Vector bionomics are important aspects of vector-borne disease control programs. Mosquito-biting risks are affected by environmental, mosquito behavior and human factors, which are important for assessing exposure risk and intervention impacts. This study estimated malaria transmission risk based on vector–human interactions in northern Ghana, where indoor residual spraying (IRS) and insecticide-treated nets (ITNs) have been deployed.Anopheles gambiae sensu lato (An. gambiae s.l.) samples were examined for parity and infectivity. The HBRs were adjusted using mosquito parity and human behavioral observations.Indoor and outdoor human biting rates (HBRs) were measured using monthly human landing catches (HLCs) from June 2017 to April 2019. Mosquitoes collected were identified to species level, and Anopheles gambiae was the main vector species in the IRS (81%) and control (83%) communities. Indoor and outdoor HBRs were similar in both the IRS intervention  and control communities . The mean proportion of parous An. gambiae s.l. was lower in IRS communities (44.6%) than in control communities (71.7%). After adjusting for human behavior observations and parity, the combined effect of IRS and ITN utilization  on reducing malaria transmission risk was 58% in IRS + ITN communities and 27% in control communities with ITNs alone . However, this also revealed that about 41% and 31% of outdoor adjusted bites in IRS and control communities respectively, occurred before bed time (10:00 pm). The mean directly measured annual entomologic inoculation rates (EIRs) during the study were 6.1 infective bites per person per year (ib/p/yr) for IRS communities and 16.3 ib/p/yr for control communities. After considering vector survival and observed human behavior, the estimated EIR for IRS communities was 1.8 ib/p/yr, which represents about a 70% overestimation of risk compared to the directly measured EIR; for control communities, it was 13.6 ib/p/yr (16% overestimation).Indoor residual spraying significantly impacted entomological indicators of malaria transmission. The results of this study indicate that vector bionomics alone do not provide an accurate assessment of malaria transmission exposure risk. By accounting for human behavior parameters, we found that high coverage of ITNs alone had less impact on malaria transmission indices than combining ITNs with IRS, likely due to observed low net use. Reinforcing effective communication for behavioral change in net use and IRS could further reduce malaria transmission.The online version contains supplementary material available at 10.1186/s13071-023-05793-2. Effective malaria vector control relies mainly on two core insecticide-based interventions: deployment of insecticide-treated nets (ITNs) and indoor residual spraying (IRS) of insecticides . IncreasSome malaria vector species that have undermined insecticidal controls such as IRS and ITN programs exhibit one or more of the following behavioral traits: feeding and resting outdoors, early exit behavior and/or opportunistic feeding upon animals rather than humans . Such feAnopheles gambiae sensu lato (An. gambiae s.l.) is the predominant vector; it has an equal tendency (i.e. 50%) to feed inside and outside at any given time when there are available hosts [In northern Ghana, le hosts , 18, andle hosts . Heterogle hosts , 21.This study was conducted to document how vector feeding behavior overlaps with human behavior and to assess its impact on malaria transmission dynamics in the northern savannah zone of Ghana where IRS and ITNs have been co-deployed for the last decade. Since 2018, the IRS intervention site has been exempt from ITN mass and school-based distribution but nets are available at health facilities through continuous distribution. We compared the human–vector interactions and estimated the transmission risk indices in two districts of northern Ghana with different IRS and ITN histories, by simultaneously monitoring hourly vector and human behavior indoors and outdoors. Findings from human–vector behavior studies can be used to determine whether outdoor biting is occurring because humans are outdoors, and to determine the protective impact of vector control tools and whether alternative tools are needed if ITNs and IRS are not protecting humans from biting/transmission.The study was conducted in four communities (sites) in northern Ghana: two IRS communities in Kumbungu district (Gbullung and Gupanarigu), where U.S. President’s Malaria Initiative (PMI) supported IRS from 2008 to 2012 and again from 2015 to the present, and two control communities (Kulaa and Tugu) located on the outskirts of Tamale metropolis, which have no history of IRS  with a residual life of 7 months, covering the entire malaria transmission season [In both 2017 and 2018 both Gbullung and Gupanarigu were sprayed with an organophosphate insecticide  and dry (December–April), with 1-month breaks in November and May of each year. A total of 10 houses per community were randomly selected for monthly human landing catches (HLCs). Of the 10 houses per community, six were selected for human behavior observations during HLCs. Direct observations of night-time activities were conducted alongside HLCs monthly.An. gambiae s.l. Mean rainfalls of 79.5 mm and 74.6 mm were recorded for the IRS and control communities, respectively, in year 1 of the study. In year 2, however, the rainfall increased to 87.5 mm in IRS communities (10% increase) and to 99.3 mm (33% increase) in the control communities.Mean daily rainfall data from the study area was obtained from weather stations of the Ghana Meteorological Services Department in Tamale and from the Savannah Agricultural Research Institute in Nyankpala. The amount of rainfall recorded 2 weeks prior to the data collections was compared with human biting rates (HBRs) to assess the correlation between rainfall and biting rates of the predominant vector, Monthly mosquito collections were conducted simultaneously across all study sites using HLCs. HLCs were performed for a total of 4 nights (collecting mosquitoes hourly from 6:00 pm to 6:00 am) in each community per month. Two mosquito collectors worked in two teams of four persons (2 indoors and 2 outdoors) per house simultaneously each night . A total of 40 man-nights (20 man-nights indoor and 20 man-nights outdoor) were used for mosquito collection per community each month. The same houses were used for data collection every month during the study. Indoor and outdoor mosquito collectors switched positions every hour and were allowed to take 10-min breaks between shifts. For the indoor collections, the collectors worked in rooms without mosquito nets and stayed in the room alone during the time of collections.Anopheles mosquitoes were preliminarily identified morphologically to species using the keys of Gillies and Coetzee [An. gambiae s.l. collected by HLCs was randomly selected and further identified to species by PCR analyses of ribosomal DNA [Anopheles gambiae sensu stricto (An gambiae s.s.) were further distinguished as either Anopheles coluzzii or An. gambiae following the restriction fragment length polymorphism–PCR procedure described by Fanello et al. [ Coetzee . A sub-somal DNA . Specimeo et al. .An. gambiae s.l. collected from HLCs monthly were sorted and tested for the presence of Plasmodium falciparum sporozoite circum-sporozoite antigens using an enzyme-linked immunosorbent assay as described by Wirtz et al. [The heads and thoraces of about 15% (average: 50 per site per month) of z et al.  to deterz et al. .An. gambiae s.l. mosquitoes from HLCs from each site every month were dissected to assess parity by observing the degree of coiling in the ovarian tracheoles [About one-third (average: 50–60 mosquitoes per site per month) of unfed acheoles .A total of 24 houses (12 houses in the IRS communities and 12 houses in the control communities) were used for the direct observations each month. Night-time indoor and outdoor activities of 394 residents (156 from the control communities and 238 from intervention communities) were directly observed. These residents comprised 51.3% females and 48.7% males. Direct human behavior observations were also conducted in the same houses where the HLCs were performed to estimate the risk/exposure to residents.One note taker was stationed per house to observe an average of 15 inhabitants. Trained note takers observed the activities of all household residents without interfering with their routine activities from 6:00 pm until 6:00 am. The direct observations included the time that people were indoors or outdoors, the types and duration of activities occurring outdoors during the night, when and where people slept and whether they were under a net, regardless of whether in/out. All observations were recorded for each hour on a household observation form  . HBRs weParity rates were estimated as:p) is estimated as:Vector survival. The probability of a mosquito surviving 1 day after a blood meal and expectation of life for a mosquito are known to affect the likelihood of being bitten by an infective mosquito. By assuming a gonotrophic cycle of 2 days, the probability of vectors surviving through 1 day  is the number of infective bites received by an individual in an area within a period . This was estimated for a specific site or district as follows:Monthly EIR = monthly HBRs × monthly sporozoite rates.Annual EIR (for March–December only) = sum of monthly EIRs.Estimating human behavior observation (HBO) and parity adjusted indicators:Directly measured HBRs indicate exposure risk but overlook the effects interventions like IRS and ITNs, which work by reducing mosquito longevity. Directly measured, HBRs estimates, which include both parous and nulliparous mosquitoes—as is the current practice—may overestimate risk and underestimate intervention effects. Since older vectors are more likely to transmit malaria, parity- and HBO-adjusted rates provide a more accurate representation of the exposure communities face.HBO-adjusted HBRs were estimated according to the method of Martin et al. , using tThe following entomological indicators were estimated for the malaria vectors collected:An. gambiae s.l. populations from IRS and control communities. Only female An. gambiae s.l. mosquitoes were considered in the analyses.Data collected were analyzed using STATA version 15 software . Linear hierarchical regression was used to calculate average differences in HBR (both directly measured and adjusted) between sprayed and control communities. In the linear hierarchical regression, the type of treatment (sprayed or unsprayed) was included as the main outcome of interest, with month of observation as a fixed effect and community and household as random effects. The Z-test for difference in proportions was also used to compare the differences in mean parity and sporozoite rates between Anopheles mosquitoes, comprising An. gambiae s.l. (93.6%), An. nili (5.40%), An. funestus sensu lato , An. pharoensis (0.56%) and An. rufipes (0.01%), were collected over the study period using HLCs. The proportions of An. gambiae s.l. collected in years 1 and 2 in the IRS communities were similar , but in the control communities a lower proportion of An. gambiae s.l. was collected in year 2 compared to year 1  while the proportion of An. nili increased from 2.1% in year 1 to 16% in year 2 . Most An. funestus s.l. (a secondary vector) collected were from IRS intervention communities  have been implicated in malaria transmission in the study area.A total of 48,735 fipes 0.0%, were cAn. gambiae s.l. that were further identified by molecular analyses revealed that An. gambiae was the majority species in both the IRS  and control  communities. Similar proportions of An. coluzzii (16%) were identified in both study areas. Hybrids of An. coluzzii and An. gambiae were only collected from the IRS communities while all of the An. arabiensis collected were from the control communities.The subset of An. gambiae s.l. females was significantly higher  in the control than in the IRS communities  compared to the control unsprayed communities  in year 1. In year 2, there was no statistically significant difference  between the HBR in the IRS (12.1 b/p/n) and control (12.9 b/p/n) communities. HBR in the IRS communities increased from 9.8 to 12.1 b/p/n in year 2 , whereas the HBR in the unsprayed areas decreased significantly from 22.2 to about 12.9 b/p/n .The cumulative average daily HBR was 9.8 (± 1.3) b/p/n for IRS communities and 22.2 (± 2.4) b/p/n for the unsprayed control communities in year 1. Controlling for fixed effects for month, random effects for community, household and place of collection (indoor/outdoor), mean HBR was significantly higher for the control communities than for the IRS communities , but in year 2 An. gambiae s.l. in the IRS communities showed slightly more exophagic tendencies . The opposite trend was observed in the control communities in both years. The differences in the proportions of An. gambiae feeding indoors versus outdoors were statistically significant in year 2 in the control communities   household residents were observed to go to bed between 9:00 pm and 11:00 pm and woke up mostly between 4:00 am and 500 a.m. in preparation for dawn prayers  were recreational (32%) and social (62%) in both seasons, suggesting an overlap between vector and human outdoor activity. Recreational activities included watching videos, storytelling and playing oware . Examples of social activities that residents were engaged in included attending funerals, community meetings and festivals, or preparing for child naming ceremonies and weddings. In general, males in most communities stayed outdoors later than females.P < 0.001)  than in the control communities (57.3%)  Table .Table 2OAn. gambiae s.l. in both the IRS and control communities, with the most intense biting activity occurring between 1:00 am and 3:00 am in both groups  indoors and 0.88 b/p/hr outdoors in the IRS communities.Comparable biting trends were observed for An. gambiae s.l. decreased to 0.33 b/p/hr  and 0.42 b/p/hr  , respectively, which were statistically significant differences. In control communities without IRS, a small decrease was observed when adjusting for parity. The average directly measured indoor biting rate decreased from 1.71 to 1.23 b/p/hr , while the outdoor biting rate declined from 1.50 to 1.12 b/p/hr .After adjusting for parity to estimate potential exposure to infectious mosquitoes, the average hourly indoor and outdoor biting rates for Four main behavioral patterns were identified during the direct observations throughout the night, including people staying inside their rooms or outside in the open courtyard of the HLC houses, their sleeping schedules and the use of ITNs. These patterns form the HBO, which we used to adjust the biting rates and estimate the number of mosquito bites experienced by different groups.By adjusting for parity and HBO, the total mean adjusted bites (for year 1 and 2)  decreased by about 61.4% in the IRS intervention communities (from 10.9 to 4.2 b/p/n), while only a 17% decrease was recorded in the control communities (from 17.6 to 14.5 b/p/n) Table . This trAn. gambiae s.l. collected by HLC and tested for the presence of sporozoites, 0.49% from the control communities and 0.26% from the IRS communities were positive for P. falciparum sporozoites.Of 7067 Anopheles gambiae accounted for most of the infections in IRS and control communities, with five infections in the IRS communities and 20 infections in the control communities. Only one An. coluzzii was positive for sporozoites in the IRS communities, while three An. coluzzii were positive in the control communities. Sporozoite-positive mosquitoes were detected in samples collected between 10:00 pm and 4:00 am  or the control communities . Fewer mosquitoes were analyzed for the IRS intervention site in the dry season because of fewer mosquitoes , hence these were excluded from the comparison in Additional file The mean annual EIR  was observed in the control communities in the second year of the study. This decline in HBR could in part be attributed to the impact of a mass ITN distribution campaign organized by the National Malaria Control Program just before the peak malaria season in June 2018. Whereas the mass ITN campaign excluded all IRS intervention communities, all households in the comparison control (non-IRS) communities were covered. ITN coverage in the IRS communities was, however, maintained through the continuous distribution in health facilities. The observed decline in HBRs in the control communities following the ITN distribution in 2018 is consistent with the findings of Bayoh et al. [An. gambiae s.s. populations following increased coverage of insecticide-treated bed nets in Kenya [An. gambiae s.l. in the IRS communities in year 2 when compared to year 1, with a significant spike in mosquito biting density in September 2018. The notable spike in mosquito biting density in the IRS communities coincided with flooding in IRS communities (under the Kumbungu district) and neighboring Savelugu, Mamprugu/Moagduri and West Mamprusi districts. The flooding, which occurred between late August 2018 and early September 2018, was caused by the spillage of the Bagre Dam in Burkina Faso [h et al.  who founin Kenya . In contina Faso , 41. Floina Faso , 43. Howina Faso . The HLCAnopheles arabiensis larval habitats which maintain vector populations, thus increasing malaria transmission risk [The general increase in EIR estimated in year 2 could be as a result of the increased vector biting rates observed in year 2. A similar observation was reported by Dia et al. in Dakar  where malaria transmission has been sustained in certain suburbs of Dakar by recurrent flooding since 2005 . These aion risk .An. gambiae has been found to be primarily endophagic and endophilic [An. gambiae tended to feed both indoors and outdoors when hosts were available. Overall, outdoor vector biting rates were slightly higher (though non-significant) in the IRS + ITN communities compared to the control (ITNs only) communities. This outdoor feeding tendencies of An. gambiae s.l. observed in IRS communities could be influenced by irritant effects of pirimiphos-methyl (in the insecticide Actellic 300CS) that was sprayed during the study period. This observation is consistent with findings in Côte d’Ivoire where pirimiphos-methyl deterred vectors from entering sprayed huts despite the commonly held belief that pirimiphos-methyl is considered to be a non-irritant insecticide [Historically, dophilic . Howeverdophilic –50, whicecticide . It alsoecticide –50,Though the overall outdoor biting rates of the vector are not as significant as reported in other studies , 52–54, Plasmodium parasites inside Anopheles vectors, mosquito survivorship largely determines the number of secondary infections that could result from the primary source [An. gambiae s.l. mosquitoes at multiple sites across the IRS and control study area showed resistance up to 10× the concentrations of alphacypermethrin and deltamethrin [Despite the increased biting density in year 2 and the plastic feeding behavior of the main vector species in the area, our results show that IRS implementation and ITN usage led to a decrease in human exposure to potentially infective mosquito bites in the IRS communities, as opposed to the control communities. This decrease was observed even with only moderate ITN usage in the IRS communities. Given the long extrinsic incubation period of y source ; there iy source . Consequy source , 37. Thiy source –58. In cy source , 60. Susamethrin . Resultsamethrin , 23, 61.At the current ITN utilization rates of 38% and 57% in the IRS intervention and control communities, respectively, the proportion of all vector bites prevented by using an ITN is 33% and 49% for the IRS and control communities, respectively. Considering that most of human bites (51% in IRS vs. 40% in control communities) occurred indoors while people were asleep, hypothetically increasing ITN utilization to 100% would significantly increase personal protection and prevent up to 85% of potentially infective bites. However important barriers, such as heat and discomfort under the ITN, room shape and size , 63, remThe findings from this study confirm that vector bionomics alone do not reveal true transmission exposure risk. HBO and parity adjustments suggest that without accounting for human behavioral patterns and the effect of interventions on vector survival we could be overestimating malaria transmission risk (EIRs) by about 70% in the IRS communities and about 17% in ITN only-control sites. Such high EIRs estimated with the directly measured HBRs may suggest that interventions might not be working and undermine the impact of existing interventions. By adjusting the HBR for human behavior and parity and using these to estimate EIRs, we determined a more accurate estimate of the effect of vector control interventions on malaria vectors and risk of transmission. The findings of the study suggest that the combination of IRS and ITNs have a greater impact on malaria transmission risk than ITNs alone. They also reveal that additional interventions would be needed to tackle possible transmission that occurs beyond the reach of IRS and ITNs, due to vector feeding plasticity and human behavior patterns. By considering this information, we can ultimately tailor vector control interventions to meet specific needs and deliver appropriate messaging to communities through innovative and effective communication strategy.The study has shown that IRS continues to have a significant impact on vector longevity and malaria transmission, as determined by entomological indicators, despite the varying degree of outdoor biting encountered in the IRS communities. By measuring multiple variables that define the dynamics of malaria transmission, namely net ownership and use, human sleeping behavior, seasonality, mosquito biting rate, infectivity and longevity, we accurately quantified exposure risk and the true impact of current vector control interventions in the study area. The results of the study indicate that ITNs are more effective when combined with IRS than when deployed alone in northern Ghana. However, we identified protection gaps due to human and vector behavior patterns that allow significant human–vector interaction beyond the current control interventions. Therefore, it is essential to consider human behavior when selecting, implementing and evaluating the effectiveness of malaria control interventions. Additionally, reinforcing effective communication on the need for behavioral change is crucial to maximize the uptake and efficacy of vector control interventions.Additional file 1. Table S1: List of study sites and their IRS history from 2008 to 2020. Table S2: Total number of Anopheles mosquitoes collected during the study period. Table S3: Proportion of parous females of An. gambiae s.l. by HLCs, June 17–April 2019. Figure S1: Estimates of monthly parity rates of An. gambiae s.l. by for IRS intervention and control communities, June 2017 to April 2019. Figure S2: Mean monthly indoor and Outdoor human biting rates of An. gambiae s.l. for IRS intervention and control sites, June 2017 to April 2019. Figure S3: Mean hourly bites per person and hourly sporozoite rates for An. gambiae s.l. in a IRS and b control sites in the rainy seasons of year 1 and 2.Additional file 2. Form S1: Household direct observation data collection form.


Results reveal systemic phonetic drift in ELoF’s L1 English vowels over the RA, and this accompanies the phonetic development occurring in the participants’ L2 French vowel system, a phenomenon we label “tandem drift.” This L1–L2 link is also supported by interspeaker variation: the individuals whose L2 French vowels shift the most are also the participants who exhibit the most substantial L1 phonetic drift in the same direction. Results for the L1 re-immersion time point suggest a partial—but not complete—reversal of phonetic drift, whereas no reversal of the L2 gains made over the RA is apparent. Nevertheless, at the individual level, the learners whose L2 gains reverse the most upon L1 re-immersion are also most likely to exhibit reverse phonetic drift in their L1. Overall, these findings indicate a relationship between L2 speech learning and L1 phonetic drift, which we argue is driven by the global phonetic properties of both L2 and L1 becoming linked at a representational level. Although these representations appear malleable, it is clear that recent changes are not guaranteed to reverse despite substantial re-exposure to L1 input. Implications for the distinction between drift and attrition are discussed.This study analyzes the production of native (L1) and foreign (L2) vowels by 42 L1 English learners of French (ELoF) at the start and end of a 6-month residence abroad (RA) in a French-speaking country. Data are also reported from a delayed post-test, which takes place 10 months after a subsection of participants ( Furthermore, although phonetic drift is deemed reversible, the persistence of these phonetic changes upon L1 re-immersion has yet to be thoroughly tested , p. 202.The present research addresses these concerns with a real-time analysis of both L1 and L2 acoustic vowel data over a residence abroad (RA) and at a delayed post-test conducted 10 months after participants return home to the L1 environment. Our results have broader implications for theories of how native and foreign sounds are stored by language learners, as well as furthering our understanding of phonetic plasticity in the face of varying ambient linguistic inputs.speech learning model (SLM)  , 2007 an) .A further open question regarding phonetic drift is the persistence of L1 changes upon returning home and whether this is affected by the reversal of L2 gains. Very few phonetic studies to date have tracked language learners as they move from an L2 environment back to their native language environment, and even fewer contain baseline data of L1 pronunciation before participants moved to the L2 environment in the first place. As such, A handful of studies have, nevertheless, focused on the reversibility of drift. One example is the early pioneering work of Results from Other studies have shown that L1 re-immersion can affect both learners’ native and foreign sounds to a similar extent. For example, in Based on this brief overview of L1 re-immersion studies, it appears that a link may exist between the two sound systems. However, it is unclear whether phonetic assimilation will occur from renewed L1 contact or whether both systems will reverse back toward the baseline an equal amount. Given the limited number of longitudinal L1 re-immersion phonetic studies, the question of how, and indeed whether, the two sound systems of language learners reorganize upon returning to the native language environment merits closer inspection.This research analyzes the English and French vowel spaces of 42 English learners of French (ELoF) at the start and end of an RA in a French-speaking country, with a subsection of the participants also recorded 10 months after returning home. Our first research question addresses whether phonetic drift and L2 phonetic development occur over the RA. In the present study, we limit our focus uniquely to systemic change in both languages.RQ1: Does a systemic phonetic change in the learners’ L1 English and L2 French vowels occur from pre-RA to post-RA?Previous comparisons of English and French vowel acoustics generally suggest that the English vowel space is lower than the French vowel space . HoweverRQ2: If ELoF’s L1 English and L2 French vowels exhibit systemic phonetic changes over the RA, does L1 re-immersion reverse these changes fully?Consistent with previous research e.g., , any phoRQ3: Does interspeaker variation support a relationship between L2 phonetic changes and L1 phonetic drift in the L2 environment and upon L1 re-immersion?Previous phonetic drift studies analyzing individual variation suggest that speakers who acquire L2 phonetic detail most successfully are also most likely to exhibit phonetic drift in their L1 English vowels in the same direction e.g., . This apFurthermore, if there is a relationship between the reversal of phonetic drift and loss of L2 gains, we hypothesize that individuals whose L2 gains are reversed upon L1 re-immersion are also the participants who exhibit most return phonetic drift in their L1. Nevertheless, previous research investigating this relationship has not reached conclusive results. For example, RQ4: Does the distance between ELoF’s L1 English and L2 French vowel systems increase, decrease, or remain constant over time?phonetic category assimilation . The majority of residences abroad lasted ~6 months and predominantly took place in France . Although 22 of these participants were recorded before their departure (in England), the other 20 were recorded at the start of their RA in Lyon, France. These groups patterned similarly across time points and so were collapsed into one experimental group  was included as a reference for the direction of any phonetic change in the learners’ L1 English or L2 French production of vowels. The majority of these participants were from the Parisian districts of France although two were also from Belgium, and the group were within their first month of an RA in the United Kingdom.The participants in the experimental group completed production tasks in both English and French, along with a French perception task. For the purposes of space in the present study, only the production tasks are reported here. A short video was watched in the language of the subsequent tasks to encourage learners to enter a more monolingual language mode  and for Experiment Builder  contexts, with the vowels surrounded by either stops or fricatives to facilitate segmentation. Although the following consonant was sometimes situated across a word-boundary due to cross-linguistic lexical constraints, lexical items were never in the phrase-initial or phrase-final position, again consistent with the original design by The stimulus phrases used to elicit productions contained one lexical item of interest, featuring one of 10 English monophthong vowels or one of 10 French monophthong vowels. Six different items for each vowel were uttered per speaker with the order of phrases pseudorandomized using  Builder  such tha Builder .3 As sExperiment Builder .At all time points, participants also answered an online questionnaire featuring a language background and engagement section based on the tool developed by Praat . This has been used as a standard method of labeling outliers and reducing formant tracking errors within bilingual speech samples  were fitted using the lme4  and lmerlmerTest  packagesrTest  and F2 (Model 4). These models were fitted with an interaction between time point  and language . Finally, Model 5 analyzed the difference between each participant’s mean F1 in English and French. Time point  was fitted as a fixed effect to determine whether ELoF’s L1–L2 distance changed over time. All models were backward reduced using the Step function from the lmerTest package (ranova) and F-tests for the fixed effects (using drop1). To determine whether other factors explained phonetic change over time . As such, we predicted that L1 drift would be upward over the RA if the learners’ English vowels shift toward the global formant values of native French. In contrast, the French comparison of y-axis marginal box plot of SE = 0.01, t = −3.06, p < .01). As such, systemic lowering in ELoF’s L2 French was expected over the RA if their vowels approximate the global formant values of the foreign language.A visual comparison between languages in SE = 0.05, t = −1.30, p = .20) and ELoF’s French vowels . No phonetic change over time was therefore expected along the front-back cline in either of ELoF’s languages.The second formant was also analyzed but no effect of Group was observed: there was little difference between the average frontness of NFS’ vowels and both ELoF’s English vowels , but also that ELoF’s L2 French vowels lowered even more than their L1 English vowel space . The second formant was also analyzed, but ELoF’s English vowels did not change over the RA  and their French vowels were no different .The systemic lowering in both vowel spaces was confirmed by statistical analyses. Model 3 analyzed the change in F1 and the fitted interaction between time point and language not only revealed that ELoF’s L1 English vowels lowered significantly from the pre-RA to post-RA . Nevertheless, pairwise comparisons suggested that ELoF’s L1 English vowels remain substantially lower at the re-immersion time point compared with the pre-RA session . As such, L1 was not entirely reset to its value at the baseline.From this graph, ELoF’s L1 English vowels appear to exhibit slight raising between the post-RA and the L1 re-immersion time points, and Model 3 confirmed that this was significant , and the vowels remained substantially lower than at the pre-RA time point . These results indicate that although there was a slight reversal of the phonetic drift after returning home in ELoF’s L1 English, limited loss of the L2 gains appears to have occurred in the learners’ French vowels.Pairwise comparisons within the Time Point × Language interaction of Model 3 suggest that ELoF’s French vowels did not significantly shift upward after L1 re-immersion , and the change in French was not significantly different from the English results .F2 was analyzed once more in Model 4, but no significant differences between the post-RA and L1 re-immersion time points were observed in English  revealed that over the RA, individuals making the most development (in terms of systemic vowel lowering) in their L2 French were also those who exhibited most substantial phonetic drift (vowel lowering) in their L1 English , reaffirming the likelihood of a link between these two systems. A second correlation (n = 27) revealed that individuals whose L2 French vowels raised the most from the post-RA session to the L1 re-immersion time point were also those whose L1 phonetic drift reversed most from the post-RA session to the L1 re-immersion session , indicating that the reversal of phonetic drift may be linked to loss of L2 gains. Both of these correlations are plotted in Subsequently, we analyzed the link between L1 and L2 phonetic change at an individual level by running Spearman’s rank correlations. The amount of lowering over the RA in ELoF’s English and French was calculated as follows: post-RA F1 minus pre-RA F1, and the amount of raising after the RA was calculated as follows: −1 × (L1 re-immersion F1 − post-RA F1). The latter calculation ensured the values for “raising” were all positive, facilitating interpretation. The first correlation . At the same time, this model suggested that the change from the post-RA to the L1 re-immersion time point did not reach significance .This revealed that the distance between learners’ L1 English and L2 French vowels diminishes and that this change was most substantial between the pre-RA test and the post-RA test. Model 5 was fitted to confirm that the distance between each participant’s English mean F1 and French mean F1 reduced significantly from the pre-RA session to the post-RA session  returns to the L1 English environment. In this section, we return to the research questions stated in Section 3 and evaluate the respective hypotheses in turn.This research analyzes the L1 English and L2 French vowel spaces of 42 L1 ELoF, before and after they spend a 6-month RA in a French-speaking country, as well as 10 months after a subsection of the participants ((RQ1) determined whether the acoustic properties of learners’ L1 English and L2 French vowels change over the RA. Baseline results for F1 suggested that the native French vowel space was higher than that of English at the pre-RA time point predicting that the learners’ English vowel system would become more raised over the RA. In contrast, the baseline results for ELoF’s L2 French vowels suggested that they were on average slightly higher than the native French vowels at the pre-RA session, predicting that the L2 vowel space would lower to approximate L2 norms over the RA. No change was expected along F2 for either of ELoF’s languages because no differences were observed between the advancement of the native French vowel space and that of both ELoF’s L1 English and L2 French vowels.Our first line of investigation Statistical analyses revealed systemic phonetic drift in the learners’ English vowel space, but not in the predicted direction: the vowel space lowered, drifting away from the norms of the L2 French input. However, as expected, the learners’ French vowels also lowered significantly in the direction of native French norms and no systemic horizontal shift in either vowel space was observed. These results indicate a relationship between acquisition of L2 phonetic detail and systemic L1 phonetic drift. Indeed, the phonetic change in the learners’ L2 French vowel space is entirely consistent with the change in the L1 English vowels. That is, although the L1 English vowels could have drifted upward to approximate the global formant values of the L2 French input , 2019a, Before elaborating on the RA results, it is important to consider why these learners exhibit a higher L2 French vowel space at the pre-RA time point compared with the NFS vowels, despite the learners’ English vowels being lower than in native French. Indeed, it is not the first time that experienced L1 ELoF have been shown to produce French vowels more raised than those of NFS, especially in the upper regions of the vowel space e.g., . AlthougThis was reemphasized by the findings in Overall, these results indicate that drift cannot always be accurately predicted “solely on the basis of cross-linguistic differences between similar sounds; rather, there are other intervening factors” , p. 196.(RQ2). Because previous research has suggested that phonetic drift is reversible . The first correlation indicated a relationship between L1 drift and L2 phonetic change: the learners whose L2 French vowel space lowered most over the RA, were also the learners who exhibited the most substantial L1 English vowel lowering . Such findings are consistent with previous research focusing on between-speaker variability in phonetic drift. For example, SLM-r  corresponded to the extent to which L2 phonetic development was reversed from the post-RA session to the L1 re-immersion time point . This finding mirrors that of (RQ4) analyzes the distance between L1 and L2 along a given acoustic dimension. Our predictions were that if L2 phonetic development occurs over the RA  phonetic change in L1 would occur to a similar extent, resulting in a sustained L1–L2 distinction  and phonetic category assimilation (reduced distinction between L1 and L2) are not mutually exclusive. In undered” , p. 40, undered” . Althougundered” .SLM-r claim that “a merger of the phonetic properties”  to determine whether L1 phonetic drift is linked to L2 phonetic development, (b) to analyze whether L1 and L2 phonetic changes over an RA were reversed upon extensive L1 re-immersion, and (c) to establish whether the distance between L1 and L2 vowel systems varies or remains constant both in the L2 environment and upon L1 re-immersion. Results revealed an apparent link between the phonetic drift in the learners’ L1 English vowels and the development of their L2 French vowel production. Concerning the persistence of phonetic drift and L2 phonetic changes, findings at the L1 re-immersion time point suggest only a partial reversal of the L1 changes which occur over the RA, and all systemic L2 developments are sustained. Finally, over time, the distance between participants L1 and L2 vowels diminishes consistent with assimilation. Overall, these results start to paint a clearer picture of longitudinal phonetic drift by highlighting L2 phonetic development as a predictive factor of this L1 change, as well as underlining the existence of a relationship between the loss of L2 phonetic gains and the reversal of L1 phonetic drift upon returning home.More broadly, these results have implications for L2 speech models and how sounds are represented in the minds of language learners. We argued that the production of L1 and L2 sounds is guided by a composite systemic representation that is characterized by the combined global acoustic distributions for both the native and foreign sound systems to which they are exposed over time. Future models of L2 speech learning may therefore not only need to be able to account for bidirectional cross-linguistic influence but also phonetic interactions both between individual segments and the wider sound systems to which those segments belong.


A causal role for TDP-43 assembly in neurodegeneration is evidenced by dominantly inherited missense mutations in TARDBP, the gene encoding TDP-43, that promote assembly and give rise to ALS and FTLD7. At least four types (A–D) of FTLD with TDP-43 pathology (FTLD-TDP) are defined by distinct brain distributions of assembled TDP-43 and are associated with different clinical presentations of frontotemporal dementia8. We previously showed, using cryo-electron microscopy, that TDP-43 assembles into amyloid filaments in ALS and type B FTLD-TDP9. However, the structures of assembled TDP-43 in FTLD without ALS remained unknown. Here we report the cryo-electron microscopy structures of assembled TDP-43 from the brains of three individuals with the most common type of FTLD-TDP, type A. TDP-43 formed amyloid filaments with a new fold that was the same across individuals, indicating that this fold may characterize type A FTLD-TDP. The fold resembles a chevron badge and is unlike the double-spiral-shaped fold of ALS and type B FTLD-TDP, establishing that distinct filament folds of TDP-43 characterize different neurodegenerative conditions. The structures, in combination with mass spectrometry, led to the identification of two new post-translational modifications of assembled TDP-43, citrullination and monomethylation of R293, and indicate that they may facilitate filament formation and observed structural variation in individual filaments. The structures of TDP-43 filaments from type A FTLD-TDP will guide mechanistic studies of TDP-43 assembly, as well as the development of diagnostic and therapeutic compounds for TDP-43 proteinopathies.The abnormal assembly of TAR DNA-binding protein 43 (TDP-43) in neuronal and glial cells characterizes nearly all cases of amyotrophic lateral sclerosis (ALS) and around half of cases of frontotemporal lobar degeneration (FTLD) Cryo-electron microscopy structures and mass spectrometry analyses show that TAR DNA-binding protein 43 (TDP-43) forms amyloid filaments with a distinct fold in type A frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP) compared with TDP-43 filaments in type B FTLD-TDP and amyotrophic lateral sclerosis. The amino-terminal part of TDP-43 includes a DIX (dishevelled and axin) domain, a nuclear localization signal, and tandem RNA-recognition motifs (RRMs). The carboxy-terminal part comprises an intrinsically disordered low-complexity domain (LCD), which contains regions enriched in glycine, hydrophobic residues, and glutamine and asparagine (Q/N). The DIX domain, RRMs and LCD all contribute to the association of TDP-43 with ribonucleoprotein granules and RNA11.TAR DNA-binding protein 43 (TDP-43) is a ubiquitously expressed RNA-binding protein with diverse roles in RNA processing. It mainly resides in nuclear ribonucleoprotein granules but also undergoes nucleocytoplasmic shuttling to participate in cytoplasmic ribonucleoprotein granules2. The assemblies have granulofilamentous morphologies, with diameters of 10–15 nm (refs. 19). They bind the amyloidophilic dye thioflavin-S poorly20. Cryo-electron microscopy (cryo-EM) structures of assembled TDP-43 from the prefrontal and motor cortices of two individuals with amyotrophic lateral sclerosis (ALS) and type B frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP) have shown amyloid filaments with an identical double-spiral-shaped fold 9. The ordered core of these filaments is formed by the N-terminal half of the LCD (G282–Q360), with the flanking regions forming a fuzzy coat.In disease, full-length TDP-43 and abnormally truncated C-terminal fragments (CTFs) assemble and are ubiquitylated and phosphorylated21. However, other studies have shown that TMEM106B filaments accumulate in the human brain in an age-dependent manner in many neurodegenerative conditions, including tauopathies and synucleinopathies, as well as in neurologically normal individuals25.The structures of assembled TDP-43 in other neurodegenerative conditions were unknown. A recent cryo-EM study involving four individuals with FTLD-TDP types A–D in the absence of ALS did not find amyloid filaments of TDP-43 and reported that filaments of transmembrane protein 106B (TMEM106B) characterize FTLD-TDP insteadHere, we use cryo-EM to determine the structures of assembled TDP-43 from the brains of individuals with the most common type of FTLD-TDP, type A. We show that TDP-43 does in fact form amyloid filaments, which are present in addition to TMEM106B filaments. The structures reveal that unlike TMEM106B, TDP-43 forms distinct amyloid filament folds in different neurodegenerative conditions and detail their structural basis.9. Four individuals carried mutations in GRN associated with type A FTLD-TDP27, whereas one individual had wild-type GRN. All individuals had abundant compact neuronal cytoplasmic inclusions, short dystrophic neurites and infrequent neuronal intranuclear inclusions of assembled TDP-43 concentrated in the second and third cortical layers, indicative of type A FTLD-TDP8 . Differences in brain extraction protocols may account for this discrepancy. We found TDP-43 filaments in the supernatant following centrifugation at 27,000g, whereas the other study examined the pellet following centrifugation at 21,000g. TMEM106B filaments have also been observed in the brains of individuals with tauopathies and α-synucleinopathies, as well as in the brains of neurologically normal individuals25. TMEM106B inclusions do not colocalize with inclusions of TDP-43, tau or α-synuclein28. In contrast to these proteins, there are no clear relationships between different TMEM106B filament folds and diseases. Available evidence is consistent with the age-dependent accumulation of TMEM106B filaments in the human brain28, which may be modified by the TMEM106B haplotype29.The presence of both TDP-43 filaments and TMEM106B filaments in type A FTLD-TDP, as well as in ALS with type B FTLD-TDPOur cryo-EM reconstructions were of sufficient resolution to visualize peptide groups and ordered solvent, enabling us to build an accurate atomic model of the TDP-43 filament fold and establish its right-handed helical twist Fig.  and 4a,bThe fold is centred around a kinked, 11-residue β-strand formed by A321–Q331 in the fourth layer, which forms steric zippers with β-strands in the neighbouring third and fifth layers and imparts an arrangement that resembles a three-bar chevron badge . Hydrophobic surface patches on amyloid filaments are thought to engage in aberrant interactions31.Less-ordered protein density extends from the N-terminal residue R272 and covers a hydrophobic patch formed by W334–A341 in the turn linking the fourth and fifth layers Fig. . This de32. We also observed non-proteinaceous densities within cavities between the first, second and third layers  classification of the cryo-EM filament segments from the individual with the largest dataset revealed alternative conformations of the N-terminal region R272–G295 and of the turn G335–Q343 linking the fourth and fifth layers Fig. . Fewer tWe determined the structures of filament segments containing the alternative conformations, with resolutions of up to 2.5 Å Fig. . In the 35. These results show that amyloid filaments do not always adopt uniform repetitive structures. The structural variability of TDP-43 amyloid filaments may, therefore, have implications for the development of diagnostic and therapeutic compounds.Mapping of the filament segment locations in the micrographs revealed that TDP-43 molecules with different local structural variations could coexist in individual filaments Fig. . SegmentThe side chain of R293 is completely buried in the main conformation of the chevron fold, and its positive charge is only partially compensated by hydrogen bonds to the surrounding peptide groups Fig. . The ove36. Methyl-R293 is incompatible with the main conformation of the filament fold, owing to steric clashes, and can only be accommodated in the alternative conformation of the N-terminal region, where its side chain is exposed  can citrullinate arginine residues in RGG motifs39, and several peptidyl-arginine methyltransferases can target this motif38. Increased PAD activity and protein citrullination have been observed in several neurodegenerative diseases40. Possible roles for the citrullination and methylation of TDP-43 at R293 in other neurodegenerative conditions, as well as in healthy individuals, remain to be determined. Together, our results indicate that post-translational modifications of R293 may influence local structural variation of TDP-43 filaments from type A FTLD-TDP by altering the charge and size of the residue.Arginine 293 is located in the sole RGG/RG motif of the TDP-43 LCD. RGG/RG motifs participate in functional interactions of RNA-binding proteins and are regulated by arginine citrullination and methylation41.The chevron fold of type A FTLD-TDP is unlike the double-spiral fold of ALS with type B FTLD-TDP, demonstrating that distinct amyloid filament folds of TDP-43 characterize different neurodegenerative conditions . These further residues may account for the presence of distinct CTFs in type A FTLD-TDP compared with ALS and type B FTLD-TDP44. Differences between the two filament folds arise from distinct structural organizations of the hydrophobic region. In the double-spiral fold, this region forms two compact hydrophobic clusters, whereas in the chevron fold it adopts an extended conformation owing to its more extensive β-structure, which is stabilized by zipper packing with β-strands of the glycine-rich and Q/N-rich regions.Residues A321–Q331 in the hydrophobic region form the nexus of both filament folds. These residues are conserved and may form cooperative α-helices and intermolecular β-sheets of functional importance45. The Q/N-rich region has an extended conformation and similar secondary structure in both folds, but the interior–exterior orientations of its side chains are reversed. This includes S347 and S350, which are buried in the double-spiral fold but solvent exposed in the chevron fold. Recently, phosphorylation of S350 was identified in assembled TDP-43 from an individual with type A FTLD-TDP but not that from individuals with ALS and type B FTLD-TDP18. It remains to be determined whether phosphorylation occurs before or after filament formation.Like the double-spiral fold, the surfaces of the chevron fold are formed of the glycine-rich and Q/N-rich regions of TDP-43 and lack charged grooves , which indicates that the local environment may influence TDP-43 filament folds. Producing disease-associated filament folds in model systems will be key to testing these hypotheses. So far, TDP-43 filaments assembled in vitro have not recapitulated structures from brain51.The differences between the two filament folds may account for the distinct seeding abilities and toxicities of assembled TDP-43 from type A FTLD-TDP and that from ALS and type B FTLD-TDP in cellular and animal models9. The chevron fold of type A FTLD-TDP is unlike the double-spiral fold of ALS and type B FTLD-TDP, demonstrating that distinct TDP-43 amyloid filament folds characterize different neurodegenerative conditions. The structures indicate a role for post-translational modifications of arginine in filament formation and in structural variation in individual filaments. This work will guide mechanistic studies of TDP-43 assembly, as well as the development of diagnostic and therapeutic compounds targeting assembled TDP-43.Here, we have established that TDP-43 forms amyloid filaments in type A FTLD-TDP, as it does in ALS with type B FTLD-TDP8, as well as a lack of comorbid neurodegenerative-disease-associated neuropathology. Individual 2 has been described before as patient 3 in ref. 52. All individuals had abundant compact neuronal cytoplasmic inclusions, short dystrophic neurites and infrequent neuronal intranuclear inclusions concentrated in cortical layers two and three that were detected using antibodies against the N terminus of TDP-43 and TDP-43 phosphorylated at S409 and S410. All individuals received clinical diagnoses of frontotemporal dementia. The clinical presentations of individuals 1, 2, 4 and 5 were consistent with non-fluent variant primary progressive aphasia, whereas individual 3 presented with clinical symptoms consistent with behavioural-variant frontotemporal dementia. These clinical presentations have previously been found to be associated with type A FTLD-TDP8. Individual 3 had wild-type GRN, whereas the remaining four individuals had mutations in GRN associated with type A FTLD-TDP. All individuals had normal C9orf72 hexanucleotide repeat numbers. Further clinicopathological details are given in Extended Data Table Human tissue samples were from the Brain Library of the Dementia Laboratory at Indiana University School of Medicine  and the Manchester Brain Bank . Their use in this study was approved by the ethical review processes at each institution. Informed consent was obtained from the patients’ next of kin. The individuals were selected based on a neuropathological diagnosis of type A FTLD-TDP, according to the criteria set out in ref. C9orf72 gene, we performed repeat-primed PCR followed by fragment length analyses as previously described53. Oligonucleotides designed to amplify the coding exons and corresponding flanking intronic regions of the GRN gene were used for PCR using 50 ng of genomic DNA extracted from brain tissue. The amplified products were purified and subjected to direct dideoxy sequencing as previously described54.Whole-exome sequencing target enrichment made use of the SureSelectTX human all-exon library , and high-throughput sequencing was carried out using a HiSeq 4000 . To screen for hexanucleotide repeat expansions in the 9. Grey matter was dissected from flash-frozen prefrontal cortex and homogenized using a Polytron (Kinematica) in 40 volumes (v/w) of extraction buffer containing 10 mM Tris-HCl pH 7.5, 0.8 M NaCl, 10% sucrose and 1 mM EGTA. A 25% solution of sarkosyl in water was added to the homogenates to achieve a final concentration of 2% sarkosyl. The homogenates were then incubated for 1 h at 37 °C with orbital shaking at 200 rpm, followed by centrifugation at 27,000g for 10 min. The supernatants were retained and centrifuged at 166,000g for 20 min. The pellets were resuspended in 6 ml g−1 tissue of extraction buffer containing 1% sarkosyl by sonication for 5 min at 50% amplitude (Qsonica Q700) then diluted fourfold with the same buffer and incubated for 30 min at 37 °C with orbital shaking at 200 rpm. The samples were then centrifuged at 17,000g for 5 min, and the supernatants were retained and centrifuged at 166,000g for 20 min. The pellets were resuspended in 1 ml g−1 tissue of extraction buffer containing 1% sarkosyl by incubation for 1 h at 37 °C with orbital shaking at 200 rpm. The samples were centrifuged at 100,000g for 20 min, and the pellets were resuspended in 30 μl g−1 tissue of 20 mM Tris-HCl pH 7.4, 150 mM NaCl by sonication for 5 min at 50% amplitude (Qsonica Q700). One to two grams of tissue was used for each cryo-EM sample. All centrifugation steps were carried out at 25 °C.Assembled TDP-43 was extracted from flash-frozen prefrontal cortex as previously describedFor histology, brain hemispheres were fixed with 10% buffered formalin and embedded in paraffin. Deparaffinized sections (8 μm thick) were incubated in 10 mM sodium citrate buffer at 105 °C for 10 min and treated with 95% formic acid for 5 min. After washing, sections were blocked with 10% fetal calf serum in phosphate-buffered saline (PBS) and then incubated overnight with primary antibodies against the TDP-43 N terminus  and pS409/410 TDP-43  in PBS containing 10% fetal calf serum. After incubation of sections with biotinylated secondary antibodies for 2 h, labelling was detected using an ABC staining kit (Vector) with DAB. Sections were counterstained with haematoxylin.For immunoblotting, sarkosyl-soluble and sarkosyl-insoluble brain extracts were resolved using 12% or 4–12% BIS-Tris gels (Novex) at 200 V for 45 min and transferred on to nitrocellulose membranes. Membranes were blocked in PBS containing 1% bovine serum albumin and 0.2% Tween for 30 min at 21 °C and incubated with a primary antibody against pS409/410 TDP-43  at 21 °C for 1 h. Membranes were then washed three times with PBS containing 0.2% Tween and incubated with fluorescent StarBright Blue 520 (Bio-Rad) or biotinylated anti-mouse IgG (Vector Lab Inc) secondary antibodies. Membranes were then washed three times with PBS containing 0.2% Tween and imaged using a ChemiDoc MP (Bio-Rad).For immunogold negative-stain electron microscopy, sarkosyl-insoluble brain extracts were deposited on to carbon-coated 300-mesh copper grids (Nissin EM), blocked with 0.1% gelatin in PBS and incubated with a primary antibody against pS409/410 TDP-43 (CosmoBio CAC-TIP-PTD-M01) at a dilution of 1:100 in 0.1% gelatin in PBS at 21 °C for 3 h. After washing with PBS, the grids were incubated with secondary antibodies conjugated to 10-nm gold particles (Cytodiagnostics) at a dilution of 1:20 in 0.1% gelatin in PBS at 21 °C for 1 h. The grids were then stained with 2% uranyl acetate. Electron micrograph images were acquired using a 120-keV Thermo Fisher Scientific Tecnai Spirit or a 80-keV JEOL JEM-1400 electron microscope equipped with charge-coupled device cameras.–1 pronase (Sigma) for 1 h at 21°C and was pelleted by centrifugation at 166,000g for 20 min. The pellet was resuspended in 100 μl hexafluoroisopropanol and incubated at 37 °C overnight. The resuspended pellets were then sonicated for 2 min at 50% amplitude (QSonica Q700), followed by incubation at 37 °C for 2 h. This sonication and incubation cycle was repeated until all material was visibly resuspended. The resuspended pellets were centrifuged at 166,000g for 15 min, and the supernatant was collected and dried by vacuum centrifugation (Savant). The dried protein samples were resuspended in 50 mM ammonium bicarbonate containing 8 M urea, reduced with 5 mM DTT at 56 °C for 30 min and alkylated with 10 mM iodoacetamide in the dark at room temperature for 30 min. Samples were diluted to 1 M urea with 50 mM ammonium bicarbonate and incubated with chymotrypsin (Promega) at 25 °C overnight. Protease digestion was stopped by the addition of formic acid to a final concentration of 0.5%. Samples were then centrifuged at 16,000g for 5 min, and the supernatants were desalted and fractionated using custom-made C18 stop-and-go-extraction (STAGE) tips (3M Empore) packed with porous oligo R3 resin (Thermo Fisher). The STAGE tips were equilibrated with 80% acetonitrile (MeCN) containing 0.5% formic acid, followed by 0.5% formic acid. Bound peptides were eluted stepwise with MeCN at concentrations increasing from 5% to 60% MeCN in 10 mM ammonium bicarbonate and partially dried down by vacuum centrifugation (Savant).Assembled TDP-43 extracted from 0.1 g of tissue was incubated with 0.4 mg ml−1. Eluted peptides were introduced directly using a nanoFlex ion source into an Orbitrap Eclipse mass spectrometer (Thermo Fisher). MS1 spectra were acquired at a resolution of 120K, mass range of 380–1400 m/z, automatic gain control target of 4e5, maximum injection time of 50 ms and dynamic exclusion of 60 s. MS2 analysis was carried out with higher energy collisional dissociation activation orbitrap detection with a resolution of 15K, automatic gain control target of 5e4, maximum injection time of 50 ms, normalized collision energy of 30% and isolation window of 1.2 m/z.Fractionated peptides were analysed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using a fully automated Ultimate 3000 RSLCnano system (Thermo Fisher). Peptides were trapped by a 100 μm × 2 cm PepMap 100 C18 nano trap column (Thermo Scientific) and separated on a 75 μm × 25 cm nanoEase M/Z HSS C18 T3 column (Waters) using a binary gradient formed of 2% MeCN in 0.1% formic acid (buffer A) and 80% MeCN in 0.1% formic acid (buffer B) at a flow rate of 300 nl minLC-MS/MS data were searched against the human-reviewed database  using Mascot . Database search parameters were set with a precursor tolerance of 10 ppm and a fragment ion mass tolerance of 0.1 Da. A maximum of three missed chymotrypsin cleavages were allowed. Carbamidomethyl cysteine was set as static modification. Methionine oxidation, arginine citrullination and methylation, and asparagine and glutamine deamination were specified as variable modifications. Scaffold  was used to validate MS/MS-based peptide and protein identifications. MS/MS spectra containing arginine citrullination and methylation were manually confirmed.−1 pronase (Sigma) for 1 h at 21 °C and centrifuged at 3,000g for 15 s. The supernatants were retained and applied to glow-discharged 1.2/1.3 μm holey carbon-coated 300-mesh gold grids (Quantifoil) and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher). Images were acquired using a 300 keV Titan Krios microscope (Thermo Fisher) equipped with a K3 detector (Gatan) and a GIF Quantum energy filter (Gatan) operated at a slit width of 20 eV. Aberration-free image shift in the EPU software (Thermo Fisher) was used during image acquisition. Further details are given in Extended Data Table Extracted assembled TDP-43 was incubated with 0.4 mg ml55). The motion-corrected micrographs were used to estimate the contrast transfer function using CTFFIND-4.1 (ref. 56). All subsequent image processing used helical reconstruction methods in RELION-4.0 (refs. 58). TDP-43 filaments were picked manually, and reference-free two-dimensional (2D) classification was performed to remove suboptimal segments. Initial 3D reference models were generated de novo by producing sinograms from 2D class averages as previously described59. Then, 3D autorefinements with optimization of the helical twist were performed, followed by Bayesian polishing and contrast transfer function refinement60; 3D classification was used to further remove suboptimal segments, as well as to separate segments with different turn conformations; and 3D autorefinement, Bayesian polishing and contrast transfer function refinement were then repeated. The final reconstructions were sharpened using the standard postprocessing procedures in RELION-4.0, and overall resolutions were estimated from Fourier shell correlations of 0.143 between the two independently refined half-maps, using phase randomization to correct for convolution effects of a generous, soft-edged solvent mask61. Local resolution estimates were obtained using the same phase-randomization procedure but with a soft spherical mask that was moved over the entire map. Helical symmetry was imposed using the RELION Helix Toolbox. Further details are given in Extended Data Table Video frames were gain-corrected, aligned, dose-weighted and summed using the motion correction program in RELION-4.0 , with appropriate symmetry constraints defined using Servalcat65. To confirm the absence of overfitting, the model was shaken, refined in Fourier space against the first half-map using REFMAC5 and compared with the second half-map. Geometry was validated using MolProbity66. ChimeraX67 was used for molecular graphics and analyses. Model statistics are given in Extended Data Table The atomic models were built de novo and refined in real-space in COOTFurther information on research design is available in the Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-023-06405-w.Supplementary InformationReporting SummaryPeer Review File


It has recently been demonstrated how the nitrogen dopant concentration in graphene can be controlled spatially on the nano-meter scale using a molecular mask. This technique may be used to create ballistic electron optics-like structures of high/low doping regions; for example, to focus electron beams, harnessing the quantum wave nature of the electronic propagation. Here, we employ large-scale Greens function transport calculations based on a tight-binding approach. We first benchmark different tight-binding models of nitrogen in graphene with parameters based on density functional theory (DFT) and the virtual crystal approximation (VCA). Then, we study theoretically how the random distribution within the masked regions and the discreteness of the nitrogen scattering centers impact the transport behavior of sharp  The electronic transport properties of graphene and 2D nanostructures based on graphene are truly remarkable. Graphene and graphene nanoribbons are quantum conductors which feature only a few electronic states/modes for transport, but with very low intrinsic electron–phonon scattering, related to the inherent symmetry properties of the quantum states in these materials. The path electrons travel on average before scattering can go beyond micro-meters, even at room temperature, and quantum wave interference effects can survive up to room temperature in graphene nanoribbons ,2. Theserations)  junctionrations) ,6, gratirations) , or noverations) . For exarations) , which irations)  or throuNitrogen-doped graphene has been focused on as a wide interest for fundamental and applied research in various fields, as outlined by several review articles ,11,12,13er scale . In FiguWhen doping graphene in the lab, the different concentrations of nitrogen are implanted by first covering parts of the graphene with a Cthe mask . This gine oxide , but lacne oxide .The doping nitrogen will induce carrier scattering, since the nitrogen atom is acting as a screened, artificial proton potential . This scIn this work, we employ transport calculations based on Greens functions of the interface between high and low N-doping regions in graphene. We consider parameters which are comparable to the actual experiments by Bouatou et al.  Figure . We firsIn order to describe the doping concentration corresponding to N–N distances on the order of Siesta [k-points in the transverse (y-) direction and an electron smearing of We first consider the screening and junction width from a DFT calculation of an ideal, straight junction between two regions corresponding to a jellium-like description where the doping is simulated using the virtual crystal approximation(VCA) . In VCA,Siesta  with a sThe results are shown in ments in . The 0.2For pristine graphene, we employ a non-orthogonal tight-binding model ,20, whertroscopy , and a seviously ,21,22. Heviously , where tThe potential landscape from Furthermore, we are here treating the N atoms as isolated and which yield n-doping, in agreement with the experiments. However, we note that, for N-atoms at much closer distances, the electronic structure may change significantly leading to p-doping instead. This has been seen in doping using solution plasma, leading to a new material , with 13.4% nitrogen .k-point sampling, TranSiesta/TBtrans [SISL [Next, we compare transport calculations based on the /TBtrans  and SISLns [SISL  tools. HSISL tool [TBtrans, and plot it using the technique and methods described in Refs. [The large-scale tight-binding transport calculations ,26,27 arin Refs. ,25. In pin Refs. ,28.We first consider how a straight/uncorrugated  STM-tip . In our E, where the current pattern displays focusing. The results indicate that a gating-potential in a device may be used to tune the Fermi energy to obtain current focusing from point injection, despite the disorder introduced by the random dopants.In When doping graphene in the lab, the different concentrations of nitrogen are implanted by first covering parts of the graphene with Cnitrogen . This giThere has been considerable interest in fabricating constrictions in graphene nanostructures to show ballistic transport and conductance quantization phenomena ,30,31,32otential .n doped region 2, In n levels, as one could expect from a leaky waveguide where parts of the injected current (from the Left) enters the barrier region and is absorbed by the CAP. However, in both “negative”/“normal” cases there is a significant additional scattering and, thus, lower transmission when the scattering potentials of nitrogen are introduced. Here, it is only the “negative” case which displays step features in the transmission function, but at about 50% reduced transmission compared to the pristine case. This comes down to the “negative” constriction having less nitrogen scatterers inside and around the constriction.In rt V1 in , indicatn-channel between the wider regions.This potential scattering is also clearly visible in the corresponding bondcurrents shown in In attering . More coIn order to analyze the step-like transmission functions,  systems , in FiguWithout any nitrogen scatterers, as shown in When nitrogen scattering potentials are introduced in the device, only the “negative” case in F is defined as the ratio of actual noise to the full shot noise I) in the device. It is given by,Finally, we consider the shot noise, as the signal-to-noise ratio is an important characterization of quantum devices, and can give insights into the contributing channels in experiments. The Fano factor, In the diffusive regime with a low number of low conducting channels, we expect ac point . Howeverac point . FurtherThe Fano factors for the “negative” and “normal” systems are seen in It is seen from The lensing shown in However, the transmission function for the constrictions shows a significant asymmetry between the electron and hole side of the Dirac point in the constriction. For clean constrictions, the quantization/step features only appear for energies starting at the constriction Dirac point going towards the Dirac point of the surroundings, corresponding to the electron  and hole (“negative”) sides, respectively. For energies opposite to the quantization region, we note that the confinement is lost. This asymmetry is known from graphene  and reflA significant degradation of quantized conductance is seen in The strength of the nitrogen scattering centers is, therefore, a crucial factor and it might be possible that steps can be taken to reduce it. For example, doping by potassium could possibly reduce the scattering. This has been shown to be experimentally feasible in graphene , and it The Fano factor from Furthermore, the results demonstrate that the current path can be controlled in-plane through the doping, while keeping the graphene sheet planar. This could be combined with out-of-plane stacking into heterostructure devices , where oWe have theoretically studied graphene systems with controlled, sharply defined patterns of different nitrogen concentrations which have recently been achieved experimentally . We used


A novel graphene antenna composed of a graphene dipole and four auxiliary graphene sheets oriented at  Terahertz (THz) communication systems, operating in the frequency range of 0.1 THz to 10 THz, have emerged as a promising solution to address the ever-increasing demand for high-speed wireless communication. This frequency range, known as the THz gap, lies between the traditional microwave and infrared bands, offering unique opportunities for a wide range of applications, including sensing , spectroGraphene is a material formed by a single layer of carbon atoms arranged in a honeycomb lattice . GraphenA review of microwave beam-switchable antennas is presented in . The metToday, the number of the papers devoted to graphene antenna technology is in the hundreds. Detailed descriptions of many graphene and graphene-based antennas are given, as in the review papers ,18. In . There, In paper , a monop2 substrate. Working in the frequency range of 1.94–2.13 THz, the antenna has four radiation operation states that can be selected by setting specific chemical potentials using the external electric field on the graphene elements. In state 1, a dipole-like radiation diagram is produced. States 2 and 3 are characterized by specific directional radiation patterns. By selecting the operation state 4, the antenna is switched off. In [The antenna in  consists off. In , a rectaRecently, an antenna containing an active radiating graphene patch and a non-radiating graphene ring was analyzed in , where trated in . The metIn , opto-elA graphene multiple-input multiple-output (MIMO) microstrip patch antenna in  containsIn , a sub-TIn this paper, we propose a very simple antenna consisting of a graphene dipole, two coplanar reflectors, and two orthogonal reflectors also made of graphene. The main peculiarity of our antenna is its multifunctionality. We will show that it can work in the omnidirectional state and in five other discrete states that differ in the form of the diagram pattern. The control of these states is fulfilled by a simple change to the chemical potential of the graphene elements. In addition to these states, due to vertical orientation of the dipole with respect to the base substrate, there is a possibility of continuous The paper is organized as follows. The antenna description is presented in Geometry of the proposed antenna in free space is presented in The device is formed by an active graphene dipole antenna  of the antenna, which is the main subject of our work, depends on the resulting symmetry of the antenna.e is the unit element; z; i is inversion through a center of symmetry; Group theory greatly simplifies the description, calculations, and analysis of the systems with high symmetry. The highest symmetry of the free-standing antenna in notation ). This ge and the plane of symmetry z. The plane of symmetry e, The group The symmetry elements of the group ment see b.If the graphene elements of the reflectors are placed on one side of the dielectric substrate, the symmetry is reduced to group etry see c. Antenne, which does not provide any information).Symmetry of the antenna system is defined not only by geometry but also by its physical parameters, in particular, by chemical potentials. First, we shall consider the antenna without dielectric substrates in free space with From the point of view of group theory, the resulting symmetry of the structure depends on the combined effect of the dielectric substrates and of the chemical potentials. This can be determined using Curie’s principle of symmetry superposition . This prNotice that with the small thickness of the substrates and low dielectric constants of the dielectric material, the influence of the substrates on the symmetry can be very small, and the resulting symmetry of the antenna will be defined mostly by the chemical potentials. Different resulting symmetries allow us to realize different RPs using the same antenna, as shown by numerical calculations in The irreducible representations (IRREPs) of group In the case of the electromagnetic fields The symmetry degeneration 2 and Cs . As a reA of A of B of A in the B corresponds to the odd symmetry with respect to the plane For example, the components belonging to the IRREPs z with the parameters z because these components change their sign on the axis. Therefore, the Poynting vector in the z-direction is equal to zero, and the antenna does not radiate in this direction. However, groups of symmetry z-direction. For example, in z, thus providing the Poynting vector in the z-direction.Qualitative analysis of both the near and far fields of the antennas can be completed using the elaborated Now, we consider the restrictions produced by the planes of symmetry. If, for example, a current or field component is odd with respect to the plane of symmetry Few studies in the literature provide equations for designing terahertz graphene dipole antennas and predicting their plasmonic resonance frequency. In , a FabryIn , semi-anling law ,43 and tling law , which wling law ,46 simulA coplanar plate capacitor is formed by the graphene dipole metallic electrodes. Its capacitance (ignoring graphene contribution) is given by ailed in ,48, in wintegral , k=dgap/ectrodes  is givene is Euler’s number and a and b are functions of As is shown in detail by , β can barameter  for caseency. In , severalc and w0 . Thus, sFor designing a pre-optimization model of our THz graphene dipole antenna, resonating at et width . As a coGraphene reflectors are passive elements that can be used to improve and control the gain of a given antenna (such as a graphene dipole). This can be achieved by placing the reflectors at specific distances For defining geometric parameters of the antenna in τ=0.5 ps ,53 and rz-direction, in the plane Symmetry of a common linear wire antenna oriented in the As a first demonstration of the effect of the reflectors in RP, we show that it is possible to transform the elliptic-like RP of the graphene antenna in The activation setup of the reflectors defines the following six antenna operation states:(state 0) coplanar and orthogonal reflectors are electrically deactivated ((state 1) a single coplanar reflector is exclusively activated ((state 2) only one orthogonal reflector is activated ((state 3) the pair of coplanar reflectors is uniquely activated ((state 4) activation is limited to the pair of orthogonal reflectors ((state 5) coplanar reflectors , where only one coplanar reflector and one orthogonal reflector are properly activated, with no elements in 2D symmetry. For instance, the main lobe azimuth angle is set to In state 1 and BSR, the dipole chemical potential Z, while 5 dB see b. In FigThe parameters ribed in  by propeThe RPs of the proposed antenna on azimuth plane z-axis  along the Similarly, for state 5, due to the simultaneous activation of coplanar reflectors and an orthogonal reflector, the main radiation lobe is oriented parallel to te 2 see .y-axis when In BSR, due to the simultaneous activation of an orthogonal reflector and a coplanar reflector, the main lobe can be rotated by Finally, for the sake of illustration of lobe rotation capabilities of the proposed device, we present in x-direction for state 3, The proposed graphene antenna electromagnetic field distribution is distinct from that of the metal wire dipole because of its different composition and geometry. Here, we consider the antenna with symmetry Analyzing these graphics, one can see that the symmetry of the field components is in accordance with the results of the group-theoretical predictions in The near field in the antenna contains all components of the electric and magnetic fields of high intensity, which vary rapidly in space. The presence of the reflectors where the excited currents produce the fields significantly changes the structure of the near field in comparison with the antenna without reflectors (not presented here). The components Circuit representations of the antenna with its reflectors can be used to study the frequency characteristics of the antenna. As demonstrated in , a graphN is the number of graphene layers composing the patch [A graphene patch surface impedance is defined by he patch . Notice tate see . Thus, trameters ,41.The graphene dipole and the activated graphene reflector act as electromagnetically coupled resonators, resonating at different frequencies. Therefore, a suitable equivalent circuit representation  for the In order to estimate circuit lumped parameters, we suppose that our device is operating in state 1. For this specific case, only a coplanar reflector is activated  in the form of, for example, Thirdly, the suggested antenna can be optimized. The optimization parameters can be the geometrical dimensions of the active dipole antenna, the dimensions of the reflectors, the gaps between the dipole and reflectors, the number of graphene layers, the chemical potentials of the dipole and the reflectors, and the dimensions of the dielectric substrates and their physical parameters. Depending on application of the antenna, the objective function (or functions) can be different for example, the parameters FBR, gain, The group-theoretical approach developed in our work allows one to reduce the volume of numerical calculus. For example, in the case of In this paper, a novel smart graphene antenna with a simple design and dynamic control of its characteristics was suggested and analyzed. Group-theoretical analysis of the antenna presented in this work allows one to predict the main properties of the radiation diagram of the antenna without solving the corresponding boundary-value problem and also to reduce the volume of numerical calculations.We have shown that, by tuning chemical potentials of the graphene reflectors and the graphene dipole, one can change the antenna radiation pattern, preserving the antenna operation band. In our work, we have calculated the main parameters of the antenna such as input reflection coefficient, total efficiency, front-to-back ratio, and gain for different operation states of the antenna. Due to the vertical orientation of the dipole with respect to the dielectric base and the presence of the four reflectors, the antenna can provide One of the key contributions of our proposed graphene antenna is its ability to achieve continuous 360In future work, we intend to perform optimization of radiation efficiency as well as other parameters and characteristics of the proposed device. We hope that the suggested antenna can find versatile applications.


There is still a lack of specific indicators to diagnose idiopathic pulmonary fibrosis (IPF). And the role of immune responses in IPF is elusive. In this study, we aimed to identify hub genes for diagnosing IPF and to explore the immune microenvironment in IPF.We identified differentially expressed genes (DEGs) between IPF and control lung samples using the GEO database. Combining LASSO regression and SVM-RFE machine learning algorithms, we identified hub genes. Their differential expression were further validated in bleomycin-induced pulmonary fibrosis model mice and a meta-GEO cohort consisting of five merged GEO datasets. Then, we used the hub genes to construct a diagnostic model. All GEO datasets met the inclusion criteria, and verification methods, including ROC curve analysis, calibration curve (CC) analysis, decision curve analysis (DCA) and clinical impact curve (CIC) analysis, were performed to validate the reliability of the model. Through the Cell Type Identification by Estimating Relative Subsets of RNA Transcripts algorithm (CIBERSORT), we analyzed the correlations between infiltrating immune cells and hub genes and the changes in diverse infiltrating immune cells in IPF.ASPN, SFRP2, SLCO4A1) were screened. We confirmed their differential expression using pulmonary fibrosis model mice evaluated by qPCR, western blotting and immunofluorescence staining and analysis of the meta-GEO cohort. There was a strong correlation between the expression of the three hub genes and neutrophils. Then, we constructed a diagnostic model for diagnosing IPF. The areas under the curve were 1.000 and 0.962 for the training and validation cohorts, respectively. The analysis of other external validation cohorts, as well as the CC analysis, DCA, and CIC analysis, also demonstrated strong agreement. There was also a significant correlation between IPF and infiltrating immune cells. The frequencies of most infiltrating immune cells involved in activating adaptive immune responses were increased in IPF, and a majority of innate immune cells showed reduced frequencies.A total of 412 DEGs were identified between IPF and healthy control samples, of which 283 were upregulated and 129 were downregulated. Through machine learning, three hub genes  were associated with neutrophils, and the model constructed with these genes showed good diagnostic value in IPF. There was a significant correlation between IPF and infiltrating immune cells, indicating the potential role of immune regulation in the pathological process of IPF.Our study demonstrated that three hub genes ( Idiopathic pulmonary fibrosis (IPF) is a kind of chronic, progressive and irreversible fibrotic interstitial lung disease (ILD) of unknown etiology . If IPF Although IPF is characterized by sustained epithelial cell damage, fibroblast activation and excessive deposition of the extracellular matrix in the lung parenchyma, the pathological mechanism of fibrosis in IPF is still poorly understood . SubstanIn this study, we searched for hub genes for IPF diagnosis, which might show promise as specific indicators of IPF. Animal models and a meta-GEO cohort were established to further verify the differential expression of these genes. Then, we constructed a diagnostic model of IPF using the hub genes through machine learning and validated the reliability of the model in all the GEO cohorts, which all met the inclusion criteria. Furthermore, the analysis of diverse infiltrating immune cells was performed to explore the immune microenvironment in IPF. By establishing links between hub genes and infiltrating immune cells, we hoped to explore which immune cells might play an important role in IPF.https://www.ncbi.nlm.nih.gov/geo/). Our inclusion criteria were as follows: more than 30 lung samples from Homo sapiens and concurrent inclusion of both IPF patients and healthy controls. According to the inclusion criteria, five GEO datasets including GSE32537 , GSE17978 , GSE53845 , GSE110147 , and GSE10667  were identified. After background calibration and normalization of the data, the differentially expressed genes (DEGs) were screened by the Limma package according to the following criteria: |log2-fold change| > 1 and adjusted P value <0.05.Our study flow chart is shown in Gene Ontology (GO) is an international standardized gene function classification system to comprehensively describe the properties of genes and gene products in an organism, including biological process (BP), molecular function (MF) and cellular component (CC) terms . The KyoAmong the included datasets, GSE32537 had the largest number of samples, so we selected it as our training set. First, LASSO regression and SVM-RFE machine learning algorithms were performed to identify candidate hub genes for a diagnostic model . Then, wP value < 0.05. We also performed correlation analysis of the different types of infiltrating immune cells in IPF  algorithm to identify the proportions of 22 immune cell types in lung samples from IPF patients and healthy controls . Correlas in IPF .Our animal experiments were approved by the Ethics Committee of Sun Yat-sen University. All C57BL/6 mice were fed in a colony room in the Sun Yat-sen University Animal Center with a 12:12 hour light/dark cycle. We injected mice with bleomycin  or an equal volume of PBS intratracheally when they were eight weeks old. On day 21 after injection, all mice were harvested, and lung samples were collected for further analysis.Mouse lung samples were embedded in paraffin and cut into sections. Then, we performed hematoxylin and eosin (H&E), Masson trichrome and Sirius Red staining to analyze pulmonary fibrosis. As previously described , the metP value < 0.05 using an independent-sample t test or the Mann-Whitney U test. All primer sequences used in this study are listed below: mouse Aspn forward 5’-TCCTCTGACAAGGTTGGACT-3’, and reverse 5’-AGAGAGTTGTCGTCATCATCGT-3’; mouse Sfrp2 forward 5’-CGTGGGCTCTTCCTCTTCG-3’, and reverse 5’-ATGTTCTGGTACTCGATGCCG-3’; mouse Slco4a1 forward 5’-CGATCTGCACAGCTACCAGAG-3’, and reverse 5’-GCTGACGAAGGTAAGGCATAG-3’; And mouse 18s forward 5’-GTGACGTTGACATCCGTAAAGA-3’, and reverse 5’-GCCGGACTCATCGTACTCC-3’.RNA was extracted from lung samples using TRIzol reagent  and reverse transcribed into cDNA with the RevertAid First Strand cDNA Synthesis Kit . Then, we performed quantitative real-time PCR (qPCR) using LightCycler480 SYBR Green I Master Mix . 18S served as the internal control. Differences were considered statistically significant with a Mouse lung samples were prepared using RIPA buffer . The total protein concentration was assessed using the Pierce BCA Protein Assay . After electrophoresis, proteins were transferred to PVDF membranes (Millipore). Then, the target proteins were immunoblotted with specific antibodies. The signal intensity of protein bands was visualized with a chemiluminescent substrate (Millipore). All protein bands from three independent blots were quantified using ImageJ software. The corresponding primary and secondary antibodies utilized are listed below: rabbit anti-Asporin , rabbit anti-SFRP2 , rabbit anti-SLCO4A1 , rabbit anti-GAPDH , and anti-rabbit IgG HRP-linked Ab .http://www.r-project.org). An independent-sample t test was applied to validate the differences between two groups if the continuous variables were normally distributed; otherwise, we used the Mann-Whitney U test. A P value < 0.05 was considered statistically significant.All statistical analyses performed in this study were conducted with R software, version 4.1.3 . After bioinformatic analysis with Limma, a total of 412 DEGs were identified between the IPF and healthy control samples, of which 283 were upregulated and 129 were downregulated , extracellular structure and external encapsulating structure organization for BP terms. With regard to CC terms, these genes were mainly involved in collagen-containing ECM and external side of plasma membrane. In the MF category, they were strongly related to ECM structural constituent     and SFRP2  had a negative relationship with neutrophils  and FCN3  had a positive relationship with neutrophils  to construct a diagnostic model for IPF. The model that incorporated the above three hub genes was developed using multivariate logistic regression analysis and is presented as a nomogram , memory B cells (P<0.001), plasma cells (P<0.001), naïve CD4 T cells (P<0.001), resting memory CD4 T cells (P<0.001), activated CD4 memory T cells (P<0.001), follicular helper T cells (P=0.024), gamma delta T cells (P=0.003), resting NK cells (P<0.001), monocytes (P<0.001), M0 macrophages (P=0.039), resting dendritic cells (P<0.001), activated dendritic cells (P=0.012), resting mast cells (P<0.001), eosinophils (P<0.001) and neutrophils (P<0.001). As shown above, the proportions of most infiltrating immune cells involved in activating an adaptive immune response were increased, and the frequencies of a majority of innate immune cells were reduced in IPF. By analyzing the correlations of different infiltrating immune cells, it was shown that resting memory CD4 T cells had the strongest negative correlation with CD8 T cells (R=-0.57) and neutrophils showed a negative correlation with resting mast cells in IPF lung samples  that were differentially expressed between IPF and healthy control samples. Then, we constructed a diagnostic model for IPF using the three hub genes. Through our validation cohorts and statistical methods to verify the reliability of the model, we demonstrated that our model had good diagnostic value. Furthermore, by analyzing the changes in infiltrating immune cells, we explored the possible roles of different immune responses in IPF. We found that there was a significant correlation between IPF and infiltrating immune cells. The levels of most infiltrating immune cells involved in activating an adaptive immune response were increased in IPF, while those of a majority of innate immune cells were reduced. We then analyzed the relationships between the hub genes and infiltrating immune cells. We found that the expression of all hub genes showed strong correlation with the decline in the neutrophil level.In this study, through combined study of a GEO cohort and an animal model, we identified three hub genes (The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The animal study was reviewed and approved by the Ethics Committee of Sun Yat-sen University.YL, XL and SH designed the research, completed most experiments and wrote the manuscript. LP and QZ analyzed the data and provided further input. WH and ZW generated the research idea and revised the manuscript. All authors contributed to the article and approved the submitted version.