Blocking the PD-1/PD-L1 pathway can diminish immunosuppression and enhance anticancer immunity. PD-1/PD-L1 blockade can be realized by aptamers, which have good biocompatibility and can be synthesized in quantity economically. For in vivo applications, aptamers need to evade renal clearance and nuclease digestion. Here we investigated whether DNA nanostructures could be used to enhance the function of PD-L1 aptamers. Four PD-L1 aptamers (Apt) were built into a Holliday Junction (HJ) to form a tetravalent DNA nanostructure (Apt-HJ). The average size of Apt-HJ was 13.22 nm, which was above the threshold for renal clearance. Apt-HJ also underwent partial phosphorothioate modification and had improved nuclease resistance. Compared with the monovalent PD-L1 aptamer, the tetravalent Apt-HJ had stronger affinity to CT26 colon cancer cells. Moreover, Apt-HJ markedly boosted the antitumor efficacy in vivo vs. free PD-L1 aptamers without raising systemic toxicity. The results indicate that multiple aptamers attached to a DNA nanostructure may significantly improve the function of PD-L1 aptamers in vivo. Cancer is a leading cause of mortality in the 21st century. There are four main treatments for cancer: surgery, radiotherapy, chemotherapy, and immunotherapy. Unlike the first three treatments that directly target the cancer cells, immunotherapy attacks the tumor through mobilization of the host′s immune system . Among IThe current ICB therapies are implemented by antibodies. There are five monoclonal antibodies targeting the PD-1/PD-L1 pathway approved by the FDA: atezolizumab, nivolumab, durvalumab, avelumab, and pembrolizumab . HoweverAn aptamer is a type of ligand with promising advantages for biomedical applications. Aptamers are short single-strand DNA or RNA oligonucleotides, which can fold into complex 3D structures and bind to target molecules with high affinity and specificity . Compared of 72nM. As a result, the aptamer could bind to murine PD-L1 expressed on CT26 or LL/2 cells, and generate significant tumor inhibition in both CT26- or LL/2-bearing mice, without observable hepatic or renal toxicity [Aptamers have been developed for PD-1/PD-L1 blockade. Lai et al. selected an aptamer for human PD-L1 with relatively high affinity. This PD-L1 aptamer could also bind to recombinant murine PD-L1 protein with a Ktoxicity . The resIt should be noted, however, that for in vivo applications, aptamers need to satisfy certain conditions. Aptamers need to have nuclease-resistance, to a certain degree, in order to survive digestion by serum nucleases. Moreover, aptamers generally have a small size and tend to leach out of the kidney , which uTo date, no report in the literature has evaluated the application of a DNA nanostructure to improve the efficacy of PD-1/PD-L1 blockade by aptamers. In this study, four PD-L1 aptamers were built into the four arms of HJ to form Apt-HJ. Compared with free PD-L1 aptamers, we found that the tetravalent Apt-HJ had stronger affinity to CT26 cells and significantly improved antitumor efficacy in vivo.Previous studies have shown that a PD-L1 aptamer can generate an anti-tumor effect in vivo . In thisIt is well known that the threshold for kidneys to clear nanoparticles is approximately 10 nm . To evalA main obstacle for in vivo application of Apt-HJ is digestion by nucleases, due to the presence of large amounts of nucleases in serum. Various chemical methods have been developed to slow down the digestion of DNA strands by nucleases ,30. Phosd values were also measured and calculated , free PD-L1 aptamers, or Apt-HJ were incubated with CT26 cells, which were subsequently fixed and imaged by confocal microscopy. As shown in It has been reported that CT26 murine colon cancer cells express PD-L1, and that the PD-L1 aptamers can bind with these cells and act as an ICB agent . Since tlculated . The Kd Although Apt-HJ had superior target affinity vs. free aptamers in vitro, it was still unclear whether Apt-HJ would generate antitumor efficacy in vivo. To address this issue, CT26 tumor-bearing mice were treated with PBS, poly-A (control DNA sequence), poly-A-HJ, free PD-L1 aptamers, or Apt-HJ. The treatments were given via intraperitoneal injection every two days, for a total of six injections. The dosage of free PD-L1 aptamers was 1.2 mg/kg. Poly-A treatment had the same molar concentration as free PD-L1 aptamers. Since Apt-HJ and poly-A-HJ were tetravalent, they were given a molar concentration that was 1/4 of the free PD-L1 aptamers, for the sake of fair comparison. As shown in In this study, a new DNA nanostructure (Apt-HJ) comprising four PD-L1 aptamers and a Holliday Junction was constructed to enhance the target affinity and the in vivo antitumor efficacy. Apt-HJ was made of four ssDNA chains which had the PD-L1 aptamer at their 3′ end and the HJ sequence at their 5′ end. These ssDNA chains were assembled into a tetravalent structure via a self-assembly process . The aveA major obstacle for in vivo applications of aptamers is their rapid renal clearance and short circulating time. To address this issue, aptamers are sometimes conjugated to carriers with a high molecular mass to increase their size . PEGylatIn this study, Apt-HJ improved the antitumor efficacy in CT26-bearing mice vs. free PD-L1 aptamers A. The meIn summary, ample evidence has shown that blocking PD-1/PD-L1 pathways can reduce immunosuppression and enhance antitumor immunity. In this study, a novel DNA nanostructure (Apt-HJ) comprising four PD-L1 aptamers and a HJ is constructed. The tetravalent Apt-HJ has higher affinity for tumor cells and improves the antitumor efficacy in vivo. Such multivalent DNA complexes may become a new strategy to further enhance the potential of PD-L1 aptamers in ICB applications.2. DMEM was purchased from the Cell Center of Chinese Academy of Medical Sciences . FBS was purchased from Biond .CT26 (mouse colon cancer cell lines) was obtained from the Cell Center of Chinese Academy of Medical Science . The cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). All cells were incubated in a 37 °C incubator with 5% COAll DNAs were synthesized by Invitrogen . The four DNA strands for Apt-HJ had the following sequences, with the underlined part indicating the sequence of PD-L1 aptamer:ACGGGCCACATCAACTCATTGATAGACAATGCGTCCACTGCCCGT-3′;5′-CGGCGATCCGGCCATAGTGGATTGCGGGCCAGTGAAAAAAACGGGCCACATCAACTCATTGATAGACAATGCGTCCACTGCCCGT-3′;5′-CTCACTGGCCCGCAATCCTGAGCACGTGGCTGACGAAAAAACGGGCCACATCAACTCATTGATAGACAATGCGTCCACTGCCCGT-3′;5′-CCGTCAGCCACGTGCTCACCGAATGCTGCGCAACCAAAAAACGGGCCACATCAACTCATTGATAGACAATGCGTCCACTGCCCGT-3′.5′-CGGTTGCGCAGCATTCGGACTATGGCCGGATCGCCAAAAAThe first nt at the 5′ end of each ssDNA for some Apt-HJ and all polyA-HJ was phosphorothioate modified.2). The concentration of each ssDNA was equal to 4 μM. The mixture was incubated at 95 °C for 5 min and slowly cooled to room temperature at a controlled rate of 0.1 °C/s.Four single-stranded DNA chains were dissolved in a saline solution  agarose and GelStain  in TBE buffer. Samples were loaded onto the gel and run for 30 min at 120 V, and analyzed under UV light. The particle size and zeta potential of the Apt-HJ were evaluated by dynamic light scattering (DLS) using Zeta Sizer Nano ZS90  at 25 °C.The prepared Apt-HJs were verified by using gel electrophoresis. The gel was made by dissolving 2.5%  were incubated in 50% fetal bovine serum (FBS) at 37 °C. At the predetermined time points, the samples were heated immediately at 95 °C for 5 min to inactivate nucleases. Thereafter, electrophoresis was utilized to analyze the degree of Apt-HJ degradation. The gray-scale value was analyzed by Quantity One software .5) were suspended in 250 μL PBS. Next, 60 pmol FAM-labeled poly-A, Apt, or Apt-HJ were added to the cells, which were shaken gently for 30 min at room temperature. The cells were washed thrice with PBS and resuspended in 250 μL PBS. The samples were analyzed directly by Accuri C6 Flow Cytometer . To evaluate the equilibrium dissociation constants (Kd), CT26 cells (2 × 105) were suspended in 250 μL PBS. Next, FAM-labeled Apt, or Apt-HJ were added to the cells. The final concentrations of Apt were 50, 100, 150, 200, 300, 600, 800, and 1000 nM, respectively. The final concentrations of Apt-HJ were 50, 100, 150, 300, 400, 600, and 1000 nM, respectively. Samples were shaken gently for 30 min at room temperature. The cells were washed thrice with PBS, resuspended in 250 μL PBS, and analyzed by flow cytometry . The Kd of PD-L1 aptamers and Apt-HJ was determined by the relationship between the fluorescence intensity and the corresponding concentration [d + X).CT26 cells in a logarithmic growth phase were incubated in PBS (plus 0.02% EDTA) for 30 s and washed gently twice with PBS. The cells  freshly prepared and pre-cooled was applied for 10 min at 4 °C to fix the cells. The cells were washed three times with PBS and stained by Hoechst  for 30 min at room temperature. Finally, cells were washed three times with PBS and incubated with 100 μL PBS. The fluorescence image was analyzed by a confocal laser-scanning microscope .CT26 cells were cultivated in a Lab-Tek Chamber #1.0 Borosilicate Coverglass System  at a density of 1.5 × 105 CT26 cells suspended in 100 μL PBS were injected subcutaneously on the right lower back. After tumor reached to a size of about 5 mm, mice were randomly divided into five treatment groups. The mice were treated with PBS, poly-A, poly-A-HJ, free-Apt, or Apt-HJ via intraperitoneal injection every two days for a total of six injections. The dosage of free PD-L1 aptamers was 1.2 mg/kg. Poly-A treatment had the same molar concentration as the PD-L1 aptamers. Since Apt-HJ and poly-A-HJ were tetravalent, the Apt-HJ or poly-A-HJ treatments were given a molar concentration that was 1/4 of the free PD-L1 aptamers. Tumor size and body weight were measured every 2 days during the experiment. Tumor volume was calculated according to the formula (a × b2)/2, where a and b represent the length and width of the tumor, respectively.Animal study and procedures were approved by the Ethics Committee of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, according to the institutional animal care and use guidelines. BALB/c mice of 6-8 w old, with an average weight of 20 g, were purchased from Charles River Corp. . To establish a colon cancer model, 5 × 10p < 0.05. Relevant data were presented as mean value and standard deviation.Statistical analyses were performed by GraphPad Prism 8 software. One-way ANOVA with Fisher’s least significant difference (LSD) comparison test was used for statistical calculation. A statistically significant difference was defined as


Zea mays L.) production. Therefore, using amplicon sequencing, we aimed to clarify how AMF communities and their diversity in maize roots vary under different cover cropping systems and two types of tillage (rotary and no tillage). Two kinds of cover crops (hairy vetch and brown mustard) and fallow treatments were established with rotary or no tillage in rotation with maize crops. Tillage and no tillage yielded a set of relatively common AMF operational taxonomic units (OTUs) in the maize crops, representing 78.3% of the total OTUs. The percentage of maize crop OTUs that were specific to only tillage and no tillage were 9.6% and 12.0%, respectively. We found that tillage system significantly altered the AMF communities in maize roots. However, the AMF communities of maize crops among cover cropping treatments did not vary considerably. Our findings indicate that compared with cover cropping, tillage may shape AMF communities in maize more strongly.Our understanding regarding the influence of intensive agricultural practices, including cover cropping and tillage, on communities of arbuscular mycorrhizal fungi (AMF) is lacking. This would prove to be an obstacle in the improvement of current maize ( In general, conservation tillage can improve soil aggregation, increase the amount of soil organic carbon in the surface layer, and reduce erosion3. Moreover, conservation tillage enhances soil microbial diversity and the abundance of beneficial functional soil microorganisms4. The soil microbial communities stimulated by conservation tillage can play important roles in soil aggregation, soil carbon sequestration, and soil nutrition; improve water use efficiencies; and influence crop yields6.Growing cover crops and practicing conservation tillage are agricultural practices worldwide that have been recommended to promote soil fertility. In particular, Williams 7, enhances soil organic matter8, inhibits weed establishment9, and increases the abundance and activity of arbuscular mycorrhizal fungi (AMF)10. Indeed, crops with AMF have a greater capacity to take up phosphorus (P), zinc (Zn), and water10 than crops without AMF. This greater capacity generally results in robust crop growth under conditions of limited nutrient availability or drought. AMF also contribute to increasing plant resistance against pathogens and help make crops generally healthier13. AMF also contribute to crop health indirectly because of their effects on soil processes in agricultural settings15. In particular, they can contribute to soil health, soil aggregate formation, and soil stability by increasing the soil’s nutrient cycling and organic matter content17. However, certain agricultural practices have negative influences on AMF abundance and functions19. For example, conventional tillage systems, including chisel plowing, rotary tillage, and disc harrowing, can disrupt the AMF hyphal network20, inhibit AMF development and decrease AMF abundance in soil21. Fallowing during the winter also decreases the AMF abundance in soil and reduces the benefit of AMF on crops23. In contrast, no-tillage or reduced tillage systems can increase the abundance and hyphal network of AMF in soil24. Growing cover crops as pre-crops can increase indigenous AMF abundance in soil and root colonization by AMF, thus improving the growth and yield of subsequent crops27. Furthermore, the introduction of mycorrhizal pre-crops combined with a no-tillage system increases early stage maize (Zea mays L.) growth and AMF colonization to a greater extent than that of mycorrhizal pre-crops combined with a rotary-tillage system28. Thus, combining cover crop systems with conservation tillage may provide many benefits that improve subsequent crop performance.The practice of cover cropping potentially reduces soil and wind erosion29. Increased AMF richness in agricultural ecosystems has been suggested to improve crop performance30, while co-inoculation of AMF taxa belonging to different families improves plant biomass and mineral nutrition more efficiently than mono-inoculation33. In terms of agricultural management practices, tillage35 and cover cropping36 can alter the AMF community structure and diversity in soil and roots. For instance, Oehl and Koch37 reported AMF spore communities in conventional tillage are distinct from those in no-tillage systems in a vineyard farm, based on AMF spore morphology. Morimoto et al.38 showed that AMF communities in the roots of subsequent soybean crops differed, depending on their rotation with winter wheat or fallow. Other studies have shown that AMF communities can be shaped in subsequent crops, and the effect of the identity of the host crop is stronger than that of the cover crops40. Aside from these conflicting results, most other studies have investigated only the impact of cover cropping on AMF communities in subsequent crops40 and there is a lack of information about the impacts on AMF communities in subsequent crops under different agricultural management systems, such as the combination of cover cropping with tillage systems. Given our lack of knowledge regarding AMF communities in combined cover crop rotations with different tillage systems, it is imperative to understand how combining cover cropping with different tillage systems change AMF communities in subsequent crops using next-generation sequencing techniques .Moreover, there is clear evidence that AMF community structure and diversity improve plant growth performanceThe AMF communities of subsequent crops in cover cropping combined with rotary tillage are distinct from those in cover cropping combined with no tillage.The AMF communities of subsequent crops in a rotary-tillage system will have relatively more taxa that can tolerate stress in the presence of disturbance compared to those in a cover cropping system combined with no tillage.It is currently unclear which factor, cover cropping or tillage, has a greater influence in the shifts in the AMF communities in the roots of subsequent crops under cover crop rotational systems. Little is also known about how the combination of cover cropping with different types of tillage (rotary tillage and no tillage) drives shifts in the AMF communities in the roots of subsequent crops. Knowing whether such management affects the soil status and its connections to AMF taxa may help to determine the proper practices under specific agricultural settings. We hypothesized that:45. Thus, we used this technique to investigate how and whether AMF communities in the roots of maize crops change in different types of cover cropping systems combined with rotary or no tillage.Illumina amplicon sequencing is a commonly used and helpful technique for AMF community analysisVicia villosa Roth., var. Fujiemon) and brown mustard (Brassica juncea (L.) Czern & Coss., var. Karajin)] and one bare fallow treatment with rotary or no tillage as our experimental design . We combined two different cover crops [hairy vetch  randomly collected using a core sampler . On May 17, 2017, we pooled the soil samples into one composite soil sample. The roots of maize crops were collected at the stage of eight fully emerged leaves (V8 stage) on July 3, 2017. We collected maize root samples from nine plants  per plot. After collecting the root samples, we stored them at −80 °C for subsequent staining and DNA extraction. The roots were stained with a 3,3′-diaminobenzidine (DAB) solution3-N) content was measured using a LAQUA Twin nitrate meter . Extractable P (available P) from all soils, regardless of tillage management, was obtained as previously described48 and measured using molybdenum blue method at 710 nm using a UV-1700 Spectrophotometer . Soil acid phosphatase (ACP), alkaline phosphatase (ALP), and β-glucosidase activities in the soils were measured according to Ishii and Hayano49 and Hayano50.Soil biochemical properties were measured after cover cropping. We analyzed soil pH (soil:water ratio of 1:2.5 w/v) with a digital pH and conductivity meter , and soil nitrate-nitrogen  region using a previously described PCR method27. We selected the primer pair AM151/NS3152 for the first PCR to amplify a partial region of the 18S rDNA of AMF taxa. The first PCR was performed in 10-μl reactions, each consisting of 2 × Platinum™ Green Master Mix (Thermo Fisher Scientific Inc), 1 µl of template DNA, and 0.4 μM of AM1/NS31 primers. For the second PCR procedure, we diluted the amplicons from the first PCR 10-fold and used these amplicons as a template for the AMV4.5NF/AMDGR53 primers that are attached via the Illumina MiSeq adapter sequences. The second PCR was performed in 10 μl reactions comprising 1 µl of template DNA, 2 × Platinum™ Green Master Mix, and 0.3 μM of AMV4.5NF/AMDGR primers. Then we added both the Illumina MiSeq adapter sequences and an 8-bp barcode sequence as an index to distinguish each PCR amplicon.We froze 100 mg of fresh maize roots from all plots and retrieved total genomic DNA from the roots using the DNA suisui-P kit et al.54. Briefly, we washed and cleaned the amplicons from the second PCR before amplicon sequencing. These products were paired-end (PE) sequenced (2 × 300 bp) using an instrument of Illumina MiSeq amplicon sequencing at the Bioengineering Lab Co., Ltd. We performed the processing of sequence reads using QIIME version 1.9.155. The PE reads were truncated at any site that received an average quality score of <20 over a 40-bp sliding window, and truncated reads shorter than 40 bp were discarded using the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html). We assembled the PE reads through their overlapping sequences that had a minimum overlap length of 10 bp and we deleted the reads that could not be assembled. We used Fast length adjustment of short reads (FLASH) ver. 1.2.11 to assess the clean sequences. Chimeric sequences were removed using UCHIME in USEARCH ver. 10.0.240. We then utilized the BLAST function in the MaarjAM database (https://maarjam.botany.ut.ee/) to determine and group the operational taxonomic units (OTUs) at 97% similarity. Additional taxonomic assignment was based on phylogenetic relationships. Representative sequences were aligned with known AMF taxa from NCBI Genbank and a neighbor-joining phylogenetic tree with 1000 bootstrap replicates was created using MEGA 756 . Before molecular bioinformatic analysis, we resampled according to the lowest number of reads to equalize the assessment among all treatments regardless of Illumina amplicon read depth. After rarefaction analysis, we calculated the Hill number 0D as OTU richness (q = 0), the Shannon index , and the Simpson index  from data on the diversity of AMF communities using the “renyi” function58 of the vegan package59. Additionally, to test whether AMF OTUs were significantly related to rotary tillage and no tillage with cover cropping, we performed a species indicator analysis. We also estimated values of indicator species using “multipatt” function60 in the indicspecies package to test for statistical significance of the highest OTU association in each plot.Root staining data reflecting AMF colonization was transformed into the arcsine-square root values for normalization. The differences in the mean values of each parameter among tillage and cover cropping systems were evaluated by Tukey’s test and then two-way analysis of variance (ANOVA) was conducted using the vegan package in R.3.6.159. Goodness-of-fit (R2) for measured factors fitted to the db-RDA ordination of the AMF communities were calculated using the “envfit” function in the vegan package with P-values based on 999 permutations59. A permutational multivariate analysis of variance (PERMANOVA) was performed with 9999 permutations using the “adonis”59 function in the vegan package to investigate if AMF communities differed significantly in tillage management and cover cropping. In addition, a multinomial species classification method61 using the package vegan were introduced to recognize “specialist OTUs” and “generalist OTUs” for tillage or no tillage.To demonstrate the variations in the AMF communities in maize crops among tillage management and cover cropping, we performed distance-based redundancy analysis (db-RDA) in the 3-N content in soil was significantly influenced by cover cropping, but soil pH was not affected by either tillage or cover crop management  .We detected a total of 148 AMF OTUs in the roots of maize Fig. . Regardlly) Fig. . The relGlomus and uncultured Glomeromycotina taxa in the rotary-tillage system  and brown mustard  significantly affected the AMF communities  did not affect the AMF communities of maize crops. Among the no-tillage treatments, only brown mustard  significantly affected the AMF communities . However, cover crops did not significantly affect the AMF communities in no-tillage treatments .Among rotary-tillage treatments, bare fallow   and no tillage  had significant effects on the AMF communities in maize crops. Moreover, PERMANOVA shows that tillage management rather than cover cropping or the interaction between tillage management and cover crop type significantly affected the AMF communities in maize crops .The db-RDA also found that tillage management alters the AMF communities of maize crops, while only brown mustard pre-crop significantly affected the AMF communities (Monte-Carlo permutation test: Glomus and Rhizophagus) and Claroideoglomeraceae (Claroideoglomus) are the main genera in maize roots  rDNA of AMF are strongly biased toward Glomeraceae. Because our results show that Glomeraceae are dominant in maize roots regardless of tillage and cover cropping, further studies should carefully select primer pairs that avoid potential PCR and primer bias as well as technical sequencing errors.Our findings indicate that representatives of Glomeraceae  may be the key in shaping AMF communities in roots.The beneficial impacts of cover cropping may differ among the AMF communities, and the question of whether cover crop type changes the AMF communities in the roots of maize crops remains to be investigated. Higo ngs Fig. . However74. Moreover, tillage management induced more changes to the AMF communities in maize roots than the type of cover crops. However, the questions of how and whether the level of tillage intensity alters the AMF communities in agricultural settings remain unanswered. Future studies on the interactions between the AMF communities and agricultural settings should focus on how tillage intensity affects the AMF communities in maize crops rotated with cover crops.In addition, AMF communities in maize crops varied distinctly, indicating that tillage practice altered the AMF communities in maize crops Fig. , in part76. In general, cover crop residues that have been incorporated into the soil surface by conventional tillage systems release allelopathic substances that can inhibit weed germination, establishment, and growth77. In contrast, in conservation tillage systems (including no tillage and reduced tillage), the nutrients from surface-broadcasted fertilizers and surface-applied crop residues accumulate and concentrate in the topsoil, where their movements are reduced78. In no-tillage systems, however, crop residue decomposition rates and nutrient release into the soil are slow compared to conventional tillage systems, including rotary tillage and chisel plow79, which may influence the soil microbial community structures80. These combined findings indicate that the differences between the effects of rotary and no-tillage management systems on the AMF communities in the roots of maize crops can be explained by cover crop residues releasing allelopathic substances and nutrients due to enhanced decomposition rates , ambient soils (20 °C), and warmed soils (26 °C)83. Heinemeyer et al.84 reported that shading and soil temperature changed AMF communities in roots, with some AMF taxa being replaced  by others and some disappearing throughout winter, summer and autumn. Higo et al.85 also reported on seasonal variation, finding a distinct difference between winter and spring AMF communities in the roots of winter cover crops. However, we did not investigate the effect of tillage systems and cover crop residues on soil temperature fluctuations. Future research should investigate how and whether AMF communities respond to environmental changes due to cover cropping and tillage management systems.Moreover, tillage can affect soil temperature by changing soil surface conditionsTillage management may be more important than cover cropping in shaping the AMF communities in maize crops. In addition, various kinds of cover cropping systems may produce various types of effects on the AMF communities in maize crop roots. The synergistic effects of tillage and cover cropping are partially responsible for shaping the AMF communities. Knowledge in this connection should be valuable in future experiments. This is because variations in the AMF communities in roots may influence crop growth performance in cropping systems with tillage management. However, we still need to clarify how the combination of tillage intensity and cover crop type is associated with individual AMF taxa in agricultural settings. Such an investigation will provide beneficial information on the appropriate cover crop selection and tillage practice for improving the functions of AMF taxa in cropping systems.Supplementary information 1.Supplementary information 2.


Computational studies of the Co-catalyzed remote nitration of 8-aminoquinolinamides have found the mechanism to operate through an unexpected high-spin induced remote radical-coupling. via a subtly different mechanism despite having a similar chelate environment.Previously, an unexpected Co-catalysed remote C–H nitration of 8-aminoquinolinamide compounds was developed. This report provided a novel reactivity for Co which was assumed to proceed through the mechanistic pathway already known for analogous Cu-catalysed remote couplings of the same substrates. In order to shed light into this intriguing, and previously unobserved reactivity for Co, a thorough computational study has now been performed, which has allowed for a full understanding of the operative mechanism. This study demonstrates that the Co-catalysed remote coupling does not occur through the previously proposed Single Electron Transfer (SET) mechanism, but actually operates through a high-spin induced remote radical coupling mechanism, through a key intermediate with significant proportion of spin density at the 5- and 7-positions of the aminoquinoline ring. Additionally, new experimental data provides expansion of the synthetic utility of the original nitration procedure towards 1-naphthylpicolinamide which unexpectedly appears to operate  However, the already known rich mechanistic diversity of Co in the field of C–H functionalisation makes this metal stand out as an exciting candidate for the basis of novel protocols.Functionalisation of quinolines beyond traditional C2 modification has recently become a topic of increasing interest10One novel approach to the functionalisation of 8-aminoquinolinamides has been the remote (non-proximate) C–H bond chlorination catalysed by Cu at the C7 position, which was originally reported by Stahl/Ertem and co-workers in 2013 .11 Non-p14tert-butyl nitrite (TBN) as nitro source  source” in the mechanism. Given the use of AcOH as solvent, the experimental conditions would allow for extensive counter-ion ligand exchange, leading to a number of potential Co(ii) species. Relative free energies for ligand exchange 2 over Co(OAc)2 in the original protocolin situ formed HNO3 in an acidic environment.The initial approach in this study was to identify the nature of the starting “Co, where this report revealed the highly complex mechanisms operative in Co-catalyzed C–H activation processes.After this initial study on catalyst species we embarked on full elucidation of the reaction mechanism. As a result of the moderate to large size of the catalytic system being studied, DFT calculations would traditionally be the method of choice. However, due to the potential for different spin states to be involved in the mechanism, which pose a significant challenge for DFT based methods, the recently implemented open shell DLPNO-CCSD(T) method was utilised.in situ formed tBuO˙ radical leading to the radical of the 8-aminoquinolinamide ligand, 1L. Coupling 1L to the [Co(AcO)2] pre-catalyst forms the initial catalytic species 1Int1·L. This species can exist in three possible spin states; low spin singlet , medium spin triplet  and high spin quintet . With the DLPNO-CCSD(T) method the high spin quintet state (5Int 1·L1) proved to be the ground-state species, which is in full agreement with previous work from Chen and co-worker.1Int 1·L1 and 3Int 1·L1 are Co(iii) complexes whereas the 5Int 1·L1 is actually a high-spin quartet Co(ii) species with the radical still residing on the quinoline moiety of the ligand. Cumulative spin densities on the aminoquinoline ligand, calculated using DLPNO-CCSD/def2-tzvp,1Int 1·L1, 3Int 1·L1 and 5Int 1·L1 are 0, 0.18 and 0.72, respectively. 5Int 1·L1. A similar spin population distribution was previously observed by Chen and co-worker leading to the description as a quartet Co(ii) ferromagnetically coupled with a ligand radical.20The first step of the reaction is hydrogen abstraction by the 1Int 1·L1 and 3Int 1·L1 being Co(iii) whereas 5Int 1·L1 being high spin Co(ii) with an associated radical ligand, based on the spin density analysis.Topological analysis using Quantum Theory of Atom in Molecules (QTAIM)2 radical coupling at the 5- and 7-positions starting from 5Int 1·L1 lie at 4.7 and 5.2 kcal mol–1 respectively  methods,–1 at most and therefore as a result we are confident this reaction proceeds via the proposed high spin pathway.The transition states for the remote NOectively . This lo1Int 1·L1. The first excited state transition, corresponding to the excitation of an electron from the 8-aminoquinolinamide ligand to the Co(iii) centre, is found to be in the range of 37–43 kcal mol–1, depending on the choice of DFT functional and amount of HF exchange included (see ESI for full detailsii) complex.1Int 1ES·L1, with the hybrid functional PBE0 provides a value of 24.4 kcal mol–1 above 1Int 1·L1. Although not directly comparable due to the differing methodologies used, both the transition energy and the optimised excited state complex is significantly higher in energy than the transition state on the proposed high-spin mechanism starting from the quintet ground state, 5Int 1·L1. As a result of the requirement for a coupling transition state after 1Int 1ES·L1, this precludes this mechanism on the grounds of it being energetically unfeasible.To ensure the previously proposed intramolecular Singlet Electron Transfers (SET) mechanism was not involved in the reaction, LR-TD-DFT  calculations were performed on 5Int 1·L1 results in the formation of the intermediate complex, 4Int 2·L1. This intermediate is 8.0 kcal mol–1 lower in energy than the preceding starting complex. The lower energy pathway leading to nitration at the 5-position forms 4Int 2·LA1 which is 3.8 kcal mol–1 more stable than the corresponding 7-substituted product (4Int 2·LB1), and 16.6 kcal mol–1 more stable than the equivalent low spin doublet mechanism (2Int 2·LA1). A number of different pathways for the deprotonation of 4Int 2·L1 at the 5- and 7-positions were explored. Addition of [Co(AcO)3]– to 4Int 2·L1 forms 5Int 3·L1, this step is exergonic by 9.7 kcal mol–1. Cobalt assisted deprotonation by acetate anion has a barrier of 9.8 kcal mol–1, leading to the final intermediate 4Int 4·L1]––– furnishes the chelate species of the final product, which can then undergo proto-demetallation by acetic acid to reform the initial Co(OAc)2 catalyst, releasing the final nitrated 8-aminoquinolinamide product.As a result of this new in-depth study, in adjustment to our previously proposed SET mechanism for the Co-catalysed remote nitration of 8-aminoquinolamide substrates, we now propose the mechanism shown in 2L,  with the quintet (S = 2) and triplet (S = 1) states being 2.2 and 3.7 kcal mol–1 higher in energy respectively  calculations suggest the ground state of the 1-naphthylpicolinamide complex is the singlet (ectively . TDDFT c5Int 1·L2 showed a significant proportion of density at the observed reactive 2- and 4-positions of the 1-aminonaphthyl moiety (via either a low spin (S = 1/2) or high spin (S = 3/2) pathway. As with the 8-aminoquiolinamide mechanism the high spin pathway, through 4TS 1-2·L2, is significantly more favourable. This suggests a Multi-State Reactivity (MSR) mechanism with crossing between potential energy surfaces,4Int 2·LA/B2, are approximately 25 kcal mol–1 more energetically stable compared to the equivalent low spin intermediate, 2Int 2·LA/B2. Interestingly the observed substitution at the 4-position is kinetically favoured, while the 2-position is the thermodynamic intermediate. This can be accounted for by the additional long range hydrogen bonding (2.45 Å) between the hydrogen at the 2-position of the nitrated naphthyl moiety and the Co bound acetate. The calculated higher energy barriers for 1-naphthylpicolinamide compared to 8-aminoquinolinamide agrees with the experimental requirement for more forcing reaction conditions . Increased spin density on the Co and decreased spin density on the 1-aminonaphthyl moiety further support this decrease in reactivity . Overall, the distinct mechanistic pathways shown in this work provide a complimentary tool for the remote C–H functionalisation of both 8-aminoquinolinamides and 1-naphthylpicolinamides. This new mechanistic understanding should now inspire others to develop new synthetic protocols for remote functionalisation using Co-catalysis.In summary, we have studied the mechanism of the remote nitration of 8-aminoquinolinamides catalysed by Co using the robust DLPNO-CCSD(T) methodology and found there to be a significant difference in mechanism to the well-studied analogous Cu-catalysed remote functionalisation protocols of 8-aminoquinolinamides. The results reported within this work identify an unusual mechanism which does not pass through the initially proposed SET process, but instead, the reaction takes place There are no conflicts to declare.Supplementary informationClick here for additional data file.


Fluorinated graphene has a tunable band gap that is useful in making flexible graphene electronics. But the carbon–fluorine (C–F) bonds in fluorinated graphene can be easily broken by increased temperature or electron beam irradiation. Here, we demonstrate that the stability of fluorinated graphene is mainly determined by its C–F configuration. The double-sided fluorinated graphene has a much stronger stability than the single-sided fluorinated graphene under the same irradiation dose. Density functional theory calculations show that the configuration of double-sided fluorinated graphene has a negative and low formation energy, indicating to be an energetically stable structure. On the contrary, the formation energy of single-sided fluorinated graphene is positive, leading to an unstable C–F bonding that is easily broken by the irradiation. Our findings make a new step towards a more stable and efficient design of graphene electronic devices. Utilizing the superior properties of this two-dimensional (2D) material, such as the exceptionally high charge mobility2, high optical transparency3, excellent flexibility4, ultrahigh mechanical strength and thermal conductivity9, pristine graphene can be used in a wide range of applications. However, the special feature of zero bandgap in graphene has greatly limited its application as a semiconductor12. Consequently, it is important to open the bandgap of pristine graphene through the bandgap engineering and explore its utilization as a novel 2D semiconductor. Fluorination has been proved to be an efficient method to modify the electronic structures of graphene. The band gap in the fully fluorinated graphene (F/C ratio is 50%) can be opened up to 3.07 eV based on the theoretical calculation13. The rise of fluorinated graphene makes the flexible and transparent all-graphene-electronics possible14. By simply changing the fluorine/carbon (F/C) ratio of this atomic layer, the electrical characteristic of fluorinated graphene can be tuned from conductive, semiconductive to insulating. The electron beam lithography (EBL) and direct electron beam irradiation have been practically used to open the nanometer-sized conductive channels in the fluorinated graphene and fabricate the all-graphene-electronic devices16.Graphene has attracted intense attention since its discovery20. The conventional synthesis methods include chemical fluorination and physical exfoliation: the former includes direct gas fluorination21, plasma fluorination24, hydrothermal fluorination25, etc.; the latter includes sonochemical exfoliation27, modified Hummer’s exfoliation28, thermal exfoliation29, etc. The F/C ratio in the fluorinated graphene depends on the fluorine agents or exfoliation solvents, the reaction temperature and time. By using the same synthetic method, higher reaction temperature and longer reaction time will result in a higher F/C ratio.Synthesis of fluorinated graphene and its stability are important for designing the flexible and printed electronic devices31 and electron beam irradiation14. A short time of annealing at temperatures below 400 °C or low-dose electron beam irradiation will partially recover fluorinated graphene to pristine graphene. A direct proof for the recovery of fluorinated graphene is the decrease in the D-band Raman spectra and significant increase in the electrical conductivity. Prolonged annealing time at high temperature and high-dose electron beam irradiation may remove both C and F atoms in the fluorinated graphene31. On the other hand, single-sided fluorinated graphene with low F/C ratio prepared by using XeF2 gas will lose 50–80% initial fluorine coverage over 10 days before reaching a saturated state32. All these facts show that the fluorinated graphene possesses a poor stability over storage time, temperature and irradiation, which severely limits the practical utilization of fluorinated graphene as a candidate of 2D semiconductor. It is interesting to know that the stability of fluorinated graphene is related to the F/C ratio of sample. A highly fluorinated graphene on substrate remains insulating after 10 days while a mildly fluorinated sample is partially recovered to its pristine state. It reflects the fact that stability of fluorinated graphene depends on the site and structure of C–F bonds, but the physical mechanism remains unclear. Understanding the stability of C–F bonding at molecular level and its electrical property is the key for the practical design of fluorinated graphene-based 2D semiconductors.So far, there are a lot of works focused on the synthesis of the fluorinated graphene, while less attention has been paid to the stability of fluorinated graphene. Recently, researchers found that the stability of fluorinated graphene is sensitive to both temperature2 gas. Subsequently, the electron beam irradiation was used to selectively remove the fluorine atoms on graphene, and the resultant variation of the sample’s electrical conductivity was monitored continuously. The measurement results show that the electrical conductivity of double-sided fluorinated graphene remained unchanged after electron beam irradiation, while the electrical conductivity of single-sided sample was significantly increased due to the loss of fluorine atoms. Such comparison demonstrates that the structure of C–F bonding at both sides of graphene has much stronger stability than that at the single side. First-principle calculations further confirmed the symmetric charge distribution and stable formation energy in the double-sided fluorinated graphene, which explains the strong stability of double-sided C–F bonding at molecular level. This work highlights the importance of double-sided fluorination to achieve stable chemically functionalized graphene samples, which can be used in the practical design of all-graphene transparent and flexible electronics.In this work, we have synthesized both single-sided and double-sided fluorinated monolayer graphene by using XeF31. At first, a monolayer graphene was grown on copper foil by using a conventional chemical vapor deposition method. Then, a thin layer of polymethyl methacrylate (PMMA) was spin-coated on the surface and used as supporting layer during the transfer process. The copper foil was etched away and the graphene/PMMA layer was transferred onto a SiO2/Si substrate. After that, the graphene was cut into micro-ribbons following the EBL patterning and O2 plasma etching. In order to measure the electrical properties of functionalized graphene sample, a thin metallic film (10 nm thick Cr and 100 nm thick Au) was deposited on the graphene ribbon by using an electron beam physical vapor deposition method. The patterned metallic electrodes were made by using EBL and a standard lift-off method. Then, the graphene was chemically modified following two schedules: (1) For the single-sided fluorinated graphene, the graphene supported on SiO2/Si substrate was directly put into a XeF2 gas etcher (BP-3F Samco Inc.). The thin SiO2 passivation layer blocked the contact between XeF2 gas and backside of graphene, thus only the top surface of graphene was fluorinated in the XeF2 gas. (2) For the double-sided fluorinated graphene, the thin SiO2 layer was removed in a buffered hydrofluoric solution. Then, the graphene ribbon partially supported on silicon was put into the XeF2 gas etcher and the silicon substrate was etched by a depth around 8 μm. Finally, the graphene ribbon was suspended between two metallic electrodes above the substrate. In this way, both top and bottom surfaces of graphene were fluorinated in the XeF2 gas. The fluorination of graphene was done at room temperature. The F/C ratio of fluorinated graphene can be tuned by controlling the exposure time in the XeF2 gas. Scanning electron microscope (SEM) images of tested samples can be found in Fig. The fabrication process of fluorinated graphene is given as following37. In the fabrication process, the SiO2 layer was removed by using a buffered hydrofluoric acid (BHF) through a micrometer sized window opened in the polymer resist layer. Due to the isotropic etching behavior of BHF, a part of SiO2 below graphene and metal was removed. The length of over-etching was measured to be 2.5 μm in the SEM image of device taken after released from the silicon substrate by using XeF2 etching. So it is seen in Fig. 2 under the metallic source, SiO2 between source and drain, and a 2.5 μm wide SiO2 section under the metallic drain were removed. But SiO2 in the middle of the drain pad remains and forms an Au/Graphene/SiO2 sandwich structure, shown as the light green part in the figure. XeF2 gas cannot permeate the SiO2 layer, so the graphene between Au and SiO2 still remains its pristine crystalline structure. In Fig. 38. This indicates that the fluorinated graphene is almost transparent in the range of visible light with energy from ~ 1–3 eV38. The absorption spectrum of partially fluorinated graphene is higher than that of the fully fluorinated graphene, and slowly increases as the photon energy increases. Hence, the fluorinated graphene with low F/C ratio appears semi-transparent under the light. As seen in Fig. Figure Raman spectra, X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) data were collected on the fabricated samples.2 gas on the top surface of supported graphene, the 2D-band peak is dramatically decreased, while the D-band peak increased and exceeds the G-band peak. All the Raman peaks of fluorinated graphene are widened and the baseline has been raised, comparing with the Raman spectrum of pristine graphene. These features indicate that the fluorination will break the symmetrical two-dimensional lattice structure of graphene and increase its defect level. In the case of DSFG, the baseline of Raman spectrum keeps increasing and the ratio between 2D-band and G-band peaks is further decreased. This indicates a higher level of fluorination to the graphene lattice.Figure 2 layer under graphene. The Si and O signals were missing for the suspended DSFG sample.Figures Figure 14. It is an effective and precise tool to investigate the stability of C–F bonding. In order to conduct in-situ electrical characterization of fluorinated graphene in the SEM chamber, four-lead wires were fed through to connect the graphene sample and two high-precision Keithley digital multimeters (2002 series). The I-V curve of fluorinated graphene was measured by using a four-probe method. Electrical measurement inside the SEM chamber (< 10–4 Pa) prevents contamination or dopant in the atmospheric environment, which may further change the resistance of graphene sample. A low energy electron beam  was used for observation in the experiment, which does not significantly affect the electrical property of graphene14.It has been reported that the electron beam irradiation can be used to remove the fluorine atoms on graphene and regain its electrical conductivityI-V curve of SSFG sample #4. The whole SSFG ribbon between source and drain was uniformly irradiated by an electron beam, with a dose gradually increased from 0 to 0.40 C/cm2. A similar experimental result14 of single-sided fluorinated graphene with a dose up to 1.00 C/cm2 is also included in Fig. I-V curve of SSFG sample shows a typical symmetric behavior with respect to the bias voltage ranging from − 9 V to 9 V. Higher electron beam irradiation dose removes more fluorine atoms from graphene surface, and the source-drain current Isd increases monotonically. Hence, the resistance per square of SSFG decreases quite rapidly with the increasing irradiation dose, as shown in Fig. 14. The fluorine atoms on graphene skeleton reduce the charge in the conducting orbitals and introduce more scattering centers. Thus, the resistivity of fluorinated graphene is much larger than its pristine state, and is highly sensitive to the F/C ratio. Although four SSFG samples have almost the same geometric size and crystalline quality as a pristine graphene before fluorination, the fluctuation in the XeF2 gas pressure and reaction time may cause significant difference in the resistances of SSFG samples. As for SSFG #3, the resistance was decreased by 200 times after electron beam irradiation and approached a saturation value of the pristine graphene. This result demonstrates that the single-sided fluorinated graphene can be easily tuned from an insulator to a conductor by simply applying electron beam irradiation. However, the electrical property of SSFG is not so stable in two aspects: (i) the resistivity of sample is highly sensitive to the synthesis process of fluorination, and (ii) the resistivity of partially fluorinated graphene may change due to various reasons, such as irradiation, temperature, dopant in the atmospheric environment or storage time32. This can be understood by the fact that even a low energy electron beam irradiation can alter the resistance of SSFG, and the other factors  at the same energy level will have the similar impact.Figure I−V curve and resistance per square of DSFG samples. The I−V curve in DSFG sample represents a typical asymmetric diode characteristic, which is totally different from the metallic symmetric electrical behavior of SSFG shown in Fig. 43, a charge rectification behavior is noticed in the metal/fluorinated graphene (MFG). The graphene below metallic film was fluorinated at the bottom surface with an opened band gap up to 2.8 eV13. The difference in the work functions between metal and fluorinated graphene causes a potential barrier at their interface, leading to the charge rectification phenomenon. Here, the thickness of MFG heterojunction is mainly decided by the metallic film, because the fluorinated graphene only has a thickness less than 1 nm. It has a potential to create a vertically stacked diode within several nanometers by reducing the thickness of metallic film. For the SSFG samples supported on SiO2/Si, no charge rectification behavior was shown in Fig. 2 has a zero band gap. This result proves the validity of building nanoscale diodes by using fluorinated graphene with a tunable band gap.Figure I-V curve of DSFG was measured from − 8 V to 8 V at each irradiation dose. Extreme caution should be paid to load the bias voltage on DSFG, and it took a relatively long time to obtain a single experimental data point in Fig. 2 dose irradiation. In contrast, the same electron beam irradiation didn’t cause any reduction in the resistance of DSFG, but instead slightly increased the resistance. These results all proves the fact that the C-F bonding in DSFG has a much stronger stability than that in SSFG.In addition to the charge rectification at the MFG interface, the main finding in Fig. 45. The electron–ion interactions are described by the projector-augmented-wave (PAW) method46. The Perdew-Burke-Ernzerhof (PBE) functional within the generalized-gradient approximation (GGA) is used to treat the exchange–correlation interaction of electrons47. A kinetic energy cutoff of 400 eV for the planewave basis and a convergence criterion of 10–7 eV for the total energies are adopted for all ab initio calculations. The van der Waals (vdW) interaction is included according to the Tkatchenko and Scheffler method48.In order to understand the mechanism responsible for the ultra-strong stability observed in DSFG, first-principle calculations were carried out by using the Vienna Ab initio Simulation Package (VASP) based on density functional theory (DFT) and planewave basiseF) by performing ab initio calculations. Take the suspended graphene for instance, the eF is calculated as49:EFG, EF and EG are the total energy of fluorinated graphene, one fluorine atom, and pure graphene sheet, respectively. NF is the number of fluorine atoms in the calculation cell.To gain insight into the stability of fluorinated graphene, we calculated the averaged formation energy per fluorine atoms  and silicon dioxide (SiO2) are used as the substrate in our calculations, where are two typical dielectric substrates in literatures51. The stability of SSFG supported on the substrate of h-BN and amorphous SiO2 was studied respectively. As shown in Fig. h-BN substrate is relatively flat, while it is notably curved on amorphous SiO2 substrate. The eF of the supported graphene was calculated similar as Eq. (3) after replacing EFG with the total energy of fluorinated graphene with substrate, and EG with the total energy of pure graphene with substrate. The eF of the supported fluorinated graphene on h-BN and SiO2 substrates are 1.4854 eV and 1.5144 eV, respectively. Obviously, the positive values of eF indicate an unstable C–F bonding. Therefore, the defluorination in the single-sided fluorinated graphene can be easily induced by the electron beam irradiation as observed in experiment. In addition, the rough surface of amorphous SiO2 substrate causes fluctuation in the supported SSFG structure, which makes the defluorination process even easier.For the case of DSFG, we consider the structure of the fully fluorinated graphene (F/C ratio is 100%). The optimized crystal structure is shown in Fig. 52. As shown in Fig. h-BN and graphene leads to the charges in graphene dangling on the substrate side, as shown in the DCD of Fig. eF between neighbor C atoms in DSFG is − 3.380 eV, which is higher than the eF between fluorinate atoms and graphene layer (− 1.0534 eV). This calculation indicates the defluorination can be achieved without damaging the graphene layer as observed in the previous study13, which further makes the resistance of fluorinated graphene approaching to the value of pristine graphene during the defluorination process.To further understand the bonding nature and stability in the fluorinated graphene, we calculated the deformation charge density (DCD)Fluorination has been proved to be an efficient way to tune the band gap of graphene and control its electrical properties. Furthermore, electron beam irradiation was proved to be an efficient method in patterning the conductive channels in the fluorinated graphene, wherein the stability of fluorinated graphene is an important factor. In this report, we have fabricated electronic device with fluorinated graphene in two different configurations, double-sided fluorinated graphene and single-sided fluorinated graphene. The results show that the double-sided fluorinated graphene has an ultra-strong stability against the electron beam irradiation, while the single-sided fluorinated graphene loses the fluorine atoms easily even at a low irradiation dose. The first-principle calculations reveal that there exists a strong covalent bonding between the fluorine and carbon atoms in the double-sided fluorinated graphene, which well explains the ultra-strong stability observed in the experiment. Furthermore, a significant charge rectification behavior was observed at the interface between metal and fluorinated graphene. Our study provides important insights to the understanding of the unique chemical bonding characteristics in suspended graphene sheet, which is crucial for the fabrication of more stable and efficient graphene electronic devices.


Background: Lineage 1 (L1) and 3 (L3) are two lineages of the Mycobacterium tuberculosis complex (MTBC) causing tuberculosis (TB) in humans. L1 and L3 are prevalent around the rim of the Indian Ocean, the region that accounts for most of the world’s new TB cases. Despite their relevance for this region, L1 and L3 remain understudied.Methods: We analyzed 2,938 L1 and 2,030 L3 whole genome sequences originating from 69 countries. We reconstructed the evolutionary history of these two lineages and identified genes under positive selection.Results: We found a strongly asymmetric pattern of migration from South Asia toward neighboring regions, highlighting the historical role of South Asia in the dispersion of L1 and L3. Moreover, we found that several genes were under positive selection, including genes involved in virulence and resistance to antibiotics. For L1 we identified signatures of local adaptation at theesxH locus, a gene coding for a secreted effector that targets the human endosomal sorting complex, and is included in several vaccine candidates.Conclusions: Our study highlights the importance of genetic diversity in the MTBC, and sheds new light on two of the most important MTBC lineages affecting humans. The global tuberculosis (TB) epidemic is a major public health emergency, disproportionately affecting vulnerable populations and worsening existing inequalities. Although the estimated incidence and the number of fatalities have slowly decreased over time, every year ten million people develop the disease, and 1.4 million TB patients die . MoreoveMycobacterium tuberculosis complex (MTBC), which includes nine phylogenetic lineages with different geographic distributions. Five of these lineages are restricted to Africa , while the remaining four  are more broadly distributed .We screened a large collection of publicly available MTBC genome sequences and selected those belonging to L1 and L3. Additionally, we newly sequenced 767 strains to cover regions that were not well represented in the public data. We applied a series of bioinformatic filters to exclude low quality sequences and mixed infections (Methods), and obtained a curated genome dataset of 4,968 strains . While the phylogenetic tree of L3 showed a ladder-like topology without an evident division in sublineages, L1 comprised five clearly distinct sublineages coalescing close to the root of the tree . Because we were interested in studying L1 and L3 in their endemic range, we excluded strains originating from North America, Europe, and Australia from the biogeographic analysis, as in these low-burden regions, most TB patients are recent migrants who were infected in their country of origin .The genome sequences included in our final dataset represented 69 countries and covered the known geographic range of L1 and L3 . Most interestingly, we found a strongly asymmetric pattern of migration. For L1, PASTML identified most migration events from South Asia toward other regions, followed by further dispersion, but almost no back migration to South Asia . When we estimated the migration rates with Mascot, we found that the forward migration rate from South Asia toward the rest of the world were 3 to 17 times larger than the migration rates in the opposite direction, confirming the results of PASTML (Extended data: Table 2). We found a similar scenario for L3: PASTML inferred the largest number of migrations from South Asia toward East Africa, further spread from East Africa toward neighboring regions, but essentially no migration back to South Asia (Extended data: Files 3–4). The Mascot analysis showed that the forward migration rates from South Asia toward East Africa were 26 times larger than the migration rates in the opposite direction (Extended data: Table 2).We performed a phylogeographic analysis using two alternative approaches (Mascot and PASTML), which are based on different models and inference methods . However, the results of a previous study suggested a relatively fast evolutionary rate for L1 . By contrast, L3 had a good temporal signal, and different methods estimated that its MRCA lived between the 2nd and the 13th century AD. However, the uncertainty around all of these estimates was very large . Together, these results corroborate the findings of previous studies .With respect to the geographical aspects, we identified several interesting instances of potential long-range dispersal. First, we found a clade of L1, composed of 11 strains sampled in five different West African countries. This was surprising because West Africa is usually not considered part of the geographic range of L1. This clade is nested within sublineage L1.1.1, which is essentially only found in mainland Southeast Asia, and the PASTML analysis inferred a direct introduction from Southeast Asia to West Africa. This introduction is unlikely to have happened before the 16Extended data: Files 1 and 2). Assuming a clock rate of 1.4×10-7, the earliest introduction was between 1620 and 1830 AD from East Africa, while subsequent introductions occurred from East Africa, South Africa, and South Asia. These results support the hypothesis that L1 was first introduced to Brazil through the slave trade from East Africa . This would be compatible with early Portuguese expeditions, which reached East Timor and Papua New Guinea in the early 16th century. An alternative explanation could be the Austronesian expansion. Austronesians are thought to have reached the Comoros islands and Madagascar between the 9th and the 13th century AD, possibly through direct navigation from Southeast Asian islands. Malagasy speak an Austronesian language, and Austronesian genetic signatures are found in human populations in the Comoros, Madagascar, and to a small extent also in the Horn of Africa . However, this difference can be explained by the different size and diversity of the two datasets . The epitope that accumulated most mutations was located at the N terminus ofesxH (Rv0288). This peptide is a T cell epitope for both classes, MHCI and MHCII; it is also a B cell epitope and was previously identified as one of the few T cell epitopes that were not hyper-conserved (Extended data: Table 7). Interestingly, we did not find this signature in L3, as all strains carried the ancestral haplotype at the N-terminalesxH epitope. Moreover, when we extended the analysis to two large genomic datasets of L2 and L4 strains, we found a much weaker signal: while 21% of L1 strains carried a derived haplotype for this epitope, only 1% of L2 and L4 strains, and no L3 strain had a derived haplotype. Despite analyzing datasets with more strains , and more polymorphic positions  compared to L1, we found only three amino acid replacements at the N-terminal epitope ofesxH in L2, and seven in L4  in at least one L1 strain, while only 20% were variable in at least one L3 strain  but not in the other lineages . PAML identified four codons that have been under positive selection in L1 (posterior probability > 0.95), all of them within the N-terminal epitope  amino acid replacements occurred in sublineages L1.1.1 and L1.2.1, which are almost exclusively present in Southeast Asia . We constructed a statistical test of association similar to phyC (esxH epitope were significantly associated with a particular geographic region (Methods). We found that South African strains were less likely to harbor a derived haplotype in the N-terminalesxH epitope than expected by chance  with different HLA-A, HLA-B and HLA-DRB1 alleles (Methods). We performed this analysis for:We hypothesized that the accumulation of missense mutations at the N-terminal epitope of1) Alleles found at high frequency (> 10%) in South- and Southeast Asia, but not in East Africa.2) Alleles found at high frequency (> 10%) in East Africa, but not in South- and Southeast Asia.3) Alleles found at high frequency (> 10%) in both regions.Extended data: Table 8).However, we found no differences in the predicted binding affinities between alleles with different geographic distributions . We found two genes coding for transmembrane proteins, members of the Esx-1 secretion system, which were under positive selection in L1 , and several genes involved in antibiotic resistance that were under positive selection in both lineages (Extended data: Information). We further characterized the profile of drug resistance mutations of L1 and L3, and found that L1 strains harbored a greater proportion ofinhA promoter mutations (conferring resistance to isoniazid) compared to L3 strains, confirming previous findings . WhileOur results highlight the central role of South Asia in the dispersion of L1 and L3. First, we confirmed that the two lineages probably expanded from South Asia . As we already mentioned, tip-dating analyses of MTBC trees with roots that are several hundreds of years old is extremely challenging, and the results of such analyses should be taken with caution . It is known that L1 strains interact differently with the infected hosts compared to other lineages. For example, L1 strains show a lower virulence in animal models . Region-specific signatures of positive selection are a hallmark of local adaptation; in this case, adaptation of L1 strains to human hosts with South- and Southeast Asian genotypes. This corroborates previous studies reporting that in Taiwan, L1 is associated with indigenous populations with Austronesian ancestry (reviewed inesxH after Southeast Asia (islands), 37.3% and 72.8% respectively, opposed to 0.3% (one single strain) in the rest of East Africa.The signatures of selection at the N-terminal epitope ofesxH were contained in one single T cell epitope, the selective pressure acting onesxH could be due to some other factor, and not to the binding of the epitope by the MHC, or to its recognition by T cells. A possible mechanism was suggested by experiments in a mouse model, showing that the N-terminal epitope of EsxH generates an immunodominant CD8 T cells response. The amino acid replacement A10T  did not change MHC binding or T cell recognition, but it accelerated the degradation of the epitope, disrupting the immunodominant response , impairing trafficking, and hindering phagosomal maturation and antigen presentation, thus contributing to the survival of the pathogen , in MTBC strains belonging to L2 . Because of these limitations, our biogeographical analyses were limited to the subcontinental level. This approach revealed the global population structure and the main macroscopic patterns of diversity and migration of L1 and L3. However, MTBC populations are diverse also within subcontinental regions. For example, the MTBC population in Southern India is dominated by L1, while the most prevalent lineage in the North is L3  of MTBC strains belonging to L1 and L3, using the diagnostic SNPs described inhttps://github.com/jstjohn/SeqPrep). The resulting reads were mapped to the reconstructed MTBC ancestral sequence (https://github.com/broadinstitute/picard). The RealignerTargetCreator and IndelRealigner modules of GATK v.3.4.0  .We applied the following filters: genomes were excluded if they had 1) an average coverage <15x, 2) more than 50% of their SNPs excluded due to the strand bias filter, 3) more than 50%  of their SNPs having a percentage of reads supporting the call between 10% and 90%, 4) contained single nucleotide polymorphisms diagnostic for different MTBC lineages  with raxml 8.2.11 . For both lineages, we built SNP alignments including only variable positions with less than 10% of missing data. We inferred phylogenetic trees with RAxML 8.2.11 , using tWe used the least square method implemented in LSD v0.3-beta  random subsampling 2) random subsampling keeping at least 25 genomes for each year of sampling . For this second subsampling strategy, we used Treemmer v0.3  prior instead of the exponential growth prior, and performed a nested sampling analysis .For the biogeography analysis, we considered only genome sequences obtained from strains for which the locality of sampling was known. When the country of sampling did not correspond to the country of origin of the patient (or was unknown), we considered as sampling locality the country of origin of the patient . Furthermore, similarly to other studies . We inferred phylogenetic trees with raxml 8.2.11 , using t-7 for L1, and 9×10-8 for L3. In this analysis, genomes for which the sampling date was not known were assumed to have been sampled between 1995 and 2018, which is the period in which all strains with known date of isolation were sampled. Importantly, using different clock rates for this analysis would only change the time scale of the trees, but not the reconstruction of the ancestral characters.Since PASTML needs a time tree as input, we calibrated the phylogenies with LSD, assuming a clock rate of 1.4×10We assigned to each strain the subcontinental geographic region of origin as character, and used PASTML  and 9×10-8 (L3). We assigned the tip sampling years to the different strains, and when the sampling time was unknown, we assumed a uniform prior from 1995 to 2018 . We further assumed the best fitting nucleotide substitution model as identified by jModelTest, a gamma prior for the standard deviation of the lognormal distribution of the clock rate [0 –infinity], and a lognormal prior for the population size with standard deviation = 0.2, and mean estimated in real space. Finally, we used an exponential distribution with mean = 10-4 as prior for the migration rates. For each analysis, we ran at least two runs. We used Tracer 1.7.1  an average coverage <15x, 2) more than 50%  of their SNPs having a percentage of reads supporting the call between 10% and 90%, or 3) contained single nucleotide polymorphisms diagnostic for different MTBC lineages (Extended data: Table 1). We reconstructed the phylogenetic tree of L2 with raxml 8.2.11 . We considered separately MHCI epitopes and MHCII epitopes, and we analyzed overlapping epitopes individually. We mapped the epitope sequences onto the H37Rv genome (GCF_000195955.2) using tblastn and excluded sequences mapping equally well to multiple loci in the H37Rv genome. Additionally, we retained only epitopes that mapped with two mismatches or less over the whole epitope length. This resulted in a final list of 539 MHCI epitopes, and 1,144 MHCII epitopes. (Extended data: Table 7). We used the datasets obtained for the biogeography analysis .We downloaded the amino acid sequence of all MTBC epitopes described forFor each lineage, we independently assembled a multiple sequence alignment for each epitope. We then translated the sequence to amino acids and used PAUP 4.a  to reconesxH (MSQIMYNYPAMLGHAGDM), which was by far the epitope with most amino acid replacements in L1. We expanded the analysis of this epitope to the L2 and L4 datasets, so that we could compare the results with L1 and L3. For the PAML analysis, we randomly selected 500 strains from each MTBC lineage. We used the phylogenetic tree reconstructed by RAxML (same settings as above), and the gene alignment to estimate the branch lengths of the tree using the M0 codon model implemented in PAML 4.9e , modeling nearly neutral evolution; M2a assumes one additional ω (ω2 > 1) compared to M1a, thus modeling positive selection. We performed a likelihood ratio test between the two models as described inExtended data. The codon under positive selection were identified with the Bayes empirical Bayes method  to PhyC  Alleles that had a frequency of 10% or more in at least one population in South- and Southeast Asia, but had frequencies lower than 10% in all East African populations.2) Alleles that had a frequency of 10% or more in at least one population in East Africa, but had frequencies lower than 10% in all populations in South- and Southeast Asia.3) Alleles that had a frequency of 10% or more in at least one population both in South- and Southeast Asia and in East Africa.in silico binding prediction with three epitopes: the ancestral epitope at theesxH N terminus (MSQIMYNYPAMLGHAGDM), and the two most frequently observed derived epitopes . For HLA-A and HLA-B alleles, we used the NetMHCPan4.1 server (https://www.iedb.org/).For all these alleles, we performedesxH. We considered as under positive selection all genes, for which the likelihood ratio test resulted in a Bonferroni-corrected p-value < 0.05.For this analysis, we used the subsets generated for the Mascot analysis. These datasets are representative of the populations of L1 and L3 in their core geographic ranges, and are computationally treatable. We generated sequence alignments for all genes individually, excluding genes in repetitive regions of the genome (see above). Because some genes are deleted in L1 but not in L3, or the other way around, we obtained a slightly different number of gene alignments for the two lineages . For each gene, we performed a test for positive selection with PAML as described above forWe considered 196 mutations conferring resistance to different antibiotics , and conhttps://www.ncbi.nlm.nih.gov/sra) under the accession number PRJNA670836.The sequence data generated by this study has been deposited on SRA . As far as I can tell, this calibration is the major driving force for the difference in L1 TMRCA from O'Neill. It would be interesting to know how the degree to which this calibration affects the TMRCA and clock rate. On a somewhat related note, this article fails to cite some key references on the phylogeography of TB. In one section the authors evaluate association between esxH haplotypes and the geographic region of origin. They calculate this association using a home-made statistical test based on phyC. I could not gather from the description exactly what they measure. It is a simulation-based test that measures "how many strains from each region resulted to have a derived state according to the simulation". Is there a test statistic being ranked here? Is it single-region vs the rest or is the statistic a composite measure of multiple regions? How is the simulation for the null distribution set up? E.g. is it a forward-in-time simulation using a specified mutation rate or a permutation or something else? I see that the code for running this test is posted on github - However, I still think a few more details in the actual paper is in order for the biologists that don't read R. Speaking of this test, It is a bit unconvential for Table 1 to highlight all results with P<0.1, especially since a large number of associations are being tested here. I would not consider p<0.1 to be particularly strong evidence of association. I'd like to commend the authors for making so much of their data publicly available. Datasets are in immediately-usable formats which makes it simple to download all the raw data, and as mentioned code is also readily available. HLA abbreviation never explained in manuscript.Tip date randomisation performed, but the results of this test is never stated.Why is PRJNA630228 posted? As far as I can tell it doesnt contain any SRA data, and the other accession contains data for all 767 strains. Very minor:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Bioinformatics, microbial genomicsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thank you very much for your comments, and for reviewing our manuscript. We address all your points below. We prepared a revised version of the manuscript based on the feedback of all reviewers.Regarding the clock analysis of L1:et al. 2019) where we analyzed a different L1 dataset and found a good temporal signal and a clock rate of ~1.4x10-7. So far Menardoet al. 2019 is the only published study that estimated the clock rate for a L1 dataset. We performed a tip dating analysis but found no temporal signal for L1. Therefore we used the clock rate estimate resulted from a previous study  were compatible with the hypothesis of a West African introduction of L1.1.1 posterior to the Portuguese circumnavigation of Africa. We found that only the largest clock rate was compatible with this hypothesis. In a second step we tested whether this clock rate and two alternative values, 5x10et al. 2019: “This discrepancy is due to different assumptions about the clock rates of L1, but none of the two hypotheses can be excluded with the available data”. We are quite explicit in the manuscript about the limitation of this analysis, and how it should be interpreted in comparison to the results of O’Neill Finally, it is possible that the West African L1.1.1 clade did not originate through a direct migration from South East Asia. However, a series of short range migrations seems less likely, as we did not find strains belonging to L1.1.1 in South and Central Asia, or in East Africa. As we write in the manuscript, the dating analyses for MTB in general, and for this L1 dataset in particular, have limitations. Here we discussed what we consider the most likely scenario given the data, we did not exclude alternative scenarios. We added a section in the discussion comparing our findings with the biogeography of other MTBC lineages, and referencing the relevant studies. We revised and expanded the Methods section regarding the phylogenetic test of association to provide more details. To answer the specific questions: the statistic ranked is the number of tips with a derived state; the test is performed for each region individually; the simulation does not use a mutation rate, but it distributes randomly a fixed number of mutations on the tree (the same number inferred from the observed data). We also removed special fonts from Table 1, now all p-values have standard formatting.Minor points: We now write HLA in full at the first occurrence. The results of the DRT are reported in the supplementary Information (now amended to enhance clarity), in Sup. Figs 8 and 9 and in Sup. Table 3. In the main text we refer more generally to the lack of temporal signal , as this reflects the results of the DRT, but also of the Beast analyses. We now refer only to PRJNA670836, which contains data for all the newly sequenced strains. Thanks. Mycobacterium tuberculosis of Lineages 1 and 3. Historically, and while forming an important part of theM. tuberculosis species diversity, the study of both lineages have been neglected in comparison to Lineages 2 and 4. The authors of this study assembled a large genomic dataset encompassing 2938 and 2030 L1 and L3 isolates, respectively, and by implementing distinct molecular evolutionary algorithms were able to put forward a scenario for the dissemination of these strains. A South Asian origin is proposed for both clades from where these strains appeared to have radiated via migration-driven dispersion towards East Africa (L3) and other regions (L1). The weakest aspect of the work is the lack of phylogeographic and historical context of L1 and L3 versus L2 and L4 dissemination, especially L4 in South-America and L2 in Asia in the context of the migratory waves.This study depicts a very interesting and relevant account on the evolutionary history and phylogeography ofesxH derived haplotypes were not randomly distributed through the geographic range of L1 and the finding of positive selection acting on vaccine candidates, such as this, is important and deserves attention. The authors used PASTML and Mascot to conduct the phylogeographic analyses, revealing the migratory pattern of these lineages from South Asia revealing the origins of these two lineages. Several genes involved in virulence and drug resistance were shown to be under positive selection in L1 and L3 strains with important adaptative consequences. Importantly,th century that impacted the dissemination of L1 in Africa and other regions. The manuscript is very well written, easy to read and understand and the methods are robust and well applied and described. Important to note the absence of a few important references to other groups work on the third wave migration of migrations in the 16Some comments that can be useful for discussion: The analysis pertaining the introduction of L1 to South America is based on a more modest number of isolates (n=77), all but one from Brazil. This limits the robusteness of the conclusions for this region. It is possible that the evolutionary history of L1 in South America is far more complex than the one outlined in the article and there may be missing links. For example, EAI (L1) strains have been detected in several South and Central American countries  and besides slave trade, the Spain-operated Manila-Acapulco Galleon trade route (est. ca. 1568) connected Southeast Asia to Central and South America. Regarding the L1.1.1 sub-lineage, what precludes these strains to have been introduced to West Africa in a more recent period other than the XVI or the XVII century? As far as we can tell, this pertains a group of 10 isolates scattered over five countries, but what is the degree of divergence between these isolates? Eventually, can this also be linked with the indentured labor promoted by the British Empire in the XIX century? On the epitope analysis, upon downloading epitopes from IEDB did the authors include only epitopes with positive assays and was there a threshold for including only epitopes validated by independent assays (more reliable)? Also, how did overlapping epitopes been handled?Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Mycobacteriology, Tuberculosis, Laboratory Diagnosis and Molecular Epidemiology.We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thank you very much for reviewing and commenting our manuscript. We prepared a revised version of the manuscript based on the feedback of all reviewers. Regarding the comparison with other lineages: in this manuscript we focused on L1 and L3 because they are understudied. We now revised the text and added a section comparing our findings with the biogeography of other MTBC lineages, and referencing the relevant studies. Additionally, it is true that the evolutionary history of L1 in South America could be more complex, and we might fail to capture it because we have samples from a single country (Brazil). We now explicitly write this.th century is the earliest possible time for the introduction of L1 in West Africa (assuming that the migration could not have happened before the circumnavigation of Africa by Portuguese), we do not exclude the possibility of a later introduction. However, this group of 11 L1 strains comprising the West African clade has a relatively old MRCA. To be compatible with an introduction in the 19th century, the molecular clock rate would need to be about 3x10-7 . This would be a fast rate for MTB , and we find this hypothesis unlikely. Nevertheless, because of the lack of a temporal signal we cannot exclude a more recent introduction of L1 in West Africa. Regarding the L1.1.1 sub-lineage: in the manuscript we write that the 16 Finally, in the epitope analysis we downloaded all T cell epitopes, to be as inclusive as possible. Overlapping epitopes were analyzed independently. We added this information to the manuscript. Mycobacterium tuberculosis complex (MTBC) strains belonging to Lineages 1 and 3, which are the most prevalent lineages in South Asian countries with a high TB burden, such as India. The authors compiled an excellent dataset of nearly 5,000 MTBC genomes from these two lineages, comprising publicly available as well as newly-sequenced MTBC strains. Their phylogenomic analyses showed that L1 comprises a further five distinct sublineages, whereas L3 does not contain distinct sublineages. The authors used two different methods (PASTML and Mascot) for conducting phylogeographic analyses, with results from both methods suggesting an interesting asymmetric pattern of migration: these lineages originated in and migrated from South Asia to other regions, with very little back-migration into South Asia. This underscores the important role South Asia has played in the dispersal of these lineages. The authors found several genes involved in virulence and antibiotic resistance to be under positive selection in L1 and L3 strains. Lastly, the authors also found evidence of local adaptation at the esxH epitope in L1 strains from South and Southeast Asia, which requires further study as this epitope is a part of several vaccine candidates. The aim of this study was to conduct a phylogeographic analysis ofStrain cultures, DNA extraction and genome sequencing" is missing citations for the methods used (eg. CTAB extraction method).The Methods section titled "Figures 1A and 2A heatmaps would look better in color than grayscale. The Supplementary Information would benefit from a thorough proofreading for grammatical and typological errors.  Overall, I found this to be a well-designed and thorough study, and a very well-written manuscript. I have no major concerns and only a few minor suggestions for aesthetic improvement:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Pathogen genomicsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thank you very much for your comments, and for reviewing our manuscript. We prepared a revised version of the manuscript based on the feedback of all reviewers. We added the missing reference for the DNA extraction method, and we revised the supplementary material for errors and typos, thanks. Regarding Figures 1A and 2A: in both figures the other three panels have the same color code, where different colors correspond to different world regions. We decided to use a gray scale to avoid potential confusion of having two different color codes in the same figure. We did not modify the figures.


Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) play important roles in the turnover of extracellular matrix and in the pathogenesis of idiopathic pulmonary fibrosis (IPF). This study aimed to determine the utility of circulating MMPs and TIMPs in distinguishing patients with IPF from controls and to explore associations between MMPs/TIMPs and measures of disease severity in patients with IPF.n = 300) came from the IPF-PRO Registry, an observational multicenter registry of patients with IPF that was diagnosed or confirmed at the enrolling center in the past 6 months. Controls (n = 100) without known lung disease came from a population-based registry. Generalized linear models were used to compare circulating concentrations of MMPs 1, 2, 3, 7, 8, 9, 12, and 13 and TIMPs 1, 2, and 4 between patients with IPF and controls, and to investigate associations between circulating levels of these proteins and measures of IPF severity. Multivariable models were fit to identify the MMP/TIMPs that best distinguished patients with IPF from controls.The IPF cohort (CO) % predicted and higher composite physiologic index (worse disease). MMP9 was associated with the composite physiologic index. No MMP/TIMPs were associated with forced vital capacity % predicted.All the MMP/TIMPs analyzed were present at significantly higher levels in patients with IPF compared with controls except for TIMP2. Multivariable analyses selected MMP8, MMP9 and TIMP1 as top candidates for distinguishing patients with IPF from controls. Higher concentrations of MMP7, MMP12, MMP13 and TIMP4 were significantly associated with lower diffusion capacity of the lung for carbon monoxide (DLCirculating MMPs and TIMPs were broadly elevated among patients with IPF. Select MMP/TIMPs strongly associated with measures of disease severity. Our results identify potential MMP/TIMP targets for further development as disease-related biomarkers. Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease associated with high mortality . Two antThe pathobiology of IPF involves excess production of extracellular matrix (ECM) and dysregulated matrix remodeling . Matrix Several basic and clinical studies have underscored the importance of MMPs and TIMPs in the pathobiology of IPF, as recently reviewed . In partIdiopathic Pulmonary Fibrosis Prospective Outcomes (IPF-PRO) Registry (NCT01915511) [The IPF cohort was drawn from the multicenter observational US 1915511)  that enrThe control cohort was drawn from the Measurement to Understand the Reclassification of Disease of Cabarrus/Kannapolis (MURDOCK) Study, a registry of adult residents of North Carolina in which self-reported health information and biological samples are collected . To ensun = 23 IPF and n = 17 control), MMP8 (n = 48 IPF and n = 63 control), MMP12 (n = 7 IPF and n = 4 control), and MMP13 (n = 10 IPF and n = 3 control) were extrapolated if feasible or assigned a concentration of half the minimum observed value. No samples fell below the standard curve for MMPs 2, 3, 7, or 9 or TIMPs 1, 2, and 4.Enrollment plasma samples were assayed for antigenic levels of MMPs 1, 2, 3, 7, 8, 9, 12, and 13 and TIMPs 1, 2, and 4 . MMPs 2, 3, 9 and TIMP1 and MMPs 1, 7, 8, 12, and 13 were quantified using multiplexed luminex immunoassays. TIMP2 and TIMP4 were quantified by ELISA. Samples that fell below the standard curve for MMP1  and test sets  using stratified sampling to retain the 3:1 ratio in each set. Three linear models  and 4 non-linear models  were fit. Covariates for age and sex were included in each model. When fitting each model, 10-fold cross validation was used to choose the optimal tuning parameter based on the area under the receiver operating curve. Operating characteristics  were determined in the training set and then evaluated in the test set. Variable importance measures were determined for the best performing models [Multivariable analyses were performed to assess whether a set of MMPs or TIMPs could differentiate patients with IPF from controls, and log10 data were centered, scaled, and Box-Cox transformed to improve model efficiency. No MMPs or TIMPs were highly correlated (Pearson correlation coefficient of ≥0.9) so all analytes were retained in the analysis. The data from the 400-patient cohort were randomly divided into training , which correlates with the extent of fibrosis on radiography in patients with IPF [1), and the reference equations developed by Crapo and Morris [For univariate analyses in the IPF cohort, linear regression models were employed on the log10 transformed data to determine the association between circulating levels of each MMP/TIMP and three measures of disease severity, analyzed as continuous variables: FVC % predicted, DLwith IPF . The Natwith IPF . were usd Morris  were useCO % predicted, and CPI were modeled separately to examine their relationship with sets of MMPs/TIMPs. Pairwise correlation analysis indicated that no MMPs or TIMPs were highly correlated (Pearson correlation coefficient of ≥0.9) so all were retained in the analysis. The performance of 2 linear models  and 4 non-linear models  was measured on training  and test  sets. Covariates for age, sex, current use of nintedanib, and current use of pirfenidone were included in each model. While fitting each model, 10-fold cross-validation was used to choose the optimal tuning parameter in the training set, minimizing the root mean squared error (RMSE). R-squared was computed.In multivariable analyses of the IPF cohort, FVC % predicted, DLn = 100), the median  age was 66.0  years, 74% were men, 68% were former smokers (Table n = 300), the median  age was 70.0  years, 74% were men, 94% were white and 67% were former smokers  and CPI was 53.5 . The lower and upper tertile cutpoints for these measures are shown in Additional file In the control cohort n = 00, the mp-value < 0.05) in patients with IPF versus controls with the exception of TIMP2  CPI score  and MMP13 (DLCO). Specifically, the estimated differences in DLCO % predicted per 10-fold increase in circulating MMP7, TIMP4, and MMP13 were − 13.41, − 11.80%, and − 6.37%, respectively. For CPI, the estimated difference per 10-fold increase in circulating MMP7 was 11.01 points, while for TIMP4 it was 7.94 points  concentrations of each MMP/TIMP by tertile of FVC % predicted, DL concentr1% predicted.Given that prior reports suggest that circulating MMP or TIMP expression can be altered in smoking-related respiratory diseases such as chronic obstructive pulmonary disease and emphysema , 22, andAs nearly all of the MMPs and TIMPs measured were elevated in patients with IPF, we sought to understand whether sets of these proteins differentiated patients with IPF from controls better than any single protein. There was no collinearity for the MMPs and TIMPs, thus a multivariable approach may more appropriately account for synergistic or antagonistic relationships that may exist between these molecules. We found that in general, linear multivariable models  had similar or better operating characteristics compared with more complex non-linear methods in the training set, with most models obtaining classification accuracies between 80 and 90% over all iterations of the cross-validation procedure Fig. . In the Multivariable models were fit to understand whether sets of MMPs/TIMPs predicted measures of disease severity in this IPF cohort. The maximum R-squared value achieved for predicting any disease severity measure was 0.16 in the training set  CPI. This finding is of interest, as TIMP4 differs from other TIMPs in that at homeostasis, its expression is restricted to cardiovascular structures [CO or CPI and peripheral blood TIMP4 expression reflects pulmonary vascular remodeling and development of pulmonary hypertension, accumulating lung fibrosis, or the effects of angiogenesis supporting pulmonary hypertension and fibroplasia.In our IPF cohort, we observed higher levels of TIMP4 to be associated with lower DLructures . Moreoveructures . It is uConsistent with our results, a prior study implicated MMP8 as a differentiating protein in a 5-protein classifier  of IPF status, although the classification model did not select MMP9 and did not consider TIMP1 . Our resWhen our data on circulating MMP or TIMP levels in patients with IPF are considered in the context of data from experimental models of fibrosis, a clearer view emerges of the potential mechanism(s) by which altered MMP/TIMP expression contributes to lung fibrosis. Both MMP3 and MMP7 appear critical to the development of experimental lung fibrosis, with rodents genetically deficient in MMP3 or MMP7 demonstrating a reduction in pulmonary fibrosis after bleomycin challenge –37. MMP7In contrast to these pro-fibrotic MMPs, animal models suggest that augmented levels of MMP1 decrease fibroplasia in liver, muscle and heart –48, whilWhile our study has several strengths, including the multicenter nature of the IPF cohort, we acknowledge that it has inherent limitations. First, although we characterized a broad array of MMPs, we did not include all described MMPs nor TIMP3. Additionally, while our MMP assay provides precise quantification of circulating MMP concentrations, MMP activity and organ specificity cannot be inferred. Second, as pulmonary function data were not available for the majority of subjects prior to the date of enrolment, we cannot ascertain whether patients were experiencing significant disease progression or were relatively stable at the time of sampling. Finally, although this work contributes important new information regarding circulating MMP and TIMP expression in patients with IPF, our study was not designed to understand whether the observed changes are specific to IPF as compared with non-IPF fibrosing lung diseases. As the IPF-PRO Registry has recently expanded to include patients with non-IPF interstitial lung diseases in the ILD-PRO Registry, we anticipate that future studies will address the specificity of candidate biomarkers for IPF as compared with other fibrosing lung diseases.The results of this study further delineate the potential value of selected MMPs or TIMPs as disease-related biomarkers in patients with IPF. Further validation will be necessary, as will extension of these analyses to examine associations between MMP/TIMP expression and clinical outcomes. Rich longitudinal data collected in the IPF-PRO Registry, including serial pulmonary function measures, hospitalization data, and information on vital status will support these analyses and further the goal of improving the diagnosis and management of patients with IPF.Additional file 1: Tertile cutpoints for disease severity metrics.Additional file 2: MMP and TIMP concentrations (pg/mL) in patients with IPF versus the control population.Additional file 3: Median  concentration for each MMP/TIMP stratified by tertile of FVC % predicted, DLCO % predicted, and CPI.Additional file 4: Association of MMPs and TIMPs with measures of IPF severity, adjusted for anti-fibrotic treatment.Additional file 5: Mean (standard deviation) log10 MMP and TIMP concentrations in the IPF and control populations as stratified by smoking status at enrollment (current/past smoker vs. never smoker).Additional file 6: Mean (standard deviation) log10 MMP and TIMP concentrations in patients with IPF with or without clinically significant emphysema on CT scan as assessed by the enrolling physician.Additional file 7: Model performance in the training  set for baseline FVC % predicted, DLCO % predicted and CPI.Additional file 8: Model performance in the test set for baseline FVC % predicted, DLco % predicted and CPI.