Recordings of Spanish and English sentences by switched-dominance bilingual (SDB) Spanish  and by L1-dominant Spanish and English controls were presented to L1-dominant Spanish and English listeners, respectively. At –4 dB signal-to-noise ratio (SNR), Spanish and English productions by SDBs were equally intelligible with both reaching L1-dominant control levels. At –8 dB SNR, SDB English intelligibility matched that of L1-dominant English controls, yet SDB Spanish intelligibility was significantly lower than that of L1-dominant Spanish controls. These results emphasize that extended (but not early) exposure is both necessary and sufficient for robust speech learning. While by definition typical late bilingual and monolingual populations confound these two factors—earlier onset typically goes with longer duration of acquisition resulting in first-language (L1) dominance—switched-dominance bilinguals (SDBs) dissociate them. With early language exposure being limited to the “home” language (the L1), these bilinguals ultimately have more extensive exposure to the language that predominates in the society-at-large resulting in a switch from L1 to L2 dominance, where language dominance is defined as the language which is used most often.Prior research on speech production by SDBs has demonstrated some benefit of early exposure on speech sound production in the non-dominant L1 relative to late learners of the language . For exIn these prior studies e.g., . Thus, iIn a companion study to the present study of intelligibility of speech produced by SDBs, early exposure on non-dominant L1 production by SDB speakers, the speech-in-noise recognition accuracy evidence .praat (praat script (written by the author). In this script, the average long-term average spectrum (LTAS) of each talker was measured across all sentences and then averaged across all talkers to produce a single LTAS-curve. White noise was then generated in praat and then filtered along this LTAS curve creating speech-shaped noise customized for this experiment .In order to highlight subtle differences in intelligibility of the sentence productions by the three talker groups , all sentence stimuli were mixed with speech-shaped noise. The noise track was created in praat  using a 2.4max/msp software . ListenListeners were instructed to type as many words as possible that they heard over the headphones, even if it was just one word. Listeners were required to type something to move on to the next sentence; as such, responses such as “I am not sure” or “yo no sé” (I do not know) were allowed but then considered wholly incorrect. Participants finished within 30 min. The typed output of each listener was then compared to the correct target sentence and with each word being scored as either wholly correct (receiving a score of “1”) or incorrect . Spanish accent marks were ignored and minor spelling errors  were considered “correct.” Scores were then averaged within each sentence  and then averaged for each talker  at each SNR to produce two speech intelligibility scores per talker and per language (SDB only): intelligibility at a relatively easy (–4 dB SNR) and at a relatively difficult SNR (–8 dB SNR). Proportional scores were then log-odd transformed log((p)/(1 − p)) where p is the intelligibility score ranging from 0 to 1 for analysis.3.r  using linear mixed effects regressions (LMERs) with random intercepts for participant. The factors of interest, signal-to-noise ratio (SNR) and participant group , were contrast coded  before building any models. The dependent variable (DV) was the log-odd transformed speech intelligibility score log((p)/(1 − p)), where p is the proportional intelligibility score ranging from 0 to 1. These scores were analyzed as log-odds in order to remove the non-linear relationship of incremental changes in proportions (expressed as percent words correctly identified) at various places on the scale . Significance was assessed using the likelihood ratio test in which the degree to which the data are fit by a full model versus a model excluding a factor (n − 1) or interaction of interest is measured. Final models for both English and Spanish converged with log-odd transformed speech intelligibility as the dependent variable, a two-way interaction, and both main effects for the two factors of interest with random intercepts for participants. Random slopes by SNR were not included as each talker has only one score associated with each level within SNR, causing the model to be over-specified if present.All data were analyzed using the software 3.1β = 0.03, χ2(1) =  86.97, t = 28.79, p < 0.01), indicating that speech intelligibility scores were significantly lower for –8 dB SNR compared to –4 dB SNR. This is a reliable effect and has been shown in many previous speech-in-noise recognition studies , indicating that the impact of SNR on English speech intelligibility scores did not differ between groups. These results are shown along with the Spanish results in Fig. The average log-odds intelligibility score was 0.43 s.d. 0.21) in –8 dB SNR and 1.22 (s.d. 0.2) in –4 dB SNR for L1-dominant English controls. The average log-odds intelligibility score was 0.37 (s.d. 0.21) in –8 dB SNR and 1.07 (s.d. 0.21) in –4 dB SNR for SDBs in English. There was a significant main effect of SNR on speech intelligibility scores   =  67.03, t = 17.47, p < 0.01], indicating that speech intelligibility scores were significantly lower for –8 dB SNR compared to −4 dB SNR. This result is consistent with previous research . These results are show in Fig. The average log-odds intelligibility score was 0.42 (s.d. 0.27) in –8 dB SNR and 1.25 (s.d. 0.34) in –4 dB SNR for L1-dominant Spanish controls. The average log-odds intelligibility score was 0.03 (s.d. 0.14) in –8 dB SNR and 1.1 (s.d. 0.19) in –4 dB SNR for SDBs in Spanish. There was a significant main effect of signal-to-noise ratio (SNR) on speech intelligibility scores [4.The aim of this study was to compare the overall intelligibility of speech produced by SDBs in their non-dominant L1 and in their dominant L2. Building on prior work that demonstrated long-lasting benefits of early exposure for segmental production accuracy, we extended the scope of investigation to intelligibility of words in sentences as presented to L1 listeners of each language under both favorable and unfavorable listening conditions.4.1The main question of this subsection is whether the delayed but extended use of language resulting in L2 (rather than L1) language dominance, was sufficient to overcome any differences that may have been initially present in SDBs' delayed onset (but ultimately dominant) L2 speech. SDB did not show speech production differences in their dominant L2 (English) as the range of speech intelligibility scores for SDB was similar to that of English controls. This result is consistent with other studies that have shown L2-dominant speakers  produce native-like speech sounds in their dominant L2 , L1-dominant Spanish listeners showed similar recognition accuracy for words in sentences produced by SDB and Spanish controls. However, the significant main effect of group  and the significant group by SNR interaction (SDB speech intelligibility was more adversely affected by the more difficult –8 dB SNR compared to that of Spanish controls), suggests that the interrupted L1 acquisition and replacement by a dominant L2 resulted in reduced resistance to signal degradation by high levels of background noise, at least for L1-dominant listeners.It may appear that these results conflict with 5.The goal of the current study was to determine how adult speech production reflects the timing  and amount  of language exposure. The SDBs in this study provided unique insight into the subtle differences in language function not readily available in monolingual or bilingual speakers for whom the first-acquired language (L1) is also the dominant language. Results showed that, for L1-dominant English listeners, intelligibility of dominant L2 English as produced by SDB Spanish speakers was indistinguishable from that of L1-dominant English speakers even under a highly unfavorable (–8 dB) signal-to-noise ratio condition. This indicates that, with respect to speech intelligibility, extended exposure can successfully provide benefit even with delayed exposure to the L2. However, the converse does not appear to hold. That is, early exposure to the L1 does not entirely guard against subtle speech production differences associated with interrupted exposure to the L1. Specifically, the data showed substantially lower intelligibility of non-dominant L1 Spanish for L1-dominant Spanish listeners under a highly unfavorable (–8 dB) signal-to-noise ratio listening condition. These results suggest that with respect to speech production learning, while early exposure has some benefits, extended exposure can compensate for later onset of acquisition.


With the aim of achieving a better control, a second path involves the functionalization of the flakes with pillars molecules, bringing a new class of materials with structure partially controlled by the size, shape, and chemical-physical properties of the pillars. We finally outline the first steps on a possible third road, which involves the construction of pillared multi-layers using epitaxial regularly nano-patterned graphene as precursor. While presenting a number of further difficulties, in principle this strategy would allow a complete control on the structural characteristics of the final 3D architecture.Graphene is the prototype of two-dimensional (2D) materials, whose main feature is the extremely large surface-to-mass ratio. This property is interesting for a series of applications that involve interactions between particles and surfaces, such as, for instance, gas, fluid or charge storage, catalysis, and filtering. However, for most of these, a volumetric extension is needed, while preserving the large exposed surface. This proved to be rather a hard task, especially when specific structural features are also required . Here we review the recent experimental realizations and theoretical/simulation studies of 3D materials based on graphene. Two main synthesis routes area available, both of which currently use (reduced) graphene oxide flakes as precursors. The first involves mixing and interlacing the flakes through various treatments , leading to disordered nanoporous materials whose structure can be characterized  Since the experimental confirmation of its existence , graphenWith little exceptions, however, these require some sort of manipulation of the sheet: in general nano-electronics requires doping to increase the density of states at the Fermi energy or to open a gap, which can be achieved by chemical substitutions , introdu2), or electro-chemical energy storage , requiratteries , and theatteries . Clearlyatteries , make itatteries  or hydroatteries , yet maia posteriori.Indeed, preserving the needed properties and possibly enhancing or tailoring them in the 2D to 3D passage has turned out extremely complex. Up to now, two main routes were considered, both using graphene flakes as precursors. In the first, these are created by graphite exfoliation  and suspended in various solvents, resulting in a mixture of flakes with randomly distributed sizes and shapes; upon dehydration, they form 3D scaffolds with random structure and porosity . These tBuilding multi-layered structures separated by molecular “pillars” is considered an alternative to control the properties of the final 3D construct: theoretically, porosity and density in such structures are determined by the size and concentration of pillars, allowing the possibility of engineering the 3D structure via the pillar molecule design. Up to now, this route, has been followed using organic molecules as pillars ,36 couplClearly, the optimal route should involve the control of the pillars positioning at the nano-scale on the precursor sheets, i.e., the combination of controlled chemical nano-patterning with the possibility of stacking the patterned multilayers in a controlled way. In 2/gr [3/g [2/g). The result are 3D structures with randomly distributed sp2 areas interconnected to form a tangled scaffold with pores of size ranging in the nanometer scale . Differuenching  (temperauenching  or densiuenching ), which uenching . Howeveruenching . This meTo the aim of combining a modest computational effort with realism of the final model, a good strategy is using as precursors already formed graphene portions ,78 inste2 adsorption capability, whose low value indicates rather a small SSA value (hundreds of m2/g). The synthesis procedure was recently optimized [2/g and pores size up to 2 nm. At the same time, it was shown that in some cases, in polar solvents these material exhibits reversible swelling, posing doubts on the complete covalent nature of the layer linkage [In disordered nanoporous materials the porosity of the final structures depends on how the flakes interlace during all the phases of the preparation, which introduces a high degree of disorder and stochasticity. In order to reduce this issue and improve the control over the outcome, the idea rose of synthesizing layered structures separated by molecular pillars, i.e., organic molecules suitably designed with given lengths, rigidity and possibly other physic-chemical properties. The size of the pillars determines the inter-layer spacing, and controls the average pore size, together with the relative distance of the pillars on the sheets. The first realization of such structures traces back to almost two decades ago when, inspired by metal-organic frameworks chemistry, layered structures separated by diboronic acid molecules were first proposed . The pilptimized , obtaini linkage . Using a linkage  resulted linkage .ex post [The optimization of these materials depends on their use: for electric energy storage, both a finely tuned pore size and the conductivity are important. Therefore different synthesis routes were explored, involving reduction of GO, either ex post  or direcex post ,85,86. Aex post . A summaAlthough steps forward have been done in the control of the functionalization, the performances of these materials are not better than those of disordered GNM: SSA is at best several hundreds, far below the theoretical limits and below the simulation predictions. In fact, both the carbon-only model systems including nanotube-pillars ,89 and t2 electronic system. Clearly, reactivity can be brought back by reintroducing defects, e.g., by nitrogen sputtering [The reason why the pillars distribution is poorly controllable is encoded in the use of GO (or rGO) flakes as precursors: the covalent bonding of pillars or anchors exploits the presence of epoxy/hydroxy groups or defects, which are reactive sites . Howeveruttering , which cuttering . However2 sites but manifests a preference for graphene on hydrophilic substrates [2 [Indeed, specific kinds of epitaxial graphene offer different possibilities, which exploit the interaction with the substrate. For instance, radicals of diazonium salts are able to attach to spbstrates , due to bstrates ,102, whebstrates , where trates [2  and on trates [2 ). In therates [2 . In factrates [2  produce 3 hybridized. The bonds and corrugations follow a moiré pattern, due to the mismatch of the two lattices, displaying a hexagonal super-lattice of ~3.2 nm side, made of sharp crests and peaks with sp3-like pyramidal configuration [2 graphene can be obtained continuing evaporation: another BL forms under the first one, which is detached and becomes the so-called Mono-Layer graphene (MLG), characterised by a corrugation pattern with the same symmetry as BL, though smother [We now focus on graphene on SiC, to further explore this concept. It is important to observe that graphene on SiC is not a single material but includes different types of 2D carbons  that canguration . Fully s smother . Alterna smother  or metal smother , obtaini smother . This is smother  with ~1. smother . The ele smother .3 cusps at the vertices of the moiré super-lattice are likely to be highly reactive sites in general, not only towards radicals, but possibly also towards e.g., dissociative chemisorption of H2. Conversely, the protruding crests, organized in diene like structures, and the intruding areas, organized in “benzene-like” rings [While all of the different carbon layers on SiC display charge inhomogeneities following a regular nano-pattern induced by the interaction with the substrate, either mediated by the hybridization, by the corrugation or by the vacancies in the intercalation coverage, only the GML was tested on its reactivity, showing selective H adhesion on the crests . On the e” rings  are likee” rings , leadingClearly, the BL functionalization should be viewed as the first, yet fundamental, step of a procedure involving the multilayer formation see c: once tIn summary, we have reported three possible routes to produce graphene-based materials with porosity on the nano-scale, ordered by increasing capability of control and tailoring of the final structure. The first class produces the disordered nanoporous scaffolds from GO or rGO flakes. These can reach large values of SSA and are, up to now, the most interesting for gas storage. However, controlling their final structure is not straightforward, because of the disordered structure of the precursors and of the stochastic nature of their combination during the production procedure. With the aim of controlling at least a part of the variables determining the porosity, the second strategy introduces on the flakes pillars molecules with pre-determined lengths and shapes. This produces a class of materials with average pore sizes at the nano-metric scale, matching with the size of electrolytes and therefore suitable for the use in electric and electro-chemical storage. However, the average value of the SSA of these materials is rather low, and the poor control over the distribution of the pillars on the sheet introduces disorder, preventing a full optimization, not only for supercapacitors and batteries, but also in catalysis and filtering applications.A third route is currently in its infancy, which would provide a full control over the distribution and location of the pillars. This considers as precursors epitaxial graphene and exploits the electronic inhomogeneities of the sheet produced by the interaction with the substrate, typically following a nano-metric moiré pattern, for the controlled chemical functionalization. Although the first timid steps  were demonstrated, the way is long towards the production of multi-layers.The support of computer modeling and simulations is essential in all cases: in the case of disordered scaffolds, the main issue is to create realistic models and to understand the relationship between production procedure and final structure, and between the latter and the adsorption performances; for the pillared (r) GO materials, the challenge is to control the concentration and location of the pillars and predict the properties as a function of the used pillar. Most of all, computer modeling will be of outmost importance in the pillared multi-layers building from epitaxial graphene. In this case, the simulation of the pillaring, stacking and cross-linking would be essential to give indications for the experimental realization of the procedure. Though extremely challenging, this strategy might give a full control over all the structural features of the resulting structure, and–acting on the nature of pillars–might allow to create brand new materials with tailored and unprecedented properties, such as locally tuned elasticity or conductivity, reactivity to external fields, optical response, and others.


We propose the monolayer graphene plasmonic waveguide (MGPW), which is composed of graphene core sandwiched by two graphene metamaterial (GMM) claddings and investigate the properties of plasmonic modes propagating in the waveguide. The effective refraction index of the GMMs claddings takes negative (or positive) at the vicinity of the Dirac-like point in the band structure. We show that when the effective refraction index of the GMMs is positive, the plasmons travel forward in the MGPW with a positive group velocity . In contrast—for the negative refraction index GMM claddings—a negative group velocity of the fundamental mode  appears in the proposed waveguide structure when the core is sufficiently narrow. A forbidden band appears between the negative and positive group velocity regions, which is enhanced gradually as the width of the core increases. On the other hand, one can overcome this limitation and even make the forbidden band disappear by increasing the chemical potential difference between the nanodisks and the ambient graphene of the GMM claddings. The proposed structure offers a novel scheme of on-chip electromagnetic field and may find significant applications in the future high density plasmonic integrated circuit technique. Negative-index materials (NIMs)—a class of photonic structures with simultaneously negative effective dielectric constant and permeability ,2—providFast light is characterized by a group velocity vg = c/ng exceeding the vacuum speed of light c or taking negative values, where ng is the group-velocity index . When thIn this work, we propose a monolayer graphene plasmonic waveguide (MGPW) made up of a graphene core embedded in the graphene plasmonic metamaterial(s) (GMMs) claddings. The GMMs are composed of graphene nanodisks arrays which are immersed in the identical sheet of graphene with different chemical potential. These arrays are arranged in a square-shaped lattice with a lattice constant of a. Negative equivalent refractive indices of the GMMs are obtained by appropriately designed the structure of the graphene plasmonic metamaterials. The plasmonic waveguide is constructed by the graphene core and GMM claddings. We explore the ‘fast’ plasmon effect in the plasmonic waveguide by modifying the width of the graphene core and the chemical potential difference between the graphene nanodisks and the ambient graphene. In contrast to the conventional materials ‘fast-light’ media, the proposed broadly tunable two-dimensional ‘fast’ plasmon device offers the practical on-chip plasmon manipulation, ultracompact footprint and two- dimensional integratively  and may As shown in q is given by0 is the permittivity of the free space. According to the Kubo formula, the surface conductivity of graphene is composed of contributions from intraband electron–phonon scattering intraσ and interband electron transitions interσ, i.e., e is the electron charge, T = 300 K is the temperature, cμ is chemical potential, ω is the angular frequency. The momentum relaxation time is chosen as τ = 1 ps. Utilizing Equation (2), we obtain the complex effective refractive index of SPPs Here, a monolayer of graphene sheet is placed on the x–y plane in r = 0.2573a), where triple accidental degeneracy of the electromagnetic field takes place at the Brillouin zone center (Γ) which is known as the Dirac-like point. kx, ky) in k for simplicity. At a given frequency, the electric (magnetic) field envelope functions of SPPs, ky direction. The effective permittivity, permeability of the s reduce to The proposed graphene metamaterial is shown in I, II in a, we getm is the mode order. eff2(ω) − nGMMs2(ω))/(n(ω)2 − nGMMs2(ω)) represents the normalized refraction index with the effective mode refraction written as neff(ω) = β /k0. When nGMMs(ω) > n(ω), the normalized frequency γ = k0δIn this section, we consider a situation that SPPs travel through the monolayer graphene plasmonic waveguide composed of the graphene core , frequency and graphene’s chemical potential determine the refractive index difference between the core and claddings r = 0.2573a, which allows us to get the Dirac-like point in the band structure. In effn versus ω) for three widths of graphene core of in two different cases, where the graphene core is embedded in the positive index and negative index claddings, respectively. For the MGPW with positive index claddings, the effective mode refraction increases in proportion to the frequency ω when m = 1. Therefore, it corresponds to the normal dispersion. In contrast, in the case of the MGPW with negative index claddings, there exist narrow spectral regions where effn(ω) is a decreasing function of frequency (that is effdn/d ω < 0), which is known as anomalous dispersion. It should be noted that the mode order m takes zero, which corresponds to the fundamental SPPs mode traveling through the plasmonic structure. In addition, the phase velocity of the SPPs sustained by the MGPW is characterized by the pv = ω/β, which is plotted in vg = g, where ng is the group-velocity index. For the MGPW of length L, it takes a propagation time L/vg = ngL/c for SPPs modes. The SPPs modes that enter the proposed MGPW possess at a moment delayed by a time difference ∆T = L/vg-L/c = (ng − 1)L/c, where L/c denotes the vacuum transit time. As shown in m = 0. The delay −∆T = (1−ng)L/c becomes larger than the L/c, meaning that the backward fundamental SPPs mode (fast light) appears with the anomalous dispersion.To demonstrate the negative group velocity of the plasmon, an incident SPPs is launched at the input of the monolayer graphene plasmonic waveguide with the eigen mode. The chemical potential of the ambient graphene is It is important to point out that, as shown in a = 60 nm. The chemical potential of the ambient graphene is r = 0.2023a. The width of the graphene core is δ = 50 nm. To compare the negative and the positive group velocities of the SPPs in the proposed MGPW, we extend the scanning frequency ranged from 55.5 to 60.5 THz. The transmission spectrum is plotted in We numerically investigate the backward (forward) fundamental SPPs in the MGPW with negative(positive) index GMMs claddings. The lattice constant of the GMMs is fs of full width at half maximum (FWHM), are modulated by sinusoidal carrier waves at the entrance of the MGPW (z = 0) and recorded numerically at the rear of the MGPW (z = L). For comparison, the identical light pulses traversing in vacuum with the same distance are also numerically recorded. Because the chemical potential difference between the nanodisks and the ambient graphene limits the “fast” plasmons propagating, we use a relative chemical potential of g of the SPPs Gaussian pulses, which is proportional to the angular frequency. The results are illustrated in g—which is obtained from the analytical Equation (6)—is included in the same diagram. It should be noted the group-velocity index ng obtained from the two methods are consistent with each other in general. The analytical n and the fluctuation of the analytical curve may be resulted from the interpolation to the effective mode refraction.To further verify the negative group velocity nature of the plasmon, we investigate the propagation of the Gaussian pulses through the proposed waveguide illustrated in Comparing with other reported structures of negative group velocity ,17,18,19In conclusion, we propose a monolayer graphene plasmonic waveguide composed of the graphene core and metamaterial claddings. Emulating the propagation waves propagating through the dielectric three-layer waveguide, this structure supports SPPs modes that can be confined in the graphene core. When the refraction index of the GMM takes positive values, the proposed MGPW supports the forward fundamental SPPs mode. In contrast, the MGPW with negative index supports the backward fundamental SPPs mode when the width of graphene is narrow enough. Additionally, a forbidden band lies between the positive and negative group velocity regions. Furthermore, forbidden band gradually enlarges with the width of graphene core. Our proposed structure may offer a novel approach to manipulate electromagnetic fields in nanoscale.


Astrocytes with cytoplasmic TDP-43 inclusions can cause motor neuron death, however, whether astroglial metabolism and metabolic support of neurons is altered in astrocytes with TDP-43 inclusions, is unclear. We measured lipid droplet and glucose metabolisms in astrocytes expressing the inclusion-forming C-terminal fragment of TDP-43 or the wild-type TDP-43 using fluorescent dyes or genetically encoded nanosensors. Astrocytes with TDP-43 inclusions exhibited a 3-fold increase in the accumulation of lipid droplets versus astrocytes expressing wild-type TDP-43, indicating altered lipid droplet metabolism. In these cells the noradrenaline-triggered increases in intracellular cAMP and Ca2+ levels were reduced by 35% and 31%, respectively, likely due to the downregulation of β2-adrenergic receptors. Although noradrenaline triggered a similar increase in intracellular lactate levels in astrocytes with and without TDP-43 inclusions, the probability of activating aerobic glycolysis was facilitated by 1.6-fold in astrocytes with TDP-43 inclusions and lactate MCT1 transporters were downregulated. Thus, while in astrocytes with TDP-43 inclusions noradrenergic signaling is reduced, aerobic glycolysis and lipid droplet accumulation are facilitated, suggesting dysregulated astroglial metabolism and metabolic support of neurons in TDP-43-associated ALS and FTD.Most cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43) in neurons and non-neuronal cells, including astrocytes, which metabolically support neurons with nutrients. Neuronal metabolism largely depends on the activation of the noradrenergic system releasing noradrenaline. Activation of astroglial adrenergic receptors with noradrenaline triggers cAMP and Ca The pathologic hallmark of ALS are cytoplasmic inclusions in motor neurons. In most cases (~95%) of sporadic and familial ALS, TAR DNA-binding protein 43 (TDP-43), encoded by the TARDBP gene, has been identified as the key component of these inclusions9. Moreover, TDP-43 has also been identified as the major protein in inclusions in frontotemporal dementia with ubiquitin-positive inclusions (FTD-U)6.Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the loss of both upper and lower motor neurons in the brain and spinal cord, and by the progressive paralysis of voluntary muscles and death13. TDP-43, an RNA-binding protein, is implicated in multiple aspects of RNA processing, including regulation of transcription, splicing, transport, and stabilization of mRNAs. It also regulates microRNA biogenesis and interacts with DNA. Therefore, its perturbance may lead to significant changes in the transcriptome and proteome17. It consists of an N-terminal domain, two RNA recognition motifs and a C-terminal prion-like glycine-rich domain that mediates protein-protein interactions with other heterogeneous ribonucleoprotein (hnRNP) family members11.TDP-43 is a highly conserved protein (414 amino acids), ubiquitously expressed in all tissues and under physiological conditions, primarily localized to the nucleus; however, low levels are also present in the cytoplasm18. Although ubiquitination targets TDP-43 aggregates for degradation, TDP-43 begins to accumulate in the cytoplasm, suggesting that additional perturbance in either the ubiquitin-proteasome system or the autophagy pathway can facilitate the accumulation of TDP-43 in ALS and FTD-U19. 25-kDa C-terminal fragments of TDP-43 (TDP-43208–414) are commonly detected in ALS and FTD-U pathologic specimens, especially in the cerebral cortex, and generation of these fragments is sufficient to initiate a number of events that mirror TDP-43 proteinopathies20.In most pathologic cases, TDP-43 is hyperphosphorylated and ubiquitinated21. Astrocytes with TDP-43 inclusions are sufficient to cause motor neuron death in animal models23 and exhibit autocytotoxicity7. Thus, astrocytes were recently proposed to play an active role in controlling ALS disease progression and may even be the primary driver of TDP-43 proteinopathies7.TDP-43-containing cytoplasmic inclusions are not restricted to motor neurons but are also found in non-neuronal cells, in particular in astrocytes24, regulating CNS metabolism25. With their numerous processes, they are in tight contact with neurons, including motor neurons, and blood vessels. They transport nutrients from the blood stream to neurons and store blood-derived glucose in the form of glycogen as the CNS fuel reserve26 and perhaps also as free glucose in endoplasmic reticulum27. Astrocytes are considered an important cellular target of noradrenaline (NA), released from the locus coeruleus (LC) noradrenergic neurons, which regulates CNS energy metabolism32. NA binds to G-protein-coupled adrenergic receptors  on the surface of brain cells, including astrocytes34, where ARs are abundantly expressed35, changing the intracellular concentration of cyclic adenosine monophosphate ([cAMP]i) and Ca2+ ([Ca2+]i)39. This activates astroglial metabolism, which is mainly controlled by β-AR/cAMP signaling, enhancing glucose uptake, glycogenolysis, aerobic glycolysis, and lactate production40. Lactate is considered to be then shuttled to neurons where it is used as fuel by being transformed to pyruvate and entering oxidative phosphorylation42.Astrocytes are an abundant and heterogeneous subtype of neuroglia in the central nervous system (CNS)In vitro and in vivo studies using ALS model systems of superoxide dismutase 1 (SOD1)-related familial ALS have provided first evidence of metabolic dysfunction in ALS astrocytes, particularly in the transporter responsible for the efflux of lactate43, dysfunctional astrocytic mitochondria44, and lower levels of intracellular lactate upon exposure of astrocytes to glutamate45. Besides glucose metabolism, lipid metabolism might also be impaired in neurons and astrocytes during neurodegeneration as observed in animal models48. If reactive oxygen species (ROS) are elevated in the brain like during neurodegeneration46, the glia-neuron lactate shuttle is believed to promote lipid synthesis in neurons. Peroxidized and potentially cytotoxic lipids are then shuttled via apolipoproteins (APO) E and D and fatty acid transport proteins, from neurons to glial cells, causing accumulation of lipid droplets (LDs) in glial cells47, dynamic organelles, undergoing regulated, stress-induced biogenesis and/or degradation50, that play an important role in intracellular lipid metabolism and storage51. Consistent with the observations in animal models, a reduced glucose uptake and increased glycogen storage have been observed in the CNS of patients with ALS52; both processes highly depend on astrocytes, suggesting alterations in astroglial metabolism. Whether TDP-43 inclusions, a hallmark of ALS and FTD-U pathologies, can in astrocytes per se alter astroglial glucose and lipid droplet metabolisms is not known, and was the subject of this study.2+, cAMP signaling and lactate production in astrocytes, we used real-time fluorescence microscopy, Ca2+ indicator Fluo-4 AM dye, and genetically encoded FRET (fluorescence resonance energy transfer)-based cAMP (Epac1-camps53) and lactate (Laconic54) nanosensors. NA induced increases in [cAMP]i, [Ca2+]i, and intracellular concentration of lactate ([lactate]i) in astrocytes expressing wild-type (WT) TDP-43 and in astrocytes with cytoplasmic TDP-43 inclusions . The amplitude and the time constant of the increase in [cAMP]i were, however, reduced by 35% and 50%, respectively, and the peak amplitude and the cumulative [Ca2+]i increase were reduced by 20% and 30%, respectively, in astrocytes with cytoplasmic TDP-43 inclusions  versus astrocytes transfected with WT TDP-43. The amplitude and the rate of increase in [lactate]i upon NA treatment were similar in WT TDP-43 expressing astrocytes and in astrocytes with TDP-43 inclusions, however the responsiveness of astrocytes containing cytoplasmic TDP-43 inclusions to NA with production of lactate was increased by 1.6-fold versus WT TDP-43 expressing cells. Astrocytes with TDP-43 inclusions exhibited a reduced expression of β2-ARs and MCT1 transporters, as determined with immunocytochemistry, consistent with reduced cAMP and Ca2+ signaling and suggesting decreased astroglial lactate release capacity. Thus, astroglial aerobic glycolysis is altered in astrocytes with cytoplasmic TDP-43 inclusions, which may affect astroglial metabolic support of neurons in ALS and FTD-U. Moreover, these cells accumulated LDs.The results revealed that overexpression of the C-terminal fragment of TDP-43 (amino acids 208–414) results in cytoplasmic TDP-43 inclusions in cultured rat cortical astrocytes, typical for ALS and FTD-U pathologies. These cells accumulated LDs. To measure the effect of TDP-43 inclusions in single cells on the dynamics of AR-induced Ca208–414), known to generate cytoplasmic inclusions in other cell types20, or with the pDNA encoding RFP-tagged WT TDP-43 (RFP-TDP-43wt). Using confocal microscopy, we observed that in astrocytes expressing the RFP-TDP-43wt construct, the RFP signal is visible mainly in the cell nucleus  with anti-TDP-43 was 71.5 ± 5.5% (n = 20) and 95.5 ± 2.1% (n = 23) in RFP-TDP-43wt- and RFP-TDP-43208–414-expressing astrocytes, respectively  affects the nuclear levels of endogenous TDP-43 in cultured rat astrocytes. To evaluate this, we performed colocalization analysis between Alexa488-labelled TDP-43 antibody and nuclear marker DAPI in control, non-transfected astrocytes, and in astrocytes expressing the RFP-tagged TDP-43 pDNA constructs. The degree of colocalization (%) with DAPI was 99.1 ± 0.4% (n = 28), 92.2 ± 2.9% (n = 20), and 33.9 ± 6.0% (n = 23) in control, RFP-TDP-43wt- and RFP-TDP-43208–414-expressing astrocytes, respectively , suggests that the C-terminal fragment of TDP-43 causes partial depletion of endogenous nuclear TDP-43. When we analyzed the extent of DAPI and TDP-43-colabeled nuclei of non-transfected astrocytes , positioned around astrocytes expressing the RFP-TDP-43 constructs , we observed a small, statistically insignificant reduction trend in percentage of DAPI and TDP-43-colabeled nuclei in the RFP-TDP-43208–414 experimental group compared to RFP-TDP-43wt and control experimental group  in astrocytes increased the amount of BODIPY493/503-positive cell cross-sectional area, indicating LD content increase, by ~4- and ~3-fold compared with control non-transfected (n = 58) and RFP-TDP-43wt-expressing astrocytes, respectively . Moreover, the average number of LDs per cell (32.0 ± 4.1 (RFP-TDP-43wt) versus 74.7 ± 6.7 (RFP-TDP-43208–414), P < 0.001), LD perimeter (1.6 ± 0.1 μm (RFP-TDP-43wt) versus 2.3 ± 0.1 μm (RFP-TDP-43208–414), P < 0.001), and LD diameter (0.50 ± 0.02 μm (RFP-TDP-43wt) versus 0.74 ± 0.03 μm (RFP-TDP-43208–414), P < 0.001), increased in astrocytes with TDP-43 inclusions  enabling the nuclear and cytosolic fluorescence signals to overlap, it is more likely, that the TDP-43 inclusions seen at the DAPI-labeled nuclear area are in fact located in the cytosol.40. Through binding to ARs on the surface of astrocytes, NA activates cAMP and Ca2+ signaling56, which may be dysregulated in various neurologic disorders60. To study whether ALS- and FTD-U-linked TDP-43 inclusions affect cAMP and Ca2+ signaling, we monitored NA-induced changes in [cAMP]i and [Ca2+]i using real-time confocal microscopy and genetically encoded FRET-based cAMP nanosensor Epac1-camps53 or Fluo-4 AM dye, respectively, in astrocytes expressing WT (RFP-TDP-43wt) or mutant TDP-43 pDNA construct  or 44.7% (n = 42/94), respectively  and RFP-TDP-43208–414-expressing astrocytes 37, indicating that the overexpression of RFP-TDP-43wt and RFP-tagging of the TDP-43wt does not affect NA-mediated cAMP signals in astrocytes. However, the NA-mediated increase in [cAMP]i was significantly reduced in RFP-TDP-43208–414- compared with RFP-TDP-43wt-expressing astrocytes, as reflected by the reduced amplitude in the FRET signal . Thus, noradrenergic stimulation leading to the β-AR-mediated increase in [cAMP]i is reduced in astrocytes expressing the ALS- and FTD-U-related C-terminal fragment of TDP-43 (amino acids 208–414) compared with astrocytes expressing WT TDP-43.The percentage of astrocytes co-expressing both Epac1-camps and RFP-TDP-43ely Fig. , not sigwt or RFP-TDP-43208–414 were labeled with Ca2+ indicator, Fluo-4 AM dye, and treated with NA  and the initial rates of the Laconic FRET signal increase (ΔFRET/Δtime [%/min]) in NA-responsive cells were similar in the RFP-TDP-43208–414 compared with astrocytes expressing RFP-TDP-43wt .However, when we looked at the whole population of astrocytes, we observed that the responsiveness of astrocytes to NA-induced lactate production increased by 1.6-fold (from 42.8% to 70.0%) in cells expressing RFP-TDP-433wt Fig. ; Table 2wt) versus 60.0% (RFP-TDP-43208–414); Table 208–414 than in the RFP-TDP-43wt-expressing astrocytes  and 12.0% ± 1.4% [n = 48] versus 10.0% ± 0.8% , respectively; Fig. 2-ARs was significantly lower in astrocytes expressing RFP-TDP-43208–414 , a major component of ALS- and FTD-U-associated pathologic cytoplasmic inclusions, affects astroglial cell metabolism. TDP-43 inclusions may compromise astroglial metabolic support of neurons in ALS and FTD-U and contribute to CNS hypometabolism observed in patients with neurodegeneration.Based on the new disease models, a number of recent studies have highlighted the involvement of non-neuronal cells in the pathogenesis of ALS and FTD-U, including astrocytes20, consistent with studies in other cell types64. Astrocytes with cytoplasmic TDP-43 inclusions had a 3-fold lower amount of endogenous nuclear TDP-43  compared with astrocytes expressing WT TDP-43, where most of the TDP-43 was present in the cell nucleus. This indicates that expression of the C-terminal fragment of TDP-43 in astrocytes affects trafficking of endogenous TDP-43 between the nucleus and the cytoplasm, consistent with reports on neurons5, glial cells from human tissue samples55, various cell lines (e.g. QBI-293 and tsBN2 cells10), and muscle cells65. The results suggest that aggregated TDP-43 prevents newly synthesized endogenous TDP-43 from being imported into the nucleus and/or inhibits the re-entry of existing cytoplasmic TDP-43 into the nucleus13. Interestingly, the transfection with inclusion-forming TDP-43, caused in neighboring non-transfected astrocytes  a small, albeit not significant, trend in reduced percentage of DAPI and TDP-43-colabeled nuclei, which suggests that the transfection with the C-terminal fragment of TDP-43 might affect the physiology of non-transfected cells. In pathological conditions, mimicked here by the expression of inclusion-forming TDP-43, astrocytes bear a significant functional plasticity, known as reactive astrogliosis66. These astrocytes secrete or distribute through gap junctions various cytotoxic factors, such as Lcn268, interleukin-6, ciliary neurotrophic factor, etc.69, which can affect neighboring cells. Even though we did not specifically test whether astrocytes expressing inclusion-forming TDP-43 transform into a reactive phenotype, this outcome is possible. If this is the case, the observed reduction in the nuclear TDP-43 staining in some non-transfected astrocytes adjacent to inclusion-forming TDP-43 astrocytes may be a consequence of altered physiological state of these cells affecting the TDP-43 gene expression level or the synthesis/degradation/distribution of TDP-43.Expression of C-terminal fragment of TDP-43 in isolated cortical rat astrocytes has led to the formation of cytoplasmic TDP-43 inclusions, which can mimic key biochemical features of TDP-43 proteinopathies3, including on cell metabolism. LD accumulation has been observed in glial cells, including astrocytes, in early stages of neurodegeneration46. Recently, alterations in lipid metabolism  in spinal cords from SOD1G93A transgenic mouse model have been reported that might be linked to astrogliosis and LD formation in astrocytes48, since a population of astrocytes isolated from the degenerating spinal cords of the same animal model exhibited significant abundance of LDs as well as autophagic and secretory vesicles, all characteristic features of cellular stress and inflammatory activation70. However, the mechanisms leading to increased LD accumulation in astrocytes are poorly understood and may, among others, rely on altered neuronal mitochondrial function and ROS as well as on astrocyte-neuron lactate shuttle47, in particular on astroglial lactate-derived lipid production in neurons and transfer of excess lipids in lipoprotein-like particles (ApoE) from neurons to astrocytes71. We show here that in astrocytes with TDP-43 inclusions the LD presence is enhanced (the size and the number of LDs) in the absence of neighboring neurons, suggesting the existence of an alternative astroglial-mediated mechanism triggering accumulation of LDs through altering the balance between biogenesis and degradation of LDs. Accumulation of LDs in astrocytes with cytoplasmic TDP-43 inclusions may be a response to cellular inflammation, which is typically found in the pathology of various neurologic disorders, including ALS72. Here, LDs are hypothesized to be an important source of energy for proliferation and may serve a protective role by gathering free fatty acids to protect cells against lipotoxicity73.Because TDP-43 is involved in multiple aspects of RNA processing, any changes in TDP-43 nuclear level may have detrimental effects on astroglial physiology2-ARs and a 35% reduction in NA-mediated increase in [cAMP]i. Consistent with our results, dysregulation of astrocytic β2-AR/cAMP signalling has been suspected to contribute to the pathology of a number of other neurologic disorders, including multiple sclerosis, Alzheimer’s disease, human immunodeficiency virus encephalitis, and others74. Reduction of β2-AR expression was reported in human white matter astrocytes obtained from post mortem brain tissue of patients with multiple sclerosis75. Moreover, it was demonstrated both in vitro and in vivo that the presence of Alzheimer’s disease associated amyloid beta peptide (Aβ) in prefrontal cortical neurons leads to internalization and degradation of β2-ARs, which leads to subsequent attenuation of cAMP signalling59. Since aerobic glycolysis in astrocytes is upregulated with β-AR/cAMP signaling, one would expect that astrocytes with TDP-43 inclusions and a reduced expression of β2-ARs will exhibit reduced β-adrenergic mediated aerobic glycolysis and lactate production. When we stimulated astrocytes with Iso, a selective β-AR agonist, although the responsiveness of cells to Iso was unchanged, there was a trend in reduction of [lactate]i increase, since the amplitude in [lactate]i increase was ~2-fold lower (P = 0.08) in astrocytes with TDP-43 inclusions, consistent with downregulation of β2-AR and cAMP signaling in these cells. In contrast to Iso stimulation, the amplitude and the rate of [lactate]i increase upon NA stimulation was unaltered in astrocytes with TDP-43 inclusions, but the probability of activating aerobic glycolysis, measured as increased responsiveness to NA, was increased by 1.6-fold in astrocytes with TDP-43 inclusions. When viewing the astroglial population as a whole, this means that glycolytic lactate production upon NA stimulation is facilitated in astrocytes with TDP-43 inclusions.Besides alterations in LD metabolism, changes in noradrenergic regulation of glucose metabolism were observed in astrocytes with cytoplasmic TDP-43 inclusions. Astrocytes with TDP-43 inclusions exhibited downregulation of β2+ signals through activation of α1-AR/Gq-protein signaling pathway have important role in regulation of NA-mediated glucose metabolism in astrocytes33. Abnormal Ca2+ homeostasis has been observed in astrocytes isolated from SOD1G93A animals. In particular, excess Ca2+ release from ER stores upon purinergic/Gq-protein signaling pathway activation has been reported due to abnormal ER Ca2+ accumulation76. If such a mechanism exists in astrocytes with TDP-43 inclusions, binding of NA to α1-AR may lead to excess Ca2+-release from ER and enhanced aerobic glycolysis despite downregulation of β2-AR/cAMP signaling pathway. However, contrary to the results obtained on SOD1G93A animal model, we observed a 31% reduction in Ca2+ signaling in astrocytes with TDP-43 inclusions, even though the level of α1-AR expression was unchanged. It has been reported that β-AR/cAMP and α1-AR/Ca2+ signaling pathways interact in astrocytes, enhancing each other39, therefore downregulation of β2-ARs may reduce the noradrenergic Ca2+ response via α1-ARs, explaining the reduction of both noradrenergic cAMP and Ca2+ signals despite an unaltered expression of α1-ARs.Besides cAMP, Cai37. If aerobic glycolysis in astrocytes displays a similar bell-shaped dependency on [cAMP]i, astrocytes with reduced β2-AR/cAMP signalling may actually attain enhanced metabolic responsiveness to noradrenergic stimulation. Moreover, perturbances in TDP-43 may change the transcriptome and proteome of a cell affecting the expression level of enzymes involved in LD metabolism and aerobic glycolysis, as was observed in other cell types with a TDP-43 knock down16. Whether expression level of metabolic enzymes is altered in astrocytes with TDP-43 inclusions and whether this contributes to the observed enhanced astroglial metabolism needs to be investigated in the future.It has been reported that morphologic changes in astrocytes exhibit a bell-shaped dependency on [cAMP]77. This is, however, contradictory to the CNS hypometabolism observed in patients with neurodegenerative diseases, including ALS45. Recently, decreased expression of lactate MCT1 and MCT4 transporters has been reported post mortem in the motor cortex of ALS patients compared to non-ALS patients78. Moreover, downregulation of MCT1 mRNA in the spinal cords of early symptomatic and endstage SOD1G93A transgenic mice model of ALS has been observed, presumably in glia (oligodendrocytes and astrocytes)78. Down-regulation of the lactate MCT4 transporter has been also demonstrated in spinal cord astrocytes from patients with ALS with SOD1 mutations and in pre-symptomatic SOD1G93A transgenic mice43. We show here that expression of C-terminal fragment of TDP-43 (TDP-43208–414) per se in astrocytes causes a reduction in the expression of astroglial lactate MCT1 transporters. Lower expression of astroglial MCTs may decrease the lactate release capacity in astrocytes. Thus, despite facilitated NA-mediated aerobic glycolysis in astrocytes with TDP-43 inclusions, lactate may accumulate inside cells, and the metabolic support of neurons in patients may be decreased significantly contributing to neuron degeneration in ALS and FTD-U. Consistent with this hypothesis, cAMP signaling and lactate homeostasis in the brain were noted to be reduced in patients with some forms of ALS45.Increased lactate production upon NA stimulation in astrocytes with TDP-43 inclusions may lead to a better metabolic support of neurons with lactate due to lactate flux generated between astrocytes and neurons2+ signaling, whereas both aerobic glycolysis and LD presence appear facilitated. Although NA-mediated L-lactate production was increased in astrocytes with TDP-43 inclusions, the expression of lactate MCT1 transporters was reduced, suggesting decreased astroglial lactate release capacity .79, and grown in high-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 2 mM l-glutamine and 25 µg/ml penicillin-streptomycin in a vaporized atmosphere containing 95% air and 5% CO2 at 37 °C until reaching 70%–80% confluence. Confluent cultures were shaken overnight at 225 rpm and the medium was changed the next morning; this was repeated three times. After the third overnight shaking, the cells were trypsinized and put in flat tissue culture tubes with 10 cm2 growth area. After reaching confluence again, the cells were subcultured onto 22-mm diameter poly-l-lysine-coated glass coverslips. This procedure yielded astrocytes with > 95% purity, determined by anti-glial fibrillary acidic protein (GFAP) antibody staining80.Primary astrocyte cultures were prepared from the cerebral cortices of 2- to 3-day-old Wistar rats, as described previously53 or lactate nanosensor Laconic54 and RFP-tagged WT TDP-43 (pTagRFP-C::TDP-43wt [RFP-TDP-43wt]) or RFP-tagged C-terminal fragment of TDP-43 (pTagRFP-C::TDP-43208–414 [RFP-TDP-43208–414]) using FuGENE 6 Transfection Reagent . Transfection medium contained no serum or antibiotics.After 1–3 days, astrocytes were co-transfected with the genetically encoded FRET-based cAMP nanosensor Epac1-campsThe experimental animals were cared for in accordance with the International Guiding Principles for Biomedical Research Involving Animals developed by the Council for International Organizations of Medical Sciences and the Animal Protection Act . The experimental protocol was approved by The Administration of the Republic of Slovenia for Food Safety, Veterinary and Plant Protection , Document No. U34401-47/2014/7, signed by Barbara Tomše, DVM. Every set of data was acquired from at least 2 independent experiments.wt or RFP-TDP-43208–414 were fixed with 4% formaldehyde in phosphate buffered saline (PBS) for 15 min and then permeabilized with 0.1% Triton X-100 for 10 min at room temperature (except for MCT1 and MCT4) before being treated with 10% goat serum for 1 h at 37 °C. Astrocytes were then stained with primary antibodies against endogenous TDP-43 , α1-AR , β1-AR , β2-AR , MCT1  and MCT4  overnight at 4 °C. After washing with PBS, cells were incubated for 1 h at 37 °C with Alexa Fluor488-conjugated secondary goat anti-rabbit IgG . Excess antibodies were washed off and the coverslips were mounted onto glass slides using SlowFade antifade reagent with DAPI  and carefully sealed. Immunolabelled cells were imaged with an inverted Zeiss LSM780 confocal microscope with a Plan apochromatic 40×/1.4 oil immersion objective  using 488-nm Ar-ion, 543-nm He-Ne and 405-nm diode laser excitation. Emission spectra were acquired sequentially with 505- to 530-nm bandpass (Alexa Fluor488), 560-nm long pass (TagRFP584) and 445- to 450-nm bandpass (DAPI) emission filters.Control (non-transfected) astrocytes and astrocytes expressing RFP-TDP-43488 (immunolabelled TDP-43), TagRFP584 (RFP-TDP-43 pDNA constructs), and DAPI fluorescence signals was performed on exported TIFF files using ColocAna software 81 that counts all red, green, blue and colocalized (green and red or green and blue) pixels within the image. The threshold for the colocalized pixel count was set at 20% of the maximal green, red or blue fluorescence intensity, respectively. Colocalization was expressed as the ratio of colocalized to green or blue pixels (as percentages). Kruskal-Wallis one-way analysis of variance (ANOVA) on ranks (followed by Dunn’s test) or Mann-Whitney U test were performed to determine statistical significance between the experimental groups. P < 0.05 was considered significant.Colocalization analysis of Alexa Fluor1-AR-, β1-AR-, β2-AR-, MCT1- and MCT4-positive green fluorescence pixels with fluorescence intensity above the threshold of 10% (α1-/β1-/β2-AR-positive cell area) or 20% (MCT1 and MCT4-positive cell area) of maximal fluorescence and the number of all pixels  per cell cross-sectional area were determined for each cell separately in RFP-TDP-43wt- and RFP-TDP-43208–414-expressing astrocytes using ZEN software . The percentage of the AR- and MCT-positive cell area relative to the total cell area was calculated for RFP-TDP-43wt- and RFP-TDP-43208–414-expressing astrocytes for individual receptor and transporter type. The Mann-Whitney U test was performed to determine statistical significance between the experimental groups. P < 0.05 was considered significant.To determine the expression level of different ARs and MCTs, the number of αwt- and RFP-TDP-43208–414-expressing astrocytes  we measured the percentage of DAPI and TDP-43-colabeled nuclei per all DAPI nuclei in neighboring non-transfected cells  in the RFP-TDP-43wt and RFP-TDP-43208–414 experimental groups. In the control non-transfected group, we analyzed the nuclei of all cells. Some partially visible DAPI-stained nuclei at the edges of the confocal images were also taken into account in the analysis, however, only if no RFP signal in or around the visible part of the nuclei was observed. Kruskal-Wallis one-way ANOVA on ranks, followed by Dunn’s test was performed to determine statistical significance between the experimental groups. P < 0.05 was considered significant.To determine the extent of DAPI and TDP-43-colabeled nuclei in non-transfected astrocytes  adjacent to the RFP-TDP-43wt or RFP-TDP-43208–414 plasmid were incubated in fresh growth medium for 24 h. Cells were then fixed in 4% formaldehyde in PBS for 5 min and stained with 1 µg/ml BODIPY493/503 , a fluorescent LD marker, for 6 min at room temperature. Excess dye was washed off and the coverslips were mounted onto glass slides using SlowFade antifade reagent with DAPI  and carefully sealed. Stained cells were imaged with an inverted Zeiss LSM780 confocal microscope with a Plan apochromatic 40×/1.4 oil immersion objective  using 488-nm Ar-ion, 543-nm He-Ne and 405-nm diode laser excitation. Emission spectra were acquired sequentially with 505- to 530-nm bandpass (BODIPY493/503), 560-nm long pass (TagRFP584) and 445- to 450-nm bandpass (DAPI) emission filters.Control (non-transfected) astrocytes and astrocytes transfected with RFP-TDP-43493/503-positive green fluorescence pixels with fluorescence intensity above the threshold of 20% of maximal fluorescence (Lipid droplet S) and the number of all pixels (Cell S) were determined for each cell separately in control, RFP-TDP-43wt- and RFP-TDP-43208–414-expressing astrocytes in ZEN software . The values obtained were normalized to the average LD content in control non-transfected cells. The mean number of BODIPY493/503-labeled LDs per cell and the mean LD perimeter were determined in cross-sections of individual cells using ImageJ, Analyze Particles function after applying 20% threshold and signal intensity (watershed) segmentation on individual images. Then, assuming that all LDs are spherical, the mean LD diameter was estimated from the perimeter values with the equation d = C/π, where d is the diameter and C is the LD perimeter. Kruskal-Wallis one-way ANOVA on ranks, followed by Dunn’s test was performed to determine statistical significance between the groups. P < 0.05 was considered significant.To determine the LD content in individual astrocytes, the number of BODIPYwt or RFP-TDP-43208–414) were examined 24–30 h after transfection with a fluorescence microscope Zeiss Axio Obsever.A1 , with a CCD camera and monochromator Polychrome V  as a monochromatic source of light with a wavelength 436 nm/10 nm. Dual emission intensity ratios were recorded using an image splitter  and two emission filters . Images were acquired every 3.5 s for Epac1-camps and 10 s for Laconic. Exposure time was 100 ms.Cells co-expressing the FRET-based nanosensor Epac1-camps or Laconic and RFP-tagged TDP-43 pDNA construct ) with a Axiocam 702 camera and Colibri.2 Lamp Module  as a source of light with a wavelength 433 nm. Dual emission intensity ratios were recorded using an image splitter  and two emission filters (469-491 nm for ECFP and 530-4095 nm for EYFP). Images were acquired at intervals of 10 s with exposure time of 100 ms.In some experiments astrocytes co-expressing Laconic and RFP-TDP-43i or [lactate]i.Coverslips with transfected astrocytes were mounted in a superfusion recording chamber on the microscope stage. Imaging was performed at room temperature (22–24 °C). One cell per experiment was recorded. The FRET signals (CFP/YFP (Epac1-camps) and mTFP/Venus (Laconic) signals) were obtained from the integration of the ratio signal over the entire cell using Life Acquisition software  or ZEN . In the graphs, the FRET signal was reported as the ratio of the CFP/YFP (Epac1-camps) and mTFP/Venus (Laconic) fluorescence signals after subtracting the background fluorescence from the individual fluorescence signals using Excel . The values of the FRET ratio signals were normalized to 1.0. An increase in the FRET ratio signal reflects an increase in [cAMP]d-glucose, 135.3 mM NaCl, 1.8 mM CaCl2, 2 mM MgCl2, and 5 mM KCl, 0.5 mM NaH2PO4·H2O, 5 mM NaHCO3), and then treated with 100 µM NA (non-selective AR agonist) or 100 µM Iso (selective β-AR agonist) following a 100- to 200-s baseline. Experiments were conducted with the addition of a bolus solution; 200 µl of extracellular solution containing NA was added by pipette to 200 µl of bath solution in the recording chamber. The application of a control bolus solution without reagents had no significant effect on the FRET signal, as reported previously83. Extracellular solution osmolality was 295–305 mOsm, measured with the Osmomat 030 freezing point osmometer .Before the experiments, astrocytes were kept in extracellular solution containing sodium bicarbonate for 10 min )) were fitted to the FRET ratio signals using SigmaPlot. The time constant (τ) and amplitude changes in the FRET ratio signal (ΔFRET [%]) were determined from the fitted curves. F is the FRET ratio signal at time t, F0 is the baseline FRET ratio signal, c is the FRET ratio signal amplitude of F − F0, and τ is the time constant of the individual exponential component. In experiments with Laconic, the maximal  rates of the FRET ratio signal increase (ΔFRET/Δt) were calculated as the slope of the linear regression function (ΔFRET [%] = slope [%/min] × Δt [min]) fitting the initial FRET ratio signal increase. In these experiments, changes in the FRET ratio signal (ΔFRET [%]) were calculated by subtracting the mean maximal FRET ratio signals from the mean baseline FRET ratio signals.In experiments with Epac1-camps, single-exponential increases to maximum functions  and then transferred to dye-free medium for at least 30 min before the experiments to allow for cleavage of the AM ester group. The cells were excited with an Argon-ion laser at 488 nm, and time-lapse images were obtained every 3 s with an inverted Zeiss LSM780 confocal microscope and an 20x objective. Emission light was acquired with a 505–530-nm band-pass emission filter. Fluo-4 AM–labeled astrocytes were after 100 s baseline stimulated with 100 μM NA. Total recording time was ~400 s (135 frames). In individual cells, Fluo-4 AM intensity was quantified within a region of interest and expressed as the relative change in fluorescence: ΔF/F0 = (F − F0)/F0, where F0 denotes the baseline fluorescence level after subtraction of background fluorescence. The peak and cumulative increase in Fluo-4 AM ΔF/F0 were determined using Microsoft Excel.Astrocytes expressing RFP-TDP-43P < 0.05 was considered significant.Mann-Whitney U test was performed to determine statistical significance between the experimental groups. Supplementary material.


Acute exacerbation (AE) is a common cause of rapid deterioration and high mortality in idiopathic pulmonary fibrosis (IPF) patients. Osteopontin (OPN) plays an important role in IPF, but the studies about serum OPN in AE-IPF are unclear. We aimed to investigate whether OPN had a potential prognostic value in acute exacerbation and mortality in IPF.  Thirty-two patients with AE-IPF, 39 with S-IPF, and 20 healthy controls were included. Serum OPN and KL-6 levels were compared between AE-IPF and S-IPF. Logistic regression analysis was applied to identify the predicted value of OPN for AE. Kaplan–Meier curves were used to display survival, and Cox proportional hazards regression was used to identify risk for mortality. p < 0.001) or healthy controls (p < 0.001) or healthy controls (p < 0.001) or healthy controls (p < 0.001) or healthy controls (p < 0.001) or healthy controls (p < 0.001) or healthy controls (p < 0.001) or healthy controls (p < 0.001) or healthy controls (p < 0.001) or healthy controls (p < 0.001) or healthy controls ( In AE-IPF patients, serum OPN levels were significantly higher than in S-IPF subjects ( Elevated OPN could be a potential serum predictor for AE status and survival in IPF patients. Idiopathic pulmonary fibrosis (IPF) presents as a progressive deterioration of dyspnea and ultimately respiratory failure , charactSo far, the etiology and mechanisms of AE-IPF occurrence have been poorly known. More and more evidence demonstrated the importance of diffuse alveolar damage and enhanced epithelial injury and proliferation during AE in IPF patients . OPN as In this study, we aimed to determine whether OPN could be a biomarker of AE-IPF occurrence and mortality by comparing serum OPN levels in AE-IPF and S-IPF and by analyzing correlation between serum OPN and other variables in IPF patients.The study population includes 71 subjects (32 AE-IPF patients and 39 S-IPF patients), who were evaluated at Nanjing Drum Tower Hospital between January 2015 and April 2018. 20 healthy subjects from the Center of Physical Examination were included as a control group. The criteria used for enrollment of patients in this study were in accordance with the criteria of IPF and AE-IPF already published , 1.001 , 1.039, 1.035 , 0.950 , and 0.929 , respectively  were significantly associated with a higher risk of AE in IPF, with odds ratios of 1.305 , 1.012 , and 1.009 . Subsequently, multivariate Cox regression was performed. Several covariates were excluded for statistical limitations. We observed that OPN was also associated with the survival of IPF in multivariate Cox models, with a hazard ratio of 1.010 . The same results were also identified for serum KL-6 .To further examine the prognostic values of OPN with regard to the survival in all IPF patients, univariate Cox regression was performed . ElevateThis was the first study to assess the significance of serum OPN levels in occurrence of AE and mortality in IPF patients. IPF patients had elevated serum OPN levels, especially in AE-IPF, compared with healthy controls. In addition, the enhanced expression of OPN was also found in alveolar epithelial cells and alveolar macrophages of the lung section from IPF patients. Compared with S-IPF, serum OPN levels in AE-IPF were significantly increased, which were associated with poor outcome. We found that the survival period was obviously shorter for patients with OPN levels above, compared with those below 3.24 ng/ml; moreover, OPN was a predictor of survival. Collectively, our results suggested that OPN could predict prognosis of patients with IPF.Acute exacerbation state of IPF is an important factor contributing to IPF mortality. Unrecognized infection, diffuse alveolar damage, mechanical procedures, and secondary pulmonary hypertension have been suggested to explain acute exacerbation of IPF , 13, butOPN is a secreted phosphoprotein produced by a variety of cells such as activated macrophages, vascular smooth muscle cells, and epithelial cells, mediating cell chemotaxis, adhesion, proliferation, and migration, and plays a vital role in the pathogenesis of liver and kidney fibrosis , 19. CarIn current study, AE-IPF patients showed higher mortality than S-IPF, consistent with the reported studies , 26. ElePrevious studies demonstrate physiologic parameters such as age, FVC%, and DLCO% have been shown to be important survival factors for IPF , 28. In In this study, LDH and CRP were significantly associated with acute exacerbation status and mortality. LDH is one of the earliest for the serum marker of disease activity in different forms of pulmonary fibrosis . LDH, asMore and more studies demonstrate that acute alveolar epithelial damage and unrecognized infection play an important role in AE of lung fibrosis. This could explain why the levels of LDH and CRP increased obviously in AE-IPF. Our results indicated that elevated levels of LDH and CRP were notably associated with survival, which was consistent with previous studies, but due to their low specificity, they were difficult to be a reasonable biomarker for mortality .There are several limitations in this study. Our ability to perform serial analysis of OPN was limited due to the small study population, which was understandable in consideration of the rarity of IPF. Furthermore, there were no data about OPN in the same patient experiencing the different stages of disease , which was necessary to be a good biomarker for OPN. A comprehensive study in a larger IPF population would be helpful for further research.In summary, to the best of our knowledge, this was the first study to demonstrate elevated serum OPN levels in AE-IPF patients, which was associated with acute exacerbation state and increased mortality risk. The same results were found for KL-6. More studies are needed to verify and extend the role of OPN, to determine whether or how OPN plays a role in the progress of AE, and to confirm the clinical usefulness of OPN as a biomarker for AE-IPF occurrence and predictor of survival in IPF patients.


Several databases of epidemiologic studies in patients with idiopathic pulmonary fibrosis (IPF) have been analyzed in the Western community. However, few studies have been reported in Asia. The objective of this study was to investigate the epidemiology of IPF in Taiwan. We collected and analyzed patients with IPF from the Taiwan National Health Insurance Research Database from 2001 to 2011. We estimated the annual incidence and cumulative prevalence of IPF and mean survival time of patients and determined the causes of death. The annual incidence rates of IPF remained stable after 2005, ranging from 0.7 to 1.3 cases per 100,000 people per year, whereas the cumulative prevalence rates increased steadily from 3.1 to 6.4 cases per 100,000 people per year during 2006–2011 based on a narrow case definition. Men older than 75 years had higher incidence compared with other age groups. The mean survival after diagnosis was 6.9 years. Old age, male sex, and respiratory hospitalization were associated with shorter survival time after diagnosis. Both the incidence and prevalence rates of IPF were lower in Taiwanese patients than Western ones. Moreover, the survival time was higher in the Asian population compared with the Western population. These results may suggest the heterogeneity of the IPF definition in different study populations and geographic locations. Idiopathic pulmonary fibrosis (IPF) is a progressive and life-threatening interstitial lung disease (ILD). Patients usually exhibit symptoms of chronic exertional dyspnea, cough, bibasilar inspiratory crackles, and finger clubbing . AccordiAlthough the disease is considered rare, it occurs with similar frequency to that of stomach, brain, and testicular cancers . The incMost epidemiologic studies ,10,11,12This is a retrospective cohort study based on analysis of the database from the NHIRD. The National Health Research Institutes (NHRI) organizes the NHIRD, which contains all claims data from the Bureau of National Health Insurance (BNHI). To protect patient privacy, all patients were scrambled by BNHI. The NHI program covers almost the entire population of Taiwan. Therefore, the NHIRD can be used as a suitable research resource for analyzing the epidemiology of IPF in Taiwan. We identified patient deaths from the registration and hospital admission files. The code of death or automatic discharge is shown in the hospitalization data, and the record of the patient’s withdrawal from the health insurance is shown in the registration file. We consulted with the institutional review board of Taipei Veterans General Hospital , which waived the informed consent for our retrospective observational cohort study.The Taiwan NHRI manages the NHIRD for research purposes. The National Health Insurance (NHI) program of Taiwan provides comprehensive medical services for all Taiwanese citizens, and it has been operational since 1995 . The NHIIn this retrospective cohort study, we analyzed the epidemiology of IPF in Taiwan. We enrolled adult patients (≥18 years old) who were diagnosed with IPF from 2001 to 2011. The inclusion criteria were patients diagnosed with IPF with at least one recorded outpatient visit, emergency room visit, or hospitalization for IPF. Patients with IPF were defined according to the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) code 516.3.We consulted with Raghu et al.  to clarip < 0.05 was considered significant. All analyses were performed using SPSS version 19.0  and STATA 12  statistical software.We counted the number of patients diagnosed with IPF in each year during 2001–2011 and calculated the incidence rate of a year as the number of newly diagnosed patients per 100,000 people ever alive in the NHI system in the year. The prevalence rate was measured as the number of patients per 100,000 people alive in the NHI system as of the end of a year. We performed the analyses separately for different age–gender groups on the basis of the broad and narrow case definitions. We measured the survival time (in years) for patients with incident IPF from the start of the index day to the date of all-cause death or the end of 2011, whichever came first. The results are shown as mean ± SDs for continuous variables and percentage for categorical and ordinal variables. We estimated the time  on the basis of a Kaplan–Meier survival estimator , with reThe total population in Taiwan is approximately 23 million. Given that the database sampling is about 1:11, approximately 2 million men and women aged 0 years and older participated in the study database that came from LHID2000 and LHID2005. A total of 1,916,514 individuals were eligible from this database. Duplicate patients were deleted from the registration dataset (ID), and 348 IPF patients aged 18 years with ICD-9 code 516.3 were obtained and evaluated between 2001 and 2011. After the exclusion of 49 patients with other codes for ILD, there were 299 patients with IPF incident by broad case definition, and after the exclusion 177 patients without the ICD code with NHI procedure for surgical lung biopsy, transbronchial lung biopsy, or computed tomography of the thorax  before oBased on the narrow case definition, the annual incidence of IPF ranged from 0.2 to 1.3 new cases per 100,000 person-years in 2001–2011, and the cumulative prevalence of IPF increased from 0.2 per 100,000 persons in 2001 to 6.4 per 100,000 persons in 2011 . Based oBased on the 2011 data, the prevalence and incidence of IPF by age and gender are shown in p = 0.122) (p = 0.089) (p < 0.001) (The mean year from IPF diagnosis to death or the last follow-up was 6.9 years (95% CI: 6–7.1 years). The results of survival analysis by gender, age, or hospitalization are presented in = 0.122) A and in = 0.089) B. Howeve= 0.089) . The mea< 0.001) . Accordi< 0.001) .During the study period, the diagnostic criteria to identify persons with IPF were lacking. We consulted with Raghu et al. to clarify the narrow and broad case definitions of IPF. Because our narrow case finding criteria and the case ascertainment methods used by Raghu et al.  probablyIn our study, we used the large nationally representative Taiwan database to investigate the epidemiology of IPF in Taiwan. According to a previous study , the heaComparing our study findings with other studies is hard due to the heterogeneity of IPF definition and in different study population, the time period of analysis, and geographic locations. In North America, Raghu et al. evaluated the incidence and prevalence of IPF by analyzing the medical claims database . AlthougIn Europe, three different research groups studied the incidence and prevalence of IPF by analyzing the nationwide primary care database. Nonetheless, the broad and narrow case definitions that were used in those studies included slightly different criteria. Navaratnam et al.  reportedIn East Asia, four studies used the insurance and claims databases, and all studies reported crude incidence rates between 1.2 and 4.6 per 100,000 persons per year. Our results suggested that the incidence might be similar to those estimated in other Asian countries. Furthermore, Lai et al.  reportedThe disease awareness of patients and physicians was expected to improve after the healthcare system released the following policies: Evidence-based guidelines for the diagnosis and management of IPF established by the Taiwan Thoracic Medical Association in 2015, which allows readers to understand the definition of IPF and the latest diagnostic criteria and treatment strategies, enhance physicians’ diagnostic vigilance and ability and show appropriate treatments to enhance patient well-being. In 2017, the BNHI launched a new classification system for medical treatment. The system encouraged patients with minor symptoms to seek treatment from small clinics and those with serious illnesses to go to major hospitals. In this system, the primary care physician advises patients with known or suspected IPF to seek a center with expertise in IPF care because delayed access is independently associated with increased risk of death. The referral provides patients with access to expertise in diagnosis and management, including initiation of disease-modifying therapy, monitoring, side-effect control, and non-pharmacological support.In our study, the incidence of IPF in males was 2.4-fold higher than that in females. This rate was higher than those reported by Raghu et al.  in the Up < 0.001). Our finding is similar to that of Brown et al. [The mean survival time was 6.9 years from diagnosis to the end of the study. This survival time varied with sex and age at diagnosis; females and patients <65 years of age seemed to have longer survival time . The median survival time was longer than those of previous studies in Western (3–5 years)  and Asian et al. , who repWe hypothesized that a better survival outcome will be anticipated if the case data were homogeneously collected. Lai et al.  reportedIn the past, the 2011 ATS/ERS practice guideline [Lung transplantation can prolong the lifespan of IPF patients. Taiwan has had successful lung transplants since 1991. IPF is also a waiting list for indications, but there are few organ donors and not many cases of lung transplantation. Therefore, IPF lung transplants are even rarer compared with other cases.The primary causes of death in IPF are the progression of IPF (41%), followed by cerebral arterial 13%) and cardiovascular diseases (9%) . Our fin% and carOur study has the following strengths. First, we examined the epidemiology of IPF in Taiwan based on NHIRD samples, which include a large representative population, and the accuracy of insurance claims data in Taiwan was 92% for patients with ≥1 hospitalization per year . Second,Despite the findings, this study has some limitations. First, patients with IPF were identified based on their access to healthcare service, and determining the exact time of IPF onset was impossible. Therefore, the date of the first encounter with inpatient or outpatient healthcare systems coincided with the first recorded medical diagnosis. Second, the case definition used to identify patients with IPF in this study was not validated with respect to medical charts, and whether or not the diagnoses were based on multidisciplinary discussions was unknown. Third, NHIRD lacked detailed clinical and laboratory data; therefore, we could not identify the severity of patients at first diagnosis, which might influence the explanation of the study results.The incidence and prevalence rates of IPF in Taiwanese patients were lower than those in Western communities. Men aged 75 years and older comprised the major group suffering from IPF. The survival time in the present study was higher than those of previous studies in the Asian population. These results may suggest the variety of IPF definitions and differences in the study population, time period of analysis, and geographic locations. In light of the rising prevalence and incidence of IPF, we hope that the next 5 years will be marked by greater recognition of the disease on the part of primary care providers, leading to earlier involvement of a multidisciplinary team to aid in diagnosis and management.