The core niche for language use is in verbal interaction, involving the rapid exchange of turns at talking. This paper reviews the extensive literature about this system, adding new statistical analyses of behavioral data where they have been missing, demonstrating that turn-taking has the systematic properties originally noted by  One of the most distinctive ethological properties of humans is that they spend considerable hours in the day in a close (often face-to-face) position with others, exchanging short bursts of sound in a human-specific communication pattern: extrapolating from This core form of language use poses a central puzzle for psycholinguistics (see Section 6), which has largely ignored this context, instead examining details of the processes of language production or comprehension separately in laboratory contexts. Yet this prime use of language involves rapid switching between comprehension and production at a rate implying that these processes must sometimes overlap. Decades of experimentation have shown that the language production system has latencies of around 600 ms and up for encoding a new word (reviewed in Section 6.3) but the gaps between turns average around 200 ms (see Section 5). This would seem to imply that participants planning to respond are already encoding their responses while the incoming turn from the other speaker is still unfinished. This in turn implies potentially long-range prediction in comprehension. A sketch model of the interleaving of comprehension and production processes is presented in Section 7.To appreciate the full nature of this puzzle, it is essential to review what we know about the turn-taking system and its temporal properties. In Section 2, we review the foundational (1)If the current speaker C selects the next speaker N, then C must stop, and N should start. (2)If C does not select N, than any participant can self-select, first starter gaining rights to that next unit.(3)If no other party self-selects, C may continue.These rules then recursively apply at each TRP.These rules predict that intra-speaker silent gaps (generated by rule 3) will be longer than inter-speaker ones, a fact shown to be correct on large samples of conversation  so that the bulk of overlaps are of short duration. ‘Interruption’ is a sanctionable breach of social mores, as every child learns. The systematic properties of all the corpora that have been studied would be entirely different if overlap was not avoided.On the basis of their dismissal of the no-gap–no-overlap target, The central plank of the dismissal of projection is that turn-taking is often not as rapid as has been claimed. “The cumulative distribution above the 200 ms threshold was also of interest, as it represented the cases where reaction to cessation of speech might be relevant given published minimal reaction times for spoken utterances . The disThere are two separate proposals here. The first is that for gaps longer than 200 ms, participants might simply react to silence. This threshold is implausible. First, silence will only become recognizable as silence after c. 200 ms . Note that although mean values vary, they do so in narrow window, roughly a quarter of a second either side of the cross-linguistic mean, and that the factors affecting response times are uniform across cultures  questions cross-linguistically In running speech, how common is overlap  compared to talk by one party alone?(2)In floor transfers, how common are overlaps compared to gaps?(3)What is the distribution of overlap duration, and where do overlaps tend to start relative to the interlocutor’s turn?(4)What is the distribution of different overlap types cf. ?gaps, pauses, between-overlaps, and within-overlaps as follows. Gaps (n = 14648) corresponded to portions of the stereo signal that contained silence in each speaker’s channel, and that involved a floor transfer between the two speakers. Between-overlaps (n = 6524) were floor transfers that occurred without a silent gap between the speakers, whereas within-overlaps (n = 3343) were parts of the signal with overlapping inter-pausal units that did not result in an effective floor transfer. Figure 1 below illustrates the operationalization of gaps, between-overlaps and within-overlaps.We analyzed a subset of 348 conversations  that were free of timing errors, and with annotations included in the NXT-Switchboard Corpus release . To estiThe recordings were divided as follows: 77% of the signal corresponded to speech by one speaker only, 19.2% to silence , and only 3.8% to simultaneous speech by both speakers (either between-overlaps or within-overlaps). If we exclude silent parts, 95.3% of the speech signal corresponded to speech by one speaker. This seems to fit well with Sacks and colleagues’ observation that “overwhelmingly, one party speaks at a time” , p. 700.Figure 2 shows the distribution of the duration of gaps and between-overlaps combined together as FTOs . Between-overlaps (negative FTOs) represented 30.1% of all floor transfers. As for the duration of overlaps, and their location within the interlocutor’s turn, we observed that between-overlaps exhibited a distribution highly skewed to the left, with an estimated modal duration of 96 ms, a median of 205 ms, a mean of 275 ms, and with 75% of the data with values below 374 ms. Within-overlaps tended to start close to the beginning of the utterances that they overlapped, with a modal offset of 350 ms, a median of 389 ms, a mean of 447 ms, and 75% of the data exhibiting offsets below 532 ms. Their duration exhibited a distribution highly skewed to the right, with an estimated modal duration of 350 ms, a median of 389 ms, a mean of 447 ms, and 75% of the data with values below 532 ms. The duration of within-overlaps is thus usually shorter than that of two syllables. This appears to fit well with With regard to how common overlaps are in terms of proportion of turn-transitions, yeah, right) and other minimal utterances that do not constitute an attempt to take the floor. The We now examine the distribution of different types of overlaps. A prediction made by the yeah, right) in either the overlapped or overlapping utterance, (b) the presence of a period of silence within 200 ms from the beginning of the overlap period, (c) the presence of a transition relevance place  in the overlapped turn within the 500 ms leading to the overlap, (d) an abandoned  utterance by any of the two speakers during or immediately following the overlap interval, and (e) the presence of repeated syllables or words in any of the two speaker’s utterances during or immediately following the overlap interval. Other recurrent features observed during or close to the overlap interval, such as laughter and disfluencies, were also annotated.To estimate the prevalence of such possible causal contexts for overlap, in a separate analysis we randomly sampled 100 between-overlaps and 100 within-overlaps from our data, and annotated them for a number of relevant features, including (a) the presence of a backchannel or brief token of agreement . Interestingly, the majority of overlap cases (73%) involved a backchannel. Backchannels, especially continuers like “mm hm” or “uh huh,” are not construed as full turns, but rather pass up the opportunity to take a turn, and are thus principled intrusions into the other’s speech , or resulted in one of the two speakers abandoning their turn prematurely before reaching a point syntactic and prosodic completion . The presence of a disfluency in the utterance of the overlapped speaker before the start of the overlap  was also common. In these cases, it seems that the recipient produced a backchannel in response to the disfluency at a point when the interlocutor had already resumed her turn, causing overlap. In total, cases exhibiting one or more of these six features accounted for 95% of the data.The second most common feature (37%) was the presence of a possible transition-relevance place  in the overlapped turn within a time window of 500 ms before the start of the overlap. Another common feature was a period of silence (29%). In cases with this feature, one of the two speakers produced an utterance briefly after her interlocutor. These cases often involved a backchannel (uhm and well) produced in overlap with the last syllable of the preceding turn, one case with a speaker talking to someone else in the room, and one case of overlap due a clear phonetic segmentation error in the annotation.The remaining 10 cases involved three terminal between-overlaps affecting the last syllable of the previous turn, two cases exhibiting laughter by one of the speakers, two cases involving a turn-initial particle , are of short duration , occur largely in principled places , and mostly involve backchannels (which do not constitute full turns). In light of these observations, we conclude that the vast majority of instances of overlap in our dyadic conversations are consistent with the turn-taking system proposed by Psycholinguistic processing puts tight constraints on any psychologically real model of turn-taking. Here we first draw attention to the early sensitivity to turn-taking in child development. Then we consider three main psycholinguistic aspects: predictive theories of language comprehension, studies of language production , and ideas about the relation between these two processes. Finally we turn to a small number of experimental studies aimed at understanding the relationship between comprehension and production processes in turn-taking.increased toward a second and a half in toddlers, presumably because of the cognitive difficulties of language production, and remained at around a second even for 5-year-olds [this slow convergence with adult norms has recently been confirmed for a larger sample by Stivers et al. (under review)].Parallel to anticipate speaker transitions  have confirmed the general pattern earlier reported, namely relatively fast transitions in the prelinguistic period, with a slowing down as language starts to be comprehended at 9 months. Using eye-tracking of infants watching dyadic interaction, several studies have shown that 3-year-olds observers of dyadic conversations between two adults can nsitions . Althougnsitions , showingnsitions . Casillaun vs. une), Adjectives (“freshly baked”) and verbs (“eat”) can predict nouns by their selectional restrictions; in language that have verbs at the end of the sentence like Japanese, participants can use the nouns to predict the verbs . These studies use the inverse correlation between the cloze probability and the amplitude of an N400 to demonstrate prediction , it takes about 1500 ms before speech output begins . InteresDuring this period of planning for language production, output processes involve the synergies between multiple speech organs. For example, breathing for speaking may need to be initiated. Earlier studies have shown that such breathing activity involves a number of latencies: first, c. 140–320 ms must be allowed for from the time the decision to inhale is made till the time the signal reaches the intercostal muscles ; second,2(n) and n is the number of equally probable choices). When one considers that in production single words have to be plucked from a word lexicon consisting of over 20,000 entries, one can see immediately the processing problems involved. Combined with the relatively slow nature of nerve conduction Which character from the famous movies, is also called 007? (Late).In a second experiment, participants heard the same questions but did not have to answer them. Instead, they only had to remember them, as prompted by later probes. The neural patterns were then compared with those in the first experiment, where participants had to verbally respond, to the second where they only had to comprehend and memorize. The results revealed a clear neural signature associated with production, localized in the appropriate areas, occurring within 500 ms of the point at which a plausible answer to the question became available. Bögels and colleagues interpreted this as showing that participants begin planning their response as soon as they can, up to a second or more before the incoming turn ends.sine qua non for planning their appropriate response. In order to overcome the production latencies, they must also start the planning and encoding of the response as soon as possible.From a psycholinguistic point of view, turn-taking presents the following puzzle: in spite of the long latencies involved in language production (600–1500 ms or more), participants often manage to achieve smooth turn transitions . As a solution to this puzzle, we suggest that comprehension is predictive, even more so than is currently thought. As soon as possible, participants extract the speech act of the incoming utterance, which is the This suggests that there is a significant overlap of comprehension and production processes. Given an average turn  of 1680 ms, somewhere in the middle response preparation may already be underway. This provides a second central puzzle: conversation involves constant double tasking, and this double tasking uses the same language system. The difficulty of the puzzle is increased when one takes into account the findings that both comprehension and production use much of the same neural circuitry . It is pminus the phonological and phonetic encoding, which account for the bulk of the production latency.In any case, regardless of how this is achieved, the experimental and corpus studies reviewed in this section converge in showing that participants in conversation often anticipate the content of the others’ turns well in advance, and that they use that information to prepare their response early.Let us now gather together how the observations and inferences discussed above constrain viable models of turn-taking. Any adequate model must be consistent with a number of observations and constraints, as originally noted by (1)Turns are mostly short , consisting of one or more interjections, phrases or clauses at the syntactic level, and one or more intonational units at the prosodic level. Turn ends typically co-occur with points of both syntactic and prosodic completion.(2)Intra-speaker gaps are longer by c. 150 ms than inter-speaker gaps , suggest(3)Figure 2). Medium gaps and short overlaps are also common, although less so than short gaps.Inter-speaker gaps are most typically short, with modal values for FTOs falling between 100 and 200 ms (cf. (4)Lengthy gaps (over 700 ms) may carry semiotic significance Overlaps, though common, are brief . Overlaps are more common at turn transitions than within turns, and mostly involve back-channels, simultaneous first-starts, disfluencies, and other features predicted by (6)Turn-taking is established early in infancy, long before full linguistic competence, which actually appears to slow down response times; adult conversation timing is not achieved till late in middle childhood.(7)Given the latencies of speech production (over 600 ms), incoming turns have to be predicted if accurate timing is to be achieved. EEG recordings suggest the production process in responsive turns starts as soon as the gist of the incoming turn can be detected.(8)Turn-final cues seem to be used to recognize that a turn is definitely coming to an end. These cues are typically prosodic  but also syntactic , and in principle could be of other types too . In the appropriate pragmatic context, these turn-final cues can trigger the decision of the next speaker to articulate. From the point of view of social interaction, it is effective articulation that constitutes a point of no return .We have outlined above the (9)D: I forgot to tell you the two best things that happen’ to me today.R: Oh super=What were they?D: I got a B+ on my math test ) and I got an athletic award.An alternative model is the turn-end signaling system proposed by We believe that the property list in Section 7 above puts fairly narrow constraints on a possible model of turn-taking. One area of particular interest is the temporal constraints that turn-taking imposes on language processing, given that conversational interchange is the core form of language use. These constraints are funneled into one crucial link in the system, namely, the current addressee preparing to respond. Here we consider the cognitive processes that must be involved.The crucial questions concern what factors govern the decision making process that lies behind the initiation and timing of response. While turn-final cues in the incoming turn seem likely to play a role, they cannot be sufficient given the long latencies in language planning and production. To overcome these long latencies, predictive comprehension must be involved, together with a strategy of early beginnings to production. Bögels et al. (submitted) suggest that production begins as soon as it can – that is, as soon as the speech act content of the incoming turn is clear. This implies of course dual-tasking, perhaps by rapid alternation (‘time sharing’). A new study using a dual-task paradigm and eye-tracking suggests that the heaviest interference is rather late , and tieFigure 3 sketches the cognitive processes that must minimally be at work in the recipient of a typical turn at talk during conversation. Predictive comprehension is underway early, and already by half way through more predictable turns will suggest a temporal envelope for completion (if or either or whenever projects a two clause structure), so offering some long distance projection. Within the last half second or so, the actual words will often be predicted . Production is, at least in the latter stages, serial, and proceeds through conceptualization, lemma retrieval, phonological retrieval, and phonetic encoding, following a time course that seems well understood , extendiFigure 3 sketches the kind of interaction between comprehension and production processes that must be involved in a typical turn transition . There is an early gist comprehension with speech act apprehension sent as soon as possible to the production conceptualizer an account of how responders can often respond with short latencies despite the long latencies of the production system;(b)why the corpus statistical results reliably show a modal response with positive offsets of around 100–300 ms, reflecting the reaction time to the turn-final prosodic cues in the incoming turn , and it poses a substantial puzzle for psycholinguistic models , which until recently have completely ignored this, the most central form of language use.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nitrogen-doped graphene decorated by iron-nickel alloy is introduced as a promising electrode material for supercapacitors. Compared to pristine and Ni-decorated graphene, in acid media, the introduced electrode revealed excellent specific capacitance as the corresponding specific capacitance was multiplied around ten times with capacity retention maintained at 94.9% for 1,000 cycles. Briefly, iron acetate, nickel acetate, urea, and graphene oxide were ultrasonicated and subjected to MW heating and then sintered with melanin in Ar. The introduced N-doped FeNi@Gr exhibits remarkable electrochemical behavior with long-term stability. Due to the intense shortage of reserves of fossil fuels, increasing the awareness of energy conservation and environmental preservation has internationally spurred unprecedented interest in developing green and renewable energy. Electrochemical capacitors (ECs), as one of the promising technology for energy storage systems, have attracted significant attention due to their high power performance, very long life cycle, and reasonable energy density. Accordingly, ECs are often favored in various applications including portable electronics, uninterruptible power sources, memory backup systems, and hybrid electric vehicles -3. It isAs for other energy devices such as fuel cells and dye-sensitized solar cells, the best performance in the electrochemical supercapacitors was achieved with precious metal-based electrodes which constraints the commercialization process ,6. Recenπ bonding [It is generally believed that pseudocapacitance in carbon-based materials is largely based on the redox reactions of surface quinoid functionalities, whose reduction requires protons to proceed . Neverth bonding ,25 and t bonding . The wor bonding .Besides, some transition metal-based materials containing manganese, silver, nickel, and iron revealed good electrochemical behavior -32. In tNitrogen-doped, FeNi alloy-doped graphene was synthesized by dissolving 250 mg of urea in 100 ml aqueous solution from iron(II) acetate (0.5 mM) and nickel(II) acetate tetrahydrate (0.5 mM); the solution was stirred for 2 h and ultrasonicated for 30 min. The obtained solution was mixed with 50 ml aqueous solution containing 200 mg of graphene oxide (GO) prepared by a modified Hummers method  using miλ = 1.54056 Å) radiation over a range of 2θ angles from 10° to 80° was utilized to get information about the phase and crystallinity. The surface morphology and elemental mapping were investigated by a JEOL JSM-5900 scanning electron microscope  and transmission electron microscope  operated at 200 kV equipped with energy-dispersive X-ray (EDX) analysis. Electrochemical analysis was carried out using a different three-electrode system: platinum wire as counter electrode, Ag/AgCl as reference electrode, and the fabricated materials as working electrode. The electrochemical impedance spectroscopy (EIS) was measured with a frequency range between 0.01 Hz and 100 kHz. This system was controlled using a VersaStat4 potentiostat device . The surface composition was detected by X-ray photoelectron spectroscopy (XPS) analysis  with the following conditions: base pressure 6.5 × 10−9 Torr, resolution (pass energy) 20 eV, and scan step 0.05 eV step−1.A Rigaku X-ray diffractometer  with Cu Kα , (200), and (220) crystal planes, respectively, indicate the formation of FeNi alloy [Figure Ni alloy . XPS speNi alloy . Figure 2SO4 as electrolyte solution. As shown in Figure −1. Figure −1. Obviously, the N-doped FeNi@Gr electrode shows redox peaks, indicating that the capacitance characteristics are mainly governed by faradaic redox reactions; thus, the curve pronounces pseudocapacitive behavior attributed to nature-doped materials with nitrogen-containing surface functional groups [−1. The specific capacitance could be determined from the following equation [For proper comparison, the electrochemical properties of the introduced N-doped FeNi@Gr electrode, pristine graphene, and N-doped Ni@graphene were investigated. Figure l groups . Noteworequation ,38:FigurCsp is the specific capacitance (F g−1), I is the response current (A), V is the potential (V), υ is the potential scan rate (V s−1), and m is the mass of the electroactive materials in the electrodes (g). As shown in Figure −1 is 254 F g−1 which is about tenfolds of the other formulations. As the introduced material is based on pristine metals and the electrochemical property investigations have been carried out in acidic media, cycling performance is important to check the corrosion resistance of the introduced alloy NPs. A long-term cycle stability test was evaluated for 1,000 cycles (Figure where Bimetallic N-doped FeNi@graphene can be prepared by multi-treatment of FeAc, NiAc, urea, and GO followed by calcination of the mixture with melanin in an argon atmosphere at 750°C. The FeNi alloy structure can distinctly enhance the electrochemical properties compared to the pristine metals. The synthesized alloy NPs have an average size of 15 nm and homogeneous distribution on the graphene sheets. The introduced electrode material provides a high specific capacitance with excellent cyclability in acidic media, so it can be recommended for commercial application.


Sarcoidosis is a granulomatous disease, the etiology of which is currently unknown. The role of mycobacteria in the etiology of sarcoidosis has been extensively investigated. In this meta-analysis, we assessed the immunological evidence of the possible role of mycobacteria in the pathogenesis and development of sarcoidosis.We performed a systematic search of relevant articles from PubMed, Embase and Cochrane Library databases published between January 1990 and October 2015. Data extracted from the articles were analyzed with Review Manager 5.3 .In this meta-analysis, 13 case-control studies (733 participants) were considered eligible according to our criteria. Methodological quality was assessed using the Newcastle-Ottawa Scale (NOS). The positivity incidence of the immune response  in sarcoidosis patients was significantly higher than that in controls, as determined using fixed-effects model. The odds ratio (OR) of the positivity incidence of T-cell response in the patients with sarcoidosis versus the controls with PPD- or unknown PPD status was 5.54 (95% CI 3.56–8.61); the ORs were 16.70 (95% CI 8.19–34.08) and 1.48 (95% CI 0.74–2.96) for the two subgroups with PPD- controls and unknown PPD status respectively. However, the OR of the positivity incidence in patients with sarcoidosis versus PPD+ controls  was 0.26 (95% 0.10–0.66). Regarding the humoral response, pooled analysis of the positivity incidence revealed an OR (95%CI) of 20.43 (5.53–75.53) for the patients with sarcoidosis versus controls; the ORs were 11.93 (95% CI 2.15–66.27) and 41.97 (95% CI 5.24–336.15) in two subgroups of controls with PPD- and unknown PPD statuses respectively. Data on heterogeneity and evidence of publication bias were examined.M.tuberculosis) and sarcoidosis. The current available evidence indicates that some insoluble mycobacterial antigens that preferentially within the body are involved in the pathogenesis of sarcoidosis rather than the whole mycobacteria and that they elicit a type IV immune response.This meta-analysis confirmed the existence of an association between mycobacteria (especially  M. tuberculosis) is the second most common infectious cause of death in adults worldwide (HIV is the most common). The human host serves as a natural reservoir for M. tuberculosis, which is an intracellular obligate and aerobic bacillus that multiplies within macrophages. The bacterium triggers the production of free radicals and avoids being killed by the same radicals [M. tuberculosis to efficiently establish latent infection has enabled it to spread to nearly one-third of individuals worldwide.Mycobacterium tuberculosis  have been suggested to be infectious factors for the pathogenesis of SA [Sarcoidosis is a systemic granulomatous disease characterized by the formation of epithelioid cell granulomas (and is accompanied by an infiltration of inflammatory cells) without caseous necrosis. Multisystem granulomatous inflammation involving the lungs, lymph nodes, skin, eyes, heart, and muscles is a hallmark of sarcoidosis. Infectious and genetic factors, as well as autoimmunity, are considered to be potential causes of SA –4. In adis of SA .M. tuberculosis was isolated from several sarcoidosis patients by Scadding [M. tuberculosis and sarcoidosis has since been explored with increasing frequency. The advent of molecular biology invigorated the search for mycobacterial DNA in sarcoidosis patients [In the middle of the 20th century, Scadding . The relpatients , 8. One patients  compiledM. tuberculosis antigens could be involved in the pathogenesis of sarcoidosis. Many studies on the T-cell response to M. tuberculosis antigens have been conducted using an interferon gamma release assay (IGRA), which has been proven to be more efficient than the TST (tuberculin skin test) [Recent studies evaluating immunological evidence of mycobacterial antigens in sarcoidosis patients has renewed interest in the role of mycobacteria in sarcoidosis , indicatin test) . We condM. tuberculosis antigens in patients with sarcoidosis. To identify relevant articles for inclusion in this review, all authors independently searched PubMed, Embase, and Cochrane Library databases for relevant studies published from 1990–2015 using the following terms: sarcoidosis AND mycobacteria (OR) mycobacterium; sarcoidosis AND mycobacterium tuberculosis; and sarcoidosis AND tuberculosis. Additional eligible studies were identified by reviewing the references of all retrieved- literatures and review articles addressing the relationship between sarcoidosis and mycobacteria. The outcome measure of all included studies was positivity incidence of the immune response to M. tuberculosis antigens. No language or geographic restrictions were imposed on identified studies.The aim of this meta-analysis was to collect all publicly available studies on the immune response to M. tuberculosis antigens were extracted onto standardized data collection forms by the two reviewers, and the verified data were entered into a Microsoft Excel spreadsheet . The following items were extracted: 1) the publication details including the study geography (country or region), year of publication, first author and other citation details; 2) the number of patients with sarcoidosis and the type of mycobacterium (M. tuberculosis or other mycobacterium) used in analysis; 3) details of the technique used to evaluate the immune response in samples; 4) the health condition of the control subjects and whether details on TST outcomes were provided and 5) the numbers of the samples with an active immune response to mycobacterial antigens for patients with sarcoidosis and control. No attempt was made to include unpublished data. Methodological quality was assessed by the Newcastle-Ottawa Scale (NOS).Paired reviewers (Chuling Fang and Hui Huang) independently evaluated studies for eligibility using a two-stage procedure. During the first stage, all identified abstracts were evaluated to ensure that they were involved in the relationship between sarcoidosis and mycobacteria. All potentially relevant studies were retrieved and selected for the second stage of the procedure, in which a full-text review was performed to determine whether the studies provided the positivity incidence of the immune response. Any disagreements were resolved by discussion and consensus. Data concerning the immune response to M. tuberculosis, binomial proportions were used in which the numerator was sarcoid samples with active immune response to M. tuberculosis antigens and the denominator was the total number of study samples. As dichotomous outcomes, the percentage of sarcoid samples with a positive immune response out of the total samples versus the same percentage of control samples was expressed as an odds ratio (OR) and a 95% confidence interval (CI). The fixed-effects model was used, and the random-effects model weighted by the Mantel-Haenszel method was utilized for data with significant heterogeneity . P<0.05 was considered statistically significant.The included articles were analyzed using Cochrane Collaboration Review Manager statistical software . To calculate the percentage of sarcoidosis samples with a positive immune reaction to 2 test . An I2 value >50% indicates significant heterogeneity.The impact of heterogeneity on the pooled estimates of individual outcomes of meta-analysis was assessed using the Chi-squared test and/or IA funnel plot test was used to assess publication bias. The funnel plot is a measurement of the log of the OR  against the standard error of the log of the OR . Each point represents a study in meta-analysis.The process of identifying eligible studies is summarized in All studies performed detailed immunology analysis and the meta-analysis included studies conducted around the world . In totaM.tuberculosis antigens. Further, nine studies [The quality scores of all 13 included studies were above five points, ranging from five to nine points. Five studies , 20, 24  studies –19, 24 uM. tuberculosis specific antigens. Among the 253 samples in the sarcoidosis group, 126 exhibited a positive immune response to M. tuberculosis specific antigens, as demonstrated by immunology. The combined results of the nine trials revealed that the sarcoidosis patients had a significantly higher positivity rate of the T-cell immune response to M. tuberculosis specific antigens compared to the controls with PPD- or unknown PPD status  (2 statistics (subgroup of PPD- controls (Q (d.f. = 5) = 4.57, P = 0.47, I2 = 0%); subgroup of controls with unknown PPD status (Q (d.f. = 2) = 1.24, P = 0.54, I2 = 0%)) (2 = 95.6%) (2 = 0%)  . Combine0.00001) . An addi0.00001) . Heterog2 = 0%)) . However= 95.6%) . Further(2 = 0%) . Publicaantigens .M. tuberculosis specific antigens. Of the100 samples in the sarcoidosis group, 55 exhibited a positive immune response to M. tuberculosis specific antigens, as demonstrated by ELISA and immunohistochemical analysis. The combined results of four trials [M. tuberculosis specific antigens than the control group . Further, the combined results of two trials [M. tuberculosis specific antigens than subgroup of PPD- controls . Similarly, combined analysis of another two trials [2 statistic (subgroup of PPD- controls (Q (d.f. = 1) = 0.28, P = 0.60, I2 = 0%); subgroup of controls with unknown PPD status (Q (d.f. = 1) = 0.14, P = 0.71, I2 = 0%)) (2 = 0%). Publication bias was not evident, as estimated by generation of a funnel plot for the studies on the T-cell immune response to M. tuberculosis specific antigens ) . The hetantigens .Meta-analysis was the main method used in this research paper. It is more accurate and reliable than regression analysis or original papers.M. tuberculosis has been a highly contested and challenging issue. Further analysis is important considering their opposite therapeutic effects In the current meta-analysis, a greater positivity incidence of the immune response to M. tuberculosis specific antigens was observed in sarcoidosis group compared to the control group of subjects with PPD- or unknown PPD status for both the T-cell immune response and humoral response, indicating that these antigens induce T-cell-specific responses in the blood or BALF of sarcoidosis patients. However, it is important to note that sarcoidosis patients may be more likely to have positive TST results (i.e. to be more sensitive to LTBI) than healthy individuals due to unknown host factors with no known etiologic link between sarcoidosis and M. tuberculosis. Notably, the positivity incidence of T-cell immune response to M. tuberculosis specific antigens in sarcoidosis group was found to be significantly lower in the sarcoidosis patients than in the PPD+ (LTBI) controls, although the incidences did not differ among the four primary trials. Further, it was hypothesized that M. tuberculosis specific antigens response would not be observed in all sarcoidosis patients, and that the specific mycobacterial antigen may represent just one of the most important factors for the pathogenesis of sarcoidosis.The relationship between sarcoidosis and In the T-cell immune response, no significant heterogeneity was observed in either the subgroup of PPD- controls or in the subgroup of controls with an unknown PPD status. Similarly, no significant heterogeneity was detected in the group of PPD+ (LTBI) controls. Similarly, no significant heterogeneity was detected in the group of PPD+ (LTBI) controls. Further, in the humoral response, no significant heterogeneity was observed in either the subgroup of PPD- controls or the subgroup of controls with an unknown PPD status. However, significant heterogeneity was detected between the two subgroups with PPD- or unknown PPD status on the T-cell immune response, indicating that PPD status strongly influenced experimental outcome.M. tuberculosis antigens are involved in the pathogenesis of sarcoidosis [M. tuberculosis antigens including catalase‑peroxidase antigen (mKatG) and Mtb 16S rRNA in nearly 40% of their sarcoidosis subjects as detected by in situ hybridization [M. tuberculosis (and not the whole mycobacteria) are involved in the pathogenesis of sarcoidosis [M. tuberculosis antigens in sarcoidosis patients; (3) development of mycobacterial disease either coincidentally, before or after development of sarcoidosis [M. tuberculosis antigens or molecular evidence was observed in some sarcoidosis subjects. Therefore, we assumed that other factors besides mycobacterial antigens contribute to the pathogenesis of sarcoidosis.A number of original studies have examined mycobacteria in samples of sarcoidosis patients , 25–32, coidosis , 34. Amodization . Furthercoidosis –37; (4) coidosis , 27; andcoidosis , 39. It coidosis , 40. HowThere are several limitations to this study. First, although data were collected from more than 700 patients, the number of incorporated studies was still small, and therefore these results may have been affected by publication bias. Second, the definite sensitivity of the immunological data is unknown, as they were not described or analyzed in detail in this study. Third, some variations may also have occurred due to differences in local environmental exposure and the fact that some sarcoidosis patients may have a disorder initiated by organisms or triggers other than mycobacteria. Therefore, additional studies would be useful to provide more concrete data to investigate the accuracy of this conclusion are needed.M.tuberculosis) and sarcoidosis. Mycobacteria was not observed in all cases of sarcoidosis, however, these bacteria indeed represent an important factor involved in the pathogenesis of this disease. It has been hypothesized that only some specific, poorly degraded mycobacterial pathogenic antigens contribute to immune-mediated granulomatous inflammation rather than the whole active mycobacteria and that they elicit a type IV immune response.In conclusion, the results of this meta-analysis have demonstrated an association between mycobacteria Click here for additional data file.


We suggest an information-theoretic approach for measuring stylistic coordination in dialogues. The proposed measure has a simple predictive interpretation and can account for various confounding factors through proper conditioning. We revisit some of the previous studies that reported strong signatures of stylistic accommodation, and find that a significant part of the observed coordination can be attributed to a simple confounding effect—length coordination. Specifically, longer utterances tend to be followed by longer responses, which gives rise to spurious correlations in the other stylistic features. We propose a test to distinguish correlations in length due to contextual factors  and turn-by-turn coordination. We also suggest a test to identify whether stylistic coordination persists even after accounting for length coordination and contextual factors. Thus, such claims are susceptible to various confounding effects. For instance, it is known that there is significant length coordination in dialogues, in the sense that a longer utterance from user m user X . Thus, itime-shifted mutual information. The proposed coordination measure characterizes the dependence between the stylistic features of the original post and the response. In addition, we provide a computational framework to account for confounders when measuring stylistic coordination.Here we attempt to remedy the problem and propose an information-theoretic framework for characterizing stylistic coordination in dialogues. Namely, given a temporally ordered sequence of utterances  by two individuals, we characterize their stylistic coordination with We revisit some of the case studies where linguistic coordination was reported and demonstrate that a significant part of the observed correlations in linguistic features can indeed be explained by length coordination rather than stylistic accommodation. In particular, most stylistic features that exhibit statistically significant correlation exhibit little to no correlation after length coordination has been taken into account.We also focus on the observed length correlations, and examine whether it is due to turn-by-turn coordination between the participants, or can be attributed to other contextual factors. We construct a statistical permutation test and demonstrate unequivocally that turn-by-turn length coordination in dialogues indeed takes place. Finally, we develop a similar test for stylistic features, and demonstrate that at least for one of the datasets, the remnant coordination (after conditioning on length) cannot be explained by contextual factors alone and has to be due to turn-by-turn level coordination between the speakers.To represent stylistic features in utterances, we use Linguistic Inquiry Word Count (LIWC) , which im, and K is the total number of exchanges in a dialogue. Since we focus on coordination between the same stylistic markers, we will drop the superscript m from now on. We use the convention O to represent the originator—the person who is producing the original utterance in a single exchange, R to represent the respondent—the person who is replying to the originator.Each dialogue is a sequence of utterance exchanges between two participants. Following , 5 we bip be the joint distribution of the random variables O and R. We characterize the amount of stylistic coordination using Mutual Information (MI) [H(O) = −∑p(o) log p(o) is the Shannon entropy of O, and H(O∣R) is the entropy of O conditioned on R. Note that in our case the arguments are temporarily ordered: O is always the initial utterance, and R is the response, so that Let ion (MI) ; see S1 m in an utterance of O’s can help to predict R’s usage of m in the immediate response. In contrast to linear correlation measures, mutual information is well suited for handling strongly non-linear dependencies.Recall that mutual information between two variables measures the average reduction in the uncertainty of one variable, if we know the other variable. Thus, in essence, the proposed measure of stylistic coordination quantifies how the use of a marker after conditioning on a third variable, Z, via Conditional Mutual Information, defined asI(O : R∣LR), where LR is the length of the utterance by user R.We measure the correlation between two variables O and R. We could do this by first calculating the empirical distribution p and then using plug-in estimator tends to underestimate the entropy of a system. Instead, here we use the statistical bootstrap method introduced by DeDeo et al. [Given a set of samples o et al. , which ao et al.  for morek-nearest neighbors to each point, and then average the mutual information estimated from the neighborhood of each point. It has been shown that this estimator is asymptotically unbiased and consistent. Discussion of different entropy estimators can be found in [While the above discrete estimator works well for evaluating mutual information between discrete stylistic variables, it is not very useful for evaluating mutual information between two length variables, due to limited number of samples we have. Instead, we will use a continuous estimator introduced by Kraskov et al. . This nofound in  and refenon-Admins. All of the users interact with each other on Wikipedia talk pages, where they discuss issues about specific Wikipedia pages. We focus on dialogues where each participant make at least two exchanges within a dialogue, which results in over 30,000 utterances.We applied our coordination measures to two datasets previously studied in : oral trO and R who have participated in a dialogue. Unfortunately, most pair-wise exchanges are rather short and do not produce sufficient samples for evaluating mutual information or conditional mutual information. Instead, we group the individuals according to their roles, and then use aggregated samples to calculate stylistic coordination between the groups. The groups correspond to Judges and Lawyers for the Supreme Court data, and Admins and non-Admins for the Wikipedia data.Ideally, we would like to calculate linguistic accommodation between any pair of individuals I(O : R) and I(O : R∣LR). The bias in estimators for conditional mutual information and mutual information are generally different. Therefore, rather than estimating mutual information directly, we use a conditional mutual information estimator where we condition on randomly permuted values for LR. We repeat this procedure for four hundred times to produce 99% confidence intervals for I(O : R) (blue bars). The green bars give the 99% confidence intervals in case there is no stylistic coordination by estimating CMI with R’s utterances permuted (erasing any stylistic coordination).Impersonal Pronoun, Article, and Auxiliary Verb are reduced by factors of ∼ 6.7, ∼ 4.8, and 5.3, respectively, after conditioning on length. For the feature Conjunction, the 99% confidence interval of coordination score is above the confidence interval of zero information before conditioning, and falls into the confidence interval of zero information after conditioning. Similarly, in Impersonal Pronoun, Article, Adverb, Preposition, Quantifier) become practically zero after conditioning, suggesting that the observed coordination in those stylistic features are due to length correlations.The blue dots show the mutual information between the corresponding stylistic features, and suggest strong linguistic correlations between the groups. This effect, however, is strongly diminished after conditioning on the length of utterances (red dots). For instance, the coordination scores on features A similar picture holds for the Wikipedia dataset shown in directionality, in stylistic coordination. For instance, by comparing Fig Another interesting observation is that there is significant asymmetry, or To conclude this section, we note that some of the correlations in stylistic features persist even after conditioning on length. One can ask whether this remnant correlation is due to turn-by-turn level linguistic coordination, or can be attributed to other confounding factors. We address this question in detail later in the text.Alice and Bob are always conversing using short statements, while in another conversation they exclusively use long statements, perhaps due to different topics of conversation. Length coordination is found if data from these two conversation is aggregated, however, this coordination only reflects Alice’s and Bob’s response to the topic of conversation. More generally, aggregating data might lead to effects similar to Simpson’s paradox [As discussed in the previous section, the observed correlations in linguistic features can be attributed to coordination in the length of utterances. Here we analyze this phenomenon in more detail. In particular, we are interested whether the observed length correlations are due to turn-by-turn coordination, or can be attributed to other contextual factors. For instance, consider a scenario that in one conversation,  paradox .contextual factors), consider the Bayesian network model that incorporates both contextual factors and length coordination, as shown in LO and LR are random variables representing the length of an utterance by the originator O and the respondent R, respectively. In the model with both solid and dashed lines in LO explicitly influences LR. While if we only have the soild lines in LR is independent of LO after conditioning on the context C. Thus, the model in C can vary within a single conversation, for example, the theme of a conversation may change as time goes by. But for simplicity, we will assume that the contextual factor C does not change within the dialogue or conversation.To understand the possible extent of various confounding factors  is given by the following conditional mutual information:OLC) asOLC captures not only the length coordination in a turn-by-turn level, but also the confounding behaviors between LO, LR and C. In fact, OLC can be decomposed into two items:interaction information [co-information [LO, LR and C.A direct measure of Turn-by-turn Length Coordination (ormation  or co-inormation ), and chTLC described in LO and LR are conditionally independent of C if and only if TLC = 0. However, direct evaluation of TLC is not possible due to the lack of sufficient number of samples, e.g., the number of exchanges within a specific dialogue. Nevertheless, it is possible to test the turn-by-turn length coordination by a non-parametric statistical test as shown below.A straightforward method to test for turn-by-turn coordination is to evaluate Our null hypothesis is that there is no turn-by-turn coordination, so that all observed correlations are due to contextual factors. We now describe a procedure for testing this hypothesis.c from originator o and respondent r as:kth exchange (two utterances) by the originator o and respondent r in dialogue c, and Kc represents the total number of exchanges in c. We also define the aggregated set of exchanges of user o ∊ O and user r ∊ R as:Co,r represents all the dialogues that involved user o and r. We can rewrite SO ← R element-wise asN = ∣SO ← R∣ representing number of samples. For each triplet of right hand side in Rk is the reply utterance to Ok in the dialogue Ck. Finally, from SO ← R we obtain the setlen (⋅) is a function representing the length of an utterance.We denote the pairwise set of exchanges in a specific dialogue r’s utterances in the set So ← r,c:LO and LR are conditionally independent from each other given C. Then, it is easy to see that under this null model, the samples L(SO ← R) and p. This observation suggests the following test: We first estimate OLC from the sample L(SO ← R) (denoted as OLC0) and then using the within-dialogue shuffled samples OLC1). Under the null hypothesis, these two estimates should coincide. Conversely, if OLC0 ≠ OLC1, then the null hypothesis is rejected, suggesting that there is turn-by-turn length coordination.Consider now another sample, which is obtained by randomly permuting the respondent The above procedure, which we call Turn-by-Turn Length Coordination Test, is a conditional Monte Carlo test . The maiOLC0) before permutation. The Turn-by-Turn Length Coordination test shows that the mutual information decreases significantly after permutation, rejecting the null hypothesis that LO and LR are independent after conditioning on the contextual factor C. In other words, the contagion of length exists from the original utterance to the reply on a turn-by-turn level.The results of this test are shown in OLC is higher for Wikipedia than for the Supreme Court. However, one cannot make a general conclusion about the exact magnitude of turn-by-turn length coordination (TLC) simply by calculating the loss, i.e., OLC0 − OLC1.For the results on the Wikipedia discussion board in Personal Pronoun in Fig We demonstrated in the previous section that strong correlations in utterance length explain most of the observed stylistic coordination. However, in some situations, there are statistically significant non-zero signals even after conditioning on length, e.g., the first feature dimension LO and LR; stylistic variables m, and the contextual  variable C. The solid arrow from LO to LR reflects our findings from the last section about the existence of turn-by-turn length coordination. The dashed arc between the features C, C, Toward this goal, consider the Bayesian network in m:m appears or not in an utterance. Also, the Overall Stylistic Coordination(OSC) is defined asOSC is exactly the conditional mutual information introduced in C.  with respect to a specific style feature  path in )TSC in TSC is hard to evaluate due to lack of sufficient samples. Furthermore, the shuffling test from the previous sections is not directly applicable here either, because it needs to be done in way that keeps the correlations between LO and LR intact: In other words, one can exchange utterances that have the same lengths. Since most dialogues are rather short, this type of shuffling test is not feasible, and one needs an alternative approach.Again, a direct measure of turn-by-turn stylistic coordination corresponds to non-zero OSC = TSC. Thus, the null hypothesis is that there is contextual level coordination in stylistic features. We emphasize that by contextual coordination, we are actually referring to the links from C to FO and C to FR in Our proposed test is based on the following idea: if we can rule out the influence of the contextual factors on stylistic correlations, then any non-zero conditional mutual information can be only explained by turn-by-turn stylistic coordination, i.e., SO ← R as:fm (⋅) is a binary function represents whether the style feature m in an utterance appears or not.We follow the same notation and methodology used in previous sections. By OSC1 using the shuffled sample OSC1 is larger than zero, then there is necessarily contextual coordination. On the other hand, if OSC1 = 0, then all the observed stylistic correlations  must be due to turn-by-turn stylistic coordination.Consider now the shuffling procedure: we randomly permute respondent’s utterances within a dialogue and obtain the set OSC1 are within the zero-information confidence intervals, indicating that non-zero conditional mutual information (OSC before shuffling) cannot be attributed to contextual factors. In other words, the remnant correlations that are not explained by length coordination must be due to turn-by-turn level coordination.Let us first consider the results of the above test for the Supreme Court data. From Fig OSC), the results of the above permutation tests are rather inconclusive. Namely, although the confidence intervals of OSC1 do overlap with the zero information confidence intervals, one cannot state unequivocally that they are zero. In other words, one cannot rule out the null hypothesis that the remnant stylistic coordination is due to the contextual factors rather than turn-by-turn coordination.The situation is different for Wikipedia data. Indeed, Fig Mutual Information(MI) as a theoretically motivated measure of dependence to characterize the amount of stylistic coordination between the originator and the respondent in the dialogue. Moreover, by introducing Conditional Mutual Information(CMI), which allows us to measure the correlation between two variables after conditioning on a third variable, we are able to more accurately gauge stylistic accommodation by controlling for confounding effects like length coordination.In conclusion, we have suggested an information theoretic framework for measuring and analyzing stylistic coordination in dialogues. We first extracted the stylistic features from the dialogue of two participants and then used We then used the proposed method to revisit some of the previous studies that had reported strong stylistic coordination. While the suggestion that one person’s use of, e.g., prepositions will (perhaps unconsciously) lead the other to use more prepositions is fascinating, our results indicate that previous studies have vastly overstated the extent of stylistic coordination. In particular, we showed that a significant part of the observed stylistic coordination can be attributed to the confounding effect of length coordination. We find that for both Supreme Court and Wikipedia data, the coordination score is greatly diminished after conditioning on length. We also find that the significant asymmetry in stylistic coordination shown in the previous study  is drastLO, LR and C, the mutual information I(LO : LR) > 0, while I(LO : LR∣C) = 0. If we only look at the aggregated data, averaging over all contexts, C, i.e., I(LO : LR), there will be artificial mutual information between LO and LR. Ideally, we could calculate I(LO : LR∣C) directly, however, there may not be enough samples for us to calculate the conditional mutual information for all values of C. How can we still determine whether I(LO : LR∣C) is zero or not while using all the data? We thus designed non-parametric statistical tests to solve this problem in general while making full use of the available data. More importantly, because these information-theoretic quantities directly reflect constraints on graphical models, the mystery of Simpson-like paradoxes is replaced with concrete alternatives for generative stories as depicted in Figs Simpson’s paradox provides a famous example of how correlations observed in a population can disappear or even be reversed after conditioning on sub-populations. In an information-theoretic framework setting, a similar “paradox” can be seen in the example illustrated by We also observed that for some of the stylistic markers, there was diminished but still statistically significant correlations even after conditioning on length. We again designed a non-parametric statistical test for analyzing this remnant coordination more thoroughly. Our findings suggest that for the Supreme Court data, the remnant coordination cannot be fully explained by other contextual factors. Instead, we postulate that the remnant correlations in the Supreme Court data is due to turn-by-turn level coordination. For the Wikipedia data, however, our results are less conclusive, and we cannot draw any conclusion about turn-by-turn stylistic coordination. Thus, caution must be taken when making general claims about the possible origin of stylistic coordination in different settings.It is possible to develop alternative tests based on a more fine-grained, token-level generative models. The main idea behind such a test is to shuffle the word tokens uttered by an individual within each dialogue, which should destroy turn-by-turn coordination. Our preliminary results based on this test suggest that most of the remnant correlations are indeed due to turn-by-turn coordination. However, we emphasize that this test requires an additional assumption whose validity needs to be verified, namely that the words used by a given speaker within a conversation are independent and identically distributed (i.i.d.). Furthermore, the test assumes stationarity, i.e., that the contextual factors do not vary within the course of the dialogue. While this assumption seems reasonable in the dialogue settings considered here, it is important to note that deviations from stationarity might be yet another serious obstacle for identifying stylistic influences , 20. Indhistorical and ahistorical mechanisms of lexical entrainment [While our work focuses on linguistic style matching, we believe that the information-theoretic method proposed can be useful for studying more general types of linguistic coordination in dialogues, such as structural priming , 22, or rainment . Indeed,In a broader context, we note that sociolinguistic analysis has been used for assessing and predicting societally important outcomes such as health behaviors, suicidal intent, and emotional well-being, to name a few examples –29. ThusS1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


LINE-1 (L1) is the most abundant mammalian transposable element that comprises nearly 20% of the genome, and nearly half of the mammalian genome has stemmed from L1-mediated mobilization. Expression and retrotransposition of L1 are suppressed by complex mechanisms, where the key role belongs to DNA methylation. Alterations in L1 methylation may lead to aberrant expression of L1 and have been described in numerous diseases. Accumulating evidence clearly indicates that loss of global DNA methylation observed in cancer development and progression is tightly associated with hypomethylation of L1 elements. Significant progress achieved in the last several years suggests that such parameters as L1 methylation status can be potentially utilized as clinical biomarkers for determination of the disease stage and in predicting the disease-free survival in cancer patients. In this paper, we summarize the current knowledge on L1 methylation, with specific emphasis given to success and challenges on the way of introduction of L1 into clinical practice.  Only about 1% of the genome is comprised of genes while the vast majority is comprised of repetitive elements—retrotransposons, transposons, satellite, and tandem repeats. The first two are also known as mobile or transposable elements, since they are capable of moving within the genome. While the more ancient class—transposons—uses the “cut-and-paste” mechanisms, retrotransposons relocate via an RNA intermediate in a “copy-and-paste” mechanism.It is becoming increasingly evident that transposable elements are tightly associated with the generation of genetic diversity and can influence the expression of numerous genes. Specifically, transposable elements can affect the integrity of the genome by retrotransposition, resulting in potential insertions and deletions within the coding sequences, as well as genomic rearrangements—by shuffling genomic fragments by 5′ and 3′ transduction and recombination between the homologous elements. Additionally, transposable elements have capacity to affect gene expression by numerous mechanisms, such as providing alternative promoters, silencing by transcriptional or RNA interference (RNAi), and creating cryptic splice sites and polyadenylation signals –4.Two major classes of retrotransposons are long-terminal repeats (LTR) and non-LTR elements. The former are named for their long-terminal repeats flanking the internal proviral sequence on both sides. LTRs are structurally related to exogenous retroviruses, although lacking the ability to move from one cell to another. Two families of these endogenous retroviruses are known to be currently active in mice . ActivitThere are about 516,000 copies of LINE-1 (L1) in the human genome prevalently located within its gene-poor regions, reaching up to 20% of the human genome . HoweverPropagation of L1 in mammalian genomes occurs via the process of autonomous retrotransposition. Current endogenous retrotransposition activity of some recent L1 elements determines the widespread genomic structural variations within and between populations and variations in normal development, neuronal differentiation, and human cancers , 19–22. cis-preference followed by the entrance of RNP into the nucleus [Transcription of the L1 full-length mRNA from the internal promoter, mediated by the RNA polymerase II, initiates L1 retrotransposition. This mRNA then is transported to the cytoplasm, where it is translated to ORF1p and ORF2p—L1-encoded proteins that preferentially associate with their encoding RNA . The L1  nucleus . Next, aMobilization of L1, thus, occurring via a “copy-paste” mechanism is associated with a number of events, including the development of novel gene promoters, splice sites, polyadenylation signals, dispersing transcription binding splice sites, linking genes in transcriptional networks, and facilitating the evolution of novel traits (reviewed in ).DNA methylation is one of the most important mechanisms for the regulation of genetic information and one of the key mechanisms for silencing repetitive elements (reviewed in ). It is Silent transcriptional status of L1 has been associated with DNA methylation, specifically within the 5′-UTR, that contains both L1 promoters and is rich in CpG sites. Demethylation of L1 by exogenous stressors, DNA demethylating agents (5-azacytidine), or in disease has been associated with its aberrant transcription –29. DNA Other epigenetic mechanisms have also been reported to be involved in the regulation of L1 expression. For example, acetylation and methylation of histones have been implicated in silencing of L1 retrotransposition in embryonic carcinoma cell lines . Accumul Factor VIII gene that prevented synthesis of functional coagulation factor [β-thalassemia, and diabetes [When the regulation of normal L1 activity is impaired, retrotransposition events may result in numerous deleterious effects. For instance, it can result in disruption of the ORF of the functional gene, genome amplification, and the development of genomic instability. The first human disease associated with L1 retrotransposition was haemophilia A, stemmed from the independent mutagenic L1 insertions into exon 14 of then factor . To datediabetes , 35, 36. APC gene, leading to the disruption of its function [ APC in colorectal cancer [It has also been hypothesized for a long time that L1 retrotransposition may be associated with cancer development and progression, but the lack of tools needed to detect novel retrotransposition events in human cancers did not allow the support of this hypothesis. The first L1 retrotransposition in cancer was reported by Miki et al. in colorectal cancer and was characterized by insertion of the 3′ portion of L1 into the last exon of thefunction . A numbefunction , 39. Howl cancer , can be l cancer  reportedLoss of global DNA methylation was the first epigenetic alteration demonstrated in human cancers , 42. SubThis hypomethylation can result in a number of unwanted effects associated with aberrant L1 activity and retrotransposition. Also, while L1 is interspersed primarily within gene-poor regions of the genome, its presence within or neighboring the coding sequences can be detrimental. For instance, loss of L1 methylation within its 5′-UTR may result in its aberrant activation and affect the expression of neighboring genes. Hypomethylation of L1 element insertions within the promoters and introns of coding genes may result in aberrant expression of these genes . On the Significant alterations of L1 in human cancers, associated primarily with its increased expression, elevated protein levels, and hypomethylation, together with the very high copy numbers of L1 in the genome, suggest that L1 can be potentially utilized as a diagnostic modality. It is becoming increasingly evident that the methylation status of L1 can be utilized as a prognostic marker in cancer. Indeed, loss of L1 methylation is usually found to be more pronounced in advanced stages of cancer and in metastasis than in the early stages of cancer, or in primary tumors, respectively. c-MET oncogene, the gene that contains an L1 insertion within its intron [Growing evidence clearly demonstrates that hypomethylation of L1 is usually associated with poor prognosis and shorter disease-free survival. For instance, a study of 211 lung adenocarcinoma patients concluded that disease-free survival in the group with hypomethylated L1 was significantly shorter than that of the nonhypomethylated group . The ress intron .Importantly, the study by Benard et al.  shows thWhile, for the vast majority of human cancers, L1 demethylation was associated with poor prognosis, some controversial results exist for melanoma, where L1 hypomethylation was associated with favorable prognosis in stage IIIc patients . HoweverAccumulating evidence suggests that the methylation status of L1 cannot only be utilized as a prognostic marker but also discriminate between the earlier and later stages of cancer. Extensive research performed in several cohorts of colorectal cancer (CRC) patients indicates that methylation of L1 not only is considerably lower in the tumor compared to adjacent stromal and normal mucosal epithelial cells , 58 but  ERBB2(HER2) amplification, and p53 overexpression [Another study by Park et al. , performpression . Similarpression .Field for cancerization is the phenomenon characterized by “the occurrence of multifocal and recurrent epithelial tumors that are preceded by and associated with widespread changes of surrounding tissue or organ fields .” The ro APC and RASSF1 were significant prognostic factors only in univariate analysis in non-small cell lung cancer, the methylation status of L1 remained significant prognostic factor in multivariate analysis that included age, gender, smoking history, histologic type, and pathologic stage. Moreover, in the same study, L1 methylation also revealed a significant prognostic value for stage IA NSCLC patients in multivariate analysis [It is becoming increasingly evident that the prognostic value of the L1 methylation status might exhibit higher potential than the methylation status of individual tumor-suppressor genes characteristic of a specific cancer. In the recent study, Saito et al.  reportedanalysis . MGMT and MLH1 genes was not associated with patient prognosis [Similar findings were reported from the cohort of 217 patients with curatively resected esophageal squamous cell carcinoma, where L1 hypomethylation was significantly associated with shorter survival, while the methylation status ofrognosis . MET, RAB3IP, and CHRMP results in their reactivation and aberrant expression in CRC metastasis. The recent study by Ikeda et al. [The role of L1 in tumor's metastatic potential is becoming increasingly recognized . Severala et al.  reportedIdeal biomarkers should be low-invasive and reflect the pathomorphological changes in the target organs. In this regard, determination of L1 methylation status in biological fluids, such as blood and saliva, is of particular interest. Up to date, a number of studies have attempted to determine the association between the methylation of L1 in leukocytes, peripheral blood mononuclear cells, and buccal DNA and the risk for certain cancers –73. DespWhile normal adult human tissues usually do not express L1 (or express at very low levels), considerable levels of L1 RNA and protein are found in cancerous tissues . ImportaOne of the major challenges is the high degree of variability in L1 methylation between the evaluated samples . InteresAnother challenge is variability and certain discrepancies between the studies. For instance, while some studies report L1 hypomethylation in leukemia patients , others In the last two decades, L1, the most abundant repetitive element in the human genome, experienced a dramatic switch from being “junk DNA” to being “an important player in the mammalian genomes .” Its in


Surfactant proteins SP-A and SP-D are useful biomarkers in diagnosis, monitoring, and prognosis of idiopathic pulmonary fibrosis (IPF). Despite their high structural homology, their serum concentrations often vary in IPF patients. This retrospective study aimed to investigate distinct compartmentalization of SP-A and SP-D in the vasculature and lungs by bronchoalveolar lavage fluid (BALF)/serum analysis, hydrophilicity and immunohistochemistry.We included 36 IPF patients, 18 sarcoidosis (SAR) patients and 20 healthy subjects. Low-speed centrifugal supernatants of BALF (Sup-1) were obtained from each subject. Sera were also collected from each patient. Furthermore, we separated Sup-1 of IPF patients into hydrophilic supernatant (Sup-2) and hydrophobic precipitate (Ppt) by high-speed centrifugation. We measured SP-A and SP-D levels of each sample with the sandwich ELISA technique. We analyzed the change of the BALF/serum level ratios of the two proteins in IPF patients and their hydrophilicity in BALF. The distribution in the IPF lungs was also examined by immunohistochemical staining.In BALF, SP-A levels were comparable between the groups; however, SP-D levels were significantly lower in IPF patients than in others. Although IPF reduced the BALF/serum level ratios of the two proteins, the change in concentration of SP-D was more evident than SP-A. This suggests a higher disease impact for SP-D. Regarding hydrophilicity, although more than half of the SP-D remained in hydrophilic fractions (Sup-2), almost all of the SP-A sedimented in the Ppt with phospholipids. Hydrophilicity suggests that SP-D migrates into the blood more easily than SP-A in IPF lungs. Immunohistochemistry revealed that SP-A was confined to thick mucus-filling alveolar space, whereas SP-D was often intravascular. This data also suggests that SP-D easily leaks into the bloodstream, whereas SP-A remains bound to surfactant lipids in the alveolar space.The current study investigated distinct compartmentalization of SP-A and SP-D in the vasculature and lungs. Our results suggest that serum levels of SP-D could reflect pathological changes of the IPF lungs more incisively than those of SP-A. The pulmonary surfactant consists of phospholipids, mainly dipalmitoyl phosphatidylcholine (DPPC), surfactant protein (SP)-A, SP-B, SP-C, and SP-D. Both SP-A and SP-D are produced by type II alveolar epithelial cells and Clara cells, and are secreted into the alveolar space, 2. UnliLCO), chronological changes in respiratory functions, and desaturation during the 6-min walk test who had a bronchoalveolar lavage (BAL) performed at the Sapporo Medical University Hospital from December 2003 to May 2012. SAR patients were used as disease controls because, in Japan, they seldom show fibrotic changes in the lung, even with pulmonary lesions, 24. TheTo compare BALF concentration of the two proteins, this study also included a control group consisting of 20 healthy subjects from our previous study in which BAL was conducted under the same conditions. They hag) to remove cell components, and this Sup-1 was cryopreserved at -80°C until use.Each participant received an intramuscular injection of atropine sulfate and hydroxyzine or pethidine, followed by local anesthesia with lidocaine. A flexible bronchofiberscope  was inserted orally to pour 50 mL of 0.9% saline (37°C) into the bronchus of the right middle lobe or the left lingula, and the BALF was recovered. This process was repeated three times and the fractions pooled together. BALF was centrifuged , named SP-A Test/Kokusai-F , as previously described. A standg; 4°C; 16 h) into a supernatant (Sup-2) and precipitate (Ppt). The Ppt was resuspended in 1.2 mL of 0.9% saline. The concentrations of SP-A and SP-D in Sup-2 and Ppt were measured by ELISA as stated above. Following which, the phospholipids were extracted from Sup-2 and Ppt by the method of Bligh and Dyer[The hydrophilicity of SP-A and SP-D in the BALF of IPF patients (N = 19) were measured after centrifugation of 1.2 mL of Sup-1  and embedded in paraffin. Then, 3-μm thick sections were deparaffinized and stained using the following protocols. The primary antibodies were mouse anti-human SP-A monoclonal antibodies , and mouse anti-human SP-D monoclonal antibodies . Thereafter, the sections were incubated with biotin-labeled secondary antibody , and stained using the DAB Peroxidase Substrate Kit . For SP-D, samples were pretreated in 10 mM citric acid solution  before being incubated with the primary antibody.Furthermore, the stained sections were boiled in 10 mM citric acid solution  to remove antibodies, and stained again with monoclonal CD34  or monoclonal D2-40  as primary antibodies. Double immunostaining was performed using the Vectastain ABC-AP Mouse IgG Kit  and the alkaline phosphatase substrate kit . CD34 stains vascular endothelium, particularly in small capillaries, whereasThe semi-qualitative analysis of immunohistochemistry was performed with concordance of three well-trained pulmonologists who had knowledge of the pathologic features of interstitial pulmonary diseases. We defined immunohistochemical intensities of normal type II epithelial cells as positive; “+”, and expressed those of each area using five grades: “-”, none; “±”, more or less; “+”, positive; “++”, more intense; and “+++”, very intense.U–test (between the two groups) or the Kruskal Wallis test (among three groups). The differences between each pair of the three groups were tested using the Mann–Whitney U–test with Bonferroni correction. The differences in sex and the smoking status were tested using the chi-square test. The correlations between the two variables were evaluated using the Spearman’s rank correlation coefficient. All P values < 0.05 were considered statistically significant.All analyses were performed using the SPSS Statistics 21 (IBM Inc.). The data of age and the BALF recovery rate with symmetric distribution are expressed as mean ± standard deviation, and the differences between the three groups were tested using one-way ANOVA. The differences between each pair of the three groups were tested using Tukey’s HSD test. The data of SP-A and SP-D levels in the serum and BALF, the BALF/serum level ratios of the two proteins, and the percentages of surfactant proteins and phospholipids fragmentation in Sup-2 or Ppt showed asymmetric distributions. These data are expressed as median (interquartile range), and tested using the Mann–Whitney The basic characteristics and recovery rates of BALF of each subject group are summarized in Table The data of SP-A and SP-D levels of each subject group are summarized in Table Measurements of SP-A in BALF revealed comparable concentrations between the three groups P = 0.245): IPF patients (median 1275.0 ng/mL), healthy subjects (median 1978.0 ng/mL), and SAR patients (median 1600.0 ng/mL)  of SP-A and SP-D were median 19.1 (SP-A) and 1.3 (SP-D) in IPF patients, and median 51.2 (SP-A) and 7.8 (SP-D) in SAR patients. These gaps were significantly smaller in IPF patients than in SAR patients (SP-A: P = 0.01 and SP-D: P < 0.001)   to generate a hydrophobic precipitate (Ppt) and hydrophilic supernatant (Sup-2). Nearly all the SP-A sedimented in the Ppt (median 97.9%), indicating high hydrophobicity. In contrast, SP-D partitioned into the two phases, but significantly more in Sup-2 (median 52.4%). Finally, nearly all surfactant phospholipids were collected in the Ppt (median 100.0%). Together, these data suggest that hydrophilicity of SP-A is weakened by binding with phospholipids in BALF. The results of centrifugation of BALF are summarized in Table The BALF supernatant fractions from IPF patients centrifuged to remove cellular components (Sup-1) were centrifuged again 33,000 3,000 g tThe tissue distribution of SP-A and SP-D was analyzed by immunostaining of surgical lung biopsies from IPF patients and peripheral noncancerous control lung tissue from lung cancer patients. Type II alveolar epithelial cells, which are known to produce pulmonary surfactant, were positive for both surfactant proteins in normal peripheral lungs Figures A, B and Semi-qualitative analysis of the immunohistochemical distribution comparing SP-A and SP-D is presented in Table It has been reported that serum SP-A and SP-D concentrations are elevated in IPF and other interstitial lung diseases, and that they can predict the progression of respiratory failure and prognosis in IPF, 6. HoweWe previously reported BALF concentrations of SP-D equivalent or lower in IPF patients than in healthy subjects. Likewis99mTc-DTPA) aerosol was exacerbated among IPF patients and suggested that the migration of substances from the alveolar space into the bloodstream was accelerated[According to the current study, the BALF/serum level ratio of SP-A was reduced from 51:1 in the sarcoidosis group to 19:1 in the IPF group. In contrast, IPF caused a 6-fold decrease in BALF/serum level ratio for SP-D, with 7.8:1 in the sarcoidosis group compared with 1.3:1 in the IPF group. These data suggest that IPF caused a preferential leakage of SP-D from the alveolar space into the bloodstream. Moreover, in this study, the SP-D concentrations in sera were correlated with those in BALF, but such a correlation was not found for SP-A. This result may be due to the higher permeability of SP-D, implying that not only BALF concentration but also serum concentration can be easily altered according to the amount of secretion of SP-D from productive cells. Mogulkoc et al. reported that the clearance of inhaled technetium 99 m-labelled diethylenetriamine penta-acetic acid (elerated. One of elerated, 38. ThiThe present study revealed dissociation between SP-A and SP-D in IPF lungs, suggesting that different protein–lipid interactions may affect their leakage into the bloodstream. Most of the SP-A found in the alveolar space is bound to DPPC, which is the main component of pulmonary surfactant. In contCD34 is known as a marker of vascular endothelium, 40. EbiThis study has some limitations. We should interpret the concentrations in BALF not only from the viewpoint of the leakage and clearance but also from that of the alterations in the production amount in IPF lungs. Because both proteins have the important function to regulate the inflammatory cell response, their productions may be altered in IPF lungs. Further examinations are required regarding these issues. We used the data of BALF from healthy volunteers who were investigated in our previous study. BecauseThe histopathological features of IPF indicate a heterogeneous appearance. Honeycombing, in which type II cells are replaced with regenerated ciliated epithelial cells, is a finding that represents the end stage of fibrotic change, and this finding seldom regresses. In contrast, alveolitis is a potentially reversible and treatable parenchymal abnormality. In such a lesion, type II alveolar epithelial cells are extremely hyperplastic. These variable features may reflect dynamic differences between SP-A and SP-D because of their hyprophilicity. If serum SP-D levels mirror the severities of moderate and reversible damage to the epithelium due to their ready leakage into the bloodstream, these levels can be used as a biomarker to predict therapeutic response. We need to assess the utilities of these biomarkers with regard to the therapeutic effects of drugs such as antifibrotic agents. Currently, KL-6, MMP-7 and CCL-18 have been also reported as blood biomarkers of IPF–22. WherAlthough SP-A and SP-D are serum biomarkers of IPF having high homology, the current study suggests that the difference in hydrophilicity of the two proteins could be the cause of their difference in migration from the air space into the bloodstream. Because SP-D leaks into the circulation more easily, serum levels of SP-D may reflect pathological changes of the disease more sharply than those of SP-A.Written informed consent was obtained from the patient for the publication of this report and any accompanying images.


Amyotrophic lateral sclerosis is a neurodegenerative disease characterized clinically by motor symptoms including limb weakness, dysarthria, dysphagia, and respiratory compromise, and pathologically by inclusions of transactive response DNA-binding protein 43 kDa (TDP-43). Patients with amyotrophic lateral sclerosis also may demonstrate non-motor symptoms and signs of autonomic and energy dysfunction as hypermetabolism and weight loss that suggest the possibility of pathology in the forebrain, including hypothalamus. However, this region has received little investigation in amyotrophic lateral sclerosis. In this study, the frequency, topography, and clinical associations of TDP-43 inclusion pathology in the basal forebrain and hypothalamus were examined in 33 patients with amyotrophic lateral sclerosis: 25 men and 8 women; mean age at death of 62.7 years, median disease duration of 3.1 years (range of 1.3 to 9.8 years).2= 17.5, p= 0.00003) and bulbar symptoms at onset , but not age or disease duration. Furthermore, TDP-43 pathology in the lateral hypothalamic area was associated with reduced body mass index .TDP-43 pathology was present in 11 patients (33.3%), including components in both basal forebrain (n= 10) and hypothalamus (n= 7). This pathology was associated with non-motor system TDP-43 pathology (ΧThis is the first systematic demonstration of pathologic involvement of the basal forebrain and hypothalamus in amyotrophic lateral sclerosis. Furthermore, the findings suggest that involvement of the basal forebrain and hypothalamus has significant phenotypic associations in amyotrophic lateral sclerosis, including site of symptom onset, as well as deficits in energy metabolism with loss of body mass index. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of upper and/or lower motor neurons, muscle wasting, and bulbar symptoms  . The patDetailed pathologic studies have also demonstrated surprisingly widespread, non-motor pathology in some ALS patients, typically in the form of ubiquinated cytoplasmic inclusions that are immunoreactive for transactivating responsive sequence (TAR) DNA-binding protein 43 kDa, or TDP-43  and extrSomewhat unique among extra-motor features in ALS are alterations in energy metabolism , autonomThis study was carried out to determine whether the basal forebrain and hypothalamus are pathologically involved in ALS. We hypothesized that pathologic TDP-43 inclusions would be identified in a subset of ALS patients given the deficits in energy metabolism and autonomic function in ALS and frequent involvement of these regions in other neurodegenerative diseases. The frequency and topography of TDP-43 inclusions were examined in 33 ALS patients within these structures. Pathology in this region also was examined with respect to disease duration, the extent of TDP-43 proteinopathy, last available BMI, which is an indirect measure of energy metabolism, and patient age, since TDP-43 pathology may be seen in older patients without ALS .Pathologic materials were reviewed for 56 consecutive patients with clinically diagnosed and pathologically confirmed ALS. Archival materials were screened for representative portions of basal forebrain and hypothalamus (defined further below). Thirty-three patients were selected for study based on the presence of appropriate anatomic regions.© platform  with appropriate positive and negative controls.Patient autopsies performed at the Houston Methodist Hospital included sections from all cerebral lobes, basal ganglia, thalamus, amygdala, hippocampus, midbrain, pons, medulla, cerebellum, spinal cord and pituitary. Immunostains performed on the majority of cases included tau , beta-amyloid , alpha-synuclein , and TDP-43 . An immunostain for ubiquitin  also was performed on several archived cases. Immunostaining was performed on brain tissue that was fixed in 20% formalin for a minimum of seven days following brain removal. Sections were further fixed in 10% formalin until processing and embedding. Formalin fixed paraffin embedded tissue was sectioned at 4 microns, mounted on plus coated slides and dried at 60°C. Immunostaining was performed on the BenchMark ULTRANeuropathologists at Houston Methodist Hospital  utilized established criteria to assess Alzheimer disease (AD) neuropathologic changes, including neurofibrillary changes ,21 and pBasal forebrain components were defined on H&E and TDP-43 stains and included ventral pallidum (VP), ventral striatum and small granule cell islands (VS/Is), the bed nucleus of the stria terminalis (BNST), lateral septal area (LSA), and other neurons of the substantia innominata (SI). VP included large pallidal neurons ventral to fibers of the anterior commissure . SI inclHypothalamic nuclear groups were defined on H&E and TDP-43 stains by cytologic characteristics and relative position using standard anatomic references ,29-32. HThese anatomic regions are shown in schematic form for basal forebrain Figure A, includ2 per HPF). Global TDP-43 pathologic burden was classified as canonical  or non-canonical .Two authors  independently rated TDP-43 pathology in the aforementioned areas. The location of pathologic TDP-43 inclusions was recorded and classified by morphologic sub-type as follows: neuronal cytoplasmic (NCI), neuronal intranuclear (NNI), glial cytoplasmic (GCI), and dystrophic neurites. For cases with TDP-43 inclusion pathology, quantitative measures were performed in the area of greatest hypothalamic pathology . For these cases, any cellular TDP-43 inclusions  were recorded within three consecutive high-power microscopic fields (HPF) at 400 × magnification (0.19625 mm2) testing was used to determine whether basal forebrain/hypothalamic pathology was associated with (a) non-canonical TDP-43 pathology and (b) bulbar/respiratory onset.Two-sided Mann–Whitney (Wilcoxon rank sum) testing was used to determine whether: (a) disease duration significantly differed between patients with and without basal forebrain/hypothalamic pathology, (b) disease duration significantly differed between patients with canonical and non-canonical TDP-43 pathology, (c) BMI significantly differed in patients with and without basal forebrain/hypothalamic pathology, (d) BMI significantly differed in patients with and without LHA pathology, and (e) age at death significantly differed between those with and without basal forebrain/hypothalamic TDP-43 pathology. Chi-squared  though both patients had ALS TDP-43 pathology outside of the hypothalamic region.Table At autopsy, all patients had pathologically confirmed ALS. Table Pathology in basal forebrain and hypothalamus was present in 11 of 33 cases (33.3%). This included components of basal forebrain in 10 patients: VS/Is, n = 9 of 26 patients with the structure present; BNSTL, n = 8 of 21; SI neurons, n = 6 of 28; typically in medium-sized neurons with magnocellular neurons having filamentous/skein-like inclusions in only two cases; LSA, n = 2 of 15; and, VP, n = 1 of 21. This also included components of the hypothalamus in 7 patients, including LHA/TMH, n = 5 of 28; PHA, n = 3 of 24; MB/LTN/TMN, n = 6 of 19; PVN/POA, n = 2 of 16; and PVL/TBL/VMH, n = 4 of 10. The SON was not involved (n = 13). Representative findings are demonstrated for basal forebrain and hypothalamus in Figures Neuronal inclusions were uniformly cytoplasmic (NCIs), while intranuclear inclusions were not identified. The density of TDP-43 cellular inclusions varied between 1/three HPFs to 72/three HPFs: the average number of inclusions was 8.42 (sd = 18.75), with a per field average of 2.8 inclusions. NCI morphology consisted of granular preinclusions , two authors  performed a post-hoc rating of gliosis (on H&E stain) as absent, mild, moderate, or severe. One patient had mild gliosis in the preoptic area of the hypothalamus and this same patient had mild to focally moderate gliosis in basal forebrain (most apparent in the BNST). Three additional patients had mild gliosis in basal forebrain only, typically most apparent medially including within BNST. A fifth patient had moderate gliosis more diffusely throughout basal forebrain ventral to the anterior commissure (this patient had 63 TDP-43 cellular inclusions/three HPFs). Neuronal loss was not evident on H&E stain in these patients.limited to these canonical regions. The remaining 16 patients had pathology in these regions plus non-canonical pathology, i.e. additional involvement of hippocampus (n = 11), amygdala (n = 11), non-frontal isocortex (n = 9), dorsal striatum (n = 10), and thalamus (n = 8).ALS pathology in typical  regions was characterized by neuronal loss and gliosis with ubiquitinated or TDP-43 immunoreactive inclusions. Brain regions implicated included spinal cord (n = 33), brainstem motor nuclei (n = 27), other brainstem nuclei (n = 17), and agranular frontal cortex (n = 22). Seventeen of 33 patients had pathologic involvement 2 = 17.5, p = 0.00003). Moreover, all but one case with hypothalamic TDP-43 pathology occurred with concurrent basal forebrain pathology. In contrast, sparse basal forebrain pathology (range of 2–3 NCIs in three HPFs) occurred in three cases without hypothalamic pathology. An additional case with dense basal forebrain pathology (63 NCIs in three HPFs) did not have hypothalamic tissue available for study.TDP-43 pathology in basal forebrain/hypothalamus was seen only with non-canonical pathology  with 71.4% of all patients having bulbar symptoms at disease onset demonstrating this pathology (compared to 28.6% of patients without bulbar symptoms at onset).There was a significant association between bulbar onset symptoms and basal forebrain/hypothalamic pathology (ΧFor patients with complete data on disease duration (n = 30), this parameter did not significantly differ between patients with basal forebrain/hypothalamic TDP-43 pathology  and without  . Likewise, the density of TDP-43 inclusions was not correlated to disease duration . Disease duration also did not significantly differ between patients with non-canonical  and canonical TDP-43 pathology  . Patient age at death did not significantly differ between those with  and without  TDP-43 pathology in basal forebrain/hypothalamus .2, interquartile range, 20.8-28.2) and without  any basal forebrain and/or hypothalamic pathology . BMI did significantly differ, however, between patients with  and without  pathology in the LHA specifically  , and their pathology was limited to canonical regions . Conversely, three different patients with disease durations less than 2.5 years had extensive TDP-43 proteinopathy, including within the regions investigated in this study. The determinants of extent of TDP-43 pathology, of which disease duration does not appear to be one, therefore remain unclear. Age has been suggested as a negative prognostic factor in ALS, but our analysis shows this was not a determinant of the extent of TDP-43 pathology. Immune dysregulation may play a role as patients with “rapidly progressive” ALS have a reduction in regulatory T-lymphocytes and reduction in transcription factors required for the function of this T-cell population  . This suThere are limitations of the present study due to the retrospective design. First, the study was conducted with archived sections available or with supplemental sections that could be generated from stored tissues. In rare instances, involving older archived cases, one or more anatomic regions were not available in the original material and no additional tissue was available for sampling. Prospective sampling in future ALS cases will ensure more uniformity in the anatomic regions available for assessment. Based on our findings, two sections are likely to yield the most valuable pathologic data for future studies. The first, inclusive of basal forebrain, could be taken at level of the optic chiasm, anterior commissure, septal area, and BNST, and would demonstrate LSA, BNST, VS/Is, VP, and rostral hypothalamus Figure A. The seThe ability to determine the clinical sequelae of the pathology described herein is also limited by the retrospective nature of the study. Future clinicopathologic studies of these clinical features with respect to the brain regions identified here will further clarify the importance of these pathologic findings in the extra-motor manifestations of ALS. This is particularly true with respect to measures of energy metabolism. The optimal clinical measures would include careful documentation of BMI at multiple time points  and measThis study has demonstrated that a subset of patients with ALS have TDP-43 pathology within basal forebrain and hypothalamus, which may contribute to the extra-motor symptoms of the disease, including alterations in energy metabolism, autonomic nervous system dysfunction, and frontal executive dysfunction. The pathologic involvement was fairly stereotypic, and in nearly all cases pathologic involvement of forebrain components  preceded hypothalamic involvement . Basal forebrain and hypothalamic pathology was associated with the presence of other non-canonical TDP-43 inclusion pathology but was not associated with disease duration. Finally, the presence of forebrain/hypothalamic pathology was associated with bulbar onset of disease, which suggests the possibility of a unique and previously unrecognized clinico-pathological relationship in ALS.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Written consent was obtained by the patients and/or their representatives to allow for their autopsy tissue to be used for research purposes. The study was also carried out with IRB approval from Houston Methodist Hospital.