In wheat, this is possible using protoplast isolation and PEG transformation with Cas9 ribonucleoprotein complexes. Therefore, we have developed a streamlined protocol for testing the accessibility of sgRNA targets in wheat. The first steps involve digesting wheat leaf tissue in an enzymatic solution and then isolating viable protoplasts using filters and a sucrose gradient. The protoplasts are then transformed using Cas9 ribonucleoprotein complexes via PEG-mediated transformation. DNA is isolated from the CRISPR-Cas-edited protoplasts and PCR is performed to amplify the gene target region. The PCR product is then used to assess the editing efficiency of the chosen sgRNA using Sanger sequencing. This simplified protocol for the isolation and transformation of wheat protoplast cells using Cas9 ribonucleoprotein complexes streamlines CRISPR transformation projects by allowing for a fast and easy test of sgRNA accessibility in vivo.The genetic engineering method CRISPR has been touted as an efficient, inexpensive, easily used, and targeted genetic modification technology that is widely suggested as having the potential to solve many of the problems facing agriculture now and in the future. Like all new technologies, however, it is not without challenges. One of the most difficult challenges to anticipate and detect is gene targets that are inaccessible due to the chromatin state at their specific location. There is currently no way to predict this during the process of designing a sgRNA target, and the only way to detect this issue before spending time and resources on full transformations is to test the cleavage ability of the sgRNA  Wheat is one of the staple food crops in the world, and currently feeds more than a quarter of the global population . MultiplGene editing is the process of making specific modifications to a known DNA sequence within a cell. These modifications can consist of insertions, deletions, or changes in the gene sequence that cause a desired change in the produced protein . CRISPR Transgenic wheat has not been deregulated by any world government, and the societal distrust of genetic engineering technologies is at an all-time high. While much research has been published on CRISPR-Cas9 mediated editing of wheat using plasmid expression vectors, the transformation of wheat using CRISPR-Cas ribonucleoproteins (RNPs) is not common. This is likely because RNPs are less stable and less efficient. Plasmids are more efficient, but integrate into the genome of the target organism, and must be removed with subsequent crossing to unmodified plants. The stigma and regulation of transgenic crops means the use of plasmids for CRISPR transformation could result in future issues for deregulation, varietal releases, and shipping overseas. Conversely, RNPs do not integrate into the genome, are only active for a short time in the target organism before being degraded by the cell’s machinery, and have not shown any integration in previous studies . Therefoin vivo at the single cell level before attempting to transform an entire organism. It is important to note, however, that the chromatin state of genes is not static and may change over a cell’s or tissue’s life span and may also differ from tissue to tissue, and this should be kept in mind when performing an in vivo test . ASN2 is an asparagine synthetase gene in the acrylamide formation pathway that was chosen for its potential as an important breeding target. Each gene target was tested in both varieties a minimum of three times.This protocol was tested on two varieties of hexaploid spring wheat: Bobwhite and Chinese Spring, and three gene targets: 1.Triticum aestivum L. cv. Bobwhite and Triticum aestivum L. Chinese Spring.Wheat seed: 2.® Mix #4 professional growing mix .Sunshine3.GW2-B, PinB-D, and ASN2-A .5.PCR thermocycler .6.Microcentrifuge tubes (1.5 ml and 2.0 ml) (Eppendorf).7.DNeasy Plant Mini Kit .8.Analog Vortex Mixer .9.Benchtop Centrifuge (Eppendorf).10.NanoDrop spectrophotometer .11.TM Spy Cas9 NLS and 10x Cas9 Nuclease Reaction Buffer .EnGen12.Agarose .13.Generuler 1 kb Plus ladder .14.TBE Buffer .15.trans-illuminator system.Gel 16.Analytic balance .17.Enzyme Solution .18.Glass beakers  (Pyrex).19.Hot plate stirrer (VWR).20.Steel scissors.21.Glass flask (250 mL) (Pyrex).22.Incubator shaker .23.Mesh snap ball tea strainer.24.Aluminum foil.25.W5 Solution .26.W5A Solution .27.Sucrose .28.WI solution .29.Pipet filler and serological pipets (10 mL and 25 mL) (Fisher Scientific).30.Nylon cell strainer (100 μm) .31.Falcon conical centrifuge tubes (50 mL) (Fisher Scientific).32.TM polysulfone centrifuge tubes (50 mL) (Thermo Fisher Scientific).Nalgene33.Evans Blue .34.GFP reporter plasmid .35.PEG Solution .36.Hemocytometer.37.Fluorescence microscope .38.Phusion High-Fidelity PCR Master Mix with HF Buffer .39.dNTPs .40.QIAquick PCR Purification Kit .41.10x NEBuffer 2 .42.T7 Endonuclease I .43.EDTA .in vivo transformation steps, a gene target must be selected and an appropriate sgRNA designed and tested in vitro. In the case of bread wheat, an allohexaploid, most genes are present in three copies, one for each genome . Homoeologs may be expressed at different levels from each other and are often compensatory, meaning if one copy is knocked out, expression in another copy will increase to make up for the loss , 0.5 μl of Cas9 nuclease (1.61 μg), and 2.4 μl of 10x Cas9 Nuclease Reaction Buffer for a total volume of 3 μl RNP per reaction.∗Note: This step should be optimized for the type of sgRNA and Cas9 used. Follow the manufacturer’s instructions accordingly.5.The RNPs are then incubated at room temperature for 10 min.6.2O to 30 μl total.The RNP complex is added to 200 ng of PCR product, 0.32 μl of 10x BSA, and RNase-Free H7.The cleavage reaction is incubated at 37°C for 1 to 2 h, followed by 80°C for 5 min.8.The cleaved PCR product is then run on a 2% agarose gel in 0.5x TBE buffer with a 1 kb Plus ladder. Cleavage activity is assessed based on the amount of digested product compared to the amount of total input DNA.Protoplast isolation begins with digesting young leaf tissue in an enzymatic solution to remove the cell walls and free the protoplast cells. The solution is then filtered through a mesh strainer to remove large pieces of tissue, washed to remove small debris, centrifuged in a sucrose gradient to separate dead and viable protoplasts, and finally brought to the desired concentration.Timing: Growth of plantlets takes up to 3 weeks. Digestion of leaf tissue takes approximately 7 h, and isolation of protoplasts takes approximately 12 h.1.® Mix #4 and grow at 21°C with a 10 h photoperiod for at least 6 days, up to 30 days.Plant wheat seeds in autoclaved Sunshine2.Cut 2 g of wheat leaves from young wheat plants and leave to soak in sterile water at 4°C while preparing the solutions.3.2 and BSA.Add enzymes to Enzyme Solution, warm to 55°C for 10 min, cool to room temperature, add CaCl∗Note: 2 g of tissue requires 100 ml of Enzyme Solution.4.D-mannitol.Remove wheat leaves from water and cut into 2 mm pieces using scissors, put cut pieces into 0.4 M 5.Once enzyme solution is ready and all leaf pieces have been cut, use a mesh snap ball tea strainer to remove leaves from mannitol solution.6.Put leaf pieces into a flask containing the enzyme solution, cover the flask completely with aluminum foil, and place on an incubating shaker at 27°C at 100 rpm for 3 h.7.Gently swirl the flask to release the protoplasts.8.Filter the solution through a mesh snap ball tea strainer into a small beaker. Rinse the flask and leaf pieces with 20 ml of W5 solution.9.Filter the liquid through a 100 μm cell strainer into 50 ml Falcon tubes. Rinse the strainer with 1–2 ml of W5 solution.10.TM centrifuge tubes.Distribute evenly into Nalgene11.g for 5 min.Centrifuge at 100 × 12.Being very careful not to disturb the pellet, remove the supernatant by pipetting.13.Resuspend the pellet in 4 ml of W5A solution.14.TM centrifuge tube.Add 8 ml of filter sterilized 21% sucrose solution to a new Nalgene15.Very slowly and carefully, layer the protoplast solution on top of the sucrose.∗Note: Always cut the ends off of the pipette tips whenever transferring protoplasts in order to prevent shearing them.16.g for 13 min.Centrifuge at 720 × 17.There should be a layer of clear W5A Solution on top, followed by a small layer of viable green protoplasts, then a large layer of sucrose, and then dead/broken protoplasts at the bottom.18.Slowly harvest the viable protoplasts by pipetting, and place in 2.0 ml microcentrifuge tubes, up to 1 ml of protoplasts per tube.19.Bring total volume in each tube to 2.0 ml with WI solution.∗Note: Without WI solution, the protoplasts may not settle to the bottom, but remain suspended in solution.20.Cover the tubes with aluminum foil and leave at 4°C overnight to let the protoplasts settle to the bottom.21.Once settled, pipette off the supernatant.22.Check protoplast concentration with a hemocytometer.23.Check protoplast viability with 1% Evans Blue dye by adding 3 μl of dye per 100 μl of protoplasts and incubating for 10 min at room temperature. Any dead tissue will be dyed blue.24.6 cells/ml).Make up to desired concentration with WI solution , 10 μl of Cas9 nuclease (32.2 μg), and 8 μl of 10x Cas9 Nuclease Reaction Buffer for a total volume of 20 μl RNP per reaction, and incubated for 10 min as room temperature.∗Note: This step should be optimized for the type of sgRNA and Cas9 used. Follow the manufacturer’s instructions accordingly.2.6 cells/ml).Add RNPs to 50 μl of protoplasts , and mix thoroughly by gently inverting the tube until homogenous.∗Note: DNA, protoplasts, and PEG solution should be added to the microcentrifuge tube in that order. Don’t delay in between adding protoplasts and PEG. Once protoplasts and DNA have been combined, add PEG immediately.4.For the positive control: Add 15 μg of GFP reporter plasmid to protoplasts instead of RNPs (15 ug of GFP plasmid in 40 μl or less + 50 μl of protoplasts at 0.7–1 × 106 cells/ml), followed by PEG solution as in step 3 above.5.Incubate mixture for 15–20 min at room temperature in the dark. Add two times the volume of WI solution to the tube  and mix well by inverting the tube to stop the transformation process.6.g for 3 min at room temperature. Remove the supernatant by pipetting.Centrifuge the tubes at 150 × 7.Resuspend protoplasts gently to 1 ml with WI solution.8.Coat 2.0 ml microcentrifuge tubes with 5% BSA (5 mg BSA in 1 ml sterile water) to prevent protoplasts from sticking to the plastic and each other. Add enough BSA to coat all surfaces then pour it out. The BSA can be re-used for multiple tubes.9.Immediately transfer all of the protoplast solution into the wet BSA-coated tubes.10.Wrap the tubes with aluminum foil, lay them on their sides, and incubate at 23°C for 24 to 48 h.a.GFP expression peaks after 24 h if using a transient plasmid.b.RNPs should be allowed to incubate for 48 h.c.Check the transformation efficiency by comparing the number of GFP-fluorescing cells to non-fluorescing cells in the positive control using a fluorescent microscope and hemocytometer.11.g for 2 min at room temperature. Remove the supernatant by pipetting.Collect the protoplasts by centrifuging at 12,000 × ∗Note: Remember to also collect the protoplasts from the negative control sample from step 24a of Isolation of Protoplasts.in vivo, it can be used for editing a whole organism using any method.After incubation with the RNPs, DNA is extracted from the protoplasts and the target sequence is amplified via PCR. The PCR product can then be tested for editing using a T7EI digestion and Sanger sequencing followed by analysis with a program such as TIDE (Tracking of Indels by Decomposition)  or ICE . TIDE anTiming: The extraction of protoplast DNA takes approximately 4 h. PCR of the extracted DNA and heteroduplex formation takes approximately 5 h. The T7EI digestion takes approximately 2 h.1.Extract genomic DNA using the DNeasy Plant Mini Kit.2.Determine the DNA concentration with a Nanodrop spectrophotometer. The usual concentration is around 30 ng/μl in a total volume of 30 μl.∗Pause point: Extracted DNA can be stored at −20°C for several months, or 4°C for several weeks.3.Set up the PCR reaction to amplify the genomic region targeted for mutagenesis .∗It is important to use high-fidelity polymerase to reduce the error rate, and to use the untransformed protoplast DNA from step 11 of PEG-Mediated Transformation as a negative control.4.Perform the PCR for amplifying the desired genomic region .5.Clean up the PCR with a kit of your choice following the manufacturer’s instructions.a. At this point, perform Sanger sequencing of each sample and use CRISPR analysis software to quantify the editing rate and determine the type of change.∗Pause point: PCR products can be stored at 4°C for several weeks.6.For the T7EI digestion, set up an annealing reaction to form DNA heteroduplexes by adding 200 ng PCR product, 2 μl 10X NEBuffer 2, and nuclease-free water up to 19 μl total.7.Run the annealing reaction in a thermocycler using the following conditions: 95°C for 5 min, 95–85°C (−2°C/s), 85–25°C (−0.1°C/s), 4°C forever.8.Set up the T7EI nuclease digestion by adding 1 μl of T7EI to 19 μl of heteroduplexed DNA.9.Mix well and spin the mixture down briefly. Incubate the reaction at 37°C for 15 min, then stop the reaction with 1.5 μl of 0.25 M EDTA.a. The incubation time can be increased if necessary.10.Run digestion products on a 2% agarose gel in 0.5x TBE buffer using standard protocols. Include a DNA ladder and negative controls on the same gel. If digested bands are only observed in the CRISPR-Cas9-treated sample, and not in the negative control, an indel mutation has occurred.1.Low protoplast concentration and/or viability after isolation. This may be due to varietal differences and will require some adjustments to the concentration of enzymes in the enzyme solution and/or the length of digestion. It may also be due to the tissue age, as levels of cellulose and lignin increase over time, potentially inhibiting enzymatic digestion. To prevent this, be sure to use leaf tissue that is less than 4 weeks old.2.Low transformation percentages of GFP plasmid control. Ensure that the PEG solution is freshly prepared and that it has completely dissolved before using. Also be sure that your ratios of DNA to protoplasts are within the recommended range, as a slightly low concentration of GFP plasmid can significantly reduce transformation efficiencies.3.Low concentration of protoplast DNA after extraction. Ensure the protoplasts being transformed are viable. The sucrose gradient separation step, refrigeration overnight, and storage in WI solution should maintain viable protoplasts. If the problem persists and protoplasts are viable, increase the overall number of protoplasts used while keeping the ratios within acceptable ranges.While this protocol was specifically adapted for the assessment of RNP transformation in wheat leaf protoplast cells, there is the potential for it to be applied to other plant species and CRISPR delivery methods, with some modifications. The protoplast isolation steps can also be followed for use with different applications in addition to transformation with PEG and RNPs.6 and 1.2 × 106 viable cells at step 22 of Isolation of Protoplasts for Bobwhite and Chinese Spring, respectively. Yields of this amount were easily brought to a concentration of 0.7 to 1.0 × 106 cells/ml for use in transformation. On average, Evans Blue assays showed 16% and 9% dead protoplasts for Bobwhite and Chinese Spring, respectively, at this stage . This ensures that even with low transformation rates, the digested bands will become concentrated enough to be visible on the electrophoresis gel. In PinB-D and ASN2-A, and quantification would be a challenge. It has been shown that the accuracy of the T7EI assay can be affected by the size of the indel, the type of nucleotides added or deleted, secondary structures, flanking sequence, and mutant sequence abundance  showed 2.7% editing efficiency in all three genomes; TaLpx-1 showed editing efficiencies of 0.3 and 6.1% in the B and D genomes for two different experiments; and TaMLO showed an editing efficiency of 4.1% in the A genome. TaABCC6 had editing efficiencies between 6.6 and 13.0%; TansLTP9.4 had editing efficiencies between 0 and 11.9%; and TaNFXL1 had editing efficiencies between 0 and 42.2%.Lox2 and CER9, which resulted in 23.8 and 33.6% editing efficiency, respectively. GW2 sgRNA sequence as this study, which resulted in an editing efficiency of 33.4% for GW2-B. The GW2-B editing efficiencies in this study of 19.2% for Bobwhite and 36% for Chinese Spring are comparable to the results from ASN2-A was lower overall for both cultivars, but still comparable to the other studies.in vivo sgRNA validation using protoplasts.While NGS techniques are the most accurate for determining sgRNA editing efficiencies, they are not the most accessible. PCR/RE assays are a faster and less expensive method for determining editing efficiencies, but they are not always practical for sgRNA design and do not give details on types or frequencies of individual indels. Sanger sequencing is much more accessible, less expensive, faster, and easier to use than NGS, and is less restrictive and more detailed than PCR/RE assays. Utilizing Sanger sequencing analysis with a program such as TIDE or ICE to determine editing efficiencies has been shown to be comparably accurate to NGS techniques, and therefore is the recommended analysis tool for in vivo transformation studies, a sgRNA with a protoplast mutation rate of at least 10% is a viable candidate for recovering edited plants using other methods (GW2-B sgRNA target as this study. They obtained mutagenesis frequencies of 2.3% with stable plasmid transformation, 2.6% with transient plasmid constructs, and 1.1% with in vitro synthesized transcripts. GW2 gene to stably transform immature embryos of the variety Fielder at an average rate of 10% using Agrobacterium. The 19.2% and 36% editing efficiencies achieved in this study for GW2-B protoplasts is well above the 10% threshold, which reinforces the use of GW2-B as a positive control gene target, as well as shows the accuracy of this protocol for screening potential sgRNA candidates in vivo to be used for a multitude of downstream applications. The 16.4% and 12.9% editing efficiencies achieved for ASN2-A are also above this 10% threshold. Therefore, ASN2-A is a candidate for CRISPR-Cas9 transformation of wheat plants to reduce acrylamide formation during baking, which is an important trait for many food crops but does not yet exist in bread wheat germplasm. The consistent 0% editing efficiency for PinB-D demonstrates the utility of this protocol for screening sgRNAs in vivo before beginning full transformations in plants. A significant amount of time and resources can be saved by discovering at this stage that a sgRNA target is inaccessible, rather than after attempting transformation and regenerating multiple plants with no positive results.Based on previous  methods . Zhang ein vivo testing of CRISPR sgRNA gene targets will also benefit from information presented in this study on expected viability of isolated protoplasts, total protoplast yield, and protoplast concentration for two common varieties. During the protoplast transformation steps, the other studies recommend only a single concentration and/or amount of cells, cell volume, plasmid/RNP amount, and plasmid/RNP volume. These recommendations are also different for each study. Concentrations and volumes of sgRNA and Cas9 can vary widely depending on the reagent manufacturer, modifications to the reagents, and in vitro synthesis yields, making exact specifications difficult to meet in every situation. The recommended ratios provided in this study will simplify this step for researchers with access to various reagents. Another aspect of CRISPR-Cas9 protoplast transformation that has been simplified in this study is the detection and analysis of editing in the cells. This research demonstrates the effectiveness of our simplified protocol for protoplast isolation from wheat leaf tissue, subsequent transformation with CRISPR-Cas9 RNPs and GFP plasmids, and analysis with online deconvolution software. While other protocols are available for protoplast isolation and transformation, there is a need for consensus and clarity in some steps. For example, vacuum infiltration of the enzyme solution was found to be unnecessary for isolation of sufficient, viable protoplasts despite being considered critical in the five studies by This study demonstrates the effectiveness of a simplified protocol for use in any lab attempting to isolate protoplasts and/or implement CRISPR transformation, which gives comparable results to the other available methods. This protocol requires less specialized equipment, provides easily replicable steps, reports more comprehensive results, and provides greater flexibility for other researchers attempting protoplast transformation and sgRNA validation, thus making implementation in any lab straightforward.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.KB, HG, NM, and RZ conceived the study. KB, HG, and NM designed the study and performed the experiments. KB and HG analyzed and interpreted the results. KB prepared the manuscript. HG, NM, and RZ edited the manuscript. All authors read and approved 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.


Bacillus strains were isolated from Miang, a culture relevant fermented tea of northern Thailand. These strains were collected from 16 sampling sites in north Thailand. In this collection 95 isolates were tannin-tolerant Bacillus capable of growth on nutrient agar supplemented with 0.5% (w/v) total tannins from tea leaves extract (TE). The strains were also positive for pectinase, xylanase and amylase activity, while 91 and 86 isolates were positive for cellulase and β-mannanase, respectively. Only 21 isolates producing extracellular tannase were selected for further characterization. Identification by 16S rRNA gene sequence analysis revealed that more than 50% (11 of 21 isolates) were Bacillustequilensis, whereas the remaining were B. siamensis (3), B. megaterium (3), B. aryabhattai (3) and B. toyonensis (1). B. tequilensis K34.2 produced the highest extracellular tannase activity of 0.60 U/mL after cultivation at 37 °C for 48 h. In addition, all 21 isolates were resistant to 0.3% (w/v) bile salt, sensitive to gentamicin, erythromycin, vancomycin and kanamycin and also tolerant to acidic condition. Cell hydrophobicity varied from 9.4 to 80.4% and neutralized culture supernatants of some Bacillus isolates showed bacteriocin producing potentiality against Samonella enterica serovar Typhimurium TISTR 292. All tested probiotic properties indicated that B. tequilensis K19.3, B. tequilensis K34.2 and B. siamensis K19.1 had high probiotic potential. This is the first report describing tannin-tolerant Bacillus and their extracellular tannase producing capability in Miang, a traditional fermented tea of Thailand.A total of 117  Camellia sinensis var. assamica that has been important in the sociocultural lifestyle of northern Thailand for over several hundred years. A typical Miang production process mainly consists of fermentation of steamed tea leaves without adding other nutritional substances in a variety of containers such as bamboo basket or clay jar for several days or up to a year without the use of any preservatives. However, the steps of fermentation process of Miang are different depending on the ethnicity of the local producer  × 100.All 21 extracellular tannase producing i et al.  with somg at 4 °C for 10 min, washed twice with PBS buffer (pH 7.2), and resuspended in 5 mL of the same buffer solution. The cell suspension was inoculated  into 5 mL of PBS solution supplemented with 0.3% (w/v) bile salts  and PBS without bile salts served as control, after which all tubes were incubated at 37 °C. Following 3 h of incubation, the viable cell count was determined, and survival rate was calculated.Bile salts tolerance of bacterial strain was determined according to the method of García-Hernández et al. . Brieflyg at 4 °C for 10 min, washed twice with PBS buffer (pH 7.2), and resuspended in 5 mL of the same buffer solution. The bacterial concentration was adjusted with PBS to OD600 = 1 (A0), and then an equal volume of toluene  was added. Toluene was chosen as a nonpolar solvent because it reflects cell surface hydrophobicity and hydrophilicity [The cell surface hydrophobicity of selected bacilli was determined in terms of the bacterial cell ability in adhering to hydrocarbons , according to the methodology described by García-Hernández et al. . Bacterihilicity . The two5–106 CFU/mL) was spread evenly on the NA plate and antibiotics discs were placed on the plates. After 24 h of incubation at 37 °C, the inhibition zone diameters were measured including the diameter of the discs. Breakpoints for the interpretation of inhibition zone were expressed as sensitive, S; intermediate, I; and resistant, R as described by CLSI [Antibiotic susceptibility of 21 extracellular tannase producing strains was determined by disk diffusion method according to the guidelines of the Clinical and Laboratory Standards Institute . Antibio by CLSI .Bacillus strains was assessed against Salmonella enterica serovar Typhimurium TISTR 292 using the well diffusion assay according to Abid et al. [6–107 CFU/mL) were spread onto agar plates. Cell-free culture supernatants (CFCS) were collected by centrifugation  and filtered through 0.22 μm membrane filter . To demonstrate the antimicrobial activity, 75 μL of pH neutralized CFCS (pH 6.5) was added to each well (cut with sterile 6 mm Cork borer) of the pathogen cells agar plates. The agar plates were incubated at 37 °C for 18 h. Growth inhibition was read by measuring the diameter of the inhibition zones. The antimicrobial activity of the isolated d et al.  with sliBacillus isolates were obtained from the 39 samples of Miang collected from 16 sampling sites within six provinces of northern Thailand  total tannins. All 117 Bacillus isolates were tested for their tolerance to tannins in TE at the final concentration of 0.5% (w/v) total tannins and it was found that 95 isolates or approximately 81% were tannin-tolerant based on their growth ability in NA supplemented with 0.5% (w/v) total tannins  exhibited the extracellular tannase producing ability. The widest clear zone (Bacillus spp. isolated from Miang (18.8%) did not show any growth on NA supplemented with 0.5% (w/v) total tannins. This suggested that these 22 tannin-tolerant negative strains may harbor tannin-tolerant properties, but at concentrations lower than 0.5% (w/v) total tannins. Field and Lettinga [w/v) and showed 100% inhibition of B. subtilis.Tannins concentration in TE prepared for this study was determined after product preparation and also prior to using in medium preparation. The TE from this experiment composed of approximately 2.5%  and 96 and 91% positive for cellulase and β-mannanase production, respectively. This indicates that the capability to produce extracellular polysaccharide degrading enzymes reflects the effort of the bacteria to find carbon source for their survival and extracellular cellulose, β-mannanase, pectinase, amylase and xylanase producing Bacillus strains have been well documented in several studies on fermented products [Bacillus spp. mentioned in this study may be involved in the Miang fermentation process.In addition to the capability for survival in high-tannin condition, 95 tannin-tolerant xylanase b. Howevexylanase b. Furtheproducts ,36,37. SBacillus isolates were identified using 16S rRNA gene analysis and the data are presented in Bacillus (11 of 21 isolates) were identified as Bacillus tequilensis while the remaining as B. siamensis (3 isolates), B. megaterium (3 isolates), B. aryabhattai (3 isolates) and B. toyonensis (1 isolate), which showed similarities in 16S rRNA gene sequence related to their closest type strain in the narrow range between 99.8% and 100.0%. The phylogenetic trees based on the sequences of the 16S rRNA gene were constructed by the neighbor-joining method comparing between each Bacillus strain  and suggested involvement in Miang fermentation process [Molecular identification techniques present an alternative to other conventional methods because the independent genome for the physiological characteristics may vary among the species. Moreover, the techniques are more reproducible, and the results are achieved more rapidly than with traditional methods. All tannase producing s strain . From thsubtilis . A previ process .w/v) tannic acid and the results are presented in B. tequilensis K34.2 showed the highest activity of 0.53 U/mL from 24 h to until the end of the experiment at 48 h (0.60 U/mL), while the lowest activity of 0.25 U/mL was observed in B. megaterium K21.4 and B. siamensis K23.3 at 48 h growth phase. Most of the reported bacterial tannase producers correspond to the presence of extracellular tannase [Bacillus spp. have been identified such as B. pumilus, B. polymyxa [B. licheniformis [B. cereus [B. sphaericus [B. massieliensis [B. subtilis [B. gotthelii [B. tequilensis and B. siamensis.The tannase activity of all 21 isolates of extracellular tannase producing strains were determined from cell-free supernatant fraction (CFSF) separated from 24 and 48 h old culture broth containing 0.5%  and 2 isolates of B. siamensis (isolate K19.1 and K25.2). It was quite interesting to note that some B. tequilensis tolerated the acidic condition at pH 2 for 3 h with survival higher than 50% , whereas some strains were rather weak in acid tolerance . Among them, B. tequilensis K19.3 exhibited most resistance to all tested acidic levels after 3 h at 75.24%. Our results are similar to the result of Parveen Rani et al. [B. tequilensis FR9 was able to survive after exposure to pH 2 and 3, with survival of 71.15% and 82.86%, respectively. Whereas, Bacillus sp. strain VS-5 was found to be the most resistant among the studied Bacillus species with survival of 98.78% after exposure to pH 1.2 for 3 h [A total of 21 tannin-tolerant  for 3 h .w/v) and was selected for this study. All of the isolates were resistant to bile salt with varying degrees of resistance after 3 h exposure. B. tequilensis , B. toyonensis (isolate K22.1), B. siamensis , and B. megaterium (isolate K21.4) were highly tolerant  to 0.3% bile salt. In comparison, B. tequilensis K24.1 was found to be the most resistant strain, with a survival of 98.65%, while B. megaterium K31.3 was found to be the least bile tolerant.Tolerance to bile salt is considered to be vital characteristic for colonization and metabolic activity of bacteria in the intestinal tract and therefore it is an important characteristic of probiotic microorganisms . The meaB. tequilensis  and B. siamensis . The high cell hydrophobicity were B. tequilensis K26.2 and B. siamensis K19.1 at 80.4 and 73.6%, respectively.Another important criterion to select bacterial candidate for probiotic use is its ability to adhere to intestinal mucosal cells . The isoBacillus isolates were found to be susceptible (data not shown) towards gentamicin and vancomycin that interrupt either protein synthesis or cell wall biosynthesis in the bacteria. At the same time, most of them were found to be susceptible toward erythromycin and kanamycin, but only B. siamensis K19.1 showed intermediate response to erythromycin and kanamycin. It should be noted that the sensitivity found in our study may be related to the concentration of each antibiotic and hence, several concentrations should be tested to confirm the results.Previous studies suggested that candidate probiotic bacteria should not act as reservoirs for antibiotic resistance genes . In thisS. enterica were used as pathogenic indicator, where most of Bacillus isolates were able to show inhibition towards this pathogenic bacterium which included B. tequilensis , B. siamensis (isolates K19.1 and K25.2), B. megaterium  and B. aryabhattai . The highest antimicrobial activities were from B. tequilensis isolates K7.2, K19.3 and K26.2.The capability to produce antimicrobial compounds is related to the metabolic products of bacteria such as organic acids, hydrogen peroxide, bacteriocins and short chain fatty acids, which are among the properties generally used to evaluate the probiotic potential of bacteria . In thisBacillus spp. diversity from traditional Miang, a fermented tea leaves product of northern Thailand was for the first time identified and characterized in this study. Among 117 Bacillus spp. isolated from Miang, 21 isolates showed the ability to produce extracellular tannase. The results of current study also showed that some tannin-tolerant Bacillus strains are potentially useful as probiotic bacteria. Some B. tequilensis and B. siamensis isolates showed high acid tolerance, bile salt tolerance, hydrophobicity and antimicrobial activity. Miang may also be considered a traditional fermented product with health-relevant functional benefits with antioxidant compounds such as high levels of phenolic acid and flavonoids [Bacillus spp. Moreover, due to the high numbers of Bacillus spp. in Miang, these bacteria in themselves may be considered as a possible probiotic. From the promising findings of this study more detailed analysis of probiotic potential of specific strains isolated from Miang should be undertaken. Further understanding of their health effects, including wider functional benefits of biotransformed tannin products should be investigated.In conclusion, avonoids , which a


Taiwan implemented version 1.0 of its 10-Year Long-Term Care Plan (TLTCP 1.0) in 2007, and version 2.0 (TLTCP 2.0) in 2017. The aim was to develop a system of home- and community-based services (HCBS) for long-term care (LTC). This study assessed and compared disparities in access to LTC services using one county’s database that claims made by 14,051 older adults who applied for LTC services from TLTCP 1.0  and TLTCP 2.0 . We assessed LTC disparities related to five sociodemographic factors  and seven types of LTC services. Older adults who lived in a city were more likely to use multiple HCBS and transportation services. Older adults who lived alone were more likely to use home care and meal services but not other types of LTC services. All disparities in service use increased from TLTCP 1.0 to TLTCP 2.0. Part of a symposium sponsored by International Comparisons of Healthy Aging Interest Group.


There were significant decreases in exhaust emissions of SO2 and NOx in Jinan during the study period. Annual reductions in ambient air pollution were 72.6% for SO2, 43.1% for PM2.5, and 34.2% for PM10. A total of 2,317  premature deaths and 15,822  related morbidity cases had been avoided in 2017, leading to a total of US$ 317.7 million (95%CI: 227.5–458.1) in economic benefits. Decreasing PM2.5 concentrations to 15 μg/m3 would result in reductions of 70% in total PM2.5-related non-accidental mortality and 95% in total PM2.5-related morbidity, which translates into US$ 1,289.5 million  in economic benefits. The national and local air pollution control measures have brought significant environmental, health and economic benefits to a previously heavy polluted Chinese city.China has made great efforts in air pollution control since 2013. However, there is a lack of evaluation of environmental, health and economic co-benefits associated with the national and local air pollution control measures at a city level. We analyzed local air pollution control policies and implementation in Jinan, one of the most heavily air-polluted cities in China between 2013 and 2017. We assessed the changes in exhaust emissions, air quality, mortality and morbidity of associated specific-diseases, and related economic benefits. We also projected the future scenarios of PM Since then, air pollution, especially the risks from PM2.5 (particle matter with aerodynamic diameter ≤ 2.5 μm), has become a growing concern in China3. Spatial and temporal distributions of air quality in China show that winter months in northern cities are the most polluted because of coal burning and other fossil fuel combustion for heating7.Air pollution is a major global health concern, especially in developing countries like China. In January 2013, a severe air pollution event affected one-third of the major cities in China2.5 on mortality and morbidity, and for both short-term and long-term exposures9. In May 2018, the World Health Organization (WHO) reported that 4.2 million deaths occur annually as a result of exposure to ambient air pollution globally with developing countries having the highest burden10. The first WHO Global Conference on Air Pollution and Health held in November 2018 emphasized an aspirational goal of reducing the number of deaths from air pollution by two-thirds by 2030 and the urgent need for bold and prompt actions to address the present health crisis caused by air pollution11.Scientific studies have provided strong evidence on the effects of PM12. In September 2013, the State Council of China issued the Air Pollution Prevention and Control Action Plan (APPCAP), which comprises ten specific tasks (Ten Tasks) with specific concentration targets to be achieved by 2017 in order to reduce air pollution13. For example, by 2017, urban concentrations of PM10 shall be reduced by 10% compared with that in 2012; PM2.5 concentrations for Beijing-Tianjin-Hebei region shall be lowered by 25%; 20% reduction in the Yangtze River Delta region and 15% reduction in the Pearl River Delta region compared with that in 2013.In order to improve air quality and to protect human health, the Chinese government has implemented a series of national policies to reduce the emissions of air pollutants after the 2013 national severe air pollution event16. A clear reduction in air pollutants has been shown in many cities and regions18. Associated mortality and related economic benefits have also been reported due to improved air quality19. Wang et al.4 reviewed the effect of PM2.5 control measures and found a 24% reduction in PM2.5 during 2013 and 2015 in 74 major cities of China. Feng et al.18 found more than seventy percent of the studied Chinese cities achieved reduced PM2.5 from 2015 to 2016, and avoided a total of 10,658 per million deaths. Huang et al.14 reported that annual average concentrations of PM2.5 decreased by 33.3% in 74 Chinese cities between 2013 and 2017, and 47,240 fewer deaths due to the reduction. Chen et al.17 projected the mortality and economic benefits from a 25% reduction in PM2.5 concentration below the baseline of 2012 to 2014 in Beijing-Tianjin-Hebei region. Gao et al.18 conducted a cost-benefit analysis of data from 31 provinces and found a total of US$ 112.6 billion saved due to reduced mortality during the national APPCAP implementation. However, there was a lack of estimation on the impacts on morbidity and corresponding economic benefits attributable to reduction in PM2.5 concentration. In order to have a better understanding of the co-benefits that occurred during the national APPCAP implementation, a comprehensive evaluation of the improvement in air quality, benefits for both mortality and morbidity, and related economic benefits could aid policy making for air pollution control.Recent studies have demonstrated achieved health and economic benefits from the national APPCAP20. In addition to national policies, localized measures are required in responding to specific air pollutants. Therefore, there is a need to examine city-level experience to reveal how local measures could benefit environment, health and economy, in addition to the national APPCAP. In particular, there is a limited understanding of what air pollution control measures have been implemented at a local level and their effectiveness and benefits.Situations of air pollution in China vary across regions and cities due to different natural and climate conditions, topography, sources of air pollution, and energy combustions2.5 historical record of 443 ug/m3 during the severe air pollution event in 201321. We selected Jinan city as the study site to quantify the co-benefits of local air pollution control measures and implementation between 2013 and 2017. The co-benefits estimated in this study focused on environmental, health and economic benefits from air pollution policies and measures. Previous local epidemiological studies have demonstrated the impacts of air pollution on mortality23 and morbidity24. Our findings will assist policy making and development to improve air quality and achieve health and economic benefits at local and national levels.Jinan is one of the most heavily air-polluted cities in China with a PM2.5 concentration down to the national ambient air quality standards Grade-II (15 μg/m3)25.The methodological framework of our study was shown in Fig. 2 with ten districts. It is located at 36.40°N latitude and 110.00°E longitude, along the coast of the Yellow River, downstream of the Central China Region . It has a stable population growth from 7.0 million in 2013 to 7.3 million in 2017, with nearly 15% of older population (65 years and older)26 26 Fig. .Figure 227 and Jinan Air Pollution Prevention and Control Action Plan (phase II) on 21 October 201628, in which specific goals and detailed control measures were introduced to achieve the national goals set in the APPCAP. Jinan has also developed a range of local policies and measures to reduce air pollutants emissions in the last few years29. We reviewed all air pollution control policies in Jinan between 2013 and 2017, including documents from Jinan Environmental Protection Bureau29, Jinan Statistical Yearbook26, Shandong Statistical Yearbook34, and National Statistical Reports35. We also collected the air pollution control measures from the transportation sector from the Yellow-labeled vehicles (the government put labels on vehicles that don’t meet exhaust emission standards) and elimination policy, exhaust limitation, and oil reformation for old vehicles.In responding to the national APPCAP and Ten Tasks, Jinan government released the Jinan Air Pollution Prevention and Control Action Plan (phase I) on 12 December 20132), nitrogen oxides (NOx), and soot from industries and households were extracted from Jinan Statistical Yearbook26.The exhaust of sulphur dioxide , stroke, lung cancer (LC), and chronic obstructive pulmonary disease (COPD), recommended by Global Burden of Disease (GBD) study were included3. We obtained the four cause-specific mortalities (age > 25 years) of Jinan between 2013 and 2017 from Jinan Municipal Center for Disease Control and Prevention, which is responsible for death surveillance.Four leading causes of mortality attributable to PM2.5-related health endpoints of morbidity, including respiratory disease hospital admission (RHA), cardiovascular disease hospital admission (CHA), chronic bronchitis (CB), asthma attack (AA), and respiratory disease emergency room visits (REV). Because of limited local data for the cause-specific morbidities, we used the national cause-specific morbidity rate of RHA, CHA, CB, AA, and REV as the reference morbidity in our estimation, which were collected from China Statistical Yearbook36.We selected five PM2.5 in Jinan in 2013 and 2017, and then calculated the reduction in mortality and morbidity between 2013 and 2017.We firstly estimated the long-term burden of mortality and morbidity attributable to PMet al. 201837 was used to examine the long-term mortality effects between 2013 and 2017. GEMM modeled the hazard ratio association between outdoor PM2.5 and non-accidental mortality using data from 41 cohorts of 16 countries, including the Chinese Male Cohort. GEMM for each of the five specific causes of GEMM 5-COD was used to estimate the combined population-attributable fraction based on five specific causes of death (age > 25 years) examined by the GBD. In our study, we estimated the IHD, stroke, LC, and COPD in 2013 and 2017. The GEMM through the Log-Linear (LL) model was used to estimate excess deaths from exposure to ambient PM2.5, the formula is shown as (f(z) = z or f(z) = log(z + 1), HRmorbidity = 1 when z = 0 for either form of f. Where, ω(z) = 1/(1 + exp{−(z−µ)/(τr)}) is a logistic weighting function of z and two parameters  with r the range in the pollutant concentrations. The parameter τ controls the amount of curvature in ω with μ controlling the shape. The set of values of  define a shape of the mortality–PM2.5 association. The estimation method is based on a routine that selects multiple values of  and given these values, estimates of θ and its standard error are obtained using standard computer software that fit the Cox proportional hazards model.The Global Exposure Mortality Model (GEMM) constructed by Burnett 2.5 in 2013 and 2017. This function has been applied for several studies in China38. The cause-specific morbidity relative risk (RRmorbidity) was calculated using the following Eq.  and C0 is the reference concentration. We used the reference concentration of PM2.5 10 μg/m3 as the morbidity estimation to perform the long-term morbidity assessment, as used in previous studies38. The β coefficient for cause-specific morbidity could be derived from RR, using the following Eq. 39, the RR of CHA was 1.013 (95% CI: 1.007–1.019)40, the RR of CB was 1.029 (95% CI: 1.014–1.044)39, the RR of AA was 1.021 (95% CI: 1.015–1.028)41, and the RR of REV was 1.010 (95% CI: 1.005–1.016)5, respectively.We used the Log-linear function to estimate the long-term morbidity attributable to PMHI) between 2017 and 2013. HI was calculated from HRmortality and RRmorbidity for cause-specific disease in 2013 and 2017 using Eq.  and cost of illness (COI) methods. The WTP method could be used in calculating the individual willingness to pay for a small reduction of mortality risk and estimating morbidity related economic costs42. The COI method calculated the disease costs in terms of medical treatment, hospitalization, and productivity loss42. The health costs for Jinan in 2016 was used and adjusted using GDP per capita from the health costs for Shandong Province in 201615. The health costs (per capita) for Jinan and Shandong Province in 2016 are shown in Table The associated economic benefits of cause-specific mortality and morbidity were further quantified. The methods used to estimate health economic costs  of PMHI with health costs. The health-related economic benefit was estimated by health costs in 2017 minus that in 2013. The total economic benefit was calculated by adding the benefits from cause-specific mortality and morbidity.Lastly, we calculated the total health costs of cause-specific mortality and morbidity in 2013 and 2017 by multiplying the cause-specific 2.5 annual concentration reduced from 2013 (110 ug/m3) to the national ambient air quality standards Grade-II (15 ug/m3)25.We further projected the co-benefits in health and economics under a future scenario of PMhttps://mirrors.tuna.tsinghua.edu.cn/CRAN/). Map was created by the packages of mapdata (https://cran.r-project.org/web/packages/mapdata/index.html), maps (https://cran.r-project.org/web/packages/maps/index.html) and ggmap (https://cran.r-project.org/web/packages/ggmap/).All statistical analyses for this study were performed using R software  Fig. To reduce emissions from transportation and vehicle exhaust, the Jinan government provided allowances for the purchase of cleaner vehicles to encourage elimination of Yellow-labeled vehicle after 2013. Further, oil and gas recovery and reformation were taken to all 582 petrol stations in both urban and rural areas Table .8 m2 of centralized heating area in 2017 compared with that in 2013. To increase the clean energy  supply for households, total natural gas supply reached 9.0 × 108 m3 in 2017 with an increasing trend each year  emissions from industry showed a greater decrease than that from households between 2013 and 2016. The soot emissions from households presented a 7.67% decrease over the same period.We found the city-level exhaust emissions from industry and households dropped significantly, except the soot (dust) emissions from industry Fig. . SO2 and2 (72.6%), PM2.5 (43.1%), PM10 (34.2%), CO (33.0%), and NO2 (28.6%) in 2017, respectively , PM2.5 3.1%, PM1nts Fig. .Figure 42.5 between 2013 and 2017. The concentration of PM2.5 was extremely high in both urban and rural areas in 2013. Among the ten administrative districts of Jinan, 9 districts had the annual concentration of PM2.5 exceed 100 ug/m3 in 2013, Licheng district became the most heavily polluted region with a PM2.5 annual average value of 120 ug/m3, while Shizhong district was featured with the lowest value of 97 ug/m3 . In 2013, a total of 12,591  premature deaths or 33.69% (95%CI: 22.69–41.07%) of the non-accidental deaths, and 37,949 (95%CI: 23349–51183) related cases or 18.76% (95%CI: 11.54–25.30%) of the total morbidity cases were attributed to PM2.5. Accordingly, a total of 10,274  premature deaths or 25.21% (95%CI: 16.51–31.74%) of the non-accidental deaths, and 22,129  related cases or 10.46% (95%CI: 6.31–14.40%) of the total morbidity cases were attributed to PM2.5 in 2017.Results showed decreased burden of cause-specific mortality and morbidity in 2017 attributed to PMA total of 2,317  premature deaths were avoided, including 669 (95%CI: 621–710) cases due to IHD, 1,075  cases due to stroke, 234 (95%CI: 158–287) cases due to LC, and 339 (95%CI: 185–444) cases due to COPD between 2013 and 2017, respectively , a reduction of 74.99%  in stroke mortality, a reduction of 72.80% (966 deaths) in COPD mortality, and a reduction of 49.87%  in IHD mortality.Furthermore, in the future scenario of PM2.5 reduced to 15 μg/m3).More than 40% of the morbidities due to RHA, CHA, CB, AA, and REV with an overall of 15,822  cases reduced between 2013 and 2017. Most importantly, nearly 95% of these morbidities could be avoided in the future scenario (PM2.5 concentration from 2013 (110 μg/m3) to 2017 (63 μg/m3). The overall US$ 317.7 million (95%CI: 227.5–458.1) of health economic benefits accounted for 0.29% (95%CI: 0.21–0.42%) of local GDP in 2017.The related economic benefit from the reduction in mortality and morbidity was presented in Table 2.5 down to 15 μg/m3, a total of US$ 1,289.5 million  saved from the reduced mortality  and reduced morbidity  could be received, accounting for 1.19% (95%CI: 0.76–1.54%) of GDP in Jinan in 2017.In the future scenario of PM2.5, avoided a total of 2,317 premature deaths and 15,822 cases of related diseases, and saved US$ 317.7 million during the study period.This is the first study to systematically examine local air pollution measures and quantify environmental, health and economic co-benefits at a city level in China. Our study has analyzed the air pollution control measures and associated co-benefits in a heavily air-polluted city of China between 2013 and 2017, which provides an insight on what and how a single-city could achieve co-benefits of national and local air pollution measures. We have found significant reductions in ambient PM16. Coal consumption, for example, became the largest atmospheric pollution source and was identified as the major energy supply in Chinese cities43. It is clear that coal production, supply and consumption were the core target control factors to the national actions43. It was estimated that in all types of air pollution sources in China, about 87% of SO2 and 67% of NOx were emitted from coal combustion44. Our study has observed dramatic changes in coal consumption and reduction in exhaust emissions in Jinan between 2013 and 2017. The percentage of decrease in SO2 emissions and NOx emissions from industry was larger in Jinan compared with other heavily air polluted northern cities over the same period, such as Shijiazhuang , Haerbin 35. In early 2018, Jinan government further issued “the Jinan Working Program to Reduce Coal Consumption from 2018 to 2020”, which required coal consumption reductions from households45. Our study proves that these measures in Jinan are effective in controlling air pollution.It is important to develop effective air pollution control policies to reduce the production and emissions and to improve public health14. Compared with national studies, the achieved reduction of air pollution in Jinan is above the national average. The 25% reduction in PM2.5 in Jinan has met the Ten Specific Tasks of APPCAP13. The WHO 2018 report on global air pollution indicates that about 91% population live in areas with an annual average PM2.5 > 10 ug/m310. We have found that nearly 7 million people exposed to severe air pollution with ambient PM2.5 > 100 ug/m3 in Jinan in 2013. Due to air pollution controls and implementation, all ten districts of Jinan have achieved consistent reduction in ambient PM2.5 concentration by 2017, with more benefits in urban areas. However, the annual concentration of PM2.5 in all districts of Jinan is still significantly higher than the national ambient air quality standards Grade-I (35 ug/m3)25, which indicates further air pollution policies and measures would be necessary for Jinan. We have also noted the significant increase in O3 during study period, which is consistent with other national studies14. It indicates that O3 and other air pollutants still pose an ongoing challenge to the health of people in Jinan and other similar cities in China.Previous national studies have shown improved air quality in China due to the APPCAP10. Our results indicate that premature deaths attributed to PM2.5 exposure were three times larger than that at national average level16. After the implementation of the air pollution controls, Jinan has achieved a 50% reduction in PM2.5 and saved a total of 18.4% premature deaths in 2017. It is worthy to note that appreciable health benefits could be reduced if PM2.5 further reduces to 15 μg/m3. Additionally, significant decrease in morbidity can be achieved as suggested in our historical and scenario-based modeling analyses. Among the five cause-specific diseases, acute bronchitis and asthma attacks affected most people, which generally agreed with previous studies15.WHO recently reported that the ambient air pollution was the leading cause of the mortality due to the chronic diseases2.5 exposure could lead to tremendous economic burdens16. Our results support this statement and indicate a reduction in GDP between 2013 and 2017. Our finding is consistent with previous study in the central area of China16. The average economic loss ranged from 0.3% to 1.0% of the total GDP of 190 Chinese cities from 2014 to 201616. Our estimation demonstrates higher economic burdens than that at the national level because of the heavy air pollution in Jinan16. Another reason is that our estimation of the economic burdens included both mortality and morbidity. Not surprising, Jinan has achieved more economic benefits due to improvement in air quality and lives saved. We have also reported more economic burden due to premature deaths (94.0% in 2013 and 95.6% in 2017), which is similar to the finding of Yin et al.38 who reported more than 80% of the overall external costs due to premature deaths in Beijing. The findings imply that we need to pay more attention to primary health care to prevent premature deaths from air pollution.Adverse health outcomes attributed to PM2.5 down to 15 μg/m3, some other factors that change with time, e.g. demographic data, socio-economic situation and climate change adaptation, could not be included in the analysis. Finally, the emission reduction estimated is not directly linked with policies because the secondary chemical formation also contributed an important fraction.Limitations of the study should be acknowledged. First, due to data unavailability, the usage of national morbidity data as a proxy of Jinan population was not ideal. Second, the air monitoring stations did not cover the rural areas of Jinan before 2016, which could affect the spatial analysis of air pollution in Jinan before 2016. Last, in the projection of the benefits under the future scenario of PMOur study provides evidence of the effectiveness and co-benefits of local air pollution control measures in the study city of China. Findings suggest that government efforts to improve air quality will lead to significant environmental, health and economic benefits. Current policies should continue to achieve better air quality standards in order to prevent more premature deaths, morbidities and economic losses. Lessons learned from the study city could be applicable to other cities and regions with severe air pollution in China and globally.Supplemental files.


ClinicalTrials.gov were used to search for relevant studies from their inception to 30 October 2020. Finally, a total of six articles were included in this meta-analysis. Review Manager 5 software was applied to examine the heterogeneity among the studies and to calculate the odds ratio with 95% CI by selecting corresponding models, in evaluating the strength of the relationship. Publication bias test was also conducted. No bias and no significant correlations were found between CLDN 18.2 and TNM stages, Lauren classification, HER2, grading, or overall survival. This meta-analysis expounded that the relationship with CLDN 18.2 and pathological features depends on the percentage of staining of tumor cells for which CLDN 18.2 is considered positive. Our pooled outcomes suggest that targeted therapy for CLDN 18.2 could be effective if certain criteria were established.An increasing number of tumor markers have been discovered to have potential efficacy as diagnostic and prognostic tools in gastric cancer. We aimed to assess putative correlations between claudin 18.2 expression and pathological or prognosis features in patients with gastric cancer. MEDLINE, Web of Science, EBSCO, and  Gastric cancer (GC) is one of the most commonly diagnosed malignancies worldwide and the second cause of cancer-related death. Despite the variability of GC incidence and mortality, an estimated 1,033,701 new stomach cancers and 782,685 deaths occurred in 2018Targeted agents approved for GC like trastuzumab (anti-HER2) or ramucirumab (anti-VEGF receptor) have shortcomings such as modest survival benefits and second resistance development. New suitable biomarkers that can serve as targets have to be found for highly effective targeted therapies for GC .Claudins are a family of at minimum 27 proteins with roles in maintaining the intercellular tight junction adhesion, which create a paracellular barrier. The impossibility of these molecules to accomplish their function is linked with tumor development and progression . DiffereIMAB362 (known as zolbetuximab or claudiximab), a novel chimeric immunoglobulin G1 antibody, is the first type of ideal monoclonal antibodies (IMAB) used for the treatment of GC. After IMAB362 binds to CLDN 18.2, immune effectors activate antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. This change induces apoptosis and promotes the inhibition of cell proliferation, with beneficial effects for patients .Our objective was to assess all available studies that involve CLDN 18.2 expression in GC and its relation to clinicopathological or prognosis features in patients with GC, in order to offer more insights on its potential as a target in future clinical trials.We used the PICOS  model and PRISMA guidelines to design our search strategy .ClinicalTrials.gov (inception to 30 October 2020) to see if they evaluated the expression of CLDN 18.2 in order to find correlations with clinicopathological patient characteristics with GC. We studied reference lists as well as published systematic review articles. The search terms included (“claudin 18.2” AND “gastric cancer”) OR (“claudin18.2” AND “gastric cancer”).To identify studies, we searched the following databases: MEDLINE, Web of Science, EBSCO, and Studies evaluating the expression of CLDN 18.2 in adults with GC were included in our meta-analysis. The inclusion criteria for selection were: (1) clear definition of scoring for CLDN 18.2 staining; (2) assessment of clinicopathological patient characteristics; (3) histologically confirmed adenocarcinoma of the stomach. Exclusion criteria were: (1) tumor types other than adenocarcinoma; (2) patients who had undergone a perioperative or neoadjuvant chemo- or radiotherapy; (3) studies as case reports, systematic reviews, abstracts.Two review authors (BSU and VMS) independently extracted all data using a standardized data extraction table. Any disagreements regarding eligible articles were resolved after consulting a third review author (AT-S). The risk of bias was assessed through a funnel plot.2 statistic, which gave us the proportion of the observed variance that reflects real differences in effect size, for quantifying heterogeneity of the results in individual studies, which combined the Chi2 statistic and the number of studies contributing to each summary estimate in the forest plot . We used both the random-effects model and the fixed-effects model based on the assessment of heterogeneity, when the inverse-variance approach was implemented. We used the Iest plot .P-values <0.05 and 95% confidence intervals (CI) that did not include 1 to be statistically significant.We used odds ratio (OR) as the effect measure for dichotomous outcomes, that is the number of participants achieving TNM clinical stage, HER2, Lauren classification, and grading. Analysis and comparisons for all outcomes were performed where data were available. We considered Time-to-event data for overall-survival (OS) were analyzed using hazard ratio (HR), which was estimated using the calculation methods described by Tierney et al. . If thesTo assure our results were robust, the presence of any publication bias was analyzed with a funnel plot, based on the visual inspection of the symmetry.The characteristics of the included studies are provided in We conducted the following analysis using the standard meta-analysis to find correlations between CLDN 18.2 and pathological features and prognosis of GC patients. Two subgroups of studies were analyzed according to the definition of CLDN 18.2's positivity and the outcomes were assessed where data were available. The two subgroups were: A (positivity was defined as CLDN 18.2 staining intensity was present in any percentage of tumor cells) and B (positivity was defined as CLDN 18.2 staining intensity was present in more than 40% of tumor cells).p = 0.12) to indicate correlation between CLDN 18.2 expression and T1 + T2 vs. T3 + T4 clinical stage, with an OR of 0.83 (95% CI 0.66–1.05). The fixed-effect model was used with an I2 of 11% (p = 0.34) indicating no heterogeneity.The results are illustrated in the forest plots in p = 0.28) to indicate correlation between CLDN 18.2 expression and T1 + T2 vs. T3 + T4 clinical stage, with an OR of 1.26 (95% CI 0.83–1.91). The fixed-effect model was used with an I2 of 0% (p = 0.42) indicating no heterogeneity.If the samples were defined as CLDN 18.2-positive showing specific staining with more than 40% of tumor cells, there was no evidence ] nor for subgroup B . We used a random-effect model for the A subgroup with an I2 of 93% (p < 0.00001) and a fixed-effect model for the B subgroup with an I2 of 65% (p = 0.09). The high heterogeneity of the A subgroup (Chi2 = 54.88) was not observed in subgroup B (Chi2 = 2.83).As demonstrated in p < 0.05 (p = 0.89) proved no correlation between CLDN 18.2 expression and the M clinical stage. The fixed-effect model was used for no heterogeneity of the two studies included in this meta-analysis . The overall effect OR was close to 1 as shown in The lack of statistical significance at p = 0.80).There were no significant differences between CLDN 18.2 positive and CLDN 18.2 negative GC patients with respect to HER2 statuses, as showed in I2 = 76%, p = 0.002). The overall effect OR was 1.12 (95% 0.47–2.63).A random-effect model was used for moderate heterogeneity of the five studies included in this meta-analysis (p = 0.74) to indicate correlation between CLDN 18.2 expression and diffuse vs. other Lauren classifications, with an OR of 0.91 (95% CI 0.54–1.56), as shown in 2 of 73% (p = 0.005) indicating moderate heterogeneity.If the samples were defined as CLDN 18.2-positive showing specific staining with any fraction of tumor cells (>5 or >10%), there was no evidence (p = 0.76) to indicate correlation between CLDN 18.2 expression and diffuse vs. other Lauren classifications, with an OR of 1.24 (95% CI 0.31–4.95). A random-effect model was used with an I2 of 88% (p = 0.004) indicating high heterogeneity.If the samples were defined as CLDN 18.2-positive showing specific staining with more than 40% of tumor cells, there was no evidence  to indicate correlation between CLDN 18.2 expression and intestinal vs. other Lauren classifications, with an OR of 0.83 (95% CI 0.51–2.47). A random-effect model was used with an I2 of 74% (p = 0.004) indicating moderate heterogeneity, as shown in In the subgroup of studies where positive CLDN 18.2 was defined as more than 5% staining of tumor cells, there was no evidence (p = 0.78) to indicate correlation between CLDN 18.2 expression and intestinal vs. other Lauren classifications, with an OR of 0.86 (95% CI 0.30–2.47). A random-effect model was used with an I2 of 79% (p = 0.03) indicating moderate heterogeneity.In the subgroup of studies where positive CLDN 18.2 was defined as more than 40% staining of tumor cells, there was no evidence .There were no significant differences between CLDN 18.2-positive and CLDN 18.2-negative GC patients with respect to grading, as I2 = 62%, p = 0.07).As for the grading, we found that CLDN 18.2 expression was almost the same in GC tumors with G1/G2 when compared with G3/G4 . The fixed-effect model was used for no heterogeneity between the three studies included in this meta-analysis (I2 = 0% and p = 0.99). No significant difference in OS was found between CLDN 18.2-positive and CLDN 18.2-negative: HR = 1.01 (95%CI 0.69–1.48), p = 0.95, as Three studies were included in the meta-analysis of assessing the hazard ratio (HR) for overall survival (OS) for patients who were CLDN 18.2-positive vs. CLDN 18.2-negative. The fixed-effect model was used . This new targeted therapy is validated in preclinical studies, and several phase I and II trials are underway with positive results published so far. The FAST study (NCT01630083)  showed tOur meta-analysis reveals that there is no significant correlation between CLDN 18.2 tissue expression and clinicopathologic features. None of the available studies showed any correlation with the TNM stage, however, in T3 + T4 we emphasized a more abundant expression than for T1 + T2, if the positivity of CLDN 18.2 was defined through a higher percentage of stained tumor cells. This was similar for the N stage showing that along with an increased positivity, no correlation was observed .While our results did not show any positive correlation with Lauren classification, Coati et al. observed that higher prevalence of CLDN 18 had a diffuse type. They also found that higher expression was found in the corpus than the antrum .Regarding the HER2+ status, CLDN 18.2 staining did not correlate with it, even though one study suggested higher expression rates for HER2+  statuses . On the in vitro diagnostics) approved test is the CLAUDETECT 18.2 Kit . The CLAUDETECT 18.2 Kit was introduced for in vitro diagnosis of expression level assessment. This immunohistochemical assay which recognizes the C terminus of claudin 18 is not specific for the isoform 18.2. However, the Anti-CLDN EPR19202 kit  is specific for a synthetic peptide within human claudin 18.2 amino acids 1-100, thus it can only detect this isoform. CLDN 18.2 histopathological staining status is important because it will validate patients for future therapy.Due to the heterogeneity of studies some questions should be raised. First, there is a need for uniformity when differentiating CLDN 18.2 from other variants, currently the only IVD  will result in better efficacy (longer OS) . On the Ethnicity represents a main factor in GC response to therapy. The percentage of positive patients varied in both European and Asian countries. While two studies from Germany showed rather similar results with 17 and 18%, Dottermuch et al.  had 42% This is the first meta-analysis on CLDN 18.2 and its expression on GC patients. Even though it may represent a new addition for current therapies, our results show a low prevalence with 34.2% in 2,055 patients. The data so far suggest that targeted therapy for CLDN 18.2 could be effective if certain criteria will be established. Clinical trials might help providing more data about the expression of CLD18.2 in assessing claudiximab productivity.Our results suggest that a new cut-off value for CLDN 18.2 positivity should be taken into account, and that computer generated analysis might be an option for further studies, as it may provide more accurate results. This was actually discussed by clinical trials which achieved better efficacy if higher expression levels were taken into account. Perhaps selecting only patients with high intensity levels and correlated with clinicopathologic data could provide more candidates to establish the therapy candidates.Our study has some limitations due to the small number of included studies, but it pooled the outcomes for a large number of patients with international findings, recruiting both Caucasians and Asians.Even though our results did not show any correlation between CLDN 18.2 staining and the patient's clinicopathologic features, we believe that more specific assays for staining and quantification, as well as a cut-off value for CLDN 18.2 level, might help solve this issue. Hopefully the available trials will shed more light on this new targeted therapy much needed for GC treatment.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.BSU and DIG: conceptualization. BSU and AT-S: methodology. AT-S: software. C-VL, DP, IML, EM, and FB: formal analysis. BSU and VMS: data curation. BSU and AT-S: writing—original draft preparation. DP, DIG, and AS: writing-review and editing. All authors contributed to the article and approved the submitted version.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.


During lockdown, daily contacts decreased by 86.9% and R0 decreased by 81.0% ; each distancing measure decreased R0 by 10%–24%. By April 26, the attack rate in Greece was 0.12% (95% CrI 0.06%–0.26%), one of the lowest in Europe, and the infection fatality ratio was 1.12% (95% CrI 0.55%–2.31%). Multiple social distancing measures contained the first epidemic wave in Greece.Greece imposed a nationwide lockdown in March 2020 to mitigate transmission of severe acute respiratory syndrome coronavirus 2 during the first epidemic wave. We conducted a survey on age-specific social contact patterns to assess effects of physical distancing measures and used a susceptible-exposed-infectious-recovered model to simulate the epidemic. Because multiple distancing measures were implemented simultaneously, we assessed their overall effects and the contribution of each measure. Before measures were implemented, the estimated basic reproduction number (R Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in China in December 2019 .We estimated the average number of contacts for the prepandemic and lockdown periods. We defined 6 age groups to build age-specific contact matrices, adjusting for the age distribution of the population of Greece, by using socialmixr in R software , the average number of secondary cases 1 case would produce in a completely susceptible population in the absence of control measures. Then, we used social contacts matrices to assess the effects of physical distancing measures on R0. Finally, we simulated the course of the epidemic using a susceptible-exposed-infectious-recovered (SEIR) model.To estimate the course of the epidemic, we first estimated the basic reproduction number . As a sensitivity analysis, we estimated R0 assuming a shorter serial interval of 4.7 days (We estimated R4.7 days  (12).Primary social distancing measures implemented in Greece began on March 11. These measures and the dates implemented were closing all educational establishments on March 11; theatres, courthouses, cinemas, gyms, playgrounds, and nightclubs on March 13; shopping centers, cafes, restaurants, bars, museums, and archaeological sites on March 14; suspending services in churches on March 16; closing all private enterprises, with some exceptions, on March 18; and, finally, restricting all nonessential movement throughout the country on March 23  Table 1.0 through the social contact matrices obtained before and during lockdown, as used in other studies .s of age  Table 2 0 in 2 periods: the period of initial measures until the day before lockdown (March 11–22), which included closure of schools, entertainment venues, and shops (reduction δ1); and the period of lockdown (March 23–April 26) (reduction δ2). Because we did not assess social contacts during the period of initial measures, we created a synthetic contact matrix by assuming no school contacts because of school closures, and a reduction in leisure and work contacts .We estimated the relative reduction in Rcontacts . To assep will eventually develop symptoms (p = 80%) (pre). Ipre cases become symptomatic infectious (Isymp) cases at a rate of σs. We assumed that infectiousness can occur 1.5 days before the onset of symptoms (1 – p) will be true asymptomatic or subclinical cases (Iasymp). We assumed that the infectiousness of subclinical cases relative to symptomatic cases was q = 50%  at a rate of γasymp  become infected at a rate β and move to the exposed state (E) as infected but not infectious. Exposed persons become infectious at a rate σ, and a proportion f γasymp .0 and parameters related to the duration of infectiousness . We modeled the effect of measures by multiplying β by the parameters δ1 and δ2; in which δ1 corresponds to the reduction of R0 in the period of initial social distancing measures, where δ1 was drawn from a normal distribution with a mean of 42.7% (SD 1.7%); and δ2 corresponds to the reduction of R0 during lockdown, for which δ2 was drawn from a normal distribution of 81.0% (SD 1.6%) estimated from the bootstrap on the contact data. To account for the uncertainty in R0, δ1, and δ2, we performed 1,000 simulations of the model and obtained median estimates and 95% CrIs.We derived the transmission rate β from Riousness . We incoWe obtained the infection fatality ratio (IFR) and the cumulative proportion of critically ill patients by dividing the reported number of deaths and of critically ill patients  Table 3.0, we used information from the contacts reported on a regular weekday in January 2020 and mimicked the impact of each intervention by excluding or reducing subsets of corresponding social contacts (t) for varying levels of infection control measures  when social distancing measures are partially lifted after lockdown . During the first period of social distancing measures, in which schools, entertainment venues, and shops were closed, R0 was estimated to decrease by 42.7% (95% CrI 34.9%–51.3%); under lockdown, R0 decreased by 81.0% (95% CrI 71.7%–86.1%). Thus, the cumulative measures implemented during lockdown would have reduced R0 to <1.0 even if the initial R0 had been as high as 5.3 (95% CrI 3.5–7.2). Estimated Rt was 1.13 (95% CrI 1.38–1.61) during the period of the initial measures but was 0.46 (95% CrI 0.35–0.57) during lockdown  for the decline in work contacts, 18.5% (95% CrI 10.7%–26.3%) for school closures, and 24.1% (95% CrI 14.8%–34.3%) for the decline in leisure activity contacts. Thus, each measure separately would have reduced R0 to <1.0 if the initial R0 had been as high as 1.11 for the decline in work contacts, 1.23 for school closures, and 1.32 for the decline in leisure activity contacts. A combination of measures could be effective if the initial R0 had been as high as 1.78 for interventions reducing work and school contacts, 1.72 for reducing work and leisure contacts, and 1.43 for reducing school and leisure contacts.We assessed the effect of each measure separately and in combinations . During 0 to <1.0 for initial levels as high as 1.32 (95% CrI 1.27–1.38). An even larger decline in leisure activities (50%) could successfully reduce an initial R0 as high as 1.48 (95% CrI 1.35–1.62). We assessed alternative scenarios with less disruptive social distancing measures. A 50% reduction in school contacts, such as smaller class sizes; 20% in work contacts, such as teleworking for part of the population or rotating weekly schedules in which employees telework some days and work onsite other days; and 20% in leisure activities could reduce Rt when measures were partially lifted after lockdown. To mimic the measures implemented after lockdown in Greece, we assumed that contacts at work would return to levels 50% lower than pre-pandemic, school to 50%, and leisure to 60%. For instance, class sizes were reduced 50% when schools reopened in May. Under this scenario, Rt would remain <1.0 assuming >20% reduction in susceptibility as a result of infection control measures, including hand hygiene, use of face masks, and maintaining physical distances >1.5 meters . ThOn the basis of the number of deaths and critically ill patients reported in Greece by April 26, and using the number of infections obtained from the model as denominator, we estimated the IFR to be 1.12% (95% CrI 0.55%–2.31%) and the cumulative proportion of critically ill patients to be 1.55% (95% CrI 0.75%–3.22%). As a validation, we estimated the number of deaths by applying a published age-adjusted estimated IFR to the number of infections predicted by the model  Table 3.Greece and other countries managed to successfully slow the first wave of the SARS-CoV-2 epidemic early in 2020. Assessing the burden of infection and death in the population and quantifying the effects of social distancing was necessary because the stringent measures taken had major economic costs and restricted individual freedom. In addition, several countries, including Greece, began seeing COVID-19 cases increase after resuming economic activities and travel, indicating the need to reimplement some types of location-specific physical distancing measures.We assessed the effects of social distancing by using a social contacts survey to directly measure participants’ contact patterns during lockdown in a sample including children. To our knowledge, only 2 other diary-based social contacts surveys have been implemented during COVID-19 lockdown, 1 in China .We estimated that R0 of ≈1.1–1.3 to <1.0. Only multiple social distancing measures would be effective for reducing an R0 at the initial level (2.38) observed in Greece. The finding concerning an 18.5% reduction in R0 related to school closures agrees with recent studies suggesting that this measure likely is much less effective for COVID-19 than for influenza-like infections . The estimated R0 aligned with estimates obtained in China (The first limitation of our study was that, due to the absence of prepandemic data on social contacts, we asked respondents to report their contacts ≈2 months prior to the survey to ensure reports were not affected by increased awareness of the pandemic. Recall bias might be observed, although to what direction is not clear. A general limitation in contact diaries is that participants record a fraction of their contacts (0 to <1.0. By the end of April, the spread of COVID-19 was contained in Greece, and the country had one of the lowest ARs in Europe after the first pandemic wave. However, as social distancing and travel restrictions are relaxed, close monitoring of Rt is essential in order to adapt interventions over time without having to resort to stringent measures. Measuring social mixing patterns and adherence to infection control measures through repeated surveys can be additional tools for real-time monitoring of the epidemic potential in the months to come.Overall, the social distancing measures Greece put in place in early March 2020 had a substantial impact on contact patterns and reduced RAdditional information and formulas used to calculate effects of social distancing measures during the first epidemic wave of severe acute respiratory syndrome coronavirus 2, Greece.


The increasing application of immune checkpoint inhibitors (ICIs) will cause more checkpoint inhibitor‐related pneumonitis (CIP), which is a common cause of ICI‐related death. The clinical management of CIP needs further optimization.Patients who were managed at Peking Union Medical College Hospital (PUMCH) between February 2017 and December 2019 with a diagnosis of CIP were retrospectively analyzed. Clinical data including clinical manifestations, radiologic data, laboratory and bronchoscopy results, treatments, and outcomes were collected and analyzed. The Mann–Whitney test was used to compare patients with and without co‐infections.p = 0.001) were associated with opportunistic infection. Chest computed tomography (CT) showed diverse and asymmetrical lesions. Twelve patients were re‐challenged, and six patients developed recurrent CIP.In total, 48 CIP cases in 42 patients were analyzed. The median time from the first dose of ICI to the onset of CIP was 1.9 months (range: 0.1–13.7). Grade 3–4 (G3–4) accounted for 30 cases (71.4%). The most common symptoms were cough (88.1%) and dyspnea (78.6%). The median starting dose of equivalent prednisone (EP) was 55 mg (range: 30–200) for all patients. The median total duration of glucocorticosteroids (GCS) treatment was 42.5 days (range: 15−89). Three patients (7.14%) died because of infection. A higher starting dose and longer duration of GCS (≥30 mg/day; The clinical and imaging manifestations of CIP are various, and differential diagnosis of exclusion is essential. GCS at 1–2 mg/kg is feasible to treat CIP, but the duration of GCS ≥30 mg/day should be used with caution, given the high risk of acquired infections. Re‐challenges of ICI are feasible, but the recurrence of CIP needs to be closely monitored. This study revealed for the first time the clinical characteristics, diagnosis, and treatment of CIPs in Chinese cancer patients. The recommended starting dose of 1–2 mg/kg GCS is feasible, but the duration of GCS ≥30 mg/day should be controlled, as acquired infections during the GCS treatment rather than refractory CIP could be the main cause of CIP‐related deaths. We conducted a detailed review and analysis of their clinical progress in an attempt to explore the optimal treatment for CIP.22.1This retrospective analysis was conducted on the patients who were managed in PUMCH due to pulmonary inflammatory lesions after ICI therapy between February 2017 and December 2019; patients who were ultimately diagnosed as CIP were included.The inclusion criteria included: patients who were pathologically diagnosed with locally advanced/advanced cancer; patients who were treated by ICI ; and patients who developed new pulmonary inflammation lesions after immunotherapy and were ultimately diagnosed as CIP after evaluation by a multidisciplinary team. The exclusion criteria included: un‐blinded patients in RCT clinical trials for whom it could not be determine whether ICI had been used; patients with other lung diseases with clear etiology; and patients whose data was incomplete or lost to follow‐up. Complete medical records of all included patients were collected.2.2The severity of CIP was defined according to the Common Terminology Criteria for Adverse Events, version 4.03.Chest CT images obtained at the time of pneumonitis diagnosis were reviewed by a consensus of radiologists with expertise in thoracic and oncologic imaging. CT findings of pneumonitis were evaluated for distributions and specific CT findings including traction bronchiectasis, consolidation, reticular opacities, ground‐glass opacities (GGO), centrilobular nodularity, and interlobular septa thickening. In each case, radiographic patterns of pneumonitis were compared with the classification of interstitial pneumonias according to the ATS/ERS international multidisciplinary approach.2.3n (%) for categorical variables and as median (range) for continuous variables. Progression‐free survival (PFS) was estimated using the Kaplan–Meier method with 95% confidence intervals. The Mann–Whitney test was used to compare treatments between patients with and without co‐infections. All reported p‐values were two‐sided. For all tests, a statistical difference was considered significant at the 5% level. Statistical analyses were conducted using SPSS Statistics for Windows .Time to the onset of CIP was defined as the time from the first dose of ICI to the first occurrence of CIP‐related symptoms or imaging findings of asymptomatic patients. The data are expressed as 33.1There were 60 suspected cases of CIP in 54 patients treated with ICI. After review, a total of 48 CIP cases in 42 patients  were admitted for analysis Figure . ExclusiThe general characteristics of these 42 patients are shown in Table ICI was used as first‐line treatment in 24 (57.1%) patients, as maintenance therapy after concurrent chemoradiotherapy in 2 (4.8%), as second‐line in 11 (26.2%), as third‐line and above in 4 (9.5%), and as adjuvant therapy in 1 (1.7%). Forty (95.2%) patients received PD‐1 inhibitors, and two received PD‐L1 inhibitor. The doses of ICIs are all according to the dosage recommended in their drug instructions.3.2The median time from the first dose of ICI to the onset of CIP was 1.9 months, with a wide range of 0.1–13.7 months  and shortness of breath/dyspnea . Seventeen (40.5%) patients had mild to moderate fever. Other symptoms included sputum , bloody sputum , and chest pain ; one patient was asymptomatic. Additionally, six (14.3%) patients experienced additional thyroiditis, and one patient showed positive in ANA, anti‐Ro52 antibody, and anti‐SSA antibody after ICI treatment. No other additional irAE was observed.3.39/L) in 29.6% (8/27) of patients. 92% (23/25) of patients had increased levels of hypersensitive C‐reactive protein (hsCRP), and 91.7% (22/24) showed elevated ESR. The levels of IL‐6, and TNFa were increased in 57.9% (11/19), and 70.6% (12/17) of patients, respectively. The levels of IL‐8 and IL‐10 increased in fewer patients (15.8% (3/19) and 26.3% (5/19), respectively).Routine blood tests showed increased total white blood cells in 37.0% (10/27) of patients and increased neutrophils in 48.1% (13/27) patients, while decreased lymphocyte counts (lowest: 0.3 × 103.4The basic lesions of patients with untreated CIP on chest CT included (GGO) in 76.2% (32/42) of patients and consolidation in 54.8% (23/42) of patients. Traction bronchiectasis was found in 33.3% (14/42) of patients, reticular opacities in 28.6% (12/42), centrilobular nodularity in 11.9% (5/42), and interlobular septa thickening in 9.5% (4/42).Inflammation lesions on chest CT showed an asymmetrical distribution in 64.3% (27/42) of patients, and the remaining 35.7% (15/42) showed a symmetrical distribution. Among the 27 patients with asymmetric lesions, 11 (40.7%) had their inflammatory lesions completely surrounding the tumor lesions, while the remaining 16 showed no relation to tumor lesions.According to the classification of Idiopathic Interstitial Pneumonias imaging patterns, 31.0% 13/42) met the pattern of COP, 14.3% (6/42) met NSIP, 4.8% (2/42) met ARDS/DAD, 4.8% (2/36) met HP, and 42.9% (18/42) were non‐specified. Representative chest CT manifestations are shown in Figure  met the 3.5Bronchoscopy was performed in 12 patients either before glucocorticosteroids (GCS) treatment (10 patients) or after worsening during GCS treatment (2 patients). A complete description of bronchoscopy results is shown in Table +/CD8+ , which indicated a predominant increase of CD8+ T lymphocytes. In two patients whose symptoms worsened after GCS treatment, bronchoscopy indicated infection, as both of their microbiological evaluations were positive for CMV‐DNA and PCP‐DNA.Bronchoalveolar lavage fluid (BALF) was detected in nine patients before GCS treatment and all cases showed T‐lymphocytic alveolitis. The median percentage of lymphocytes was 52% (range: 11%–95.5%), with a decreased CD43.6All patients had stopped ICI treatment definitively. The detailed GCS therapy for each of different grades of CIP is shown in Table p = 0.016). The duration of GCS ≥30 mg was also longer   were totally or partially recovered after GCS withdrawal. However, there were three (7.1%) patients who died of acquired opportunistic infections (Table 9/42 were) Figure .3.7Twelve patients were re‐challenged after recovery from initial CIP . Six patients developed recurrent CIP; the comparison of initial and recurrent CIP is shown in Table 3.8Regardless of the type and specific regimen of ICI, among these 42 patients with CIP, the objective response rate (ORR) of ICI was 47.2% (17/36) and the disease control rate (DCR) was 91.7% (33/36), with six patients un‐evaluable. Twenty‐two patients have not progressed to date. The median PFS was 9.7 months (95% CI: 4.8–14.6) among the 34 patients with NSCLC (16 patients have not progressed yet).4Naidoo et al. first described the clinical, radiologic, and pathologic features of CIP based on data from Memorial Sloan Kettering Cancer Center and The Melanoma Institute of Australia.A CIP diagnosis is not always straightforward, as the symptoms and radiological appearances are not specific for CIP.CIP can occur at any time after the initiation of ICI treatment. The median onset time of CIP in our cohort was 1.8 months, while the latest onset time was 13.7 months. A whole course monitoring for CIP is needed. In terms of severity, CIP of G3–4 accounted for 71.4% of these cases, and only 1 G1 CIP was included in our cohort, as most patients came to our clinic because of definitive symptoms who need treatment. Oncologist need to do more work on the early diagnosis of CIPs.Cough and shortness of breath  were the most common symptom, and fever occurred in 40.5% of patients. Inflammatory factors such as hsCRP and ESR were highly sensitive for inflammatory diseases that were either infectious or non‐infectious. White blood cell and neutrophil counts can also rise in CIP. The lymphocytes in blood usually showed normal or decreased numbers. Other inflammatory factors such as IL‐6 and TNFa can also indicate the inflammatory nature of CIP.According to the classification criteria of IIP, CIP was first classified into five subtypes according to radiological manifestations: cryptogenic organizing pneumonia (COP)‐like, GGO, interstitial, hypersensitivity, and pneumonitis not otherwise specified.Pneumocystis jiroveci pneumonia therapy should be given. For patients who are relieved after GCS therapy but became worse later, infection should be highly suspected first. In addition to finding lymphocytic alveolitis, bronchoscopy can also yield accurate etiological results in patients with suspected infection.GCS is the basic therapy for CIP. However, the exact details for GCS administration are unclear, especially in terms of initial dose, the tapering process, and the overall course.Re‐challenging is feasible in some patients, but there is still a chance of irAE recurrence.The main limitation of this study is the nature of the retrospective single‐center study itself. The sample size is relatively small. However, as all the patients came from a single center, we have very detailed information about the diagnosis and treatment of every case of CIP, and their management was relatively uniform, so the results in term of treatment and outcomes are more reliable than multicenter retrospective studies.In conclusion, our retrospective study revealed for the first time the clinical characteristics, diagnosis, and treatment of CIPs in Chinese cancer patients. CIP has unique imaging manifestations, and its asymmetry should be noted. The recommended starting dose of 1–2 mg/kg GCS is feasible, but the duration of GCS ≥30 mg/day should be controlled, as acquired infections during the GCS treatment rather than refractory CIP could be the main cause of CIP‐related deaths. Re‐challenges of ICI are feasible, but the recurrence of CIP needs to be closely monitored.The authors declare that they have no potential conflicts of interest, financial interests, relationships, or affiliations relevant to the subject of their manuscript.Hanping Wang, Juhong Shi, Haitao Zhao, and Li Zhang contributed to conception and design. Hanping Wang, Yanwei Zhao, Xiaoyan Si, Xiaotong Zhang, Peng Song, Yi Xiao, Xu Yang, Juhong Shi, Haitao Zhao, and Li Zhang contributed to provision of study materials or patients. Hanping Wang, Yanwei Zhao, Xiaoyan Si, Xiaotong Zhang, Peng Song, Yi Xiao, Lan Song, Xu Yang, Juhong Shi, Haitao Zhao, and Li Zhang contributed to collection and assembly of data. Hanping Wang, Yanwei Zhao, Peng Song, Lan Song, Xu Yang, Juhong Shi, Haitao Zhao, and Li Zhang contributed to data analysis and interpretation. All authors wrote the manuscript and approved the final manuscript.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Table S1Click here for additional data file.


There are limited contemporary data on the influence of primary payer status on the management and outcomes of ST-segment elevation myocardial infarction (STEMI).To assess the influence of insurance status on STEMI outcomes.Adult (>18 years) STEMI admissions were identified using the National Inpatient Sample database (2000–2017). Expected primary payer was classified into Medicare, Medicaid, private, uninsured and others. Outcomes of interest included in-hospital mortality, use of coronary angiography and percutaneous coronary intervention (PCI), hospitalization costs, hospital length of stay and discharge disposition.p<0.001). The Medicare and Medicaid population had higher rates of cardiogenic shock and cardiac arrest. The Medicare cohort had higher in-hospital mortality (14.2%) compared to the other groups (4.1–6.7%), p<0.001. In a multivariable analysis (Medicare referent), in-hospital mortality was higher in uninsured (adjusted odds ratio (aOR) 1.14 [95% confidence interval {CI} 1.11–1.16]), and lower in Medicaid , privately insured (aOR 0.73 [95% CI 0.72–0.75]) and other insurance (aOR 0.91 [95% CI 0.88–0.94]); all p<0.001. Coronary angiography (60% vs. 77–82%) and PCI (45% vs. 63–70%) were used less frequently in the Medicare population compared to others. The Medicare and Medicaid populations had longer lengths of hospital stay, and the Medicare population had the lowest hospitalization costs and fewer discharges to home.Of the 4,310,703 STEMI admissions, Medicare, Medicaid, private, uninsured and other insurances were noted in 49.0%, 6.3%, 34.4%, 7.2% and 3.1%, respectively. Compared to the others, the Medicare cohort was older (75 vs. 53–57 years), more often female (46% vs. 20–36%), of white race, and with higher comorbidity (all Compared to other types of primary payers, STEMI admissions with Medicare insurance had lower use of coronary angiography and PCI, and higher in-hospital mortality. Ischemic heart disease and specifically acute myocardial infarction continue to be leading cause of cardiovascular admissions in the United States –4. ST-seIn the United States, prior studies in cardiac and non-cardiac patient populations have demonstrated that insurance status may have an association with clinical outcomes –9. In paIn the current era, it is unclear if insurance status continues to play a role in the management and outcomes of STEMI . Using aThe National (Nationwide) Inpatient Sample (NIS) is the largest all-payer database of hospital inpatient stays in the United States. NIS contains discharge data from a 20% stratified sample of community hospitals and is a part of the Healthcare Quality and Utilization Project (HCUP), sponsored by the Agency for Healthcare Research and Quality . InformaS1 Table) ), and more often in all other populations–private insurance (OR 1.25 [95% CI 1.24–1.26]), uninsured (OR 1.09 [95% CI 1.07–1.10]) and other insurance (OR 1.14 [95% CI 1.12–1.16]); all p<0.001 (S2 Table). Early coronary angiography and PCI were performed less frequently in the Medicare population, and CABG was performed less frequently in the Medicare and uninsured admissions (Table 2). During this 18-year period, there was a steady increase in early coronary angiography and PCI use across all 5 cohorts; however the Medicare group had consistently lower utilization . The Medicaid cohort had higher rates of mechanical circulatory support use compared to the other groups (Table 2). There was a steady decline in CABG use across all sub-groups and an initial peak till 2009 followed by a decline in mechanical circulatory support use across all cohorts .Coronary angiography was used less frequently in the Medicare population (59.6%) compared to the other cohorts (77.4–82.2%) during this 18-year period. There was a steady increase in the use of coronary angiography across all insurance sub-groups; however the Medicare cohort had consistently lower use (Table 2). The Medicare population had consistently higher in-hospital mortality during the 18-year study period, despite a comparable decline in 2017 compared to 2000 across all cohorts . In a multivariable logistic regression analysis with Medicare as the referent population, in-hospital mortality was higher in the uninsured (OR 1.14 [95% CI 1.11–1.16]), and lower in all other populations–Medicaid , privately insured (OR 0.73 [95% CI 0.72–0.75]) and other insurance (OR 0.91 [95% CI 0.88–0.94]); all p<0.001 (S3 Table).The unadjusted all-cause in-hospital mortality was the highest in the Medicare cohort (14.2%) compared to the other groups (4.1–6.7%) (Table 3). The Medicare and Medicaid populations had longer lengths of hospital stay, and the Medicare population had the least hospitalization costs and fewer discharges to home (Table 2). Palliative care and DNR status use were low (<3%) with the highest rates in the Medicare population (Table 2).Multiple sensitivity analyses were performed to confirm the results of the primary findings. The primary results were consistent in all sub-groups except those aged >75 years and with use of palliative care referral and DNR status use Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.