The response of titin to mechanical forces is a major determinant of the function of the heart. When placed under a pulling force, the unstructured regions of titin uncoil while its immunoglobulin (Ig) domains unfold and extend. Using single-molecule atomic force microscopy, we show that disulfide isomerization reactions within Ig domains enable a third mechanism of titin elasticity. Oxidation of Ig domains leads to non-canonical disulfide bonds that stiffen titin while enabling force-triggered isomerization reactions to more extended states of the domains. Using sequence and structural analyses, we show that 21% of titin’s I-band Ig domains contain a conserved cysteine triad that can engage in disulfide isomerization reactions. We propose that imbalance of the redox status of myocytes can have immediate consequences for the mechanical properties of the sarcomere via alterations of the oxidation state of titin domains. Titin regulates myocyte stiffness through uncoiling and unfolding but these two processes cannot fully explain its elasticity. Here, the authors use atomic force microscopy to study the properties of titin disulfide bonds, showing that disulfide isomerization represents a third mode of titin elasticity. In the extensible I-band portion of sarcomeres, the titin filament is composed of unstructured polypeptide regions and tandem immunoglobulin (Ig) domains  range. Hence, disulfide bonds reduce the contour length of proteins, making them stiffer11. Similarly, engineered disulfides have been shown to accelerate mechanical folding of Ig domains, leading also to stiffer domains12. In this regard, the proposal by Mayans et al.13 that disulfides could be present in several domains of the I-band of titin was very attractive. Using sequence similarity analyses, these authors proposed that 40% of the I-band Ig domains contain cysteines at positions that form the classical disulfide bond connecting β-strands B and F in extracellular Ig domains such as antibodies. However, deficient disulfide formation in vitro has so far precluded examination of the functional role of disulfides in titin domains14.Titin-based myocardial stiffness is modulated by isoform switching and by posttranslational modifications, such as phosphorylation and 15), which shows six Ig domains containing cysteines in geometries that are compatible with intradomain disulfide bond formation, we decided to investigate the mechanical effects of disulfides in the Ig domains of titin. We developed in vitro protocols to introduce disulfide bonds in I65–I70, and examined its mechanics using single-molecule atomic force microscopy (AFM) in force-clamp mode. Unexpectedly, we found that the preferred disulfide in domains that contain more than two cysteines is not the classical disulfide characteristic of extracellular Ig domains, but a titin-specific disulfide between β-strands B and G. This disulfide configuration enables mechanically induced isomerization reactions, which in turn allow fine-tuning of the response of titin to a stretching force.Prompted by the availability of the crystal structure of segment I65–I70 of rabbit titin (pdb: 3B4315. Human I65–I70 (residues 7945–8511 in uniprot Q8WZ42-1) is 95% identical to rabbit I65–170 and all 14 cysteines are conserved , which corresponds to the predicted mechanical extension of single disulfide-free Ig domains . Mechanical unfolding trajectories of single polyproteins unambiguously reveal the existence and position of disulfide bonds that cannot be cleaved by force, resulting in limited polypeptide extension under forceAFM Fig. . When pu21. Storage of I65–I70 for 2 weeks at 4 °C did not increase the proportion of shorter unfolding steps coming from disulfide-containing domains. Slightly better oxidation yield was obtained by incubation with 0.3% H202 for 72 h, which is a efficient oxidation protocol for exposed cysteine residues10. Hence, disulfide bond formation appears unfavorable once the Ig domains in I65–I70 are folded, suggesting that the cysteines are buried and not accessible to oxidants in solution3. We found that incubations at higher temperatures and in the presence of CuCl2 improved oxidation yields. Under the optimized oxidation conditions, only 22% of the AFM steps correspond to the mechanical unfolding of reduced domains (26 nm steps). The remaining steps are shorter due to the presence of disulfide crosslinks that block mechanical extension of the amino acids trapped behind the disulfides11 8 is expressed in standard conditions as a fully reduced protein that shows an average mechanical unfolding step size of 26 nm AFM Fig. . Hence,  nm Fig. , which m23. Upon mechanical unfolding of I69oxidized, CysF is located in the loop connecting CysB and CysG, in a geometry that is compatible with isomerization reactions , and the speed of appearance of the 4 and 17 nm steps at 100 pN  is obtained from the ratio of detected 4 and 17 nm steps.The relative frequency of 4 and 17 nm steps defines the regiospecificity of the nucleophilic attack of CysF on disulfide CysB–CysGoxidized in the presence of the reducing agent Tcep. We find that I69 domains remain oxidized and extend with the same 6 nm steps, even after incubation with 10 mM Tcep for 1–4 h, demonstrating that disulfide CysB–CysG remains inaccessible to the solvent in the folded state of I69 , isomerization of disulfide CysB–CysG (4 nm and 17 nm) and intermolecular reduction of disulfide CysB–CysG by Tcep (19 nm). In the experiments where Tcep is present, we also expect a fraction of the 4 and 17 nm steps to correspond to reduction of newly isomerized disulfides CysB–CysF and CysF–CysG by Tcep, which produce steps of similar size as the isomerization reactions, making it challenging to discriminate between them  while I69oxidized unfolds at an average force of 114 pN (N = 534)  Fig. . Fitting3. In the case of disulfide-containing domains, mechanical unfolding enables intermolecular reduction events by exposing disulfides to the solution. Hence, the speed at which disulfide-containing domains are able to refold is a key factor controlling the endurance of titin disulfides in the reducing environment of the cytosol.During contraction–relaxation cycles of myofibrils, refolding of Ig modules maintains titin-based stiffness26. Domains that unfold in the probe pulse mark those ones that managed to refold during the quench time. The ratio between the number of unfolding events observed in the probe pulse and those observed in the unfolding pulse reports the refolding fraction for a given quench time3. It has been observed before that Ig modules from titin refold efficiently. Indeed, the proximal I4 domain and the well-characterized distal I91, both located in the I-band, refold with rates of 0.3 s−1 and 1.2 s−1, respectively27. In contrast, we only detected rare refolding events of I65–I70reduced   . Single-molecule events are fingerprinted by the same extension of the polyprotein at the end of the unfolding and probe pulses83) Fig. . Similarly) Fig. . I69reduoxidized)8. However, these experiments bring new challenges to the identification of single-molecule events due to isomerization reactions that modify the contour length of the protein 2 that contains only 3 cysteines in the central I69 domain and is fingerprinted by the cysteine-free I91 modules, whose mechanical properties are equivalent to wild-type I913. This construct enabled us to monitor the disulfide status of domain I69 in fingerprinted single-molecule folding traces. In the typical recordings obtained with (I91ΔCys)2-I69oxidized-(I91ΔCys)2 in the absence of isomerization, one I69oxidized domain unfolds producing a 6 nm step, while the fingerprinting I91ΔCys unfolding is marked by distinct steps of 25 nm  2 also allowed us to explore the contribution of disulfides CysB–CysF and CysF–CysG to I69 refolding. With this aim, we focused on single-molecule traces where the unfolding pulse contains the signature of specific isomerization of CysB–CysG to CysB–CysF  . Overall, our results show that disulfide bonds in titin domains greatly accelerate protein folding.Experiments with (I91ΔCys)31) Fig. . In cont28. The canonical sequence corresponding to the long N2BA cardiac isoform (uniprot Q8WZ42-1) contains 34,350 amino acids arranged in series of 163 Ig and 132 fibronectin domains. In the N2BA variant, titin contains 459 cysteines. All cysteines in the I-band, except for six cysteines that belong to the unstructured N2B region10, are located in Ig domains. There are no cysteines in the PEVK unstructured region or the interdomain linkers in the I-band. Titin’s I-band Ig domains can contain up to 5 cysteines, whereas cysteines become less common in the Ig domains of the Z-disk, A- and M-bands  and use the nomenclature Cxx’ to refer to them  structure of I69, oxidized in the crystal, with the other five reduced domains of I65–I70, shows a perfect conservation of the backbone structure (root mean square deviation < 2 Å). In all domains, there is no noticeable change in the structure of the mechanical clamp motif established by the hydrogen bonds between β-strands A’ and G, which is responsible for mechanical stability of Ig folds . In long titin isoforms, the proportion of disulfide-containing domains in the I-band exceeds 60% . Nevertheless, a few domains are missing in this annotation. For instance, the best-characterized Ig domain of titin, I91, is not annotated. Such additional domains can be verified by comparing their sequence to other Igs and checking the presence of the strictly conserved tryptophan 35. Accordingly, we manually added 12 Igs to the Uniprot-annotated domains. These domains correspond to positions 2704–2794, 3145–3233, 9581–9670, 12,041–12,133, 12,321–12,411, 12,674–12,765 (I91), 12,854–12,943, 13,030–13,119, 13,837–13,926, 14,319–14,411, 20,618–20,710 and 31,553–31,646 . Following this manual annotation, we report a total of 163 Igs in the canonical human titin (Supplementary Note 3).8 and (I91ΔCys)2-I69-(I91ΔCys)2 were engineered by iterative cloning using BamHI, BglII and KpnI restriction sites27. Final cDNAs were cloned into expression vector pQE80L using BamHI and KpnI (Qiagen). Full protein sequences are provided in Supplementary Note 2-I69-(I91ΔCys)2 were expressed in BLR E. coli while the expression of (I69)8 was enhanced by the use of strain RB79147 (24 h expression at 16 °C). Cultures are induced with 1 mM isopropyl-d-1-thiogalactopyranoside after growth at 37 °C to reach OD600 = 0.6–0.8. Extracts from harvested cells are purified in two steps. We use an immobilized metal ion affinity chromatography (IMAC) followed by a size exclusion chromatography in a fast protein liquid chromatography (FPLC) machine using a Superdex 200 10/300 column . The polyprotein is eluted in Experimental Buffer . Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis confirmed size and purity of the samples. Oxidation of the substrates requires an additional step in which the samples eluted from IMAC are incubated overnight in an oxidizing buffer containing 0.3% H2O2 and 1 mM CuCl2 at 4 °C for (I69)8 and (I91ΔCys)2-I69-(I91ΔCys)2, and at room temperature for I65–I70. Before the last FPLC step, the samples are incubated during 30–60 min on ice with a large amount of reducing agent (20 mM Tcep). This last step is critical for the AFM experiments. The reduction of the accessible terminal cysteines is required for the covalent attachment to the gold surface while keeping the buried disulfide bonds protected from the solvent. Tcep treatment may reduce some of the preformed disulfide bonds in oxidized I65–I70, which would prevent their detection in the AFM. However, the absence of 10 nm (signature of CysB–CysF), 23 nm (signature of CysF–CysG) and 26 nm (signature of any reduced disulfide) unfolding steps shows that the only disulfide formed in oxidized I69 is CysB–CysG, and that this disulfide is not accessible to Tcep.We used synthetic complementary DNAs (cDNAs) coding for rabbit I65–70 (Genscript) and I69 (Genewiz). Polyproteins (I69)48. A total of 5–20 μl of purified polyprotein solution is absorbed on an ~40 nm gold-coated coverslip. Veeco silicon nitride MLCT cantilevers  are mounted on a fluid cell chamber and calibrated using the equipartition theorem49, obtaining typical spring constants of ~14–17 pN nm−117. We carry out all experiments in Experimental Buffer. We could maintain all protein samples at 4 °C and obtain productive traces within 2–3 weeks following purification. Experimental AFM cycles involve approaching the gold surface to the cantilever to establish protein tethers and retraction until the set force is reached. Cycles run continuously until a tether is formed. Tethers break spontaneously during the experiment or by pulling at high forces at the end of the experimental protocol. AFM traces were selected according to two criteria: (1) for the determination of mechanochemically induced changes in contour lengths of protein domains   was calculated with a bootstrap method based on traces that show the same extension in the first and pulse probe (before isomerization)12. To measure isomerization rates accurately, we only consider traces that are longer than 20 s, which favor completion of isomerization reactions  and the Ig domains using the Freely Jointed Chain model of polymer elasticityAll relevant data supporting the findings of this study are available from the authors on reasonable request.Supplementary InformationPeer Review File


With the increasing number of patients affected with metabolic diseases such as type 2 diabetes, obesity, atherosclerosis and insulin resistance, academic researchers and pharmaceutical companies are eager to better understand metabolic syndrome and develop new drugs for its treatment. Many studies have focused on the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ), which plays a crucial role in adipogenesis and lipid metabolism. These studies have been able to connect this transcription factor to several human metabolic diseases. Due to obvious limitations concerning experimentation in humans, animal models—mainly mouse models—have been generated to investigate the role of PPARγ in different tissues. This review focuses on the metabolic features of human and mouse PPARγ-related diseases and the utility of the mouse as a model. Peroxisome proliferator-activated receptors (PPARs) are ligand-inducible transcription factors of the nuclear receptor superfamily . There aWithin the last two decades, PPARγ became a focus of attention as a transcription factor implicated in metabolic syndrome. Metabolic syndrome is a concerning public health issue worldwide, which is characterized by a cluster of different symptoms, including obesity, insulin resistance, hyperglycemia, hypertension, hypertriglyceridemia and decreased serum HDL cholesterol levels . All of There are several mutations described in the PPARγ gene that affects metabolism traits in humans. These mutations have been classified previously as followed: common polymorphisms , dominant-negative , haploinsufficient mutations , gain of function mutations (Pro115Gln) and promoter variants  . In 1997The association between five PPARγ promoter variants and T2D has been described in T2D postmenopausal women . In addiHypercholesterolemia and hypertriglyceridemia have also been associated with PPARγ polymorphisms in a large meta-analysis in 2012 by Asselbergs et al. . MoreoveEpigenetic changes of the PPARγ gene locus have also been found in metabolic syndrome-related diseases. Recently Kokosar et al. investigate methylation and gene expression in adipose tissue in women with PCOS. Methylation and gene expression of PPARγ was inversely correlated in this study . FurtherPPARγ loss of function mutations have been reported in colorectal cancers. Not surprisingly in 2010 a novel germline mutation in this gene (S289C) was found in a patient with dyslipidemia, obesity, and hypertension not associated with T2D and a large intestine polyp that progressed to adenocarcinoma .Lipodystrophy is a syndrome characterized by adipose tissue deficiency; this results in ectopic lipid accumulation in organs and causes non-alcoholic fatty liver disease (NAFLD), reduced blood leptin insulin resistance and T2D ,61. HumaThere have been approximately 60 patients in the world identified with FPLD3. The most recent reported mutations in the PPARγ gene that has been found in patients with FPLD3 are summarized below.The PPARγ mutation D424N is located in the ligand-binding domain, and the patients carrying this mutation exhibited a loss of function; which is partially restored by adding the PPARγ agonist rosiglitazone during in vitro analysis using luciferase assays . PPARγ HFurther studies investigating PPARγ binding and general gene expression are needed in patients with partial lipodystrophies and human common polymorphisms. Also iPS technology should be used to the generation of patient-specific cell lines and the differentiation of such cells to adipocytes and other cell types should allow disease-in-a-dish type experiments and molecular dissection of the mutant receptor to cellular processes.The first attempts to generate whole body PPARγ knockout (KO) mice showed that loss of PPARγ caused impaired terminal differentiation of the trophoblast and placental vascularization resulting in utero lethality of null embryos tetraploid-rescued PPARγ-null mice survived and showed lack of adipose tissues, which established the essential role of PPARγ in adipogenesis  68]. Th. Th68]. Expounding on these systems, Sox2Cre is another type of recombination technology for generating epiblast-specific conditional KO mice . Sox2Cre+/− mice, which was abolished by thiazolidinedione treatment, thereby linking regulation of the metabolic rate and physical activity to PPARγ [Mice heterozygous for PPARγ showed increased insulin sensitivity instead of the expected insulin resistance. These mice showed decreased triglyceride content in metabolic relevant organs due to elevated leptin expression and induction of fatty acid metabolism . Heterozto PPARγ .hyp/hypPPARγ) mice are born normally, indicating that the PPARγ2 isoform may not be required for placental development. However, these animals present growth retardation, severe lipodystrophy and about a 40%–50% mortality rate before the age of five weeks. Neonatal hyp/hypPPARγ mice have insulin resistance, hyperinsulinemia, hyperglycemia and fatty liver, which resembles to human CGL. In contrast, adult mice overcome the fatty liver and hyperlipidemia. However, the skeletal muscle and the heart accumulated more lipids and it was associated with glucose intolerance. The PPARγ agonist, rosiglitazone, reversed glucose intolerance, but not the insulin resistance in homozygote mice. Adipogenic markers and PPARγ target genes were reduced. The mild insulin resistance was explained by an up-regulation of β-oxidation in muscle. However, lipid metabolism and β-oxidation genes in the liver remained unchanged. This model demonstrates the compensatory mechanisms in the absence of WAT [Targeting the exon B of adipose tissue specific PPARγ2 isoform generated the PPARγ KO in WAT . These me of WAT .Two PPARγ2 KO mouse models were generated . PPARγ2 ob/ob backgrounds [Modeling the human PPARγ dominant negative mutations is important due to its impact in human metabolic diseases. Therefore, researchers generated mouse lines carrying similar dominant negative mutations in the PPARγ gene. Tsai et al.  generatekgrounds . Anotherkgrounds . Moreovekgrounds . One of kgrounds .Importantly, the well-known PPARγ2 P12A mutation in human populations was also generated in mice as a P12A knockin model. Homozygous Ala/Ala mice are viable, however they have lean phenotype, improved insulin sensitivity and plasma lipid profiles on chow diet. Heikkinen et al. demonstrates that P12A variant of PPARγ2 is an important modulator in metabolic control, but the effects depend on the metabolic context and gene–environment interactions .ldi allele. The PPARγldi/+ mouse show reduced fat mass and insulin sensitivity giving a unique model of human conditional lipodystrophy [Inducible PPARγ knockin mouse model was also developed in which the endogenous PPARγ gene was substituted with recombinant inducible PPARγystrophy .Imai et al. selectively deleted PPARγ in adipocytes of adult mice using the tamoxifen-dependent Cre-ERt2 recombination system . The matTwo similar adipose-specific PPARγ KO mice have been published both KO mice use floxed PPARγ knock-in mice crossed with transgenic aP2-Cre mice model. He and colleagues report that WAT and BAT decreased in young mice, has decreased leptin and adiponectin plasma levels, increased circulating FFAs and triglycerides; therefore, developing liver steatosis. However, adipose-specific PPARγ KO mice have insulin resistance in adipose tissue and liver, but not in skeletal muscle when challenged with high-fat diet. Administration of TZDs to these mice improves insulin sensitivity in skeletal muscle and liver, but not in adipose tissue . Jones eRecently, Jonker and colleagues identified the fibroblast growth factor 1 (FGF1) as a critical transducer in the process of adipose tissue sensing nutrients and it stay under the regulation of PPARγ via the promoter of FGF1 gene. Interestingly, FGF1 KO have no significant phenotype under standard laboratory care, these mice develop severe diabetic phenotype and impaired adipose tissue expansion with multiple pathologies when challenged with a HFD. The phenotype of FGF1 KO mouse establishes the PPARγ–FGF1 axis as critical for maintaining metabolic homeostasis and insulin sensitization . An in vSkeletal muscle is one of the main insulin responsive tissues in the body. Although PPARγ is expressed to a much smaller extent in muscle than in adipose tissue , it is aob/ob mice results decreased hepatic triglyceride accumulation, but elevated serum lipid levels and insulin resistance [PPARγ is expressed most highly in adipose tissue, but is also detectable in many other tissues such as liver , where Psistance . These rsistance .ob/ob mice with genetic ablation of PPARγ2, known as POKO mice failed to enlarged its β-cell mass. They identified genes that regulate β-cells proliferation and survival and identified some PPARγ dependent pathways , which are differentially regulated in POKO mice [Pancreatic β-cells also express PPARγ  100,101101, wherOKO mice . HoweverOKO mice .fl/fl conditional KO mice have spontaneous lung inflammation and emphysema. Using genome wide microarray analysis, they identified potential PPARγ regulated genes in emphysema [PPARγ has an important role in many immune cell types . Many stmphysema . Schneidmphysema .Novel compounds have been developed in the last few years, which might be potential modulators of PPARγ. One of them is Z-551 that has both PPARα agonistic and PPARγ antagonistic activities. The effects of Z-551 were examined in wild type mice on HFD and it could suppress body weight gain, ameliorated insulin resistance and abnormal lipid metabolism, significantly reducing the plasma levels of glucose, FFAs, insulin and leptin . AnotherSeveral years ago Heikkinen et al.  already In this current review, we provide an update and focused on human and mouse experiments regarding metabolic syndrome, summarizing the earlier findings and more recent studies as well. As PPARγ effects occur in a tissue specific manner and the different PPARγ full agonists have severe side effects, also suggested the need of tissue selective PPARγ modulation. PPARγ allelic variants are the most common cause of metabolic traits related to the PPARγ gene. However, knockin mice (P465L) carrying a similar human mutation P467L have normal insulin sensitivity in contrast with the severe insulin resistance in these patients. This clearly indicates a difference in the response of the genetic variants between human and mouse. The Pro12Ala mutation of PPARγ2 is a risk factor of weight gains in human obese patients; in contrast, the Ala12 allele improves insulin sensitivity and has protective effect against obesity and type 2 diabetes mellitus in lean patients ,119. Theob/ob model may provide some insight into the human lipodystrophy [Mouse full body PPARγ KO MORE-PG does not mimic the human lipodystrophy findings . Althougystrophy .On the other hand, Gray et al. hypothesize that manipulations of PPARγ gene in mice generates very similar defects than in humans but these can only be seen when these mice are challenged with a HFD, exposure to low temperature, during exercise or food deprivation ,125 arguAnother important recent development is the discovery of PPARγ biallelic mutations in a human patient. These mutations are rare; therefore, fibroblasts and/or tissues derived from these patients are usually limited. Better full body PPARγ KO mice models are needed to elucidate PPARγ related CGL .knock out or knock in genes in whole body animal models and in human cell lines; allowing researchers to study at molecular and physiological level the effect of this gene disruption. This can also lead to identification of the tissue and cell type specific roles including tissue-specific gene expression. The extensive usage of genomic and epigenomic approaches are also going to help dissect the gene expression networks coordinated by the receptors. The ultimate goal of therapy should be to develop tissue selective PPARγ modulators to avoid side effects. For this to happen, mouse and human need to go forward hand in hand in an even more intertwined manner.In summary it is likely that a better and more strategic integration of mouse and human phenotypes and data will be possible by direct comparison of disease-in-a-dish type experiments on human derived iPS cell lines and mouse cellular models. As far as animal models are concerned more “dynamic” models allowing recombination of PPARγ in a temporal and tissue specific manner can accelerate the rate of discovery. CRISPR/Cas9 technology can be used to Mice have many genes in common with humans (99% of human genes are conserved in mouse genome) and show many similarities in organ physiology, metabolic processes and pathogenesis of different diseases. Mice are excellent model organisms for other reasons as well. First of all, they are small in size and have a short generation time, which makes breeding and housing relatively simple and cost-effective. Second, since the mouse genome is known, the use of genetically modified mouse models in research and preclinical studies has increased. Furthermore, the mouse is the only mammalian model in which it is technically possible to replace a particular mouse gene with its human counterpart. These so-called “humanized” mouse models are able to produce the human version of the protein of interest, or it can be created to carry a mutated version of the human gene, which is known to be associated with a human disease.Comparing the findings from human and mouse PPARγ related metabolic diseases, we can conclude that mice models can generally be used to investigate and more deeply understand the processes of human diseases. However, it is important to know that despite genetic and physiological similarities, mice have a lot of specific features, which make it difficult to extrapolate mouse results to human. Moreover, there are different conditions in mice, such as genetic background, gender, age, diet and environmental conditions, which could further modify the results. In the last decade, genome-wide studies have changed the epidemiological and functional research of PPAR variants in both human and mouse. However, more systemically used epigenetic and transcriptomic analyses are necessary in the different PPARγ mouse mutants for elucidating PPARγ and its cistrome’s role in metabolic syndrome. The future of PPARγ research relies on using humanized mouse models coupled with human iPS cells derived tissues and genome-wide studies for not only clarify the molecular mechanism of PPARγ in its target genes that have an impact on metabolic syndrome conditions, but also to find suitable PPARγ modulators for human insulin resistance and diabetes treatment.


Aspergillus flavus keratitis patients and uninfected controls. Proteome was separated into glycosylated and non-glycosylated fractions using lectin column chromatography before mass spectrometry. The data revealed the major processes activated in the human host in response to fungal infection and reflected in the tear. Extended analysis of this dataset presented here complements the research article entitled “Aspergillus flavus induced alterations in tear protein profile reveal pathogen-induced host response to fungal infection [1]” . The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE:PXD003825.Fungal keratitis is one of the leading causes of blindness in the tropical countries affecting individuals in their most productive age. The host immune response during this infection is poorly understood. We carried out comparative tear proteome analysis of  Specifications TableValue of the Data•This dataset on tear proteins will be of value for those who are employing tear proteomics approach to understand the fungal and bacterial infections of the human eye.•The data shows how fungal infection modulates host immunity, which could be studied using readily available tear.•This dataset provides an insight into the various antifungal defense mechanisms elaborated in the tear which forms the first line of defense against infections.122.1Aspergillus flavus keratitis patients and healthy individuals. The samples were centrifuged briefly to remove cellular debris and snap-frozen in liquid nitrogen until further analysis. All tear samples were collected from patients before the start of anti-fungal treatment. Tear samples were pooled and the protein concentration was estimated using the Bradford׳s method Reflex tears (50–150 µl) were collected from 2.2Tryptic peptides resuspended in 0.1% formic acid and 5% acetonitrile were analyzed by nano-RPLC-MS/MS using an Easy nLC 1000  coupled to Orbitrap Velos Pro mass spectrometer . A pre-column  followed by a capillary RSLC column  were used for the separation of peptides using a linear gradient programme (5%–30%. ACN) at a constant flow rate of 200 nL/min.2.3Positive mode electrospray ionization with an ion spray voltage of 2.4 kV and a capillary temperature of 200 °C with an RF lens voltage of 69 and maximum injection time of 50 ms. The LTQ was calibrated using the positive ion calibrant solution (Pierce) and tuned to optimize the response of ions at m/z of 524 for the tetrapeptide Met–Arg–Phe–Ala. The acquisition was performed using Nth Order Double Play mode with Xcalibur software . Full scan profile mass spectra were acquired over an m/z of 400–2000 Da at a frequency of 1 spectrum every sec. Top 10 intense ions were targeted for MS/MS under an isolation width of 2 m/z units in CID mode with a collision energy of 35. Switching criteria were set to include ions greater than m/z 400 and smaller than m/z 2000 with the charge state of 2–5 and an abundance threshold of more than 500 counts and the target ions were excluded for 30 s with a repeat duration of 30 s and repeat count set one.2.4PXD003825.All MS/MS raw data acquired from Orbitrap Velos Pro mass spectrometer were analyzed by Proteome Discoverer v1.4 following the workflow is shown in 2.5After combining the results of the individual replicates in the control and keratitis samples by summing up the PSMs. The missing values were replaced with a constant value of 0.1. The PSM values were log2 transformed after z-score normalization of the data. The normalized PSM values were then taken for hierarchical clustering using Euclidean distances.2.6The significantly enriched GO terms and pathways from KEGG and REACTOME was obtained using the g:Cocoa multi-query algorithm from g:profiler. The “Best per parent (moderate filtering)” option under “Hierarchical filtering” was selected in order to select only the most statistically significant term from a particular hierarchical group. The “order of total significance” option under “Comparison methods” was selected to evaluate the total statistical significance between the control and keratits datasets.


Escherichia coli. To study DSB processing in vivo, we induced DSBs into the E. coli chromosome by γ-irradiation and measured chromosomal degradation. We show that the DNA degradation is regulated by RecA protein concentration and its rate of association with single-stranded DNA (ssDNA). RecA decreased DNA degradation in wild-type, recB, and recD strains, indicating that it is a general phenomenon in E. coli. On the other hand, DNA degradation was greatly reduced and unaffected by RecA in the recB1080 mutant (which produces long overhangs) and in a strain devoid of four exonucleases that degrade a 3′ tail (ssExos). 3′–5′ ssExos deficiency is epistatic to RecA deficiency concerning DNA degradation, suggesting that bound RecA is shielding the 3′ tail from degradation by 3′–5′ ssExos. Since 3′ tail preservation is common to all these situations, we infer that RecA polymerization constitutes a subset of mechanisms for preserving the integrity of 3′ tails emanating from DSBs, along with 3′ tail’s massive length, or prevention of their degradation by inactivation of 3′–5′ ssExos. Thus, we conclude that 3′ overhangs are crucial in controlling the extent of DSB processing in E. coli. This study suggests a regulatory mechanism for DSB processing in E. coli, wherein 3′ tails impose a negative feedback loop on DSB processing reactions, specifically on helicase reloading onto dsDNA ends.Double-strand breaks (DSBs) are lethal DNA lesions, which are repaired by homologous recombination in  A DSB is an adverse DNA lesion that has to be repaired in order for a cell to survive. DSBs are repaired in all living organisms by either mutagenic nonhomologous end joining or by much more universally distributed and precise homologous recombination (HR). During HR, a single 3′-terminated strand is produced from each of two double-strand DNA (dsDNA) ends of a DSB by a process called DNA end resection, wherein a combination of helicase and nuclease activities result in degradation of complementary 5′-terminated strands . The 3′-Since evolutionarily conserved recombinase proteins  have a lower affinity of binding to ssDNA than their cognate ssDNA-binding proteins SSB/RPA, a recombination-mediator class of proteins  facilitates recombinase polymerization on ssDNA  from the 5′-end, and RecFOR proteins mediating RecA polymerization onto the unwound 3′ overhangs. This pathway is operative only when ssExos that degrade 3′-terminated overhangs  are inactive, which enables stabilization of the recombinogenic substrate  (recA mutant (recA mutant population is devoid of a chromosome due to its complete degradation (An ld-type) . This reld-type) , which iA mutant . In thisradation .E. coli chromosome by γ-irradiation. Ionizing radiation, such as γ-rays, induce DSBs in DNA by either directly transferring energy to it or indirectly, by creating reactive oxygen species in the cell’s cytoplasm that then damage DNA; during repair of these clustered lesions, DSBs are produced  were mixed with serially diluted phage T4 2 stock and incubated for 15 min at 37°. Soft LB agar was added to each mixture, the mixtures were poured onto LB plates, and incubated for 24 hr at 37°. Determinations were repeated three times.Exponentially growing bacteria (OD3H]thymidine) and their chromosomes were visualized in three independent experiments by combined phase-contrast and fluorescent microscopy, as described earlier , serially diluted in 67 mM phosphate buffer (pH 7.0), and aliquots spread onto LB plates. The plates were immediately irradiated at room temperature and then incubated at 37° for 24–48 hr.Bacteria were exposed to various doses of γ-rays from a −1 [3H]thymidine  and 100 µg ml−1 deoxyadenosine. Unincorporated [3H]thymidine was washed out with phosphate buffer; the cultures were suspended in a double volume of fresh LB medium and divided into two counterparts. One served as an unirradiated control, while the other was irradiated with 400 Gy at 0°. After irradiation, the cultures were incubated at 37° and at intervals duplicate samples were spread onto Whatman filters pretreated with 0.3 M NaOH. The filters were allowed to dry at room temperature, and then were suspended for 30 min in 10% trichloroacetic acid and twice in 5% trichloroacetic acid at 4°. Trichloroacetic acid precipitates high molecular weight DNA, while the low molecular weight DNA is washed away. The filters were then washed in 1:1 solution of ether and ethanol at 4° for 30 min, and then in ether at room temperature. Acid-precipitable radioactivity of the filters represents the amount of the intact chromosomal DNA, and was measured by scintillation counting . On average, 1500–3000 cpm was measured in unirradiated samples.We used the procedure described earlier . The celE. coli chromosome. This is in accord with a measured rate of DSB induction in the E. coli chromosome of 2.7 DSBs per 100 Gy (in vivo using a fluorescent DSB binding protein.The frequency of genomic DSBs inflicted by γ-irradiation ranges from 0.004 to 0.01 DSBs/Gy/Mbp in various organisms . The dos3H]thymidine) were split into two parts, one of which was irradiated with 400 Gy at 0°. After irradiation, both cultures were incubated at 37° for an additional 35 min. Cell-free extracts were prepared as previously described . The membrane was stained with Amido Black  for protein visualization. This was a loading control and allowed determination of protein transfer efficiency across the blot. Western analysis performed as described  showed much stronger, continuous DNA degradation that resulted in degradation of ∼95% of its cellular DNA after ∼120 min postirradiation incubation  compared to the unirradiated control  concentration of RecA protein  DNA degradation compared to that in the lexA3 mutant , the T4 2 phage plating efficiencies in the recA730 and wild-type strains were 0.0019 ± 0.001 and 0.0017 ± 0.0007, respectively, which is significantly different  from the recB derivative of recA730 mutant that had 0.94 ± 0.19 of recB’s titer (n = 3), suggesting that RecBCD inhibits phage propagation in the recA730 mutant and that phage genome metabolism is otherwise not impaired in that mutant. Hence, we infer that RecAE38K protein does not interfere with degradation of purely dsDNA.Next, we wanted to determine how the RecA rate of association with ssDNA influences DNA degradation in irradiated cells. For that, we used a to ssDNA . When in1.5-fold . This efe strain . We meas3 mutant . The lex3 mutant , revealirecA730 and recA730 lexA3 mutants  and ectively . On the cubation . These dcubation  is refleheir DNA .recA mutant showed an extreme sensitivity to γ-rays; at 90 Gy dose, its survival was more than three orders of magnitude lower than that of the wild-type strain AB1157 , whereas the o281recA mutant had the same survival as the wild-type strain  mutant, whereas it displayed almost identical γ-survival  and SOS induction .A comparison of γ-survivals shows that the important factors for higher survival of γ-irradiated cells are: higher RecA protein concentration  (see Introduction). Since we have shown that RecA’s function in inhibition of DNA degradation does not necessarily correspond to its role in DSB repair and SOS induction, we wanted to determine its relationship with PolV activation. For that goal, we determined DNA degradation and γ-survival of a PolV-deficient umuDC mutant and its recA derivative. As shown in umuDC recA and recA mutants  also showed constitutive DNA degradation; ∼40% of its DNA was degraded after 2 hr of incubation, thus exceeding the degradation level observed in the wild-type strain , which is not significantly different . This result indicates that RecBCD is proficient in binding to and degrading phage DNA in the mutant lacking 3′–5′ ssExos.Since the mutant lacking four 3′–5′ ssExos is an ExoVrecA derivative of the quadruple ExoI− SbcCD− ExoVII− ExoX− mutant showed about the same, low-level ∼10% degradation as its RecA+ counterpart  dsDNA end unwinds the DNA duplex and degrades both of the unwound strands. Only after interaction with the χ sequence does the enzyme starts a resection process, meaning that it continues its degradation of the 5′ strand while ceasing trimming of the 3′ strand. Therefore, DNA degradation is an essential and indivisible part of DSB processing in E. coli (there is no situation in which RecBCD activity on DNA is nuclease-free), and by assessing DNA degradation one can indeed get an insight into DSB processing. RecBCD enzyme is the strongest DNase in E. coli  ss overhangs is the main mechanism for RecA inhibition of DNA degradation. A recent study indicated that a RecA-ssDNA complex is resistant to degradation by nucleases  protein], while having similar γ-survival. This phenotype is not unique as the recB mutant has it too; it also has RecA-inhibited DNA degradation and extremely poor survival. Thus, our results suggest that the RecA role in DNA degradation regulation is less challenging than its role in DSB repair; the former is about preventing 3′–5′ ssExos from degrading the 3′ tail , while the latter requires production of a functional RecA nucleofilament. Furthermore, recB and recD null mutants share poor DNA degradation (suppressed by RecA), while the former is deficient for DSB repair and the latter is proficient. Also, recA730 lexA3 and o281 lexA3recA mutants have about the same amount of DNA degradation, while their γ-survivals greatly differ.Interestingly, our results show that the ability of the RecA protein to protect DNA from degradation differs from its role in DSB repair. The E. coli. Namely, inactivation of four ssExos  that degrade a 3′ tail . Since RecBCD poorly degrades fragmented chromosomes in cells lacking 3′–5′ ssExos while retaining ExoV activity, we infer that the enzyme is affected in binding to DNA, i.e., creation of blunt DNA ends is suppressed. A previous study showed that a strain lacking three 3′–5′ ssExos  retains nearly wt capacity for DNA repair, whereas its recA derivative had inhibited DNA degradation (ExoV−), thus indicating that these ssExos are not required for blunting the initial irradiation-produced dsDNA ends although being critical for “reckless” DNA degradation , which unsurprisingly correlates with the activities of their respective DSB processing helicases RecBCD, RecBC, and RecQ. The RecBCD helicase/nuclease processes at least 30 kb of DNA at ∼1000–2000 bp s−1in vitro  bind exclusively onto a (nearly) blunt dsDNA end , 3′–5′ ssExos may be actively trimming those oligonucleotides, thus making them acid-soluble. In this way, low acid-soluble DNA content observed in irradiated 3′–5′ ssExos-deficient cells would mask ongoing DNA degradation in them. However, this hypothesis may be ruled out since both the un- and UV-irradiated ExoIE. coli is inhibited by either DSB-processing helicase inactivation , or by preservation of 3′ tails, again leading to the inability of these helicases to reload on the processed dsDNA ends. Our results therefore indicate that repeated helicase loading is the main determinant of the extent of DSB processing, with 3′ overhang metabolism being the crucial factor in helicase reloading. We describe three ways by which the availability of dsDNA ends to a helicase is controlled by 3′ tails: (i) RecA polymerization onto 3′ tails either directly inhibits RecQ loading onto them in the recB null mutant or inhibits their degradation by the 3′–5′ ssExos, thus preventing creation of blunt dsDNA ends and, consequently, reloading of RecBC(D) onto them; (ii) the appearance of blunt dsDNA ends is prevented by inactivation of the 3′–5′ ssExos, hence making shielding RecA binding onto such a protected 3′ overhang dispensable; and (iii) in the recB1080 mutant, lengthy DSB-derived 3′ overhangs are protected from 3′–5′ ssExos in a RecA-independent manner , hence preventing recreation of blunt dsDNA ends. Similarly, excessively long 3′ overhangs produced during resection inhibit meiotic DSB repair in eukaryotes  3′ tails protection from degradation by 3′–5′ ssExos, by either “insulating” them with the RecA protein or by inactivation of these 3′–5′ ssExos, and (ii) their excessive length.Our collective results indicate that resection of a dsDNA end in E. coli  directing a resected end toward HR and microhomology-mediated end joining pathways and away from the nonhomologous end joining repair pathway (E. coli . FurtherE. coli .Hence, one can note a common regulatory mechanism for DSB processing, with a 3′ tail produced during end resection acting as the main supervisory element by imposing a negative feedback loop on subsequent processing reactions. This mechanism ensures that processing of dsDNA ends, continues until stable and utilizable 3′ tails are produced that enable the efficient repair of DSBs in both bacteria and eukaryotes.


Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease with high mortality, uncertain cause, and few treatment options. Studies have identified a significant genetic risk associated with the development of IPF; however, mechanisms by which genetic risk factors promote IPF remain unclear. We aimed to identify genetic variants associated with IPF susceptibility and provide mechanistic insight using gene and protein expression analyses.We used a two-stage approach: a genome-wide association study in patients with IPF of European ancestry recruited from nine different centres in the UK and controls selected from UK Biobank (stage 1) matched for age, sex, and smoking status; and a follow-up of associated genetic variants in independent datasets of patients with IPF and controls from two independent US samples from the Chicago consortium and the Colorado consortium (stage 2). We investigated the effect of novel signals on gene expression in large transcriptomic and genomic data resources, and examined expression using lung tissue samples from patients with IPF and controls.AKAP13; rs62025270, odds ratio [OR] 1·27 [95% CI 1·18–1·37], p=1·32 × 10−9) and confirmed previously reported signals, including in mucin 5B  and desmoplakin . For rs62025270, the allele A associated with increased susceptibility to IPF was also associated with increased expression of AKAP13 mRNA in lung tissue from patients who had lung resection procedures (n=1111). We showed that AKAP13 is expressed in the alveolar epithelium and lymphoid follicles from patients with IPF, and AKAP13 mRNA expression was 1·42-times higher in lung tissue from patients with IPF (n=46) than that in lung tissue from controls (n=51).602 patients with IPF and 3366 controls were selected for stage 1. For stage 2, 2158 patients with IPF and 5195 controls were selected. We identified a novel genome-wide significant signal of association with IPF susceptibility near A-kinase anchoring protein 13 (AKAP13 is a Rho guanine nucleotide exchange factor regulating activation of RhoA, which is known to be involved in profibrotic signalling pathways. The identification of AKAP13 as a susceptibility gene for IPF increases the prospect of successfully targeting RhoA pathway inhibitors in patients with IPF.UK Medical Research Council, National Heart, Lung, and Blood Institute of the US National Institutes of Health, Agencia Canaria de Investigación, Innovación y Sociedad de la Información, Spain, UK National Institute for Health Research, and the British Lung Foundation. Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic lung disease of unknown cause with a poor prognosis. The incidence of IPF in the UK is about 4·5–7·5 per 100 000 and is increasing,MUC5B]), telomere maintenance (such as telomerase reverse transcriptase [TERT] and CST complex subunit [STN1]) and cell–cell adhesion (such as desmoplakin [DSP] and dipeptidyl peptidase 9 [DPP9]).MUC5B and Toll interacting protein (TOLLIP) variants are associated with reduced disease progression.The current theory suggests that IPF is characterised by initial damage to the alveolar epithelium, which then signals to various cell types, predominantly fibroblasts and macrophages, promoting tissue damage and extracellular matrix synthesis that leads to parenchymal destruction and alveolar replacement by dense fibrotic tissue.Evidence before this studyWe searched Web of Science between Aug 21, 2015, and June 7, 2017, with the search terms “pulmonary fibrosis” and “genome wide” with no restrictions on publication date or language. Previous genome-wide association studies (GWAS) have reported 16 independent signals associated, at genome-wide significance, with susceptibility to idiopathic pulmonary fibrosis (IPF). Although previous studies have shown that epithelial cell function, lung defence, cell–cell adhesion, and telomere maintenance might play important roles in IPF, the precise mechanisms through which the genes identified in previous GWAS promote IPF are still poorly understood.Added value of this studyAKAP13) as an IPF susceptibility gene. AKAP13 is a Rho guanine nucleotide exchange factor (RhoGEF) that regulates activation of Rho A. RhoA is a molecule with a known role in profibrotic signalling pathways; however, AKAP13 has not previously been implicated in the pathogenesis of IPF. These studies provide genetic veracity for targeting Rho signalling in IPF.To our knowledge, this study is the largest genetic study of IPF done to date, bringing together 2760 patients with IPF and 8561 controls. We present a novel genome-wide significant genetic association signal and novel gene expression data that implicate A-kinase anchoring protein 13  in stage 1 were then further analysed in independent samples of patients with IPF and controls from two US samples, the Colorado consortium and the Chicago consortium (stage 2). We defined statistically significant associations with susceptibility to IPF as variants that met genome-wide significance (p<5 × 10−8) after meta-analysis of stages 1 and 2 together. Variants that became less significant in the meta-analysis than in stage 1 alone were not reported as showing an association.This GWAS used a two-stage approach to identify novel genome-wide significant signals associated with susceptibility to IPF. In stage 1, a GWAS was done using patients with IPF from nine different centres in the UK and controls from UK Biobank. Variants showing a significant association . The remainder of the controls were genotyped using the Affymetrix Axiom UK Biobank array . Genotyping and imputation procedures for studies contributing to stage 1 and 2 are described in the −3, the analysis was rerun using the Firth test using EPACTSThe genome-wide association analysis of IPF susceptibility was done assuming an additive genetic effect and conditioning on age, sex, and the first ten principal components to adjust for ancestry.−6 in association testing in stage 1 were followed up in stage 2. Conditional analyses were used to identify additional independent signals in the same genomic region  association with IPF risk in stage 1, available association test statistics from stages 1 and 2 were meta-analysed using a fixed-effects model. Signals that had genome-wide significance (p<5 × 10−8) when meta-analysing stages 1 and 2 were reported as significantly associated with susceptibility to IPF. A Bayesian approach was used to fine-map those signals to create 95% credible sets  were investigated in three expression quantitative trait locus (eQTL) datasets: a lung eQTL database comprising individuals from three cohorts , a blood eQTL database, and in the Genotype-Tissue Expression project (GTEx) cohort  expression in controls and patients with IPF while also controlling for age, smoking (ever or never and pack-years), and sex.RT-PCR gene expression analysis was done on RNA extracted from human lung tissues of 46 patients with IPF and 51 controls from the Lung Tissue Research Consortium using standard methods . InitialFormalin-fixed paraffin-embedded human lung tissue sections were obtained from tissue distant from the tumour that was obtained during lung resection from controls and tissue from patients with IPF taken at either post-mortem examination or lung transplantation. Immunohistochemistry was done using standard methods .We performed in-silico analyses to establish whether known drugs target the proteins identified in the genome-wide and expression analyses or the proteins that interact with them .The funders had no role in the study design. GlaxoSmithKline Research and Design participated in collection of data and had access to the raw data from a subset of the UK IPF patient data. LVW and RGJ were involved in all stages of study development and delivery, had full access to all data in the study, and had final responsibility for the decision to submit for publication.−6) and were followed up in stage 2  were included in the stage 1 analysis of 13 076 821 variants . The pat stage 2 .Figure 1−3, after Bonferroni correction for 27 tests) in stage 2, and had genome-wide significance (p<5 × 10−8) in the meta-analysis of stage 1 and 2 together . This SNP was also significant in stage 2 alone after Bonferroni adjustment for 27 tests . The 95% credible set  produced for this signal contained 113 variants (MUC5B (rs35705950) and DSP (rs2076295).12A total of 2158 patients with IPF and 5195 controls were included in stage 2 , table 1together . A novel KLHL25; , had genvariants . The othce p<5 × −8 in the95% CI 1·–1·33, p=vs 1231 controls); the results were consistent with the original analysis . We reimputed the region with only variants genotyped on both arrays using the new Haplotype Reference Consortium imputation reference panel;−6; meta-analysis OR 1·27 [95% CI 1·19–1·35], p=1·55 × 10−9).We explored whether genotyping array differences could have influenced the association test result for SNP rs62025270. In the control–control comparison , rs62025−3) with a consistent direction of effect to previous GWAS; namely variants in MUC5B, DSP, family with sequence similarity 13 member A (FAM13A), TERT, mucin 2 (MUC2), DPP9, and TOLLIP . In non-diseased whole-lung tissue from the lung eQTL databases , the minor allele A of rs62025270, associated with increased susceptibility to IPF, was associated with increased expression of AKAP13 (r2=0·60) with rs17636666, the variant that was most significantly associated with expression of AKAP13 in lung tissue. This co-localisation of IPF susceptibility association signal and strongest AKAP13 gene expression association signal (eQTL) in lung tissue  was also associated with expression of AKAP13 in whole blood  with the SNP most strongly associated with AKAP13 expression in blood (rs870689). This finding suggests differences in regulation of AKAP13 expression between lung and whole blood, which have different cell-type compositions. Furthermore, the minor allele A of rs62025270 was associated with decreased expression of AKAP13 in blood, rather than increased expression as seen in lung tissue. The SNP rs62025270 was also found to be associated with expression of RNA genes RP11–158M2.3, RP11–158M2.4, RP11–158M2.5, RP11–815J21.3, and RP11–815J21.4 in a range of tissues in GTEx .Having identified that the minor allele A of rs62025270 was associated with increased susceptibility to IPF and increased expression of ithelium  and alveithelium  in contrithelium . High AKithelium  than in ithelium . Real-tiWe tested the association of rs62025270 with survival time in all patients from stage 1 for whom survival data were available . The median survival time after diagnosis of IPF was 1621 days . rs62025270 showed no association with survival time .11 proteins that interact with AKAP13 were identified as known targets for existing drugs or compounds in development, including aspirin, dextromethophan, and GSK-690693 .AKAP13 as a potential driver of IPF pathogenesis. We showed that the minor allele (A) of rs62025270 , associated with increased susceptibility to IPF, was associated with increased expression of AKAP13 in lung tissue. Morphological analysis of control and diseased lung tissue identified AKAP13 expression primarily in epithelial cells, with some additional expression in lymphoid tissue in patients with IPF. Furthermore, we showed increased expression of AKAP13 in fibrotic lung tissue from patients with IPF compared with samples from healthy controls.We studied 2760 patients with IPF and 8561 controls and identified a novel genome-wide significant association of variant rs62025270 that implicates AKAP13-null mice are embryonic lethal because of arrested cardiac development at embryonic day 10.NCT01371305).The high AKAP13 expression in epithelial cells further supports the hypothesis that IPF is a disease characterised by epithelial susceptibility to injury, consistent with findings of other genetic risk factors associated with epithelial cell function.We also identified AKAP13 in lymphoid follicles within fibrotic regions of lung tissue from patients with IPF. Lymphoid follicles without germinal centres are well described in IPF and are thought to be composed primarily of B cells and dendritic cells.Because of the known function of AKAP13, it probably has some potential downstream molecular interactions that could be targeted therapeutically, most notably RhoA and Rho kinase.AKAP13 at genome-wide significance in IPF susceptibility.MUC5B promoter (stage 1 p=1·86 × 10−37) was the strongest genetic risk factor for susceptibility to IPF with an OR of 4·11,DSP (stage 1 p=4·14 × 10−12), MUC2 (stage 1 p=1·33 × 10−5), TOLLIP (stage 1 p=1·60 × 10−5), TERT (stage 1 p=8·25 × 10−5), DPP9 (stage 1 p=4·17 × 10−4), and FAM13A (stage 1 p=0·002). 17 variants were identified in stage 1 that have not been previously reported to be associated with IPF (of which 14 were low frequency or rare), but we could not investigate all of these in the stage 2 analyses because not all data were available. These could be additional true positive signals of association with susceptibility to IPF that will reach genome-wide significance in larger and more densely imputed studies.To our knowledge this is the first GWAS implicating A strength of our study is that we were able to bring together the largest sample size of patients with IPF and controls available to date and apply a robust and widely used two-stage study design−3), and two additional signals that reached genome-wide significance (MUC5B p=1·86 × 10−37 and DSP p=4·14 × 10−12). In addition, the diagnostic practices between the UK and USA might be different but the concordant results from the three case-control studies reassures us that these observations are robust.This study also has some limitations. Although clinical investigators were careful to exclude other fibrotic lung diseases, a small number of non-IPF cases of pulmonary fibrosis might have been included. However, the advantage of collating a large dataset is that the effects of misclassification on the analysis are reduced. Furthermore, we expect that misclassification would lead to attenuation of signals rather than produce false-positive findings, which was supported by our replication of nine previously reported signals of association with IPF in our previously unreported stage 1 data. This included five signals that reached a Bonferroni-adjusted significance threshold for 16 tests (p<3·13 × 10AKAP13 expression in patients with IPF compared with controls. The discordance between these results could be due to a number of factors, including the difference in normalisation strategies between the two techniques and sample heterogeneity. The eQTL data that we present from control lung tissue shows that the minor allele A of rs62025270, associated with susceptibility to IPF, is also associated with increased expression of AKAP13 in three independent studies.AKAP13 mRNA. Therefore, these data provide strong independent evidence that increased AKAP13 expression in alveolar cells has a role in IPF, and the cellular heterogeneity within the fibrotic lung samples used for RNA analysis is likely to explain this discordance.A previous study of 119 patients with IPF and 50 donor lung controls from the Lung Tissue Resource Consortium reported a small decrease of AKAP13 expression in patients with IPF compared with controls using gene expression data from the Affymetrix Gene ST1·0 array;AKAP13. AKAP13 is a RhoGEF that is known to interact with a central fibrogenic pathway—G protein-coupled receptor activation of RhoA—and expression is increased in fibrotic regions of lungs from patients with IPF. Expression of AKAP13 occurs primarily in the epithelium thereby reinforcing the idea that epithelial processes are central to IPF pathogenesis. The identification of a RhoGEF that regulates a pathway known to be involved in the pathogenesis of IPF, a pathway for which drugs are in development, could potentially provide a novel target for antifibrotic therapy for IPF.In summary, we report a novel genome-wide significant association for IPF susceptibility with SNP rs62025270 and present evidence that this SNP might exert its effect via expression of the nearby gene


Jc, targets for the Future Circular Collider (FCC), the planned replacement for the Large Hadron Collider (LHC), the high field performance of Nb3Sn must be improved, but champion Jc values have remained static for the last 10 years. Making the A15 phase stoichiometric and enhancing the upper critical field Hc2 by Ti or Ta dopants are the standard strategies for enhancing high field performance but detailed recent studies show that even the best modern wires have broad composition ranges. To assess whether further improvement might be possible, we employed Extended X-ray Absorption Fine Structure (EXAFS) to determine the lattice site location of dopants in modern high-performance Nb3Sn strands with Jc values amongst the best so far achieved. Although Ti and Ta primarily occupy the Nb sites in the A15 structure, we also find significant Ta occupancy on the Sn site. These findings indicate that the best performing Ti-doped stand is strongly sub-stoichiometric in Sn and that antisite disorder likely explains its high average Hc2 behavior. These new results suggest an important role for dopant and antisite disorder in minimizing superconducting property distributions and maximizing high field Jc properties.To meet critical current density,  In particular, Nb3Sn wires are being used to wind magnets for the Hi-Lumi upgrade of the Large Hadron Collider3 and they are the most technologically ready candidates for the LHC replacement, the envisioned Future Circular Collider (FCC)4. However, the performance of these wires must be improved to meet the FCC requirements5, so there is an important need to understand the limitations of the best currently produced wires. One area of uncertainty is how close we are to full optimization of the wires because Nb3Sn is in fact a compound with a range of Sn composition and the critical temperature, Tc, upper critical field, Hc2, and critical current density, Jc, are all sensitive to the composition, which is never without variation within real wires. In binary Nb3Sn, Tc and Hc2 increase as the ratio of Nb:Sn approaches the stoichiometric value of 36. However, dopants  are required for high field operation. In fact these dopants can raise the Hc2(0) by ~3 T7 )8. For the strand design closest to meeting the FCC critical current target , we have previously shown that doping with Ti rather than Ta produces a more homogeneous A15 composition and a tighter distribution of Tc. Furthermore, Ti appears to accelerate the diffusion reaction that creates the A15 phase from its Sn and Nb constituents9. An early EXAFS (Extended X-ray Absorption Fine Structure) study of Ta doped samples10 and an ALCHEMI  study of both Ti-doped and Ta-doped samples11 using older generation strands indicated that both dopants substitute on the Nb sites. However, more recent studies hypothesized that Ti might substitute on the Sn site13. This hypothesis seemed to better fit the normal and superconducting properties of Ti-doped rather than Ta-doped Nb3Sn and their average Sn chemical compositions. In ref.9 we found that the Ti-doped wire had very high Jc values (3035 A/mm2 at 12 T and 4.2 K) and a relatively narrow specific heat Tc-distribution at 15 T despite measuring only 23.1 at%Sn in the A15 layer. Ti doping on the Nb site would lead to a strongly off-stoichiometric A15, whereas the Flukiger et al.13 suggestion of Ti being on the Sn site would lead to a composition much closer to stoichiometry. This apparent inconsistency between studies persuaded us to revisit the early EXAFS study with our modern high-Jc wires with Ta, Ti and Ta + Ti-doping.Nbχ(k), can be expressed as:The extended x-ray absorption fine structure (EXAFS) is sensitive to the local environment of the dopant atoms. It occurs at x-ray energies above the absorption edge energy of the element under study. Thus, by tuning the x-ray energy to different absorption edges, the local structure of individual elements in a complex material can be determined. In general terms, considering only the dominant single scattering paths, the EXAFS function, 0 is the smoothly varying absorption of an isolated atom, E0 is the absorption edge energy of the element under study, and k is the wave vector of the photoelectron that is ejected when the absorption edge energy is crossed. The sum is over the local coordination shells of atoms, where N is the number of atoms in the shell, R is the distance from the absorbing atom, ψ(k) is a phase shift term, and A(k) is an overall amplitude factor that includes a Debye-Waller like factor, σ2 is a measure of the disorder over the distance R. The disorder can be structural due to a rearrangement of the atoms around the dopant or vibrational due to the natural vibrations of the atoms about their lattice sites. Because each shell of atoms give rise to a distinct frequency, 2kR + ψ, a Fourier transform of the spectrum can be used to separate the contributions of the different shells.where μ is the x-ray absorption coefficient, μ3Sn structure there is a distinct difference in the local environment of the two sites. The Nb atoms have three closely spaced nearby coordination shells, while the Sn site has a single nearest neighbor shell  and the composition was verified by EDS (Energy Dispersive Spectroscopy) to be between 24.3 and 25.3 at.% Sn. The descriptions and basic properties of the wires under investigation are summarized in Table 3Sn is formed by diffusion of Sn from central Sn cores to surrounding Nb filaments through a Cu matrix) manufactured by the Restacked Rod Process (RRP®) by Oxford Superconducting Technologies (now Bruker-OST). The 54 internal-Sn sub-elements were hexagonally stacked around 7 Cu blanks inside an outer Cu tube to create the “54/61” multifilamentary composites. In the Ti-doped strand (OST billet #9415-BE), ~2 at% Ti was introduced by inserting 30 Nb-47wt.%Ti rods into the filament packs within each sub-element. This sample is referred to here as Ti#1. For the Ta-doped strand (OST #8781) the filaments and the diffusion barriers were 4 at% Ta instead of pure Nb. The Ta + Ti composite (OST #9362-5) used a combination of the 4 at.% Ta alloy filaments and barrier with 16 Nb-Ti filaments produced a composition of ~1 at.% Ti + 4 at.% Ta. The samples were part of an extensive strain study16 using a common reaction heat treatment (HT) of 40 h at 640 °C. They were slightly underreacted to avoid any Sn diffusion into the stabilizer Cu. In addition, a second similarly made RRP® “108/127” wire series was included: a Ti-doped (Ti#2) and a Ta-doped (Ta#2) wire from our earlier Tc-distribution comparison study9 (OST #14895 and #12879). These samples were heat treated by Arup Ghosh at Brookhaven National Laboratory at 662–665 °C for 48 h and reacted close to their maximum Jc as is reflected in the ≈3000 A/mm2  performance. Because of the different heat treatments, the two series show differences in both Jc and the extrapolated Kramer field: Jc ~ 2620–2870 A/mm2, µ0Hk ~ 22.7–24.6 T for the 54/61 series and Jc ~3130–3210 /mm2, µ0Hk ~ 23.8–25.5 T for the 108/127 series (µ0Hk was estimated by the linear extrapolation of the Kramer function fk = Jc1/2H1/4)17. Noticeable is also the larger Hk of the Ta + Ti strand, despite its inferior Jc(12 T) performance when compared with both Ti#1 and Ta#1. For completeness the “54/61” series was analyzed by specific heat to determine the 0–15 T Tc-distributions, in the same way that we previously characterized the “108/127” wires9. This characterization is shown in Fig. Tc-distribution than both Ta- or Ti-doped samples, despite a quite broad zero-field Tc-distribution. The WHH extrapolations18 of Hc2(0) obtained for all the samples from the Tc-distributions are reported in Table Hk, also for Hc2(0) we found a higher value in the double-doped Ta + Ti wire than with Ta#1 and Ti#1.A binary Nb19. This provided a 3 µm beam that could be focused onto the narrow Nb3Sn regions in the strands. Figure 20, which allows the dopants to enter either site. Because the fitting range is limited, it was not possible to vary all of the parameters corresponding to the 4 separate scattering paths of the two sites 18. Since γ is proportional to the density of states NF and ρ0 is proportional to 1/NFτ (with τ being the scattering rate), Hc2 can be written as η by the relation 21, so Hc2 should follow the relation x on the Sn site obtained by EXAFS is taken into account, the resulting ratio (Nb + Ti + (1−x)Ta)/(Sn + xTa) is in most cases even more strongly off-stoichiometry: this is particularly evident in the Ti-doped samples where it exceeds 3.4. This off-stoichiometry implies that the dopants are not the only source of disorder in the A15 structure but either vacancy (i.e. empty site) or antisite (i.e. atom occupying the “wrong” site) disorder has to be considered. By ab initio calculations, Besson et al.22 estimated the defect structure in Nb3Sn finding that it is of antisite nature: in fact, their results indicate that the fraction of defects by vacancies is at least 7 orders of magnitude smaller than by antisite disorder at 1000 K (more than 20 orders of magnitude at 300 K). This means that vacancies are extremely rare in Nb3Sn and that off-stoichiometry occurs by antisite disorder.At the root of our concern for the site occupancy is consideration of whether there might be an effect on A3B) two distinct LRO parameters have to be defined as: ck (α) is the fraction of the α sites occupied by the κ element23. The LRO parameter is then defined by the fractional difference between elements siting on the “right” sites and those sitting on the “wrong” sites and in general it varies between 1 (perfectly ordered system) and 0 (a completely disordered system with random site occupancy). With ternary additions (X), as in our case, the LRO is always less than 1, requiring use of the parameters cX (A) and cX (B). To calculate the LRO parameters ηNb and ηSn for our samples we can take into account the global composition determined by EDS . It is important to notice that this evaluation will underestimate the disorder because disorder induced by antisite exchange (i.e. Nb and Sn atoms exchanging sites) is unknown. Although this contribution might be limited for single doping, it is likely important for the Ta + Ti sample (because Ta and Ti favor opposite antisite disorder in the 54/61 series). In this case, we include a small amount of exchange antisites proportional to the amount of antisites that Ti generates in the Ti-doped samples reduced by the amount of Ta on the Sn sites . The resulting values are reported in Table Hc2 of Ta + Ti). In both series, Ta-doped samples are the more ordered  whereas Ti-doped samples are more disordered than Ta-doped ones. We also found that Ta + Ti sample from the 54/61 series is more disordered than either Ta#1 or the Ti#1.For an alloy with two sublattices , in order to test the presence of the 3Sn magnets for important applications in physics, chemistry and medical devices means that a better understanding of the high-field limitations of doped wires is highly desirable. Our recent studies showed that even the highest Jc Nb3Sn strands still suffer from composition gradients that lead to property gradients9. For the HiLumi LHC upgrade, Nb3Sn wires were optimized for 12 T use by not pushing the heat treatment to maximum extent so as to prevent Sn leakage out of the sub-bundles into the surrounding high purity Cu. This involves some compromise to the irreversibility field and Hc2 but also has the benefit of maintaining a high grain boundary density essential for strong vortex pinning. However, higher field applications such as the proposed FCC require optimization of Jc for fields closer to Hc2 (for the FCC the Nb3Sn target is 16 T) thus shifting the balance towards increasing Hc224 and making the A15 layer more uniform. Until now it has been thought that the route to this goal requires maximum Sn stoichiometry. However, a full understanding of how Ta and/or Ti dopants act to enhance Hc2 was not clear and several contradictory hypotheses remained in play. To clarify the dopant site occupancy, we performed EXAFS characterizations of the highest performance Nb3Sn strands available that surprisingly revealed that Ta atoms not only substitute on the Nb sites but also occupy a significant fraction (~21–32%) of Sn sites in the Nb3Sn, whereas Ti sits only on the Nb site. These findings raise the question whether or not the ability of Ta to occupy either site makes it compete with Sn for the Sn sites, slowing down the Sn diffusion that drives A15 phase conversion and producing Sn gradients at both layer and filament levels as observed in ref.25. On the other hand, the strong preference of Ti for the Nb sites might favorably contribute to the Sn diffusion on its own sites, helping produce a more homogeneous phase with tighter distribution of properties9. The reasons for these site occupancies of the two dopants do not appear to be straightforward, for instance, the atomic radii are similar for all 4 elements and the oxidation states are 5 for Nb and Ta and 4 for Sn and Ti. Ab-initio density functional theory calculations will probably be necessary to provide a better understanding of this behavior and could prove useful in predicting the behavior of other potential dopants. These new insights suggest further studies to understand if and to what extent the choice of heat treatment temperature influences not only the distribution of the Sn but also the dopants distribution. Because of the evident off-stoichiometry of these strands, doping cannot be the only source of disorder in the A15 phase and antisite disorder seems to contribute significantly in increasing Hc2. In particular, the antisite disorder induced by Ti appears more effective than Ta in driving up Hc2. Interestingly, despite the lower 12 T performance, the double-doped (Ta + Ti) sample showed higher Kramer field (Hk) and upper critical field (Hc2) extrapolations with respect to similar single doped strands. This suggests that double-doped strands should be re-explored for 16–20 T applications, like FCC, re-investigating both the absolute dopant level and the optimal heat treatment.The drive to higher magnetic field superconducting NbJc ~5.2 kA/mm2, 4.2 K, 12 T) indicate that further investigation of global and local properties are needed in what will probably always be compositionally inhomogeneous wires. The striking possibility suggested by this site occupancy study, coupled to the compositional and high field specific heat study, is that antisite disorder in Ti-doped wires mitigates degradation of Hc2 in the Sn-deficient regions of the wires. We conclude that improvements of Jc properties in the >16 T range at 4.2 K are still possible, also perhaps by using longer and higher HT temperatures to make the layers more compositionally uniform (recognizing of course that more effective diffusion barriers must be provided to protect the outer stabilizing Cu too). Although high field Jc can also be enhanced by increasing the density and effectiveness of flux pinning sites by decreasing the grain size, adding pinning centers27 or introducing point pinning by irradiation29, these techniques do not raise Hc2. Thus, consideration of these surprising site disorder results should also be incorporated into any enhanced pinning center approach in order to raise the high field Jc.These considerations and the markedly sub-stoichiometric Sn composition of the best performing Ti-doped internal-Sn strands (layer Constituent powders were first densified using a cold isostatic press (CIP) at 40 kpsi and then subjected to two separate heat treatments (HT) in a hot isostatic press (HIP) at 29 kpsi. The first was a two-step HT (650 °C for 16 h +1200 °C for 72 h). The second HT occurred at 1800 °C for 24 h. The composition was verified by EDS (Energy Dispersive Spectroscopy) and confirmed to be between 24.3 and 25.3 at.% Sn.−4 in fluorescence mode using a 4-element vortex detector for the Ti K edge, and a bent Laue analyzer for the Ta L3 edge. Since the filaments are surrounded by Cu, the bent Laue analyzer30 was necessary to distinguish the Ta Lα1 line at 8146 eV from the Cu Kα lines near 8040 eV. The Nb3Sn standard was measured at beamline 20-BM also using a Si(111) monochromator. A focused beam of 0.5 mm2 was provided by a toroidal mirror. The measurements were made in fluorescence using a 13-element Ge detector. Since the sample was thick, the results were corrected for self-absorption using the FLUO algorithm31 in the Athena analysis software20. Because of this correction, the 3Sn measured in transmission mode.The EXAFS measurements of the wires using the 20-ID-B microprobe station were done using a Si 111) monochromator with energy resolution ΔE/E of 1.4 × 1011 monochSupplementary Information


Macrophages (MØs) are a key cell type of both the innate and the adaptive immune response and can tailor their response to prevailing conditions. To sense the host’s status, MØs employ two classes of receptors: Toll-like receptors (TLRs), which are sensors for pathogen-derived material, and Fcγ receptors (FcγRs) that are detectors of the adaptive immune response. How MØs integrate the input from these various sensors is not understood and is the focus of active study. Here, we review the recent literature on the molecular mechanisms of TLR and FcgR crosstalk and synergy, and discuss the implications of these findings. This overview suggests a multilayered mechanism of receptor synergy that allows the MØ to fine-tune its response to prevailing conditions and provides ideas for future investigation. Macrophages (MØs) are among the key effector cells of host defense and have the ability to tailor their response to the host’s immune status. To accomplish this feat, MØs use two classes of immune receptors: Toll-like receptors (TLRs), which are sensors that drive the innate immune response, and Fcγ receptors (FcγRs) that are detectors of the adaptive immune response. How MØs integrate the inputs from these two distinct signaling pathways is not fully understood and is the focus of active investigation.in vitro studies directly engaging single receptor types with saturating doses of strong ligands . However, during infection, it is likely that conditions will lead to less than 100% receptor engagement, meaning that physiologically MØs will be responding to submaximal receptor signaling. Moreover, the presence of natural antibodies able to opsonize incoming pathogens3 suggests that most pathogens will engage both TLRand FcγR, even in naïve hosts. As the host response is thus driven by the integration of the TLR and FcγR inputs, our comprehension of the mechanisms of TLR–FcγR crosstalk is critical to our understanding of host defense.Much of our knowledge of the signaling pathways activated by TLR and FcγR has been determined fromIn broad terms, in the issue of receptor signal integration, crosstalk between the two receptor-driven signaling pathways could regulate messenger RNA (mRNA) transcription or mRNA translation into protein or both. In the case of transcriptional control, integration of TLR and FcγR signals would regulate the levels and activity of transcription factors that control the production of cytokine mRNA. Alternatively, signal integration could occur post-transcriptionally, whereby signals from either of the two pathways would modulate the efficiency of mRNA translation. Because of the need for tight regulation of MØ activation and cytokine production, it would not be surprising if both pre- and post-transcriptional mechanisms were at work.Although the mechanisms of TLR–FcγR signal integration are incompletely understood, recent advances provide clues as to how MØs might integrate these signals for the tight regulation of cytokine production. With respect to FcγR, we will focus on crosstalk involving the activating (as opposed to inhibitory) FcγR.4. In contrast, signaling by activating FcγR occurs via src family kinase-mediated phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs), which results in the recruitment of the tyrosine kinase Syk for triggering of downstream pathways, including activation of phospholipase C, generation of an intracellular calcium flux, and NADPH activation6.Although it is well accepted that there is crosstalk between the TLR and FcγR signaling pathways, the underlying cellular and molecular mechanisms are far from clear. A primary signaling pathway for most TLR is through MyD88, TIRAP, and TRAF6, which ultimately leads to the activation of transcription factors such as nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1)7, suggesting multiple nodes for TLR regulation of FcγR signaling.Even though there are shared signaling molecules between the two pathways, how these two distinct signaling tracks communicate is not completely clear. Interestingly, a recent unbiased analysis of the TLR2/4-induced phosphoproteome in MØs revealed multiple targets within the FcγR-mediated phagocytosis pathway that are phosphorylated in response to TLR2/4 ligation and demonstrated an ability of TLR2/4 signaling to augment FcR-mediated phagocytosis8. Here, the authors were able to co-immunoprecipitate TLR4 and Syk8, suggesting either a direct interaction between the two molecules or the presence of a signaling complex. Subsequent publications have demonstrated a similar TLR–Syk interaction in MØs  in, or immediately adjacent to, the TLR transmembrane domain  or filipin profoundly changes  the MØ interleukin-6 (IL-6) cytokine response to TLR2–FcγR co-engagement . Thus, although there is intriguing evidence that TLR–FcγR crosstalk may occur at a receptor proximal stage of signaling, perhaps through assembly of signaling platforms within lipid rafts, the molecular basis for that interaction and the mechanism of TLR–FcγR–Syk communication require much additional investigation.Although TLR–FcγR crosstalk may occur at the level of shared downstream signaling molecules such as MAPK and components of the NF-κB pathway, it is also possible that the interaction occurs more proximal to the receptor at the level of Syk activation. Though the role of Syk in FcγR signaling is well known, Syk’s involvement downstream of TLR engagement is less well appreciated. The first evidence linking Syk and TLR was from Arndt and colleagues, who reported the LPS–TLR4-driven activation of Syk in neutrophils19. Subsequent studies, recently summarized by Suthers and Sarantopoulos20, have identified several potential points of communication between TLR signaling pathways and BCR ITAM signaling. For example, one aspect of TLR–BCR crosstalk involves Syk-driven upregulation of the TLR signaling molecule TRAF621. However, this mechanism requires induction of new gene expression, a process that would take minutes to hours and thus cannot account for receptor synergy that occurs within seconds of receptor engagement. However, a recent publication by Schweighoffer and colleagues22 revealed a faster-acting potential molecular mechanism. Here, it appears that in B cells TLR4 engagement drives Syk activationthrough the BCR (in the absence of a BCR ligand) via a mechanism that doesnot involve MyD88. Thus, ligand binding to the TLR is influencing the neighboring unligated BCR via an unknown mechanism to drive Syk activation. It is possible that a similar mechanism underlies TLR–FcγR synergy.FcγRs are not the only ITAM-bearing receptors that can interact with TLR, as both the B-cell receptor (BCR) on B lymphocytes (discussed below) and IgE FcR on basophils/mast cells . SCIMP is a member of the family of transmembrane adaptor proteins (TRAPs) and is known to be involved in Src family kinase signaling in B cellsTaking all of this into consideration, we would argue that TLR ligation drives two distinct downstream signaling pathways. One is the canonical MyD88-dependent pathway that leads to the activation of NF-κB. The second is a Src–Syk-dependent pathway downstream of an ITAM. In this model, the strength of the MyD88 versus ITAM inputs would tailor the cell’s ultimate response. “Strength of signal” can vary in response to changes in parameters such as the percentage of bound receptors , the degree of pathogen opsonization, the avidity/affinity of the TLR for the pathogen-associated molecular pattern (PAMP) or FcγR for the opsonizing IgG subtype or the size of the particle or both. For example, conditions of partial TLR engagement, such as would occur at the early stages of infection with few bacteria avidly binding to TLR but only low circulating IgG levels, could lead to robust activation of the MyD88-dependent arm of TLR signaling but weak activation of Syk-dependent signaling. This would lead to partial/incomplete MØ activation. In contrast, MØ interaction with the same pathogen opsonized with many IgG molecules (at the peak of infection when IgG levels are high) would promote TLR–FcγR synergy to drive robust MØ activation. Here, bacterial PAMPs such as LPS would engage the TLR to drive MyD88-dependent signaling, while IgG binding to FcγR would directly drive ITAM-dependent Syk signaling. This model of TLR–FcγR synergy in MØs is derived from information gained from many experiments using disparate cell types and receptor agonists and provides a framework for future studies.Regulation of mRNA translation/stability is a general mechanism to fine-tune cellular responses such as cytokine production, and microRNAs (miRNAs) are one tool that the cell can use to this end. Although to the best of our knowledge there are no published studies on the role of miRNA in the crosstalk between MØ TLR and FcγR, recent reports on the interplay between miRNA and TLR/FcγR signaling raise some interesting possibilities for future investigation. Here, we will discuss those findings in a general framework of MØ IL-6 and tumor necrosis factor-alpha (TNF-α) production, as these cytokines are two key MØ-produced inflammatory mediators.25.miRNAs are short noncoding RNAs of about 21 nucleotides in length and can regulate protein production in multiple ways. In addition to binding mRNA molecules and targeting them for degradation via the RNA-induced silencing complex (RISC), miRNAs can interfere with mRNA translation as well as nascent polypeptide stabilityEscherichia coli LPS, numerous miRNAs are downregulated26, including those that inhibit production of the inflammatory cytokines TNF-α (miR-24) and IL-6 (miR-24 and Let-7c)28. Consequently, robust TLR signaling would lead toboth the upregulation of cytokine mRNAs via a MyD88/NF-κB-dependent pathwayand the downregulation of miRNAs that would block the translation of cytokine mRNA into protein. One of the questions raised by this observation is how these miRNAs might block cytokine protein production. Here, recent work from the Nares lab suggests an interesting possibility29. Their report shows that, in human monocytic cells, ectopic overexpression of a miR-24 mimetic results in a significant decrease in both TNF-α and IL-6 cytokine production butno downregulation of either mRNA species. This indicates that the blockade in inflammatory cytokine production is most likely occurring via a miRNA-based inhibition of mRNA translation. In regard to the role of miRNA in TLR signaling, it is currently unclear whether the signals that elicit TLR-driven miRNA downregulation are MyD88 or Syk dependent. However, the finding that FcγR signaling can also lead to the downregulation of the same miRNA species (see below) suggests that it may be the Syk-dependent pathway.When MØs are activated with strong TLR ligands such as purified30. In addition, it has been shown in tumor cells that Src kinase signaling can drive the downregulation of Let-7a 31, suggesting that FcγR ITAM Src signaling in MØs might also downregulate levels of Let-7 miRNAs. This would mean that MØ interaction with even low numbers of antibody-opsonized bacteria could result in robust inflammatory cytokine production. Here, TLR signaling would be responsible for driving the transcription of cytokine mRNA molecules, while FcγR ITAM signaling (driven by FcγR recognition of the opsonizing antibodies) would downregulate miR-24/Let-7 miRNA to allow efficient cytokine mRNA translation. Hence, TLR–FcγR synergy may be occurring at a post-transcriptional stage in the cytokine response and be mediated by miRNA. However, this intriguing possibility requires additional investigation.Under physiological conditions of TLR engagement, such as MØ interaction with an individual bacterium, it is unclear whether the levels of induction of cytokine mRNA and downregulation of miRNA would be similar. It is possible that some conditions of TLR engagement lead to robust cytokine mRNA induction but little or no downregulation of the miRNA that would block the translation of these mRNA molecules, resulting in little or no cytokine production. Under these conditions, TLR–FcγR synergy could become very important to the development of a robust cytokine response. In human monocyte-derived MØs, FcγR signaling has been shown to downregulate the level of miR-24, which is known to block the translation of IL-6 and TNF-α mRNAThe ability of MØs to integrate and respond to multiple signals from their ever-changing environment is critical for MØ function and the health of the host. Although it is clear that TLR and FcγR are two key MØ receptors that allow the cell to discern the immune state of the host, the molecular mechanisms that underlie this feat are incompletely understood. At the pre-transcriptional step, there are multiple nodes of interaction between the TLR–MyD88 signaling pathway and FcγR–ITAM–Syk signaling pathways that represent potential points of crosstalk. At the post-transcriptional step, TLR and FcγR regulation of inhibitory miRNA are also likely involved. This sophisticated regulatory web would allow precise tailoring of the MØ response to environmental cues. Dendritic cells also express TLR and FcR, and it is known that crosstalk occurs in these cells as well. However, the underlying molecular mechanisms and impact of crosstalk on immune function appear to be different (recently reviewed in