The synthesis of metal-free but electrochemically active electrode materials, which could be an important contributor to environmental protection, is the key motivation for this research approach. The progress of graphene material science in recent decades has contributed to the further development of nanotechnology and material engineering. Due to the unique properties of graphene materials, they have found many practical applications: among others, as catalysts in metal-air batteries, supercapacitors, or fuel cells. In order to create an economical and efficient material for energy production and storage applications, researchers focused on the introduction of additional heteroatoms to the graphene structure. As solutions for functionalizing pristine graphene structures are very difficult to implement, this article presents a facile method of preparing nitrogen-doped graphene foam in a microwave reactor. The influence of solvent type and microwave reactor holding time was investigated. To characterize the elemental content and structural properties of the obtained N-doped graphene materials, methods such as elemental analysis, high-resolution transmission electron microscopy, scanning electron microscopy, and Raman spectroscopy were used. Electrochemical activity in ORR of the obtained materials was tested using cyclic voltamperometry (CV) and linear sweep voltamperometry (LSV). The tests proved the materials’ high activity towards ORR, with the number of electrons reaching 3.46 for tested non-Pt materials, while the analogous value for the C-Pt (20 wt% loading) reference was 4. Therefore, over the past decades, graphene’s potential uses have been examined within many fields5: fields emission (FE) devices6, sensors and biosensors7, optoelectronic devices (transparent electrodes for solar cells), liquid–crystal displays (LCD)9, and electrode materials in batteries and supercapacitors11. Graphene can exist in the form of zero-dimensional (0D) dots , one-dimensional (1D) fibers (ribbons)14, two-dimensional (2D) sheets15, and three-dimensional (3D) foams16. 3D graphene foam is a network of connected graphene sheets and, just as 2D graphene, can be widely used to improve the electrical, mechanical, or thermal properties of electrochemical devices. The size, quality, and type of graphene sheet synthesis determines the properties and potential application of the 3D materials17. The synthesis of a three-dimensional graphene structure is based on either the assembly of two-dimensional graphene flakes or on a direct synthesis. Many papers describe 3D structure synthesis based on chemical vapor deposition (CVD) of graphene on a nickel foam matrix21. A relatively new synthesis method is the 3D printing of graphene materials24. Well-known methods of synthesis produce three-dimensional graphene materials effectively, but they are too expensive and require specialized equipment. The assembly of graphene flakes26 by the hydrothermal method27 is also popular, although not without disadvantages; self-organization of graphene sheets is the most common problem that researchers want to eliminate28. The microwave method30 supports the synthesis of graphene, combining many advantages: it is fast, simple, does not require specialized equipment, and the starting material for foam production can be expanded graphite31 or sucrose32.Graphene possesses excellent mechanical and thermal properties34. Hence, to further improve their properties, increase electrode capacity, and power or energy density, many researchers try to dope graphene materials with metal compounds or metal oxides, or to create composites with other active materials38. These solutions involve increased production and disposal costs. The fact that these substances are not environmentally friendly leads researchers to try to introduce heteroatoms (mainly nitrogen) into the carbon structure using natural reagents40, thus increasing the range of potential applications of 3D graphene materials.Graphene foams, due to their excellent porous structure, are a light material that combines the properties of two-dimensional graphene sheets and the three-dimensional system they form when joined together. The large pore volume and subsequent high specific surface area of the material, its thermal stability, high electronic conductivity, and high ion transfer rate all contribute to graphene foams’ functionality in energy storage devicesChlorella vulgaris as an N-precursor. Different solvents  were applied in the microwave reactor study. After the microwave process, the samples were carbonized. The study investigated the influence that the microwave (duration time) and carbonization processes had on graphene structure and nitrogen content.The synthesis of any metal-free but electrochemically active electrode material is an important contribution to protecting the environment and the key motivation for engaging this research approach. To this end, we present a fast and facile method of producing N-doped graphene foam using a microwave process. In the proposed process, expanded graphite was used as a starting material and green algae The proposed method to obtain N-doped graphene foam using a pressure microwave reactor was confirmed to be effective using instrumental methods. Figure Chlorella vulgaris. Heteroatoms were successfully introduced into the graphene structure using the natural reagent. For samples synthesized in different solvents with N-reagent, the percentage by mass of nitrogen was high, at 2.44 wt% for the 1A-10 sample. Along with extending the reaction time for the 1A-T series produced in the ethyl alcohol solvent, a significant decrease of the nitrogen content, down to 0.65 wt%, was noted. In the case of the 1B-T series, where DMF was the solvent, an increase in the samples’ nitrogen content was observed when holding time was increased. The highest nitrogen content for the 1B-T series was recorded for a sample maintained for 90 min, at 0.89 wt%.A basic premise of this method of enriching the final material with nitrogen is the use of green algae. After carbonization, it can join graphene flakes  into a durable, porous 3D structure resembling a foam or sponge. Aside from providing amorphous carbon matter adhering graphene flakes, green algae is a source of nitrogen. It was selected due to its useful features, such as ease of mixing with graphene flakes in EtOH or DMF and thermal degradation to a non-porous nitrogen-rich matrix. A low carbon matrix yield from the starting green algae mass makes it possible to avoid soaking the graphene flakes and retains the graphene flakes’ surface accessibility to chemical reagents, e.g., electrolytes. The green algae-originated carbon phase is assumed to be present in the form N-rich bridges between graphene flakes which enable electron transfer throughout the whole mass of material. The effective exfoliation of materials obtained with a holding time of 10 min in the reactor led to the investigation of the effect that extending holding time had on the structure and elemental composition of graphene foams. The influence on the nitrogen content was examined in two different electrolytes, while the carbonization process was used to change the structure of graphene by creating exfoliated graphene foam with embedded nitrogen functional groups. The elemental analysis was used to determine the composition of the N-doped graphene foams. The influence of the elemental composition for materials in series 0A/B-T and 1A/B-T obtained in different solvents is shown in Table 41. On the Raman spectra for graphene, three characteristic bands can be observed, D, G, and 2D, for which the shift at the 532 nm laser line is 1347 cm−1, 1577 cm−1, and 2698 cm−142 respectively. Using Raman spectroscopy analysis, it is possible to estimate the number of graphene sheet layers that have been laid, as well as determine defects in the material obtained. Raman spectra were compiled for the N-doped graphene foams carried out in two selected solvents, EtOH , C–C (sp3), C–O–C, C=O, and O–C=O, respectively. The total content of carbon for all samples, determined by XPS measurement, was in the range from 90.2 to 93.2 at.% , C–C sp, C–O–C, 43. Modified graphene structures subjected to microwave heating facilitated nitrogen substitution in the graphite plane. The effect of high carbonization temperatures on doped structures was the formation of mainly quaternary-N functional groups, responsible for the materials’ effective catalytic activity in the oxygen reduction reaction44. Due to access to an electron pair in the graphene structure, the pyrolytic N-group is active in response to the oxygen reduction reaction, which leads to an increase in the catalytic activity of N-doped materials45. The overall content of nitrogen atoms for all samples in the 1A-T series was between 0.6 and 2.7 at.%. The XPS results show that nitrogen doping in graphene is successful; there were two characteristic types of nitrogen functional groups that improved catalytic properties in response to ORR results, the highest content belonging to the pyrrolic-N groups. Deconvolution of the high-resolution O1s spectrum fit into three bands at energy values of 531.6 eV, 533.2 eV, and 536.0 eV which correspond to C–O–C, O–C–O, and absorbed H2O bonds, respectively46.After deconvolution of the high-resolution spectrum of N1s, two characteristic types of bonds were determined for nitrogen in the form of pyrrolic-N and quaternary-N in binding energies of 399.1 eV and 400.8 eV, respectively48. In this case, graphene structures served as a matrix for the introduction of nitrogen, using the natural substrate Chlorella vurgalis as the source. As the literature reports, N-precursor is transformed into conductive carbon forms under the influence of high carbonization temperatures, 800–900 °C, which increase electrocatalytic properties50. The reference material in electrochemical tests was a carbon material based on platinum (20 wt%). All measurements were made in an oxygen-saturated 0.1 M KOH electrolyte for the materials from the 1A-T and 1B-T series, however the plots are presented for the 1A-T series only  at one rotation speed, 1600 rpm. Sample 1A_60 exhibits the highest current density among the samples obtained, but it is still lower than a commercial material based on platinum. This proves that, despite sample 1A-60 having the lowest nitrogen content at 0.65 wt%, the material shows good catalytic properties in ORR. The value of the onset potential for the 1A-60 sample, shown in Fig. 51. ORR performance parameters for the 1A-T series and commercial platinum-based carbon are presented in Table 52. The LSV results show that the resulting materials have good catalytic properties. This is proof that the electrocatalysts doped with heteroatomic nitrogen, and mainly nitrogen functional groups in the graphene structure, are responsible for catalyzing the ORR reaction. The products are completely free of any metals and can potentially achieve wide application in the field of electrochemistry. Remarkably, the 1A-60 and 1A-90 exhibited prominent ORR activity with the most positive onset potential (Eonset) at 0.91 V and 0.92 V. The half-way potential (E1/2) was also highest for 1A-60 and 1A-90 compared to other samples. The prominent ORR activities of 1A-30 and 1A-60 in an alkaline medium may be benefitting from the synergistic effect of the structure and elemental composition. First, the unique few-layer architecture of graphene is conducive to exposing more of the active sites and facilitates the accessibility of oxygen. Second, the nitrogen heteroatoms can induce charge redistribution and facilitate the adsorption of oxygen and the consequent reduction reaction on carbon. The proposed materials’ composition is their advantage, because generally structures described for ORR application tend to be decorated with expensive, precious, or environmentally-unfriendly metals or metal oxides, e.g., Pd, Ru53, perovskie oxides54, Fe(II)55, Fe7C356, or Au57.In order to check the catalytic activity of the obtained materials in the oxygen reduction reaction, cyclic voltammetry and linear voltammetry measurements were taken. The ORR reaction is distinctive in metal-air batteries and fuel cells, therefore examining the catalytic activity of the produced nitrogen-doped materials was an important aspect of the work. In recent years, heteroatom-doped graphene has become an alternative to platinum-based materials owing to the heteroatoms’ improvement of electrochemical propertiesnly Fig. . Figure Chlorella vurgalis. Research indicates that the use of a natural N-reagent during synthesis in a microwave reactor in various solvents can contribute to the incorporation of nitrogen atoms into the graphene structure at a level of several percent. It can be concluded that ethyl alcohol and dimethylformamide are good solvents which contribute to the exfoliation of graphene layers and the production of pure graphene material. The high quality of the obtained material, without defects, shows that the method is a promising way of producing heteroatom-doped graphene foams. The optimal time for synthesis in the microwave reactor was 30 min and introduced mainly pyrrolic-N groups descend from N-precursor Chlorella vurgalis. The best electrochemical results were obtained for materials using ethanol as a reaction medium. The solvent also influenced the introduction of nitrogen functional groups into the graphene structure. The products showed good catalytic activity owing to the nitrogen functional groups introduced into the graphene structure, which are responsible for the catalytic properties in the oxygen reduction reaction. The tests proved their high activity towards ORR, i.e., the number of electrons n reached 3.46 for tested materials (no Pt added), while the analogous value for the C-Pt (20 wt% loading) reference was only slightly higher and equal 4. For pristine, i.e., non-N-doped graphene, the n value was only 2. It has to be underlined that the progress in electrochemical performance was achieved in an environment-friendly way, not only without the use of noble metals, but also with a reduction in energy consumption. Heteroatom doping of graphene materials is an alternative to more expensive materials based on metals, an alternative that can have promising properties for electrochemical applications and a huge impact on the potential use the composite materials in supercapacitors, metal-air batteries, or fuel cells.In summary, an effective method of obtaining N-doped graphene in a microwave reactor was developed, one which uses expanded graphite and Chlorella vulgaris. The compounds were mixed in a solvent with a 1:4 mass ratio of graphene to Chlorella vulgaris, then sonicated to obtain a better dispersion. The samples were loaded into a microwave reactor (Microwave 400) and heated under pressure to 180 °C, with the power of about 300–600 W. The microwaving duration was either 10, 30, 60, or 90 min; after this time, the samples were stirred. The obtained mass was then washed with distilled water to remove the solvent and dried at 120 °C overnight. Finally, samples were carbonized at 900 °C with a heating rate of 3 °C min−1 and then heated for 4 h under the flow of nitrogen (to provide an inert environment during carbonization). Reference samples were made using the same procedure, but without the addition of an N-precursor nor the carbonization process. For all samples without the addition of Chlorella vulgaris, the process in the reactor was maintained for 10 min. The reference samples obtained without carbonization, only the microwave process, are designated as 0A/B-T, where A and B describe the solvent and denote EtOH or DMF, respectively, while T is the time in minutes that the sample was held in the microwave reactor. Samples obtained with the addition of the N-precursor and using carbonization are designated as 1A/B-T, where A and B describe the solvent and mark EtOH or DMF, respectively, and T is the duration of time spent in the microwave.The production of N-doped graphene foams was based on a one-step process using microwave radiation in two dispersed solvents: ethyl alcohol (EtOH), and dimethylformamide (DMF). The starting materials used were expanded commercial graphite  and the natural green algae N-precursor, The atomic structure of the obtained materials was analyzed using a high-resolution transmission electron microscope  at an accelerating voltage of 200 kV. A scanning electron microscope operating at 30 kV  was used to determine the structure of the samples. The elemental composition of the materials was analyzed by means of a combustion elemental analyzer . Raman-microscopic spectroscopy analysis was performed using Renishaw InVia , excitation wavelength 532 nm at an ambient temperature. Nitrogen sorption isotherms were determined through nitrogen physisorption at 77 K in a volumetric apparatus ASAP 2010 . The attachment, bonding configuration, and compositional analysis of the material was carried out by X-ray photoelectron spectroscopy measurements . A monochromatic Al-Kα X-ray (1486.6 eV) was used as the operating excitation source. Atomic Force Microscopy (AFM) investigations were conducted using a Scanning Probe Microscope (SPM) apparatus produced by Veeco , working under ambient conditions.−2. Before electrochemical testing, the alkaline solution was saturated with O2/N2 for 20 min. The activity of the samples in an oxygen reduction reaction was evaluated by measuring cyclic voltammetry (CV), carried out at a scanning speed of 10 mV s−1, and linear voltammetry (LSV), at a scanning speed of 5 mV s−1 and rotation speed of 800–2800 rpm, in a solution of 0.1 M KOH at room temperature. The number of electrons (n) directly participating in ORR was calculated using the Koutecky–Levich equation (K–L):L is the current density limiting diffusion, and JK is the kinetic current density; ω is the angular velocity of the electrode; n is the number of electron transfers in the reaction; F is the Faraday constant ; C0 is the concentration of dissolved oxygen (1.2 × 10–6 mol L−1 in 0.1 M KOH); D0 is the diffusion coefficient of dissolved oxygen (1.9 × 10–5 cm2 s−1 in 0.1 M KOH); ν is the kinetic viscosity of the electrolyte for 0.1 M KOH, 0.01 cm2 s−1. The slope of the K-L graph can be obtained using Koutecky–Levich Eqs. . The standard measuring setup was made up of three electrodes—an Ag/AgCl electrode in 3 M KCl was the reference, a Pt electrode the counter, while the working electrode was glassy carbon (GC rotating disk electrode with a 5.0 mm diameter) onto which ink, with the catalyst, was applied. The catalyst ink was prepared basing on the dispersion of 2.5 mg of the test sample in a mixture of distilled water and ethanol (ratio 1:4) and Nafion (0.5 wt% Nafion). As a point of comparison, electrochemical studies were also performed on a commercial catalyst from Sigma Aldrich, platinum on graphitized Pt/C carbon (20 wt% Platinum). The prepared ink was pipetted onto glassy carbon in an amount of 15.63 µl and the electrode was dried afterwards. Catalyst content was about 0.4 mg cm


Correction to: J Foot Ankle Res (2020) 13: 44https://doi.org/10.1186/s13047-020-00411-1Following publication of the original article  the authIncorrect values: 20.3 ± 5.1(4–15) 11.0 ± 2.6(16–43)Correct values: 20.3 ± 5.1(16–43) 11.0 ± 2.6(4–15)


Here, we examined the KCa3.1 channel expression and its role in rat bone marrow‐derived MSC (BMSC) proliferation in response to mechanical stretch. Application of mechanical stretch stimulated BMSC proliferation via promoting cell cycle progression. Such mechanical stimulation up‐regulated the KCa3.1 channel expression and pharmacological or genetic inhibition of the KCa3.1 channel strongly suppressed stretch‐induced increase in cell proliferation and cell cycle progression. These results support that the KCa3.1 channel plays an important role in transducing mechanical forces to MSC proliferation. Our finding provides new mechanistic insights into how mechanical stimuli regulate MSC proliferation and also a viable bioengineering approach to improve MSC proliferation.Mechanical stimulation is an important factor regulating mesenchymal stem cell (MSC) functions such as proliferation. The Ca Here, we showed that application of mechanical stretch to rat bone marrow‐derived MSCs (BMSCs) significantly increased cell proliferation, mainly via altering cell cycle progression. Such mechanical stretch also up‐regulated the KCa3.1 expression at mRNA, protein and functional levels. Importantly, mechanical stretch‐induced stimulation of BMSC proliferation and alteration in cell cycle progression were prevented by pharmacological inhibition of the KCa3.1 channel or siRNA‐mediated knockdown of the KCa3.1 expression. Taken together, our results provide compelling evidence to support an important role of the KCa3.1 channel in mechanical stimuli‐induced stimulation of MSC proliferation, thus revealing a new molecular mechanism in transducing mechanical forces to regulate MSC proliferation and identifying a viable bioengineering approach to improve MSC proliferation.Mesenchymal stem cells (MSCs) have many promising applications in regenerative medicine.22.1Ca3.1‐specific siRNA (siKCa3.1) or control siRNA (siCTL) was described previously,Isolation of BMSCs from 30‐day‐old male Sprague‐Dawley rats, BMSC characterization and culture were previously described.2.25 on silicone chambers pre‐coated with collagen I (Becton Dickinson). When cells reached 80% confluence, the silicone chambers were mounted to a stretch device  and were exposed to stretch by 2.5%, 5%, 10% and 15% for 6, 12 and 24 hours. Cells cultured without stretch were used as static control (SC). To block the KCa3.1 channel, 100 nmol/L TRAM‐34 (Alomone) was added into the culture medium.Cells were seeded at density of 1 × 102.3Total RNA extraction and RT‐PCR were described in the Supplementary File. The primers used were listed in Table 2.4Ca3.1 protein expression was determined using fluorescence‐activated cell sorting (FACS) as previously described.Ca3.1 (Alomone). Isotype control IgG was used as control. The fluorescence intensity was determined by FACSCalibur (Becton Dickinson) and analysed using CellQuest software.The cell surface K2.5Cell proliferation was examined by determining the number of living cells using CCK‐8 kits according to the manufacturer's instructions (Dojindo). The absorbance at 450 nm was measured using a microplate reader (Thermo Scientific).2.6Cells, after being synchronized for 24 hours in serum‐free medium, were subjected to mechanical stretch for further 24 hours in media containing 10% foetal bovine serum. The cell cycle distribution was determined by FACSCalibur (Becton Dickinson) and analysed by ModFit software as previously described.2.7+ currents was performed using a HEKA amplifier (Lambrecht) as described previouslyCa3.1 channel currents were derived from TRAM‐34‐sensitive current components.Whole‐cell patch‐clamp recording of the K2.8post hoc Fisher's test as indicated. P < .05 was considered statistically significant.Data are presented as mean ± standard deviation, where appropriate. Statistical analysis was conducted using Student's test or one‐way ANOVA and 3We started with examining the effects of mechanical stretch on BMSC proliferation. We chose mechanical stretch from 2.5% to 15%, the most commonly used range in study of mechanical stretch‐induced regulation of MSC functions.Ca3.1 channel is critically engaged in MSC proliferation.Ca3.1 channel expression. Exposure to mechanical stretch of 5%‐15%, but not 2.5%, for 24 hours increased the expression of KCa3.1 at the mRNA level shown by RT‐PCR (Figure + currents was enhanced by membrane stretch induced using hypotonic solution (Figure Ca3.1 channel expression and activity. We finally investigated whether the KCa3.1 channel plays a role in mechanical stretch‐induced stimulation of BMSC proliferation. Treatment with TRAM34, a KCa3.1 channel‐specific inhibitor, prevented mechanical stretch‐induced increase in cell proliferation, without effect on cell proliferation under normal control condition (Figure Ca3.1 expression (Figure Ca3.1 channel or genetic depletion of the KCa3.1 expression prohibited mechanical stretch‐induced arrest of cell cycle in the G0/G1 phase (Figure Ca3.1 channel in mediating mechanical stretch‐induced stimulation of BMSC proliferation.As introduced above, the Kn Figure  suppress4Ca3.1 expression in BMSC (Figure Ca3.1 channel activity (Figure Ca3.1 channel with TRAM‐34 (Figure Ca3.1 expression (Figure Ca3.1 channel inhibited mechanical stretch‐induced alteration in cell cycle (Figure Ca3.1 channel in cell proliferation.Ca3.1 channel in rat BMSC had no effect on cell proliferation (Figure 2+‐activated K+ channels, including KCa3.1, in BMSCs is strongly species‐dependent,2+ increase and cell proliferation in BMSCs.[Ca3.1 channel activity (Figure Ca3.1 channel plays a critical role in coupling mechanical stimulus‐induced Ca2+ signal to activation of the downstream signalling pathways to stimulate MSC proliferation. It has been also shown that the KCa3.1 channel can regulates extracellular Ca2+ entry and thereby MSC proliferation.Ca3.1 channel is critically engaged in the transduction of mechanical forces to intracellular Ca2+ signalling and cell proliferation in MSCs.We here show that exposure to mechanical stretch stimulates BMSC proliferation Figure A, in supC Figure A‐D and, y Figure E. More i4 Figure F or siRNn Figure G strongle Figure H‐K. Prevn Figure F. It is in BMSCs., 20, 21 Ca3.1 channel in mediating mechanical stretch‐induced stimulation of BMSC proliferation, thus revealing a new molecular mechanism in the transduction of mechanical forces to MSC proliferation. Such a finding should be helpful in developing new strategies to maintain and stimulate the capacity of MSC proliferation and increase the cell number required for MSC‐based applications in regenerative medicine.In summary, this study shows a critical role of the KThe authors confirm that there are no conflicts of interest.YF developed and organized this paper. XJ, HS, XC, YH and YZ carried out experimental work. XJ and HS mainly drafted the paper and created the figures. PJ, CG, XG and YH assisted in data analysis and interpretation. LH J critically reviewed the manuscript. This manuscript is not under review elsewhere, and all authors read and approved the final manuscript. Click here for additional data file.


Lipomyces, the most 5' terminal intron position is occupied by one of three complex intervening sequences consistent of differently nested U2 intron units, as demonstrated in L. lipofer, L. suomiensis, and L. starkeyi. In L. starkeyi, the donor elements of the constituent introns are abutting and the complex intervening sequence can be excised alternatively either with one standard splicing reaction or, as a [D] stwintron, by two consecutive reactions. Our work suggests how [D] stwintrons could emerge by the appearance of new functional splice sites within an extant intron. The stepwise stwintronisation mechanism may involve duplication of the functional intron donor element of the ancestor intron.In primary transcripts of eukaryotic nuclear genes, coding sequences are often interrupted by U2-type introns. Such intervening sequences can constitute complex introns excised by consecutive splicing reactions. The origin of spliceosomal introns is a vexing problem. Sequence variation existent across fungal taxa provides means to study their structure and evolution. In one class of complex introns called [D] stwintrons, an  U2 intron is nested within the 5'-donor element of another  U2 intron. In the gene for a reticulon-like protein in species of the ascomycete yeast genus  Few U12-type introns are excised by the minor spliceosome). Differently from spliceosomal introns, group I and group II introns are ribozymes, which may or not need accessory proteins for self splicing. In eukaryotes, group I and group II introns are primarily restricted to mitochondrial and chloroplast DNAs.In the eukaryote nuclear genome, coding sequences are often interspersed with intervening sequences (introns). Introns have to be precisely excised from the primary transcript to generate the open reading frame that translates into the appropriate peptide. Excision of U2-type introns and splicing of the bordering exons is catalysed by the major spliceosome6. Previously, we have described a particular class of nested U2 introns, the stwintron, for which the excision of the internal intron is required to accomplish the subsequent excision of the external intron because the former interrupts one of the three conserved intron splicing elements of the latter, the 5'-donor, the sequence element around the lariat branch point adenosine, or the 3'-acceptor11 . Correct stwintron excision requires intron definition whereby the nearest 5’- and 3'-splice sites are paired across the intron prior to splicing16. This results in excision of the smallest possible U2 intron; in the case of a stwintron, this is thus the internal intron. In fungi, spliceosomal introns are generally quite small – often <100 nucleotide (nt). Nevertheless, one of the fungal stwintrons we have described is crucial to an authentic alternative splicing event11.Intervening sequences of any of the three main classes – group I, group II and spliceosomal – can be made up of more than one functional intron unitPezizomycotina taxa8. One possible mechanism by which such a stwintron could emerge involves the stepwise formation of new 5'- and 3' splice sites within an extant intron of sufficient size to evolve into two U2 introns 18 could occur during repair of an asymmetric DNA double-strand break (DSB) which would leave a 3' overhang. In an independent event, mutations in the central region of the host intron could result in functional 3'-splice sites. The new internal splice sites may pair to define the new U2 unit, whose subsequent excision is necessary for the formation of a functional donor for the second U2 definition and standard splicing reaction to properly remove all intervening sequences.Two [D] stwintrons conserved across whole classes of filamentous fungi are located at exactly the same intron position as a canonical U2 intron present in early divergent 11, we identified a  stwintron in the gene encoding a reticulon-like protein in the Pezizomycotina, one of the three Ascomycota subphyla. The gene product is a “protein of unknown function which associates with the endoplasmic reticulum” (protein family PF02453). While assessing the ancestry of this  stwintron, we encountered different complex intervening sequences (CIS) consistent of nested U2 introns in the orthologous gene in seven species of Lipomyces, a taxon of the Saccharomycotina, another subphylum of the Ascomycota. The parallel evolution of these three different CIS at the very same intron position in species of the same fungal genus supports the stwintronisation mechanism of stwintron evolution.In a previous paperTaphrinomycotina and in the family of the Lipomycetaceae (Saccharomycotina) is consistent with their reticulon-like genes being genuine orthologues of the one in Aspergillus nidulans (rtnA) and in other Pezizomycotina11. Figure Lipomyces genus (nine species) drawn from an extensive maximum likelihood tree of the reticulon-like protein in all Ascomycota (see Methods section for details). Lipomycetaceae occupy a unique phylogenetic position at the basis of the Saccharomycotina clade19. The species Lipomyces starkeyi harbours many more ORF-interrupting introns than ascomycete yeasts outside the Lipomycetaceae20. The intron density of L. starkeyi resembles that seen in Pezizomycotina rather than that apparent in other Saccharomycotina.The conservation of intron positions in multiple species of Saccharomycotina, the primary transcript of the gene for the reticulon-like protein harbours multiple introns in eight genome-sequenced Lipomyces. However, the intron content varies considerably from the maximum of five introns in L. starkeyi and three closely related species, as well as in L. lipofer , or species of Taphrinomycotina (position 3), or species from both these other subphyla (positions 4 and 5). The observed simplification of the intron-exon structure of the gene encoding the reticulon-like protein suggests that some early divergent Lipomyces species have lost introns rather than that new introns have been gained in the other taxa, completely independent of the massive intron loss observed in all other families of Saccharomycotina21.Exceptionally within the fer Fig. . L. japoLipomyces species. The second exon is either 162 or 165 nt (L. japonicus & L. lipofer) long. The extra codon is located upstream of the codon of the absolutely conserved Trp of the PF02453 domain  is disrupting the 5'-donor element of an 162-nt long external intron (5' GUGA | GU – 137-nt – ACUAAC – 10-nt – UAG) between A4 and G5. The splicing scheme is shown in Fig. L. lipofer stwintron is the first donor-disrupted stwintron of the  type as well as the first ever stwintron (sensu stricto) described outside the Pezizomycotina subphylum.In L. suomiensis and L. japonicus, we detected two potential canonical 5' donor sequences separated by one nt at the 5' extremity of the predicted CIS. Furthermore, a fully canonical 3'-acceptor and associated lariat branch point element could be identified within the CIS in both species. Sequence analysis of the mature messenger in L. suomiensis . Similarly, in L. japonicus, we predict that an 162-nt long canonical U2 intron (5' GUAAGU – 135-nt – ACUAAC – 12-nt – UAG) is disrupting an 124-nt long canonical U2 intron (5' GUAAGUG | A – 97-nt – AUUAAU – 10-nt – UAG) between G7 and A8. The whole CIS is 286 nt long and the two functional donor elements are identical in sequence (5'-GUAAGU) . A difference between the two species is that the constituting U2 introns in L. suomiensis are half the size of those in L. japonicus.In two species, sensu stricto as previously defined7, since the internal intron does not interrupt the donor element of the external intron; the two canonical donor elements are actually one nt apart from each other. In the primary transcript of the gene for the reticulon-like protein in L. suomiensis, the primary selected 5'- and 3'-splice sites are clearly those that define the smallest possible internal intron (intron definition). We can therefore consider such CIS of nested introns examples of a new class of fungal stwintron, the “stwintron sensu lato”.To excise these  CIS, two standard splicing reactions are necessary. This intron nesting does not constitute a [D] stwintron in L. starkeyi and the three closely allied species  whether the intervening sequence could be alternatively excised using either the one or the other donor element. Amongst the cDNA clones of matured mRNA generated, we indeed found two different ORFs, one of them containing six nt (GTACGT) more than the other (GenBank MN689086 & MN689087), demonstrating alternative splicing. Under our experimental conditions, fewer cDNA clones contained the GTACGT sequence. Subsequent inspection of the intervening sequence suggested the presence of a coupled lariat branch point element and 3'-acceptor roughly in its centre, of which the lariat branch point element could be the imperfect 5'-AUUCAU or the equally imperfect 5'-UUUAAU, associated with a 5'- CAG shortly downstream. If the internal splice signals were recognised by the spliceosome, one would expect a complex  intervening sequence of 252 nt, where an 120-nt long internal U2 intron (5' GUAAGU – 75-nt – AUUCAU – 14-nt – UUUAAU – 10-nt – CAG) is disrupting an 132-nt long external intron (5' GUACGU | A – 109-nt – GCUGAC – 7-nt – UAG) between U6 and A7 . However, if the spliceosome would ignore the imperfect internal 3' splice site halfway the intervening sequence but not the fully canonical proximal donor, one would expect that a 246-nt long canonical U2 intron (5' GUAAGU – 224-nt – GCUGAC – 7-nt – UAG) is excised in one standard splicing reaction , which is poorly conserved both in length and in sequence in ascomycete reticulon-like proteins.In the genes for the reticulon-like protein of genome-sequenced species of the 22. Some modes of autonomous intron gain could involve repair of double-strand breaks (DSB) by an end-joining mechanism26. The formation of a new stwintron from the sequences of an existing canonical intron can occur similar to the intronisation mechanism17, by the formation of new 5'- and 3'-splice sites within the parental intron at the chromosomal locus  could represent an intermediate stage in which the CIS can be removed by one- as well as by two subsequent U2 splicing reactions, as the internal imperfect 3' lariat branch point element is apparently functionally weak.The origin of spliceosomal introns is a vexing problem. Some commonly purported mechanisms involve mobile elements like transposons or group II introns of mitochondrial or bacterial origin, while other proposed mechanisms  are autonomous and have a strictly endogenous originpomyces D,5-,  stwintron in the gene for the fungal reticulon-like protein, we searched the DNA databases at the National Center of Biotechnology Information (NCBI) for homologous genes in other fungi. Additional homologues were found in the Saccharomycotina and Taphrinomycotina subphyla of the Ascomycota, the Blastocladiomycota, the Glomeromycota, the Mucoromycotina and the Mortierellomycotina , to verify the status of the Lipomycetaceae as the earliest divergent family of Saccharomycotina20. Most of these analyses yielded monophyletic clades for the nine sequenced Lipomyces: A representative maximum likelihood subtree is shown in Fig. Lipomyces genome sequence resources are: L. starkeyi (GenBank Whole Genome Shotgun Master accession: LSGR00000000.120), L. mesembrius (PPJS00000000.219), L. arxii (PPJT00000000.219), L. kononenkoae (PPJW00000000.119), L. lipofer (PPJX00000000.119), L. doorenjongii (PPJU00000000.119), L. suomiensis (PPJQ00000000.219), L. oligophaga (PPJR00000000.219) and L. japonicus (PPJV00000000.119).Species of Lipomyces clade shown in Fig. 28, applying the L-INS-i iterative refinement method trained on recognising one conserved domain with long extensions – appropriate for the PF02453 domain – with the BLOSUM80 scoring matrix. This MAFFT alignment was trimmed to 136 informative residues with Block Mapping and Gathering with Entropy software (BMGE version 1.12)29, using the BLOSUM40 similarity matrix with the block size set at 4. A maximum likelihood tree was then calculated from the trimmed alignment by PhyML (version 3.0) with automatic substitution model selection30 in default mode. The Smart Model Selection (SMS) software selected LG + G + I + F as the substitution model31 using the Akaike Information Criterion. Branch support was estimated by approximate likelihood-ratio tests32 implemented by the PhyML webserver. The inferred tree was drawn from the PhyML Newick output with FigTree (version 1.4.3: http://tree.bio.ed.ac.uk/software/figtree). The Lipomyces subtree was selected in FigTree (select clade option) and saved in a separate file, then exported as an interactive pdf. Final annotation was done in Adobe Illustrator.The phylogenetic tree at the basis of the monophyletic Lipomyces lipofer CBS 944 , Lipomyces suomiensis CBS 7251 , and Lipomyces starkeyi CCY 33-1-1  were used to verify the existence of a particular CIS in the primary transcript of the orthologous genes encoding a reticulon-like protein. Lipomyces kononenkoae CBS 2514 was also assessed in support of results obtained for L. starkeyi. All yeasts were maintained and cultivated on rich growth media listed in Supplementary Table –1 (rpm) for 16 h. The biomass was harvested by centrifugation at 10,000 rpm for 5 min then transferred to tubes with ceramic beads  and homogenised at 4000 rpm for 30 s with the help of a MagNA Lyser Instrument (Roche). Macherey-Nagel NucleoSpin kits were used for the extraction of genomic DNA (NucleoSpin Plant II) and total RNA (NucleoSpin RNA Plant) from the homogenate. Nucleic acids were quantified using NanoDrop technology (Thermo Scientific).For isolation of nucleic acids, yeast cultures were grown in 500-mL Erlenmeyer flasks with 100 mL medium in an orbital shaker (Infors HT Multitron) at 200 revolutions minReverse transcription was primed off 1 μg of total RNA with Oligo(dT) as the primer using the First Strand cDNA Synthesis Kit (Thermo Scientific). PCR reactions were performed in a 25 μL volume containing 4 μL of single strand cDNA, using gene-specific oligonucleotides  and cloned . Plasmid DNA was isolated using the NucleoSpin Plasmid EasyPure kit (Macherey-Nagel). Independent clones were sequenced over both strands using universal primers hybridising to the vector . All sequences determined were deposited at GenBank. For convenience, determined sequences of RNA stwintron splicing intermediates are also given in the Supplementary Information.Accession numbers for sequences determined during this study: MN689081–MN689090.Supplementary Information.


While control cells displayed robust puromycilation, TDP-43-ΔNLS transfected cells exhibited reduced ongoing protein synthesis. Next, by using a transgenic mouse overexpressing cytoplasmic TDP-43 in the forebrain (TDP-43-ΔNLS mice) we assessed whether cytoplasmic TDP-43 regulates global translation in vivo. Polysome profiling of brain cortices from transgenic mice showed a shift toward non-polysomal fractions as compared to wild-type littermates, indicating a decrease in global translation. Lastly, cellular level translational assessment by SUNSET was performed in TDP-43-ΔNLS mice brain slices. Control mice slices incubated with PURO exhibited robust cytoplasmic PURO signal in layer 5 neurons from motor cortex, and normal nuclear TDP-43 staining. Neurons in TDP-43-ΔNLS mice slices incubated with PURO exhibited high cytoplasmic expression of TDP-43 and reduced puromycilation respect to control mice. These in vitro and in vivo results indicate that cytoplasmic TDP-43 decreases global translation and potentially cause functional/cytotoxic effects as observed in ALS/FTD. Our study provide in vivo evidence (by two independent and complementary methods) for a role of mislocalized TDP-43 in the regulation of global mRNA translation, with implications for TDP-43 proteinopathies.TDP-43 is a major component of cytoplasmic inclusions observed in neurodegenerative diseases like frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). To further understand the role of TDP-43 in mRNA/protein metabolism and proteostasis, we used a combined approach with cellular and animal models overexpressing a cytoplasmic form of human TDP-43 (TDP-43-ΔNLS), recapitulating ALS/FTD features. We applied in HEK293 cells a method for labeling  TAR DNA-binding protein 43 (TDP-43) is a ubiquitously expressed, predominantly nuclear protein with multiple roles in RNA processing  recapitulates multiple features of TDP-43 proteinopathies of the ALS/FTD spectrum, including progressive neurodegeneration, gliosis, global gene expression changes and behavioral abnormalities encompassing the motor, cognitive and social domains  are virtually unknown. In this work, we investigated both in vitro and in vivo if cytoplasmic expression of TDP-43 modifies global protein synthesis, using two different approaches, polysome profiling and surface sensing of translation (SUnSET) technique.While most studies have focused on the regulation of global or specific translation using cell lines or primary neurons in culture, the consequences of 2. Cells (5 × 105 per well) were plated on poly-D-lysine-coated 12 mm glass coverslips in a 6-well plate, and grown for 24 h before transfection. Lipofectamine 2000 reagent (Invitrogen) was used for transfection of constructs (empty vector as control or hTDP-43-ΔNLS), according to the manufacturer's instructions. After 48 h, medium was replaced with fresh medium with or without puromycin  for 20 min and washed with PBS.Myc epitope-tagged hTDP-43-ΔNLS construct was generated in pcDNA 5/To plasmid (Invitrogen) using restriction sites BamHI and XbaI, as previously described  for 10 min, permeabilized with 0.2% Triton X-100 (Sigma) in PBS for 10 min, blocked with 5% powdered milk in PBS for 2 h, and incubated overnight with primary antibody at 4°C. Primary antibodies used in this study (diluted in 5% powdered milk in PBS) were as follows: anti-total-TDP-43  at a ratio of 500 μl buffer /10 mg tissue. Homogenates were then placed on ice for 10 min and centrifuged at 5,000 rpm for 10 min at 4°C. Supernatant, consisting in cytosolic extract containing polysomes, was collected and protein concentration was determined by bicinchoninic acid (BCA) method (Pierce). Sucrose gradient, ranging from 15 to 50%, were prepared in gradient buffer . Briefly, 2.4 ml 50% sucrose solution was added to a tube and chilled for 30 min at −80°C. The procedure was repeated with layers of sucrose solution at lower concentration  and stored at 4°C if used the next day. 0.5 to 1 mg of cytosolic protein extract were added to each gradient, and centrifuged in a SW40 rotor at 35,000 rpm for 2–4 h, at 4°C. Five hundred microliters aliquots were separated and absorbance at 260 nm was measured . A peak area ratio between polysomal (P) and non-polysomal  fractions was determined for each group.Cortices of control (non-transgenic) or hTDP-43-ΔNLS animals were homogenized in cold lysis buffer [15 mM Tris-HCl, pH 8.0, 300 mM NaCl, 15 mM MgClFive hundred microliters of sample from each fraction was subjected to methanol/chloroform protein extraction method and resuspended in sample buffer. Equal amounts of samples were resolved on 10% sodium dodecyl Sulfate polyacrilamyde gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes . After transfer, membranes were stained with Ponceau S for total protein load assessment, then blocked in 5% powdered milk in 0.1% Tween-TBS, and incubated in anti-total TDP-43 primary antibody overnight . Detection of primary antibody was performed with horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch), and ECL was used for blot development. Digital images were acquired using a G-BOX Imager (Syngene). Ponceau S staining was used as a control for equal protein loading in our immunoblot analysis of polysomal fractions, since typical housekeeping genes are barely detectable in polysome fractions . The brain was next glued onto a vibratome stage and acute sagittal slices (400 μm) were cut. Then, the slices were recovered at room temperature for an hour in ACSF . After recovery, slices were placed on a fine grid mesh in a petri dish and incubated in ACSF (C- group) or ACSF supplemented with 5 μg/μl puromycin  at a final volume of 30 ml. Incubation time was 45 min under continuous aeration with carbogen (95% O2/ 5% CO2). Slices were then rinsed in ACSF and fixated in 4% paraformaldehyde (Sigma-Aldrich Inc.) in 0.1 M PBS for 24 h at 4°C. Next day, slices were washed three times in 0.01 M PBS for 10 min each under continuous agitation in an orbital shaker, incubated with 10% sucrose in 0.01 M PBS for 2 h at 4°C and stored in 30% sucrose-0.01 M PBS at −20°C until use.SUnSET labeling in brain slices was adapted from Cook et al. , with soBrain slices containing the motor cortex [approximately bregma 1.18 mm, . In non-transgenic  mice incubated with puromycin (C+), each cellular area was defined in the green channel corresponding to puromycin signal, as well as background areas between cells; the same approach was employed in hTDP-43-ΔNLS animals incubated with puromycin (ΔNLS+) but in the red channel . For non-transgenic mice incubated in ACSF (C-), a circular area centered in each red nucleus and background areas between cells were quantified. Background signal was normalized to individual cell areas, and a ratio between raw cell intensity signal and normalized background signal was calculated. The signal from individual cell populations and averaged signal per animal were compared between experimental groups.t-test when comparing only two groups on one measure. A one-way ANOVA followed by Tukey's multiple-comparison post hoc test was used when comparing three groups. Prism Version 8 (GraphPad Software) was used for statistical analysis and differences were considered to be statistically significant when the probability value was < 0.05.Data are expressed as means ± SEM. Statistical analysis was performed using a standard unpaired two-tailed student's +) show a decrease in ongoing protein synthesis  in ALS/FTD pathogenesis.Protein synthesis is an essential process for all cellular types, but neurons are particularly sensitive to proper control of this metabolic process due to their high polarization and the demands of plasticity mechanisms  as demonstrated by immunoprecipitation, and that TDP-43-ΔNLS co-localizes in the cytoplasm with RACK1 and rpS6, suggesting that TDP-43 associates to translational machinery  leading to translational modulation events such as eIF2α phosphorylation .in vitro and in vivo SUnSET studies, image processing, and data analysis. MB provided key reagents, funding, and helped design and analyze the in vitro experiments. SC and LI wrote the manuscript. LI conceived and supervised all aspects of the project and obtained study funding. All authors read and approved the final manuscript.SC, LL, and LI designed the study. SC and AV performed the cell culture experiments. LL performed the polysome profiling and immunoblot experiments. SC performed 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.


TBX5), which is an important regulator in heart development. Knock‐down of lncRNA TBX5‐AS1:2 via promoter hypermethylation reduced TBX5 expression at both the mRNA and protein levels by affecting its mRNA stability through RNA‐RNA interaction. Moreover, lncRNA TBX5‐AS1:2 knock‐down inhibited the proliferation of HEK293T cells. In conclusion, these results indicated that lncRNA TBX5‐AS1:2 may be involved in TOF by affecting cell proliferation by targeting TBX5.Tetralogy of Fallot (TOF) is the most common complex congenital heart disease (CHD) with uncertain cause. Although long non‐coding RNAs (lncRNAs) have been implicated in heart development and several CHDs, their role in TOF is not well understood. This study aimed to investigate how dysregulated lncRNAs contribute to TOF. Using Gene Expression Omnibus data mining, bioinformatics analysis and clinical heart tissue sample detecting, we identified a novel antisense lncRNA TBX5‐AS1:2 with unknown function that was significantly down‐regulated in injured cardiac tissues from TOF patients. LncRNA TBX5‐AS1:2 was mainly located in the nucleus of the human embryonic kidney 293 (HEK293T) cells and formed an RNA‐RNA double‐stranded structure in the overlapping region with its sense mRNA T‐box transcription factor 5 ( We further demonstrated that lncRNA TBX5‐AS1:2 expression was markedly decreased in injured heart tissue from patients with TOF. In vitro, lncRNA TBX5‐AS1:2 down‐regulation, mediated by DNA hypermethylation in the promoter region, significantly suppressed cell proliferation by reducing the expression of TBX5 at both the mRNA and protein levels, the reason of which was that lncRNA TBX5‐AS1:2 affected the stability of TBX5 mRNA through the formation of an RNA‐RNA duplex.In the current study, we characterized the lncRNA and mRNA profiles in human foetal and adult heart tissues by Gene Expression Omnibus (GEO) data mining and bioinformatics analysis, and focused on a previously unreported antisense lncRNA TBX5‐AS1:2 with unknown function. LncRNA TBX5‐AS1:2 was significantly up‐regulated in foetal heart and was predicted to adjust the expression of its sense gene 22.1https://www.ncbi.nlm.nih.gov/geo/) using the keywords lncRNA, human, heart development or CHD. Differentially expressed lncRNAs were analysed using the DESeq package, and WikiPathways database was applied to screen mRNAs related to heart development or CHD with differential expression. A coding and non‐coding co‐expression (CNC) network was established followed by these procedures: (a) data pre‐processing: for same gene, median value of different transcripts for same genes represents gene expression value; (b) data screening: assessing differential expression of lncRNA and mRNA; (c) calculation and removal of subset of data based on Pearson's correlation coefficient (PCC) and calculation of correlation coefficient of PCC between lncRNA coding genes using R values; (d) screening with a standard of PCC ≥0.9 or ≤−0.9 and P ≤ .05 as meaningful subset and constructing CNC network using Cytoscape. Meanwhile, mRNAs adjacent to lncRNAs in the CNC network (≤10 kbp on the genome) were annotated. Target lncRNA was selected based on the following requirements: (a) in the CNC network; (b) adjacent mRNA was one of the differentially expressed in relation to heart development or CHD; and (c) adjacent mRNA co‐expressed with the lncRNA.Online data mining was performed in the GEO database (2.2® RNA Stabilization Solution (Thermo Fisher Scientific) after surgery or autopsy and were stored at −80°C.This study was reviewed and approved by the Institutional Research Ethics Committee of the Children's Hospital of Fudan University (2016‐56). Cardiac tissue samples were obtained from 53 patients with TOF from the Biobank of the Children's Hospital of Fudan University, Shanghai, China. The diagnosis of TOF was based on echocardiography carried out at our hospital. None of the patients included in the study had been diagnosed with extracardiac anomalies or had any common chromosomal anomalies, such as 22q11 microdeletion. Cardiac tissues were removed from the blocked right ventricular outflow tract during surgery. Thirteen normal cardiac tissues samples were acquired from the Department of Forensic Medicine, Fudan University, Shanghai, China. They died as a result of traffic accidents and had no abnormal cardiac structures at autopsy. All tissue samples for RNA extraction were maintained in RNAlater2.3GAPDH) and analysed according to the relative quantification method (2−ΔΔCt). The 2−ΔΔCt was used to calculate the relative expression of RNA. Three repeated measurements were carried out for each sample.Total RNA was extracted from cardiac tissues and cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The quantity and quality of the extracted RNA were assayed using a NanoDrop ND‐2000 spectrophotometer (Thermo Fisher Scientific) and agarose gel electrophoresis. A total of 1000 ng of RNA from each sample was used to synthesize cDNA using a Prime Script RT Reagent Kit (Takara), followed by quantitative polymerase chain reaction (qPCR) with SYBR Premix Ex Taq™ (Takara) on a StepOnePlus™ Real‐Time PCR System (Thermo Fisher Scientific). Gene expression was normalized to the housekeeping gene glyceraldehyde 3‐phosphate dehydrogenase  cells were cultured in a mixture of Dulbecco's modified Eagle's medium , 10% foetal bovine serum  and 1% penicillin‐streptomycin  at 37°C in 5% CO2.5https://www.ncbi.nlm.nih.gov/). Empty vector (pcDNA 3.1) was used as a negative control (NC). When the cells plated in 6‐cm dishes reached about 75% confluence, they were transfected with plasmids using Lipofectamine 3000 (Invitrogen). The cells were harvested 48 hours after transfection.Human lncRNA TBX5‐AS1:2‐pcDNA 3.1 and human TBX5‐pcDNA 3.1 were obtained from GeneRay. The plasmids were sequenced and shown to be consistent with the sequence in the National Center for Biotechnology Information database (2.6TBX5) were designed and cloned into the lentiviral vector PGMLV‐SC5‐GFP by Genomeditech. The lentivirus was amplified in HEK293T cells and concentrated using polyethylene glycol (System Biosciences). The interference efficiency was detected by qPCR. HEK293T cells infected by the most interference‐efficient lentivirus were used to establish stable cell lines by selection with puromycin (Sigma‐Aldrich).Three short hairpin RNAs (shRNAs) targeting lncRNA TBX5‐AS1:2  was added to each well followed by incubation for 3 hours at 37°C. Cell viability was used to represent for proliferation and evaluated by the absorbance at 450 nm. All samples were prepared in triplicate and normalized to blank controls. Three time‐points were set at 40, 56 and 72 hours after transfection or incubation. The experiments were repeated three times.Cell proliferation was determined using a Cell Counting Kit‐8  according to the manufacturer's protocol. A total of 1 × 102.86 HEK293T cells and staining using an Annexin V‐fluorescein isothiocyanate (FITC)/propidium iodide (PI) Kit (Dojindo). Cells were stained successively with 5 µL FITC reagent and 5 µL PI reagent, and incubated in the dark for 15 minutes at room temperature each time. Flow cytometry analysis was performed using a FACSCalibur (BD Biosciences) and apoptosis was analysed using FlowJo software.Cell apoptosis was detected by harvesting 1 × 102.9Nuclear and cytoplasmic fractions were isolated from HEK293T cells using a PARIS kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Actin was used as a cytoplasmic control, and U1 was used as a nuclear control. RNA levels of lncRNA TBX5‐AS1:2, Actin and U1 in the cytoplasm and nuclear components were measured by qPCR after RNA extraction and reverse transcription.2.10Cells were grown in a 4‐chamber slides for 24 hours, fixed with 4% paraformaldehyde for 20 minutes and dehydrated in an ascending series of ethanol solutions. Cells were hybridized overnight at 42°C with probe. Non‐specific probe was removed using 0.5× saline sodium citrate containing 50% formamide at 37°C. Biotin‐labelled lncRNA TBX5‐AS1:2 was detected using anti‐biotin monoclonal antibody and secondary antibody. Finally, the slides were stained with DAPI  and subjected to fluorescence signal detection under Leica TCS SP8 laser confocal microscope (Leica).The probe of lncRNA TBX5‐AS1:2 used was as follow:CY3‐GGUUUCGAUUAAGAUACACCAUAGGCUCUACACGAUCGGC.2.11Human embryonic kidney 293 cells were collected and lysed with RIPA buffer (Yeasen) containing a protease inhibitor cocktail (Sigma‐Aldrich). Proteins were determined using a BCA Protein Assay Kit (Pierce) and equivalent amounts of proteins were separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes . The membranes were blocked with 5% milk in Tris‐buffered saline Tween for 1 hour at room temperature and then incubated with specific primary antibodies at 4°C overnight, followed by secondary antibodies at room temperature for 1 hour. The primary specific antibodies used were anti‐TBX5  and anti‐Actin , and the secondary horseradish peroxidase‐conjugated antibody was antimouse IgG . The proteins were then visualized on X‐ray film using Clinx ChemiScope (Clinx Science Instruments).2.12TBX5 employing two sets of distinct primers designed to target the overlapping region of the TBX5 sense and antisense transcripts, and the non‐overlapping region of TBX5, respectively. The primer sequences were as follows:Each RNA sample from HEK293T cells was incubated for 1 hour at 37°C and then treated with RNAse A+T cocktail (Ambion) to digest single‐stranded but not duplex RNAs. After incubation for 30 minutes at 37°C, samples were dealt with proteinase K (Yeasen). A NC without RNAse treatment was operated in the same way. After ribonuclease protection assay (RPA), reverse transcription (RT)‐PCR and gel electrophoresis were used to detect TBX5‐overlapping region‐F: CCCTCATTCCTCCGGAGAAAG,TBX5‐overlapping region‐R: GCCGGGTCTGCGCAGCCACAG;TBX5‐non‐overlapping region‐F: ACATCGTGAAAGCGGATGAA,TBX5‐non‐overlapping region‐R: GTGATCTTGTGGTTCTGGTAGG.2.132, 0.5% NP‐40, RNase Inhibitor 160 U/mL cocktail). The supernatant was mixed with biotinylated lncRNAs and antisense transcripts for 2 hours at 4°C, followed by the addition of Dynabeads MyOne Streptavidin T1 (Invitrogen) and incubation overnight at 4°C. After washing beads four times, RNA was extracted using TRIzol reagent. The lncRNA‐associated RNAs were subjected to cDNA synthesis and qPCR using the following primers:Full‐length biotinylated lncRNAs and antisense transcripts were transcribed using an AmpliScribe™ T7‐Flash™ Biotin‐RNA Transcription Kit (Epicentre Technologies). After purification, HEK293T cells were lysed for 40 minutes with RNA pull‐down buffer  for 4, 6, 8 and 10 hours, respectively. Total RNA was extracted after treatment at each time‐point and subjected to qPCR for TBX5 mRNA quantification.RNA stability was measured by plating HEK293T cells at 2.5 × 102.15https://www.ncbi.nlm.nih.gov/nuccore/NC_000012.12?from=114408195&to=114412832&report=genbank), and CpG islands were predicted using MethPrimer (http://www.urogene.org/cgibin/methprimer/methprimer.cgi). The DNA methylation statuses of the selected CpG islands were analysed by bisulphite sequencing PCR (BSP). Genomic DNA extracted from six TOF and five normal cardiac tissues was subjected to bisulphite conversion using an EpiTect Fast DNA Bisulfite Kit (Qiagen) according to the manufacturer's instructions. LncRNA TBX5‐AS1:2 CpG islands from bisulphite‐modified DNA were then amplified by EpiTaq HS DNA polymerase (Takara), and the PCR products were purified and cloned into the T/A cloning vector pGEM T‐Easy (Promega). Ten positive clones were isolated and sequenced. Methylation analysis was performed using BiQ Analyzer 2.0 and QUMA (http://quma.cdb.riken.jp/). The primers used were as follows: lncRNA TBX5‐AS1:2‐I2‐F1: TTTTAGTAAAATAAAGAGGTAATTAGG,The DNA sequence of the lncRNA TBX5‐AS1:2 regulatory region was obtained from the GenBank database . The recombinant plasmid was treated with CpG methyltransferase (M.SssI) (New England Biolabs) for 2 hours at 37°C and then purified using an AxyPrep DNA Gel Extraction Kit (Axygen) to generate a patch‐methylated construct. Whether the plasmids were methylated was detected by methylation‐sensitive restriction enzymes (MSREs)  (New England Biolabs) digestion and DNA gel electrophoresis assay. Methylation efficiency was evaluated by BSP and the PCR products were used for direct pyrosequencing. The primers used were as follows: lncRNA TBX5‐AS1:2‐I2‐F2‐1: TGTGAATYGATAGTATTAATATAYGTTT,lncRNA TBX5‐AS1:2‐I2‐R2‐1‐tail:aaccttcaacaccccaaccatataTAATTTATATCTTTATTTATTCCCRAAACC;lncRNA TBX5‐AS1:2‐I2‐F2‐2: TTAGTGTAAGTGTAGGTGTTAGAATATT,lncRNA TBX5‐AS1:2‐I2‐R2‐2‐biotin: aaccttcaacaccccaaccatata..2.174 cells per well were seeded in 96‐well plates and incubated at 37°C overnight. The respective methylated and unmethylated reporter plasmids were co‐transfected with pGL3‐Renilla vector into HEK293T cells using Lipofectamine 3000. After transfection for 48 hours, the HEK293T cells were treated with a Dual‐Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. Both firefly and Renilla luciferase activities were measured using an EnSpire plate reader (PerkinElmer). All samples were prepared in triplicate. The firefly luciferase activity normalized to the Renilla luciferase activity represented the transcriptional activity of the lncRNA TBX5‐AS1:2.Human embryonic kidney 293 cells at 1 × 102.18t tests with GraphPad Software. Data were shown as mean ± standard error (SEM), and a P value < .05 was considered statistically significant.All experiments were repeated three times. All statistical analyses were performed using paired two‐tailed Student's 33.1https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE68279) was filtered out, which was from an article related to the lncRNA profile in human foetal and adult hearts.TBX5   in HEK293T cells with down‐regulated and up‐regulated lncRNA TBX5‐AS1:2. TBX5 mRNA and protein levels were both significantly down‐regulated after lncRNA TBX5‐AS1:2 knock‐down   Figure , but the) Figure .3.9Hypermethylation of CpG islands can inhibit the transcriptional activity of lncRNAs. We therefore transfected methylated and unmethylated lncRNA TBX5‐AS1:2 reporter plasmids containing CpG island 2 into HEK293T cells. The unmethylated construct was digested by MRSEs whereas the methylated construct was not Figure C. BSP re4CHD is resulted from abnormal cardiogenesis during early embryonic development. Cardiogenesis involves the orderly proliferation, apoptosis, migration and differentiation of a variety of cells, requiring tight and precise regulation by appropriate spatiotemporal gene expression.TBX5. Antisense lncRNAs account for approximately 50%‐70% of all lncRNAs and are transcribed from the opposite strand of their endogenous sense counterparts.TBX5 were down‐regulated in injured heart tissue from TOF patients. We therefore performed in vitro experiments to clarify the molecular mechanism underlying this clinical phenomenon. Given that no human myocardial cell lines are available and lncRNA TBX5‐AS1:2 has no homologous sequence in mice, we used the tool cell line HEK293T, as in previous studies of CHD or human heart development.TBX5 mRNA and protein abundances in vitro. LncRNA TBX5‐AS1:2 and TBX5 formed an RNA duplex by RNA‐RNA interaction, thus preventing TBX5 mRNA degradation and increasing the mRNA stability of TBX5. In addition, lncRNA TBX5‐AS1:2 knock‐down significantly inhibited HEK293T cell proliferation, which could be rescued by overexpression of TBX5. These results showed that the influence of lncRNA TBX5‐AS1:2 on cell proliferation may be mediated by its interaction with TBX5. TBX5 is a member of the T‐box transcription factor family primarily known for its role in cardiac development.TBX5 can increase the risk of CHD, including TOF.TBX5.TBX5 plays a critical regulatory role in cell proliferation during cardiogenesis.Among the differentially expressed lncRNAs, we focused on the novel antisense lncRNA TBX5‐AS1:2, which showed a relatively high fold change in foetal compared with adult heart, and predicted to target its cognate coding mRNA TBX5 mRNA and protein expression via an RNA‐RNA interaction regulatory mechanism to inhibit cell proliferation in TOF. However, further studies are needed to determine the detailed mechanisms by which lncRNA TBX5‐AS1:2 modulates cardiac development and its function. Moreover, if there are ideal human cardiomyocyte lines or cardiac stem cells and the parallel data can be obtained from in vitro experiments with these cell lines, the conclusions will be more credible. In summary, this study identified the novel lncRNA TBX5‐AS1:2 as being down‐regulated in human TOF heart tissue and further explored its part of function in cardiac development by in vitro experiments.We therefore suggest that the previously unknown non‐coding antisense transcript lncRNA TBX5‐AS1:2 may be involved in TOF. Hypermethylation could mediate the down‐regulation of lncRNA TBX5‐AS1:2, leading to decreased The authors declare no conflict of interest.JM, DM and GH were responsible for the idea, project design and concept of the paper. JM performed bioinformatics analysis. WS, WC and XM collected the clinical samples and information. SC, JM, LH and BZ performed the clinical sample detection and in vitro experiments. JM, SC, DM and GH wrote, edited and revised the manuscript. All authors read and approved the manuscript.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.


Hypercalcemia is a common clinical laboratory abnormality with a majority of cases attributed to malignancy or hyperparathyroidism. Although hypercalcemia is a common manifestation of sarcoidosis, it is rarely the initial presentation. Here we present a case of acute hypercalcemia in a 60-year-old gentleman, which was diagnosed as sarcoidosis following an elaborate workup, including radiological assessment and multiple organ biopsies. This case highlights the diagnostic dilemma of sarcoidosis due to varying clinical presentation that can mimic multiple conditions, including malignancy. Biopsy showing noncaseating granulomas is pathognomic of sarcoidosis. Due to its multisystemic and nonspecific presentation, selecting an appropriate biopsy site is key to diagnosis. Sarcoidosis is an inflammatory multisystem disease, histologically characterized by noncaseating granulomas. Over 90% of cases have lung involvement; however, 30% of patients can have extrapulmonary manifestations . PresentA 60-year-old Caucasian gentleman with a past medical history significant for hypertension and diabetes presented to his primary care physician with abdominal pain for one month. The pain was insidious in onset and associated with poor oral intake, nausea, and vomiting. His diet had been restricted to clear liquids due to unremitting nausea and vomiting, resulting in 32 pounds of weight loss. He denied recent sick contacts, diarrhea, fever, chills, dysuria, or hematuria. Family and social history were insignificant. Examination revealed a blood pressure of 177/94 mmHg, a pulse rate of 87 beats per minute, afebrile, and epigastric tenderness with no other focal systemic findings.Initial laboratory workup revealed hemoglobin of 10 mg/dL, platelet count of 250,000 cell/µL, creatinine of 3.32 mg/dL (baseline creatinine 1.13 mg/dL), calcium of 13.8 mg/dL, and phosphorous of 2.5 mg/dL. Initial evaluation for hypercalcemia revealed low parathyroid hormone (PTH) level of 11.3 pg/mL, parathyroid hormone-related peptide (PTHrP) of 1 pmol/L, 25-hydroxyvitamin D level of 17 ng/mL, and a 1,25-dihydroxyvitamin D level elevated to 68 pg/mL. The constellation of abdominal pain, anemia, acute kidney injury, and hypercalcemia guided further evaluation for multiple myeloma. Serum protein electrophoresis and urine protein electrophoresis revealed no monoclonality. Serum immunofixation revealed imperceptible free lambda light chains and elevation in both kappa and lambda free light chains with a ratio of 1.73. A bone marrow biopsy revealed normal cellularity, no significant increase in plasma cells, and no plasma monoclonality. He was started on intravenous fluids, pamidronate, and calcitonin for hypercalcemia. Due to worsening renal function, a renal biopsy was obtained which revealed acute tubular injury with chronic and subacute thrombotic microangiopathy, but no evidence of multiple myeloma or sarcoidosis. He was briefly started on steroids for possible sarcoidosis with improvement in serum calcium; however, a normal serum angiotensin-converting enzyme (ACE) level and lack of granulomas on biopsy arrested concerns for sarcoidosis. Due to microangiopathy on renal biopsy, workup including autoimmune antibody, rheumatoid factor, anti-double-stranded DNA antibody, anti-Sjogren's syndrome A (anti-SSA) antibody, anti-Sjogren's syndrome B (anti-SSB) antibody, and lactate dehydrogenase were all negative. CT of his chest revealed small bilateral pleural effusions and pulmonary edema  scan was obtained, which revealed multiple tiny fluorodeoxyglucose (FDG)-avid lymph nodes in the chest, abdomen, and pelvis, as well as abnormal FDG labeling in the liver and spleen. Due to concerns for lymphoma, he underwent biopsy of an inguinal lymph node which revealed noncaseating granulomas with giant cells Figure . He was Calcium ions play an important role in cellular function, physiological processes, cardiac contractility, and blood coagulation. Therefore, the extracellular concentration of calcium is tightly regulated through feedback mechanisms that involve PTH and 1,25-dihydroxyvitamin D. Integrating signals from the kidney, parathyroid gland, bone, and intestine orchestrate the regulating mechanisms of calcium.In healthy individuals, vitamin D is stored as a provitamin D in the skin within the epidermis and dermis. Ultraviolet light from the sun converts it into a biologically inactive previtamin D. This previtamin D is enzymatically activated by a two-step hydroxylation reaction in the liver and kidney respectively to 1,25-dihydroxyvitamin D. Vitamin D and its potent metabolites increase extracellular calcium concentration by increasing intestinal epithelial calcium absorption and increased bone resorption through osteoclast production. Though PTH and hypophosphatemia stimulate and enhance renal 1-hydroxylase enzyme; 1, 25 dihydroxyvitamin D in turn inhibits the secretion of PTH, providing a negative feedback regulation.The cause and pathogenesis of hypercalcemia can be understood based on derangements in normal physiological regulation. Broadly based on the inciting mechanism, hypercalcemia is classified into PTH-dependent and independent causes. The most common causes of hypercalcemia include PTH-dependent hyperparathyroidism and malignancy which causes a PTH-independent hypercalcemia through PTHrP. Although rare, chronic granulomatous diseases can cause a PTH-independent hypercalcemia through enhanced conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D. Sarcoidosis alone has been found to be the cause of hypercalcemia in only 1% of patients [Sarcoidosis is a multisystem inflammatory disease histologically characterized by noncaseating granulomas. The disease is seen worldwide with an estimate prevalence of 20 to 60 per 100,000 population . DemograDespite advances in immunopathogenesis, the exact etiology of sarcoidosis remains elusive. The formation of the characteristic sarcoid granuloma involves a complex interplay between immune cells and their mediators. These granulomas can produce 1,25-dihydroxyvitamin D independent of PTH. This has been shown through studies of increased calcitriol in an anephric patient with sarcoidosis, in vitro studies of alveolar macrophages and lymph node tissues which showed increased 1,25-dihydroxyvitamin D, as well as increased 1-hydroxylase RNA in alveolar macrophages from patients with sarcoidosis . In sarcDiagnosis of sarcoidosis can be challenging when hypercalcemia is the presenting symptom. Although hypercalcemia is a well-studied electrolyte abnormality in sarcoidosis, it is seen in 10% of cases, with severe hypercalcemia of more than 14 mg/dL rarely reported . The incTreatment of hypercalcemia in sarcoidosis consists mainly of corticosteroids. Furthermore, hypercalcemia is an indication to start steroid therapy even in the presence of only mild symptoms. However, other causes of granulomatous diseases including histoplasmosis and tuberculosis should be ruled out prior to steroids. Although steroids are the mainstay of treatment in sarcoidosis, they play an important role in managing hypercalcemia by inhibiting macrophage 1-hydroxylase activity . They reThis case shows an unusual presentation of sarcoidosis, as acute hypercalcemia is rarely the initial manifestation. This highlights the importance of including sarcoidosis in the differential diagnosis, as it has a wide and varying presentation. As biopsy is key to diagnosis, locating an appropriate site to biopsy is crucial as sarcoidosis can have a multisystemic involvement. In the setting of high clinical suspicion along with elevated 1,25-dihydroxyvitamin D and corticosteroid response, treatment can be initiated while awaiting histological confirmation.


Finally, we evaluate the therapeutics that may best prevent the onset or spread of pathology in ALS and discuss what lies on the horizon for treating this currently incurable disease.Amyotrophic lateral sclerosis (ALS) is characterized by the progressive degeneration of the motor neurons that innervate muscle, resulting in gradual paralysis and culminating in the inability to breathe or swallow. This neuronal degeneration occurs in a spatiotemporal manner from a point of onset in the central nervous system (CNS), suggesting that there is a molecule that spreads from cell-to-cell. There is strong evidence that the onset and progression of ALS pathology is a consequence of protein misfolding and aggregation. In line with this, a hallmark pathology of ALS is protein deposition and inclusion formation within motor neurons and surrounding glia of the proteins TAR DNA-binding protein 43, superoxide dismutase-1, or fused in sarcoma. Collectively, the observed protein aggregation, in conjunction with the spatiotemporal spread of symptoms, strongly suggests a prion-like propagation of protein aggregation occurs in ALS. In this review, we discuss the role of protein aggregation in ALS concerning protein homeostasis (proteostasis) mechanisms and prion-like propagation. Furthermore, we examine the experimental models used to investigate these processes, including  A major pathological component of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, frontotemporal lobar degeneration (FTLD), and amyotrophic lateral sclerosis (ALS) is the prion-like propagation of misfolded and aggregated proteins in the central nervous system ; however, it can be converted into a β-sheet-rich pathogenic conformation (PrPSc) that can recruit and convert normal PrP to become pathogenic PrPSc through a template-directed manner  containing many chaperones due to the role it plays in protein synthesis. Protein disulfide isomerases (PDI) are a family of molecular chaperone proteins, localized primarily to the ER, that have oxidoreductase activity, which allows them to reduce and oxidize disulfide bonds in the proteins they are chaperoning  is characterized by the progressive degeneration of motor neurons in both the motor cortex, brain stem, and spinal cord  repeat expansions in the C9ORF72 gene, which result in the transcription of large (tens to thousands of repeats) hexanucleotide repeat RNA molecules and subsequent repeat-associated non-AUG (RAN) translation into dipeptide repeat (DPR) proteins , Pro-Gly (PG), Pro-Arg (PR), Gly-Ala (GA), and Pro-Ala (PA) polypeptides. These DPRs are thought to elicit different levels of toxicity and react differently within the cell based on their physicochemical properties , accounting for roughly 90% of cases. The remaining cases are familial (fALS) and, although some of the underlying genetic causes remain unidentified, are typically associated with a family history of the disease . UBQLN2 is an fALS-associated protein responsible for many interactions that facilitate protein degradation in vitro study of purified proteins and cultured cells, as well as in vivo in invertebrate models  and mouse models. For reviews that incorporate zebrafish (Danio rerio) models, see Babin et al.  and the proteins  in an assay, a more fundamental understanding of the processes underlying protein misfolding and prion-like propagation can be achieved.E. coli) or yeast  and electrostatic loop (loop VII), being responsible for the coordination of metals or guidance of superoxide substrate to the enzymatic site, respectively. Furthermore, a conserved intramolecular disulfide, that stabilizes the tertiary structure and promotes dimerization, is formed between residues Cys57 and Cys146  prevents excessive FUS fibril formation in condensation experiments in vitro  is resistant to detergent denaturation and proteolytic degradation would be useful to determine if purified FUS can form pathogenic amyloids. For a visual summary of the assays that can be performed with purified protein, see Indeed, current understanding of ALS as a prion-like disorder is supported strongly by experimentation of both SOD1 and TDP-43, however, experimentation with FUS is in early stages , HeLa cells, SH-SY5Y cells, Neuro-2a, and NSC-34, however, other cell lines are used too. NSC-34 cells are a hybrid cell line generated from a fusion of murine neuroblastoma cells and murine embryonic spinal cord enriched in motor neurons  disease-associated mutant forms of SOD1 are significantly more aggregation-prone than wild-type SOD1  provide unique opportunities to investigate the pathological role of ALS-associated protein aggregates. Here we will discuss models for neurodegeneration using C. elegans has been used for decades in the study of neuronal development  with expression restricted to specific cells or tissues, including the remarkable ability to express transgenes in individual neurons. Also, the transparent nature of C. elegans enables imaging of fluorescence-tagged proteins in specific tissues/cells within a whole organism  C. elegans transgenic lines to investigate the mechanisms through which ALS-associated mutations in SOD1, TDP-43, and FUS contribute to protein aggregation, neurodegeneration, and behavioral defects concerning proteostasis mechanisms and prion-like propagation. Other aspects of ALS (and other neurodegenerative disease) pathology are covered in detail in reviews elsewhere  or mutant SOD1 (hSOD1) tagged with fluorescent proteins to facilitate visualization of protein localization . These studies have shown that expression of disease-associated mutant forms of SOD1  is associated with increased aggregation and behavioral deficits. The formation of aggregates during aging is correlated with defects in the proteostasis network  despite all hSOD1(mutant) lines showing clear aggregate formation  including heat shock factor 1 (HSF1), Hsp110, a DnaJ family member A2 (DNJ-19), an Hsp70 family member (STC-1), E3 ubiquitin ligase components SEL-10 and RBX-1, and proteins involved in SUMOylation [small ubiquitin-like modifiers (SUMOs)] UBC-9 and GEI-17. Several of these hits were validated through genetic crosses  confirmed earlier findings, showing that expression of GFP-tagged hSOD1-G93A is correlated with reduced locomotion and the formation of large aggregates  via Mos1-mediated single copy insertion (MosSCI). Toxic effects were more subtle in MosSCI transgenic lines compared with conventional multi-copy arrays expressing hSOD1 : single-copy insertions of mutant ceSOD1 showed significantly higher hSOD1WT-YFP inclusions compared with ceSOD1WT or the empty vector control. The authors theorize that the hSOD1WT-YFP inclusions were possibly seeded by misfolded mutant C. elegans SOD1 protein in MosSCI models, suggesting a prion-like propagation effect , ALS-associated mutant forms (Q331K and M337V), or the C-terminal 25 kDa fragment  led to severe locomotor defects  receptor DAF-2, which requires HSF1 for pro-longevity effects  and hTDP-43 transgenic lines improved locomotion and reduced hTDP-43-YFP aggregation  in all transgenic lines  lines was able to partially rescue the locomotion defects observed in G290A and M337V transgenic animals  hTDP-43 also showed similar locomotion defects, with some evidence of aggregate formation in the nucleus of C. elegans led to adult-onset, age-dependent paralysis and progressive motor neuron degeneration in hTDP-43(mutant) lines  hTDP-43 to GABAergic motor neurons of C. elegans ortholog of TDP-43 , the authors showed that ceTDP-43 knockout was linked to sensitivity to oxidative and osmotic stress, as well as increased lifespan at moderate (20°C) temperatures. Unlike effects on hTDP-43 transgenic animals, the effects of the IIS pathway on ceTDP-1-mediated stress responses and lifespan are less clear—two distinct DAF-2 insulin receptor mutants have opposing effects, making it more difficult to derive a conclusive mechanistic link. Nonetheless, ceTDP-43 does appear to be required for the stress resistance in DAF-2 mutants as well as age-dependent proteotoxicity . The authors conclude that mutated FUS is likely to be pathogenic through a gain-of-function effect, rather than through titrating WT-FUS from the nucleus. In future work, it would be interesting to assess whether the colocalization of FUS with stress granules can be initiated by stressors other than acute heat stress  in e Olsen, .C. elegans model expressing wild type and S57Δ mutant hFUS in GABAergic motor neurons, hFUS transgenic lines showed adult-onset, age-dependent locomotion deficits  and complex array of behaviors that can be rapidly assessed experimentally, provides a powerful model animal to pursue investigations into the mechanisms of neuronal dysfunction  system , and is a more practical and ethical system than mammalian models to perform large-scale genetic or pharmacological screens . Interestingly, although the hSOD1-G85R line showed the most significant locomotor defect, this did not correlate with a gross reduction in the number of motor neurons in the thoracic ganglia or with the formation of insoluble species in these neurons  in the eyes leads to significant levels of tissue degeneration. This toxicity was completely reverted by the co-expression of EGFP-tagged 12xQ/N. The authors proposed that dTDP-43 may be sequestered in aggregates formed by EGFP-12xQ/N, thereby titrating excess dTDP-43  of TDP-43 in the Drosophila models expressing FLAG-tagged hTDP-43WT, hTDP-43 with a defective NLS  or hTDP-43 with a defective NES . Neuron-specific expression of these transgenes was larval lethal, so an inducible system was used to bypass developmental effects  species, although hTDP-43ΔNES and hTDP-43ΔNLS expression showed more HMW species compared with hTDP-43WT. When probed with an antibody against phospho-TDP-43(S409/410), hTDP-43ΔNES HMW species, but not those of hTDP-43WT or hTDP-43ΔNLS, were strongly labeled by the pTDP-43 antibody. Interestingly, immunostaining did not reveal the presence of inclusions formed by the expression of any of these transgenes, suggesting that toxicity  does not require the formation of inclusions. This study also demonstrated cell-type-specific toxicity resulting from the expression of hTDP-43 transgenes: in adult neurons, both nuclear and cytoplasmic accumulations of TDP-43 result in toxicity, whereas in muscle and glial cells, only the accumulation of cytoplasmic species of TDP-43 was toxic ] as a modulator of hTDP-43 protein levels  in its 3′ UTR  is highly expressed in neurons and functions in synaptic vesicle exo- and endocytosis  or microautophagy functions (Hsc70–43KA). This suggests that hTDP-43 compromises both chaperone and microautophagy activities of Hsc70–4  do not develop an ALS phenotype  of doxycycline-regulatable transgenic mice expressing a human TDP-43Δ NLS in forebrain neurons (under a CAMKIIa promoter). Recipient mice displayed pTDP-43 inclusions within the ipsilateral injection site within 1-month post-injection, with relatively few inclusions observed on the contralateral side. Furthermore, spatiotemporal immunohistochemical analysis of mice injected with TDP-43 seeding material showed a transneuronal-like spreading pattern of TDP-43 pathology in the 1–9 months following injection. Unsurprisingly, a large majority of TDP-43 aggregates were observed within forebrain neuronal cells where the CAMKIIa promoter controls the expression of TDP-43Δ NLS. However, TDP-43 aggregates were also found within the subcortical regions including the nucleus accumbens and caudate-putamen, suggesting regional spreading.Observations from ALS and FTLD post-mortem tissue suggests that TDP-43 also exhibits prion-like propagation and spreading throughout neuroanatomical tracts. Until recently there has been a lack of a et al.  were ablin vivo evidence for the prion-like characteristics of TDP-43, further research is required to identify the specific regions of TDP-43 that are important for seeding. As mentioned previously, full-length TDP-43 and its CTFs have multiple amyloidogenic regions  overexpression mouse models under various promoters. Mitchell et al.  were theFUS mutations are inherited in an autosomal dominant fashion, with FUS-R521C mutations commonly observed in FUS-associated fALS patients. Qiu et al.  developeRecently, Zhang et al.  employedTo distinguish the pathological role of FUS aggregates without the confounding effects on RNA metabolism, Shelkovnikova et al., developed a transgenic mouse expressing a truncated human FUS protein (residues 1–359), which lacked the RNA recognition motif and NLS but still retained an N-terminal QGSY prion-like domain , locomotor deficits measured by rotarod test (140 days vs. 120 days), and mortality , however the mechanism was not described -1-piperazinyl]phenyl]-2-thiophenecarboxamide. In vivo experiments using a SOD1-G93A mouse model showed that the drug was well tolerated, with toxicity estimated to be 75 mg/kg. These mice also showed a significant decrease in SOD1 protein levels, but aggregation was not described  in a randomized double-blind clinical trial in Poland, led to a reduction in ALS severity  and median survival time (212–248 days depending on dose) in SOD1-G37R mice compared to vehicle control . Administration of riluzole in conjunction with CuATSM did not exhibit any additive effect , a continuation of the study ongoing (NCT04313166), and a placebo-controlled study currently recruiting (NCT04082832) at the time of publication.Anti-sense oligonucleotides (ASO) can bind to mRNA and reduce protein expression, representing a potential therapeutic strategy  over 11.5 h in phase I, randomized trial of SOD1 fALS was demonstrated to be well tolerated, however, the therapeutic benefits were not measured   is an important chaperone that prevents protein aggregation  in Huntington’s disease patients (Reilmann et al., Years of intensive study into the disease mechanisms of ALS strongly implicates a pathological role for both declining proteostasis and prion-like propagation. There are intimate links between both of these concepts that explain much of the pathological manifestation of ALS. For example, the observation of proteinaceous inclusions in affected tissues, the apparent spread of pathology through neuroanatomically connected tracts, and the preferential degeneration of motor neurons over other cell types. SOD1 and TDP-43 can misfold and aggregate into conformations that can propagate within and between cells in a prion-like manner, as evidenced by the studies mentioned above ranging from biophysical assays of protein stability to whole-organism studies of aggregate seeding. Indeed, this knowledge of proteostasis dysfunction and protein aggregation has led to the development and examination of multiple therapeutics for ALS. The questions that remain, however, are what the conformation of these spreading strains are, the precise mechanism by which they contribute to pathology, and if targeted therapies can be designed to prevent their formation or spread. Also, a question of significant importance to the field is whether FUS can spread pathology from cell-to-cell in a pathological prion-like manner. This knowledge would unify our understanding of the underlying pathomechanisms in ALS.LM directed the manuscript. All authors wrote and edited the manuscript. NRC and JJY conceptualized the manuscript. All authors contributed to the article and approved the submitted version.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.