTnt1, in M. truncatula and Lore1 in L. japonicus are now routinely used for identification of mutants for genes of interest through reverse genetics (Latirus odorata and M. truncatula (M. truncatula, L. japonicus and chickpea (In addition, different reverse genetic and genomic tools that can be used to validate the function of candidate genes for architectural traits are now available in several model and non-model legume species. First, mutant populations for the retrotransposons genetics . Second,uncatula . VIGS isuncatula . Finallyuncatula . TILLINGuncatula . These pchickpea , and wilArabidopsis and rice, where it was possible to engineer site-directed mutations in the genes of interest (Finally, “designer” alleles also appear to be within reach through the recently developed genome editing techniques mediated by the CRISPR-Cas system. CRISPR has been showed to efficiently work in several plants like interest . HoweverIn summary, the increasing availability of genomic tools and resources offers a unique opportunity to accelerate breeding of inflorescence architecture and, in general, of agronomic important traits in legumes. The combination of the increased understanding of the genetic networks controlling legume inflorescence architecture and the use of these genomic tools and resources, promises a rapid progress in obtention of new legume varieties with improved performance, which will be instrumental in developing a sustainable agriculture for the future.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To determine whether people with central field loss (CFL) from macular degeneration have improved ability to recognize a particularly difficult spatial configuration embedded in noise, the peripherally-viewed ‘ladder contour’. The visibility of these configuration has been linked to general contour integration ability and crowding limitations in peripheral vision.We used a trial-based yes-no task. CFL patients and normally-sighted controls performed the task, looking for ladder contours embedded in a field of randomly oriented Gabor patches, at a range of stimulus presentation times (varying stimulus difficulty). Viewing eccentricity in CFL patients was set by their preferred retinal loci (PRLs) and matched artificially in the control group. The contours were presented so as to be tangent to the CFL region, given a patient’s PRL location.CFL and normally-sighted groups performed similarly on the task. The only significant determinant of performance was the viewing eccentricity.CFL patients do not seem to develop any improved ability to recognize ladder contours with their parafoveal retina, which suggests that there is no underlying improvement in contour integration or reduction in crowding limitations in the region of the PRL despite extended daily use. Central field loss (CFL) usually occurs well after the critical phases of visual development, and it seems that the deafferented portion of visual cortex is not recruited by other parts of the visual system. There have been some reports of “reorganization” of deafferented visual cortex –5, but icus (PRL), to explcus (PRL)–10. Labocus (PRL),12. The ladder contours in CFL patients. We chose this task because an earlier report showed that these stimuli are not recognized in the visual periphery [crowding make ladder detection difficult or impossible in the periphery [Here, we report a study of peripheral detection (or more accurately recognition) of eriphery ; a plauseriphery –16. We rSubjects included 11 individuals with CFL  and 8 normally-sighted subjects . All participants gave informed consent in written form to participate in the study; the study and the consent procedure were approved by the Schepens Eye Research Institute IRB (Mass Eye and Ear human subjects protocol 12-188H-2010-09) and the Declaration of Helsinki.2 = 0.678, p < .001). The control subjects all had normal  acuity and no known visual impairments. Some of the subjects in each group had prior experience in psychophysical tasks, but none were expert observers.Patient etiologies were as listed in train any subjects on the task beyond a few practice trials so that the rhythm of stimulus presentation could be learned. Training may or may not be critical to measuring “effects” in this domain; in a replication of the original May and Hess study ’s second experiment  . We presWe started with longer presentation times and worked down to the shorter; however, many of the CFL patients were unable to progress to the shortest presentation durations (250 msec), finding the timing too difficult to coordinate with their relatively unstable fixation patterns (according to their accounts of the difficulty). Except for one patient (C10), data collection began with a block of 3000ms trials, after an instruction and practice period, typically around 10–15 minutes, with untimed presentations (C10 began at 1000ms). Normally-sighted controls foveally fixated a spot at variable distance from the center of the Gabor array, and mostly succeeded in performing the task at the shortest presentation times; we distributed the fixation distances over control subjects so that the contours (when present) would fall at similar eccentricities to the CFL patients . Depending on whether a patient's PRL was left/right of or above/below the scotoma, vertical  or horizontal  contours were presented. The controls fixated either below the Gabor arrays, looking for horizontal contours  or to the left of the arrays, looking for vertical contours .d' = z(H)—z(FA), where z is the inverse of the cumulative normal distribution at H or FA. The display was a Trinitron CRT, running at 100 frames per second. The display mean luminance was 44 cd/m2. Gamma correction was performed through the video software and the experiments were written in Matlab , using Psychophysics Toolbox functions [We used a yes-no experiment design to measure sensitivity to the ladder contours : half the trials (randomly selected) contained ladder contours embedded in an array of randomly oriented Gabors  while thunctions ,18.d' as a function of contour eccentricity. Different symbols denote different presentation durations (which can be read as task difficulty), different colors to different individuals. The black 'X' symbols represent the PRL eccentricities of the patients who were unable to complete a single block of the experiment due to complete inability to recognize the targets.Results are as shown in There is a trend towards lower sensitivity at larger PRL eccentricities, represented by the thick dashed line, which is a power function fit to the patient data averaged over presentation duration . The thin dashed lines are half and double the mean trend, a range that encompasses all the between- and within-subject variation. The right panel shows the normal control data, with the same conventions as the patient data, but retaining the fit-to-patient lines to help with comparison of the two groups.A statistical analysis of the data—ANCOVA with stimulus duration and participant group as discrete factors, and with eccentricity as a continuous factor, excluding the participants who could never see the targets  to make the task possible for all the CFL patients, we obtained the same effect of visibility in normally-sighted controls. Our conclusion is that there appears to be no significant difference between CFL and normally-sighted individuals when it comes to perception of these difficult stimuli. This, combined with the likely common neural substrate underlying both the difficulty of seeing ladder contours and the phenomenon of crowding ,16, suggA recent study by Chung  only parThe fact that we found no difference in performance on this task between the two groups does not mean there is no difference. Our normally-sighted controls did not only have normal retinas, but as a consequence they had different initial contact with the stimuli—i.e. the controls were allowed to foveally identify the ladder contours, a much easier task, before performing the difficult peripheral task. The CFL patients, meanwhile, had no such step-up, and had to bootstrap the task. The fact that, excepting their greater difficulty in seeing the brief presentations , the CFL patients performed similarly with the normally-sighted controls might indicate the presence of some other compensatory visual advantages. A negative result in this experiment also should not be interpreted as meaning that perceptual learning cannot occur near the PRL; learning can surely be induced by repeated practice on specific tasks designed to test individual psychophysical mechanisms. Our result means that, whatever learning has taken place in CFL patients near the PRL, it is either not at the level of contour integration mechanisms (i.e. crowding), or is of such small magnitude that we could not detect an effect.S1 AppendixSubjects in this experiment were normally-sighted, and did not overlap with the control subjects in the main study. The important difference between the replicated study and the main study is in the size of the stimulus: when the stimulus is large enough, ladder contours are indeed visible in peripheral vision.(DOC)Click here for additional data file.S1 Figst vs 2nd interval) is not too large.Blue and red symbols are for snakes and ladders, respectively. We calculated d’ for the May & Hess data by treating the overall p(correct), which they reported, as the hit rate, and 1-p(correct) as the false alarm rate. This is an accurate means of d’ estimation if the response bias (1(TIF)Click here for additional data file.


N-acyl homoserine lactones (AHLs). We report here the identification and characterization of AHLs produced by bacterial strain ND07 isolated from a Malaysian fresh water sample. Molecular identification showed that strain ND07 is clustered closely to Pseudomonas cremoricolorata. Spent culture supernatant extract of P. cremoricolorata strain ND07 activated the AHL biosensor Chromobacterium violaceum CV026. Using high resolution triple quadrupole liquid chromatography-mass spectrometry, it was confirmed that P. cremoricolorata strain ND07 produced N-octanoyl-l-homoserine lactone (C8-HSL) and N-decanoyl-l-homoserine lactone (C10-HSL). To the best of our knowledge, this is the first documentation on the production of C10-HSL in P. cremoricolorata strain ND07.Quorum sensing (QS) is a bacterial cell-to-cell communication system controlling QS-mediated genes which is synchronized with the population density. The regulation of specific gene activity is dependent on the signaling molecules produced, namely  The regbacteria ,5.To date, the most commonly studied autoinducers are the AHL molecules produced by most Gram-negative bacteria. The AHL molecule side chain length varies from short chain (C4) to long chain C18) carbon chains . When at carbon cPseudomonas syringae and Erwinia carotovora [Pseudomonas cremoricolorata strain ND07.QS bacteria have been isolated mainly from clinical and some other habitats ,5 while rotovora ,10. Fresrotovora  and henc2.2.1.The collection site of water samples was at the Sungai Ampang waterfalls, Malaysia (GPS coordinate: N 03°12.69′ E 101°47.72′) in 2013. Water samples were collected at the depth of 12–17 cm using sterilized plastic tubes. The temperatures and pH of the samples were recorded. Water samples were kept in an icebox and brought to the laboratory for further analysis [2.2.Overnight culture was serially diluted and plated on Reasoner's 2A (R2A) agar . The pla2.3.® DNA Mini Kit  was used to extract and purify bacterial genomic DNA of strain ND07 and the resulting DNA was used as template for PCR. The forward primer 27F and the reverse primer 1525R were used to amplify the 16S rDNA gene [The QIAampDNA gene . The PCRDNA gene .2.4.Chromobacterium violaceum CV026, was used to detect the presence of short chain AHLs [Erwinia carotovora GS101 were used as positive control while Erwinia carotovora PNP22 was negative control [An AHL biosensor, namely ain AHLs  whereby ain AHLs ,16 on Lu control ,18.2.5.N-morpholino)propanesulfonic acid  at 28 °C with shaking (220 rpm). An equal volume of acidified (0.1% v/v acetate acid) ethyl acetate was used to extract twice the spent supernatant [The bacterial colony of strain ND07 showing positive results for the detection of AHL was grown overnight in LB broth buffered to pH 6.5 with 3- equipped with an Agilent ZORBAX Rapid Resolution High Definition SB-C18 Threaded Column . Mass sp of AHLs . Analysi2.7.600 of 0.1, and the diluted culture was added with LB medium supplemented with catechin  [590 with microplate reader. All experiments were performed in triplicate.The biofilm assay was performed as described previously ,22. Brieompound)  and the 3.3.1.Pseudomonas cremoricolorata strain IAM 1541. The result obtained was based on a web search and phylogenetic analysis of 16S rDNA nucleotide sequence  (m/z 228.2000) and N-decanoyl-l-homoserine lactone (C10-HSL) (m/z 256.5000) were detected (ain AHLs . To furtdetected .P. cremoricolorata strain ND07, which is the first report of such a finding. According to Uchino and colleagues, P. cremoricolorata sp. nov. has been identified as a new species [P. cremoricolorata will pave a way to a deeper understanding of the QS system in this bacterium.The MS results shown in  species . Our wor3.3.Pseudomonas possess the ability to form biofilms [P. cremoricolorata strain ND07 has been shown to be able to form biofilms (P. cremoricolorata strain ND07 (Biofilm formation is often a QS-regulated phenotype ,30. The biofilms . P. crembiofilms . In our ain ND07 .P. cremoricolorata strain ND07 aiming to study the AHL synthase and receptor genes that will provide more insight into the QS regulatory system in this bacterium.Since QS regulates a battery of bacterial virulence factors , hence t4.P. cremoricolorata strain ND07 showed QS activity with the production of C8-HSL and C10-HSL according to LC/MS analysis. To the best of our knowledge, this is the first report of such a finding. P. cremoricolorata, ND07 has also been shown to form biofilms which could be inhibited by anti-QS compounds, suggesting that it is QS-dependent trait. Our group is currently performing whole genome sequencing in order to identify the luxI/luxR genes in this isolate.In conclusion,


Since adaptive immunity is thought to be central to immunity against influenza A virus (IAV) pneumonias, preventive strategies have focused primarily on vaccines. However, vaccine efficacy has been variable, in part because of antigenic shift and drift in circulating influenza viruses. Recent studies have highlighted the importance of innate immunity in protecting against influenza.Granulocyte-macrophage colony stimulating factor (GM-CSF) contributes to maturation of mononuclear phagocytes, enhancing their capacity for phagocytosis and cytokine production.Overexpression of granulocyte macrophage-colony stimulating factor (GM-CSF) in the lung of transgenic mice provides remarkable protection against IAV, which depends on alveolar macrophages (AM). In this study, we report that pulmonary delivery of GM-CSF to wild type young and aged mice abrogated mortality from IAV.We also demonstrate that protection is species specific and human GM-CSF do not protect the mice nor stimulates mouse immunity. We also show that IAV-induced lung injury is the culprit for side-effects of GM-CSF in treating mice after IAV infection, and introduce a novel strategy to deliver the GM-CSF to and retain it in the alveolar space even after IAV infection. Influenza pandemics are also lethal and have killed up to 50 million people [Seasonal influenza is one of the most important community acquired pneumonias in the U. S., causing 36,000 deaths and 200,000 hospitalizations annually  in the lung provides remarkable protection against seasonal influenza H3N2), PR8 (H1N1), pandemic flu (H1N1) and secondary bacterial pneumonia due to , PR8 (H1nfection . In thesnfection . In thisDelivery of high doses of GM-CSF (~100X of systemic dose) to the lungs is needed to be able to activate AMs. High concentrations of GM-CSF in the alveolar space are well tolerated as lungs are naturally semi-permeable to therapeutic proteins ,8. HowevGM-CSF is associated with inflammation and asthma in some experimental systems –11. HoweEight 10-week-old wild type C57BL/6 and Balb/c mice were purchased from Jackson laboratory and 20-months old C57BL/6 mice were purchased from National Institute of Aging. All mice were maintained in the vivarium at the University of Texas Health Science Center at Tyler.All experimental animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Texas Health Science Center at Tyler. Mice were observed at least once daily by authorized personnel for signs of distress, weight loss, ruffled fur, hunched back posture and lack of movement. Mice that lost more than 20% of their bodyweight were evaluated by veterinary staff. Experimental mice were euthanized by Beuthanasia or Euthasol which were administered intraperitoneally (ip) at a dosage of 5ul/g body weight. Any intranasal treatment was performed under deep anesthesia with Ketamine HCL (100mg/kg) and Xylazine (8.5 mg/kg) ip. Experimental mice were euthanized if lost more than 30% of their weight and were distressed with ruffled fur, hunched back posture and lack of movement.The mouse-adapted influenza virus A/Puerto Rico/8/34 (PR8) (H1N1) strain was used in all experiments. Mice were intranasally inoculated with 50 μl of PBS containing the H1N1 PR8 strain under light general anesthesia with a combination of Ketamine/Xylazine. All infected mice were monitored for weight loss and mortality on a daily basis.Mice were treated intranasally daily with recombinant murine GM-CSF (Invitrogen) or recombinant human GM-CSF (LEUKINE) for 7 days, prior to infection with PR8 H1N1 influenza. Seven days treatment regimen has been shown to be optimal in our previous publication .Mouse bone marrow cells from C57BL/6 mice were incubated with plain or conjugated mouse GM-CSF. Ninety six hours later, WST-1 reagent  was added (10 μl/well) to the plate and incubated for 4 hrs and the absorbance was measured. WST-1 is a colorimetric assay to assess the number of viable cells by the cleavage of tetrazolium salts added to the culture medium.Mice were treated with human or mouse GM–CSF or PBS as described above. After seven days of treatment, lungs and bronchoalveolar lavage (BAL) fluids were collected and stored at -80°C. Levels of TNFα and Amphiregulin in supernatants of BAL fluid and level of MCP-1 in supernatants of lung homogenate were measured by enzyme-linked immunoassay kit .Mice were treated with either mouse or human GM-CSF as described in 2.3 for 7 days. Broncho alveolar lavage was collected on day 8 and the number of alveolar macrophages was counted for each individual mouse. We stained Bronchoalveolar lavage cells with anti-F4/80 to identify AM, and found that over 95 percent of BAL cells were AMs.Lungs from the mice treated with either mouse or human GM-CSF as described in 2.3 were collected and lung homogenates were prepared. Thirty μg of lung homogenate from mGM-CSF-treated mice, hGM-CSF-treated mice and un-treated control mice were electrophoresed through 4–15% SDS polyacrylamide gel. Samples were transferred onto the PVDF membrane and developed using rat anti-mouse PU.1/Spi-1 antibody . Density of the bands were quantified using Image lab software version 5.0 .Commercially available Qdots are between 10–20 nm in diameter . Thus, Qdots were used as crude molecular rulers to measure the pore size of the permeable lung vasculature after infection. The Qdots used in this experiment were either 13 nm (Qdot 525) or 20 nm (Qdot 625) in diameter and emitted light at 525 nm and 625 nm wavelength, respectively, allowing clear discrimination of particle size from various samples using a standard fluorimeter. Several initial experiments were conducted to optimize the Qdot dose. Intravenous injection of 50 μl of each Qdot was sufficient for detecting Qdots up to 4 h after intravenous administration in urine, lung homogenate and BAL.Qdots (625 nm) were activated with 20 μM EDC/NHS for 15 min at room temperature. The activation reactions were terminated using DTT, and mGM-CSF was then added  and amine coupling allowed to proceed for 2 h at room temperature before quenching with 5 mM ethanolamine. Conjugated mGM-CSF to Qdot samples were filtered and concentrated using a 30,000 MWCO Centricon to separate conjugated material from non-conjugated mGM-CSF in solution. Qdot-mGM-CSF were recovered and subjected to protein concentration analysis by ELISA and determination of biological activity by cell proliferation assay. The efficiency of the conjugation reactions was determined by the ratio of conjugated to non-conjugated GM-CSF after centrifugation.Mice were infected with IAV and imaged post infection using a GE eXplore Locus micro CT scan . The exposure time was 90 ms per projection and the gantry was rotated over 200 degrees while 200 two dimensional projections were taken.GraphPad Prism software was used for statistical analysis. Survival curves were analyzed by chi-square test. Comparisons between two groups were calculated by unpaired Student’s t test and p<0.05 was considered statistically significant. All measures of variance are presented as SEMs.Biological activity of recombinant mouse and human GM-CSF  was evaluated with respect to protection against influenza virus. We treated C57BL/6 mice with either mGM-CSF or hGM-CSF intranasally daily for one week (1.34 μg/g body weight). A control group was treated with PBS. All mice were then infected with lethal dose (2 LD50) of mouse-adapted influenza A virus PR8. Infected mice were monitored daily and their weight loss and mortality were recorded. All C57Bl/6 mice treated with PBS or hGM-CSF and infected with PR8 lost more than 25% of their body weight whereas mGM-CSF treated mice showed less than 15% weight loss . Next, wTo confirm our finding is not specific in C57Bl/6 genotype, we repeated the experiments carried out in Fig Next we evaluated the effects of mGM-CSF and hGM-CSF on total cell counts of bronchoalveolar lavage (BAL) that were over 95% alveolar macrophages and an indicator of in vivo immune cell proliferation in the alveolar space. Overexpression of mGM-CSF in the lungs in mice significantly increases the numbers of alveolar macrophages . Wild tyThis is a well-established fact that GM-CSF is a pleiotropic cytokine. Its effects on hematopoietic cells consist of stimulation, proliferation and differentiation. These functions are mediated either directly through binding to GM-CSF-receptor or indirectly through the network of numerous cytokines, chemokines and growth factors . Hence, It is known that elderly individuals are at higher risk for complications from influenza, and vaccination is less effective in this population . Thus, ie have shown that pulmonary infections such as influenza induce lung injury and disrupt the barrier integrity of lung vasculature and results in increased albumin levels in the alveolar space . On the When WT mice were infected with 1.0 LD50 of PR8 and then treated intranasally with GM-CSF, there was a significant increase in the concentration of GM-CSF in the blood of PR8-infected mice only three hours after treatment with GM-CSF at 3 and 6 days post-infection indicating that lung injury induced by influenza infection induces a porous lung that facilitates the escape of GM-CSF from alveolar space .We next used a novel approach to confirm the lung injury of IAV-infected mice and crudely assess the pore size of the permeable IAV-injured lung vasculature/epithelium. The endothelial cell lining of the pulmonary vasculature forms a semi-permeable monolayer barrier between the blood and the interstitium of the lung. Pulmonary infections such as influenza disrupt the barrier’s integrity, resulting in facile movement of fluid and macromolecules into and out of alveoli. We used Qdot ITK carboxyl quantum dots that are fluorescent semiconductor-based nanocrystals (Life Technologies). These nanocrystals show a direct, predictable relationship between their physical size and the energy of the excitation, and consequently display a different wavelength of emitted fluorescence from different sizes. We used commercially available Qdots in this experiment that were either 13 nm (Qdot 525) or 20 nm (Qdot 625) in diameter and emitted light at 525 nm and 625 nm wavelength, respectively, using a standard fluorimeter. Qdots were administered intravenously into the tail vein of either naïve mice or IAV-infected mice seven days after infection. Samples were collected an hour later. Shortly after intravenous injection of Qdots, the 13-nm Qdots passed through glomerular capillaries of kidneys and were found in abundance in urine samples of naïve mice whereas the 20-nm Qdots did not . SamplesWe decided to take a novel approach and conjugate mGM-CSF to Qdot particles to increase its size and facilitate retention of delivered GM-CSF in the alveolar space. Recombinant mGM-CSF was conjugated to Qdot 625 particles using standard amine-coupling chemistry . ConjugaIn order to test the efficacy of Qdot-mGM-CSF in protecting IAV-infected mice, wild type C57BL/6 mice were infected with a sub-lethal dose (0.2 LD50) of influenza strain PR8 and then divided into four groups. IAV-infected mice were treated with PBS or 12.5 μg of either mGM-CSF, Qdot-mGM-CSF or Qdot only at 3 h and 24 h post-infection. There was no significant difference in the weight loss among the four groups . HoweverGM-CSF is among the first cytokines identified, is a pleiotropic cytokine and its biological activity is mediated by binding to specific cell surface receptors . GM-CSF GM-CSF has been used by our lab and others to protect against influenza, secondary bacterial pneumonia, and acute lung injury –15,26–28We have provided the first evidence that pulmonary GM-CSF provides extraordinary protection against different IAV strains by stimulating pulmonary innate immunity through alveolar macrophages . We haveOne of the controversies is heterologous use of GM-CSF to target the innate immunity of lung . The preA recent publication demonstrated that treating mice intranasally with human GM-CSF conferred 80% protection against lethal influenza H1N1 . These rElderly population is significantly more vulnerable to seasonal influenza infections and approximately 90% of influenza related deaths in the USA occurs among elderly population of 65 years or older ,41. On tOur data and previous publication from our labs and others show that delivering GM-CSF to the lungs before infection protects mice against lethal pneumonia due to different strains of IAV Figs  and 3 anThe covalent attachment of nanoparticles to therapeutic proteins enhances their bioavailability, prolongs half-lives and reduces immunogenicity . Ten pro


The aim of this study was to analyze the state of activity and levels of complement in the cerebrospinal fluid (CSF) of patients with various prion diseases (PrDs).The proteomic data emphasized the levels of 20 known complement components found in the CSF of the sCJD panel that were lower than those found in the non-PrD panel. 50 % of the complement hemolytic activity (CH50) assays revealed significantly lower activity of complement in the CSF of the sCJD panel. The decreased levels of three key complement subunits, C3a/α, C4β, and C9 in the CSF of the sCJD panel were verified by Western blots. Furthermore, the CH50 values in the CSF of 136 sCJD, 39 gCJD, 22 FFI and 145 non-CJD patients were individually tested. Compared with the control of non-PrD, the CH50 value in the CSF specimens of various PrDs, especially in three subtypes of inherited PrDs, were significantly lower. Relationship analysis identified that the CH50 activity in the CSF was negatively associated with the protein 14–3–3 positive in the CSF.These results indicate a silent complement system in the CSF of PrD patients.The online version of this article (doi:10.1186/s40249-016-0128-7) contains supplementary material, which is available to authorized users. Please see Additional file Human prion diseases (PrDs) are a category of fatal neurodegenerative disorders including Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI) and Kuru . SurrogaThe complement system, which consists of at least 30 kinds of soluble and membrane-bound proteins, is an immune-defense mechanism with a wide range of effects that take place in the central nervous system (CNS). Evidence suggests that the activation of complement components occurs in patients with various neurodegenerative disorders , and thaThis study was approved by the Ethical Committee of the National Institute for Viral Disease Control and Prevention, China CDC, under protocol 2009ZX10004–101.To verify the changes of complement in the CSF of sCJD patients according to the proteomic code, we reviewed 37 CSF samples from probable sCJD patients who tested positive for 14–3–3 protein in their CSF and typical periodical sharp-wave complexes (PSWC) in their EEG. We also analyzed 36 CSF samples from non-CJD patients, who were CSF 14–3–3 negative and had normal levels of EEG. These samples were taken from the CSF bank in the China CJD Surveillance Center  in CSF (50 μl of each tested sample) was derived from the commercial double-antibody sandwich enzyme-linked immunosorbent assays . Western blots for the complement components C3a/α, C4β and C9 in the panels of the pooled CSF (20 μl of each tested sample) were conducted using anti-C3a/α mAbs (sc-47688), anti-C4β pAbs (sc-25816) and anti-C9 mAbs (sc-390000) (Santa Cruz biotechnology Inc). The detailed procedures of CH50 ELISA and Western blot are described elsewhere [P < 0.01). 5 out of 20 complement components were statistically different at the 95 % confidence interval . Furthermore, the decreased levels of three key subunits of complement C3a/α, C4β, and C9 in complement cascades in the CSF panel of sCJD were observed by Western blots  were analyzed using a commercial indirect ELISA kit. Compared with the panel of non-CJD, CH50 in the sCJD panel was significantly lower . Further multivariate logistic regression proposed that only the presence of 14–3–3 in CSF showed the notable correlation with CSF CH50 activity (P = 0.004). To further evaluate the relationship between CH50 activity and 14–3–3 positive CSF, all enrolled cases were grouped depending if they were 14–3–3 positive or negative. As shown in Fig. P < 0.0001). Subsequent assays based on various diagnoses illustrated lower CSF CH50 activity in the sCJD cases that were 14–3–3 positive (P < 0.0001). Non-CJD cases that tested 14–3–3 positive, in contrast, tested relatively higher without statistical difference (P = 0.545) . This demonstrates a negative correlation of lower CSF levels and CH50 activity with 14–3–3 positive PrD patients.To address the potential factor(s) correlated with the CSF CH50 activity, some clinical and laboratory parameters from the enrolled patients were statistically analyzed. Univariate analysis illustrated a significant correlation between 14–3–3 and CH50 , are lower than their non-CJD counterparts are. Additionally, subjects whose CSF samples tested 14–3–3 positive exhibit lower complement activity in sCJD patients.


However, despite extensive experimental results supporting the formation of adenosine heteromers in heterologous systems, the existence of such heteroreceptor complexes in the brain remains largely unknown, mainly because of the lack of appropriate methodology. Also no systematic study was carried out on heteromers form by adenosine receptor subtypes alone. In this study, we used several experimental approaches2 to investigate whether adenosine receptor A2A and A1 subtypes can form heteromers among themselves. In situ PLA clearly demonstrated that adenosine receptors  exist as homo/heteroreceptor complexes in rat brain. In the hippocampus, A2A-A1 heteroreceptor complexes are mainly localized to the pyramidal cell layer of the Ammon´s horn and the hilo (PoDG). The complex was also observed throughout the piriformis layer. Several distinct differences were apparent between the distribution of the A2A-A1 heteroreceptor complexes and that of the A2A-A2A homoreceptor complex, which could have important functional consequences. Furthermore, bioluminescence resonance energy transfer analysis of adenosine A2A receptors established that they can physically interact in HEK293T27 cells, as both homomers and heteromers. In addition, static/non-dynamical human GPCR data derived from this and other interaction studies were integrated in a large scale graph, called the GPCR heterodimer network (


In contrast, a combination of three non-cross-competing antibodies completely neutralizes GM-CSF activity in vitro by sequestering the cytokine in high-molecular-weight complexes, and in vivo promotes the rapid degradation of GM-CSF-containing immune complexes in an Fc-dependent manner. Taken together, these findings provide a plausible explanation for the severe phenotype of PAP patients and for the safety of treatments based on single anti-GM-CSF monoclonal antibodies.Pulmonary alveolar proteinosis (PAP) is a severe autoimmune disease caused by autoantibodies that neutralize GM-CSF resulting in impaired function of alveolar macrophages. In this study, we characterize 21 GM-CSF autoantibodies from PAP patients and find that somatic mutations critically determine their specificity for the self-antigen. Individual antibodies only partially neutralize GM-CSF activity using an  Autoimmune pulmonary alveolar proteinosis is caused by autoantibodies to GM-CSF. Here the authors show that the individual autoantibodies only partially neutralize GM-CSF and that antibodies to at least three different epitopes are required to block GM-CSF bioavailability. Autoantibodies against cytokines have been frequently reported both in healthy individuals and in patients with autoimmune or infectious diseases1235in vitro using cell proliferation bioassays with cytokine-dependent cell lines. Interestingly, however, several studies with toxins910in vivo. In addition, it has been reported that monoclonal antibodies to IL-2, that neutralize the cytokine activity in vitro, can paradoxically enhance and extend IL-2 activity when injected in vivoThe reason why cytokine autoantibodies may or may not cause pathology is not entirely clear. The prevailing view is that, when of enough affinity and present above a certain threshold of concentration, an autoantibody can neutralize the biological activity of the cytokine by simply binding and preventing its interaction with the cognate cellular receptor, a mechanism that can be recapitulated −1, and that bind and neutralize GM-CSF at high titresAutoimmune PAP is a rare and severe disease caused by the production of autoantibodies that neutralize the biological activity of GM-CSF. GM-CSF is essential for the terminal differentiation and function of alveolar macrophages that are responsible for the catabolism of surfactant lipids and proteins in the lungsin vitro, depending on the experimental conditions of the bioassay, whereas three non-cross-competing antibodies completely neutralize GM-CSF activity. In addition, we show that, in vivo, single antibodies enhance the levels of bioavailable GM-CSF, while three non-cross-competing antibodies induce a rapid Fc-dependent clearance of the cytokine. Finally, we show that in most cases somatic mutations are critical for binding to GM-CSF, suggesting that clones that give rise to autoantibodies derive from memory rather than naive B cells.In this study we isolate a panel of monoclonal autoantibodies from PAP patients and show that single antibodies can only partially neutralize GM-CSF activity −1 , ranged from 0.2 to 5.1 nM, consistent with high-affinity binding. Of notice, the kinetics values were heterogeneous. For instance, antibodies GCA7 and GCE402, which had comparable EC50 and KD values, showed different kinetics, being GCA7 characterized by a low on-/low off-rate and GCE402 by a high on-/high off-rate. The properties of the monoclonal antibodies isolated are consistent with a polyclonal response of somatically mutated B cells.Peripheral blood mononuclear cells (PBMCs) and sera were collected from five chronic PAP patients of age ranging from 32 to 69 years. All patients had high serum titres of GM-CSF autoantibodies, ranging from 46 to 805 μg ml−1 . Memory −1 . The EC5The role of somatic mutations in the development of GM-CSF autoantibodies was investigated by testing versions of the autoantibodies in which the somatic mutations of the whole V(D)J gene were removed. We refer to these antibody variants as the unmutated common ancestors (UCA). We also produced shuffled variants in which only the VH or the VL were reverted to the UCA sequence. When analysed using SPR and ELISA, five out of the seven UCA antibodies tested did not bind to GM-CSF, while the remaining two showed a reduced, but still detectable, binding . ShuffliUsing SPR cross-competition experiments we identified multiple antigenic sites on GM-CSF and a complete antigenic map based on a matrix of cross-competition experiments was generated . Sites I−1 and from 0.018 to 0.181 μg ml−1, respectively (−1). These findings are consistent with previous reports17The neutralizing activity of the autoantibodies was assessed by measuring their ability to inhibit the proliferation of TF-1 cells in response to recombinant GM-CSF. Polyclonal IgG and autoantibodies purified from the sera of PAP patients showed potent and complete neutralizing activity, with IC90 values ranging from 0.53 to 36 μg mlectively . From th−1, while the therapeutic antibodies Namilumab and MOR103 , which was lower than that of the therapeutic antibodies MOR103 and Namilumab. A strong synergy between three non-cross-competing antibodies was observed for most combinations tested, with IC90 values comparable to those of affinity-purified antibodies from PAP sera  combined with GCB9 and GCA21 antibodies . These rin vitro using cell proliferation bioassays. The second is the in vivo Fc-dependent degradation of immune complexes formed between GM-CSF and multiple autoantibodies.Our study identifies two mechanisms by which polyclonal anti-GM-CSF antibodies produced by PAP patients inhibit the biological activity of GM-CSF. The first is the irreversible sequestration of GM-CSF in high-molecular-weight immune complexes that can be readily detected The autoantibodies isolated show affinities in the nanomolar range and distinct kinetics of binding, differing in particular in their off-rates. Overall, they were comparable to those reported in a recent study242223in vivoin vivo in an Fc-dependent manner. The degradation of GM-CSF induced by polyclonal antibodies is reminiscent of a previous study where three antibodies were shown to cause degradation of IL-6 in vivo27A key observation in this study was that, when injected in mice, single antibodies led to the accumulation of a large pool of long-lived GM-CSF that was still able to dissociate and trigger the receptor, as shown by the capacity of the sera to stimulate proliferation of TF-1 cells. The capacity of a single antibody to build up a large cytokine reservoir, as we observed for GM-CSF, could be the basis of the enhancing activity of monoclonal antibodies to common gamma-chain cytokines An interesting observation of this study is related to the role of somatic mutations and the presence of low levels of GM-CSF antibodies in healthy individuals. The finding that the UCA of the autoantibodies did not bind, or showed minimal binding to GM-CSF, is consistent with previous studies on the role of somatic mutations in determining the specificity of autoantibodies to desmogleins or DNA in patients with pemphigus or lupus29303233GM-CSF autoantibodies have been described not only in PAP patients but also in the serum of healthy donors or in patients with inflammatory bowel disease with normal pulmonary function21−1 of recombinant human GM-CSF (Miltenyi Biotec), blocked with blocking solution Stabilcoat  and incubated with titrated sera , followed by incubation with horseradish peroxidase-conjugated anti-human IgG F(ab′)2 fragment. Plates were then washed, substrate  was added followed by 1 N H2SO4 and the plates were read at 450 nm. Serum concentration of GM-CSF autoantibodies was determined from a GM-CSF-autoantibody polyclonal reference standard by quadratic regression analysis using Microsoft Excel . The Disease Severity Score, a combination of presence and absence of symptoms and degree of PaO2, was determined according to the classification previously describedPeripheral blood samples were obtained from five PAP patients with high levels of GM-CSF autoantibodies. Determination of the serum level of GM-CSF autoantibodies was performed in the Laboratory of the Rare Lung Disease Consortium at the Cincinnati Children's Hospital Medical Center, as described17The usage of VH and VL genes and the amount of somatic mutations were determined by analysing the homology of VH and VL sequences of mAbs to known human V, D and J genes by the IMGT database−1 of recombinant human GM-CSF (Gentaur), blocked with 1% BSA and incubated with titrated antibodies, followed by AP-conjugated anti-human IgG secondary antibodies (SouthernBiotech). Plates were then washed, substrate  was added and plates were read at 405 nm. EC50 (ng ml−1) was calculated for every sample by nonlinear regression analysis using the GraphPad Prism 5 software.Human mAbs and total IgG from PAP sera were purified by protein A or protein G chromatography . Total IgG from healthy donors were purified using the HiTrap Protein A HP columns  and concentrated using Amicon Ultra filter units . Total GM-CSF antibodies were affinity-purified from PAP sera using magnetic beads (Invitrogen) conjugated with human GM-CSF. Total IgGs were quantified using ELISA plates coated with anti-human IgG (SouthernBiotech) using Certified Reference Material 470  as standard. Binding to GM-CSF was tested by ELISA using 384-well SpectraPlates (PerkinElmer) for primary screenings or 96-well MaxiSorp plates (Nunc) for any following test. Briefly, ELISA plates were coated with 1 μg ml−1. Monoclonal antibodies were diluted in HBS (200 nM) and injected on the protein A-coated chip for capturing, followed by injection of different concentrations of human GM-CSF ; one channel of the chip was injected with HBS and used as reference for the analysis. Injection time and dissociation time were 120 and 600 s, respectively. Each binding interaction of mAbs with GM-CSF was assessed using a ProteON XPR36 instrument (Bio-Rad) and data processed with the ProteOn Manager Software. Ka, Kd and KD were calculated by applying the Langmuir fit model. To determine the epitope specificity, GM-CSF autoantibodies (150 mM) were directly immobilized on a sensor chip, followed by injection of GM-CSF (100 nM) and autoantibodies (200 nM). To assess simultaneous binding to GM-CSF, different mAbs (200 nM each) were serially injected after the GM-CSF capture (50 nM). Injection time and dissociation time were 60 and 20 s, respectively.Protein A (450 nM) was stabilized in 10 mM acetate buffer, pH 4.5, and immobilized on a EDC/NHS pre-activated ProteOn sensor chip (Bio-Rad) through amine coupling; unreacted groups were blocked by injection of ethanolamine HCl (1 M). HEPES-buffered saline  was used as running buffer. All injections were made at the flow rate of 100 μl min−1). A universal solvent 2-μm filter (Agilent) was put between injector and column. Detection was performed using a Variable Wavelength Detector (Agilent) with ultraviolet absorption at 220 nm.Three non-cross-competing mAbs were diluted in PBS singularly or as a three-antibody-combination  and were mixed with GM-CSF (1:1 or 10:1 molar ratios) for 1 h at room temperature (RT). Samples were analysed with the Agilent 1100 HPLC machine using TSK-GEL G3000SW columns  with PBS as the mobile phase (flow rate: 1 ml min−1 human GM-CSF (Gentaur) and 10 ng ml−1 human IL-3 (ImmunoTools). Cells were grown at 37 °C in a humidified incubator with 5% CO2. A GM-CSF neutralization assay was performed by serially diluting mAbs  in growth medium with neither GM-CSF nor IL-3, adding GM-CSF at a concentration of 100 pg ml−1, and preincubating in 96-well flat-bottom cell culture plates (Costar) at 37 °C for 1 h. TF-1 cells were washed five times, diluted in the growth medium with neither GM-CSF nor IL-3, and 10,000 cells per well were seeded . In other tests, GM-CSF was used at the final concentration of 500 and 5,000 pg ml−1, and 1,000 cells per well were seeded. Cells with or without GM-CSF in absence of antibodies were used as control to determine maximum and minimum levels of cell proliferation. Plates were incubated at 37 °C in a humidified incubator with 5% CO2 for 72 h, and cell proliferation was measured after 6-h incubation with 0.2 μCi per well of [3H]-thymidine (PerkinElmer). GM-CSF neutralization was calculated as percentage of inhibition of TF-1 growth with the following formula: [1−(CCPM of a single well−average CCPM of control cells grown without GM-CSF) × (average CCPM of control cells grown with GM-CSF−average CCPM of control cells grown without GM-CSF)−1] × 100 (CCPM=corrected counts per minute). IC90 (μg ml−1) was calculated for every sample by a nonlinear regression analysis using the GraphPad Prism 5 software. In some experiments mouse sera were titrated in TF-1 growth medium and preincubated at 37 °C for 30 min. TF-1 cells were washed and seeded . A titration of GM-CSF  was added as growth control. CCPM of each single well were plotted against the serum titration.TF-1 cells (Cell Lines Service) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Hyclone), 1% GlutaMAX, 1% Penicillin/Streptavidin, 1% non-essential amino acids, 1% sodium pyruvate, 1‰ 2-mercaptoethanol , 5 ng ml−1 of an antibody that bound to site II of GM-CSF was used to coat 96-well Maxisorp plates (Nunc), which were then blocked with PBS+10% FBS (Gibco). All sera and GM-CSF, which was used as standard , were titrated and tested in parallel under different conditions: in one plate all samples were supplemented with 25% (vol/vol) of an alkaline dissociation buffer , in the other plate all samples were supplemented with 25% (vol/vol) of PBS+10% FBS. Plates were left overnight at RT. Detection of captured GM-CSF was made with 1 μg ml−1 of a biotinylated antibody that bound to site I of GM-CSF for 1 h at RT, followed by binding of 0.5 μg ml−1 streptavidin-AP (Jackson ImmunoResearch) for 1 h at RT. Plates were then washed, substrate  was added and plates were read at 405 nm.BALB/C mice were supplied by The Jackson Laboratory, USA; Harlan, Italy; Charles River, Germany, Italy. Animal experiments were approved by the Cantonal Autorithies of Cantone Ticino. Groups of 6- to 8-week-old female BALB/c mice were injected intravenously with 100 μg of purified mAbs or 2 mg of total IgG purified from PA96 patient. After 16 h, 2 μg of human GM-CSF were injected. Serum samples were collected on day 1 and day 5. GM-CSF was quantified with a sandwich ELISA. Briefly, 10 μg ml−1 Gentamicin and 20 μg ml−1 Blasticidin. Untransfected TZM-bl cells were used as negative control and were maintained in DMEM medium supplemented with 10% fetal bovine serum (Hyclone) and 2% Penicillin/Streptavidin. Cells were grown at 37 °C in a humidified incubator with 5% CO2. Expression of specific FcγRs was assessed by staining TZM-bl cells with FITC-conjugated anti-CD64 (anti-FcγRI), anti-CD32 (anti-FcγRIIa and anti-FcγRIIb) and anti-CD16 (anti-FcγRIIIa) antibodies . Untransfected and transfected TZM-bl cells were washed with staining buffer  and seeded in 96-well plates at a density of 50,000 cells per well. A single anti-GM-CSF mAb (GCA21) or a combination of three non-cross-competing mAbs  at a final concentration of 2.5 μg ml−1 were mixed with 0.05 μg ml−1 GM-CSF, or staining buffer . The LALA versions of all antibodies and a mAb with a different specificity were included as controls. Samples were incubated at 37 °C for 30 min to allow the formation of immune complexes and then cooled down to 4 °C before adding them to TZM-bl cells for 30 min. Cells were washed twice and stained with anti-human IgG Fcγ fragment specific F(ab′)2 fragment (Jackson ImmunoResearch). Samples were analysed on BD FACSCanto (BD Biosciences) and the median intensity fluorescence was analysed and compared between samples.Four TZM-bl cell lines (NIH AIDS Research & Reference Reagent Program) each transfected with a specific Fcγ receptor  were maintained in DMEM medium supplemented with 10% fetal bovine serum (Hyclone), 0.025 M Hepes, 10 μg mlHow to cite this article: Piccoli, L. et al. Neutralization and clearance of GM-CSF by autoantibodies in pulmonary alveolar proteinosis. Nat. Commun. 6:7375 doi: 10.1038/ncomms8375 (2015).Supplementary Figures 1-3, Supplementary Tables 1-2


The cerebrospinal fluid (CSF) circulatory system is involved in neuroimmune regulation, cerebral detoxification, and delivery of various endogenous and exogenous substances. In conjunction with the choroid plexuses, which form the main barrier site between blood and CSF, this fluid participates in controlling the environment of the developing brain. The lack of comprehensive data on developmental changes in CSF volume and distribution impairs our understanding of CSF contribution to brain development, and limits the interpretation of blood-CSF permeability data. To address these issues, we describe the evolution of the CSF circulatory system during the perinatal period and have quantified the volume of the different ventricular, cisternal and subarachnoid CSF compartments at three ages in developing rats.Immunohistofluorescence was used to visualize tight junctions in parenchymal and meningeal vessels, and in choroid plexus epithelium of 19-day fetal rats. A quantitative method based on serial sectioning of frozen head and surface measurements at the cutting plane was used to determine the volume of twenty different CSF compartments in rat brain on embryonic day 19 (E19), and postnatal days 2 (P2) and 9 (P9). Blood-CSF permeability constants for sucrose were established at P2 and P9, following CSF sampling from the cisterna magna.Claudin-1 and claudin-5 immunohistofluorescence labeling illustrated the barrier phenotype acquired by all blood–brain and blood-CSF interfaces throughout the entire CNS in E19 rats. This should ensure that brain fluid composition is regulated and independent from plasma composition in developing brain. Analysis of the caudo-rostral profiles of CSF distribution and of the volume of twenty CSF compartments indicated that the CSF-to-cranial cavity volume ratio decreases from 30% at E19 to 10% at P9. CSF compartmentalization within the brain changes during this period, with a major decrease in CSF-to-brain volume ratio in the caudal half of the brain. Integrating CSF volume with the measurement of permeability constants, adds to our understanding of the apparent postnatal decrease in blood-CSF permeability to sucrose.Reference data on CSF compartment volumes throughout development are provided. Such data can be used to refine blood-CSF permeability constants in developing rats, and should help a better understanding of diffusion, bulk flow, and volume transmission in the developing brain.The online version of this article (doi:10.1186/s12987-015-0001-2) contains supplementary material, which is available to authorized users. The cerebrospinal fluid represents 50% of the brain extracellular fluid in adult mammals including humans. Recent findings indicate that the CSF circulatory system is not a mere passive drainage system for the brain, but is involved in neuroimmune regulation -3, cerebThe CSF is secreted and protected from peripheral harmful molecules by the choroid plexuses which develop from the neuroepithelium to form the major site of the blood-CSF barrier. Choroid plexuses also have the capacity to secrete into CSF different bioactive substances including guidance molecules as well as growth and differentiation factors. Owing to the early fetal development of the choroidal tissue, the choroid plexus-CSF system is likely to fulfill essential functions for brain development -12. The Animal care and procedures were conducted according to the guidelines approved by the French ethical committee (decree 87–848), and by the European Community (directive 86-609-EEC). Sprague–Dawley rats, either pregnant time-dated females, or females with their litter, were obtained from Janvier . All animals were kept under identical conditions in standard cages, with free access to food and tap water under a controlled environment (12 h day-light cycles). Timed-pregnant female rats were anesthetized with inhaled isoflurane (5%) and body temperature was maintained with a heated pad. E19 animals were removed one by one from the mother and frozen in 2-methylbutane cooled at −50°C, in a position that allowed serial sectioning of the brain. P2 and P9 animals were decapitated and the severed head immediately frozen in 2-methylbutane cooled at −50°C. Limited expansion of the brain at the time of freezing led occasionally to a partial crack in the cranial bones. These brains were discarded from the analysis. Animal heads were kept at −80°C until use for serial sectioning.Immunohistochemical analysis of claudins in E19 rat brains (n = 3) was performed as previously described , using aThe whole head  with intact meninges and CSF spaces was cut into 35-μm-thick serial sections using a CM 3050S cryostat . After every fifth section, a photograph of the remaining tissue block in the cryostat was taken with a sharp focus on the cutting plane using a VR-320 camera . On these photographs, the tissue could be easily distinguished from the crystal-clear CSF present in the cisternal, subarachnoid, and ventricular spaces. Each photograph included a scale bar added in the cutting plane to enable quantification of the surface area of the cerebral structures and spaces of interest. In addition, every fifth section was collected on a glass slide and stained with hematoxylin and floxin. Micrographs of the sections were taken using a AxioCam ERc5s camera (Zeiss) connected to a SMZ800 stereotaxic microscope . The surface areas of the cranial cavity and of the CSF spaces of interest were delineated on the photographs and determined using the ZEN 2012 software (Zeiss). Micrographs of the stained sections were used to determine the surface area of the smaller intraparenchymal CSF-filled compartments (such as the third ventricle), when the borders were not sharp enough to be outlined in the tissue block photographs.The total brain tissue volume and the volume of each fluid compartment were calculated by building the area under the surface area-distance curve, between the section where the given structure appears and the section where it disappears. Calculation was done using the composite trapezoidal rule for integration. CSF compartment volumes and tissue volumes were corrected by factors of 0.910 and 0.920, respectively, to account for water expansion at the time of freezing. The correction factor for tissue was adjusted for a water content in neonatal rat brain tissue of 88.5% ,15. The Choroid plexuses from individual animals (n = 6 for both P2 and P9) were micro-dissected under a stereomicroscope and digested in 1 M sodium hydroxide. Protein concentration was determined by the method of Peterson  using bo14C]-sucrose  was administrated by intraperitoneal injection (12.5nCi/g) under slight gas anaesthesia, to prevent backflow. Blood was sampled from different animals of the same litter (n = 5 for P2 and n = 9 for P9) by cardiac puncture under pentobarbital anesthesia at times ranging from 3 to 30 minutes after injection. Additional animals from the litter were injected with 50nCi/g [14C]-sucrose. In these animals, blood was sampled twenty minutes after injection, rapidly followed by CSF sampling as follows. The skin was incised above the cistern magna, and a glass pipette was introduced in the cistern for CSF collection. The CSF was transferred in a microtube and the sample volume measured. Blood was collected in a heparinized tube and centrifuged at 5000 rpm for 5 min. Plasma and CSF samples were analyzed for radioactivity in a 1600 TR Packard scintillation counter, using Ultima-gold  as scintillation cocktail.Radio labelled [14C]-sucrose concentration-time curve (AUC) [in csf and Kw csf were calculated as follows:A litter-based area under the plasma [ve (AUC)  was built is the [14C]-sucrose concentration in CSF at the time of sampling t (ranging from 20 to 23.5 minutes), AUC 0→t the AUC recalculated from the litter-based experimental AUC and from the plasma concentration measured before CSF sampling. Kin csf is similar to an influx constant as defined previously for brain tissue.where Ccsf is the total volume of CSF in the brain. Kw csf represents the plasma volume cleared in the whole CSF of the animal.where VAntibodies against claudin-5, the hallmark constituent of tight junctions at the blood–brain barrier stained the entire microvascular network throughout the brain as well as endothelial junctions of meningeal vessels in E19 rat fetuses , CSF flow is very limited between the pia-covered cerebral cortex and the arachnoid/dura mater. This is likely to be different in human, in which cortical foldings develop mainly between week 25 and 30 of gestation , resultiFinally the data show that the size of the ventricular system remains modest at all stages. The volume of the ventricles changed only moderately between E19 and P9, with a slight decrease for the lateral ventricles, an increase for the third ventricle and no change for the fourth ventricle  is representative of the overall total CSF concentration. Kin csf values did not differ significantly between P2 and P9 animals , was 0.17 × 10−3 min−1 -sucrose permeability constants measured in 2- and 9-day-old rats confirm that the brain fluid environment is controlled and independent from plasma composition during development. Volumes of the different CSF fluid compartments measured during pre- and postnatal development in rat indicate not only a decrease in CSF-to-brain volume ratio, but also a geographical redistribution of the fluid especially around birth in rat. The volume data can be used to refine blood-CSF permeability constants in developing animals. The description of the different ventricular, cisternal and subarachnoid spaces and their respective developmental volume profile compared to total brain volume can be used to better understand cerebral fluid dynamic and diffusional/bulk flow movement of solutes in the developing brain. These fluid spaces are involved in volume transmission, clearance of potentially deleterious molecules, and immune cell migration within the brain These results will enable a better appreciation of the role of fluid compartments in brain development, neuroimmune surveillance of the infant, and in neonatal injuries.The presence of tight junctions at blood–brain/CSF interfaces in 19-day rat embryos and the [


Post-traumatic stress disorder (PTSD) is a debilitating anxiety disorder that may develop after an individual has experienced or witnessed a severe traumatic event. It has been shown that the 18 kDa translocator protein (TSPO) may be correlated with PTSD and that the TSPO ligand improved the behavioral deficits in a mouse model of PTSD. Midazolam, a ligand for TSPO and central benzodiazepine receptor (CBR), induces anxiolytic- and anti-depressant-like effects in animal models. The present study aimed to determine whether midazolam ameliorates PTSD behavior in rats as assessed by the single prolonged stress (SPS) model. The SPS rats received daily Sertraline (Ser)  and midazolam  during the exposure to SPS and behavioral assessments, which included the open field (OF) test, the contextual fear paradigm (CFP), and the elevated plus-maze (EPM). The results showed that, like Ser , midazolam  significantly reversed the behavioral deficiencies of the SPS rats, including PTSD-associated freezing and anxiety-like behavior but not the effects on spontaneous locomotor activity. In addition, the anti-PTSD effects of midazolam  were antagonized by the TSPO antagonist PK11195 , the CBR antagonist flumazenil  and the inhibitor of steroidogenic enzymes finasteride , which by themselves had no effect on PTSD-associated freezing and anxiety-like behavior. In summary, this study demonstrated that midazolam improves the behavioral deficits in the SPS model through dual TSPO and CBR and neurosteroidogenesis. Post-traumatic stress disorder (PTSD) can develop following exposure to severe traumatic events, such as the events that occur during war, after a violent personal assault, or following natural disasters As a general principal, the selective serotonin reuptake inhibitors (SSRIs), such as sertraline and paroxetine, are a first-line treatment option for the core PTSD symptoms The 18 kDa translocator protein (TSPO), a promising target for treating neurological disorders without benzodiazepine-like side effects The physiological and behavioral changes observed in animals exposed to the single prolonged stress (SPS) model can represent the pathophysiological process and core symptomatology of PTSD, including anxiety behavior and cognitive impairments Currently, no reports have examined the therapeutic effects of midazolam on PTSD in pre-clinical animal model experiments. Midazolam's direct interact to CBR and high affinity for TSPO lead to the anxiolytic-like effects and induce the hypothesis Male Sprague-Dawley rats were obtained from the Beijing SPF Animal Technology Company . These animals were selected because Sprague-Dawley rats exhibit PTSD-like symptoms Based on previous studies, Sertraline (Ser)  was administered at a dose of 15 mg/kg (p.o.) as a positive control in the behavioral tests The SPS model was performed as previously described with minor modifications We investigated whether the SPS model and midazolam treatment affected locomotor activity. The OF test was performed as previously described with minor modifications The CFP test is based on previous studies that showed a freezing response upon re-exposure to the traumatic context can be used as a measure of conditioned associative fear memory, which reflects the response to trauma-related cues, a symptom of PTSD The EPM is one of the most widely used assessments to evaluate the PTSD-associated anxiogenic-like behavior in rodents p<0.05 were considered significant for all the tests.Unless otherwise specified, statistical analysis was performed by GraphPad Prism 5.0 . All data are presented as the mean±S.E.M. The statistical significance of experimental observations was determined by one-way analysis of variance (ANOVA) or two-way ANOVA followed by Bonferroni's multiple comparison tests, as indicated in the results section. Differences with p>0.05, p>0.05, p>0.05, The effects of repeated administration of midazolam on locomotor activity are shown in p<0.01, p<0.01, p<0.01, p<0.01, The effects of midazolam on the contextual freezing behavior of the rats are shown in p<0.05; p<0.01; p>0.05; p>0.05; p<0.01; p<0.01; p>0.05; p>0.05; As shown in The present study was performed to investigate the possible mechanism of the anti-PTSD effects of repeated midazolam treatment using the SPS model. Similar to Ser, midazolam significantly ameliorated the PTSD-associated freezing and anxiety-like behaviors without affecting spontaneous locomotor activity, and these effects were antagonized by PK11195, flumazenil or finasteride. We found that the anti-PTSD effects of midazolam were mediated by TSPO, CBR and neurosteroidogenesis.Furthermore, the results in the present study indicate that SPS-induced freezing and anxiety, which is consistent with previous studies showing that the SPS model produces enhanced contextual freezing and increases anxiety-like behavior in the elevated plus maze (EPM) test and stress-induced analgesia compared with rats subjected to a sham treatment We also found that Ser improved the behavioral deficits of this SPS model, which is consistent with a previous study A receptor through a neurosteroid recognition site directly and different to CBR A receptor-mediated tonic inhibition facilitates phasic increases in glutamatergic and GABAergic activity, which could disrupt the frontal lobe-mediated inhibition of the amygdale A receptor at neurosteroid recognition sites  The anti-PTSD effects of midazolam might also be associated with the neurosteroidogenesis of allopregnanolone, which is known to interact with GABAA receptor inhibition by direct actions on central benzodiazepines receptor. Therefore a specific antagonist of the central benzodiazepines receptor, flumazenil On the other hand, as a typical benzodiazepines drug, midazolam was known as enhancing GABAThe present study demonstrated that repeated administration of midazolam ameliorated behavioral deficits in the SPS model without affecting spontaneous motor activity. The anti-PTSD effects of midazolam were mediated at least partially by TSPO and CBR and neurosteroidogenesis.


Capsicum annum) response to Ralstonia solanacearum inoculation (RSI), but the underlying mechanism remains largely unknown. Here, we functionally characterize CaCDPK15 in the defense signaling mediated by CaWRKY40. Pathogen-responsive TGA, W, and ERE boxes were identified in the CaCDPK15 promoter (pCaCDPK15), and pCaCDPK15-driven GUS expression was significantly enhanced in response to RSI and exogenously applied salicylic acid, methyl jasmonate, abscisic acid, and ethephon. Virus-induced gene silencing (VIGS) of CaCDPK15 significantly increased the susceptibility of pepper to RSI and downregulated the immunity-associated markers CaNPR1, CaPR1, and CaDEF1. By contrast, transient CaCDPK15 overexpression significantly activated hypersensitive response associated cell death, upregulated the immunity-associated marker genes, upregulated CaWRKY40 expression, and enriched CaWRKY40 at the promoters of its targets genes. Although CaCDPK15 failed to interact with CaWRKY40, the direct binding of CaWRKY40 to pCaCDPK15 was detected by chromatin immunoprecipitation, which was significantly potentiated by RSI in pepper plants. These combined results suggest that RSI in pepper induces CaCDPK15 and indirectly activates downstream CaWRKY40, which in turn potentiates CaCDPK15 expression. This positive-feedback loop would amplify defense signaling against RSI and efficiently activate strong plant immunity.CaWRKY40 is a positive regulator of pepper ( Exposure to biotic or abiotic stresses triggers transcription reprogramming of defense-associated gene expression and subsequent biochemical and physiological responses, which leads to plant adaptation1242+) is a ubiquitous second messenger involved in plant responses to abiotic and biotic stresses2+ flux across the plasma membrane, leading to rapid increases in the cytoplasmic Ca2+ concentration, with stress-dependent variations in frequency, amplitude, and duration72+ concentration are sensed and decoded by different Ca2+ sensors and/or Ca2+ -binding proteins, including calmodulins (CaMs), calmodulin-like proteins (CaMLs), calcineurin B-like proteins (CBLs), and calcium-dependent protein kinases (CDPKs), which subsequently activate different downstream defense responses562+ sensors and effectors, thereby allowing individual CDPK proteins to relay specific Ca2+ signatures to downstream components via CDPK-dependent protein phosphorylation. CDPKs are encoded by a large gene family, including 34 CDPK genes in ArabidopsisPopulusArabidopsis have been reported or predicted to be membrane-associated proteinsCDPK genes exhibit inducible expression patterns; have pivotal roles in plant responses to abiotic stresses such as drought1718171818Calcium  families, characterized by their highly conserved WRKY domains and indiscriminate binding to their cogent W-box26Capsicum annuum) is one of the most important vegetables worldwide. Pepper is a typical Solanaceae and is susceptible to various soil-borne diseases such as Ralstonia solanacearum inoculation (RSI) and Phytophthora capsici, which is aggravated by high temperature and high humidity. These diseases cause heavy losses in crop yield and quality. Pepper germplasm evolved under these stresses, and may have developed unique mechanisms for disease tolerance and/or resistance, and tolerance to high temperature and high humidity. Our previous study reported that CaWRKY40 functioned as positive regulator in pepper’s response to Pseudomonas solanacearum attack and heat stress under high humidityCaWRKY40 was transcriptionally modulated by CaWRKY6, another member in the WRKY TF family in pepperPseudomonas solanacearum attack by indirectly activating CaWRKY40 expression, and CaWRKY40 in turn activated transcriptional expression of CaCDPK15 by binding to the CaCDPK15 promoter, thereby establishing a positive-feedback loop.Pepper , suggesting a possible role in pepper immunity to R. solanacearum. In the present study, the CaCDPK15 promoter (pCaCDPK15) was identified, along with the cis-elements within the promoter  , methyl jasmonate (MeJA), abscisic acid (ABA), and ethephon (ETH). The results showed that GUS expression was upregulated by RSI, SA, MeJA, ABA, and ETH, with different temporal expression patterns  . A pCaCDpatterns .Nicotiana benthamiana leaves by agroinfiltration. The subcellular locations of CaCDPK15-GFP and GFP control were visualized with laser scanning confocal microscopy. The results revealed that CaCDPK15-GFP was localized in both the plasma membrane and the nucleus, whereas the GFP control was localized in multiple subcellular compartments including the cytoplasm and the nucleus . The vectors TRV1 (PYL192) and TRV2:CaCDPK15 (PYL279) were separately transformed separately into Agrobacterium tumefaciens GV3101, and the two resulting GV3101 strains were mixed and co-infiltrated into pepper seedling leaves. The infiltrated seedlings were incubated at 16 °C for 56 h without light, and then were kept at 25 °C. Six independent experiments were performed, and we obtained approximately 100 plants of TRV:00 and 100 plants of TRV:CaCDPK15. A TRV:PDS control construct was used in the same way to monitor gene silencing by the resulting photobleaching phenotypeCaCDPK15 pepper plants, CaCDPK15 transcript levels were reduced to ~30% of those in TRV:00 plants  and trypan blue (indicator of cell death or necrosis). Strongly polymerized DAB (dark brown) and hypersensitive response (HR)-mimicking cell death were detected in control leaves at 48 h post inoculation (hpi), whereas the intensities of DAB and trypan blue staining were distinctly reduced in CaCDPK15-silenced leaves  compared with that in control plants  into pepper leaves  experiments. A specific primer pair was designed based on the flanking sequences of each typical W-box in the promoters of d CaDEF1 . For propression .N. benthamiana leaves with the tagged constructs 35S:CaCDPK15-HA and 35S:CaWRKY40-Flag, and the positive control constructs 35S:CaPIK1-Flag and 35S:CaSGT1-HAIf CaCDPK15 acts as an upstream modifier of CaWRKY40 signaling, one possibility might be that CaWRKY40 is a target of CaCDPK15. We tested this hypothesis by performing co-immunoprecipitation (co-IP) analyses to evaluate possible interactions between the two proteins. These experiments employed a transient coexpression system in CaCDPK15 promoter, suggesting that CaWRKY40 might act as a regulator of CaCDPK15. To test this hypothesis, the transcriptional expression of CaCDPK15 was determined in pepper leaves transiently overexpressing CaWRKY40 or its repressor version CaWRKY40-SRDX by performing real-time RT-PCR analysis. The results showed that transient CaWRKY40 overexpression significantly activated the expression of CaCDPK15 and the immunity-associated marker genes CaNPR1, CaPR1, and CaDEF1. By contrast, the expression of CaCDPK15, CaNPR1, CaPR1, and CaDEF1 was significantly reduced by CaWRKY40-SRDX overexpression  leaves, which were harvested at 48 hpi for chromatin isolation. The isolated chromatin was randomly sheared into fragments with lengths of 300−500 base pairs, and chromatin fragments that bound to CaWRKY40 were immunoprecipitated using the HA antibody. The resulting DNA fragments were isolated and used as templates for PCR analysis with specific primer pairs. The results showed that only the primer pairs flanking the fifth and sixth W-boxes produced amplified products, suggesting that CaWRKY40 directly binds to the CaCDPK15 promoter  TaCPK2-A in rice (Oryza sativa) promoted OsWRKY45-1 expression, which is a TF involved in resistance to fungi and bacteria, by regulating genes involved in JA and SA signalingArabidopsis, the CDPK4/5/6/11 isoforms phosphorylate a specific subgroup of WRKY TFs (WRKY8/28/48) that regulate crucial transcriptional reprogramming that restricts pathogen growthOur previous work showed that CaWRKY40 expression. Unexpectedly, we found that CaCDPK15 expression in pepper plants was transcriptionally upregulated by transient CaWRKY40 overexpression, whereas it was downregulated by transient overexpression of CaWRKY40-SRDX (repressor) and by CaWRKY40 silencing. This suggests that there is a positive-feedback loop between CaCDPK15 and CaWRKY40. The presence of 7W-boxes in the CaCDPK15 promoter indicated that WRKY TFs might directly transcriptionally regulate CaCDPK15 expression. Our ChIP analysis data revealed that CaWRKY40 binds to the CaCDPK15 promoter, which was significantly enhanced by RSI. These results strongly suggest that CaWRKY40 acts as a direct TF in the transcriptional modulation of CaCDPK15 expression. Similar positive-feedback loops in plant responses to stresses including pathogen attack have been reported5253CaCDPK15 expression, which is consistent with their effects on CaWRKY40 expressionWe showed that CaCDPK15 modulates CaCDPK15 in pepper immunity, there were indications that CaCDPK15 might be involved in thermotolerance under high humidity. For example, we identified an HSE element in the CaCDPK15 promoter, determined that pCaCDPK15-driven GUS expression also was activated by heat stress treatment. and found that CaCDPK15 silencing impaired plant thermotolerance and downregulated the expression of the thermotolerance-associated marker gene CaHSP24. CaHSP24 expression was consistently upregulated by CaCDPK15 expression (data not shown).In our previous study, CaWRKY40 positively regulated pepper response to RSI and plant thermotolerance under high humidity, which is important for plant adaption to conditions that promote the invasion and growth of soil-borne pathogens. Although the present study focused on the role of CaCDPK15 expression is upregulated by RSI, which indirectly activates the transcriptional expression of downstream CaWRKY40. Likewise, transcriptional expression of CaWRKY40 directly activates the transcriptional expression of CaCDPK15. This generates a positive-feedback loop that would rapidly amplify plant signaling in response to RSI and efficiently activate plant defense responses.Collectively, our data indicate that Capsicum annuum) cultivar GZ03 and Nicotiana benthamiana were provided by the pepper breeding group at Fujian Agriculture and Forestry University. The seeds were sown in a soil mix  in plastic pots, and were placed in a greenhouse at 25 °C, 60–70 μmol photons m−2 s−1, 70% relative humidity, and a 16-h light/8-h dark photoperiod.Seeds of the pepper  with 10 mM MgCl2. Pepper plants were inoculated by infiltrating 10 ml of the resulting R. solanacearum suspension into the third leaves from the apical meristem using a syringe with a needle. The leaves were harvested at the indicated time points for the analysis of GUS activity, ChIP, or RNA.2O. To study the relative CaCDPK15 transcript levels in response to R. solanacearum infection, pepper plants were inoculated at the eight-leaf stage by injecting 10 ml of bacterial suspension (108 cfu ml−1) or distilled sterile 10 mM MgCl2, and then harvested at different time points.Pepper plants at the four-leaf stage were sprayed with 1 mM salicylic acid, 100 μM methyl jasmonate, 100 μM abscisic acid, or 100 μM ethephon. Mock-treated plants were sprayed with a corresponding solvent or sterile ddHCaCDPK15 was cloned into the plant expression vector pMDC83 downstream of the two CaMV35S promoters and in-frame with GFP using the Gateway cloning system (Invitrogen). The 35S:CaCDPK15-GFP and 35S:GFP (used as a control) constructs were transformed into Agrobacterium tumefaciens strain GV3101, which was cultured in induction medium , harvested when the OD600 was approximately 1.0, and diluted to OD600 = 0.8. Bacterial suspensions expressing p35S:CaCDPK15-GFP and p35S:GFP were injected into Nicotiana benthamiana leaves using a syringe without a needle. GFP fluorescence was imaged using laser confocal fluorescence microscopy (Leica TCS SP8) with an excitation wavelength of 488 nm and a 505–530 nm band-pass emission filter.The full-length cDNA of et al.−1 DAB. The stained leaves were cleared by boiling in lactic acid:glycerol:absolute ethanol [1:1:3 (v/v/v)], and then destained overnight in absolute ethanol. Representative images of DAB and trypan blue staining were photographed with a light microscope .Staining with trypan blue and DAB was performed according to the previously published method of Choi CaCDPK15. The PYL192 and PYL279 VIGS vectors were described previouslyCaCDPK15 was amplified using gene-specific primers (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTTTTCTTTTCGC CCTTTA-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCAATGAACT CCATCCAGCA-3′), and cloned into the PYL279 VIGS vector using the Gateway cloning system (Invitrogen). The PYL192 and PYL279 vectors were with or without CaCDPK15, respectively. PYL279 contained a 250- or 500-bp PDS fragment. The PYL192 and PYL279 vectors were transformed into A. tumefaciens strain GV3101. Agrobacterium harboring PYL192 with PYL279 (PYL192 vector with PYL279 as TRV:00), PYL279-CaCDPK15 (PYL192 with PYL279-CaCDPK15 as TRV:CaCDPK15), or PYL279-PDS (OD600 = 1.0) were mixed at a 1:1 ratio, and the mixture was infiltrated into cotyledons of 2-week-old pepper plants using a 1 ml sterile syringe without a needle. The Agrobacterium-inoculated pepper plants were grown for 2–3 weeks in a growth chamber at 16 °C (in darkness for the first 56 h) with 45% relative humidity, and then transferred into a growth room at 25 ± 2 °C, 60–70 μmol photons m−2 s−1, 70% relative humidity, and a 16-h light/8-h dark photoperiod.The tobacco rattle virus (TRV)-based VIGS system was employed to silence Agrobacterium tumefaciens strain GV3101 harboring the pK7WG2-CaCDPK15 vector was cultured to OD600 = 1.0 in induction medium  and diluted to OD600 = 0.8. The diluted culture was injected into pepper or Nicotiana benthamiana leaves using a syringe without a needle. The plants were kept in a growth room for 2 days, and then the injected leaves were harvested for further use.CaCDPK15 or CaWRKY40 in the pDONR vector was directly introduced into the destination vectors p35S:HA (pEarleyGate 201) and p35S:Flag (pEarleyGate 202) to generate p35S:CaCDPK15-HA and p35S:CaWRKY40-Flag by the LR reaction (Invitrogen). Plasmids were transformed into Agrobacterium strain GV3101, and cells harboring p35S:CaCDPK15-HA and p35S:CaWRKY40-Flag were simultaneously infiltrated into leaves of N. benthamiana plants. Leaves were harvested at 48 h after infiltration (hai), and total proteins were extracted using protein extraction buffer To construct vectors for co-IP analysis, −ΔΔCT) of the respective genes. The relative transcript levels were normalized with respect to the transcript levels of CaActin and 18 s rRNA. In each case, three technical replicates were performed for at least three independent biological replicates.To determine the relative transcript levels of selected genes, real-time PCR was performed with specific primers  accordinCaWRKY40-HA and 35S:CaCDPK15-Flag constructs were generated by Gateway cloning (Invitrogen)Agrobacterium strain GV3101. GV3101 cells containing 35S:CaWRKY40-HA and 35S:CaCDPK15-Flag were co-infiltrated at a ratio of 1:1 or infiltrated individually into pepper leaves, which were harvested at 48 hpi for chromatin preparation. ChIP was performed according to standard protocols. Briefly, approximately 2 g of pepper leaves was treated with either 10 mM bithionol sulfoxide or DMSO (solvent control) for 16 h and subsequently fixed with 1.0% formaldehyde for 5 min. Antibody against HA or FLAG (Santa Cruz Biotechnology) were used for immunoprecipitation. Protein-A-agarose beads were blocked with salmon sperm DNA and used to pull down the protein-DNA complex. Equal amounts of starting plant material and the ChIP products were used for PCR or real-time PCR with specific primers for the promoters of CaCDPK15, CaPR1, CaNPR1, and CaDEF1 .