Staphylococcus aureus is recognized as one of the most frequent causes of biofilm-associated infections. The recently identified phenol-soluble modulin (PSM) peptides act as the key molecular effectors of staphylococcal biofilm maturation and promote the formation of an aggregated fibril structure. The aim of this study was to evaluate the effect of various pH values on the formation of functional amyloids of individual PSM peptides. Here, we combined a range of biophysical, chemical kinetics and microscopic techniques to address the structure and aggregation mechanism of individual PSMs under different conditions. We established that there is a pH-induced switch in PSM aggregation kinetics. Different lag times and growth of fibrils were observed, which indicates that there was no clear correlation between the rates of fibril elongation among different PSMs. This finding confirms that pH can modulate the aggregation properties of these peptides and suggest a deeper understanding of the formation of aggregates, which represents an important basis for strategies to interfere and might help in reducing the risk of biofilm-related infections.The pathogen  Salmonella Tafi protein, Pseudomonas fluorescens Fap proteins, E. coli curli, Bacillus subtilis TasA protein, and fungal hydrophobins are examples of such functional bacterial amyloids [A range of microbes are known to form biofilms by assembling into well-organized and complex communities on surfaces to endure different environments ,2. Biofiamyloids . S. aureus is recognized as one of the most frequent causes of biofilm-associated infections [S. aureus are composed of small amphipathic peptides known as phenol soluble modulins [fections . The funmodulins ,12. PSM modulins ,14. PSMsmodulins . PSM pepmodulins , with PSmodulins , while Pmodulins . Staphylococcus aureus [Despite the high sequence similarity between the PSM peptides, some of the peptides—namely PSMα2, PSMα4, and δ-toxin—do not aggregate at quiescent conditions without a facilitating agent . As the s aureus . EnhanceIn this study, we explored the effect of altering pH, focused on all seven PSM peptides, and dissected the propensity for individual monomers to form aggregated fibril structures. Using chemical kinetics, as well as biophysical and microscopic techniques, we demonstrate that despite their sequential and structural similarities, all peptides are highly sensitive to changes in pH and serve as an on/off switch for kinetics at different pH values. The results reveal how a subtle change in pH can amend the delicate kinetic balance and change the overall mechanistic character of the aggregation process of PSM peptides.Peptides, reagents, and solutions: N-terminal modified (Formylation) PSM peptides at >95% purity were purchased from GenScript Biotech, Leiden, The Netherlands. Hexafluoroisopropanol (HFIP), trifluoroacetic acid (TFA), and Thioflavin T (ThT) were purchased from Sigma-Aldrich Ltd., St. Louis, MO, USA. Dimethyl-sulfoxide (DMSO) was purchased from Merck, Darmstadt, Germany. Ultra-pure water was used for the entire study. For measuring kinetics at different pH values, experiments were performed in the pH range of 1.0–12.0 with the following 20 mM buffers: pH 1 (KCl-HCl), pH 2 and pH 3 (Gly-HCl), pH 4 and pH 5 , pH 6–8 (phosphate), pH 9–11 (Gly-NaOH) and pH 12 (KCl-NaOH). The ionic strength of the different pH buffers was calculated and found to be 0.10935 M (pH1), 0.0198 M (pH 2–3), 0.00168 M (pH 4–5), 0.03257 M (pH 6), 0.05598 M (pH 7), 0.09254 M (pH 8), 0.0198 M (pH 9–11), and 0.01935 M (pH 12), respectively. Buffers and stock solutions were filtered using PVDF 0.45 µm syringe filters (Millipore Milex-HV) before use.Peptide pre-treatment: Each dry lyophilized PSM peptide stock was dissolved to a final concentration of 0.5 mg/mL in a 1:1 mixture of trifluoroacetic acid (TFA) and hexafluoroisopropanol (HFIP), followed by a 5 × 20 s sonication with 30 s intervals using a probe sonicator, and incubation at room temperature for one hour. Further, the stock solution was divided into aliquots, and the organic solvent was evaporated by speedvac at 1000 rpm for 3 h at room temperature. Dried peptide stocks were stored at −80 °C prior to use. Preparation of samples for kinetic experiments: ThT fluorescence was observed in clear-bottomed half-area 96 well black polystyrene microtiter plates  with a non-binding surface. We used a Fluostar Omega  plate reader in bottom reading mode. Aliquots of purified PSMs were thawed and dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mg/mL prior to use. Concentrated peptide aliquots were diluted in MilliQ water and passed through a 0.22 μm filter. Freshly dissolved peptides were further diluted to the required concentrations into various buffers KCl-HCl for pH 1, Gly-HCl for pH 2–3, Na-acetate and Glacial acetic acid for pH 4–5, phosphate buffer for pH 6–8, Gly- NaOH for pH 9–11, and KCl-NaOH for pH 12. ThT was added to the protein solutions to a final concentration of 40 μM. Further, samples were loaded (100 μL) in a 96-well plate and sealed to prevent evaporation. For each peptide, different concentrations—0.25 mg/mL , 0.5 mg/mL (PSMα3), and 0.3 mg/mL (δ-toxin)—were used for kinetic measurements. The plates were incubated at 37 °C under quiescent conditions  and ThT fluorescence was measured every 10 min with an excitation filter of 450 nm and an emission filter of 482 nm. The ThT fluorescence was followed by three repeats of each monomeric concentration. The kinetic experiments with increasing peptide concentration were performed at 37 °C, monitored at every 10 minutes with an excitation filter of 450 nm and an emission filter of 482 nm under acidic and basic pH conditions. Synchrotron radiation circular dichroism spectroscopy (SRCD): The SRCD spectra of the various PSM fibrils were collected at the AU-CD beamline of the ASTRID2 synchrotron, Aarhus University, Denmark. To remove DMSO from the solution, fibrillated samples were centrifuged , supernatants discarded, and the pellet resuspended in the same volume of milliQ water. Three to five successive scans over the wavelength range of 170 to 280 nm were recorded at 25 °C, using a 0.2 mm path length cuvette at 1 nm intervals with a dwell time of 2 s. All SRCD spectra were processed and subtracted from their respective averaged baseline , smoothing with a 7 pt Savitzky-Golay filter, and expressing the final SRCD spectra in mean residual ellipticity. The SRCD spectra of the individual PSM fibrils samples were deconvoluted using DichroWeb  [don, UK)  to obtaidon, UK) , and an Circular dichroism analysis: Far-UV CD spectra were measured in a JASCO-810  spectrophotometer equipped with a Peltier thermally controlled cuvette holder at 25 °C. Aggregated peptides were centrifuged and traces of DMSO were removed to avoid any noise in the CD signal. Spectra were obtained by averaging five individuals spectra recorded between 250 and 190 nm, at 0.1 nm intervals, 1 nm bandwidth, a scan speed of 100 nm/min, and a response time of 1 s. Spectra of the buffers’ only controls were subtracted from the spectra of the proteins.Fourier Transform Infrared (FTIR) Spectroscopy: Fourier transform infrared spectroscopy was recorded on a Tensor 27 FTIR instrument  equipped with an attenuated total reflection accessory with a continuous flow of N2 gas. To remove DMSO from the solution, fibrillated samples were centrifuged , supernatants discarded, and the pellet resuspended in MilliQ water. Prior to measurement, 5 μL samples were dried under a stream of N2 gas, and 64 interferograms were accumulated with a spectral resolution of 2 cm−1 in the range of 1000–3998 cm−1. OPUS 5.5 software  was used to process the data, which included baseline subtraction, atmospheric compensation, and a second derivative analysis. For comparative studies, all absorbance spectra were normalized. Only the range of 1600–1700 cm−1 comprising information about the secondary structure is shown. ANS fluorescence: ANS fluorescence spectra were measured on an LS55 luminescence spectrophotometer  using excitation and emission slit widths of 2.5 nm. The experiments were performed in a black 3 mm × 10 mm quartz cuvette at 25 °C. A hydrophobic dye 1-anilino-8-naphthalene sulfonate (ANS) was dissolved in MilliQ water, filtered with a 0.45 µm filter. Furthermore, its concentration was determined with Nanodrop  using a molar extinction coefficient of 4990 M−1 cm−1 at 350 nm [t 350 nm . FibrilsTransmission electron microscopy: For the TEM analysis, each PSM (one acidic and one basic pH) peptide sample (5 μL) was deposited onto carbon coated formvar EM grids  and incubated for 120 s at room temperature. The buffer was subsequently removed from the grid by blotting the grid into Whatman filter paper. Further, the grid was washed with MilliQ water (5 μL) and stained with 1% uranyl acetate for 120 seconds, and the excess of staining solution was removed from the grid with Whatman filter paper. Finally, the grid was washed twice with a drop of MilliQ and blotted dry on filter paper for 10 minutes. Images were recorded using Morgagni 268 , equipped with a CCD digital camera, and operated at an accelerating voltage of 80 kV. To investigate how different pH values alter the kinetic behavior of PSM peptides, monomers of all seven PSM peptides  were incubated at a pH range from 1 to 12, and the aggregation kinetic was monitored using ThT. Fibril formation processes generally exhibit a sigmoidal kinetic behavior characterized by a lag-phase, growth, and a stationary phase , and difThe isoelectric point for all PSMα’s and δ-toxin is above pH 7, and hence changes in the aggregation behavior at acidic pH are not caused by changes in the charge of the peptide from positive to negative. For PSMβ2, this switch in the aggregation behavior coincides with the introduction of an overall negative charge by pH values above the pI of PSMβ2 (pI of 5.69). However, for PSMβ1 the changes in aggregation behavior do not coincide with the pI of the peptide of 4.89, as aggregation occurs at pH 1, 2, and 7. Hence, the switch from an overall positive charge to an overall negative charge is not the sole reason for the changes in aggregation propensity.At basic pH, several of the PSM peptides show a lack of ThT fluorescence. For PSMα2, PSMα4, and δ-toxin, no significant increase in ThT fluorescence is observed at any of the basic pH values tested here . BesidesFor PSMα3, the lack of aggregation at pH 9 does not exactly coincide with the pI of 9.53, but does coincide with the pH value with the lowest numerical value of the charge of the peptide, and hence can be linked to the charge status of the peptide. This is not the case with the PSMα1with a pI of 9.72, which, contrary to PSMα3, coincides with the aggregation curve seen at both pH 9 and pH 10. Moreover, we observed significant differences in ThT fluorescence intensity between all the peptides at the said conditions, which might be due to differences in the degree of formation of aggregates that bind to ThT. Further, the morphological analysis (discussed later) surprisingly indicated that δ-toxin forms aggregates irrespective of the insignificant fluorescence intensity, as observed by ThT kinetics g at basit1/2 with increasing peptide concentrations, as observed for PSMα1 and δ-toxin , as observed for other systems [Previously, it has been shown that changes in the protein concentration can lead to fibrillar polymorphism due to an increased chance of association at increased concentrations (due to solubility limits), as well as a decreased aggregation due to the crowding effect ,37. Usin δ-toxin . At basiormation . However δ-toxin . These o systems ,40. This systems . The conformational changes of all PSM peptides following aggregation at acidic and basic conditions were monitored using synchrotron radiation circular dichroism (SRCD) spectroscopy and Fourier transform infrared (FTIR) spectroscopy. The CD spectrum of the native peptide of all PSMs at the beginning of the time course shows α-helical characteristics with a double minimum at 208 and 222 nm . Despite−1 for PSMα1, PSMα4, PSMβ1, and δ-toxin is indicative of intermolecular β-sheets packed into the characteristic amyloid fibrils structure [−1 corresponding to β-turn conformations [−1, attributed to intermolecular amyloid-like β-sheets and a band at ~1645 cm−1, corresponding to a random coil conformation [−1 by PSMα2 and δ-toxin, typically assigned to a helical/random conformation, arises at basic conditions. This may reflect an equilibrium between residual helical soluble states and a predominant aggregated assembly. However, as the FTIR analysis is carried out on a pelleted sample, the contribution from the soluble peptide is not expected to be significant. The characteristic high frequency band at ~1625 cm−1 region suggests a high content of calculated β-sheet (~71%) for PSMα1 in an acidic pH range, whereas in basic conditions, the peak was less sharp, and the calculated β-sheet content was found to amount to ~48%. Additionally, PSMα3 did not experience any significant conformational change during the same incubation period in acidic conditions  . ANS binnd PSMβ1 e. Additind PSMβ1 e. This mnd PSMβ1 e. Similand PSMβ1 f. Furthend PSMβ1 f. Howevend PSMβ1 f. OveralThe macromolecular morphological structures of PSM peptide solutions were examined by transmission electron microscopy (TEM). In good agreement with the recorded aggregation kinetics and structural data, ordered fibrils were observed for some PSM peptides at acidic (pH 2) and basic (pH 10) conditions . The PSMStaphylococcus aureus virulence [S. aureus biofilms [S. aureus PSMs’  amyloid formation mechanism using a range of pH (1–12) to dissect the contribution of these short peptides to the biofilm structure. The extracellular matrix of S. aureus is highly complex, so the interaction between PSMs peptides with other matrices or matrix-like components, e.g., eDNA, heparin, exopolysaccharides, and alginate, must be considered to completely reveal the effect of pH on aggregation under in vitro as well as in vivo conditions. Our present study of PSM behavior with different pH values was intended to mimic variable biofilm conditions, and we confirmed that all PSMs fibrillate to a greater extent at moderate and high pH values. However, some of them also tend to fibrillate at low pH values. This indicates that the presence of different PSM peptides ensures the formation of aggregated functional amyloids at a large range of pH values that could be encountered by the bacteria during biofilm formation in vivo.PSM peptides are key determinants of irulence . Among tirulence ,48. In rirulence ,50. Remabiofilms ,16. In oPseudomonas, are highly resistant to acidic conditions [Earlier, computational and experimental analyses already suggested that the peptides in this family might exhibit differential self-assembly properties . These wnditions , so incra (presence of different amino acid as well as charges), that plays an important role in determining its propensity towards aggregation [a shift is sensible, given that it would be more favorable to have neutral rather than charged groups buried within the hydrophobic core, and hence protonation is facilitated upon aggregation [S. aureus with impairment of the maturation of biofilms [S. aureus biofilms [Despite the high sequence similarity between PSMα1 and PSMα2, the fibrillation differences between these two peptides are distinct. The sequences of all peptides are summarized in regation ,35. Thisregation . Interesbiofilms , PSM pepbiofilms . PSM peptides are not only related to the formation of amyloid-like fibrils in the biofilm, but also to biofilm disassembly ,54,55, lS. aureus PSMs are strongly dependent on pmt (PSM transporter) and provide a promising therapeutic target, because their inactivation would avert the translocation of PSM peptides to the extracellular space [S. aureus upon the blockage of their secretion would be essentially exerted by more soluble PSMs (αPSMs), and not by βPSMs that would likely aggregate into inert inclusions. Interestingly, when PSMs that accumulated in the cytosol of a pmt deletion mutant were analyzed, only αPSMs could be identified but not βPSMs, i.e., PSMβ1 and PSMβ2 [The secretion of all ar space . This lear space . Our resnd PSMβ2 ,66. It wnd PSMβ2 ,66. Howend PSMβ2 ,68 in a nd PSMβ2 ,70. Marind PSMβ2 . These vnd PSMβ2 . Additiond PSMβ2 ,72. Furtnd PSMβ2  thought S. aureus. These results indicate the likely existence of different pathways in fibril formation and serve to shed light on mechanisms of aggregation in these complex peptides. Most importantly, they will provide new targets that can be exploited for therapeutics, as PSMs that possess a critical and diverse role during infection signify a promising target for anti-staphylococcal therapy [S. aureus peptides that may generate various virulence activities encoded by diverse aggregate morphologies when secreted concurrently at numerous subcellular surroundings. These insights are highly valuable in the development of antibiotic alternatives for combating biofilms and ultimately reducing the burden of hospital-acquired infections. However, more research is required to evaluate the role of solution conditions along with other extracellular matrix components, to clarify how these effects contribute to the formation and stabilization of biofilm formation of S. aureus under in vitro as well as in vivo conditions.In conclusion, we show that aggregation kinetics was largely exaggerated by solution conditions and confirms the formation of aggregates in PSMs that are responsible for biofilm formation in  therapy . Togethe


Saccharina cultivar produced by crossing the cultivars ‘Zaohoucheng’ (♂) and ‘Pingbancai’ (♀). Circular mapping revealed that the mitogenome was 37,657 bp in length and had an overall AT content of 64.66%, including 35 protein-encoding genes, three ribosomal RNA genes (rRNA), 25 transfer RNA genes (tRNA) and three open reading frames (ORF). A phylogenetic tree constructed from the amino acids separated the six cultivars into two groups; ‘Hainong No.1’ had a closer evolutionary relationship with the cultivars ‘Zaohoucheng’, ‘Pingbancai’ and ‘Ailunwan’, whereas ‘Rongfu’ and ‘Fujian’ formed a distinct cluster. Further comparison between the ‘Hainong No.1’ and the parental mitogenomes displayed that ‘Pingbancai’ and ‘Zaohoucheng’ were the female and male parents, respectively.Here, we sequenced the complete mitogenome of ‘Hainong No.1’, a hybrid  Saccharina  is an economically important macroalgal in China that is highly valued for use in foodstuffs and industrial raw materials of extractions of iodine, algin, mannitol among others  , and stored at −80 °C in the Culture Collection of Seaweed at the Ocean University of China) via the homologous PCR amplification method described by  and ‘Pingbancai’, and the transfer RNA genes were identified using tRNAscan-SE v.1.21 software , with a nucleotide composition of 28.48% A , 14.73%C , 20.61% G  and 36.18% T, and had an overall AT content of 64.71%. The mitogenome encoded 35 protein-coding genes, three rRNA genes , three ORFs , and 25 tRNA genes that were scattered throughout the entire genome. The coding region consisted of 28,969 bp, accounting for 76.9% of total genome length. All protein-coding genes started with an ATG codon, and terminated with TAA, TAG and TGA codons. In addition, all the tRNA genes were encoded on an H-strand and most likely possessed clover-leaf-shaped secondary structures. Gene types and numbers showed a high level of conservation when compared to previously published mitogenomes of other S. japonica and Saccharina longissima, were selected to construct a Bayesian phylogenetic tree. Phylogenetic analysis was based on the polymorphisms of the amino acid sequences of 16 shared protein-encoding genes , with Laminaria digitata used as the outgroup. The results indicated that six cultivars were divided into two branches , as well as branches , and thanad6 gene in ‘Hainong No.1’ were the same  as those of ‘Pingbancai’ as opposed to those of ‘Zaohoucheng’ . From these results, we concluded that ‘Pingbancai’ is the female parent of ‘Hainong No.1’ and ‘Zaohoucheng’ is the male parent, and confirmed the previous speculation regarding parental identification.Mitogenome analysis is a maternally inherited model that aids in identifying parental sources. A comparison of the complete ‘Hainong No.1’ mitogenome with those of its parents revealed a high degree of similarity among the three, except that the 25th and 81st nucleotide sites  of the


Nitrospira are key players of the biogeochemical nitrogen cycle. However, little is known about their occurrence and survival strategies in extreme pH environments. Here, we report on the discovery of physiologically versatile, haloalkalitolerant Nitrospira that drive nitrite oxidation at exceptionally high pH. Nitrospira distribution, diversity, and ecophysiology were studied in hypo- and subsaline , highly alkaline (pH 8.9–10.3) lakes by amplicon sequencing, metagenomics, and cultivation-based approaches. Surprisingly, not only were Nitrospira populations detected, but they were also considerably diverse with presence of members from  Nitrospira lineages I, II and IV. Furthermore, the ability of Nitrospira enrichment cultures to oxidize nitrite at neutral to highly alkaline pH of 10.5 was demonstrated. Metagenomic analysis of a newly enriched Nitrospira lineage IV species, “Candidatus Nitrospira alkalitolerans”, revealed numerous adaptive features of this organism to its extreme environment. Among them were a sodium-dependent N-type ATPase and NADH:quinone oxidoreductase next to the proton-driven forms usually found in Nitrospira. Other functions aid in pH and cation homeostasis and osmotic stress defense. “Ca. Nitrospira alkalitolerans” also possesses group 2a and 3b [NiFe] hydrogenases, suggesting it can use hydrogen as alternative energy source. These results reveal how Nitrospira cope with strongly fluctuating pH and salinity conditions and expand our knowledge of nitrogen cycling in extreme habitats.Nitrite-oxidizing bacteria of the genus  Nitrospira [Nitrospira beyond nitrite oxidation, such as aerobic growth on hydrogen or formate [Chemolithoautotrophic nitrite-oxidizing bacteria (NOB) are key players of the nitrogen cycle in virtually all oxic habitats including soil, freshwater and marine ecosystems, engineered environments, and geothermal springs –9. By catrospira , 10–13. trospira , 14, 15. formate , 17 and, formate , 19.Nitrobacter alkalicus, was isolated and analyzed regarding its morphology and tolerance toward elevated pH of around 10 [Haloalkaline systems are highly productive environments that harbor diverse, haloalkaliphilic microbial communities capable of rapid biogeochemical cycling –28, but round 10 .Shallow, saline-alkaline lakes are a characteristic of the Pannonian steppe in Central Europe—an ecosystem which extends into eastern Austria and is protected in the national park “Neusiedler See-Seewinkel”. The salinity of these lakes varies within the hyposaline range and the pH is generally above 9 , 32. TheNitrospira in haloalkaline systems. An investigation of the NOB community structure in sediments of saline-alkaline lakes in the national park “Neusiedler See-Seewinkel”, Burgenland, Austria  from nine saline-alkaline lakes in the national park “Neusiedler See-Seewinkel”, Burgenland, Austria  and a pH meter . Physiological tests were performed with selected Nitrospira enrichment cultures to determine their pH tolerance  and nitrite concentration optimum of growth (with tested concentrations ranging from 0.15 to 1 mM NO2−). A detailed description of the cultivation procedure and physiological experiments is provided in the Enrichment cultures of alkalitolerant NOB were established in mineral nitrite medium with a pH of 9–10.2 at 28 °C. The medium was composed to reflect the chemical properties of the saline-alkaline lakes; however, trace elements were added as in Koch et al. [nxrB sequences, as well as rRNA-targeted fluorescence in situ hybridization (FISH), were performed as described in the DNA extraction, PCR amplification, cloning, Illumina amplicon sequencing, and phylogenetic analyses of 16S rRNA gene and Ca. Nitrospira alkalitolerans” enrichment culture were harvested by centrifugation at 20.000 × g for 15 min and the cell pellet was used for DNA extraction according to Angel et al. [Cells of the “l et al. . MetagenCa. N. alkalitolerans”, which are likely important for its adaptation to haloalkaline conditions, were identified by comparison to previously sequenced genomes of Nitrospira and Nitrospina that did not originate from haloalkaline habitats by using the OrthoFinder software [Nitrospira moscoviensis [N. defluvii [N. inopinata [Ca. N. nitrosa” and “Ca. N. nitrificans” [Nitrospina gracilis [c were reconstructed as described in the Specific genomic features of “software  with defoviensis  and N. ddefluvii  . In sediment samples from nine lakes, we detected phylogenetically diverse Nitrospira phylotypes which were affiliated with Nitrospira lineages I, II and IV  operon. Therefore, our further assessment of the Nitrospira community structures relies on the 16S rRNA gene amplicon datasets.The genomes of sequenced trospira . This laNitrospira 16S rRNA gene phylotypes was compared across the nine examined lakes . The decrease of Nitrospira diversity with increasing pH may indicate that only specific Nitrospira phylotypes tolerate highly alkaline conditions.The estimated alpha-diversity of kes Fig. . The invNitrospira communities clustered into two distinct major groups  than the mean pH of 9.37 ± 0.26 and salinity of 2.74 ± 0.88 g/l in the group 2 lakes . Members of the genera Nitrotoga and Nitrospina were screened for by FISH or PCR, but were not detected.Following the inoculation of mineral nitrite medium flasks with sediment and/or water samples from four saline-alkaline lakes  to determine the pH range for activity of the enriched Nitrospira members. Enrichment cultures EN_A and EN_C contained phylotypes from Nitrospira lineages I and II, while EN_B contained phylotypes from lineages I, II, and IV as determined by 16 rRNA gene amplicon cloning and Sanger sequencing  and nitrate production  over time for any of the three enrichments  and observation by FISH.Further analyses focused on one additional enrichment, which had been inoculated with sediment from lake Krautingsee, belonging to the group 2 of the analyzed lakes  [[+ and H+, they are important for survival under saline and/or alkaline conditions [E. coli, NhaA is regulated by the cytoplasmic pH and it catalyzes the import of 2H+ with the concurrent export of one Na+. This electrogenic activity is driven by ΔΨ and maintains pH homeostasis at elevated external pH [[Ca. N. alkalitolerans” remain unknown, the Mrp- and Nha-family antiporters most likely exhibit important physiological roles in this organism and support its survival under haloalkaline conditions. Possibly, “Ca. N. alkalitolerans” can even combine its growth in dense flocks with the extrusion of protons by its numerous proton transporters thereby lowering the pH inside the flock [At high pH, alkaliphilic and alkalitolerant microbes maintain a higher transmembrane electrical potential (ΔΨ) component of the proton motive force (PMF) than usually found in neutrophiles. The high ΔΨ is required to maintain PMF, because the ΔpH component of the PMF is reversed when the extracellular pH is higher than the intracellular pH . Like iners Fig.  includintiporter , 58 encogle gene . The Mrp~ 10.5) [ and refe~ 10.5) . The Mr~ 10.5) [, 57. Nha~ 10.5) [. Being inditions . In E. crnal pH [ and refernal pH [, 59. Althe flock .nhaB genes present in the “Ca. N. alkalitolerans” genome is located in an interesting genomic region that also contains all genes encoding the group 3 Mrp-type Na+/H+ antiporter  subunits NuoM and NuoL. However, based on the genomic context they are more likely additional mrpA- and/or mrpD-like genes, as these Na+/H+ antiporter subunits are evolutionary related to NuoM and NuoL [nuo operons  and the haloalkalitolerant, sulfur-oxidizing Sulfuricella denitrificans (88.2% AA identity), respectively. Hence, in “Ca. N. alkalitolerans”, the N-ATPase may contribute to the maintenance of ΔΨ, the generation of a sodium motive force (SMF), and salt resistance , “liphiles , ATP synase Fig. . In addiN-ATPase   . They are present in one to three copies each. The nuo genes are mostly clustered at several genomic loci (Table nuo operons present in Nitrospira defluvii [nuoL/M-like genes at loci without other nuo genes might represent subunits of cation antiporters.The genome of “in) Fig. . Firstlyci Table  and are defluvii , with AA+-dependent NADH:quinone oxidoreductase (Nqr or type III NAD dehydrogenase) .The genome furthermore contains a locus encoding all six subunits of a Nase) Fig. . The loclocation , is mostN. defluvii [+-dependent complex I (Nuo) are used for forward or reverse electron transport, respectively. Nitrospira possess a canonical Nuo that is likely used for PMF generation during the forward flow of low-potential electrons from the degradation of intracellular glycogen or from hydrogen as an alternative substrate . In addition, reverse electron transport is essential in NOB to generate reducing power for CO2 fixation. In Nitrospira, a second (modified) form of Nuo with duplicated proton-translocating NuoM subunits might use PMF to lift electrons from quinol to ferredoxin [2 fixation via the rTCA cycle. As expected, “Ca. N. alkalitolerans” possesses these two Nuo forms that are conserved in other characterized Nitrospira members. In addition, the Na+-dependent Nqr complex might function in two directions in “Ca. N. alkalitolerans” as well. During forward electron flow, Nqr would contribute to SMF generation  to adjust the cytoplasmic ion concentrations and the membrane potential in response to the environmental salinity and pH.The metabolic model for defluvii  assumes rredoxin . The redion Fig. . Reversenol Fig. . Hence, bd-like” cytochrome c oxidase, which is commonly found in Nitrospira genomes [Ca. N. alkalitolerans” contains a locus with fused genes for a cbb3-type cytochrome c oxidase hydrogenase that also occurs in other NOB and in comammox Nitrospira [2 utilization as an alternative energy source and electron donor by “Ca. N. alkalitolerans” is pending. However, we assume that this capability would confer ecophysiological flexibility, especially if nitrite concentrations fluctuate and H2 is available at oxic-anoxic boundaries in biofilms or upper sediment layers. While electrons from the group 2a hydrogenase are probably transferred to quinone [+ [Interestingly, “ion Fig. . First, y source . Second,trospira , 41 but  quinone , the groCa. N. alkalitolerans” has the genetic capacity to synthesize or import the compatible solutes trehalose, glycine betaine, and glutamate (Fig. treS of trehalose synthase (Table opuD and opuCB for glycine betaine import (Table Nitrospina gracilis [Nitrospira species. For glutamate synthesis, the genes gltB and gltD were identified (Table Ca. N. alkalitolerans” to the low availability of iron and the presence of toxic arsenite in saline-alkaline systems (The intracellular accumulation of compatible solutes is an important mechanism allowing microorganisms to withstand the high osmotic pressure in saline habitats . “Ca. N.ate Fig. . For trese Table , which ert Table  have beegracilis , but noted Table . They co systems .Nitrospira phylotypes are able to colonize saline-alkaline lakes, and that members of these lineages can carry out chemolithoautotrophic nitrite oxidation under strongly alkaline conditions up to pH 10.5. The genomic analysis of the newly cultured Nitrospira species “Ca. Nitrospira alkalitolerans” has revealed several adaptive features, many of which are also found in other haloalkalitolerant or –philic microorganisms but are missing in other characterized NOB. These results extend our picture of nitrogen cycling in extreme habitats and push the known limits of nitrite oxidation to an unusually high pH. The presence of hydrogenase genes in “Ca. N. alkalitolerans” suggests that alkalitolerant NOB can utilize alternative energy metabolisms and thus share at least part of the physiological versatility known from their neutrophilic relatives [This study shows that diverse elatives , 17, 72.Supplemental textFigure S1Figure S2Figure S3Figure S4Figure S5Figure S6Figure S7Figure S8Figure S9Figure S10Table S1Table S2Table S3


We found little evidence of an overall effect of ozone on the DNA methylome but some suggestive changes in PLSCR1, HCAR1, and LINC00336 DNA methylation after ozone exposure relative to clean air. We observed some participant-to-participant heterogeneity in ozone responses.We used a randomized crossover experiment to estimate the effects of ozone (vs. clean air) exposure on genome-wide DNA methylation of target bronchial epithelial cells, using 17 volunteers, each randomly exposed on two separated occasions to clean air or 0.3-ppm ozone for two hours. Twenty-four hours after exposure, participants underwent bronchoscopy to collect epithelial cells whose DNA methylation was measured using the Illumina 450 K platform. We performed global and regional tests examining the ozone versus clean air effect on the DNA methylome and calculated Fisher-exact  Numerous randomized controlled human exposure studies have shown that ozone exposure causes decrements in lung function and increased pulmonary inflammation  with considerable heterogeneity across people, and a randomized study has described cardiovascular changes in humans exposed to ozone7. Multiple studies have reported ozone-induced changes in pro-inflammatory cytokines in the lung of humans exposed to ozone8, as well as changes in cytokine mRNA expression in bronchoalveolar lavage cells obtained following exposure of humans to ozone9. In addition, ozone has been shown to alter upstream mitogen-activated protein (MAP) kinase pathways that control cytokine gene expression11.Ozone is a ubiquitous air pollutant that has been studied more extensively than perhaps any other environmental toxicant. Both short-term12. Exposure to air pollution has been associated in several recent non-randomized studies with epigenetic changes, especially DNA methylation. In one study13, exposure to traffic-related pollutants was associated with reduced lung function in elderly men and epigenetic changes in genes related to inflammation and immunity were proposed to modify the air pollution-lung function association. Increased air pollution was significantly associated with changes in DNA methylation of several genes, including those involved in inflammation14. A relationship between particulate air pollution and hypomethylation of the inducible nitric oxide synthesis gene was reported in a cohort of children15.It has been proposed that adverse health effects caused by exposure to air pollutants may be mediated by epigenetic changesassociated with epigenetic changes, it is difficult to claim a causal relationship between air pollutant exposure and epigenetic changes. Furthermore, these studies measured epigenetic changes in blood cells rather than the primary targets of inhaled pollutants . To show a direct causal link between air pollutant exposure and biologic changes in humans, randomized controlled human exposure studies are considered to be the “gold standard”. A recent randomized controlled human exposure study measured DNA methylation changes in T helper cells found in blood and reported changes in methylation in genes involved in mitochondrial oxidative energy metabolism in subjects exposed to particulate air pollution16. Here, we report the results of a random exposure study in which human volunteers were randomly exposed to either 0.3-ppm ozone or clean air on two occasions separated by several weeks. Bronchoscopy was performed 24 h after each exposure to remove cells directly targeted by ozone; i.e., airway epithelial cells. DNA methylation following each person’s clean air exposure was then compared with DNA methylation following that person’s ozone exposure. Because of the small sample size of the experiment, we eschew standard asymptotic inference assuming Student’s t-distributions under the null hypothesis, and instead capitalize on exact randomization-based inference.Because all these studies are non-randomized cohort studies in which changes in ambient pollutant levels are associations between ozone and the health outcomes are confounded by other environmental exposures correlated with ozone . In this randomized setting, we can directly estimate, with uncertainties expressed by the results of the randomization tests, the effect of ozone on the DNA methylome of bronchial cells (another “gold standard” for the field of air pollution epigenetics).In our study, we go beyond the current state of knowledge on the effect of ozone on DNA methylation. In observational studies, we do not know whether the estimated 7, who estimated the causal effect of ozone versus clean air on the cardiovascular system. A total of seventeen healthy individuals (see their characteristics in Table 1) of less than 80% predicted from their height and age. Prior to enrollment, all participants were informed of the study procedures and potential risks, and all provided a written informed consent. The consent forms and protocol were approved by the University of North Carolina School of Medicine and the US Environmental Protection Agency. The study was registered on ClinicalTrials.gov (NCT01492517).The study population and exposure design have been described in detail previously by Devlin et al.2, SO2 concentrations were below detection limit) or 0.3-ppm ozone (2015 US 8-h ozone standard: 0.07 ppm). The exposures were only for 2 h, in contrast to the standard, which is for 8 h. The actual number of ozone molecules to which a person was exposed in this study was about what they would have breathed for 8 h at the current standard. The exposure sessions were separated by a minimum of 13 days in an attempt to avoid carry-over effects associated with the first exposure. The exposure chamber and pollutant generation system are described elsewhere7. During the 2-h exposure, participants alternated between exercising for 15 min on a cycle ergometer and resting for 15 min. Minute ventilation was measured during each exercise session, and exercise levels were adjusted to obtain a minute ventilation of approximately 25 L min−1 m−2 body surface area. All exposures were conducted at the same time of the day and on the same day of the week for all participants between July 2010 and June 2011. The meteorological conditions in Chapel Hill during the time of the study17 are presented in Table We conducted a randomized, single-blind, crossover study in which each participant was exposed twice, for 2 h to clean air , DNA extracted using the Gentra Puregene Buccal Cell Kit . DNA samples were stored frozen at − 80 °C until analysis. DNA extracted from the bronchial epithelial cells was sent to a commercial laboratory  for DNA methylation assessment using the Illumina HumanMethylation 450 K BeadChip platform. The extracted DNA samples were placed on four chips  method20. Although the modes of probe-type DNA methylation distributions were different, they were well aligned after the BMIQ procedure —ignoring the crossover time period, and the seventeen ones observed after exposure to ozone, denoted by Yiobs(wi = 1), are shown in Fig. k after clean air and ozone exposures by m0,k = , and m1,k = , respectively. The full set of K = 484,531 CpG sites includes locations near promoter regions. Although the empirical distribution of m0,k was bimodal among the overall set of 484,531 CpGs, the empirical distribution of m0,k was unimodal among 91,795 CpGs sites near promoter regions }, among both sets of methylation sites (results not shown). The boundaries displayed in Supplemental Fig. The seventeen DNA methylome distributions observed after exposure to clean air, denoted by Yiobswi = , are shoHere, we perform statistical analyses on four sets of CpG sites: the epigenomic set of K = 484,531 CpG sites, and the sets of CpG sites with low, medium, and high methylation.0,k, m1,k)k=1:484,531, also suggests two modes, around low and high methylation levels (data not shown). The Q–Q plot between the empirical epigenomic participant mean methylation distribution after ozone versus after clean air, fk=1:484,531 versus fk=1:484,531 suggests that these two distributions are similar (results not shown). However, the participant-specific Q-Q plots show some deviations between the empirical epigenomic participant methylation distributions after ozone versus after clean air, Yiobs(wi=2) vs. Yiobs(wi=0), with the largest deviation for Participant 4  to denote the empirical distribution of the generic K-component vector D. The joint bivariate epigenomic distribution of: (1) the participant mean methylation distribution under clean air, and 2) the mean methylation distribution under ozone exposure, f Σi,j:{wi,j=1}=0 Yi,j=1,k + (O3-CA) Σi,j:{wi,j=2}=0 Yi,j=2,k, and m1,k = (CA-O3) Σi,j:{wi,j=2}=1 Yi,j=2,k + (O3-CA) Σi,j:{wi,j=1}=1 Yi,j=1,k, where NCA-O3 and NO3-CA are the number of participants who were first exposed to clean air (CA) or ozone (O3), respectively. The stochasticity of the exposure assignment mechanism resides in the order each participant is exposed to clean air or ozone, which, if we assume a Bernoulli (with probability 1/2) assignment mechanism, implies the existence of 217 = 131,072 possible allocations. Using the Fisherian procedure to test the sharp null hypothesis (H00) stating for each participant i, for each CpG site k, Yi,j=1,k = Yi,j=1,k and Yi,j=2,k = Yi,j=2,k, as described by Bind and Rubin21, we performed four randomization tests that compared the empirical participant mean methylation distributions under ozone versus clean air exposures for the four sets of CpG sites: (1) fk=1:484,531 versus fk=1:484,531, (2) fk=1:197,754 versus fk=1:197,754, (3) fk=1:112,190 versus fk=1:112,190, and (4) fk=1:174,587 versus fk=1:174,587. We chose the Kolmogorov–Smirnov (KS) distance as the test statistic to compare the two mean distributions of clean air versus ozone. Then, we calculated the KS distance for all possible allocations of the N × 2 matrix of exposure assignment w =  assuming the sharp null hypothesis (H00). Here, note that because the test statistic is symmetric, the number of unique values of KS distances under H00 is 217/2 = 65,536 (assuming no ties).The mean participant methylations at site i–j unit causal effect (UCE) for site k, τi,j,k, as the unobservable contrast τi,j,k = Yi,j,k − Yi,j,k  causal effect (PCE) for site k, τi,k, as the average of the two unit-level causal effects, τi,1,k, and τi,2,k, i.e., τi,k = , andthe participant k, τk, as the average of the N individual-level causal effects τi,k’s, τk =  for site We define three causal estimands, none of which, as with all causal estimands, is directly observable:k, di,k, is: Yi,j=2,k − Yi,j=1,k if participant i is exposed to clean air first , and Yi,j=1,k − Yi,j=2,k if participant i is exposed to ozone first  .We consider the marginal average participant causal effect (APCE) along the entire epigenome, and three conditional APCEs defined by boundaries of mean participant methylation at site k after clean air exposure, mlow, τmed, τhigh) as a permutation of τ such that:low) is the density of the klow-dimension vector of APCEs such that m0,k ≤ 0.2%5mC,f(τmed) is the density of the kmed-dimension vector of APCEs such that 0.2%5mC < m0,k < 0.8%5mC,f(τhigh) is the density of the khigh-dimension vector of APCEs such that 0.8%5mC ≤ m0,k.f denotes the density of the K-dimension vector of APCEs. Slightly abusing notation, we write τ = (τlow), (2) f(τmed), and (3) f(τhigh).We examined the characteristics of the empirical distributions of the APCEs for all sites, that is, f(τ), and for the three subsets of CpG sites , that is, (1) f(τComb-p (Python), which groups neighboring Fisher-exact p-values that are small and weighted according to their observed autocorrelation22. We calculate the univariate Fisher-exact p-values based on the standard paired statistic and on the 2To identify potential differentially methylated regions (DMRs), we used p-values to identify DMRs associated with ozone exposure with a minimum distance of 500 base pairs and a required p-value of 10−3 to start a region. For each CpG site within regions suggested by the Comb-p analysis, we report the estimated APCE and the associated Fisher-exact p-value.We use these Fisher-exact DMRcate23. Briefly, this algorithm for regional DNA methylation analyses fits linear regression models via limma for each CpG and subsequently applies a Gaussian kernel smoothing function to test-statistics grouping significant probes based on genomic proximity. We modeled the mean DNA methylation difference between the clean air and ozone exposure at each CpG with an intercept only. For the discovery of candidate regions, given the multiple comparison issue, we chose the 10−6 threshold for statistical significance for the asymptotic, meaningless, p-values provided by limma.We also examined averaged regional DNA methylation changes after ozone exposure using the R Bioconductor package k, we compared the participant methylation mean after clean air exposure  versus the participant methylation mean after ozone exposure  using univariate Fisher-exact two-sided tests, similarly as in Bind and Rubin21. We then constructed Q–Q plots of observed versus “expected” p-values, Manhattan24 and Volcano25 plots, i.e.,10 versus “expected” − log10,quantile–quantile of observed − log10 versus chromosome number, and,− log10 versus APCEs, respectively.− logFor each CpG p-values calculated with the Fisher-exact and approximating asymptotic paired Student’s tests.We compared (2) and (3) plots for i “responded” to ozone (with respect to the epigenome) by calculating the Cook’s distance26 of the estimated individual causal effect for the 484,531 CpG sites. Participant i was defined as an ozone “epigenomic responder” at CpG k if the Cook’s distance of its estimated individual causal effect was greater than four times the mean of the Cook’s distances of the estimated individual causal effects across the 17 participants. For each participant, we compare “responsiveness” counts, which are bounded by 484,531, the total number of CpG sites. As sensitivity analyses, we also increased the “epigenomic responsiveness” threshold from four to five and six times the mean of the Cook’s distances.We estimated whether each participant missMethyl R package while adjusting for varying gene size to avoid bias from large genes being overrepresented among the CpGs. We used the corrected p-values using the false discovery rate (FDR) to identify possible enriched pathways at a significance level of 0.05.Based on the Cook’s distances described in the previous section, we identified (1) the individual(s) with a large number of responsive CpGs and describe them as “most-extreme responder(s)”, and (2) the individual(s) with a low number of responsive CpGs, the “least responder(s)”. We compared them in enrichment analyses. For each responder, we took the list of CpGs that were responsive in that individual, then removed from that list all the CpGs that were also responsive in the non-responders and performed enrichment analysis on the final gene list. Thus, enrichment analyses were performed for each responder based on an inter-individual comparison of responsive CpGs for responder(s) versus non-responder(s). We examined enrichment for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using the All methods were performed in accordance with the relevant guidelines and regulations.p-values ≤ 0.21, Table p-values, all less than 10−10, are deceptive.According to the four randomization tests comparing the participant mean methylation distributions under clean air versus ozone, we found some evidence against one or two of the four sharp null hypotheses (0.07 ≤ k)k=1:484,531, appears to follow roughly a double-exponential distribution k=1:197,754, appears to follow roughly a double-exponential distribution k=1:112,190, appears to follow a bell-shaped distribution k=1:174,587, appears to follow a roughly double-exponential distribution  and the positive APCEs across 484,531 CpG sites  for all loci, f(dPhospholipid Scramblase 1 (PLSCR1) gene, nine CpGs in the Hydroxycarboxylic Acid Receptor 1 (HCAR1) gene, and eight CpGs in the Long Intergenic Non-Protein Coding RNA 336 (LINC00336) gene after exposure to ozone compared to clean air  compared to the “expected” − log10  could be explained by the CpG dependence, which is not taken into account for the construction of the “expected” − log10. The Manhattan and Volcano plots are presented in Figs. p-values. We observed that the minimum possible p-value 2/217  = 4.8) was subceeded by some univariate asymptotic p-values. The Manhattan plot also indicates that the Bonferroni adjustment  offers no power in this high-dimensional setting with small sample size. The asymptotic and Fisher-exact (two-sided) p-values do not agree in this crossover experiment  are designed to be unconfounded, (2) are based on DNA methylation of respiratory tract cells, primary targets of inhaled ozone, and (3) do not assume approximating asymptotic distribution that can be inaccurate in small studies21. Airway epithelial cells present in where we obtained the biopsies consist of at least three cell types: ciliated cells, Clara cells (mucin secreting), and basal cells. In this study, we cannot determine the contribution of each cell type to the methylation results.We found some modest evidence for regional changes in the DNA methylome of bronchial cells after ozone exposure compared to clean air exposure. In contrast to observational studies examining the healthp-values, which is an issue in human experimental settings with small samples. Here, we perform statistical tests of the ozone effect on the DNA methylome and on site-specific tests using randomization-based inference to compute Fisher-exact p-values. Global tests did not reveal substantial changes in DNA methylome measured after ozone versus clean air, but there were some regional differences, as well as inter-individual differences. By comparing inter-individual differences, we were able to discover pathways that are potentially related to the “epigenomic responsiveness” to short-term ozone exposure.The thorough description of the characteristics of: (1) the DNA methylation distributions after exposures to clean air and ozone, (2) the empirical distributions of the average participant causal effects for the sites with low, medium, and high methylations, should motivate future statistical work. Traditional epigenome-wide analyses rely mostly on approximate asymptotic responsiveness, in particular in high-dimensional settings. A study should further examine the estimated individual causal effects, especially whether the observed heterogeneity can be explained by background covariates such sex, age, race, DNA methylation under clean air. Future statistical research should also focus on the understanding of the high-dimensional characteristics of estimated average causal effects and should explore whether these distributions approximately follow double-exponential distributions, which has been used to model noise accumulation27. Other strategies that focus on computing accurate and robust p-value adjustments should also be explored28.To our knowledge, this is the first time that personalized epigenomic responsiveness is suggested. It is interesting that healthy participants are not responding homogeneously to short-term ozone exposure. New statistical methods should be developed to investigate 229. In the same participants of our study, fatty acid metabolism and amino acid metabolism were found to be enriched after ozone exposure compared to before the exposure30. Particulate matter-associated amino acids were also strongly associated with markers of inflammation and lung function31.Using the number of CpGs per individual with substantial responsiveness to ozone based on their Cook’s distance, we were able to identify individuals who had an apparent excess, or deficit, of responsive CpGs. Four individuals were highly responsive to ozone from the perspective of DNA methylation loci changes, whereas one individual was “under-responsive”. Although there was no overlap in the individual CpGs among the responders, there was substantial overlap in the pathways enriched for altered CpGs among the most-extreme responders. Among “responsive” CpGs identified in the responders, 70% were present in all “ozone responders” and linked to pathways related to lipid and fatty acid metabolism and amino acid metabolism, among others. Long-chain fatty acids have been previously shown to be responsive to short-term exposure to NOHCAR1, PLSCR1, and LINC00336) that were uncovered in both the DMRcate and Comb-p analyses. HCAR1 is located in an ozone exposure DMR on chromosome 12. HCAR1 is an important lactic acid receptor molecule, which itself has a variety of known functions. Some functions are mediated by HCAR1, including inhibition of pro-inflammatory and cytotoxic responses. In the brain, HCAR1 mediates angiogenesis32 and is related to wound healing33. These functions have not been examined in the lung but may be a key function linking HCAR1 and ozone given the tissue damage induced by ozone exposure. PLSCR1 is related to epidermal growth factor (EGF) and its receptor (EGFR) signaling pathway. This gene is closely linked with apoptosis and cellular growth. In a mouse model, cells with elevated PLSCR1 expression showed an eightfold decrease in growth34. Cellular growth is an important component of epithelial-layer revival after exposure to ozone. LINC00336 is a long non-coding RNA that is involved in ferroptosis  in the lung35.At the pathway level, there were three genes (i.e., Short-term exposure to ozone may alter the epigenome in young, healthy individuals who had epigenomes with variable responsiveness to an identical exposure to ozone. The most responsive individuals had several shared enriched pathways that mirrored effects of short-term exposure to ozone on fatty acid and amino acid metabolism as seen in metabolomics data from the same individuals and independent cohorts. Future studies are needed to further expand on the effects observed here and to integrate data to obtain a multiomic understanding of short-term ozone exposure effects.Supplementary Information.


In 2019, the Chinese government implemented the first round of the National Centralized Drug Procurement (NCDP) pilot  in mainland China, in which 25 drugs were included. We conducted this study to examine the impacts of NCDP policy on drug utilization and expenditures, and to clarify the main factors contributing to drug expenditure changes. This study used drug purchasing order data from the Centralized Drug Procurement Survey in Shenzhen 2019. Drugs related to the “4 + 7” policy were selected as study samples, including 23 “4 + 7” policy-related varieties and 15 basic alternative drugs. Driving factors for drug expenditures changes were analyzed using A.M. index system analysis (Addis A. & Magrini N.’ method). After the implementation of the NCDP policy, the volume of “4 + 7” policy-related varieties increased by 73.8%, among which winning products jumped by 1638.2% and non-winning products dropped by 70.8%; the expenditures of “4 + 7” policy-related varieties decreased by 36.9%. Structure effects (0.47) and price effects (0.78) negatively contributed to the increase in drug expenditures of “4 + 7” policy-related varieties, while volume effects (1.73) had positive influence. NCDP policy successfully decreased drug expenditures of “4 + 7” policy-related varieties with structure effects playing a leading role. However, total drug expenditures were not effectively controlled due to the increasing use of alternative drugs. Many countries worldwide are facing the challenge of ever-increasing pharmaceutical expenditures ,2,3,4, aGlobally, centralized drug procurement successfully saves drug costs ,11. WithIn January 2019, the General Office of the State Council of the People’s Republic of China implemented the National Centralized Drug Procurement (NCDP) policy, aiming at cutting drug costs and improving the drug procurement mechanism. In the first round of the NCDP pilot, 11 cities were selected as pilot cities to carry out drug volume-based purchasing, including four municipalities  and seven sub-provincial cities  in mainland China. Therefore, the first round of NCDP pilot is also known as the “4 + 7” pilot or “4 + 7” policy. The highlight of the “4 + 7” policy lies on the implementation of “volume-based procurement”, which indicates that the tenderee (government representative) clarifies the procurement volume  when conducting tendering, and the tenderers  quote according to this specific volume. The significance is to build up the contractual relationship between price and volume. “4 + 7” policy successfully tendered 25 drug varieties, of which 23 were generic products and 2 were branded products. In April 2019, the pilot cities successively implemented the tendering results of the “4 + 7” policy.The “4 + 7” policy directly resulted in an average price reduction of 52% and a maximum price reduction of 96% for 25 winning products. However, a growing body of literature has clarified that price reduction alone does not necessarily lead to a decrease in drug expenditures. This is the so-called “bypass effect” which is very common in pharmaceutical policies ,22; thatThis study used data from the Centralized Drug Procurement Survey in Shenzhen 2019 (CDPS-SZ 2019). In China, the CDPS-SZ 2019 was organized and conducted by the Global Health Institute of Wuhan University between December 2019 and January 2020. The survey aimed to evaluate the effect of drug-related policies in Shenzhen, China, and collected monthly drug purchase order data between 2017 and 2019. In the CDPS-SZ 2019 database, each purchase order record included purchase date, generic name, dosage form, specification, pharmaceutical manufacturer, price per unit, purchase volume, purchase expenditures, etc. A general database containing 963,127 monthly aggregated purchase order records was established, involving 1079 drug varieties (by generic name), 346 medical institutions, 857 pharmaceutical manufacturers. The total purchase expenditures reached RMB 20.87 billion.This study aimed to examine the general effects of the “4 + 7” policy. Thus, we included samples with the following criteria: (a) the medication covered by “4 + 7” policy-related varieties and their basic alternative varieties ; (b) theThe “4 + 7” policy-related drugs and their basic alternative varieties were included as study subjects. The “4 + 7” policy-related varieties were sorted into winning and non-winning products based on the bidding results of “4 + 7” policy. Winning products referred to products that won the tender in “4 + 7” policy, otherwise they were deemed to be non-winning products. Besides, in terms of the original intention of policy design, “4 + 7” policy was trying to promote the replacement of branded drugs with generic drugs, so as to archive the goal of reducing drug costs. Thus, this study also dichotomized our samples into branded and generic products.This study assessed the effect of “4 + 7” policy on both expenditure and volume of policy-related drugs. Expenditure data were reported in Chinese yuan, i.e., RMB. Volume was measured using the Defined Daily Dose (DDD), a measurement developed by the World Health Organization (WHO) to compare drug consumption. DDD refers to the average maintenance dose per day for a drug used for its main indication in adults, and is regarded as the international standard for assessing drug consumption across countries . In thisDescriptive statistics were used. We first described the volume and expenditures of “4 + 7” policy-related drugs in the same period before (April to December 2018) and after (April to December 2019) the implementation of “4 + 7” policy. Then, we created graphical displays of the monthly purchase volume and expenditure of each drug group in order to observe and describe patterns over time from January 2018 to December 2019.The A.M. index system analysis method (Addis A. & Magrini N.’ method) was used to assess different factors explaining the change in drug expenditures . This meE = Expenditures index, refers to the variations of drug expenditures in period 1 compared with period 0;P = Price effects, refers to the pure price change in period 1 compared with period 0;V = Volume effects, refers to the pure volume change in period 1 compared with period 0;S = Structure effects, refers to the change in the mean price per DDD from shifts toward more or less expensive drugs;P0, P1 = price per DDDs of each product in period 0, 1;V0, V1 = DDDs of each product in period 0, 1.In this study, period 0 is the base period and was assigned to April to December 2018, period 1 is the reporting period and was assigned to April to December 2019. In this model:P > 1, price effects increase the expenditure index. If P = 1, price effects have no impact on the expenditure index. If P < 1, price effects decrease the expenditure index.If V > 1, volume effects increase expenditure index. If V = 1, volume effects have no impact on expenditure index. If V < 1, quantity effects decrease expenditure index.If S > 1, structure effects increase the expenditure index. If S = 1, structure effects have no impact on the expenditure index. If S < 1, structure effects decrease the expenditure index.If A total of 38 drug varieties (by generic name) were included in this study, including 23 “4 + 7” policy-related drugs (72 products) and 15 basic alternative drugs (34 products). The total purchase volume was 311 million DDDs, and the total purchase expenditure was RMB 1347.5 million. Of those, the volume and expenditures of “4 + 7” policy-related drugs were 225.0 million DDDs and RMB 1046.7 million, respectively. The volume and expenditures of basic alternative drugs were 86.1 million DDDs and RMB 300.8 million, respectively.According to the monthly trends , the expS = 0.47) and price effects (P = 0.78) contributed negatively to the increase in drug expenditures, while volume effects (V = 1.73) had a positive contribution. In addition, volume effects (V = 15.52) contributed to the increase in drug expenditures for winning products and structure effects (S = 1.38) contributed to the increase in drug expenditures for non-winning products. For basic alternative varieties, volume effects (V = 1.26) and structure effects (S = 1.02) positively contributed to the increase in drug expenditures, while price effects played negative roles (P = 0.97).V = 4.12) contributed to the increase in expenditure for generic products and structure effects (S = 1.19) had a positive contribution for branded products. For basic alternative varieties, the decomposition analysis results of generic products and branded products were basically consistent with the whole basic alternative varieties.The results of A.M. index system analysis for generic and branded products are shown in This study examined the impacts of NCDP policy on drug utilization and drug expenditures. The results suggested that NCDP policy exerted little influence on basic alternative varieties, whose drug utilization and drug expenditures held a mild growth trend throughout the study period. However, “4 + 7” policy-related varieties were obviously affected. The total drug utilization of “4 + 7” policy-related varieties increased, of which winning products soared and non-winning products declined sharply. The total drug expenditures of “4 + 7” policy-related varieties decreased significantly. Structure effects and price effects had negative influence on drug expenditures, while volume effects had a positive influence.Containing costs is one of the inherent requirements of drug procurement . Our resThis study explored the role of price effects, volume effects, and structure effects in the changes in drug expenditures. For “4 + 7” policy-related varieties, structure effects acted as the key role, which was consistent with previous conclusions ,28,29. SInitiatives to contain pharmaceutical expenditures can be divided into supply- and demand-side ,32. SuppIn this study, “4 + 7” policy-related varieties were also divided into generic products and branded products. Our results demonstrated that NCDP policy brought about cheaper generic products replacing branded products in abundance and ultimately led to a drop in drug spending, in line with Haas et al.  and AaltAlthough rules have been perfected in new rounds, NCDP policy still needs improvements. For one thing, in the long run, a sustainable centralized drug procurement project needs a solid legal basis, or it will be questioned by stakeholders ,47,51. FSeveral potential limitations should be mentioned regarding the present study. Firstly, this study only considered basic alternative varieties provided by the monitoring program, rather than all the drugs which could replace the “4 + 7” policy-related varieties. Secondly, confounding factors such as the potential impact of other reform policies in Shenzhen have not been controlled, so there may be some deviation in the results. Thirdly, caution should be exercised in generalizing the findings. Only one of the 11 pilot cities was included in the study, so the results should be carefully extrapolated. Despite these limitations, the present study quantitatively evaluated the general effect of Chinese NCDP policy, and is the first to explore the influencing factors of drug expenditure changes under NCDP policy. The research results might be valuable references for the follow-up policy promotion and improvement.After the implementation of the “4 + 7” policy, the winning products significantly replaced non-winning products, indicating a notable direct policy effect. The “4 + 7” policy-related drugs increased in volume and decreased in expenditures, while both the volume and expenditure of alternative drugs increased. The price of all drug categories dropped after the “4 + 7” policy was introduced, which played a positive role in drug cost control. The reduction in volume of non-winning products and branded products also played a positive role in drug cost control. The irrational use structure of non-winning products, branded products in “4 + 7” policy-related drugs, and alternative drugs contributed to the growth of drug expenditures. In order to perfect the policy, a monitoring mechanism for drug use needs to be established, especially for non-winning products, branded products, and alternative drugs.


Age is closely related to the efficacy of treatment for non-small cell lung cancer (NSCLC) patients. Latest clinical trials have proved the better overall survival (OS) for the use of immune checkpoint inhibitors verse chemotherapy in NSCLC patients. However, we had no clear idea of the efficacy of them in elderly patients. So we conducted a meta-analysis to compare the efficacy of immune checkpoint inhibitors for NSCLC patients of different age groups and summarized overall treatment-related adverse events.PubMed, EMBASE, Web of Science and the Cochrane Library were searched for all clinical trials in NSCLC until 30th of April 2019. Eligible studies included randomized controlled trials (RCTs) comparing immune checkpoint inhibitors with chemotherapy in NSCLC patients. The hazard ratio (HRs) and 95% confidence intervals (CIs) of OS, progression-free survival or adverse events (AEs) were used.A total of 4994 patients from 8 RCTs were included. Immune checkpoint inhibitors significantly prolonged the OS  versus chemotherapy in NSCLC patients who were less than 65 years old. Also, they prolonged the OS  in NSCLC patients who were more than 65 years old. However, there was no statistical significance of OS  among NSCLC patients who were more than 75 years old. It also showed that the single use of immune checkpoint inhibitors had fewer all-grade AEs.Regardless of the NSCLC patients who were less or more than 65 years, immune checkpoint inhibitors could achieve better OS than chemotherapy. But there was no significant difference when NSCLC patients who were more than 75 years old. Older patient should be offered immune therapies if it is possible and the mechanism in old age treatment should be further studied. Recently, with the increasing use of immune checkpoint inhibitors in large randomized controlled trials, the immune therapy has gradually turned into mainstream cancer therapy , 2. The However, very few studies have researched whether age would affect the effectiveness of immune therapy. As we all know, the immune checkpoint inhibitors attack tumor cells by activating our own immune system. Yet the elderly patients often have the problems of immunosenescence  and autoThis meta-analysis and systematic review were reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Statement . PubMed,We searched studies from these databases in all fields with“Nivolumab” OR “Opdivo” OR “ONO-4538” OR “MDX-1106” OR “BMS-936558” OR “Ipilimumab” OR “Yervoy” OR “MDX-010” OR “MDX-CTLA-4” OR “Pembrolizumab” OR “Keytruda” OR “Lambrolizumab” OR “MK-3475” OR “Atezolizumab” OR “MPDL3280A” OR “Tecentriq” OR “RG-7446” OR “Durvalumab” OR “MEDI-4736” OR “Imfinzi” OR “Avelumab” OR “MSB0010718C” OR “PD-1” OR “PD-L1” OR“PD-1/PD-L1” OR “programmed cell death 1” OR “programmed cell death ligand 1” AND “Carcinoma, Non-Small Cell Lung” OR “Lung Carcinoma, Non-Small-Cell” OR “Non-Small Cell Lung Cancer” as the keywords. Articles that were not published in English were excluded.The inclusion criteria were listed as follows: ① The trial should enroll stage IIIB or IV NSCLC patients. ② The intervention should include PD-1, PD-L1 or CTLA-4 inhibitors. ③ The control group should be treated with chemotherapy. ④ The outcome of overall survival (OS) or progression-free survival (PFS) for NSCLC patients with different age groups should be reported. ⑤ The trials should be Phase II or III randomized controlled trials (RCTs). The following excluded criteria were used: ① Studies not in English. ② The control group contained radiotherapy or targeted drugs. ③ Studies only had abstract without full text.The data weres extracted by two authors (SYZ and HD) independently. The following information was extracted from the trials: first author, year of publication, histology of lung cancer, therapeutic line, trial phase, number of patients, experimental arms, control arms, hazard ratio (HR) of PFS or OS. The third author (HJC) assessed the data and resolved the disagreement.The risk of bias was assessed according to the Cochrane Handbook for Systematic Reviews of Interventions , which iP value was less than 0.05, the difference between two arms had a statistical significance. The heterogeneity would be low, moderated and high, if the I2 value was less than 25%, 25–50% and over 50%, respectively. In this analysis, the null hypothesis that the studies were homogenous would be rejected if P for heterogeneity was less than 0.10 or I2 > 50%. When there was significant heterogeneity among the results of included study, the random-effects model was used to calculate summary estimate [Stata SE 12.0 was used to conduct our systematical review, making forest plots and describe publication bias. HR and confidence interval (CI) were used as effect sizes. If the estimate . OtherwiOf the 864 identified trials, 8 were included. The screening flow chart was shown in Fig. P = 0.212) by Begg’s test and the funnel plot was shown in Fig. Some of the included studies had a high , 20 or uWe explored the heterogeneity via subgroup analysis based on the type of experimental arms, the class of immune target, different tumor stage and the line of therapy. And the analysis outcomes using both random-effect and fixed-effect model were shown in Tables I2 = 82.2%) analysis. Hence, random-effect model was used in this analysis. The meta-analysis showed  analysis. Hence, fixed-effect model was used in this analysis. The meta-analysis showed  analysis. Hence, random-effect model was used in this analysis. The meta-analysis showed  analysis. Hence, random-effect model was used in this analysis. The meta-analysis showed  analysis. Hence, random-effect model was used in this analysis. The comparison showed  in NSCLC patients compared with chemotherapy, while combination of immune therapy and chemotherapy had similar AEs  as chemotherapy.There were seven trials , 21, 22 wed Fig.  that theCancer is a disease of the elderly with more than 50% of new cases occurring in adults older than 65 years . And in Our study included 4994 patients from 8 RCTs , covering the immune checkpoint inhibitors used in more than phase II clinical trials. Based on the data provided by the included trials, we classified the patients into different subgroups, which were less than 65 years old, over 65 years old, from 65 to 75 years old and over 75 years old. The meta-analysis showed that no matter immune therapy or the combination of immune therapy and chemotherapy both prolonged the OS  versus chemotherapy in NSCLC patients who were less than 65 years old. And to explore the heterogeneity, we made detailed subgroup analyses which showed all the subgroups other than the immune target (CTLA-4) achieved a better OS than chemotherapy. Also, they prolonged the total OS  compared with chemotherapy in NSCLC patients who were more than 65 years old. The result is consistent with the observational study  in ItalyHowever, when we made detailed subgroup analyses of the data from patients aging more than 65, we found there were no differences as to the subgroups of using immune therapy alone, the immune target being PD-L1, tumor stage IIIB or IV, the using in the subsequent line of therapy. And there was no statistical significance of OS  in the comparison among NSCLC patients who were more than 75 years old, which was similar to the results in one cohort . It is wCytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein-1 (PD-1) are the two most well studied immune regulatory checkpoint pathways in cancer . So manyIt is known that an age-related immune dysfunction, which is often called immunosenescence, may influence the anti-tumor effect of these drugs . It has Although we strictly followed the PRISMA statement, our study still had several potential limitations. First, some of the included studies had a high , 20 or uIn conclusion, regardless of the NSCLC patients who were less or more than 65 years old, immune checkpoint inhibitors could achieve better OS than chemotherapy. But there was no significant difference when NSCLC patients who were more than 75 years old. So if the patients were older than 75, they should weight the advantages and disadvantages for the best therapeutic schedule. Older patient should be offered immune therapies if it is possible and the mechanism of treating the elderly with them should be further studied.


Facial (lip and jaw) tremor (FT) is associated with Parkinson’s disease (PD) but few studies have been conducted to explore its clinical profile. We performed this study to investigate the prevalence and clinical correlates of FT in PD, and further to evaluate its effect on disease progression.n = 2224) and longitudinal (n = 674) study was conducted. The presence of FT was based on a ≥ 1 score in the United PD Rating Scale (UPDRS) item 20A. Group comparisons were conducted, followed by a forward binary logistic regression analysis. Inverse probability of treatment weighting (IPTW) based on the propensity score and weighted or unweighted Cox regression models were used to explore the impact of FT on five clinical milestones including death, UPDRS III 11-point increase, Hoehn and Yahr (H&Y) stage reaching 3, dyskinesia development, and Montreal Cognitive Assessment 3-point decrease.A retrospective, cross-sectional (P < 0.001), female (P < 0.001), disease duration (P = 0.001), speech (P = 0.011), rigidity (P = 0.026), rest tremor on limbs (P < 0.001), kinetic tremor on hands (P < 0.001), and axial symptoms (P = 0.013) were independent factors associated with FT. Both unweighted and weighted Cox regression models indicated that baseline FT and FT as the initial symptom were not associated with the five outcomes.FT was presented in 403 patients (18.1%), which showed increasing trends with disease duration and H&Y score. Age (Our study suggested that FT was not uncommon and provided a deeper insight into the characteristics of FT in PD. The predict value of FT on long-term progronis of PD may need future longer follwe-up study.The online version contains supplementary material available at 10.1186/s12883-021-02105-y. Parkinson’s disease (PD) is a chronic, progressive neurodegenerative disease mainly characterized by rest tremor, rigidity, bradykinesia, and postural and gait disorders . As one The exact functional anatomy of rest tremor in PD remains unclear. It is probably related to a combined impairment of the cerebello-dentato-thalamocortical and the basal ganglia–thalamocortical circuits . Rest trTo date, data on the relationships between FT with other PD motor and non-motor symptoms are scarce, and the prevalence of FT is based on a few studies with a small sample size. Although previous analyses of some studies reported that patients with tremor-predominant (TD) subtype have a more benign course  and shown = 2224).All procedures of the current study were approved by the Ethics Committee of West China Hospital, Sichuan University. Initially, we enrolled 4332 PD patients from the Department of Neurology, West China Hospital of Sichuan University between March 2009 and July 2019. All participants have provided written informed consent and met the Unified Kingdom PD Society Brain Bank Clinical Diagnostic Criteria for PD . To inven = 725): 1) assessment at “off” medication; 2) disease duration < 3 years; 3) Hoehn and Yahr (H&Y) stage < 3; 4) absence of motor complications; and 5) absence of dementia. During the follow-up visit, 21 patients withdraw informed consent and 30 patients lost contact, resulting in the remaining 674 patients who provided information on clinical outcomes were included in the longitudinal data analysis.To explore the influence of FT on the disease progression, participants who met the following inclusion criteria were followed up at least once (range 1–10) . For those without an “off” score, we estimated an “off” medication score by adding the difference value of the study population’s mean “off”- and mean “on”-scores to the patient’s “on” medication score, as reported by a previous study [Trained neurologists completed standardized assessments of all patients. Demographic and clinical data including sex, age, age at onset, disease duration, education, smoking and drinking history, hypertension, and diabetes mellitus (DM) history, motor complications, and total levodopa equivalent daily dosage (LEDD) were collected. The LEDD was calculated based on a previous systematic review . The Unius study .≥ 1 score on UPDRS-13  or a ≥ 3 score on UPDRS-14 .The global non-motor symptoms (NMS) was assessed using the Chinese version of the Non-Motor Symptoms Scale (NMSS) . In addiThe diagnosis of FT was established by a neurologist trained in movement disorders, and presence or absence explicitly marked on the UPDRS-III score sheet .Mortality surveillance was performed mainly throughout the continuing active follow-up of patients and their families. It lasted until July 1, 2020, which was 11 years after our study began (2009), with as many as 14 years of passive follow-up for mortality for patients first diagnosed in 2006.Since a change of 2.5–5.2 points on the UPDRS III score represent a clinically significant difference , we thusTime to transfer to H&Y stage 3 was defined as the time from the baseline to the first follow-up examinations in which the patient scored at least stage 3.Time to dyskinesia development was defined as the time from the baseline to the first follow-up examinations in which the patient reported dyskinesia.Cognitive decline was defined as a 3-point decrease from baseline MoCA score and time to event as the time from the baseline to follow-up examinations in which a 3-point decrease was first measured.T test, Wilcoxon rank-sum test, or chi-square test were applied as appropriate.Continuous variables were presented as mean ± standard deviation if normally distributed and as median (interquartile range) if non-normally distributed. Categorical data were shown as number (percentage). To identify the differences in demographic and clinical features at baseline between patients with and without FT, the Student’s P < 0.1) or those that were possibly related to FT were chosen as independent variables. The Hosmer and Lemeshow test was implemented to examine the goodness of fit, with P value > 0.05 suggesting high goodness of fit of the model. The tolerance and variance inflation factor (VIF) that were calculated based on a multiple linear regression analysis were used to diagnose the multicollinearity among each independent variable, with tolerance < 0.2 or VIF > 5 suggesting the presence of multicollinearity.To explore the potential clinical factors related to FT, a binary logistic regression model was used. The presence or absence of FT was set as the dependent variable and the variables that reported differences between the two groups , a propensity score (PS) weighting method was served to balance the differences. The PS model was developed by constructing a logistic regression model in which patients with FT vs. patients without FT  were regressed on baseline characteristics related to the outcome variables. The estimated PS was achieved as the predicted probability of having FT in each subject. The inverse probability of treatment weighting (IPTW)  was thenFurthermore, to verify the results from IPTW, a sensitivity analysis was conducted. Multivariate Cox proportional hazards regression models were conducted to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) of five clinical outcomes across either presence or absence of FT or FT as the initial symptom or not. Two models were conducted. In model 1, sex, age, and age at onset were adjusted. In model 2, sex, age, age at onset, BMI, LEDD, UPDRS III score, MoCA score, and NMSS score were adjusted.P values < 0.05 were regarded as statistically significant.Statistical analyses were conducted by R version 4.0.0 using “Matching”, “survey”, “reshape2”, “survival”, and “reportReg” packages. All statistical tests were two-tailed, and In total, 2224 PD patients  were included for retrospective analysis. Among these patients who were assessed UPDRS at “off” medication, 403 patients reported FT (18.1%). The smooth curves indicated that the prevalence of FT along with limbs tremor at rest or action/postural tremor of hands showed an increasing tendency with increased disease duration and H&Y stage   Table .Table 1P < 0.001), females , disease duration , speech , rigidity , limbs tremor at rest , action or postural tremor of hands , and axial symptoms  . The percentage accuracy in the classification of the model was 81.9%.Forest map indicated that FT was association with age , with a mean time to death or censoring from baseline of 5.7 ± 2.3 years; 203 (30.1%) reported a decrease of at least 11 points in UPDRS III score after 3.7 ± 2.2 years of follow up; 119 (17.7%) patients reached at least H&Y stage 3 after a mean disease duration of 4.9 ± 2.0 years; 161 (23.9%) patients reported a decrease of at least 3 points in MoCA score after a mean 3.6 ± 2.1 years of follow-up; and 94 (13.9%) reported new occurrence of dyskinesia after a mean 5.1 ± 2.1 years of follow up. The mean change in the UPDRS III score from baseline to follow-up visit was − 7.9 ± 10.8 points, while the mean change in the MoCA score was − 0.7 ± 3.1 points, after excluding the patients who had died.IPTW was formed by those with complete data on clinical outcomes. A total of 18 covariates at baseline including sex, age, age of onset, disease duration, drinking, smoking, hypertension, DM, BMI, education, LEDD, UPDRS III score, H&Y stage, MoCA score, FAB score, HDRS score, HARS score, and NMSS score were included for estimating the propensity score Fig. . CompareP = 0.006 and P < 0.001, respectively), but this association disappeared after using the weighted method by IPTW  (Table P > 0.05)   subtypes. It is reported that patients with the TD subtype have a more benign course compared to those with the non-TD subtype . Based oActually, a recent study  verifiedSome limitations should be considered. First, we focused on a group of patients who were assessed at “off” medication and therefore selection bias cannot be ruled out. Second, the relatively short observation of FT on clinical outcomes is not enough to conclude the influence of FT on long-term outcomes such as death. Third, the assessment of FT was based on UPDRS, a scale that does not distinguish between lips and chin tremor, resulting in these variables could not be used for stratification.FT is not rare in patients with PD, which can be affected by sex, age as well as disease duration and motor severity. Baseline FT and FT as the initial symptom are not associated with the disease progression of PD. The predictive value of FT on long-term progronis of PD may need to clarify by future longer follow-up studies.Additional file 1: Supplementary Table 1. Diagnosis of multicollinearity for each candidate variate. Supplementary Table 2. SMD of baseline covariates before and after IPTW between groups