Granulin (FTLD-TDP-GRN) or C9orf72 repeat expansion mutations (FTLD-TDP-C9+). We demonstrate that intracerebral injections of biologically active pathogenic FTLD-TDP seeds into transgenic mice expressing cytoplasmic human TDP-43  and non-transgenic mice led to the induction of de-novo TDP-43 pathology. Moreover, TDP-43 pathology progressively spreads throughout the brain in a time-dependent manner via the neuroanatomic connectome. Our study suggests that the progression of FTLD-TDP reflects the templated cell-to-cell transneuronal spread of pathological TDP-43.The stereotypical distribution of TAR DNA-binding 43 protein (TDP-43) aggregates in frontotemporal lobar degeneration (FTLD-TDP) suggests that pathological TDP-43 spreads throughout the brain via cell-to-cell transmission and correlates with disease progression, but no in vivo experimental data support this hypothesis. We first develop a doxycycline-inducible cell line expressing GFP-tagged cytoplasmic TDP-43 protein (iGFP-NLSm) as a cell-based system to screen and identify seeding activity of human brain-derived pathological TDP-43 isolated from sporadic FTLD-TDP and familial cases with  Cell-to-cell transmission of TDP43 occurs in cell cultures and may contribute to pathological TDP43 propagation in FTLD-TDP. In this study, the authors demonstrate using mouse models that a single intracerebral injection of human brain-derived pathological TDP43 from FTLD-TDP cases initiates the process of seeding and spreading of TDP43 pathology in a spatio-temporal dependent manner in the brain. The stereotypical distribution of TDP-43 pathology as disease progresses suggests a hierarchical regional spreading of TDP-43 aggregates over time6. The cell-to-cell templated propagation of Aβ, tau, and α-synuclein (α-syn) aggregates offers a common pathophysiological hypothesis for disease progression in TDP-43 proteinopathies and other neurodegenerative diseases, including AD, Parkinson’s disease, and related synucleinopathies12. Although the mechanisms underlying progression in these diseases are unknown, increasing evidence supports a model where misfolded proteins released from a cell harboring pathological inclusions act on recipient cells to form de novo pathology by corrupting endogenous normal proteins to adopt pathological conformations. The reiteration of this process leads to cell-to-cell propagation of pathological proteins throughout the brain14.Deposition of TAR DNA-binding 43 protein (TDP-43), especially phosphorylated TDP-43 (pTDP-43), into cytoplasmic and intranuclear inclusions is the pathological hallmark of frontotemporal lobar degeneration (FTLD-TDP) and amyotrophic lateral sclerosis (ALS), as well as a common comorbid pathology in other neurodegenerative diseases, such as Alzheimer’s disease (AD)17. Furthermore, anterograde and retrograde transport of TDP-43 oligomers in cell culture provide evidence for neuron-to-neuron transmission of pathological TDP-4315. Currently, however, no evidence exists to demonstrate TDP-43 spreading in animal models. Here we show that pathological TDP-43 derived from FTLD-TDP brains induces formation of de novo TDP-43 pathology with subsequent spreading throughout the central nervous system in a regional- and time-dependent manner in experimental animal models.Cell-to-cell transmission of TDP-43 aggregates occurs in cell culture, and exosomes have been proposed to contribute to pathological TDP-43 propagation18. Both GFP fluorescence and immunofluorescence (IF) using an anti-TDP43 antibody -inducible QBI-293 stable cell lines expressing either green fluorescent protein (GFP)-tagged wild-type TDP-43 (iGFP-WT) or mislocalized cytoplasmic TDP-43 (iGFP-NLSm) due to inclusion of mutations within the bipartite nuclear localization signalody Fig.  showed tody Fig. . Densitoody Fig. . MoreoveUsing iGFP-WT and iGFP-NLSm cell lines, we evaluated whether TDP-43 intracellular aggregates can be induced when treated with sarkosyl-insoluble extracts containing pathological TDP-43 from FTLD-TDP postmortem frontal cortex  or C9orf72 repeat expansion mutations (FTLD-TDP-C9+) genes  23. With respect to pathological TDP-43 within FTLD-TDP and ALS tissue extracts, propagation has been demonstrated only in cell culture models24. Thus we tested the hypothesis that human brain-derived pathological TDP-43 could induce the templated propagation and spreading of TDP-43 pathology in mouse models. We injected brain-derived FTLD-TDP-GRN extracts, with proven seeding activity in vitro, into the brains of previously characterized Dox-regulatable transgenic (Tg) mice expressing a cytoplasmic NLS mutant of human TDP-43 (hTDP43NLSm) in forebrain neurons (CamKIIa-hTDP43NLSm)26. These CamKIIa-hTDP43NLSm mice develop little to no pathological TDP-43 aggregates in the brain even late in their lifespan26. Transgene expression was suppressed by providing Dox-containing chow, and 1 week prior to brain extract injection, mice were switched to standard chow to induce transgene expression. Brain-derived FTLD-TDP-GRN extracts were stereotaxically injected into the neocortex, hippocampus, and thalamus and sacrificed 1 month post-injection (mpi). Brain sections were subjected to immunohistochemistry (IHC) to detect pathological TDP-43 using the phospho-specific p409-410 antibody  in the neocortex Fig.  and hippThe hippocampus also exhibited pTDP-43 aggregates in neuronal perikarya and their processes with significantly greater abundance ipsilateral compared with contralateral side Fig. . PyramidNLSm  promoter (rNLS8)27. Using the same dosing and timing schedule as described above  alone  and p409-410 antibodies confirmed the presence of p409-410-positive NCIs that occasionally co-localizes with full-length TDP-43 protein in the hippocampus of injected CamKIIa-hTDP-43NLSm mice, we constructed heat maps to visualize the spatial distribution. NCIs and neuritic pTDP-43 immunostaining in gray and white matter tracts were graded on a scale from 0 to 3 to represent absent to the most abundant pathology and color-coded in six rostral-to-caudal coronal sections at 1, 3, 5, and 9 mpi , astrocytes ), microglia (Iba1), and oligodendrocytes (Olig2)  that could trap RNA-binding proteins such as TDP-43 into the cytoplasm38. While the C-terminal region of TDP-43 is essential for aggregation and self-templating, the N-terminus of TDP-43 is also important in regulating its oligomerization and biological function41.In FTLD-TDP brains, CTFs are a major component of cytoplasmic TDP-43 pathology42 ice Fig.  with cytice Fig.  and a feice Fig. . This coice Fig.  and hippice Fig.  of the iine Fig. 42.Fig. 6To characterize whether 208-CTFs were the principal components of the pTDP-43-positive NCIs, we used a polyclonal antibody that preferentially recognizes hTDP-43  at 1 and 9 mpi  with a close correlation between pathologic subtype and genetic risk factors or clinical phenotypes52. Moreover, the resistance and banding pattern of CTFs after PK digestion suggested that distinct conformational TDP-43 species recovered from FTLD-TDP brains may be more potent. Indeed, synthetic peptides corresponding to specific sequences in the extreme C-terminus of TDP-43 form more active amyloid-like fibrils seeding TDP-4317. However, more extensive biochemical and biophysical studies are needed to demonstrate the presence of distinct pathogenic TDP-43 strains in FTLD-TDP brains due to assembly of different C-terminal TDP-43 regions. Such studies would strengthen the hypothesis that clinical heterogeneity in FTLD-TDP patients is due to the heterogeneity of TDP-43 neuropathology resulting from different TDP-43 strains.Biochemical analysis of pathological TDP-43 from human FTLD-TDP brain extracts revealed a correlation between reduced seeding activity and lack of two lower Mr CTFsNLSm mice resulted in widespread cortical, hippocampal, and subcortical pTDP-43 accumulation in both ipsilateral and contralateral hemispheres in a time-dependent manner, consistent with cell-to-cell transmission of TDP-43 pathology which follows the neuroanatomical connectome from the injection site. For example, soon after injections of pathological brain-derived TDP-43 lysates into the anteromedial visual cortex, pTDP-43-positive NCIs were found within deep neocortical neurons within layer VI of the lateral visual cortex and lateral and dorsal auditory cortex. Anterograde tracer studies have demonstrated that pyramidal cells in anteromedial visual cortex indeed project their axons to the lateral visual cortex and the lateral and dorsal auditory cortex via deep cortical neurons within layer VI (Allen Mouse Brain Connectivity Atlas and ref. 53), consistent with the hypothesis that pathological TDP-43 species could propagate via axonal connections5.Remarkably, our study demonstrates that brain-derived FTLD-TDP extracts not only efficiently induce seeding of de novo TDP-43 pathology when injected into the brains of TDP-43 Tg mouse models expressing cytoplasmic hTDP-43 protein but also to a lesser extent in WT mice. More importantly, our data demonstrate that TDP-43 pathology spreads throughout the brain in a spatio-temporal manner. The propagation of TDP-43 pathology in injected CamKIIa-hTDP-4354.The pattern of TDP-43 pathology in the hippocampus also supports the hypothesis of axonal spreading of pathology. The absence of pTDP-43 in the CA3 layer at early time points followed by progressively increased numbers of TDP-43 aggregates in CA3 neurons over time supports the hypothesis of cell-to-cell transmission of pathology either by anterograde transport from DG granule cells via MF or retrograde transport from CA1 pyramidal neurons. These observations can be extended to include the spread of pTDP-43 pathology over time in dorsal hippocampus, pre- and para-subiculum, and lateral and medial entorhinal cortex. Thus the spatio-temporal pattern of TDP-43 pathology spread in the hippocampus and related structures appears to be related to their neuronal connectivity56. This suggests that select neuronal populations may be particularly vulnerable to TDP-43 aggregates. Moreover, we also found that white matter oligodendrocytes and astrocytes are capable of developing pTDP-43 aggregates over time. This is particularly interesting since the CamKIIa promoter should direct transgene expression only in neurons. How TDP-43 pathology spreads to glial cells is not clear but could be due to neuron-to-glial transfer of TDP-43 pathology. Glial cells do accumulate TDP-43 inclusions in FTLD and ALS brains57, while astrocytes accumulate TDP-43 pathology in Alexander’s disease58. Further studies are required to better understand the relationship between neuronal and glial TDP-43 and how it relates to cell-to-cell transmission of TDP-43 and other neurodegenerative disease pathologies.While the spreading of TDP-43 pathology in our animal models is partly dependent on the distribution of transgene expression, TDP-43 pathology was found to progressively affect subcortical areas such as nucleus accumbens, the basolateral amygdala, and the caudate/putamen, areas also affected in FTLD-TDP brains41, our data indicate that N-terminal sequences of TDP-43 may play an important role in self-templating and aggregating TDP-43 in vivo. Injections of pathological TDP-43 into the brains of CamKIIa-208 mice that overexpress cytoplasmic 208-CTFs did not efficiently seed and transmit TDP-43 pathology compared to CamKIIa-hTDP-43NLSm mice. While the expression levels of cytoplasmic 208-CTF and hTDP-43NLSm proteins are comparable, the high insolubility of 208-CTF protein42 when compared with hTDP-43NLSm26 may also determine the accessibility of the pathological TDP-43 seeds, thereby affecting the efficiency of seeding and propagation of TDP-43 pathology in CamKIIa-208 mice.Notably, as has been described in vitro59, defects in the nucleocytoplasmic function have been proposed as a common pathogenic mechanism in ALS and FTLD60. C9orf72 animal models demonstrate impaired nucleocytoplasmic transport63 and a link between cytoplasmic poly-GA dipeptide, a major aggregating protein in C9orf72 patients, and impairment of TDP-43 nuclear import has been reported64. Furthermore, TDP-43 aggregates in cultured cells causes the disruption of nuclear membrane and nuclear pore complexes, reducing both nuclear protein import and mRNA export65. Overall these results suggest that TDP-43 aggregates may result in a positive feedback loop due to nucleocytoplasmic transport defects.Our study also demonstrates the seeding of endogenous TDP-43 and propagation of TDP-43 pathology over time in non-Tg mice injected with brain-derived FTLD-TDP-GRN seeds, albeit at a very low rate. Our findings suggest that pathophysiological conditions, such as aging or cellular stress affecting homeostasis and accumulation of cytoplasmic TDP-43, may promote the seeding and propagation of TDP-43 pathology in the brain. Although disease-causing missense mutations promoting cytoplasmic mislocalization of TDP-43 are uncommon in ALS casesAdditional studies are required to better elucidate the mechanisms that affect the cell-to-cell transmission of misfolded TDP-43 as well as the processes that affect TDP-43 homeostasis. The mouse models we have developed here provide compelling systems for the interrogation of molecular mechanisms and physiological consequences of transneuronal spreading of TDP-43 disease pathology. This raises the intriguing idea that inhibiting the cell-to-cell transmission of TDP-43 pathology may mitigate disease progression and that blocking transmission of TDP-43 pathology may have therapeutic potential for the treatment of TDP-43 proteinopathies.18 into the enhanced GFP-C1 vector . cDNA for GFP-WT and GFP-NLSm were subcloned into the tetracycline (Tet)-inducible expression vector, pLVX-Tight-Puro, using the BamHI and NotI restriction sites to generate the pLVX-GFP-WT- and pLVX-GFP-NLSm-expressing vectors.N-terminal GFP-tagged TDP-43: GFP-WT and GFP-NLSm, were generated by subcloning the cDNA encoding WT TDP-43 and site-directed mutagenized NLS sequences of TDP-43 −1, Calbiochem, La Jolla, CA). Cells were maintained at 37 °C in humidified atmosphere containing 5% CO2. For transfection, cells were plated at 3 × 105 cells in 6-multi-well plates the day before and medium was replaced with 500 µl of pre-warmed growth medium immediate before transfection. Transfection mixture (500 µl) contained 15 µl of FuGENE® 6 Transfection Reagent  and 3.0 µg of either pLVX-GFP-WT or pLVX-GFP-NLSm vectors in Opti-MEM® . 175 µl of Opti-MEM® was removed from each well and was replaced with 175 µl of transfection mixture. After 4 h incubation, transfection medium was replaced with growth medium. Cells were selected with puromycin  and drug-resistant cells were subcloned by limited dilution in 96-well plates. iGFP-WT (clone #12.5) and iGFP-NLSm (clone #6.B7) individual subclones were expanded and transgene expression was validated by IF and immunoblot.iGFP-WT and iGFP-NLSm cells were generated by transient transfection of pLVX-GFP-WT and pLVX-GFP-NLSm expression vectors into QBI-293 stable cells expressing the reverse tetracycline transcriptional activator rtTA (QBI-293rtTA cells (clone #6.10)) generated in the Center for Neurodegenerative Disease Research (CNDR). QBI-293A cells were originally purchased from Quantum (AES0506). QBI-293rtTA cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% of tetracycline-screened fetal bovine serum , 1% Penicillin–Streptomycin , and L-glutamine  with G418  and CamKIIa-rtTA-rev (CATGTCCAGATCGAAATCGTC); NEFH-rTA-frw (ATTGAGGGCTTGACCACCAGGAA) and NEFH-rTA-rev (GCATGTCCAGGTCAAAATCATCCAAG) and for hTDP-43NLSm and 208 transgenes; TDP-43-frw (TTGGTAATAGCAGAGGGGGTGGAG) and MoPrP-rev (TCCCCCAGCCTAGACCACGAGAAT)42.Monogenic tetO-hTDP-43−1, Dox Diet, S3888; Bio-Serv., Flemington, NJ). Transgene expression was induced by switching to standard chow lacking Dox . WT B6C3HF1 mice were purchased from the Jackson Laboratories . Both male and female adult mice (3–5 months old) were used for this study. All breeding, housing, and procedures were performed according to the NIH Guide for the Care and Use of Experimental Animals and approved by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC).Mice were maintained with chow containing Dox . Immediately before transduction, cells were washed twice with Opti-MEM® medium, which had been pre-equilibrated in the cell culture incubator overnight. Protein–Bioporter complexes were added to the cells and incubated for 4 h. Cells were placed back on fresh medium in the absence or presence of Dox  and cultured for 3 additional days (3 dpt). To compare the seeding activities of sporadic FTLD-TDP, FTLD-TDP-GRN, and FTDL-TDP-C9+ brain extracts, equivalent amounts of insoluble TDP-43 (250 pg) according to ELISA (C89) measures , sarkosyl-insoluble extracts from a non-pathological case were used to account for differences in protein content . Equivalent amounts of sarkosyl-insoluble extracts from non-pathological cases were used as a negative control . All cell culture experiments were blinded with respect to the patient-derived brain extracts used for transduction and posterior analysis.For the analysis of the efficiency of seeding of iGFP-WT and iGFP-NLSm cells, the same amounts of sarkosyl-insoluble extracts were used for transduction; if not specified in the text, 1.0 µg total protein per well (24-MW plates) or 4.0 µg total protein per well (6-MW plates) were used for transduction (Supplementary Table 5 cells), and after 24 h, growth medium was replaced with pre-warmed growth medium with Dox (Dox+) or without Dox (Dox−) for an additional 72 h. Cells were washed with ice-cold dPBS and extracted with 300 µl of ice-cold RIPA buffer ) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and a mixture of protease inhibitors and phosphatase inhibitors by sonication using a hand-held probe  and centrifuged at 100,000 × g for 30 min at 4 °C. The supernatants were saved for analysis as RIPA fraction (R) and pellets were re-sonicated in 60 µl of Urea buffer . After centrifugation at 100,000 × g for 30 min at 22 °C, the supernatant was saved as the Urea fraction (U). For evaluating the formation of insoluble TDP-43 aggregates after transduction, iGFP-WT and iGFP-NLSm cells were washed with ice-cold dPBS and protein was extracted with 300 µl homogenization buffer  and 1 mM PMSF and protease inhibitor cocktail. Sarkosyl was added to the lysates to a final concentration of 1% and incubated for 10 min at room temperature. Protein extracts were sonicated and centrifuged at 100,000 × g for 30 min at 4 °C. The supernatant containing the sarkosyl-soluble protein was saved, whereas pellet was washed with 300 µl of dPBS, sonicated, and centrifuged again at 100,000 × g for 30 min at 4 °C. The final pellet was resuspended in 50 µl of dPBS as sarkosyl-insoluble fraction. Protein concentrations of the RIPA fractions were determined using the BCA .To evaluate the expression levels of induced GFP-WT and GFP-NLSm fusion proteins, iGFP-WT or iGFP-NLSm cells were plated in 6-MW plates  using MOPS-SDS as a running buffer. Proteins were transferred onto 0.45 µM nitrocellulose membranes and blocked with Odyssey blocking buffer . Membranes were immunoblotted overnight at 4 °C with primary antibodies diluted in Odyssey blocking buffer (Supplementary Table −1), xylazine (10 mg kg−1), and acepromazine (0.1 mg kg−1) and immobilized in a stereotaxic frame (David Kopf Instruments). Human sarkosyl-insoluble extracts were sonicated  and aseptically delivered unilaterally in the thalamus (rate of 0.6 μl min−1), dorsal hippocampus (rate of 0.6 μl min−1), and overlying cortex (rate of 0.4 μl min−1) 69 . Each injection site received 2.5 µl of sarkosyl-insoluble human extracts (Supplementary Table Adult mice (3–5 months old) were deeply anesthetized with ketamine hydrochloride  for 20 min. The brains and spinal cord were dissected and immersion-fixed in NBF overnight at 4 °C. Post-fixed tissues were rinsed in Leaching buffer  and brains were cut in 2-mm-thick coronal slices using a mouse brain slice matrix . Brain and spinal cord slices were processed for paraffin embedding.TM containing DAPI . Images were obtained in a high-resolution Leica DMI6000B microscope using the Leica LAS-X software.Distribution of GFP-WT and GFP-NLSm proteins and the presence of p409-410-positive TDP-43 aggregates (pTDP-43) in iGFP-WT and iGFP-NLSm stable cells were evaluated by IF. Cells were plated on coated poly-D-lysine coverslips and transduced as described above. Cells were washed once with dPBS at room temperature and fixed with 4% paraformaldehyde (4% PFA) for 15 min and washed three times with dPBS at room temperature. For experiments where the percentage of area occupied by p409-410 immunostaining was quantified, cells were fixed in 4% PFA containing 1% Triton X-100 to remove soluble proteins and to visualize the insoluble TDP-43 aggregates. After incubation in blocking buffer  for 30 min at room temperature, cells were incubated with specific primary antibodies  by microwaving for 15 min at 99 °C. Slides were then washed with 0.1 M Tris buffer, pH 7.6 for 5 min. To reduce nonspecific signals, brain sections were blocked with 2% FBS–0.1 M Tris buffer and immunostained with the corresponding primary antibody . For double-labeled IF, deparaffinized sections were blocked as described above and incubated with different combinations of primary antibodies overnight at 4 °C  to more abundant  and color-coded into six representative coronal sections from rostral to caudal brain levels . Brain regions were marked according to the anatomical patterns in The Mouse Brain Atlas in stereotaxic coordinates69. After grading individual brain regions for each mouse, averaged values for each time point analyzed (1 mpi (n = 3), 3 mpi (n = 2), 5 mpi (n = 3), and 9 mpi (n = 3)) were used to generate heat maps using the in-house software to graphically show distribution of pTDP-43 pathology based on a scale-color system71.The semiquantitative heat maps for injected CamKIIa-hTDP-4372 was quantified and used as a measure of the seeding activity in our cell-based system. The number of DAPI-positive nuclei (#Dapi µm−2) was quantified using the Indica Labs HALO software.To quantify the extent of TDP-43 aggregation in iGFP-WT or iGFP-NLSm cells transduced with brain-derived human extracts, iGFP-WT or iGFP-NLSm stable cells were plated into coverslips and fixed 3 dpt with 1% Tx–4% PFA to remove soluble proteins and immunostained using the p409-410 antibody. Whole coverslips were scanned and images were digitalized using a Lamina Multilabel Slide Scanner . The images were processed using the Indica Labs HALO software and the percentage of area occupied by p409-410 immunopositive stainingTo evaluate the extent and spread of TDP-43 pathology in CamKIIa-208 and WT (B6C3HF1) mice injected with FTLD-TDP-GRN extracts, bright-field images of brain sections immunostained with p409-410 antibody were acquired at ×20 magnification and digitized using a Lamina Multilabel slide scanner . Cells containing cytoplasmic p409-410-positive staining were manually counted in the cortex and hippocampus using the CaseViewer software (3DHistech). For each animal, p409-410-positive cells were counted in a total of 50–60 coronal brain sections separated by 120 µm and covering rostral to caudal areas of the brain.t test was performed when two groups were compared and one-way analysis of variance (ANOVA) or two-way ANOVA was performed when multiple groups were compared, according to different conditions. Statistical analysis was performed using the Prism software 6.0 .For each experiment, the corresponding statistics test is indicated in the figure legend. Number of samples for each group or how many times the experiment was repeated independently is indicated in the figure legend. Paired and unpaired two-tailed Student’s Supplementary InformationPeer Review File


Micro-supercapacitors have recently emerged as promising microscale power sources for portable and wearable microelectronics. However, most reported planar micro-supercapacitors suffer from low energy density and the complexity of fabrication, which calls for their further development. In recent years, the fortification of graphene has enabled the dramatic improvement of planar micro-supercapacitors by taking full advantage of in-plane interdigital architecture and the unique features of graphene. The development of viable printing technologies has also provided better means for manufacturing, bringing micro-supercapacitors closer to practical applications. This review summarizes the latest advances in graphene-based planar micro-supercapacitors, with specific emphasis placed on formulation of graphene-based inks and their fabrication routes onto interdigital electrodes. Prospects and challenges in this field are also discussed towards the realization of graphene-based planar micro-supercapacitors in the world of microelectronics. The push towards internet of things (IoT) may become a key technological and economical driver for global development in the near future. IoT allow for sensors and actuators (devices) to deploy over a large area, connect into large databases and networks (internet), and autonomously operate in correlation with computing systems . The matUnlike batteries, which store energy and produce electricity through chemical reactions ,7, super2·g−1 and exceptional carrier mobility of up to 2 × 105 cm2·V−1·cm−2 [−2 (~550 F·g−1) [Since the discovery of graphene a decade ago, a great numbers of its potential uses have been proposed ,16,17. OV−1·cm−2 ,19, grapV−1·cm−2 ,21. In rV−1·cm−2 . The qua0 F·g−1) , which i0 F·g−1) . The mac0 F·g−1) , porous 0 F·g−1) , graphen0 F·g−1) , honeyco0 F·g−1) , graphen0 F·g−1) , and 3D 0 F·g−1) , have be0 F·g−1) ,32, whic0 F·g−1) ,34. Grap0 F·g−1) ,36. The To realize the commercial application of graphene-based micro-supercapacitors, it is necessary to develop a facile, reliable, and cost-effective technique for scalable fabrication of graphene electrodes. Among the available processing techniques, printing of graphene inks offers a simple and effective route for production of interdigital electrodes that can be adapted into an industrially accessible scale . In thisFor supercapacitor devices, the capacitance  [The basic structure of micro-supercapacitors consists of four main components—a substrate, current collectors, electrodes, and electrolytes . The energetic performance of micro-supercapacitors is mainly dependent on the intrinsic electrochemical properties of the electrode materials . Based ond RuO2) ,46,47 annd RuO2) ,49 can pnd RuO2) ,51.Another key factor affecting the performance of micro-supercapacitors is the electrolytes, which provide ions for the charge-storage mechanisms and define the operating voltage window of the devices ,53. LiquOther components, such as substrates and current collectors, may also affect the flexibility and reliability of micro-supercapacitors, but the intrinsic capacitance and the amount of power it can deliver are among the most important facets when considering the performance of micro-supercapacitors.2) thin films were sandwiched between lithium phosphorousoxynitride (LiPON) solid electrolytes, and exhibited a volumetric capacitance of ~380 µF·cm−3. This conventional sandwich structure is preferable for cost-effective mass production, since it inherited the fabrication technologies from thin-film microbatteries [The basic design of micro-supercapacitors can be divided into two categories: sandwich and planar configuration . The stratteries . Howeveratteries . It is aatteries . On the atteries  in 2003 atteries ,37, whicatteries , smalleratteries ,65. As aatteries , or can atteries . All the2). Kyeremateng and colleagues [For benchmarking of micro-supercapacitors, the traditional yardstick to evaluate their performance is calculating their capacitance, energy, and power densities based on the weight and/or volume of the devices. However, unlike conventional supercapacitors, the mass of the electrode materials in micro-supercapacitors is almost negligible compared to the weight of the devices. As the active electrode layers are too thin  and their thicknesses may not be uniform throughout the devices, volumetric measurements may provide misleading information about their performance. Hence, neither gravimetric nor volumetric properties should be used to evaluate the performance of micro-supercapacitors, especially for those with planar architecture. In contrast to the weight and volume, the footprint area of the devices is actually the key concern for micro-supercapacitors. Therefore, the proper way for reporting micro-supercapacitors performance is to normalize its features  by the footprint area of the devices  that showed excellent performance in screen printing. The printed patterns with line resolutions of ~40 µm were dried at 100 °C for only 5 min and exhibited excellent sheet resistances of 30 Ω/sq at 25 μm thickness. Hyun et al. [In 2016, Arapov and co-workers  describen et al.  also forWater has long been established as the most preferred solvent due to its low boiling point and non-toxic nature. As graphene cannot be dispersed in water alone due to its hydrophobicity, surfactants are usually used to tailor their interfacial energy and stabilize the exfoliated graphene flakes against aggregation ,86,87. BGraphene oxide (GO) is produced by the oxidative treatment of graphite via either Brodie , Stauden2 atmosphere, forming conductive graphene electrodes with the spatial resolution of ~50 μm. Likewise, Shin and co-workers used an inkjet printer for micropatterning of aqueous GO inks with different concentrations onto poly (PET) substrates. The printed GO patterns were reduced in a chamber containing hydrazine and ammonia solution at 90 °C for 1 h, which resulted in conductive graphene electrodes with excellent conductivity of ~65 Ω/sq. These works paved a new avenue for the fabrication of graphene electrodes for micro-supercapacitors.The formulation of GO inks is simple and straightforward as GO can be easily dispersed in the most preferable solvent, water. In 2011, Le et al.  demonstr2/Ar atmosphere, which showed good electrochemical performance and achieved a capacitance of up to 140 F·g−1.For 3D printing, the formulation of graphene inks is relatively different, as a printable ink solution required a high viscosity and shear-thinning behavior. In 2017, Rocha and co-workers  formulatInterestingly, among all studies on the chemistry of GO, the largest portion is focused on its reduction routes back to graphene ,98. As G2 laser, conductive rGO arrays with porous structure were formed and served as active electrodes, while the intact GO served as electrolyte. Micro-supercapacitor devices with both sandwich and in-plane architecture were fabricated and showed comparable performance with those using external electrolytes. This work is significant since it removed the need of additional electrolyte and established a new approach for scalable fabrication of all-carbon, high-precision, and lightweight micro-supercapacitors.By nature, hydrated graphene oxide is simultaneously an electrical insulator and a good ionic conductor, allowing it to serve as electrolyte and separator in energy devices. Hence, it is possible to produce all-graphene micro-supercapacitors by employing rGO as electrodes and GO as electrolyte. In 2011, Gao and co-workers  demonstrGraphene is not only advantageous by itself but is also promising for combining with other materials to boost their superior performance. Intensive research has been devoted to the design and synthesis of graphene hybrid complexes for enhancing their electrochemical properties ,108. By 2) to form the test electrodes. A supercapacitor device was fabricated and exhibited specific capacitance of 326.5 F·g−1 with ultrahigh energy and power densities . One of the most apparent challenges when processing graphene inks is the restacking of graphene layers, which leads to lower active surface area and the degradation of power capacity. Yang et al.  have suc−2, which is among the highest value achieved on graphene-based micro-supercapacitors.Poly : poly (styrenesulfonic acid) (PEDOT:PSS) is an important conductive polythiophene derivative and a favorable electrode material for supercapacitors. Liu and colleagues  have suc−1. A supercapacitor cell was fabricated using the printed electrodes and yielded a maximum specific capacitance of 82 F·g−1, power density of 124 kW·kg−1, and energy density of 2.4 Wh·kg−1. By growing vertically aligned pseudo-capacitive PANi nanorods on both sides of the GO surface and subsequent reduction in the presence of PEDOT:PSS, Liu and co-workers [−2 and volumetric capacitance of 19.2 F·cm−3. By adapting an asymmetric design, the printed micro-supercapacitor with extended operating voltage window from 0.8 to 1.2 V achieved an improved energy density (from 3.36 to 4.83 mWh·cm−3) and power density (from 9.82 to 25.3 W·cm−3).Polyaniline (PANi), a typical conducting polymer with pseudocapacitance, is commonly used as electrode material for supercapacitors. In 2014, Xu et al.  formulat-workers  formulat2, MoO3, Co3O4, NiO, and RuO2) [There is still a scarcity of literature of graphene composite inks for printing of micro-supercapacitors. In fact, numerous pseudocapacitive materials have been reported to have higher capacitance when made of composites with graphene, including conductive polymers  ,49 and mnd RuO2) ,46,47. HPrinting technologies have been widely employed for fabrication of microelectronics . Compare2 and the thickness of 10 μm, which were further coated with a layer of PVA-H3PO4 as solid-state electrolyte. The printed micro-supercapacitor delivered a high specific areal capacitance of 3.4 mF·cm−2 with good rate capability and cycling stability . This technique is significant since it removed unnecessary steps, provided a promising route for scalable and fully automated fabrication of micro-supercapacitors.Unlike screen printing, inkjet printing works without the need of a physical printing mask . The basInkjet printing of graphene inks has attracted enormous attention due to its high resolution and versatility. The most simple and straightforward formulation of graphene inks is based on graphene oxide (GO inks) due to its hydrophilic nature to form stable dispersion in water and solvents, as in a report by Le et al.  in 2011.−1) are required for the formation of ink droplets [In recent years, research efforts have been emphasized on the modification of the fluidic characteristic of graphene inks, since they are crucial factors for printable ink systems. Generally, ink dispersion with low viscosity (0.004–0.03 Pa·s) and high surface tension  is required for stabilizing graphene, which usually does not exhibit any interesting electrochemical properties. Therefore, the combination of pristine graphene with other materials , which can both stabilize graphene and exhibit pseudocapacitance, can be a good direction for future development. Further research effort should also be devoted to remedy the printability of the devices and enhance the printing resolution. More advances still need to be made in the engineering of compatible graphene inks and the printing protocols for cost-effective fabrication, as the nanostructures of graphene in the printed electrodes play an important role in the performance of micro-supercapacitors. Further research should also be devoted to the investigation of various graphene nanostructures in the formulation of new graphene inks and their electrochemical performance. Research in this field is still at the nascent stage, but there is no doubt that the printing of graphene inks can be a promising key to open the door for graphene-based planar micro-supercapacitors in the future.


Among these photocatalysts, the r-Cu2O-G possesses the best photocatalytic performance of 98% removal efficiency of methyl orange (MO) with outstanding kinetics for 120 min of visible light irradiation. This enhancement is mainly due to the dangling “Cu” atoms in the highly active {110} facets, resulting in the increased adsorption of negatively charged MO. More importantly, the unique interfacial structures of Cu2O rhombic dodecahedra connected to graphene nanosheets can not only decrease the recombination of electron-hole pairs but also stabilize the crystal structure of Cu2O, as verified by a series of spectroscopic analyses , X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and transmission electron microscopy (TEM)). The effective photocatalysts developed in this work could be applied to the efficient decolorization of negatively charged organic dyes by employing solar energy.Three morphologies  of crystal Cu Howr energy ,5,6,7,8.catalyst ,11,12,13h) pairs  and has h) pairs . Therefoh) pairs ,19,20,21h) pairs ,23,24, ah) pairs ,26. Moreatalysis ,28,29. Tl orange . In addi} facets . In comptivities . Howeverperation .2O nanocrystals, one of the possible methods was carried out by the incorporation of carbonaceous materials onto Cu2O [2 g−1) [5 cm2 V−1 s−1) [2O-graphene composites [2O-graphene nanoheterostructures on their visible light-responsive activity and corresponding durability.To further increase the stability of Cunto Cu2O ,34,35. Gnto Cu2O , which h [2 g−1) , high inV−1 s−1) , and excV−1 s−1) ,40,41. Omposites ,43 for f2O crystals with low-index facets  decorated with graphene sheets were prepared via simple wet-chemical methods. These as-synthesized Cu2O crystals were thoroughly characterized by a variety of analytical spectroscopies and used as visible light-driven photocatalysts in the degradation of methyl orange (MO).In this research, three different morphologies of Cu2O [2O-graphene, ca. 88 mL of deionized water or 1% GO solution was mixed with ca. 1 mL of copper(II) chloride solution and 0.87 g of sodium dodecyl sulfate (SDS) solids under vigorous stirring until the dissolution of the SDS powder. Then, 8.5 mL of 0.2 M NH2OH·HCl and 2.5 mL of 1.0 M NaOH solution were consecutively added to the resulting mixture. Lastly, the precipitate was treated by centrifugation and dried under a vacuum. The aforementioned photocatalysts prepared by using deionized water and 1% GO solution in the synthesis process were labelled as o-Cu2O and o-Cu2O-G, respectively.Typically, the synthesis of graphene oxide (GO) was carried out by referring to a modified route described earlier . For pre2O  and octa2O [2O-graphene, 48 mL of 1% GO solution was mixed well with 1 mL of copper(II) chloride solution. Afterward, 40 mL of ethylene glycol (EG) was introduced to the aforementioned mixture. Then, 24 mL of 0.1 M NH2OH·HCl was added to the above solution for 10 min. The resultant solution was moved to a Teflon-lined stainless steel autoclave, followed by ramping from room temperature to 180 °C for 1 h. The solid products were filtrated, washed and dried at ambient temperature. The aforementioned samples prepared without and with 1% GO solution in the synthesis process were denoted as h-Cu2O and h-Cu2O-G, respectively.In terms of the synthesis of hierarchical facets of Cu2O  and hier2O [2O-graphene, ca. 34.6 mL of deionized water or 1% GO solution was heated to 34 °C in a water bath. Then, ca. 2.5 mL of copper(II) chloride solution (0.1 M) and 0.44 g of SDS solids were added into the above mixture with continuous stirring. While the SDS was dissolved, 2.5 mL of NaOH solution was introduced, followed by adding 12 mL of 0.1 M NH2OH·HCl to the resulting mixture. Finally, the precipitated products were separated by centrifuge, washed by using a water–ethanol mixture and dried. The aforementioned samples prepared by using deionized water and 1% GO solution in the synthesis process were denoted as r-Cu2O and r-Cu2O-G, respectively.For the preparation of rhombic dodecahedral Cu2O  and rhomλ = 1.541 Å). The elemental compositions of samples were analysed by X-ray photoelectron spectroscopy (XPS) using a Kratos AXIS Ultra DLD spectrometer  and monochromated Al Kα X-ray source. The morphologies of the photocatlysts were observed through a scanning electron microscope  with an accelerating voltage of 20 kV. In addition, to further study the detailed surface characteristics of the Cu2O nanocrystals, a high-resolution transmission electron microscopy  was carried out. The UV-visible (UV-Vis) diffuse reflection spectra of the samples were collected on a UV-Vis spectrophotometer .Powder X-ray diffraction (XRD) patterns of the samples were examined by using a PANalytical X’Pert PRO diffractometer with Cu Kα radiation (−1). Prior to irradiation, the aqueous solution was kept stirring in the dark for 2 h to establish the adsorption equilibrium. The pH value of the solution was 5.6. Then, the photocatalytic reaction was performed by using a 300 W Xe lamp combined with a 420 nm cutoff filter as the light source. About 2 mL of aliquots was periodically withdrawn for every 30 min, centrifuged to separate solid samples, and the variations of concentration were measured by using a Hitachi UV-Visible (UV-Vis) spectroscope (Model U-2910).Photocatalytic degradation tests of MO were performed at 25 °C in a photoreactor (100 mL) described previously . The tes2O- and Cu2O-incorporated graphene samples possess five characteristic reflections at 2θ = 29.6°, 36.4°, 42.3°, 61.4° and 73.6° which are attributed to the (110), (111), (200), (220), (311) planes of cuprous oxide. This result shows the synthesized samples are classified to the cubic phase Cu2O (JCPDS No. 78-2076). The sharp diffraction peaks indicate that the high crystallinity of Cu2O with different morphologies in all samples can be prepared by using wet-chemical methods. In the previous study [I(220)/I(200)) is able to be used to evaluate the degree of crystal structure. For instance, the index of I(220)/I(200) is nearly 0.79 in terms of rhombic dodecahedra Cu2O. As can be seen in 2O , the value of I(220)/I(200) is practically unchanged, indicating that no apparent perturbation of crystal structure is observed. This result implies that the introduction of graphene may cause little impact on the growth of nanocrystal Cu2O in the samples. As displayed in us study , the int2O, o-Cu2O-G, h-Cu2O-G and r-Cu2O-G were investigated by using field emission scanning electron microscopy (FESEM). As can be seen in 2O possesses octahedron morphology of eight {111} facets. 2O with randomly crosslinked polyhedrons which are composed of {110} and {111} facets. In terms of r-Cu2O, a 12-facet polyhedral with mostly {110} facets can be observed, as displayed in 2O-G, h-Cu2O-G and r-Cu2O-G are calculated to be 315 ± 35, 218 ± 19 and 289 ± 38 nm, respectively. Upon incorporating graphene onto various crystals of Cu2O , the morphologies and crystal structure Cu2O are almost the same as those without graphene, as shown in 2O nanocrystals were additionally identified by TEM. It can be observed that the TEM images  images of o-Cu2O-G, h-Cu2O-G and r-Cu2O-G in 2O. Moreover, selected area electron diffraction (SAED) and HRTEM suggest that these crystal Cu2O samples possess a single-crystal structure, which matches well with the high crystallinity explored by XRD. Therefore, different shapes of highly crystallized Cu2O decorated with graphene nanosheets have been fabricated by using simple wet-chemical methods.The morphologies of the crystal CuM images  show tha2O-G, h-Cu2O-G and r-Cu2O-G samples are decreased slightly in comparison to the pure graphene oxide sheets, suggesting that the graphene sheets may maintain the reduced states even with the existence of Cu2O nanocrystals in the samples. The XPS of spectra of Cu2p (3/2 and Cu(I) 2p1/2 peaks [2O-G and r-Cu2O-G composites are much higher, indicating that Cu2O nanoparticles may exist stably when dispersed on graphene nanosheets, which can maintain the photocatalytic performance during photodegradation process [The surface atomic compositions and interfacial electronic states of the samples can be investigated by XPS analysis, as displayed in  of Cu2p b indicat/2 peaks , respect process . Moreove2O-G, h-Cu2O-G and r-Cu2O-G nanocomposites, diffused reflectance UV-Vis (DR UV-Vis) absorption spectroscopies were carried out. 2O-G possesses a band edge of ca. 580 nm in the visible region, suggesting that o-Cu2O-G photocatalysts having the bandgap of ca. 2.2 eV  and absorption edge positions as compared to o-Cu2O-G and h-Cu2O-G. Moreover, the band gap of these photocatalysts can be obtained by plotting transformed Kubelka–Munk functions, as illustrated in 2O-G, h-Cu2O-G and r-Cu2O-G photocatalysts are ca. 2.20, 2.12 and 2.06 eV, respectively. This finding may be due to their different crystal sizes and distinct exposed facets in the photocatalysts. Combining the outcomes of the XRD, TEM, SEM, XPS and DR UV-Vis spectra, various morphologies of crystal Cu2O incorporated with graphene nanosheets may be able to take advantage of natural light for decontamination and also be recycled for reuse after photocatalytic reactions.To understand the optical properties of o-Cu2 eV see b should 2O, h-Cu2O and r-Cu2O) and various graphene-incorporated cuprous oxides  were studied by the photocatalytic oxidation of the MO solution under visible light illumination at room temperature. About 8.3, 4.5, 17.7, 21.2, 37.4 and 20.2% of MO were adsorbed by o-Cu2O, h-Cu2O, r-Cu2O, o-Cu2O-G, h-Cu2O-G and r-Cu2O-G after 120 min in the dark, respectively, indicating that the presence of a high surface area of graphene can increase MO adsorption. 2O (79.4%) > h-Cu2O (35.4%) > o-Cu2O (26.9%) can be observed after 120 min of visible light irradiation. Based on this result, it is concluded that the rhombic dodecahedra Cu2O nanocrystals which expose mainly {110} facets exhibit superior photocatalytic activity. However, the octahedral Cu2O composed of {111} facets have the lowest photocatalytic activity. The hierarchical facets of Cu2O exposing both {110} and {111} facets possess moderate photodegradation activity. Upon the incorporation of graphene sheets onto the aforementioned Cu2O crystals, all the photoactivities of prepared catalysts are highly enhanced, suggesting that the existence of graphene owing to the unique interface contact between Cu2O facets and graphene [2O-G photocatalysts. It has been reported that the level of conduction band (CB) potential for Cu2O (−3.0 eV vs. vacuum) [2O can barely happen, i.e., the MO sensitization during photocatalysis should hardly occur. Moreover, kinetic studies can be used to confirm the photocatalytic activities of various catalysts. As can be seen in 2O, h-Cu2O, r-Cu2O, o-Cu2O-G, h-Cu2O-G and r-Cu2O-G are found to be ca. 0.0026, 0.0036, 0.0131, 0.0043, 0.0089 and 0.0292 min−1, respectively. It should be noted that the photodegradation rate of r-Cu2O-G is 6.8 and 3.3 times higher than those of o-Cu2O-G and h-Cu2O-G, respectively.The photocatalytic performance of pure cuprous oxides (o-Cugraphene  can enha vacuum)  was grea vacuum) . As a re2O and r-Cu2O-G as the example photocatalysts to test the stability of MO degradation. As can be seen in 2O after three consecutive tests. However, only ca. 10% of efficiency decline has been observed for r-Cu2O-G, which is probably due to the assistance of graphene. It is noteworthy that no obvious dissimilarity can be found in the fresh and used r-Cu2O-G photocatalysts  upon the presence of ultra-low content of samples (0.06 g L−1) using visible light illumination. Moreover, these r-Cu2O-G photocatalysts can be obtained via a facile and low-cost liquid-phase method. Above all, our r-Cu2O-G nanocomposites also have a remarkable enhancement of their long-term durability by the assistance of 1 wt % of graphene. In comparison to previously reported Cu2O-based photocatalysts, the synthesized r-Cu2O-G photocatalysts with dominant {110} facets of crystal Cu2O exhibit an excellent degradation kinetic in a low concentration of photocatalysts under visible-light irradiation that could be practically used to make the best use of daylight for the remediation of organic wastewater.It is crucial to explore the durability and reusability in terms of practical applications of these photocatalysts. We take r-Cumediates  during p2O-graphene photocatalysts can be proposed. Upon visible-light illumination, the crystal Cu2O  is excited to produce electrons (in the conduction band (CB)) and holes ). Because of the stronger contact between crystal facets and the graphene surface, the photogenerated electrons can be rapidly transported to the graphene nanosheets. As a consequence of the small difference between the Fermi potential of graphene and the reduction potential of O2/O2− [2− and H2O2 can be observed after the reaction of high-energy electrons with the dissolved oxygen. In this way, the recombination of electron/hole pairs can be greatly prohibited. In addition, compared to octahedral Cu2O (mainly {111} facets), the holes (h+) with positive charge generated on the valence band ) of rhombic dodecahedra Cu2O (mainly {110} facets) also can degrade more MO molecules (ca. 1.48 eV vs. NHE) [+ may be the dominant species governing the photodegradation of MO under visible-light irradiation. It should be noted that the graphene sheets not only serve as acceptors of the photogenerated electrons from Cu2O but also as stabilizers to prevent the crystal Cu2O from structural destruction under irradiation over a long period.As shown in f O2/O2− , O2− andvs. NHE) ,52 sincevs. NHE) . Therefo2O photocatalysts decorated with graphene sheets were synthesized by using simple solution-phase methods. The order in terms of photocatalytic MO degradation was observed to be as follows: rhombic dodecahedral Cu2O-graphene (r-Cu2O-G) > hierarchical Cu2O-graphene (h-Cu2O-G) > octahedral Cu2O-graphene (o-Cu2O-G), which was demonstrated to be related to the morphologies and crystal structure of facets . In earlier reports regarding Cu2O-based photocatalysts, the r-Cu2O-G photocatalysts possessed high kinetics of photocatalytic degradation, i.e., by using an ultra-low content of samples (0.06 g L−1) to reach 98% of MO photodegradation under 120 min irradiation of visible light. More importantly, the cycling tests indicate that the resulting r-Cu2O-G composites show a surpassing durability compared to pure r-Cu2O nanocrystals. These significant enhancements are possibly because of the unique interfacial interaction of rhombic dodecahedra Cu2O (more positively charged {110} facets) with the graphene nanosheets, which could lead to the effective isolation of electron/hole pairs, stabilization of the crystal Cu2O, and an increase of MO adsorption. Consequently, the development of a cost-effective and facile method to prepare r-Cu2O-G composites with mostly {110} facets and graphene sheets, which exhibit superior photocatalytic performance (kinetics and stability), offers the potential for a promising application in the treatment of organic wastewater by utilizing natural sunlight.In this study, three different crystal facets of Cu


Infection by Zika virus (ZIKV) is linked to microcephaly and other neurological disorders, posing a significant health threat. Innate immunity is the first line of defense against invading pathogens, but relatively little is understood regarding host intrinsic mechanisms that guard against ZIKV. Here, we show that host tripartite motif-containing protein 56 (TRIM56) poses a barrier to ZIKV infection in cells of neural, epithelial and fibroblast origins. Overexpression of TRIM56, but not an E3 ligase-dead mutant or one lacking a short C-terminal portion, inhibited ZIKV RNA replication. Conversely, depletion of TRIM56 increased viral RNA levels. Although the C-terminal region of TRIM56 bears sequence homology to NHL repeat of TRIM-NHL proteins that regulate miRNA activity, knockout of Dicer, which abolishes production of miRNAs, had no demonstrable effect on ZIKV restriction imposed by TRIM56. Rather, we found that TRIM56 is an RNA-binding protein that associates with ZIKV RNA in infected cells. Moreover, a recombinant TRIM56 fragment comprising the C-terminal 392 residues captured ZIKV RNA in cell-free reactions, indicative of direct interaction. Remarkably, deletion of a short C-terminal tail portion abrogated the TRIM56-ZIKV RNA interaction, concomitant with a loss in antiviral activity. Altogether, our study reveals TRIM56 is an RNA binding protein that acts as a ZIKV restriction factor and provides new insights into the antiviral mechanism by which this E3 ligase tackles flavivirus infections. The E3 ligase TRIM56 was previously shown to inhibit the replication of several viruses in the family Flaviviridae, including dengue virus serotype 2, yellow fever virus and bovine viral diarrhea virus, but had not demonstrable antiviral effect against hepatitis C virus, a hepatotropic virus in the same family. Nonetheless, the antiviral mechanism remains unclear and whether TRIM56 restricts other flaviviruses remains to be determined. In this study we demonstrated that TRIM56 inhibits ZIKVs of Asian and African lineages and a dengue virus serotype 1 replicon. We additionally uncovered that TRIM56 is an RNA-binding protein and that a portion of the C-terminal NHL-like domain mediates the association of TRIM56 with ZIKV RNAs in infected cells. Importantly, the RNA-binding activity of TRIM56 was found to be required for its antiviral function, although it alone is insufficient. In contrast, TRIM56 restricted ZIKV in Dicer-deficient cells, indicating an antiviral mechanism independent of miRNA regulation, a function known to be associated with NHL-containing proteins. In aggregate, our work identifies TRIM56 as a novel restriction factor of ZIKV and sheds new lights on the antiviral mechanism of TRIM56 against flaviviruses. Zika virus (ZIKV) is a small, enveloped RNA virus classified within the family Flaviviridae, genus flavivirus, which also includes medically important pathogens such as dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV) , among oetc… [Based on the phylogeny of viral sequences, ZIKV is classified into two major lineages, African and Asian . First ietc… , 16–18. Ifnar1 or triply deficient for Irf3/Irf5/Irf7 supported heightened ZIKV replication, concomitant with developing neurological diseases [Host cells are equipped with exquisite, intrinsic mechanisms to fend off invading viruses, which could be harnessed for developing antivirals against flaviviruses, including ZIKV. The mammalian innate immune system constitutes the first line of defense against invading microorganisms including viral pathogens. A hallmark of the intrinsic antiviral responses is the rapid induction of interferons (IFNs). Once produced, IFNs act in a paracrine and/or autocrine fashion, signaling through cell surface IFN receptors and downstream Jak-Stat pathway, culminating in the upregulation of hundreds of IFN-stimulated gene (ISG) products that collectively establish an antiviral state, reining in viral replication and spread . Flavividiseases . Of the diseases , 23. HowWe have previously demonstrated that TRIM56, a RING-type E3 ligase of the tripartite motif protein family, puts a check on intracellular RNA replication of DENV serotype 2 (DENV2), YFV, and bovine viral diarrhea virus (BVDV) in cell culture , 25. AltIn this study, we demonstrate that TRIM56 exerts a direct antiviral effect on ZIKV infection in human cells of fibroblast-, epithelial-, and neural-origins and that both the E3 ligase activity and C-terminal portion of TRIM56 are critical for restricting ZIKV. Although the C-terminal region of TRIM56 bears sequence homology with the NHL repeat motif of several other TRIM proteins  that binE. coli for polyclonal antibody production and protein-RNA interaction assay, we inserted a cDNA fragment encoding the C-terminal 392 aa of human TRIM56 (T56-C392) into pMAL-c4x (New England Biolabs). The resultant plasmid vector, designated pMAL-c4x-T56-C392, allowed expression and subsequent purification of an MBP-T56-C392 fusion protein in E. coli. The plasmid encoding serotype 1 DENV replicon, pACYC-DENV1-Rluc2A-Rep [The retroviral vectors encoding C-terminally Flag-tagged, wild-type (WT) human TRIM56, the E3 Ub ligase-deficient CC21/24AA mutant, and a deletion mutant lacking C-terminal aa 693 to 750, respectively, in the pCX4bsr backbone, have been described previously . N-termiuc2A-Rep , was obtuc2A-Rep .Human embryonic kidney (HEK) 293, HeLa, SV40 T antigen-transformed human fetal glial cell line SVGA ), human neuroblastoma SK-N-SH , human hepatoma cell lines Huh7 and HLCZ01  , SV40 T HeLa-Flp-In T-REx-ACE2 (referred to as HeLa-FitA2) cells with tetracycline (Tet)-inducible expression of 2xHA-tagged, WT, E3 Ub ligase-deficient CC21/24AA mutant, or C-terminal 693–750 aa deletion mutant of TRIM56 have been described in a previous study . Tet-ind2 and plated on ultra-low attachment plates at 2.5 x 104 cells/10 cm2 and incubated at 37°C, 5% CO2, 95% humidity. Half of the media were exchanged with fresh 37°C CO2 equilibrated NpGrow every 3–4 days; neurospheres are present at 4–7 days. To differentiate NPCs into primary neuronal cultures, neurospheres from day 7 cultures were dissociated in DS for 10 min at 30°C, centrifuged at 200 x g, and DS was replaced with NbActiv4 neuron culture medium (BrainBits). Cells were counted and seeded into 24-well plates at 2 x 105 cells/well and incubated at 37°C, 5% CO2, 95% humidity. Half of the media were exchanged with fresh NbActiv4 media every 3–4 days; axons and dendrites are observable by day 4 while synapses and action potentials have been reported at 7 days [Generation of primary mouse neuronal cultures was conducted as follows. A pair of combined cortex and hippocampus with subventricular zone was obtained from E18 C57BL/6 mice . Neuroprogenitor and differentiated cultures were generated based on established protocols , 36. Spet 7 days , 36.50) assay in 96-well plates [ZIKV MR766 strain (BEI Resources # NR-50065) and PRVABC59 strain  were propagated in C6/36 cells. For infections of various cell types, cells were inoculated with ZIKV in 2% FBS-containing DMEM at indicated multiplicities of infection (MOIs) for 1–2 h. Subsequently, viral inoculum was removed, and cells were washed three times in PBS, refed with complete culture medium and cultured for the indicated time periods. Infectious virus titers in cell-free culture supernatants were determined by an endpoint dilution-based 50% tissue culture infective dose  or infected with ZIKV-PRVABC59 at a MOI of 1 in NbActiv4 media for 2 h at 37°C; duplicate wells for each time point were used. Inoculum was then removed, cells washed twice with NbActiv4, and then incubated in 0.5 ml of NbActiv4 media at 37°C, 5% COHeLa cells with stable, shRNA-mediated knockdown of TRIM56 (HeLa-shT56) and control cells stably transduced with a non-targeting shRNA (HeLa-shCtrl) were described in a previous study . To deplTLR3, IFNB, IL29, RANTES and ISG56 have been described [28S rRNA [4 to 107 copies/ml.Extraction of total cellular RNA by TRIzol (Invitrogen), cDNA synthesis by reverse transcription, and quantitative PCR (qPCR) were implemented as described elsewhere , 41. Theescribed , 42. The28S rRNA . Copy nui.e., U6 snRNA. The primers for qPCR detection included a miRNA/snRNA-specific forward primer and a universal reverse primer, as follows. miR-92a, 5’-TATTGCACTTGTCCCGGC-3’ (forward); miR-21, 5’-TAGCTTATCAGACTGATGTTG-3’ (forward); U6 snRNA, 5’-CTGCGCAAGGATGACACGC AA-3’ (forward); and miR-Rev-universal, 5’-GAACATGTCTGCGTATCTC-3’ (reverse).For miRNA detection, we synthesized oligonucleotide primers and adopted the protocol as described in the qSTAR miRNA qPCR detection system (Origene). In brief, after a poly(A) tailing procedure, a miR-oligo-dT primer with a sequence of 5’-GAACATGTCTGCGTATCTCAGACTTCTGATTCACGCTTTTTTTTTTTTTTTTTTTVN-3’, was used for reverse transcription of total small RNAs. Subsequently, SYBR green-based qPCR was performed to measure the levels of miRNAs of interest relative to that of an internal control, E. coli); peroxidase-conjugated secondary goat anti-rabbit and goat anti-mouse pAbs (Southern Biotech); FITC-conjugated secondary goat anti-mouse pAb (Southern Biotech). Specifically, FH-T56 was detected by mouse anti-Flag-tag M2 mAb (Sigma); rabbit anti-TRIM56 S4091 pAb was used to detect endogenous T56 protein in HeLa cell lysates; and rabbit anti-TRIM56 pAb (Bethyl Labs) was used for other T56 immunoblotting experiments.Cell lysates were prepared in RIPA buffer and subject to SDS-PAGE and immunoblotting analysis were described previously , 43. ImmZIKV-infected HEK293 cells expressing control vector, or Flag-tagged, WT or mutant TRIM56, respectively, were washed twice with ice-cold PBS and lysed in a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, and protease inhibitor cocktail (Sigma). The cell lysates were incubated at 4°C for 30 min, clarified by centrifugation at 12, 000 g at 4°C for 15 min, and their protein concentrations were adjusted to 1 μg/μl. For RNP-IP, 400 μg of cell lysates were incubated with the anti-Flag mAb at 1:500 at 4°C overnight. Subsequently, RNA-protein-antibody complexes were captured by incubation with protein A/G agarose beads (Santa Cruz Biotech). After three washes, the beads were divided equally into two fractions, which were subjected to RNA isolation by TRIzol and protein extraction by boiling in SDS-sample buffer, respectively. The qPCR analysis of ZIKV RNA levels and immunoblotting of TRIM56 were performed as described above.E. coli using the pMAL Protein Fusion and Purification System . To produce a control protein, we purified MBP along with a short stretch of the downstream polylinker (MBP-polylinker) from E. coli transformed with the empty vector pMAL-c4x. ZIKV RNA was extracted using TRIzol from virions in high-titer ZIKV stocks. Subsequently, two micrograms of MBP-polylinker or MBP-T56-C392 protein were incubated with 0.5 μg of ZIKV RNA at room temperature for 30 min, followed by pull-down of MBP-tagged proteins using amylose resin. After three washes, the resins were divided equally into two fractions, which were subjected to RNA isolation by TRIzol and protein extraction by boiling in SDS sample buffer, respectively. Quantification of ZIKV RNA levels was performed by qPCR as described above. MBP-polylinker and MBP-T56-C392 in the protein samples were probed by immunoblotting using rabbit anti-MBP pAb (New England Biolabs) and rabbit anti-TRIM56 pAb (Bethyl Labs).To assess whether TRIM56 directly interacts with ZIKV RNA, we first purified MBP-tagged T56-C392  protein from P value of < 0.05 were deemed statistically significant.All results are presented as means ± standard deviations. For analysis of statistical differences, two-tailed student t-test  was used to compare the means of two groups and one-way ANOVA followed by Tukey or Dunnett test  was applied for comparing the means of more than two groups. Differences with a i.e., the neuroblastoma line SK-N-SH and SVGA, a fetal glial cell line, and found both to be permissive for ZIKV infection  exhibited limited permissiveness, if any, for ZIKV, harboring mid-105 to ~106 copies/μg total RNA range viral RNA at 24 h.p.i. Moreover, viral RNA levels did not increase in hepa1-6 and MEFs, while had a mere ~2-fold uptick in L929 cells, at 48 h.p.i. Collectively, these data demonstrate that ZIKV infects and replicates in a broad range of human and animal cell lines, although the susceptibility of different cell lines vary considerably.To study host factors that impact ZIKV replication, we first surveyed a number of human and animal cell lines of various tissue origins for ZIKV susceptibility. Cells were infected by MR766, a prototype African strain of ZIKV, followed by immunofluorescence staining of viral E protein expression at 72 h.p.i. Three human hepatocyte cell lines, including Huh7, PH5CH8, and HLCZ01, were all susceptible to ZIKV infection, albeit to different degrees . The hepnfection . Apart fnfection . We alsoOur previous studies have shown that TRIM56 is an antiviral host factor against several different RNA viruses, among which include 3 members of the Flaviviridae, DENV2, YFV, and BVDV , 25. To Since the recent ZIKV outbreaks were caused by viruses of Asian lineage, we sought to verify the anti-ZIKV effect of TRIM56 using the PRVABC59 strain, an Asian lineage ZIKV . When chTo determine whether TRIM56 expressed at physiologic levels impedes propagation of ZIKV, we depleted endogenous TRIM56 in HeLa cells by shRNA-mediated knockdown and evaluated the changes in viral protein expression and progeny virus production. As shown in To understand how TRIM56 exerts its antiviral action, we determined the domains or activities that are critical for TRIM56 to restrict ZIKV infection. The main functional domains of TRIM56 and its various mutants are illustrated in To corroborate this finding, we evaluated ZIKV propagation efficiency in HEK293 cells stably transduced with an empty retroviral vector (Bsr), WT or TRIM56 mutants (AA and Mut N), respectively. Compared with that in Bsr control cells, ZIKV E protein abundance was reduced in cells overexpressing WT TRIM56  cells by retroviral-mediated gene transfer, and there was comparable expression of Flag-TRIM56 between the two cell lines . The samSince the NHL repeat motif of TRIM71 has been shown to bind to mRNA, we inveE. coli a TRIM56 fragment comprising the C-terminal 392 aa fused to MBP (MBP-T56-C392), and as a negative control, an MBP-polylinker protein. Their quality and purity were verified by Coomassie blue staining . As shown in Trim56. In line with the expression pattern of TRIM56 transcript in SVGA cells, Trim56 mRNA abundance was upregulated by ~ 2-fold at 48 h.p.i. In aggregate, the experiments show that TRIM56 is expressed in human and mouse neural cell types, which moderately upregulate the expression of this antiviral protein in response to ZIKV infection.To investigate if TRIM56 expression changes in neural cell types following ZIKV infection, we first examined the abundance of endogenous TRIM56 protein in SVGA cells by immunoblotting . At 72 hNA level . To deteet al. reported that in mouse macrophages Trim56 deletion did not significantly alter viral RNA levels following ZIKV infection [In this study we demonstrate that TRIM56, an E3 ubiquitin ligase ubiquitously expressed in human tissues , poses anfection . It shounfection , 21. Coni.e., the RIG-I-like receptors  and TLR3 [i.e., the immune stimulus) levels. Third, although ZIKV may elicit TLR3 signaling, as suggested by a recent study showing that ZIKV could disrupt neurogenesis through TLR3 activation [A subset of TRIM proteins has been shown to modulate innate immune signaling during viral infections . Flaviviand TLR3 . While Tand TLR3 . It shouand TLR3 . ZIKV enand TLR3 –56. Constivation , and TRItivation , the E3 tivation  was incaFlaviviridae, HCoV-OC43 from the family Coronaviridae [Orthomyxoviridae [Flaviviridae, in permissive hepatoma Huh7 cells [Previously, TRIM56 has been shown to possess direct antiviral activities against distinct RNA viruses, including BVDV, YFV and DENV2 from the family aviridae , 25, andoviridae . Howeveroviridae . In addih7 cells , 25, theh7 cells . Notablyh7 cells , 25, andh7 cells , 25, TRIImportantly, the current study significantly advances our understanding of the antiviral mechanism of TRIM56 by showing that TRIM56 is an RNA-binding protein and that its C-terminal region mediates the association with ZIKV RNA in infected cells. In addition, we revealed that host miRNAs are not essential for ZIKV replication and that the restriction of ZIKV by TRIM56 is independent of miRNA biogenesis or its regulation. TRIM56 is currently classified within the subgroup C-V of TRIM proteins , becauseWhile future studies are needed to define the boundary of the RNA-binding motif(s) within TRIM56, we favor a model in which the structured NHL-like domain mediates the interaction with viral RNA. Lending support to this notion, our previous studies have shown that the integrity of the TRIM56 C-terminal portion is essential for inhibiting viral RNA replication of BVDV, DENV2 and YFV, and that two different C-terminal deletion mutants, Δaa 621–695 and Δaa 693–750, each lacking a separate portion of the NHL-like domain, invariably lost antiviral activity , 25. WheIn summary, the present study identifies TRIM56 as a novel restriction factor of ZIKV, deepening our understanding of host intrinsic mechanisms that fend off this medically important flavivirus and broadening the antiviral spectrum of TRIM56. Moreover, the revelation that TRIM56 is an RNA-binding protein that associates with viral RNAs in ZIKV-infected cells and in cell-free reactions unravels new insights into the molecular mechanisms by which TRIM56 restricts flaviviruses. Further investigations that elucidate the precise action mechanism of TRIM56 may expose therapeutic targets that can be harnessed to combat ZIKV and possibly other flavivirus infections.S1 FigZIKV E protein was immunostained with green fluorescence, and nuclei were counterstained blue with DAPI. Images were representative of two independent experiments.(TIF)Click here for additional data file.S2 FigZIKV E protein was immunostained with green fluorescence, and nuclei were counterstained blue with DAPI. Images were representative of three independent experiments.(TIF)Click here for additional data file.S3 FigZIKV E protein was immunostained with green fluorescence, and nuclei were counterstained blue with DAPI. Images were representative of two independent experiments.(TIF)Click here for additional data file.S4 FigZIKV E protein was immunostained with green fluorescence, and nuclei were counterstained blue with DAPI. Images were representative of two independent experiments.(TIF)Click here for additional data file.S5 FigThe indicated cell lines were infected by ZIKV MR766 strain (MOI = 1) for 24 h or 48 h, followed by qPCR analysis of intracellular viral RNA levels. Data were representative of two independent experiments.(TIF)Click here for additional data file.S6 FigReplication of a luciferase-encoding DENV-1 RNA replicon in HEK293-FIT-T56 cells repressed (Dox-) or induced (Dox+) for HA-TRIM56 expression at different times post electroporation. Student t-test, **P<0.01. Results were representative of three independent experiments.(TIF)Click here for additional data file.S7 FigRANTES (A), ISG56 (B), IFNB (C) and IL29 (D). Results were representative of three independent experiments.HEK293-T3Y cells with and without expression of Flag-HA-TRIM56 (FH-T56) were infected by ZIKV for the indicated times, followed by qPCR analysis of the expression of (TIF)Click here for additional data file.S8 FigTLR3 mRNA (A) and intracellular viral RNA levels (B) were quantified by qPCR. Student t-test, **P<0.01, ***P<0.001. Results were representative of two independent experiments.HEK293 cells expressing control vector (Bsr) or Flag-T56 were transfected with non-targeting control siRNA or TLR3 siRNA for 24 h, followed by infection by ZIKV-MR766 for additional 48 h. The expression of (TIF)Click here for additional data file.S9 FigTRIM56 binds to ZIKV RNA via its C-terminal portion, in ways that involve its E3 ligase activity to impede viral RNA replication.(TIF)Click here for additional data file.


Caenorhabditis elegans development, providing an in vivo model to study proliferative capacity after quiescence. Hatching under starvation provokes the arrest of blast cell divisions that normally take place during the first larval stage (L1). We have used a novel method to precisely quantify each stage of postembryonic development to analyze the consequences of prolonged L1 quiescence. We report that prolonged L1 quiescence delays the reactivation of blast cell divisions in C. elegans, leading to a delay in the initiation of postembryonic development. The transcription factor DAF‐16/FOXO is necessary for rapid recovery after extended arrest, and this effect is independent from its role as a suppressor of cell proliferation. Instead, the activation of DAF‐16 by decreased insulin signaling reduces the rate of L1 aging, increasing proliferative potential. We also show that yolk provisioning affects the proliferative potential after L1 arrest modulating the rate of L1 aging, providing a possible mechanistic link between insulin signaling and the maintenance of proliferative potential. Furthermore, variable yolk provisioning in embryos is one of the sources of interindividual variability in recovery after quiescence of genetically identical animals. Our results support the relevance of L1 arrest as an in vivo model to study stem cell‐like aging and the mechanisms for maintenance of proliferation potential after quiescence.Cells can enter quiescence in adverse conditions and resume proliferation when the environment becomes favorable. Prolonged quiescence comes with a cost, reducing the subsequent speed and potential to return to proliferation. Here, we show that a similar process happens during  Working model of the regulation of L1 aging and its effect on the initiation of postembryonic development. Reduced insulin signaling increases vitellogenin provisioning in embryos. After hatching, these L1 larvae age at a slower rate, allowing fast initiation of larval development. Increased L1 aging leads to a delay in the reactivation of proliferation, represented by the delayed initiation of seam cell divisions. Development proceeds at similar speed, independently of the rate of L1 aging. In the absence of growth‐sustaining factors, cells can enter quiescence, a reversible, nonproliferative state. Interestingly, cellular quiescence also occurs in normal development, with many cells spending most of their lifetime in this state. Under certain conditions, quiescent cells re‐enter the cell cycle and resume proliferation  have 558 nuclei, 53 of which are in somatic blast cells that divide further to reach the 959 somatic cells of the adult   needed to reach the first molt when time in arrest (x) equals 0. This provides an estimate of the bonafide duration of L1 for nonstarved larvae of ca. 10 hr , daf‐2(e1370), and daf‐16(mu86);daf‐2(e1370) double mutant. After one day of starvation, the recovery time of the daf‐16 mutant was similar to that of the wild‐type strain. However, after four days of starvation, the daf‐16 mutant showed a remarkable delay in recovery. For some animals, recovery took more than 50 hr  mutants, leads to enhanced reactivation of proliferation. During prolonged L1 arrest, DAF‐16 relocalizes to the cytoplasm  and acridine orange (AO) , DNA damage, aggregated proteins, and they show mitochondrial misfunction  mutants, which present a constitutive DAF‐16 activation, show lower levels of ROS and faster recovery. Although FoxO deficient hematopoietic stem cells present increased ROS  to the arrested L1, to have a final concentration of 10 g/L E. coli OP50‐1.For recovery experiments, we resumed development by adding one volume of 20 g/L 4.3m Luciferin. After all animals were placed in the wells, we added 100 µl of S‐basal containing 20 g/L E. coli OP50‐1 per well to resume development simultaneously for all the animals. We used sample sizes >20 individually quantified animals in at least three biological replicates. We alternated the samples across the plate to avoid local effects .We measured recovery time and developmental timing using a bioluminescence‐based method  or 6.5–20.5 hr (H seam cells and M cells). Sampling times were displaced in time among independent experiments to maximize the information to be obtained from the time course. We calculated the percentage of animals with divisions over a population of at least 40 animals per time point and condition.For the experiments with the single reporters, we obtained about 300 µl of L1 larvae suspension of the strains GAL69, JR667 (seam cell reporters), or PD4666 (M‐cell reporter) arrested for one day or four days. On the day of the experiment, we added bacteria to all samples simultaneously to initiate recovery. At the indicated times, we collected 30 µl to a clean 1.5 ml tube and centrifuged for 1 min at 850 For the experiments with the double‐seam and M‐cell reporter, we prepared embryos twice per condition, separated by 12 hr. This way we could resume development of these animals with a 12‐hr difference, allowing monitor divisions in the first 24 hr over an experimental period of 12 hr. We obtained L1 larvae of the strains MOL198 and MOL253 arrested for one day or four days and then resumed development by the addition of food, as indicated above. We calculated the percentage of animals with divisions over a population from at least 40 animals per time point and condition. In order to calculate the time at which 50% of the developing population had divisions, we fitted the data to a cumulative Gaussian distribution and calculated the mean value. In both cases, we monitored the animals using a Leica scope DMi8 using GFP excitation/emission filters.4.5m Levamisole. We used a reduced concentration of Levamisole in order to avoid artificial translocation of DAF‐16. We visualized the localization of DAF‐16 in a Leica scope DMi8 using GFP excitation/emission filters and counted the animals with nuclear, cytoplasmic, or intermediate localization. Since the localization of DAF‐16 can be affected by temperature, the temperature of the room was maintained at 20°C during visualization of the animals. We categorized animals as having nuclear, intermediate, or cytoplasmic localization, in a population of at least 100 larvae. Since this categorization is somehow subjective, the experimenter was blind to the condition tested.We obtained L1 larvae of the strain TJ356 arrested for one day or four days and initiated the recovery by adding food. At the indicated times, we took an 18 µl aliquot of the suspension and placed it on a microscope slide with 2 µl of 10 m4.6g. We removed 50 µl of the supernatant and added 1 µl of 100 mm Levamisole. We transferred the sample to a glass slide and cover and visualized the GFP signal in a Leica scope DMi8 using GFP excitation/emission filters. We counted the fraction of animals with GFP signal in the seam cells (categorized as induced cki‐1), in a population of at least 50 larvae.We obtained L1 larvae of the strain VT825 and MOL270 arrested for one day or four days. When relevant, we resumed development by addition of food. At the indicated times, we transferred 50 µl of the suspension to a clean tube and centrifuged 1 min at 850 4.7m in 30 µl of arrested L1. After incubation for 2 hr at 20°C, we centrifuged the sample, removed 25 µl of supernatant, and added 1 µl of 100 mm Levamisole. We placed 5 µl of the anesthetized L1´s on a microscope slide with a cover slip. We imaged the animals in a Leica scope DMi8 at 200x magnification using transmitted light and mCherry excitation/emission filters. For quantification, we drew ROIs around the head of the larvae using the image from transmitted light and then quantified pixel intensity in the red channel using Image J. We subtracted the background for each image. We measured DHE of at least 15 animals per conditions in 3–4 biological replicates.We added Dihydroethidium  at a final concentration of 10 µFor the longitudinal analysis of recovery, we stained L1 larvae arrested for 8 days with DHE, as above, and then categorized them into animals with low or high signal. We collect animals from both categories to measure recovery and developmental timing as above.4.8m (0.1% DMSO in M9 buffer) to an aliquot of 30 µl of arrested L1. After incubation for 2 hr at 20°C, we centrifuged the sample, removed 25 µl of supernatant, and washed with 25 µl of M9 buffer to remove the excess NIAD‐4. After centrifugation, we removed again 25 µl and added 1 µl of 100 mm Levamisole. We placed 5 µl of the anesthetized L1 on a microscope slide for analysis. We imaged the animals in a Leica scope DMi8 at 200× magnification using transmitted light and mCherry excitation/emission filters. Quantification was performed as for DHE staining. We measured NIAD‐4 of at least 15 animals per conditions in four biological replicates.We added NIAD‐4  at a final concentration of 1 µ4.9m ammonium chloride (NH4Cl) or 100 mm acridine orange (AO) for eight days. These chemicals were diluted >104 times during the preparation of the plate for the luminometer, to avoid possible effects of the compounds on larval development.After hypochlorite treatment to obtain embryos, we incubated them in M9 containing 5 m4.10To obtain embryos from mothers in their first, second, and third day of egg laying, we prepared synchronized populations allowing 20 gravid adults to lay eggs on NGM plates for two hours. After this period, we removed the gravid adults and allowed the progeny to grow at 20°C. After approximately 80, 88, 104, 112, 128, and 136 hr, the progeny were, respectively, at what we have named day 0.5, 1, 1.5, 2, 2.5, and 3 of egg laying. Day 0.5 refers to the time when the first embryos have been produced, while at day 1 all animals  have started egg laying.4.11rme‐2 RNAi from the Ahringer library. We cultivated rme‐2 RNAi bacteria overnight and diluted 1/10 with control bacteria HT115 containing the empty plasmid pL4440. We added 500 µl of the diluted RNAi or control bacteria on NGM plates with 1 mm IPTG and 100 μg/ml ampicillin. We let the bacterial lawn dry and incubated the plates for 5 hr at 37°C and overnight at room temperature. We transferred 20 gravid adults of the strain MRS387 per plate and let them lay eggs for 2 hr before retiring them from the plates. We grew them for 4 days at 20°C before proceeding with the hypochlorite treatment. We maintained the animals in L1 arrest for 8 days at 20°C. Development was re‐initiated by adding food, and we monitored development as previously described. As a control for the RNAi treatment, we used the strain RT130 to monitor VIT‐2::GFP transport into oocytes.We obtained embryos with reduced yolk by alkaline hypochlorite treatment of gravid adults grown on 4.12. For all luminometry experiments, we have plotted the values for each individual animal and also the average of the values for each biological replicate. For statistics, we have used the averages of independent biological replicates to avoid the inflated N value from using individual animals. The averages of most groups were normally distributed but in cases when N was too low to assess normality, we visually inspected the values to discard important deviations from normality and variances were similar. We used unpaired two‐tailed t test to compare the means of two groups and one‐way ANOVA to compare more than two groups. ANOVA was followed by Bonferroni´s post hoc to compare all groups or by Dunnett´s test to compare groups to a control condition (mutants vs. wild‐type). For the analysis of DAF‐16 localization in wild‐type and daf‐2 mutants over time in arrest, we have performed Two‐way ANOVA.We analyzed luminometry data as previously described (Olmedo et al., The authors declare no competing financial interests.M.O., A.M‐C., M.J.R‐P., S. G‐S., and A. F‐Y. carried out the experiments; M.O. and A.M‐C. designed the experiments; M.O., A.M‐C., M.J.R‐P., M.M., and M.A‐S. interpreted results; and M.O., A.M‐C., M.M., and M.A‐S. wrote the manuscript. Click here for additional data file.