Peritoneal ultrafiltration (pUF) in refractory heart failure (HF) reduces the incidence of decompensation episodes, which is of particular significance as each episode incrementally adds to mortality. Nevertheless, there are insufficient data about which patient cohort benefits the most. The objective of this study was to compare pUF in HFrEF and HFpEF, focusing on functional status, hospitalizations, surrogate endpoints and mortality.n = 37, 25.9%) or HFrEF  and who received pUF due to refractory HF.This study involves 143 patients, who could be classified as either HFpEF (P <  0.001) and HFrEF . CRP decreased in HFrEF  and HFpEF . Body weight was significantly reduced in HFrEF  and HFpEF . LVEF improved only in HFrEF . BCR decreased significantly in HFrEF and HFpEF . Number of hospitalization episodes as well as number of hospitalization days decreased significantly only in HFpEF .Baseline eGFR was similar in HFrEF (23.1 ± 10.6 mg/dl) and HFpEF (27.8 ± 13.2 mg/dl). Significant improvements in NYHA class were found in HFpEF (3.19 ± 0.61 to 2.72 ± 0.58, pUF offers various benefits in HFpEF and HFrEF, but there are also substantial differences. In particular, hospitalization rates were found to be significantly reduced in HFpEF patients, indicating a greater medical and economical advantage. However, LVEF was only found to be improved in HFrEF patients. While pUF can now be regarded as an option to supplement classical HF therapy, further studies are desirable to obtain specifications about pUF in HFpEF, HFmEF and HFrEF patients. The global prevalence of heart failure (HF) is increasing, due to ageing populations, insufficiently controlled cardiovascular risk factors as well as prolonged survival in consequence of evidence-based treatments . The risEspecially in patients over 60 years, HF is the main reason for hospitalization . In the HF can be classified as heart failure with reduced left ventricular ejection fraction (HFrEF) LVEF < 40%), heart failure with preserved ejection fraction (HFpEF) and recently heart failure with mid-range ejection fraction . Prognosis of HF is known to be rather poor: Overall 1-year mortality is 8.8% in HFrEF and 6.3% in HFpEF 0%, heart, while HThe cardiorenal syndrome is an overarching pathophysiology in HF, irrespective of EF , 8–11. IDue to critical changes in intraglomerular filtration pressures, renal venous congestion and arterial underfilling both lead to “excretory renal insufficiency” with an inadequate volume control triggering recurring hydropic decompensations , 14–21. Peritoneal UF (pUF) in patients with in end stage HF and refractory congestion can offer an additional treatment option  and is aTherefore, the aim of this study was to determine possible advantages and disadvantages of pUF treatment in HFpEF versus HFrEF (according to ESC 2012) . Interestingly, patients with pUF often display a mild hypokalaemia, which may represent an additional advantage over haemodialysis as it offers the chance to reach a dosage of RAAS blockers or MRAs that would accord to the guidelines . In our Charlson Comorbidity Score that revealed a halving of the expected mortality. Moreover, the reduced hospital admissions may have been counterbalanced by the complexity of pUF therapy, which should have been controlled for by a standardized quality of life assessment. Our previous study, however, invalidates this objection by demonstrating an improved quality of life with pUF [Finally, some limitations should be noted. The study comprises a relatively small patient cohort, while cardiorenal patients were included from 18 different centres. This all-comers population may thus have resulted in a highly heterogeneous collective that does not allow the exclusion of potential biases. Heterogeneity, however, is a problem always inherent to the HF syndrome which is driven by the complex situation of multiple comorbidities. In our recent publication  we therewith pUF . FinallyTo conclude, in our study all congestive HF patients clinically improved. While HFpEF patients might in particular benefit the most with respect to hospitalization, HFrEF patients experienced an improvement concerning EF. This study thus warrants larger controlled studies in order to elaborate the differential effects of pUF as an adjunct palliative therapy in end stage HF. With regard to HFpEF patients it may be especially rewarding to gain more insight concerning the specific cardio-pulmo-renal interactions as this clinical entity still is in search for evidence based therapeutic approach.


Tomato yellow leaf curl virus (TYLCV), the V1 protein is known to bind and shuttle viral genomic DNA, however, the role of the V2 protein in this process is still unclear. Here, we report that the V1 protein is primarily localized in the nucleus when expressed but the nucleus-localized V1 protein dramatically decreases when co-expressed with V2 protein. Moreover, the V2-facilitated nuclear export of V1 protein depends on host exportin-α and a specific V1-V2 interaction. Chemical inhibition of exportin-α or a substitution at cysteine 85 of the V2 protein, which abolishes the V1-V2 interaction, blocks redistribution of the V1 protein to the perinuclear region and the cytoplasm. When the V2C85S mutation is incorporated into a TYLCV infectious clone, the TYLCV-C85S causes delayed onset of very mild symptoms compared to wild-type TYLCV, suggesting that the V1-V2 interaction and, thus, the V2-mediated nuclear export of the V1 protein is crucial for viral spread and systemic infection. Our data point to a critical role of the V2 protein in promoting the nuclear export of the V1 protein and viral systemic infection, likely by promoting V1 protein-mediated nucleocytoplasmic transportation of TYLCV genomic DNA.Geminiviruses are an important group of circular, single-stranded DNA viruses that cause devastating diseases in crops. Geminiviruses replicate their genomic DNA in the nucleus and the newly synthesized viral DNA is subsequently transported to the cytoplasm for further cell-to-cell and long-distance movement to establish systemic infection. Thus, nucleocytoplasmic transportation is crucial for successful infection by geminiviruses. For  Geminiviruses are a group of plant viruses with a circular, single-stranded DNA genome. Viruses in this family cause devastating diseases in crop plants, leading to worldwide agricultural losses . While vGeminiviruses can be divided into two major groups based on their genomic components: one group is the monopartite geminiviruses, while the other group is the bipartite geminiviruses . The movMaize streak virus (MSV) and Tomato yellow leaf curl virus (TYLCV) (Tomato leaf curl Yunnan virus (TLCYnV)  . It has  (TYLCV) . It was  (TYLCV) , HSP70 ( (TYLCV) , and exp(TLCYnV) . In addi(TLCYnV) . HoweverTomato yellow leaf curl virus is a typical monopartite begomovirus in the family Geminiviridae. The single-stranded (ss) DNA genome has six open reading frames (ORFs) and an intergenic region (IR). Two ORFs (V1 and V2) are located on the viral strand and the other four ORFs  are located on the complementary strand , which often mediates the nuclear export of proteins. A specific interaction between V2 and V1 proteins has been identified by co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC). Substitutions for cystine 85 in V2 protein inhibit the V1-V2 interaction, block the effect of V2 protein on the subcellular localization of V1 protein, and cause delayed and mild symptom in plants. Our results indicate that the V2 protein interacts with V1 protein, promotes the nuclear export of V1 protein, and plays an important role in viral systemic infection.In this study, we demonstrate that V2 protein affects the subcellular localization of V1 protein by dramatically decreasing the nucleus-localized V1 protein in Tomato yellow leaf curl virus V1 protein is known as a nucleocytoplasmic shuttle protein that facilitates the transport of viral genomic DNA into and out of the nucleus. When expressed in cells of N. benthamiana by agroinfiltration as a YFP-tagged protein, V1-YFP, the signal was found in both the nucleus and cytoplasm at 40 h post agroinfiltration (hpai) (n (hpai) , consistn (hpai) .N. benthamiana cells via agroinfiltration. The fluorescence signal was observed under a laser confocal microscope at 40 hpai. Large aggregates of V2-YFP were easily observed when high concentrations of agrobacteria were used for infiltration . In about 87% of cells, only a weak fluorescence signal of the V1 protein was found in the nucleus compared to that of V1 protein alone (Brome mosaic virus (BMV)  with V1-YFP. Interestingly, a strong nuclear YFP signal was only detected in ∼13% of cells  . BMV 1a us (BMV) . Howevern = 100, , indicatTo confirm our visual observations, we performed a fractionation assay to separate the nucleus from the cytoplasm  and testN. benthamiana. Total protein extracts were subject to immunoprecipitation by using FLAG-trap beads, and the resulting precipitates were analyzed by an anti-GFP antibody or an anti-FLAG antibody. Although a similar amount of FLAG-V2 was pulled down with FLAG-trap beads, only V1-YFP, and not YFP, was pulled down along with FLAG-V2  assay because V1 protein is self-activating in the yeast two-hybrid (Y2H) system. FLAG-tagged V2 protein (FLAG-V2) was co-expressed with YFP or V1-YFP in  FLAG-V2 .The fact that the V1 protein was co-precipitated with V2 protein suggests that V2 protein may bind to V1 protein to form a V1-V2 protein complex. To confirm the V1-V2 interaction and identify the location where V1 and V2 proteins may form a complex, we used a bimolecular fluorescence complementation (BiFC) assay. A positive interaction between nYFP-V1 and cYFP-V2 was observed in both the cytoplasm and perinuclear region, as indicated by the presence of reconstituted fluorescence . We alsoN. benthamiana plants expressing V2-YFP with or without LMB treatment. As shown in The fact that V2 protein hanged the nuclear localization of the V1 protein, raised the possibility that V2 protein might help V1 protein to export from the nucleus to the cytoplasm or block the entrance of V1 protein into the nucleus. Because the nuclear export of proteins is often mediated by exportin-α, we tested the subcellular localization of V2 protein upon treatment with leptomycin B (LMB), an inhibitor of exportin-α . As expeWe also checked whether V2-mediated V1-YFP relocalization can be affected by the LMB treatment. Co-expressed with FLAG-V2, V1-YFP had very low accumulation in the nucleus , which makes LMB unstable and thus, LMB becomes inactive . As expeTo verify the nucleocytoplasmic shuttling of the V1-V2 complex, we performed a BiFC assay applying the same treatments as above. In the presence of LMB only, the reconstituted YFP signal was strongly detected in the nucleus , indicatC85A mutant, which has a cysteine to alanine substitution in the residue at position 85, was not pulled down well along with FLAG-V1 because only a much weaker band was detected compare to that of wt V2-YFP  was expressed as a positive control  further confirmed that similar levels of GFP transcripts were accumulated in the plants expressing wt V2 or the V2C85A mutant (It is well-known that V2 protein is involved in PTGS by binding to tomato SGS3 (SlSGS3), an ortholog of the Arabidopsis SGS3 protein . It was ilencing . Given tH system , indicata plants . To indudetected . These G control . As expe control . ExpressA mutant , consistC84AC86A  in N. benthamiana cells. In 56% of cells expressing V2C85A-YFP  and N. benthamiana plants.To assess the role of the V1-V2 interaction in viral infection, we incorporated a substitution in the C85 of the V2 ORF in the backbone of an infectious TYLCV clone. As the V2 ORF overlaps with the V1 ORF in the TYLCV genome, mutations in V2 may affect V1 amino acid sequence. To ensure that a specific change in C85 has no effect on the V1 protein in TYLCV genome, the C85S mutation, instead of the C85A mutation, was introduced into a TYLCV clone to generate TYLCV-C85S. It needs to be addressed that the V2 protein , but int protein , and in  protein . Most imf V1-YFP , similarFifteen tomato plants were inoculated with either wt TYLCV or TYLCV-C85S. Symptoms such as chlorosis on leaves were obviously observed at 13 days post-infection (dpi) on tomato plants inoculated with wt TYLCV but not in the plants inoculated with TYLCV-C85S. The vast majority of TYLCV-C85S-inoculated tomato plants remained symptomless even at 33 dpi and only 1-2 plants among 15 eventually developed mild symptoms, such as leaf yellowing . In addiN. benthamiana plants. All wt TYLCV-inoculated plants showed symptoms as early as 13 dpi, such as leaf yellowing and curling as shown in Similar results were also obtained in TYLCV-C85S-inoculated Collectively, these results showed that the mutation at C85 of the V2 protein causes significantly low levels of virus accumulation in the systemic leaves and a dramatic decrease in the infection efficiency with delayed and mild symptoms.Because genome replication of geminiviruses takes place in the nucleus of the infected host cells , it is cIn this study, we found that the V2 protein localized primarily in the perinuclear region and the cytoplasm . A very Our work found that V1 protein was primarily localized in the nucleus when expressed alone, but the nucleus-localized V1 disappeared when co-expressed with V2 protein , suggestCysteine85 of V2 protein was found to be crucial for the V1-V2 interaction because substitutions of Cys85 with alanine  or serinIn monopartite geminiviruses, V2 protein is a multifunctional protein that is involved in suppressing host PTGS and TGS, pathogenicity and systemic infection , 2018. SOur data also showed that the V2 C84AC86A double mutant interacted with V1  but not Agrobacterium concentration used for infiltration   and confirmed by sequencing  , using cU111505) . Site-spquencing . For morC84AC86A-YFP, V2C85A-YFP and V2C85S-YFP were individually introduced into A. tumefaciens strain GV3101 through electroporation. Leaves of 4-week-old N. benthamiana were infiltrated with A. tumefaciens harboring the designated constructs. At 40 hpai, leaves were excised and YFP fluorescence was examined in epidermal cells using confocal microscopy (Zeiss LSM 710). The microscope was configured with a 458–515 nm dichroic mirror for dual excitation and a 488-nm beam splitter to help separate YFP fluorescence.The expression vectors p1300-YFP, V2-YFP, V1-YFP, V2N. benthamiana. After agroinfiltration, N. benthamiana were grown in a growth chamber with a 16 h light/8 h dark cycle. YFP fluorescence was observed and photographed using confocal microscopy (Zeiss LSM 710) at 48 hpai. YFP was observed under a mercury lamp light using a 488-nm excitation filter. Photographic images were prepared using ZEN 2011SP1.Bimolecular Fluorescence Complementation experiments were performed as previously described  with minN. benthamiana leaves were harvested at 40 hpai. Proteins were extracted in IP buffer . After centrifugation, the supernatant was mixed with FLAG-trap beads . After 1 h incubation at 4°C, the beads were washed with IP buffer and resuspended in 2 × SDS loading buffer. The samples were loaded onto a 12% (vol/vol) SDS/PAGE gel and target proteins were detected using a polyclonal anti-GFP antibody  or a monoclonal anti-FLAG  antibody.The Co-IP assay was performed as previously described . The infC85A, V2C84AC86A and SlSGS3. V2, V2C85A, and V2C84AC86A were cloned into the activation domain (AD) vector and SlSGS3 was cloned into the vector harboring the DNA binding domain (BD). Both constructs were transformed into the yeast strain AH109. The plasmid pair of BD-53 and AD-recT served as a positive control, while BD-Lam and AD-recT was used as a negative control. Transformants were grown at 30°C for 72 h on synthetic defined medium lacking Histine, Leucine and Tryptophan (SD/-His/-Leu/-Trp) to test protein-protein interactions.The yeast two-hybrid system was used to examine interactions between V2, V22, 25% glycerol, 250 mM Sucrose, 5 mM DTT, 10 mM protease inhibitor). The centrifuged pellet was resuspended with 500 μL of NRB2 and overlaid on top of 500 μL NRB3. The final nuclear pellet was resuspended in lysis buffer. As quality controls for the fractionation assays, PEPC protein and H2B-RFP were used as a cytoplasmic and a nuclear marker, respectively. For more details, see the electronic supporting information.Nuclear-cytoplasmic fractionation assays were performed as described previously  with minin vivo treatment of N. benthamiana leaves, stock solutions were diluted in water to prepare a working solution of 10 nM LMB. Agroinfiltrated N. benthamiana leaves expressing the protein of interest at 40 hpai were infiltrated with 10 nM LMB. Two hours after LMB treatment, the infiltrated leaves were cut and mounted on a glass slide for confocal imaging. When needed, DMSO was further infiltrated into LMB-treated leaves and tissues were harvested at the specified time points.Leptomycin B (LMB) Treatment Assays were performed as previously described  with minC85S, a full-length TYLCV mutant, TYLCV-C85S was synthesized . The TYLCV-C85S and wt TYLCV infectious clone were constructed as previously described (To make a DNA clone of TYLCV containing V2escribed . For morAgrobacterium cultures harboring TYLCV constructs were injected into the stem of tomato and N. benthamiana with a syringe. Inoculated plants were grown in an insect-free cabinet with supplementary lighting corresponding to a 16-hour light and 8-hour dark schedule.Agrobacterium-carrying empty vector)-, wt TYLCV- or TYLCV-C85S-infiltrated tomato or N. benthamiana leaves at different time points. RCR reaction mixes consisted of 6 μl of SYBR Green supermix , 0.10 μl of each primer (10 pmol) and 1.5 μl of DNA sample (10 ng/μl) in a total volume of 12 μl.Total DNA was extracted from mock  real-time PCR detection system. GU111505.The coding sequences of TYLCV V2 and V1 can be accessed at the NCBI database with the accession number YZ, YJ, XW, and WZ designed the project. WZ, SW, and EB conducted experiments. All authors analyzed the data and reviewed the manuscript. WZ, YJ, and XW wrote the manuscript.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.


Random sampling is an important approach to field vegetation surveys. However, sampling surveys in desert areas are difficult because determining an appropriate quadrat size that represent the sparse and unevenly distributed vegetation is challenging. In this study, we present a methodology for quadrat size optimization based on low-altitude high-precision unmanned aerial vehicle (UAV) images. Using the Daliyaboyi Oasis as our study area, we simulated random sampling and analyzed the frequency distribution and variation in the fractional vegetation cover (FVC) index of the samples. Our results show that quadrats of 50 m × 50 m size are the most representative for sampling surveys in this location. The method exploits UAV technology to rapidly acquire vegetation information and overcomes the shortcomings of traditional methods that rely on labor-intensive fieldwork to collect species-area relationship (SAR) data. Our method presents two major advantages: (1) speed and efficiency stemming from the application of UAV, which also effectively overcomes the difficulties posed in vegetation surveys by the challenging desert climate and terrain; (2) the large sample size enabled by the use of a sampling simulation. Our methodology is thus highly suitable for selecting the optimal quadrat size and making accurate estimates, and can improve the efficiency and accuracy of field vegetation sampling surveys. Random sampling survey is one of the most important methods for conducting ecological research; it is widely used in studies of plant species diversity, species association analysis, vegetation spatial pattern research, biomass estimation, and other areas –3. In fiThe concept of spatial scale is among the most fundamental concepts in ecological research, making quadrat size a vital component of field survey design –8. No moAs a result of spatial heterogeneity, any adjustment of the quadrat size in a given study design strongly modifies the variance of the samples. Some studies have shown a nonlinear relationship between quadrat area and sample variance. Research on pasture crops shows that increasing quadrat area can effectively suppress the variance of samples, reducing it by 68% or more . AnotherCurrently, most methods use the frequency of species as the key indicator for determining the appropriate quadrat size, but this method is not without its limitations, particularly in desert areas. The species-area relationship (SAR) is one of the general methods; when the SAR is used, the species-area curve is drawn on the basis of field survey data, and the inflection point of the curve is taken as the quadrat size. It is generally believed that a quadrat should cover 63%–86% of the species in the survey area; for areas with fewer species, some researchers consider 20% to be a reasonable lower limit . HoweverPopulus euphratica is an important dominant desert species, widely distributed in Xinjiang, north-western China; it plays an indispensable role in protecting oasis ecosystems [Populus euphratica populations have been carried out on the basis of field-measured data, but the basic and important issue of choosing the optimal quadrat size for field surveys has remained unaddressed. Frequently, the choice of quadrat size relies on the subjective experiences of the researchers, and the question of whether it is appropriate is rarely asked. This can result in the negative effects of an inappropriate quadrat size on sampling results being ignored.Furthermore, osystems . SeveralFractional vegetation cover (FVC) is a comprehensive parameter used to characterize the extent of land surface vegetation cover –22. FVC In summary, the sampling design of vegetation surveys in sparse desert areas faces difficulties in determining the appropriate quadrat size. Our study aimed to solve this problem by proposing a new methodology for quadrat size optimization in desert areas. Our methodology consists of three steps: (1) simulating random sampling via the Monte Carlo method using low-altitude unmanned aerial vehicle (UAV) images to establish sample sets; (2) calculating the FVC of the sample sets; and (3) analyzing the frequency distribution and the variation of FVC to determine the optimal quadrat size.2 [Populus euphratica and Tamarix chinensis [We selected the Daliyaboyi Oasis as our study area, located in Xinjiang Province in the northwest of China , as it is representative of a desert ecosystem , 24. Aft2 , the ann2 , the weahinensis . Due to hinensis . The stuData were collected in July 2018, using the DJI Phantom 4 UAV. The heading and lateral overlap ratio was set to 80%, the flight height was approximately 60 m, and the imaging focal length was 4 mm. The Pix4D software was used for correction and stitching processing. After stitching the images, the spatial resolution was 0.026 m.Extract with the cropping method set to the random mode; this causes the software to apply the Monte Carlo method to generate random numbers that determine the cropping positions. In this procedure, the process of random cropping can be regarded as a random sampling process, and the size of the cropped pictures can be regarded as the quadrat size, while the number of pictures obtained by cropping is the sample size  that corresponded to seven different test plots . For eacThereafter, we calculated the FVC under different quadrat sizes and counted the distribution frequency. We used the vegetation index threshold method with IDL software version 8.5 to calculate the FVC of the samples in batches.There are various methods for estimating the FVC from low-altitude high-precision UAV RGB images; we chose the vegetation index threshold method due to its clear meaning and simple steps. Low-altitude UAV images have a high spatial resolution that can reach sub-centimeter levels. There are very few mixed pixels in the images, so they can be regarded as pure pixels; this means that a single pixel only belongs to one ground object category—either vegetation or non-vegetation . TherefoThe threshold segmentation method known as the Otsu method (named after the Japanese researcher Nobuyuki Otsu) is widely used in image processing for cluster-based image thresholding, which converts a gray level image to a binary image via the maximization of inter-class variation . It is ct) separated the pixels into either foreground or background categories. The foreground pixels accounted for a certain percentage (w0) of the image, and the average gray level for a foreground pixel was denoted by u0; the background pixels accounted for w1 percent, and the average gray level for a background pixel was u1. The overall average gray level for the image was denoted by u, and we used g to represent the inter-class variance.Assuming that the grayscale image contained a number of gray levels (denoted by L), a threshold   2) we obOr:L, let t vary to update the wi and ui, such that the inter-class variance g achieved its maximum value, and the corresponding t of the maximized g was the optimal threshold for the image. The Otsu method for image segmentation was carried out in IDL 8.5.For each gray level There are many vegetation indices that can be used to estimate the FVC from RGB images; the conditions under which each of them is most effective are different. Therefore, we performed a test to determine the optimal vegetation index for our study area. After consulting relevant literature, we selected three vegetation indices  with supAfter calculating the three vegetation indices, we used the Otsu method to determine the vegetation and non-vegetation thresholds and calculated the corresponding FVC values. Two methods were used to verify the accuracy of the vegetation indices: (1) the Support Vector Machine (SVM) method in ENVI 5.3 to classify and calculate the FVC of the test image; (2) using the ArcGIS 10.2 software to randomly generate 400 samples from the original image and visually interpreting whether each pixel was a vegetation pixel.From the three vegetation indices tested, the ExGR had the best separation effect and extracted the most complete vegetation information . The FVCa is the peak of the fitted curve, μ is the mean, σ is the standard deviation. R2 represents the correlation coefficient of the Gaussian function when fitted to the frequency curve.The Gaussian function is also known as the normal distribution function, and many ecological data can be described by it . We usedFirst, we used OriginPro 2016 to count the frequency of FVCs and plot the frequency distribution histogram. Next, using the Gaussian function  to fit the frequency curve of the sample FVCs. Finally, we analyzed the changing characteristics of frequency curves created with different quadrat sizes.The mean and variance of the samples are important indicators that can be used to judge the efficacy of the sampling unit. Thus, we used Microsoft Excel 2016 to calculate the mean and variance of the FVC of the samples under different quadrat sizes, and using OriginPro 2016, we drew scatter diagrams of quadrat size versus FVC variance and then fit quadrat size-FVC variance curves. When the variance reaches a minimum, it indicates that the corresponding quadrat size gives the best sampling results, and that quadrat size (inflection point) can be taken as the optimal one.2 of the fitted curve was more than 0.8, the fitting curve had two peaks, the mean values (μ) were 0.044 and 0.52, the variance (σ2) values were 0.029 and 0.033, and the relative frequency values were 0.14 and 0.07 (true = 0.05) in plot A. This implies that when a quadrat size of 20 m × 20 m is adopted for field vegetation surveys in areas that are similar to this plot, the quadrats were too small to cover both the sparse and dense vegetation areas. Since a given quadrat may happen to exclusively contain sparse or dense vegetation, this can lead to the occurrence of extrema; thus, this quadrat size is likely to skew the sampling results, making them unlikely to be representative. The frequency distribution of the FVC gradually concentrated around the true value as quadrat size increased  was defined as the true FVC. In plot A, when the quadrat size was 20 m × 20 m, the Rand 0.07 . The FVCncreased . When thσ2 of the FVC gradually decreased, and the frequency distribution gradually concentrated within a smaller range. When quadrat size was 50 m × 50 m, the μ was 0.2, the σ2 was 0.02, and the maximum of the relative frequency was 23%. According to the frequency count, 77.8% of the FVC sampling results were distributed within 16%-32%, which is significantly better than the result from a quadrat size of 20 m × 20 m.The frequency distribution features of the FVC in plot E were significantly different from those in plot A. Under a quadrat size of 20 m × 20 m, the FVC sampling results were evenly distributed between 0 and 0.68, with a wide range of variation and a relatively low frequency with a maximum of only 7%. This indicates that adopting a quadrat size of 20 m × 20 m, when carrying out field vegetation surveys in areas with similar characteristics to this plot would lead to strong fluctuations in the sampling results; thus, this quadrat size was not representative. As quadrat size increased, the μ and σ2 of FVC varied slightly , 18.4% (30 m × 30 m), 30.2% (40 m × 40 m), 31.6% (50 m × 50 m), and 31.6% (60 m × 60 m). This shows that the sampling results tended to be relatively stable and only gradually increase with quadrat size; however, the slightly .μ and σ2 changed with increasing quadrat area.The ideal result of a field vegetation survey is a sample value that is concentrated within a small range, and a high frequency, in which case the sampling results are representative and can reflect the overall vegetation characteristics well. We conducted the sampling simulation in plots with different distribution characteristics. Our results showed that the sampling results tended to be gradually more stable and approach their true values as the quadrat size increases. However, while theoretically a larger sample area improves the sampling design, this is often not practically achievable. Therefore, to determine the optimal quadrat size, we needed to further observe how the variation in σ2 seen in the seven test plots was 0.055, when the quadrat size was 10 m × 10 m  thought that the value of a small quadrat was absorbed as a component of the large quadrat, which meant that the extrema would manifest in a large quadrat. Therefore, the value of μ remains unchanged while σ2 decreases.In our sampling simulations, the frequency distribution of the FVC and its fitted curve varied with increases in quadrat size. When quadrat size increased, the ficantly . Regardiσ2 of samples and reduce the frequency of extrema. A large quadrat is more representative in vegetation surveys, but when the quadrat area was expanded beyond a certain size, the σ2 of samples did not decrease much further. This indicates that at that point, the quadrat area is sufficient to effectively filter the differences in the small-range distribution of vegetation, and that this quadrat size is appropriate for the sampling survey.In desert areas, sparse, unevenly distributed vegetation results in samples displaying high variance . Previouσ2 of samples approached its minimum value. Our conclusion that 50 m × 50 m is the appropriate quadrat size for vegetation surveys at this site is validated by previous studies conducted in very similar conditions. Specifically, the ecosystem of the Daliyaboyi Oasis is similar to the lower reaches of the Tarim River, with the plant communities in both locations dominated by Populus euphratica and Tamarix chinensis. Li, Tang [2, 3600 m2, and 4500 m2, respectively. This similarity in the results achieved using different methods lends support to the methodology proposed by our study.The application of our methodology to the Daliyaboyi Oasis showed that when the quadrat size was 50 m × 50 m, the frequency distribution of the FVC was concentrated within a small range, and the Li, Tang  investigLi, Tang  also invIn ecological studies, both remote sensing data and ground truth data are required. The ground truth data can verify the accuracy of remote sensing data, but more often, ground data and remote sensing data together constitute data sets to solve ecological problems. However, due to the shelter effect of the plant canopy, it is difficult to obtain some vegetation parameters by remote sensing, such as the number of trees, diameter at breast height (DBH), first branch height. In addition, some parameters can only be measured manually by using specific instruments or tools, such as collecting plant leaves to detect their nutrient content, the number of seeds, and the measurement of soil moisture. Therefore, selecting an appropriate quadrat size for performing ground surveys is imperative. Meanwhile, UAV technology plays an increasingly important role in vegetation studies, and has been widely applied in vegetation mapping, plant conservation, retrieval of vegetation structural parameters and other studies –45. It iIn fieldwork, it is often difficult to establish a large sample size based on measured data that could then be used to estimate the appropriate quadrat size. Besides, desert areas are especially notorious for their harsh climate and poor transportation, making them difficult to research. The application of UAV technology can help researchers to overcome severe weather conditions and investigate complex terrains, while retaining full control of spatial resolution. We believe that the use of UAV to acquire high-precision images of study areas for random sampling simulations is a significant strength of our methodology, which can thus be applied to other desert areas to estimate optimum quadrat size with limited labor, thus improving fieldwork efficiency, and achieving high survey accuracy. In addition, the simulation covers multiple sampling possibilities, and has good stability, which is conducive to acquiring accurate sampling results. Thus, this study exploited the advantages of UAV to determine the optimal quadrat size for ground surveys, which is an attempt at a partially UAV-assisted ground survey.In this study, our objective was to propose a methodology for choosing a suitable sampling scale for obtaining accurate sampling estimation in areas with sparse vegetation cover, and test it in a desert study site. We specifically aimed to overcome the difficulties in vegetation survey that are caused by the discontinuous distribution of vegetation, which if unaddressed can lead to large discrepancies in survey results. These difficulties include the dependence of vegetation surveys on vegetation spatial distribution patterns, and the potential under- or overestimation of sampling requirements. If appropriately selected, the right quadrat size will match the characteristics of such sites and overcome the differences caused by the clumped vegetation distribution, benefitting the overall estimation. However, it must be noted that our study aimed to demonstrate the suitability of our proposed methodology, using the Daliyaboyi Oasis as a test site; therefore, 50 m × 50 m size should not be taken as the default best quadrat size for other locations. Instead, to account for the spatial heterogeneity and the unique characteristics of each site, we propose that the appropriate quadrat size for other desert areas with sparse vegetation should be determined by this method. In addition, we adopted FVC as a suitable metric for determining the optimum survey quadrat; this method for determining optimal quadrat size is only suitable for surveys of sparse-vegetation areas such as deserts. This conclusion is based on the observation that the variation in FVC in a high FVC area is very slight, and therefore cannot be used to determine the appropriate quadrat size.To address the issue of determining the optimal quadrat size for surveys in desert areas with sparse vegetation, we proposed a method based on low-altitude UAV images and simulated random sampling. The simulated random sampling was used to establish the sample sets and calculate their FVC. This was followed by an analysis of the frequency distribution and the variation in FVC, which facilitated determination of the appropriate quadrat size for our study area. Compared with the traditional methods, our methodology is both simpler and more robust. It can effectively reduce the field workload, provide quantitative references for the design of field vegetation surveys, and effectively improve the efficiency and accuracy of field vegetation investigations.S1 File(ZIP)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Ischaemic heart disease is the world’s leading cause of mortality. Survival rates from acute myocardial infarction (MI) have improved in recent years; however, this has led to an increase in the prevalence of heart failure (HF) due to chronic remodelling of the infarcted myocardium, for which treatment options remain poor. We have previously shown that inhibition of isoform 4 of the plasma membrane calcium ATPase (PMCA4) prevents chronic remodelling and HF development during pressure overload, through fibroblast mediated Wnt signalling modulation. Given that Wnt signalling also plays a prominent role during remodelling of the infarcted heart, this study investigated the effect of genetic and functional loss of PMCA4 on cardiac outcomes following MI. Neither genetic deletion nor pharmacological inhibition of PMCA4 affected chronic remodelling of the post-MI myocardium. This was the case when PMCA4 was deleted globally, or specifically from cardiomyocytes or fibroblasts. PMCA4-ablated hearts were however less prone to acute arrhythmic events, which may offer a slight survival benefit. Overall, this study demonstrates that PMCA4 inhibition does not affect chronic outcomes following MI. Of these, ischaemic heart disease accounts for nearly half of all deaths due to CVD3, and is the leading cause of death worldwide4. Whilst mortality from acute myocardial infarction (MI) has fallen in recent decades in the developed world, thanks in large part to the rise in primary percutaneous coronary interventions5, this has led to a concomitant rise in the prevalence of heart failure (HF) as a result of chronic post-infarct left ventricular remodelling6.Cardiovascular diseases (CVD) account for 45% of all mortality across Europe7. During this time, large numbers of seemingly promising approaches, such as stem cell and gene-based therapies, have failed to significantly reduce mortality or improve cardiac function upon reaching clinical trials9. Consequently, frontline prevention and management of HF remains centred around targeting the renin–angiotensin–aldosterone system and sympathetic nervous system to delay disease progression10, rather than intrinsic cardiac factors which regulate the remodelling process.Despite the significant improvements in survival from infarction, only a handful of new pharmacological therapies aimed at preventing and treating heart failure have been approved in the past two decades11. In cardiomyocytes we and others have shown PMCA4′s contribution towards global calcium extrusion is minimal during excitation–contraction coupling13; however, through the control of local calcium and protein–protein interactions, it plays a key role in a regulating a number of pathways such as nitric oxide15, calcineurin-NFAT16 and tumour suppressor RASSF1-Erk17 signalling. Importantly, these roles have been shown to have functional implications in PMCA4 ablated or overexpressing mice, including the regulation of cardiac contractility19, hypertrophy20, blood pressure21, angiogenesis22 and sperm motility23.One promising target for cardiac remodelling that has emerged in recent years is the calcium signalling protein plasma membrane calcium ATPase isoform 4 (PMCA4). PMCA4 is a ubiquitously expressed pump which extrudes calcium from the cytosol to the extracellular space, and in non-excitable cells it can act as a major regulator of intracellular calcium concentration24, we have shown PMCA4 to be a druggable candidate in a number of cardiovascular settings including the lowering of blood pressure25, promotion of angiogenesis26, the treatment of cardiac hypertrophy and the prevention of HF20. Our previous study found that PMCA4 inhibition reversed cardiac remodelling in mouse hearts subjected to pressure overload, through a paracrine mechanism involving the secretion of the Wnt inhibitor sFRP2 from cardiac fibroblasts20. Given that Wnt activation also contributes towards remodelling and HF development following MI27, and that sFRP2 treatment has produced beneficial outcomes in the infarcted rat heart28, we conducted a study into the effects of PMCA4 inhibition after MI. Surprisingly, we found that PMCA4 inhibition did not affect chronic outcomes after MI, and therefore does not present a new target to prevent HF development in the infarcted heart.Through the development of a specific inhibitorPmca4 genetic ablation following myocardial infarction (MI), we performed surgery to permanently ligate the left anterior descending coronary artery in our global PMCA4 knockout mice (PMCA4−/− mice). During 6 weeks follow-up, PMCA4−/− mice experienced a 79% survival rate post-MI, compared to 50% survival in wild type (WT) littermate controls . Examining the data further by gender, the survival benefit appeared restricted to female PMCA4−/− mice, which showed 100% survival versus 50% in WT , a common marker for pathological cardiac remodelling, was increased in MI infarct and border regions compared to remote regions and sham hearts in both genotpyes 20. Therefore, to investigate whether a similar signalling mechanism was active following MI we also examined regional expression of sFRP2 and Wnt/β-catenin target gene Axin2. In general sFRP2 levels were higher in border and infarcted regions; however matched regional sFRP2 expression was comparable in PMCA4−/− and WT hearts  staining of heart sections 2 days after MI Fig. A. Similaice Fig. , indicat30. We therefore performed 3 lead ECG analysis on the first day after MI surgery, prior to the occurrence of sudden death in WT mice. Traces were recorded for a period of 15 min in mice maintained under general anaesthesia, and assessed for the presence of arrhythmic beats. All sham mice were found to have a regular sinus rhythm throughout the period of recording. In contrast, 56% of WT MI traces exhibited ectopic beats during the 15 min period, significantly higher than the rate of 19% observed in PMCA4−/− traces after MI  and chronic (6 week) stages post-MI. Expression of pro-inflammatory mediators tumour necrosis factor-α (TNFα), interleukin-1β (Il-1β), Il-6 and monocyte chemoattractant protein-1 (MCP-1) was increased in MI compared to sham hearts 2 days after surgery, but did not differ between WT and PMCA4−/− hearts . Expression of fibrosis markers Col1a1 and Col3a1 was increased in the border region and to a greater extent the infarct of MI hearts compared to sham and remote regions, whilst Tgfb levels were also elevated in border and infarct regions . Previously, we found that Pmca4 deletion specifically from cardiac fibroblasts protected the myocardium during pressure overload through increased secretion of sFRP2, a factor previously shown to improve outcomes after MI28. We therefore performed LAD ligation in our fibroblast-specific PMCA4 knockout mice to investigate whether survival benefits were manifested through deletion from this cell type. We had previously generated this mouse line using Cre-recombinase under the control of the promoter for Periostin, and shown deletion of PMCA4 from cardiac fibroblasts without affecting expression in cardiomyocytes20.Our previous work has suggested that PMCA4 might have distinct functions in different cell types, such as cardiac fibroblastsPMCA4fko mice and PMCA4flox/flox controls experienced a 92% survival rate following 6 weeks of MI, with only 1 death per group  also has an effect on outcomes after LAD ligation. We have previously generated PMCA4cko mice, and confirmation of the specific ablation of PMCA4 from their cardiomyocytes has been described20. PMCA4cko mice were subjected to MI for 6 weeks. PMCA4cko mice exhibited a 40% 6 week survival rate, which did not differ significantly from that of their littermate PMCA4flox/flox controls  as a potent inhibitor of PMCA4Pmca4 silencing does not affect chronic cardiac remodelling following myocardial infarction, when induced by permanent ligation of the left anterior descending coronary artery. These results were consistent amongst global and tissue-specific knockout models, and confirmed by pharmacological targeting with the specific PMCA4 inhibitor ATA. Global ablation of Pmca4 did however reduce the occurrence of ectopic beats acutely post-MI, which may be associated with a small but non-significant survival benefit during this period.Overall this study shows that Pmca4 inhibition to treat remodelling after MI. Our previous work in the pathological setting of pressure overload had shown that Pmca4 deletion protected the myocardium from the development of cardiac hypertrophy and remodelling20. Moreover, deletion was able to preserve long term function and prevent heart failure development, whilst pharmacological inhibition was able to reverse established hypertrophy. These beneficial outcomes were achieved through the paracrine secretion of the Wnt inhibitor sFRP2 from Pmca4-silenced fibroblasts, with a subsequent reduction in canonical Wnt signalling20.It was important to conduct an investigation into the therapeutic potential of using 33. In the healthy adult heart the Wnt pathway is quiescent; however it is reactivated in response to pathological stress and contributes towards adverse cardiac remodelling34, including following myocardial infarction38. Moreover, it has been shown that sFRP2 treatment is capable of reducing infarct size, attenuating cardiomyocyte apoptosis and improving long term cardiac function in experimental MI and hypoxia models40. This was found to be the case both when administered exogenously28, and upon its paracrine release from mesenchymal stem cells40.Wnt signalling plays an essential role in cardiac development, supporting the proliferation of cardiac progenitor cells and controlling their migration and differentiation during heart formationPMCA4−/− mice, which have elevated levels of sFRP2 in their cardiac fibroblasts20, exhibited similar scar size and cardiac function to controls throughout the 6 week period after MI. This could be due to endogenous upregulation of sFRP2 in WT fibroblasts, which has been noted to occur during the first week after MI35. Indeed, we found sFRP2 levels to be dramatically increased in both control and PMCA4−/− tissue at the whole heart level 6 weeks post-MI, particularly in the infarct and border regions. Analysis of β-catenin target gene Axin2 expression suggested a possible reduction of Wnt signalling in infarcts of PMCA4−/− mice, but this didn’t translate to any observable physiological effects. It should also be noted that sFRP2-null mice, and hamsters treated with antibody-mediated sFRP2 blockade, have been shown to have protection from fibrosis and improved cardiac function after MI42, indicating that sFRP2’s role in the infarcted heart may be multi-faceted, and more complex than in the TAC heart. Furthermore, Wnt signalling has been shown to be highly active in a number of other cell types in the infarcted myocardium including in progenitor, endothelial cell and leucocyte populations38. Therefore, it is also possible that regulating sFRP2 signalling in cardiac fibroblasts is not sufficient to affect Wnt activity at the organ level.It was therefore surprising to find that Pmca4-null hearts after TAC and MI also likely reflects significant distinctions between the pathological processes that drive concentric hypertrophic remodelling following pressure overload, compared to eccentric remodelling that occurs in volume overloaded MI hearts44. Pressure and volume overload trigger different biomechanical and neurohumoral pathways, with a far greater extent of protein synthesis occurring in pressure overloaded TAC hearts43. In contrast, post-MI remodelling is largely driven by an extensive inflammatory response to widespread necrosis, aimed at repairing the damaged myocardium45. This involves the recruitment of large numbers of immune cells to clear debris from the infarct46, and fibroblasts, whose proliferation and transformation into myofibroblasts leads to reparative fibrosis and scar maturation47. Ultimately, infarct size and healing are thought to be the major determinants of adverse remodelling after MI47. In TAC hearts, while there is evidence of a macrophage-led inflammatory response, this is more transient and to a lesser extent than after MI48, and is not associated with vast fibroblast recruitment.The differences in phenotype between Pmca4 inhibition after MI went beyond its regulation of Wnt signalling in cardiac fibroblasts. PMCA4 has been shown to play an important role in mediating angiogenesis through its actions in endothelial cells, with genetic ablation and pharmacological inhibition each enhancing cell migration and improving reperfusion following hindlimb ischaemia26. These studies also contributed towards our hypothesis of beneficial outcomes post-MI, given that stimulation of angiogenesis in the infarcted heart has been shown to improve chronic left ventricular remodelling and function49. Although we did not measure vessel formation in this study, we did not see any evidence of improved chronic outcomes. It may be that PMCA4’s regulation of angiogenesis after MI would be better studied in a model of ischaemia with reperfusion rather than following permanent coronary artery ligation, but this was beyond the scope of the current study.The rationale to investigate the role of 32. This was the case both when expressed from birth, or inducibly 4 weeks after MI, and was associated with a reduction in infarct size and improved survival32. The authors found PMCA4′s regulation of nNOS mediated nitric oxide signalling to be responsible for these protective effects32. In the present study, we found that specific deletion of Pmca4 from cardiomyocytes did not have a significant effect on outcomes following MI. This was also the case in our previous study during pressure overload, in which cardiac hypertrophy was not affected when PMCA4 levels were reduced only in cardiomyocytes20. This is likely due to differences in the mechanism of nNOS regulation between PMCA4 overexpression and knockout models. We have found that increased expression of PMCA4 inhibits nNOS activity through reducing local calcium19, whereas nNOS is delocalised from the membrane compartment in PMCA4−/− hearts12.Despite our findings in this study, it is likely that PMCA4 does play a role in regulating the response to ischaemic injury in the heart. A recent study has found that transgenic mice overexpressing human PMCA4b specifically in cardiomyocytes displayed less cardiac hypertrophy and improved fractional shortening following LAD ligationPmca4 silencing in other cell types involved in the response to MI. We did note significantly fewer instances of premature atrial/junctional contractions upon ECG examination on the day following LAD ligation in PMCA4−/− mice compared to controls, which to some extent may have protected against sudden death during the first 72 h post-MI. Indeed, premature atrial complexes are associated with increased mortality from sudden cardiac death50; however telemetric ECG recordings would need to be conducted in order to verify whether lethal arrhythmias were the cause of excess deaths amongst WT mice in our experiments.This does not rule out an effect of 45. Infiltration of leucocytes and their adherence to endothelial cells amplifies the inflammatory response and allows for clearance of necrotic cells45; however, overactive inflammation during the early stages after MI can cause electrophysiological remodelling, arrhythmia and sudden death29. In addition, suppression of inflammation is important in order to proceed to the reparative phase of infarct healing, which involves fibroblast proliferation, extracellular matrix secretion and eventually fibrotic scar maturation45. In the present study we did not find Pmca4 global ablation to modulate myocardial inflammatory or fibrotic gene expression at either 2 days or 6 weeks post-MI, which may suggest that regulation of arrhythmia is more likely to contribute to the phenotype than inflammation and fibrosis. However we would not completely rule out a role in the remodelling process, as it is possible that PMCA4 could regulate processes at another time point, for example the transition to the reparative and maturation phases of infarct healing. Furthermore, in order to fully rule out any involvement in inflammation, it would be pertinent to investigate the immune cell response in our mice.The acute response to infarction begins with an immune response to necrotic signals, sparked by the release of inflammatory cytokines and chemokines46. Thereafter, M2 macrophages, monocyte and T-cell populations act to supress inflammation46. Therefore it could be that PMCA4 regulates inflammatory processes following MI in one or more cell types not examined during our study, such as endothelial cells, macrophages, or T-cells, which may account for the slight improvement in survival and protect from the development of acute arrhythmic events in our Pmca4 global knockout mice. Indeed, PMCA4 has been previously shown to play an important role during T-cell activation52. However, given the minimal effect of global genetic and pharmacological Pmca4 inhibition on overall outcomes post-MI, it would be beyond the scope of this study to produce further tissue-specific knockouts to investigate such a role.Studies of the immune cell response following MI in mice have shown that after early neutrophil infiltration on day 1 post-MI, the macrophage population predominates by day 3, and in particular M1 inflammatory macrophagesPmca4 deletion may be restricted to female mice, which may account for comparable survival rates amongst vehicle and PMCA4 inhibitor-treated mice as these experiments were performed exclusively in males. However, it should be noted that when performing our 2 day MI experiments we observed deaths in female PMCA4−/− mice at a rate comparable to WT, and so further investigation would be required in order to confirm a sex-dependent survival benefit.We also examined whether sex played a role in determining post-MI outcomes in each knockout strain in our study. This suggested that improved survival in mice with global PMCA4fko-floxed controls, to 50% in PMCA4cko-floxed controls and WT strains. It is likely that the different responses are due to background strain, which has been shown to play a significant role in the response to MI in terms of survival, cardiac rupture, dilatation and function. A study comparing 5 different mouse strains found survival rates after MI to vary greatly by strain, ranging from 33% in 129S6 mice to 85% in BalbC mice53. Each of the 3 lines used in our study are bred on different mixed backgrounds, including C57Bl/6J for PMCA4fko mice54 and ICR for PMCA4cko mice55, which likely accounts for the response to MI in each strain.An additional point of interest in our study is the variation in survival rate between WT mice and targeted flox strains, which ranges from 92% in 20, the promotion of angiogenesis26 and the reduction of blood pressure25. Further studies looking to develop PMCA4 inhibitors as a translational therapy may want to focus on these processes, along with alternative models such as ischaemia with reperfusion.Overall these results demonstrate that PMCA4 ablation and inhibition do not significantly affect chronic outcomes in a model of permanent myocardial infarction, but may reduce the susceptibility to acute arrhythmia and sudden death. In addition, PMCA4 inhibition presents a promising target in a number of other cardiovascular areas including pressure overload induced cardiac hypertrophyPMCA4−/− mice with targeted deletion of exons 2–3 of the Pmca4 gene used in this study were previously generated23. PMCA4flox/flox mice, cardiomyocyte-specific PMCA4cko and fibroblast-specific PMCA4fko mice were also previously generated in our laboratory, with Cre-mediated excision of exons 2–3 of the Pmca4 gene driven by the αMHC and Periostin promoters in cardiomyocytes and fibroblasts, respectively20. 8–12 week old mice of both sexes were used in each strain, with subjects age and sex matched between knockouts and littermate controls. 8 week old male C57BL/6 mice weighing 20–23 g were used for ATA experiments.56. This study does not have any implications on replacement, refinement and reduction.Animals were housed in a specific-pathogen free environment and studies were performed in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. All studies were approved by the University of Manchester Ethics Committee. Researchers were blinded to genotype and treatment during data collection and analysis. All animal experiments were conductance in accordance with the ARRIVE guidelines2. A left-sided thoracotomy was performed and the ribs retracted to expose the heart. The LAD artery was ligated above its bifurcation using #8.0 suture, approximately 1 mm below the left atrial appendage. Visual confirmation of ischaemia was observed by a paling of the ventricular tissue distal to the ligation. In sham operations, a suture was passed through the myocardium but withdrawn without ligation. The thorax and chest were then sutured shut, and mice allowed to recover at 30 °C before return to normal housing.Myocardial infarction was induced by permanent ligation of the left anterior descending (LAD) coronary artery. Mice received pre-operative analgesia via subcutaneous injection of buprenorphine (0.1 mg/kg body weight). They were induced and intubated under 5% isofluorane, and thereafter mechanically ventilated  and maintained in a stable plane of anaesthesia with 3% isofluorane in 100% OImmediately following sham or coronary artery ligation surgery, osmotic minipumps (Alzet) containing either ATA or vehicle  were implanted subcutaneously. A small skin incision was made dorsally to create a pocket into which the minipump was implanted, following which the skin was sutured closed. ATA mice received a dose of 5 mg/kg BW/day for a period of 6 weeks.To evaluate cardiac function during 6 weeks follow up to MI induction, transthoracic echocardiography was performed using a Visualsonics Vevo 770 imaging system fitted with an RMV-707B scanhead. Mice were anaesthetised using 1.5% isofluorane, and images were acquired in parasternal long and short axis views to measure wall thickness and chamber dimensions, from which ejection fraction was calculated. Researchers were blinded to mouse genotype and treatment during the procedure. Measurements were obtained using the leading-edge method over a minimum period of three cardiac cycles.max and dP/dtmin, respectively.Analysis of haemodynamic function was performed via invasive catheterization of the left ventricle under terminal anaesthesia (250 mg/kg body weight tribromoethanol injected i.p.). Mice were placed in a supine position and a midline cervical incision made to expose the right carotid artery. This was tied at its bifurcation and occluded proximally to allow an incision to be made, through which a 1.4F pressure–volume catheter  was inserted, and fed down the ascending aorta into the left ventricle. Recordings were collected using a PowerLab system once traces had stabilised. Inotropic and lusitropic function were assessed by the maximal and minimal rates of pressure change, dP/dt3-lead ECG recordings were collected 24 h post-MI under anaesthesia with 1.5% isofluorane. Mice were placed on a heat pad set to 37 °C and electrodes inserted into the muscle of the right and left forelimb, and right hindlimb. ECG traces were recorded for a period of 15 min on a PowerLab system, and examined for the occurrence of ectopic beats during this time using LabChart software (AD instruments).Mice were euthanised under terminal anaesthesia by injection of 1 M KCl solution directly into the LV chamber to arrest the heart in diastole. Hearts were then flushed of blood with PBS. For 2 day experiments hearts were collected onto dry ice for subsequent analysis of area at risk by triphenyltetrazolium chloride (TTC) stain. For 6 week experiments, hearts were sliced into transverse sections 1 mm in thickness using a mouse heart slicing matrix (Harvard Apparatus). Sections were embedded in OCT compound and frozen using a bath of 2-methylbutane and dry ice, following which they were stored at − 80 °C.57. 1 mm thick OCT embedded slices taken at 5 levels of the ventricle were sectioned on a cryostat at a thickness of 6 µm and mounted onto glass slides. After equilibrating to room temperature, sections were fixed in 4% PFA for 15 min and then stained with Masson’s trichrome using a standard protocol. Slides were imaged on a 3D Histech Panoramic slide scanner in the University of Manchester bioimaging facility, and infarct size was measured using Image J software. The LV midline circumference was determined for each section by drawing a line equidistant to the epicardium and endocardium. The infarct length in each section was determined by drawing a midline through the infarct, where the scar constituted > 50% of the myocardial wall thickness. Infarct size as a percentage of the left ventricle was calculated as the sum of the infarct lengths at each level divided by the sum of the total LV circumferences at each level, × 100.Infarct size was determined following Masson’s trichrome staining of transverse sections using the midline length measurement technique as described by Takagawa et al.−ΔΔCT method58, using GAPDH and β-actin as endogenous controls.Following cryosectioning, the remaining heart tissue was retrieved from the OCT, divided into infarct, border and remote regions and placed in RNA later solution (Qiagen) for storage as per manufacturer instructions. Sham tissue was also OCT embedded, frozen and retrieved into RNA later. For RNA extraction, tissue was manually dissociated in a dounce homogenizer using Trizol reagent (Thermo), and then processed according to the manufacturers’ instructions. RNA concentration was determined using a Nanodrop 8000 spectrophotometer, following which 1 µg total RNA was reverse transcribed to cDNA using a high capacity cDNA reverse transcription kit (Thermo). qPCR was conducted on a 7500 Fast RT-PCR system (Applied Biosystems) using Brilliant III Ultrafast SYBR green (Agilent) and Quanitect Primer Assays (Qiagen). Samples were loaded in triplicate and gene expression was determined using the 2Hearts were stored on dry ice to keep tissue metabolically active. They were then sectioned into 1 mm transverse slices at 5 levels through the ventricle using a mouse heart slicing matrix. Sections were incubated in 0.5% TTC solution in pH7.4 buffered PBS for 15 min at 37 °C. Sections were then washed in PBS and immediately imaged using a stereomicroscope. Area at risk was determined using Image J software, by measuring the sum of the white (infarcted) area at each level and dividing by the sum of the total LV area at the 5 levels, multiplied by 100.p-values < 0.05 were considered statistically significant.Data was analysed using Microsoft Excel and GraphPad Prism software, and are presented as mean ± SEM. Data was analyzed using one- or two-way ANOVA followed by Bonferroni post hoc tests where appropriate, and comparisons between two groups were performed using Student’s t-test. Survival analysis was performed using the Log-rank (Mantel-Cox) test. Fisher’s exact test was used to calculate contingency of arrhythmia. Supplementary Information.


N on the escape dynamics of a classical open system is also discussed.We study the effect of self-oscillation on the escape dynamics of classical and quantum open systems by employing the system-plus-environment-plus-interaction model. For a damped free particle (system) with memory kernel function expressed by Zwanzig ), which is originated from a harmonic oscillator bath (environment) of Debye type with cut-off frequency  The study of open systems, which may trace back to the pioneering studies on Brownian motion ,2, has bN is presented here. The effect of self-oscillation caused by many frequencies missing in the interval of interest in a finite bath on escape dynamics is also analyzed.The aim of this paper is to analyze the effect of self-oscillation ,20,21 ca systems ,25,26. W systems , i.e., γime, τe,  and the ime, τe, . CompariThe paper is organized as follows. In N) are, respectively, the test particle and the ith oscillator coordinates and momentums. M and ith oscillator. ith oscillator. The coupling parameter In the classical regime, the starting point for our analysis is the Hamiltonian of a particle plus environment consisted of N-independent harmonic oscillators ,7,8, whiIn the quantum regime, the total system-bath Hamiltonian can be written as ,26,30=1A satisfies A is an ergodic variable; ergodicity breakdown in the classical and quantum systems is observed because of frequencies cut-off. Notably, the ergodicity is exhibited when As ex plane , it is cEquation , the chaU is the well depth. We use the second-order Runge–Kutta algorithm ,35,36 wigiven by (11)r(t)variance , 〈V2〉=ℏwPanels  in u can be determined by u numerically.The escape behavior of a self-oscillated system is analyzed by two timescales: the correlation time of fluctuations, xp(UkBT) , and a cxp(UkBT) . For the02wd2=ξ0 . On the ff≫2wa).  From Kraff≫2wa). , the ste formula ,29 can bEquation . Under tOn the other hand, when ’ theory , the steComparing the steady escape rate of a quantum open system to a classical open system, it is no difficult to find that quantum fluctuation enhances the steady escape rate. It may be easy to verify that the QGLE in c numbers reduces to the GLE in the thermal limit ℏwi≪kBT , where {N from small to large. The equations of motion are given byMany examples for non-Markovian ergodicity breaking in a finite-size bath ,19,24,31N. We consider a statistical average over 200,000 test particles. Each of the test particles is coupled to a bath composed by N-independent harmonic oscillators. The initial velocity distribution of test particles is assumed to be Gaussian with zero-mean and variance, T is the temperature of the bath. Moreover, the vibrational frequencies of oscillators are chosen randomly from a frequency distribution of Debye type. The frequency distribution function is N, we fix values of We use fourth-order Runge–Kutta algorithm with a small time-step, N when we fix N in panel (c) in N increases gradually.Panels  in N varying from small to large in N is low and the dynamics of system is non-Markovian process, which means that the friction exerting on the particle is strong. As N increases gradually, the friction becomes weak. In the limit of In our approach, due to the finite number of oscillators, the spectral density is always structure for low values of N. By plotting the frequency distribution We have analyzed ergodicity breakdown in classical and quantum open systems described, respectively, by a GLE and a QGLE in c numbers, both analytically and numerically, which is caused by a harmonic oscillator bath of Debye type. The VAF has been shown to oscillate in cosine function for asymptotic time. Escape of a self-oscillated open system from a metastable potential has shown interesting phenomena. On the one hand, the steady escape rate depends non-monotonically on The effect of self-oscillation on escape dynamics of open systems can be presented more intuitive through the present work. We believe that the present study will provide useful information about the study of the escape processes of open systems. Thus, some surprising findings may be revealed.


TNF is a highly pro-inflammatory cytokine that contributes not only to the regulation of immune responses but also to the development of severe inflammatory diseases. TNF is synthesized as a transmembrane protein, which is further matured via proteolytic cleavage by metalloproteases such as ADAM17, a process known as shedding. At present, TNF is mainly detected by measuring the precursor or the mature cytokine of bulk cell populations by techniques such as ELISA or immunoblotting. However, these methods do not provide information on the exact timing and extent of TNF cleavage at single-cell resolution and they do not allow the live visualization of shedding events. Here, we generated C-tag TNF as a genetically encoded reporter to study TNF shedding at the single-cell level. The functionality of the C-tag TNF reporter is based on the exposure of a cryptic epitope on the C terminus of the transmembrane portion of pro-TNF on cleavage. In both denatured and nondenatured samples, this epitope can be detected by a nanobody in a highly sensitive and specific manner only upon TNF shedding. As such, C-tag TNF can successfully be used for the detection of TNF cleavage in flow cytometry and live-cell imaging applications. We furthermore demonstrate its applicability in a forward genetic screen geared toward the identification of genetic regulators of TNF maturation. In summary, the C-tag TNF reporter can be employed to gain novel insights into the complex regulation of ADAM-dependent TNF shedding. TNF (Tumor necrosis factor) is a pro-inflammatory cytokine involved in the regulation of immune responses, inflammation, and cell death , 2. The TNF is produced as a type II transmembrane protein that assembles into trimers on the cell membrane. To be released, TNF needs to be cleaved by membrane metalloproteases, mainly ADAM17  , 5 but aTNF production is typically assessed by end point analysis techniques of bulk cell cultures or tissues, detecting the matured version of the cytokine. Although well established, these approaches do not provide single cell resolution, which precludes them from being employed in genetic screens or kinetic analyses of TNF production at the cellular level. Flow cytometry or immunofluorescence microscopy techniques, on the other hand, can be used to determine pro-TNF abundance at the single cell level, yet they cannot inform on the maturation status of the cytokine. Moreover, relying on the fixation and permeabilization of cells, these techniques are disruptive. To circumvent these limitations, we here generated a genetically encoded reporter system that allows the study of the dynamics of TNF shedding at single cell resolution.A, B). Introducing the C-tag at positions 73-76 of TNF creates a new recognition site, in which the naturally occurring positions P4, P3 and P2 are changed. However, the newly introduced amino acids are well compatible with the experimentally determined recognition sites of ADAM17 or ADAM10 (B). To test this hypothesis, we set out to study the activation of the C-tag TNF reporter in HEK293T cells. Both ADAM17 and ADAM10, the two main sheddases of TNF, were found to be expressed in HEK293T cells (C). To address their involvement in TNF maturation, we generated ADAM17−/−, ADAM10−/− and ADAM10−/− × ADAM17−/− HEK293T cells (C). Because HEK293T cells do not express TNF, we transiently transfected wild-type (WT) TNF and studied its shedding by ELISA and immunoblotting. As expected, TNF cleavage was mainly dependent on ADAM17, whereas ADAM10 contributed to a minor extent (D). We then analyzed the cleavage of C-tag TNF using the same approach. Soluble TNF levels (sTNF) in the supernatant of C-tag TNF transfected cells were slightly lower compared with what observed on transfection with WT TNF, suggesting that the C-tag TNF is a less efficient substrate for metalloproteases than its WT counterpart. However, C-tag TNF cleavage was also mainly ADAM17-dependent, and also showed a partial reduction in ADAM10-deficient cells (E). The N-terminal fragment of C-tag TNF  was detectable in the lysate of transfected WT HEK293T cells by immunoblotting using the C-tag nanobody. In line with the ELISA results, the C-tag and the sTNF signals were mainly dependent on ADAM17 and to a lesser extent on ADAM10 (E). Deletion of both ADAM17 and ADAM10 led to an almost complete loss in sTNF or C-tag signal. Further, introduction of the C-tag cleavage site did not interfere with pro-TNF binding to TNFR2  directly before the characterized ADAM17 cleavage site A, B. Intr ADAM10  (17). Ber ADAM10 , we hypor ADAM10 B. To tes3T cells C. To add3T cells C. Becausr extent D. We thent cells E. The N-n ADAM10 E. Deletito TNFR2 . Taken t−/−, ADAM10−/− and ADAM10−/− × ADAM17−/− HEK293T cells with a plasmid that encodes for the C-tag TNF reporter and for a blue fluorescent protein (BFP) (A). Gating on BFP-positive cells, we first employed an antibody that detects pro-TNF and assessed whether ADAM deficiency would increase the pro-TNF signal in these cells. Comparing cells of different ADAM genotypes for their pro-TNF expression level, increased pro-TNF levels were indeed observed for ADAM17−/− and ADAM10−/− × ADAM17−/− cells . However, the individual populations displayed overlapping peaks, so that they could not be clearly separated from one another. We assessed the discriminatory performance of the pro-TNF fluorescence intensity to separate TNF shedding cells from non-shedding cells, as determined by their ADAM genotype, using a receiver operating characteristic (ROC) analysis . This analysis confirmed the notion that determining pro-TNF levels was not suited to separate shedding from non-shedding cells (ROC area under curve (AUC) = 0.567). However, using the C-tag nanobody to identify cells for TNF maturation, a much better separation of the different cell populations was achieved . WT cells displayed a strong C-tag signal and this signal was decreased in ADAM17−/− cells, indicating a lack of TNF processing . ADAM10 deficiency on its own only showed a slight phenotype, yet its contribution to TNF maturation was more evident in ADAM10−/− × ADAM17−/− cells that showed almost half the C-tag TNF signal compared with ADAM17−/− cells. Indeed, the discriminatory performance of the C-tag signal to separate shedding from non-shedding cells (WT versus ADAM10−/− × ADAM17−/− cells), as determined by ROC analysis, was much better compared with measuring pro-TNF levels (ROC AUC = 0.929) . To further enhance the discrimination of cells that have cleaved TNF, we additionally stained pro-TNF on the cell surface. Based on their pro-TNF expression levels, we separated pro-TNFlow, pro-TNFmed and pro-TNFhigh cells . Analyzing the C-tag positivity as a function of their genotype, the best separation of WT cells from all other genotypes was observed for the pro-TNFhigh population . In particular, the peaks of cleaved TNF  of WT and ADAM10−/− × ADAM17−/− cells were almost completely separated, with a ROC AUC of 0.997 . Again, a stronger contribution of ADAM17 and a redundancy with ADAM10 was observed. At lower levels of pro-TNF expression, the discriminatory capacity of WT versus ADAMs-deficient cells by C-tag positivity decreased . Of note, the residual C-tag signal observed in ADAM10−/− × ADAM17−/− cells is most probably attributable to TNF shedding by other proteases. As such, the expression of an uncleavable C-tag TNF reduced the C-tag signal to background levels  A. Gatingd levels . In summA, Movie S1). The accumulation of cleaved TNF on shedding allows the anti-C-tag nanobody to bind to its target, leading to a gain of fluorescent signal at the cell membrane . Importantly, the C-tag positivity depended on the presence of ADAM proteases, as demonstrated by a diminished fluorescent signal observed in ADAM17−/−, ADAM10−/− and, most strikingly, in ADAM10−/− × ADAM17−/− cells . These results are consistent with data obtained by ELISA, immunoblotting and flow cytometry . In summary, these data demonstrate that the C-tag reporter can be utilized in live cell imaging experiments to follow ADAM proteases-dependent shedding events over time.To further explore the range of applications of the C-tag TNF reporter, we tested the possibility to visualize TNF shedding over time by live cell imaging. For this purpose, we generated a variant of the C-tag TNF reporter bearing an mCherry tag linked to its cytosolic tail (C-tag TNF linker mCherry). This way, both the total amount of TNF as well as cleaved TNF can be visualized at the same time during the analysis. Transfecting cells with this reporter, we conducted a series of live cell imaging studies of WT cells by confocal microscopy over a period of 7 h. These studies revealed that TNF shedding can be monitored on addition of the fluorescently labeled anti-C-tag nanobody to the medium . With this gating, 96% of the ADAM10−/− × ADAM17−/− cell line could be identified as non-shedding cells. We generated HEK293T cells stably expressing Cas9 and transduced these cells with a genome-wide gRNA lentiviral library. To avoid multiple gRNA integrations per target cell, we infected cells with a multiplicity of infection (MOI) of 0.3. After antibiotic resistance selection and expansion, successfully transduced cells were transiently transfected with the C-tag TNF construct and protein expression induced by doxycycline treatment. On the day of sorting, a fraction of unsorted cells was kept as a negative control and the rest were stained for pro-TNF and C-tag. On FACS analysis, we pre-gated on pro-TNF high cells  and subsequently sorted for non-shedding cells as identified as cells with a low C-tag signal (B and S4A). This resulted in the selection of ∼1% of cells of the parental cell population. Next, we performed deep sequencing of unsorted control and sorted non-shedding cells to identify gRNAs enriched in the non-shedding population. 98.7% of the original gRNAs were represented in our unsorted control population, confirming a very high representation of the library in our screen . Although RHBDF2−/− cells phenocopied the deficiency for ADAM17, FURIN−/− cells exhibited a less pronounced reduction of their C-tag signal, in line with the more modest enrichment of FURIN targeting gRNAs in the non-shedding population observed in the screen. Of note, ADAM10 was not ranked as a hit in our screen, consistent with the minor reduction of C-tag signal observed for ADAM10−/− cells in previous experiments. Next, we conducted a STRING analysis to retrieve potential protein-protein interactions within the group of proteins that were scored as hits, as defined by an enrichment of their mean fold change >2  for the detection of TNF cleavage. This reporter exploits the introduction of the C-tag as a cryptic epitope at the TNF cleavage site of ADAM17. On exposure of the membrane portion of pro-TNF that is generated on shedding, the C-tag epitope is efficiently and specifically detected by a nanobody. We could demonstrate that this reporter is compatible with different techniques, namely ELISA, immunoblotting, flow cytometry and live cell imaging. The cleavage of WT and C-tag TNF was mainly dependent on ADAM17, whereas ADAM10 showed only a minor impact on TNF maturation. Residual TNF shedding could be detected in the ADAM10−/− and ADAM10−/− × ADAM17−/− deficient cells in flow cytometry experiments. This discriminatory capacity allowed us to perform a FACS-based genetic screen for the identification of factors involved in TNF shedding. The top ranked hits of the screen were known regulators of TNF shedding, namely ADAM17, RHBDF2 and FRMD8, confirming the validity of our approach. In addition, a STRING analysis highlighted other potential regulators of TNF shedding, in particular a factor involved in vesicle trafficking (AP2M1) and another metalloprotease (ADAMTS1). However, further studies are required to investigate their involvement in TNF maturation.The ability to monitor the shedding activity by flow cytometry and live cell imaging enables the investigation of shedding events at single-cell resolution in real time. Indeed, the C-tag signal was superior to assessing the pro-TNF signal in its ability to separate WT cells from ADAM17At present, TNF shedding is often indirectly studied by quantification of the mature form of the cytokine in bulk cell cultures or tissues. Despite the existence of excellent anti-TNF antibodies that can be reliably employed in bulk techniques, these often only allow for an end point quantification of the amount of pro-TNF and matured TNF in a certain sample, but do not contribute to detecting the interaction between the enzyme and the substrate or the shedding event itself. The same limitation applies to alkaline phosphatase (AP) reporters, where AP, fused to the extracellular domain of the substrate under investigation, is released in the supernatant on cleavage by membrane metalloproteases . In the e.g. fluorescent colors or enzymes) that could impact the shedding process. In addition, because the C-tag is retained on the membrane fragment of pro-TNF, its shedding leaves the mature, soluble cytokine untouched. This is of importance if the activity of the shed TNF needs to be retained. Of note, the uncleaved C-tag TNF also retains its binding capacity to TNFR2, which allows the employment of this reporter in studies where the effects of both soluble and membrane-bound TNF are investigated. In light of their relaxed cleavage site specificities, we speculate that the C-tag would also be compatible with other metalloproteases, which would make this approach a general blueprint for generating reporter systems for these enzymes. However, it should be considered that this approach is only applicable to substrates that expose a free C terminus on the cell membrane after cleavage.The C-Tag TNF reporter can be employed to visualize TNF shedding at single cell level in living cells. Moreover, the C-tag TNF reporter has additional advantages compared with previously described methods for the detection of TNF maturation or ADAM activity. First, it requires little genetic manipulation. Only three amino acids need to be exchanged in the WT TNF sequence, whereas there is no need to introduce additional functional domains . Cells were split every 2–3 days or on plating.HEK293T cells were cultured in DMEM Medium (Life Technologies) supplemented with 10% FCS (v/v), 100 U penicillin-streptomycin and 1 m6 cells were plated in each well of a 6-well plate and transfected the day after with 3 μg of the respective plasmid . Twenty-four hours after transfection, cells were treated with doxycycline in 2% FCS containing medium for 7 h before harvesting. For flow cytometry analysis, 0.2 × 106 cells were plated in 24-wells and transfected the day after with 800 ng of the indicated plasmid (pLI_BFP_C-tag TNF). Cells were treated with doxycycline for 36 h before the analysis. For confocal microscopy, 2.5 × 104 cells were plated in collagen R (Serva) coated Ibidi 8-well u-Slides (Ibidi) using FluoroBrite DMEM medium (Gibco) supplemented as described above, with addition of 10 mm Hepes (Sigma-Aldrich). Cells were transfected with 50 ng each of pLI_C-tag TNF linker mCherry and pEF-BOS_nBFP for a total of 100 ng plasmid/well and treated with doxycycline at the same time. Twenty-five hours after transfection/induction, cells were analyzed via confocal microscopy as described.HEK293T cells of indicated genotypes were transfected in different formats and with different plasmids according to the experimental need. Transfections were always performed with GeneJuice transfection reagent (Merck Millipore) according to manufacturer's instructions. Doxycycline (Sigma-Aldrich) was always used at the final concentration of 1 μg/ml. For immunoblotting and ELISA experiments, 0.7 × 106 cells were plated in each of 4 T175 flasks and infected with the gRNA library at a MOI of 0.3. The day after, virus was removed and cells were split into 12 T175 flasks. Twenty-four hours later, puromycin was added (3.3 μg/ml) to select for successfully infected cells. Four days later, 1.5 × 106 cells were plated in 20 × 10 cm dishes and transfected the day after with 7.2 μg pLI_C-tag TNF plasmid/dish with GeneJuice. Seven hours after transfection cells were treated with 1 μg/ml doxycycline for 36 h. On the day of sorting, 300 × 106 cells were detached from dishes, pooled and stained with CaptureSelectTM biotin anti-C-tag conjugate  and PE anti-human TNF  for 30 min on ice. FITC-streptavidin (Biolegend) was employed to detect the anti-C-tag conjugate . A sample of 20 × 106 cells was left unstained and used as not sorted control. Both stained and not stained cells were fixed with 4% PFA for 10 min at room temperature. Cells were pre-gated for singlets with high PE-pro-TNF expression and ∼1% of cells with the lowest FITC-C-tag signal were sorted (non-shedding population) on a BD FACS Aria Fusion. Unsorted and sorted cells were then lysed as previously described  . Cells wescribed  and lysaescribed . For theSamples were deep sequenced on an Illumina's Hi-Seq platform (HiSeq1500) with a 50 base single read. Data were analyzed by the online tool PinAPL-Py  for gRNATM biotin anti-C-tag conjugate (Thermo Fisher Scientific). Antibodies were used at 1:1000 dilution overnight, anti-C-tag conjugate was used at 1:800 dilution and incubated 36 h at 4 °C. Correspondent HRP-conjugated secondary antibodies (streptavidin-HRP in case of the biotin anti C-tag conjugate) were used for 1 h at room temperature to allow detection of chemiluminescent signals via a CCD camera . When necessary, the contrast of images was enhanced in a linear fashion.Protein precipitation from cell supernatants was performed with methanol/chloroform extraction. Cell pellets were lysed on ice in RIPA buffer containing a mixture of protease inhibitors (Roche). Quantification of proteins in cell lysates was performed via bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). Cell lysates and protein precipitated from supernatant were denatured in Laemmli Buffer for 10 min at 95 °C before separation by denaturing SDS-PAGE. Equal amount of proteins from lysates (10 or 15 μg/lane) and equal volumes of precipitated supernatants (8-12 μl) were loaded in each lane. Proteins were transferred on 0.2 μm nitrocellulose membranes for 75 min at 100 V. After transfer, membranes were blocked in 5% milk and incubated with the indicated primary antibodies: TNF , ADAM17 , ADAM10 , ACTB , CaptureSelecthTNF quantification in cell supernatant was performed with a hTNF ELISA kit  following manufacturer's instruction.m EDTA) and stained with PE anti-human TNF  and CaptureSelectTM biotin anti-C-tag conjugate  for 30 min on ice. After washing in FACS buffer, samples were stained with FITC-streptavidin (Biolegend) for 30 min on ice (1:100) to detect the anti-C-tag conjugate. Cells were subjected to a further wash before analysis on a BD LSR Fortessa. Flow cytometry data were analyzed using FlowJo 10.7. All histograms are shown normalized to mode.HEK293T cells were trypsinized, washed once with FACS buffer  used in live cell imaging experiments, to facilitate cell identification on analysis. The nBFP sequence was inserted by employing XhoI and BamHI restriction sites.−/− × ADAM17−/− cells (determined as non-shedding cells). To this end, the individual fluorescence intensity values of 1000 randomly chosen events of each data set were obtained and a ROC curve analysis was conducted using GraphPad Prism 8. As a quantifiable measure, the area under the ROC curve is provided for each data set.To determine the discriminatory capacity of either pro-TNF or C-tag staining to identify shedding or non-shedding cells, ROC curve analyses were conducted, comparing WT (determined as shedding cells) and ADAM10TM Alexa488 anti-C-tag conjugate (Thermo Fisher Scientific) was added to the cell supernatant at a final dilution of 1:480. Time series of confocal sections were acquired using a LEICA SP8 confocal microscope with a 40× (1.1 NA) water-immersion objective. Five regions of interest per genotype with a time resolution of one image per 30 min, over a period of 7 h, were scanned. Time series of confocal images were then processed by using the Definiens XD 2.0 image analysis platform as follows. First, the contours of the nuclei were identified based on the nBFP signal in channel one. These contours were then used as seeds to identify the entire cell bodies based on the mCherry signal  in channel 2. Finally, the average fluorescence intensity corresponding to the C-tag signal  and the total TNF signal  was computed for each of the resulting segmented patterns. The analysis was performed for 4 regions of interest per genotype in each of 3 independent experiments. The average signal of all analyzed single cells from each replicate was computed and used to calculate the final mean ± S.E.M of 3 independent experiments.Before imaging, the CaptureSelectGene deficient HEK293T cells were generated as previously described. Briefly, gRNA specific for the genes of interest were selected from a genome-wide library . Cells wp < 0.001, ** p < 0.01, * p < 0.05, ns= not significant.Data were analyzed for statistically significant differences using One-way ANOVA with Dunnett's correction for multiple testing. The number of repetitions (n) is indicated in the Fig. legends. The analysis was performed with GraphPad Prism 8. *** PRJNA644482.Primary sequencing data from the genetic screen are available in the Sequence Read Archive (SRA) with the following accession ID:


To explore the mechanism of action of Bu-Fei-Yi-Shen formula (BFYSF) in treating chronic obstructive pulmonary disease (COPD) based on network pharmacology analysis and molecular docking validation.  First of all, the pharmacologically active ingredients and corresponding targets in BFYSF were mined by the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database, the analysis platform, and literature review. Subsequently, the COPD-related targets (including the pathogenic targets and known therapeutic targets) were identified through the TTD, CTD, DisGeNet, and GeneCards databases. Thereafter, Cytoscape was employed to construct the candidate component-target network of BFYSF in the treatment of COPD. Moreover, the cytoHubba plug-in was utilized to calculate the topological parameters of nodes in the network; then, the core components and core targets of BFYSF in the treatment of COPD were extracted according to the degree value  to construct the core network. Further, the Autodock vina software was adopted for molecular docking study on the core active ingredients and core targets, so as to verify the above-mentioned network pharmacology analysis results. Finally, the Omicshare database was applied in enrichment analysis of the biological functions of core targets and the involved signaling pathways.  In the core component-target network of BFYSF in treating COPD, there were 30 active ingredients and 37 core targets. Enrichment analysis suggested that these 37 core targets were mainly involved in the regulation of biological functions, such as response to biological and chemical stimuli, multiple cellular life processes, immunity, and metabolism. Besides, multiple pathways, including IL-17, Toll-like receptor (TLR), TNF, and HIF-1, played certain roles in the effect of BFYSF on treating COPD.  BFYSF can treat COPD through the multicomponent, multitarget, and multipathway synergistic network, which provides basic data for intensively exploring the mechanism of action of BFYSF in treating COPD. Chronic obstructive pulmonary disease (COPD) is a common clinical disease characterized by respiratory system symptoms and flow limitation . It is gModern medicine can not attain satisfactory therapeutic effect or safety on stable COPD patients, and the limited efficacy must be maintained by long-term inhalation of bronchodilators and glucocorticoids –11. By cChinese medicine formula is the organic whole constituted by multiple traditional Chinese herbal medicines, which has a complex chemical composition . Nonethehttps://tcmspw.com/index.php) database and the analysis platform [http://bionet.ncpsb.org/batman-tcm/) [Using the Traditional Chinese Medicine Systems Pharmacology   accordinhttp://db.idrblab.net/ttd/, Last update by June 1, 2020) [https://www.disgenet.org/, v7.0) [https://www.genecards.org/, version: 5.0) [http://ctdbase.org/, Last update by June, 2020) [The targets related to the COPD pathogenesis or therapeutics were obtained through retrieving the TTD database  , DisGeNe/, v7.0) , GeneCaron: 5.0) , and CTDe, 2020)  using thhttps://www.uniprot.org/) [https://bioinfogp.cnb.csic.es/tools/venny/, version: 2.1) to acquire the common targets, which were the candidate targets of BFYSF in treating COPD. Thereafter, the Cytoscape software (version 3.7.2) [First of all, the UniProt database (ot.org/)  was selen 3.7.2)  was empln 3.7.2)  was emplhttp://www.rcsb.org/) [The interactions between compounds and targets were validated using the Autodock vina software (version: 1.1.2) . The molsb.org/) . The MOEsb.org/)  were usehttps://www.omicshare.com/) was used for GO-BP and KEGG enrichment analyses of the targets at the threshold of p < 0.05.To further explore the biological processes (BPs) and the regulatory signaling pathways involved in the core targets of BFYSF in treating COPD, the screened core target information was imported, and the Omicshare online software  were the candidate active compounds of BFYSF.COPD is a polygenic genetic disease, and its pathogenesis can be potentially illustrated by studying the gene-gene or gene-environment associations . ThroughLater, the Cytoscape software was employed to construct the candidate component-target network of BFYSF in treating COPD . To furtThe associations of components with targets were assessed through molecular docking study, which reduced the network complexity and improved the accuracy. Virtual screening performed to determine the binding affinity between protein models and 9 core active compounds , namely, atractylenolide I, quercetin, formononetin, kaempferol, beta-sitosterol, stigmasterol, gallic acid-3-O--glucoside, icariresinol, and ginsenoside rh2. Upon molecular docking, 4 targets  together with 9 compounds were identified .355, PHE518, SER353, and GLN192 of COX-2 and VAL352, TYR347, and GLN263 of iNOS. At the same time, quercetin also formed a strong hydrophobic interaction with residues LEU352 and VAL523 of COX-2 and π-anion bonds with residues GLU377. The gallic acid-3-O--glucoside-PPARγ complex might be stabilized through H-bond as well as π-cation bonds with CYS285, TYR327, SER289, and LYS367 . The most significantly enriched GO-BP items were response to biological and chemical stimuli, multiple cellular biological process, cell proliferation, immunity, metabolism, exercise, and biological regulation  four oxidative stress (OS) and inflammation related pathways, including the hypoxia-inducible factor 1 (HIF1), Fork head transcription factor O (FoxO), TNF, and JAK-STAT; (3) one protease/antiprotease imbalance related pathway, which was the transforming growth factor β (TGF-β) signaling pathway; (4) two pathways which were related to airway remodeling and airway mucus hypersecretion, including the MAPK and VEGF signaling pathway; and (5) one pathway which was related to cell growth and death, which was cell apoptosis.To further understand the multitarget and multipathway mechanism of BFYSF in treating COPD, this study utilized the Omicshare online tool for the GO-PB and KEGG enrichment analyses of core targets, so as to mine the biological processes and signaling pathways involved in BFYSF in treating COPD (gulation . The abogulation , which rBFYSF is a cipher prescription used in the Department of Respiration at our hospital to treat COPD, which is most suitable for patients with lung and kidney deficiency. In the clinical treatment for COPD, BFYSF has significant therapeutic efficacy, but its active ingredients and mechanism remain unclear, which has hindered the further development and utilization of this prescription.Network pharmacology is a new strategy of drug design and development proposed based on the rapid development of systems biology and polypharmacology. Hopkins first put forward this concept in 2007 ; in 2008It was discovered in this study that all the six single herbal medicines in this formula contained large amounts of components, and the components acted on multiple targets, or multiple components acted on the same target, which reflected the multicomponent and multitarget synergistic features of BFYSF. At the same time, the targets might be regulated by single herbal medicine or multiple herbal medicines of BFYSF, which showed that in a formula, both the unique contribution of single herbal medicine and the synergy of multiple herbal medicines existed. As discovered from the component-target network of BFYSF in treating COPD, 48 components in this prescription might act on 65 targets to exert the therapeutic effect. Furthermore, based on the topological parameters of nodes in the component-target network, it was speculated that PTGS2, PPARG, NOS2, and NR3C1 were the core targets for BFYSF in treating COPD. Besides, the interactions between core components and core targets were verified through molecular docking study. The subsequent GO-BP and KEGG enrichment analyses revealed that these targets mainly participated in regulating inflammatory immune response, OS, suppressing protease/antiprotease imbalance, improving airway remodeling and airway mucus hypersecretion, energy metabolism, and modulating the related signaling pathways.α levels in blood and airway elevate, the neutrophil elastase level in serum elevates, α1-AT level decreases, and the protease/antiprotease balance is destroyed [Airway and pulmonary inflammation, protease/antiprotease imbalance, and oxidation/antioxidation imbalance are the major pathogenic mechanisms of COPD. As suggested in research, the Th17 cell proportion and COX-2 expression in the lung tissue and peripheral blood of COPD patients significantly increase, the inflammatory cytokine TNF-estroyed –53. In testroyed . Regulatestroyed , 56. It estroyed , 57.Airway remodeling and airway mucus hypersecretion are the major pathological features of COPD, which are mainly manifested as airway wall structural changes, luminal stenosis, and flow limitation , 59. ResTo sum up, the mechanism of action by which BFYSF treats COPD may be related to the regulation of immune inflammatory response, antioxidation, suppression of protease/antiprotease imbalance, and improvement of airway remodeling and airway mucus hypersecretion.