The aim of this paper is to prepare nano-functionalized ceramic foam filters from carbon-bonded alumina. The carbon-bonded filters were produced via the Schwartzwalder process using a two-step approach. The prepared ceramic foam filters were further coated using graphene oxide. Graphene oxide was prepared by the modified Tour method. The C/O of the graphene oxide ratio was evaluated by XPS, EDS and elemental analysis (EA). The amount and type of individual oxygen functionalities were characterized by XPS and Raman spectroscopy. The microstructure was studied by TEM, and XRD was used to evaluate the interlayer distance. In the next step, filters were coated by graphene oxide using dip-coating. After drying, the prepared composite filters were used for the purification of the water containing lead, zinc and cadmium ions. The efficiency of the sorption was very high, suggesting the potential use of these materials for the treatment of wastewater from heavy metals. Usually, a C/O ratio from 1.8 to 2.5 is achieved. Since graphite is a very inert mineral, it can undergo oxidation only with very strong oxidation agents. Basically, two oxidizing agents are used to prepare graphite oxide: potassium permanganate  and potaGraphite oxides and graphene oxides can be used as precursors for graphene synthesis or the synthesis of graphene derivatives such as hydrogenated and halogenated graphenes ,24,25,26Graphene oxide itself is a suitable material for the fabrication of membranes or foils . The preIn this paper, the potential of ceramic foam filters was combined with graphene oxide in order to evaluate the possibility of using this composite material for the purification of polluted water from selected heavy metals in low concentrations.2SO4 (98%), H3PO4 (85%), KMnO4 (99.5%), H2O2 (30%) and Ba(NO3)2 (99.5%) were obtained from Penta, Prague, Czech Republic. For sorption experiments, we used Pb(NO3)2, Cd(NO3)2 and Zn(NO3)2.6H2O from Lach-Ner, Neratovice, Czech Republic.For the graphene oxide suspension (GO), pure graphite microparticles  were used. H2O3, d90 ≤ 3.0 μm) in combination with several carbon sources. As a binder, a modified coal tar pitch powder  was selected. Furthermore, fine natural graphite  and carbon black powder  were added in small amounts. For the dispersion in water and the stabilization of the water-based slurry, the following additives were introduced: ligninsulfonate  as a wetting agent and temporary binder, modified polycarboxylate ether  as a dispersing agent, and alkylpolyalkyleneglycolether  as an antifoam agent.The carbon-bonded filters were prepared from fine aluminum oxide  suspension was prepared according to the modified Tour’s method. This method is described in detail in the 3 polyurethane foams with an average porosity of 10 ppi (pores per inch) were selected. The foams were first impregnated by a thick water-based slurry and squeezed to remove the excess material. Afterwards, the same samples were cold spray-coated with a second, thinner slurry to achieve the desired wall thickness [The carbon-bonded filters were produced via the Schwartzwalder process , by using a two-step approach. As substrates, 100 × 100 × 22 mmhickness . After d0 = 0.3.A home-made dip-coating device was used for the coating of the ceramic foam filter using a graphene oxide suspension. The GO/water suspension with a concentration of 20 g/L was sonicated using an ultrasonic needle immediately before the dip-coating process. After this process, coated filters were placed into a vacuum dryer for 24 h at 30 °C at p/p2+, 1 ppm of Pb2+ and 1 ppm of Cd2+  was synthesized using the modified Tour method. This sample was analyzed by TEM, EDS, EA, XPS, XRD, BET and Raman spectroscopy. GO was used for the nano-functionalization of carbon-bonded alumina-based ceramic foam filters. Coated filters were used for the purification of water contaminated by lead, zinc and cadmium ions. The morphology of GO was investigated by SEM and TEM see . SEM micThe chemical composition was also measured by EA see . From thGO was also analyzed by XPS. In the XPS survey spectra . To consider the level of oxidation of GO, the D/G ratio was calculated (intensities ID/IG). The obtained ID/IG ratio was 1.02, suggesting a high concentration of oxygen functionalities [The structure of GO was investigated by X-ray powder diffraction and Raman spectroscopy . While g348 cm−1 ,42. The nalities . 2O3-C filters was previously confirmed [Ceramic foam filters were prepared by the Schwartzwalder process. Photographs of the filters before and after the coating are shown in onfirmed . 2O3-C filters for water treatment. In addition, after the experiment the solution was analyzed to check whether some GO was released during the sorption process. The solution was filtered using a Nylon filter membrane and no graphene oxide was collected, suggesting sufficient adhesion of GO to the substrate.After the coating, the efficiency of the sorption was analyzed by AAS. A comparison of the obtained concentrations is shown in 2+/1 g of GO, 265 mg of Pb2+/1 g of GO and 11 mg Zn2+/1 g of GO, respectively. These results are also in good agreement with the data published for graphite oxides prepared by Hummers’ method [The maximal sorption capacity of GO was measured by EDS obtained as an average from five measurements. The obtained values were 35 mg of Cd’ method . In this paper, graphene oxide was prepared and characterized. Graphene oxide with a high number of oxygen functionalities was obtained. In the next step, graphene was used for the coating of carbon-bonded ceramic foam filters that were prepared by the Schwartzwalder process. The coated filters were then used in a sorption experiment, where the uptake efficiency for lead, cadmium and zinc ions was tested. The results of the research are very promising, since the efficiency was very high. The concentrations of metals dropped from 1 ppm to several ppb in all cases. Our composite filters can thus be used for water treatment where the concentration of waste metals is not very high, otherwise the maximum capacity of the filter would soon be reached. Similar materials could be used in building applications for self-cleaning surfaces. Another potential application of such composites is in the steel industry, where the modification of the ceramic foam surface can influence microroughness and, therefore, also the efficiency of metal melt filtration.


In this context, the melt ponds impart increasing unique footprints on the periglacial rivers due to their hydrodynamic connection. Given that bacterial communities control numerous ecosystem processes in the glacial ecosystem, exploring the fate of bacterial communities from melt ponds to periglacial rivers yields key knowledge of the biodiversity and biogeochemistry of glacial ecosystems. Here, we analyzed the bacterial community structure, diversity, and co-occurrence network to reveal the community organization in the Zhuxi glacier in the Tibet Plateau. The results showed that the bacterial communities in melt ponds were significantly lower in alpha-diversity but were significantly higher in beta-diversity than those in periglacial rivers. The rare sub-communities significantly contributed to the stability of the bacterial communities in both habitats. The co-occurrence network inferred that the mutually beneficial relationships predominated in the two networks. Nevertheless, the lower ratio of positive to negative edges in melt ponds than periglacial rivers implicated fiercer competition in the former habitat. Based on the significantly higher value of degree, betweenness, and modules, as well as shorter average path length in melt ponds, we speculated that their bacterial communities are less resilient than those of periglacial rivers.Since the early 21 Glacial ecosystems, at the interface of the cryosphere, hydrosphere, pedosphere, and the biosphere, cover approximately 10% of the Earth’s surface . Owing tDistributed over glacial landscapes and key components of glacial ecosystems are melt ponds, which collect meltwater on the glacial surface in visible pools . To dateThe bacterial communities of melt ponds play crucial roles in influencing glacial mass balance. For instance, heterotrophic bacteria and their products reduce the surface albedo and accelerate the melting of snow and ice , which aAlthough the crucial role of bacterial communities on melt ponds has been increasingly recognized, their patterns of composition and structure are rarely studied. Alternatively, much attention has been gathered for bacterial communities in cryoconite holes, which are physically analogous to melt ponds. Phylogenetically, four major groups are represented in the communities: Proteobacteria, Actinobacteria, Bacteroides, and Cyanobacteria ,20,21. WOn the glacier forefronts, periglacial rivers (namely glacier-fed rivers), which integrate upstream catchment processes, also constitute a prominent geomorphological and ecological component in the glacial ecosystem . Owing tIn the last 30 years, atmospheric warming has been aggravated, causing climate-induced glacier retreat. This in turn changes the influences of melt ponds on their periglacial rivers in terms of speciation of nutrient, hierarchical habitat template, and ecosystem services ,27,30. HTo uniquely characterize the glacial bacterial communities in glacial ecosystems during deglaciation succession, we comprehensively characterized and compared the bacterial communities from melt ponds to periglacial rivers, regarding their structure, diversity, and co-occurrence network. This investigation sheds new insight on the ecological succession for bacterial communities under the global warming scenario. Specifically, we hypothesized that: (1) the rare sub-community contributes to the bacterial community in the glacial ecosystem; and (2) the bacterial communities and their co-occurrence networks are distinct between melt ponds and their periglacial rivers.Our field work was carried out on the Zhuxi Glacier , which lies at an elevation of 3500 m on the Tibetan Plateau in October 2017 . The IndWater samples  were collected with a 5 L Schindler sampler, then were placed in sterile polyethylene bottles, and were stored in a frozen condition. A 1500 mL subsample for 16S rRNA gene analysis was filtered onto a 0.2 μm polycarbonate membrane  under the vacuum pressure of < 15 mm Hg. In situ measurements of water temperature (Temp), turbidity (Turb), and dissolved oxygen (DO) were measured by a multi-parameter water quality sonde . Total nitrogen (TN), dissolved total nitrogen (DTN), total phosphorus (TP), and dissolved total phosphorus (DTP) were determined, according to the standard methods [4 was determined using the indophenol blue method and NO3 with the cadmium reduction method. The dissolved organic carbon (DOC) was determined using a total organic carbon analyzer . methods , using a® cycle-Pure kit . The V4 region of the 16S rRNA genes were amplified using the primers 515F (GTGYCAGCMGCCGCGGTAA) and 806R (GGACTACNVGGGTWTCTAAT) [2 (25 mmol L−1), 0.5 μL of each primer (10 μmol L−1 each), 30 ng quantified template DNA measuring by Pico green, and 0.4 μL of Taq polymerase . The PCR cycling was conducted in a thermocycler  by a touchdown program: denaturation at 94 °C for 5 min, 11 cycles of denaturation at 94 °C for 1 min, annealing at 65 °C for 1 min (temperature was decreased by 1 °C every cycle until 55 °C was reached), and extension at 72 °C for 1 min. Nineteen additional cycles were performed at an annealing temperature of 55 °C, followed by a final extension at 72 °C for 10 min.Crude DNA extracts were purified by the E.Z.N.AWTCTAAT) . Polymerwww.arb-silva.de). The representative sequence of each phylotype was selected and aligned by RDP Classifier (version 16) against the SILVA 16s rRNA database (release 132) with a confidence threshold of 80%. We excluded from downstream analyses low confidence singletons that had a sequence count smaller than 2.Clean amplicon pools for 22 samples were paired-end sequenced (2 × 250) on an Illumina Miseq platform at Magigen Biotechnology . We removed sequences if they: (i) contained more than one ambiguous nucleotide, (ii) lacked a complete barcode and primer at one end, and (iii) were shorter than 200 bp after removal of the barcode and primer. Sequences were merged and denoised with USEARCH (version 10.0.240)  and thenp-values from multiple interaction metrics were merged using the Simes method [We constructed two individual co-occurrence networks of the bacterial communities for melt ponds and periglacial rivers using the CoNet (v.1.1.1.beta) plugin in Cytoscape (v3.5.1), as previously described . We incls method  and a fas method . igraph” package [The network was explored by the “ package . To desc package . The key package .vegan [ggplot2 [http://www.r-project.org). Data sets of multivariate statistical analyses, diversity estimates, and co-occurrence network construction were rarefied according to the lowest numbers of reads among all samples. Previous studies have claimed that abundant phylotypes are defined as having relative abundance >0.1% at the local level (one sample) [All analyses and visualizations were prepared by vegan  and ggpl[ggplot2  in the R sample) . However sample) . To dete sample) ,46. As tkruskal.test function. To compare the relative abundance of bacterial taxa, STAMP (version 2.0.9) was used. To determine the beta-diversity of the bacterial community, we conducted non-metric multidimensional scaling (NMDS) using the metaMDS function based on the four distance matrices: Bray-Curtis distance, Jaccard dissimilarity, and (un)Weighted UniFrac distance. The variations in bacterial communities between habitats were compared by betadisper function. The statistical differences between bacterial community composition were determined by the PERMANOVA test using the adonis function. We determined four measures of the alpha-diversity of the bacterial community: Chao1, Faith’s phylogenetic diversity (Faith’s PD), Shannon index, and Simpson index. Faith’s PD was based on the phylogenetic tree built with representative OTUs by using FastTree (version 2.1.10). The alpha-diversity and environmental properties between melt ponds and periglacial rivers were compared by Kruskal-Wallis test through the p = 0.01 and p < 0.001, respectively). Furthermore, the concentrations of nutrients, including TN, NH4, NO3, DTN, and DOC, were significantly higher in periglacial rivers than in melt ponds . In contrast, TP was comparable between two habitats (p = 0.92). Turbidity increased from 26.37 ± 13.53 NTU to 31.20 ± 9.00 NTU, but with no significance (p = 0.44). DTP was below the detection limitation in both habitats.Our results showed that the aquatic environments were inherently distinct between melt ponds and periglacial rivers . The watAfter quality filtering and removing chimeras, the Illumina sequencing yielded an average of 88,767 high-quality sequences with 235 bp for each site. The Good’s coverage was 98.69%–99.78%, suggesting that the sequencing effort captured most of the bacterial diversity. This implication was also supported by the rarefaction curves, which approached the saturation plateau in all 22 communities . The resp < 0.001, p = 0.02, p < 0.001, and p = 0.04 for Chao1, Simpson, Shannon, and Faith’s PD, respectively) . The beta-diversity pattern of the bacterial communities in melt ponds and periglacial rivers were also evaluated. The NMDS plot showed a clear segregation between the bacterial communities in the two habitats (p = 0.001). Based on the four distance matrixes, the permuted analysis of betadispersion of pairwise similarities showed that the bacterial communities in melt ponds were significantly more variable than those in periglacial rivers (p < 0.001). Furthermore, to explore the influence of the rare sub-communities on their whole communities, we calculated the bacterial community variation along a sequence of an accumulation of rare OTUs  . Within habitats , which ware OTUs . The step < 0.001, We compared the taxonomic structure of bacterial communities in melt ponds and periglacial rivers . The seqp = 0.04), while Alphaproteobacteria, Spartobacteia, and Verrucomicrobiae were significantly higher in periglacial rivers , followed by Betaproteobacteria (18.12%), Sphingobacteriia (14.06%), and Bacilli (15.31%). In contrast, in periglacial rivers, the dominant class was Alphaproteobacteria (26.65%), followed by Gammaproteobacteria (19.81%), Flavobacteriia (14.45%), and Bacilli (12.54%). STAMP analysis indicated that only three classes were significantly different between the two habitats, with Betaproteobacteria being significantly higher in melt ponds (Exiguobacterium (14.56%), followed by Flavobacterium (11.26%), Ferruginibacter (8.86%), and Polaromonas (7.62%). In periglacial rivers, the most abundant genera were dominated by Flavobacterium (14.45%), Sphingorhabdus (14.18%), Exiguobacterium (11.95%), and Acinetobacter (5.75%). STAMP analysis showed that 10 of the 83 genera significantly differed between the two habitats. Polaromonas, Rhizobacter, Sediminibacterium, and Variovorax were higher in melt ponds  was The co-occurrence network of bacterial communities was more complex in melt ponds than in periglacial rivers . For melThe black dashed line represents the variation of whole bacterial communities of melt ponds and periglacial rivers.p = 0.002), higher levels of betweenness , and shorter average path length (3.29 versus 4.84 p = 0.03) than that of periglacial rivers. Additionally, the network of melt ponds was divided into less modules (64) than that of periglacial rivers (148) (p = 0.001). We also explored the keystone nodes by GIANT plugin in the Cytoscape. The results identified 7 and 12 keystones for melt ponds and periglacial rivers, respectively. For the melt ponds, the keystones were affiliated with Enterobacteriaceae (five nodes) and Polaromonas (two nodes). In contrast, the keystone nodes in the periglacial rivers were more phylogenetically diverse, including Acinetobacter (one node), Exiguobacteriummexicanum (five nodes), Flavobacterium (two nodes), Terrimonas (two nodes), and Xanthomonadaceae (two nodes). The network inference showed that the network of melt ponds had significantly higher degree , and fish species [Remarkably, previous studies have documented that Cyanobacteria commonly dominate in glacial habitats, such as Arctic and Antarctic  and other alpine regions (Tibetan Plateau and high-mountain Asia) ,19,20,22 species ,11) has et al. demonstrated that the rare biosphere even determined the succession strategy of the bacterial communities during early ontogeny of meltwater-lakes [et al. also reported that the rare microbiome exhibited sensitive responses to glacier retreat in Hailuogou Glacier in the Tibetan Plateau [Bacterial communities commonly show a skewed species abundance distribution with only a few dominant phylotypes coexisting along with an extremely high number of rare species. This pattern is in line with our results that in each habitat, more than 90% of the total OTUs made up the rare sub-communities, which accounted for 64.82% and 54.26% of the whole reads for melt ponds and periglacial rivers, respectively. Additionally, the stepwise inclusion of rare OTUs significantly stabilized the bacterial communities in melt ponds and periglacial rivers, implying that the rare biosphere is crucial for these bacterial assemblages. Recent advances in molecular biology and cultivation techniques provide an increasing body of evidence that potentially supports this implication. For instance, Peter  Plateau . An important trait of the rare biosphere is the extremely high diversity, which facilitates their contributions to the entire communities  and has In our present study, co-occurrence networks in melt ponds and periglacial rivers suggested that most of the involved nodes belonged to the rare sub-communities. This pattern has previously been reported from other glacial environments in Tibet Plateau  and fromBacteria live in a complex network through various types of interactions, which can be categorized into mutually beneficial and exclusive interactions. Within melt ponds and periglacial rivers, network inference indicated that positive correlations prevailed in the bacterial communities. This correlative pattern is in line with that reported from other habitats, including soil , rivers With the edges only predicting biotic interactions, other emergent properties are more reliable, such as degree, betweenness centrality, modularity, and average path length . The netThe bacterial communities are significantly different between melt ponds and periglacial rivers in the Zhuxi Glacier on the Tibet Plateau. Specifically, owing to the pristine condition and smaller size of metacommunities, the bacterial communities in melt ponds are less diverse than those of periglacial rivers. In contrast, the bacterial communities of melt ponds are more variable across all sampling sites than are those of periglacial rivers. The co-occurrence network infers that the positive correlation is mainly the intra-taxon pair, while the negative correlation is mainly the inter-taxon pair, and that the rare sub-community significantly stabilized the whole bacterial community in both habitats. Moreover, the bacterial communities in melt ponds may be more competitive and more connected than those in periglacial rivers. In short, these observations suggest the bacterial communities of melt ponds may be more sensitive to environmental change than those in periglacial rivers. This study provides a substantial framework to guide the study of micro-ecology in glacier ecosystems.


This herbal medicine has been successfully used to treat patients with diabetic foot ulcers and pressure sores in Taiwan for several decades. We previously examined its biological activities on cell-based in vitro assay platforms. Because some patients refused to use animal-derived ingredients ointment during our clinical practice, the efficacy of plant oil-based reconstituted “Jinchuang ointment” was also investigated.A porcine excisional wound model was established and used to evaluate its efficacy in vivo in this study. Besides, an unusual clinical case is also present.As judged from the wound appearance of animal studies on day 14 and the results of blood flow flux at the wound sites on day 28, “Jinchuang ointment” accelerated wound closure significantly better than the control group.The results from clinical treatment, histopathological evaluation, and the animal study showed that “Jinchung ointment” promotes wound healing significantly better than the control group. Also, sesame oil-reconstituted ointment can be a choice for patients who refuse to use lard-containing ointment. Medicine Comprehended, published in 1732. This herbal medicine has been successfully used to treat patients with diabetic foot ulcers and pressure sores for several decades in Taiwan [“Jinchuang ointment” is a traditional Chinese herbal medicine for external incised wounds. Its recipe is recorded in the ancient Chinese medical book, “Jinchuang ointment” is composed of lard, wax, starch, borneol, camphor, frankincense, dragon’s blood, myrrh, and catechu. In general, we can classify its components into three categories: (i) chemically synthesized compounds, including camphor and borneol; (ii) excipient-like materials, including lard, wax, and starch; (iii) natural products, including frankincense, dragon’s blood, myrrh, and catechu. For quality control, our group established a set of different chemical and biological assay methods to analyze each of the components. For instance, HPLC was used to determine the content of dracorhodin in dragon blood, catechin and epicatechin in catechu, and acetyl-11-keto-β-boswellic acid in frankincense , 4. A maThe weight percentage of lard is as high as 67% in this ointment. During our clinical practice, some patients refused to use animal-derived ingredients ointment for personal or religious reasons. It is reminiscent of the fact that Shiunko ointment is a famous traditional Chinese herbal medicine for wounded skin caused by cuts, abrasions, frost or burn. Sesame oil and beeswax are included in its recipe , a porciBoswellia sacra Flueck) was the product of Daily Health Co., Ltd.  (Batch number: BCGY161229). Myrrh (resin from Commiphora myrrha Engler) was the product of Hou Chuia Biopharm Co., Ltd.  (Batch number: ULS031). “Bao chu Brand” dragon’s blood (dried fruit powder from Daemonorops draco) was the product of Meida Co., Ltd. (Singapore). Catechu (dried evaporated decoction from Uncaria gambir Roxb) was the product of Hing Zong Co., Ltd.  (Batch number: 213N-107-02). Identifications of above plant-based materials by HPLC were reported below. Vascular endothelial growth factor (VEGF) was bought from B&D Systems . Neomycin ointment was obtained from Genuine Chemical Pharmaceutical Co., Ltd. . Food-grade lard was obtained from President Nisshin Corporation . Food-grade sesame oil was obtained from Fwusow Industry Co. Ltd. . Beeswax  was obtained from KahlWax . Paraffin wax was obtained from Chenyi Chemical Co. . The composition of the three different types of Jinchuang ointment that used in this study is listed in Table Chemically synthesized borneol (purity: 98%) and camphor (purity: 96%) in compliance with the Chinese Pharmacopoeia were bought from Cheng Yi Chemical Co., Ltd. . Frankincense  was used for analysis. Sample preparation, and HPLC analytical conditions were reported previously [The clinical case that presents in this study took place in an emergency room. Because using this herb medicine to treat wounds is a routine treatment method in this hospital, the Institution granting permission for the treatment is not required. Both the consent form from the patient and the Institution granting permission (Number: 1090114004) from China Medical University Beigang Hospital for using exist data in case report were included.2) with polished concrete floors at 21 ± 2 °C and 30–70% humidity under a 12-h light and dark cycle. The room was cleaned twice a day. The animal filed was sterilized by 0.06% sodium hypochlorite once per week. Animals will be sacrificed prior to, during, or after the experiments, if animals cannot eat for 5 days, cannot stand for 24 h, or body temperature is lower than 35 °C.The Institutional Animal Care and Use Committee, National Laboratory Animal Center, Taiwan, ROC, approved all experimental animal procedures (Permission number: NLAC(TN)-107-M-010). Three female Lee-Sung pigs without skin disease were used in this study. They were obtained from the Department of Animal Science and Technology, National Taiwan University, Taiwan. The pigs were 4.5 months old with similar body shapes and an average weight of 15.8 kg. Three weeks before the experiments, the pigs were moved from an animal room located in the Department of Animal Science and Technology, National Taiwan University, by trunk to a laboratory. Each animal was maintained in an isolated room  and Xylazine 2.2 mg/kg) were intramuscularly injected and Atropine (0.05 mg/kg) was subcutaneously injected for sedation. Five minutes later, Isoflurane (0.4–2.0%) inhalation and Lidocaine (8%) spray anesthesia were started and continued for the duration of the operation Fig. a. Subcut.2 mg/kg The dermal wound tissue was sampled via a 6 mm biopsy punch  and preserved in 10% neutral buffered formalin (NBF) at 7-, 14- and 28-days post-surgery. After fixation, the tissues were trimmed, embedded, and sectioned 4–6 mm thick onto glass slides . For the hematoxylin and eosin (H&E) stain, the procedures were carried out using automated staining equipment  with routine protocols. Meanwhile, the modified Masson’s trichrome (MT) stain was conducted manually following the manufacturer’s instructions . A five-phase scoring system for semi-quantitative evaluation of histopathological observations was employed in this study  stain, the slides were dewaxed in xylene and rehydrated in PBS. The antigen retrieval was performed by incubating the slides in a pressure cooker utilizing commercial unmasking buffer . Sections were treated with endogenous peroxidase and ALKP blocking solution  in order to block endogenous peroxidase activity for 10 min at room temperature and with protein blocker  for 30 min. Slides were incubated with 10 μg/mL rabbit anti-VEGF serum  in primary antibody diluent  for 1 h at room temperature, followed by 15 min incubation with a goat anti-rodent/rabbit polymer visualization system . Additionally, 3, 3′-diaminobenzidine  was used as a chromogen for color development for 6 min. Slides were counterstained with hematoxylin and observed under light microscopy. Slides were scored based on the signal intensity in the section: − (negative), + (weak), ++ (moderate) and +++ (strong). A modified four-point scoring system for qualitatively recording IHC observations was used  compared with DMSO and represents two reproducible results.Cytotoxicity assay was performed using a 24-well plate. 4 * 10The participant, Miss Su, was a 25-year-old female nurse working at China Medical University Beigang Hospital. On September 5, 2015, 1:47 PM in the hospital during work, her four fingers were accidentally and simultaneously cut by a scalpel  or antibiotics , 14. TheThe skin structure and function of pigs are closest to those of humans among animals, so a porcine excisional model was used in this study . Small mWound healing is a complicated and intricate process . ThroughThe efficacy of “Jinchuang ointment” that observed in this study might be further supported by following in vitro and in vivo studies. Dracorhodin perchlorate (DP) is the reference standard compound of dragon blood. It can promote NIH-3T3 fibroblast cell proliferation in vitro through the activation of ERK and induce rat wound healing in vivo . DP was From the results of the clinical case and animal experiments, we verify the efficacy of the traditional Chinese medicine “Jinchuang ointment” in promoting wound healing. Excisional wounds on pig skin were treated using three different recipes of “Jinchuang ointment”: lard-based ointment , sesame oil-reconstituted ointment (TA2), and sesame oil and beeswax-reconstituted ointment (TA3) with 28-days of observation. All three test materials significantly increased the speed of healing on day 14. The lard-based ointment, TA1, showed the most healing with the most collagen deposition. However, the sesame oil-reconstituted ointment also showed significant improvement in wound healing compared to the control and would be the right choice for patients who refuse to use lard-containing ointment.Additional file 1: Table S1. Semi-quantitative evaluation criteria for histopathological observations. Table S2. Dermal wound healing categories. Table S3. Qualitative evaluation criteria for histopathological observations. Table S4. Qualitative evaluation criteria for immunohistochemistry observations. Table S5. HPLC calibration curves of reference compounds including regression equations, the coefficients of determination (R2) and calibration ranges. Table S6. Individual histopathological data of TA 1, 2, and 3 on day 7. Table S7. Individual histopathological data of TA 1, 2, and 3 on day 14. Table S8. Individual histopathological data of TA 1, 2, and 3 on day 28. Table S9. Individual histopathological data of healthy skin. Figure S1. HPLC separation of reference compounds present in extracts of herbal components. Figure S2. In vitro tube formation assay displaying the stimulation of angiogenesis by “Jinchuang ointment” on HMEC-1 cells.


Mastomys coucha as preclinical model, this is the first study demonstrating seroconversion against different L1 isoforms during the natural course of papillomavirus infection. Intriguingly, positivity with the cutaneous MnPV was accompanied by a strong seroresponse against a longer L1 isoform, but to our surprise, the raised antibodies were non-neutralizing. Only after a delay of around 4 months, protecting antibodies against the short L1 appeared, enabling the virus to successfully establish an infection. This argues for a novel humoral immune escape mechanism that may also have important implications on the interpretation of epidemiological data in terms of seropositivity and protection of PV infections in general.Although many high-risk mucosal and cutaneous human papillomaviruses (HPVs) theoretically have the potential to synthesize L1 isoforms differing in length, previous seroepidemiological studies only focused on the short L1 variants, co-assembling with L2 to infectious virions. Using the multimammate mouse  Cancer is not one disease but rather a collection of disorders. As such there are many reasons why someone may develop cancer during their lifetime, including the individual’s family history, lifestyle and habits. Infections with certain viruses can also lead to cancer and human papillomaviruses are viruses that establish long-term infections that may result in cancers including cervical and anal cancer, and the most common form of cancer worldwide, non-melanoma skin cancer.The human papillomavirus, or HPV for short, is made up of DNA surrounded by a protective shell, which contains many repeats of a protein called L1. These L1 proteins stick to the surfaces of human cells, allowing the virus to get access inside, where it can replicate before spreading to new cells. The immune system responds strongly to HPV infections by releasing antibodies that latch onto L1 proteins. It was therefore not clear how HPV could establish the long-term infections and cause cancer when it was seeming being recognized by the immune system.Mastomys coucha, as a model system for an HPV infection to uncover how papillomaviruses can avoid the immune response. This African rodent is naturally infected with a skin papillomavirus called MnPV which, like its counterpart in humans, can trigger the formation of skin warts and malignant skin tumors.Now, Fu et al. have used the Southern multimammate mouse, Fu et al. took blood samples from animals that had been infected with the virus over a period of 76 weeks to monitor their immune response overtime. This revealed that, in the early stages of infection, the virus made longer-than-normal versions of the L1 protein. Further analysis showed that these proteins could not form the virus’s protective shell but could trigger the animals to produce antibodies against them. Fu et al. went on to show that the antibodies that recognized the longer variants of L1 protein where “non-neutralizing”, meaning that could not block the spread of the virus, which is a prerequisite for immunity. It was only after a delay of four months that the animals started making neutralizing antibodies that were directed against the shorter L1 proteins that actually makes up the virus’s protective coat.These findings suggest that virus initially uses the longer version of the L1 protein as a decoy to circumvent the attention of the immune system and provide itself with enough time to establish an infection. The findings also have implications for other studies that have sought to assess the success of an immune response during a papillomavirus infection. Specifically, the delayed production of the neutralizing antibodies means that their presence does not necessarily indicate that a patient is not already infected by a papillomavirus that in the future may cause cancer. Epidermodysplasia verruciformis patients) (Human Papillomaviruses (HPVs) are widely distributed in nature and more than 220 types were sequenced up to date (PaVE: Papillomavirus Episteme). They cannot only be divided in mucosal and cutaneous types , but alsatients) , commensatients) .Mastomys coucha represents a unique model system to investigate the consequences of a natural PV infection in the context of skin carcinogenesis  soon after birth  induces a long-lasting neutralizing antibody response that completely prevents the appearance of skin lesions both under immunocompetent and immunosuppressed conditions  protein together with up to 72 molecules of the minor (L2) capsid protein . The L1 Considering the cross-talk between viral infections and the immune system, PVs have developed multiple strategies to escape from immune surveillance . While tIn the present study, we show that MnPV, as a rodent equivalent for cutaneous PVs in humans, induces a strong seroconversion in its natural host early after infection. However, the raised antibodies are non-neutralizing and directed against a longer isoform of the L1 protein which is unable to assemble into viral particles. Only after a delay of around 4 months after infection, protecting antibodies appear. This argues for a novel PV immune escape mechanism, probably providing a selective advantage to establish an efficient infection. We characterized this mechanism in greater detail since it may also have important implications in understanding the humoral immune response during a normal infection cycle in general.7YLPP motif within the N-terminal region -capture ELISA  encompassing an age between 8 and 76 weeks. Since most serological detection methods developed to date are based on the L1re ELISA . Notably68 weeks . Convers76 weeks . In the 76 weeks . Seroreaeen both  and conseen both , developeen both . Conversh  produced via baculovirus expression system  lack neutralizing capability   . NotablySLPASRN)  that forSLPASRN)  and are LONG antibodies lack neutralizing capacity, we dissected the seroresponse against L1 with respect to the 31 amino acids present only at the N-terminus of L1LONG , the number of seropositive animals strikingly increased (SHORT) obtained in a previous study  were performed. For this purpose, a panel of monoclonal antibodies raised against MnPV VLPs was used. These antibodies differ in terms of neutralization and sensitivity in VLP and GST-ELISAs  and denatured-GST-L1 ELISA (281 sera). Interestingly, denaturation of both VLPs and GST-L1 antigens abrogated the reactivity of all sera  were found   . This inSHORT-transfected cells both mAb 2D11 and serum mix detected bands between 100 and 130 kDa and around 250 kDa, which disappeared in lysates treated with additional DTT, β-mercaptoethanol and extended heating prior to SDS-PAGE separation  could be favored over L1SHORT (RI = 0.17)  that have the potential to encode both L1 L1SHORT . Using t L1SHORT , it turn = 0.17) . Indeed,onstruct .stratum corneum) of the epidermis. Here, L1 and L2 are assembled to capsomers and virus particles  of certain human and animal papillomaviruses  potentiaMastomys coucha is a reliable preclinical model that mimics many aspects of the situation found in humans infected with cutaneous HPVs. Since the animals are immunocompetent and become naturally infected early in life  but required its N-terminal truncation   , generatL1SHORT)  and simiL1SHORT) . This L1SHORT cannot be efficiently translated from polycistronic transcripts coding for E1^E4, L2 and L1  at 22+/- 2°C and 55+/- 10% relative humidity in a light/dark cycle of 14/10 hr. Mastomys were fed with mouse breeding diet and allowed access to water ad libitum. For the follow-up experiment, animals were monitored for the duration of their lifetime until they had to be sacrificed due to tumor development or decrepitude. Blood was taken in intervals from 2–8 weeks by puncturing the submandibular vein of anesthetized animals (3% isoflurane), starting at the age of eight weeks.The 2 and 95% humidity and regularly checked for Mycoplasma via PCR. Sf9 and TN-High Five insect cells were kept as described elsewhere , 1% Penicillin/Streptomycin and 1% L-glutamine. Media of HeLaT and 293TT cell were further supplemented with Hygromycin B (125 μg/ml) to maintain additional SV40 large T-antigen expression. All cell lines were kept at 37°C, 5% COlsewhere .Mastomys sera diluted 1:50 in CBB containing GST-SV40-tag were incubated for 1 hr at RT to remove unspecific reaction against bacterial proteins or the GST-SV40-tag fusion protein. Afterwards, ELISA plates were washed four times with PBST and incubated with pre-incubated sera for 1 hr at RT. After washing four times, 100 µl/well HPR-conjugated goat anti-mouse IgG (H+L) antibody  were applied for 1 hr at RT. Antibodies were quantified colorimetrically by incubating with 100 μl/well substrate buffer for 8 min . The enzymatic reaction was stopped with 50 μl/well 1 M sulfuric acid. The absorption was measured at 450 nm in a microplate reader . To calculate the serum reactivity against the respective antigen, sera were tested in parallel against the GST-SV40-tag fusion protein and the reactivity was subtracted from the reactivity against the GST-antigen-SV40-tag. Each ELISA was performed in duplicates at least. The cut-offs were calculated individually for each antigen by measuring sera of virus-free animals.As previously described , glutathSHORT-VLPs were coated overnight in 50 mM carbonate buffer pH9.6 and blocked with CBB the next day. After incubation for 1 hr at RT with three-fold dilutions of sera in CBB, plates were washed four times with PBST and incubated with goat anti-mouse IgG-HRP . After four washes, color development and measurement was performed as described for the GST-ELISA. Antibody titer represents the last reciprocal serum dilution above the blank.VLP-ELISAs were performed as described elsewhere . BrieflySHORT antibodies mAb2E2, mAb2D6, mAb2D11, mAb5E5 and mAb3H8  as controls for denaturation conditions.VLPs and GST fusion antigens were denatured at 95°C for 10 min in coating buffer (50 mM carbonate buffer pH9.6) prior to coating to ELISA plates overnight at 37°C. For the denatured VLP-ELISA, further steps were carried out according to the VLP-ELISA protocol described above. Denatured GST-ELISA antigens were then directly coated onto the ELISA plates overnight at 37°C and further steps  were carried out according to the GST-ELISA protocol. The five monoclonal MnPV anti-L1Mastomys serum mix at a dilution of 1:300 in 10% RBB in PBST. The array was washed three times for 1 min with PBST and then incubated for 1 hr at RT with 0.2 µg/ml 10% RBB in PBST goat anti-mouse IgG (Fc) DyLight680 . Subsequently, the array was washed three times for 1 min with PBST and rinsed with 1 mM TRIS-HCL pH7.4. As peptide controls, HA peptide spots were stained with monoclonal mouse-anti-HA IgG antibody  conjugated with DyLight800 , followed by washing as described above. The antibody was diluted to 1 µg/ml in 10% RBB in PBST and staining was performed for 1 hr at RT in the dark followed by washing as described above. After drying of the array, fluorescence images were acquired with an Odyssey Infrared Imager  at a resolution of 21 µm. Scanner sensitivity was set to 7.0 for the 700 and 800 nm channels respectively, the focal plane was set to +0.8 mm. Quantification of spot intensities, based on 16-bit gray scale tiff files and microarray image analysis, via PepSlide Analyzer . A software algorithm breaks down fluorescence intensities of each spot into raw, foreground and background signal, and calculates averaged median foreground intensities and spot-to-spot deviations of spot duplicates. Averaged spot intensities of the assays with the sample were plotted against the antigen sequence from N- to C-terminus to visualize overall spot intensities and signal-to-noise ratios (intensity plot).The peptide array  was produced as previously described . The amiLONG, L1MIDDLE and L1SHORT), L2 and a reporter plasmid encoding Gaussia luciferase. The 2nd and 3rd ATG of L1LONG and the 2nd ATG of L1MIDDLE were mutated to GCG  to exclusively guarantee L1LONG or L1MIDDLE expression. Transfected cells were incubated for 48 hr, harvested and resuspended in an equal volume of PBS and supplemented with 0.5% Brij 58 (Sigma) and 1% RNase A/T1 mix (Thermo Fisher Scientific). Cells were lysed for 24 hr under rotation at 37°C to allow pseudovirion maturation prior to adjustment with 5 M NaCl to 0.85 M NaCl and treatment with 700 U Benzonase (Merck) for 1 hr at 37°C. For purification, the lysate was transferred on top of a three-step gradient of 27%, 33% and 39% Iodixanol  diluted in 0.8 M NaCl/DPBS. and centrifuged at 37,000 rpm for 5 hr at 16°C in a swinging bucket rotor. Fractions of 500 µl each were collected in siliconized LoBind tubes (Eppendorf) and quantity and quality of pseudovirions in each fraction was assessed by electron microscopy (EM) and Gaussia luciferase reporter activity after infection of HeLaT cells.Pseudovirions were produced as previously described . 293TT c5 HeLaT cells/ml were seeded to the pseudovirion-serum mixture and cultured for 48 hr at 37°C. The activity of secreted Gaussia luciferase was measured 15 min after adding coelenterazine substrate and Gaussia glow juice  according to the manufacturer’s instructions in a microplate luminometer reader . The neutralization titer represents the reciprocal of the highest dilution that reduces the signal by at least 50%.As previously described , animal LONG, L1MIDDLE and L1SHORT were inserted into the Multibac vector pFBDM using EcoRI/HindIII and XmaI/SphI. Comparable to the VLP production, to ensure that only L1LONG and L1MIDDLE are expressed in the Multibac expression system, the 2nd and 3rd ATG start codons of L1LONG and the 2nd ATG of L1MIDDLE were mutated to GCG . The recombinant MultiBac bacmids were generated by electroporation (1.8 kV pulse) of DH10MultiBacCre E. coli with the generated plasmids, followed by selection with antibiotics and blue/white screening  and added dropwise to Sf9 cells. After incubation at 27°C for 5 hr, cells were washed twice and then cultured for 6 days in supplemented TNM-FH medium (Sigma). One ml supernatant was used for generation of a high-titer baculovirus stock by infecting 2 × 106 Sf9 cells in a T25 flask followed by virus amplification for 6 days. This step was repeated with the obtained supernatant two times with increasing cell numbers (3 ml supernatant for 1 × 107 cells in a T75 flask and 5 ml supernatant for 2.5 × 107 cells in a T175 flask). TN-High Five cells were cultivated to a density of 2.5 × 106/ml in 250 ml suspension culture, which were then pelleted and resuspended in 42 ml EX-CELL 405 serum-free medium (Sigma) and 8 ml high titer virus stock. The cells were shaken at a low speed for 1 hr at RT and then incubated within 250 ml final volume of medium for 3 days at 27°C. Cell pellets were harvested by centrifugation  and washed in pre-chilled PBS for two times. Dry pellets were resuspended in 10 ml VLP extraction buffer  containing 200 μl 100 mM PMSF, and then followed by three times sonication. A two-step gradient consisting of 7 ml of 40% sucrose on top of 7 ml CsCl solution was prepared. Clear cell lysate was obtained by centrifugation  and carefully loaded onto the top of the CsCl layer. After centrifugation , the interphase between sucrose and CsCl together with the complete CsCl layer was transferred into a Quickseal tube. The fractions were collected in 1 ml aliquot after 16 hr centrifugation at 48,000 rpm at 20°C in a Beckman 70Ti rotor and analyzed by Coomassie blue dye and Western blot. Small aliquots from the fraction with highest and lowest protein yield were dialyzed against H2O on a membrane filter and analyzed by EM.Recombinant baculoviruses were generated as previously described  with som2, 2% sucrose in 100 mM calcium cacodylate, pH7.2), followed by post-fixation in buffered 1% OsO4, graded dehydration with ethanol and resin-embedding in epoxide . Ultrathin sections at nominal thickness 60 nm and contrast-stained with lead-citrate and Uranylacetate were observed in a Zeiss EM 910 at 100 kV  and micrographs were taken with image-plates, scanned at 30 µm resolution .VLP and PsV preparations or tissue were fixed with buffered aldehyde solution  and the ORFs were cloned downstream of an artificial Kozak sequence. Alternatively, the complete genuine viral L1 ORF encoding start codons and Kozak sequences of all isoforms was cloned and termed pPK-CMV_MnPV-vL1.Variants of L1 ORFs were cloned into pPK-CMV-E3 expression plasmids. For exclusive and strong eukaryotic expression of the respective L1 isoform, viral codons were humanized, unwanted start codons mutated  according to the manufacturer’s protocol. Cells were collected 48 hr after transfection, washed in PBS and lysed for 30 min on ice in 1.25x Laemmli buffer . Lysates were then heated at 95°C for 5 min, chilled on ice and treated with 100 U/ml Benzonase (Millipore) for 5 min at RT. Protein concentrations were measured using a NanoDrop spectrophotometer. Forty μg lysate/lane were loaded to 8% SDS-PAGE. To guarantee complete denaturation of the samples, additional DTT and β-mercaptoethanol were added to a final concentration of 100 mM and 4%, respectively, prior to incubation for 1 hr at RT and heating at 95°C for 10 min. After blotting, proteins were detected with anti-HA , anti-vinculin , anti-MnPV-L1SHORT  or Mastomys serum mix (1:1000) prior to detection with goat anti-mouse-HRP  or goat anti-rat-HRP . For Coomassie staining, gels were incubated overnight in Coomassie stain and then destained in 20% methanol.MaFi132 cells  were transfected with 5 µg pPK-CMV_MnPV-L1SHORT , Mastomys serum mix (1:1000) or anti-HA  and the respective secondary goat anti-mouse or donkey anti-rat IgG . Nuclei were stained with DAPI. Cover slides were mounted with Faramount Aqueous Mounting Medium (Dako) and imaged with a Cell Observer (Carl Zeiss).MaFi132 cells were transfected with L1 isoforms as described above and seeded on glass cover slides after 24 hr. Additional 48 hr later the cells were washed with PBS and fixed for 10 min in 4% PFA. Cells were blocked in 10% goat serum/0.3% Triton X-100 in PBS for 1 hr and stained with anti-MnPV-L1Mastomys (LONG in the OVX313 platform (2O2 substrate solution (Sigma) was stopped with distilled water followed by counterstaining with hemalum solution . Fluorescence stainings were detected with anti-mouse-IgG1-Alexa594 or anti-guinea-pig-Alexa488 (Invitrogen) and nuclei were stained with DAPI. Sections were mounted with Dako Faramount Aqueous Mounting Medium.Staining of formalin-fixed, paraffin-embedded tumors was performed as previously described . BrieflyMastomys , serum oplatform  or the rplatform  or serumLONG- and L1SHORT- positive animals was calculated and compared with a two-tailed McNemar’s test at an alpha level of 5% to assess significance.Data analyses and graphic representations were performed with GraphPad Prism 6.0 Software and the respective statistical test indicated in the figure legends at 95% confidence interval and an alpha level of 5% to assess significance. For time course analyses, individual time points were compared to the eight-week starting time point. The rate of L1 In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Mastomys. Three isoforms of L1 can be expressed L1 long, L1 middle, L1 short. This study and others confirm that these isoforms are differentially expressed both temporally and spatially; only L1 short is expressed late in the infectious cycle in the upper layers of the epithelium and elicits neutralising antibody. L1 long and middle are expressed early in the infectious cycle in the lower epithelial layers but induce predominantly non neutralising antibody. The authors propose that L1 long and middle antibody responses are a viral immune evasion strategy. While no mechanisms for this are described, the study is well-conceived, the findings are novel and unexpected and will provoke interest and further experimentation.This study investigates the antibody response to the major coat protein L1 throughout the life cycle of a natural papillomavirus infection the multimammate mouse Decision letter after peer review:Mastomys natalensis papillomavirus as mechanism to circumvent adaptive immunity" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Margaret Stanley as the Reviewing Editor and Reviewer #3, and the evaluation has been overseen by Satyajit Rath as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Neil Christensen (Reviewer #2).Thank you for submitting your article "Expression of different L1 isoforms of The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:Mastomys. Three alternative translation initiation codons of the L1 ORF are found in Mastomys. Depending upon which ATG is used three isoforms of L1 can be expressed L1 long, L1 middle, L1 short. This study and others confirm that these isoforms are differentially expressed both temporally and spatially; only L1 short is expressed late in the infectious cycle in the upper layers of the epithelium and elicits neutralising antibody. L1 long and middle are expressed early in the infectious cycle in the lower epithelial layers but induce predominantly non neutralising antibody. The authors propose that L1 long and middle antibody responses are a viral immune evasion strategy but no mechanisms for this are described. However the study is well-conceived, the findings are novel and unexpected and will provoke interest and further experimentation.This study investigates the serological response to the major coat protein L1 throughout the life cycle of a natural papillomavirus infection the multimammate mouse Revisions:eLife but the authors should address the following comments and clearly identify in the text that their assertions that the responses they observe are an immune evasion mechanism is a speculation not an established fact.The reviews are favourable and the paper can be published in 1) In the correlation analyses in Figure 3 and 4, please indicate the animal's age for the sera used.2) In Figure 4B, there appear to be only 8 data points, while the figure legend states that the sera from 60 animals were evaluated. Please reconcile.3) Subsection “Mapping of immunodominant epitopes in MnPV L1”: Why were sera from tumor bearing animals used here? Is there any data on whether tumor-bearing and tumor free animals differ in virus antibody responses?4) Figure 6 legend, last sentence: change to "GST-L1LONGaa1-41”, as indicate correctly in the figure.5) Discussion paragraph seven: the N-terminus in long L1 must do more that block capsomer assembly because capsomers can induce neutralizing antibodies but long L1 cannot. It must induce a remarkable change in L1 monomer conformation.6) Discussion paragraph nine: it would be helpful to clarify whether "cutaneous papillomaviruses” refers to just MnPV/MmuPV or also skin HPVs.7) What are the authors views as to why a long/middle L1 is not capable of triggering a neutralizing ab response?8) Figure 10B is not of publishable quality Revisions:The reviews are favourable and the paper can be published in eLife but the authors should address the following comments and clearly identify in the text that their assertions that the responses they observe are an immune evasion mechanism is a speculation not an established fact.Following this advice, we changed our conclusions accordingly:“In conclusion, it is tempting to speculate that our results show that early synthesis of alternative immunogenic L1 isoforms represents a novel mode of humoral immune escape mechanism, favoring persistent infections and viral spread due to a delay of immune recognition by the host.”1) In the correlation analyses in Figure 3 and 4, please indicate the animal's age for the sera used.As already stated in Figure 3, all 682 sera were used and therefore represent the complete range of the animal age. However, we added the information:“All graphs include the analysis of all 682 sera taken during the study”.The correlation analyses in Figure 4 contain fewer animals that nevertheless again represent the complete age range. We added this information:“Correlation analyses contain sera from animals representing the complete age range”.2) In Figure 4B, there appear to be only 8 data points, while the figure legend states that the sera from 60 animals were evaluated. Please reconcile.We understand the confusion which is attributable to a technical issue: The 60 sera were applied in serial dilution steps of one to three in both Pseudovirion-based Neutralization Assay (PBNA) and VLP-ELISA. Since the titers represent the last reciprocal serum dilution above the blank (for VLP-ELISA) and the reciprocal of the highest dilution that reduces the signal by at least 50% , several sera end up at the same titer. In the graph showing the correlation between the VLP-ELISA and the neutralization assay , these 60 data points subsequently overlay, ending up with only eight visible points. However, to avoid confusion, we added following statement to the legend of Figure 4:“Please note, that for both assays all 294 sera were diluted in three-fold dilution steps. Since the titers were calculated from the dilution, data points of different sera overlay when having the same titer in both assays.”Furthermore, we also added the following sentence in the Materials and methods section.“The neutralization titer represents the reciprocal of the highest dilution that reduces the signal by at least 50%.”3) Subsection “Mapping of immunodominant epitopes in MnPV L1”: Why were sera from tumor bearing animals used here? Is there any data on whether tumor-bearing and tumor free animals differ in virus antibody responses?LONG/SHORT and the presence of skin lesions in which infectious progeny viruses are formed.The answer of the latter question is “yes” and this was the reason why sera from tumor-bearing animals were used. As shown in our previous studies , there is a quite obvious relationship of the higher levels of antibodies against L14) Figure 6 legend, last sentence: change to "GST-L1LONGaa1-41”, as indicate correctly in the figure.LONGaa1-41 accordingly.We apologize for the typo and changed it to GST-L15) Discussion paragraph seven: the N-terminus in long L1 must do more that block capsomer assembly because capsomers can induce neutralizing antibodies but long L1 cannot. It must induce a remarkable change in L1 monomer conformation.SHORT obviously leads to a distinct folding of L1LONG, thereby forming a new conformational epitope which is not present in L1SHORT-derived VLPs and natural virions that do not induce such antibodies upon immunization or infection .”Yes, we mentioned that point already: “Hence, the addition of N-terminal residues to L1To emphasize this finding, we included the following statement: “Additionally, the extended N-terminus not only inhibits assembly, but also causes a complete new folding of L1, thereby exposing a novel immunogenic conformational epitope that induces non-protective antibodies”.6) Discussion paragraph nine: it would be helpful to clarify whether "cutaneous papillomaviruses” refers to just MnPV/MmuPV or also skin HPVs.We added this information:“Indeed, in contrast to mucosal types , cutaneous papillomaviruses  initiate their late functions in the lower epithelial regions ”.7) What are the authors views as to why a long/middle L1 is not capable of triggering a neutralizing ab response?As already stated (see point 5), our data suggest that an extended N-terminus abrogates L1 self-assembly and prevents the formation of repetitive, highly immunogenic structures usually found on native capsids. Consequently, an extended N-terminus of L1 leads to an alternative dominant immunogenic conformation, which is not present in infectious particles. Therefore, antibodies raised against this structure have no neutralization capability when PsVs as equivalents for mature MnPV virions were tested.8) Figure 10B is not of publishable qualityOf course, this is due to embedding the figure in a.pdf-file. For publication, an original.tiff-file will be provided.


Cytoplasmic aggregation of TDP-43 characterizes degenerating neurons in most cases of amyotrophic lateral sclerosis (ALS). Here, we develop an optogenetic TDP-43 variant (opTDP-43), whose multimerization status can be modulated in vivo through external light illumination. Using the translucent zebrafish neuromuscular system, we demonstrate that short-term light stimulation reversibly induces cytoplasmic opTDP-43 mislocalization, but not aggregation, in the spinal motor neuron, leading to an axon outgrowth defect associated with myofiber denervation. In contrast, opTDP-43 forms pathological aggregates in the cytoplasm after longer-term illumination and seeds non-optogenetic TDP-43 aggregation. Furthermore, we find that an ALS-linked mutation in the intrinsically disordered region (IDR) exacerbates the light-dependent opTDP-43 toxicity on locomotor behavior. Together, our results propose that IDR-mediated TDP-43 oligomerization triggers both acute and long-term pathologies of motor neurons, which may be relevant to the pathogenesis and progression of ALS. Optogenetic approaches for inducing TDP-43 aggregation have been described previously in cellular models. Here the authors develop an approach to optogenetically induce TDP-43 aggregation in vivo using zebrafish to model ALS pathologies. TDP-43 aggregation characterizes almost all cases of sporadic ALS4, which accounts for greater than 90% of ALS. Moreover, mutations in the TARDBP gene encoding TDP-43 are linked to certain fraction (~4%) of familial ALS5. Despite its correlation with and causation of ALS, the role of TDP-43 in ALS pathogenesis has been largely unknown at the mechanistic level.Amyotrophic lateral sclerosis (ALS) is a neurological disorder in which the upper and lower motor neurons progressively degenerate, leading to muscular atrophy and eventually fatal paralysis. Trans-activation response element (TAR) DNA-binding protein 43 (TDP-43), a heterogeneous nuclear ribonucleoprotein, is mislocalized to the cytoplasm and forms pathological aggregates in the degenerating motor neurons in ALS8. At the C-terminus TDP-43 contains an intrinsically disordered region (IDR) with prion-like glutamine/asparagine-rich (Q/N) and glycine-rich regions, which can undergo liquid–liquid phase separation (LLPS) to form dynamic protein droplets9. The TDP-43 IDR mutations that are linked to familial ALS cases enhance intrinsic aggregation propensity and protein stability of TDP-4311 and result in altered phase separation9, which could contribute to disease propagation through acceleration of the formation and accumulation of pathological aggregates14. The modular architecture of TDP-43 has led to several hypotheses that its N-terminus-dependent oligomerization modulates C-terminal IDR-mediated aggregation either by enhancing15 or hindering IDR interactions between adjacent TDP-43 molecules16.Multimerization of TDP-43 underlies its physiological and pathological roles. Under normal physiological conditions, homo-oligomerization of TDP-43 occurs through its N-terminal domain and is necessary for its RNA regulatory functions, such as splicing26. However, cytoplasmic TDP-43 aggregation is not always detectable in these models. Moreover, in a certain type of degenerating upper motor neurons, loss of nuclear TDP-43 can occur without the accumulation of cytoplasmic aggregates27. Therefore, it has been difficult to evaluate how TDP-43 aggregation contributes to TDP-43 toxicity. Under these circumstances, it is necessary to develop a system to induce TDP-43 aggregation conditionally. Recently, light-dependent aggregation of Arabidopsis cryptochrome-2 was applied to the formation of IDR droplets via LLPS in a light illumination-dependent manner28. This optogenetic approach has been successfully extended to the induction of cytotoxic TDP-43 aggregates formation in cultured cells30. However, interconversion of normal and toxic TDP-43 forms with spatiotemporal precision has not been achieved in animal models yet, which is central for the understanding of TDP-43 toxicity in vivo.The severity of TDP-43 toxicity is correlated with the levels of wild-type and mutant TDP-43 expression in the various cellular and animal modelsIn the present study, we develop an optogenetic TDP-43 variant (opTDP-43) carrying a light-dependent oligomerization module of cryptochrome-2 attached to the IDR, and analyze the mechanisms of TDP-43 toxicity in spinal motor neurons in vivo. Transgenic expression and light stimulation of opTDP-43 in transparent zebrafish larvae show that oligomerization and aggregation of opTDP-43 is inducible and tunable in vivo by external light illumination. We reveal that, in the spinal motor neurons, short-term light illumination reversibly increases the cytoplasmic opTDP-43 pool and elevates myofiber denervation frequency in the absence of distinct aggregate formation. Furthermore, longer chronic light stimulation eventually leads to accumulation of cytoplasmic opTDP-43 aggregates that further seed aggregation of non-optogenetic TDP-43, which is accompanied by motor decline. The sequential pathological alterations of spinal motor neurons triggered by opTDP-43 oligomerization may provide clues about how motor neuron degeneration progresses at both molecular and cellular levels in a prodromal phase of ALS.31. We generated a Gal4-inducible transgene of the zerbafish tardbp, encoding one of the two zebrafish TDP-43 paralogues, tagged with mRFP1 at its N-terminus (mRFP1-TDP-43z)  of zebrafish, which innervate a ventral third of the myotome and are present uniquely in every spinal hemisegment Fig. 31. We ge3z) Fig. . To testhpf Fig.  and theihpf Fig. , showinghpf Fig. . These o10. Therefore, we reasoned that cytoplasmic TDP-43 aggregation might effectively occur when the proximity between IDRs was increased by addition of an exogenous multimerization tag, such as Arabidopsis cryptochrome CRY2. We first assessed the oligomerization capacity of mRFP1-tagged CRY2olig in the skeletal muscle cells by taking advantage of their relatively large nucleus and cytoplasm. We created a transgenic zebrafish line carrying a fusion of mRFP1 and CRY2olig, a point mutant version of CRY2 (E490G) that exhibits significant clustering upon blue light illumination34, under the UAS sequence   Fig. . When exNext, we tested the feasibility of CRY2olig oligomerization in spinal motor neurons in vivo. When driven by TgSAIG213A] driver, mRFP1-CRY2olig was dispersed throughout the CaPs at 30 hpf under dark conditions   cells, by combining both Tg[mnr2b-hs:Gal4]ish Fig. . Upon blish Fig. , in contish Fig. . The grava) Fig. . Unexpecva) Fig. , the cytTo explore the impact of light-induced cytoplasmic opTDP-43z mislocalization at the whole cell level, we restricted opTDP-43z expression to CaPs by using TgSAIG213A] driver. We devised a protocol by which Tg[SAIG213A] Tg[UAS:EGFP] Tg[UAS:opTDP-43z] triple transgenic fish were raised under continuous dark conditions until 48 hpf except being illuminated for 3 h during 28–31 hpf Fig. . Under t3A drivermnr2b promoter, and established a Tg[SAIGFF213A] Tg[UAS:opTDP-43z] Tg[mnr2b-hs:EGFP-TDP-43z] triple transgenic fish. Under dark conditions at 28 hpf, both opTDP-43z and EGFP-TDP-43z exhibited nuclear localization with subnuclear distribution patterns similar to each other   Fig. . We shifCT) Fig. . Live imCT) Fig.  and formCT) Fig. , indicatCT) Fig. . On the CT) Fig. . RemarkaCT) Fig. , and 44%CT) Fig. , demonst37. We found that, in both wild-type and opTDP-43z expression conditions prior to light stimulation, the DCCT axon terminals were decorated by Vamp2-Venus, and the Vamp2-Venus signals were well colocalized with dT-chrnd , respectively Fig. 37. We fornd Fig. , indicatrnd Fig. , demonstrnd Fig. , left. Omnr2b-BAC transgene (Tg[mnr2b-hs:opTDP-43h])  from an h]) Fig.  and estah]) Fig. . We founh]) Fig. . The proh]) Fig. . These oh]) Fig.  left. Uph]) Fig. , showingh]) Fig.  right, e38. To prove contribution of the IDR to oligomerization-dependent toxicity of opTDP-43h, we created an opTDP-43 mutant with an IDR mutation (A315T) linked to familial ALS (opTDP-43hA315T), occurring in the residue implicated in irreversible aggregation13, and expressed opTDP-43hA315T widely in the spinal motor neurons from Tg[mnr2b-hs:opTDP-43hA315T]  and autophagy43. Since the light-dependent cytoplasmic opTDP-43 mislocalization commences without affecting non-optogenetic TDP-43 localization, the toxicity accompanied by opTDP-43 mislocalization may not be caused by a global shutdown of nucleocytoplasmic transport or proteolysis systems for TDP-43, but rather by dysregulation of RNAs and/or proteins being bound by opTDP-43. TDP-43 can associate with more than 6,000 RNA targets47, and RNA-binding is antagonistic to toxic TDP-43 oligomerization in an optogenetic cellular model29, implying that light-dependent opTDP-43 oligomerization would profoundly affect its RNA-binding capacity, thereby influencing the expression of a myriad of genes. In terms of the axon outgrowth defect, whether toxicity is ascribable to dysregulation of specific key proteins48 or to a widespread translational abnormality49, which could lead to stress-inducing misfolded protein accumulation, remains to be investigated. Nevertheless, the normal motor axon pathfinding and unaffected branching frequency suggest a certain specificity of opTDP-43 toxicity and would favor the idea that the cellular growth pathway is primarily affected by the toxicity. It should also be noted that our current illumination paradigm encompasses not only the somas but also the motor axons. Therefore, “resident” cytoplasmic opTDP-43, such as that included in mRNP granules undergoing axonal transport50 and in mitochondria at the axon terminals52, could also be photoconverted in situ, thereby contributing to the acute toxicity that involves neuromuscular synapse destabilization. Spatially-resolved light stimulation, a major advantage of optogenetics, could identify such potentially multiple pathogenic origins in the future.We discovered that the reversible cytoplasmic opTDP-43 mislocalization induced by short-term light illumination was sufficient to cause defective motor axon outgrowth accompanied by enhanced myofiber denervation. The physiological nuclear-enriched TDP-43 localization is sustained by nucleocytoplasmic transport system53 emphasize the importance of studying vulnerable cell types in vivo for accurately disclosing the mechanisms underlying TDP-43 localization, and thereby toxicity.The light-dependent cytoplasmic opTDP-43 mislocalization provides unexpected insight into the relationships between TDP-43 multimerization, localization, and toxicity, given that a toxic level of mRFP1-TDP-43 overexpression led neither to cytoplasmic mislocalization nor aggregation. The persistent nuclear localization of overexpressed mRFP1-TDP-43 indicates a robustness of the nucleocytoplasmic transport and cytoplasmic protein degradation systems against proteostatic perturbation of TDP-43. On the other hand, these TDP-43 surveillance systems appear to be inert against the IDR-mediated TDP-43 oligomers, as evidenced by the light-dependent cytoplasmic opTDP-43 mislocalization. These manipulations of proteostasis and multimerization revealed that a dosage increase of TDP-43 does not immediately lead to IDR-mediated oligomerization in the spinal motor neurons, and therefore TDP-43 toxicity associated with proteostatic abrogation could be mechanistically distinct from that caused by IDR-mediated oligomerization. Importantly, our animal model approach explicitly revealed a striking cell-type variation of opTDP-43 mislocalization and aggregation, such that the neuronal cells are less prone to accumulate opTDP-43 aggregates compared to the differentiating muscle cells, while both of these cell types are inherently more competent for cytoplasmic mislocalization than the epithelial cells. Although the mechanisms underlying this cell-type specificity remain unknown, the present and previous observationsA315T that was expressed less and formed fewer cytoplasmic aggregates was more toxic than the opTDP-43h expressed in larger amounts , TDP-43 displays distinct aggregate assemblies and toxic effects in disease-subtype-specific manners54. Consistent with these results, we found that cytoplasmic opTDP-43 aggregates were heterogeneous as the phosphorylation at S409/410 and recruitment of G3BP and TIAL proteins occurred with varying degrees. Although TDP-43 toxicity can be independent of phosphorylation at S409/41029 and recruitment of SG components55 in cellular models, the toxicity of opTDP-43 aggregates should be evaluated by considering such heterogeneity, and therefore remains as a challenging but important question to be addressed. Alternatively, the seeding capacity for non-optogenetic TDP-43 aggregation suggests that the toxicity of opTDP-43 aggregates can be exerted as a long-term effect through gradual depletion of the available nuclear and cytoplasmic TDP-43 pools into these aggregates, which would manifest as a TDP-43 loss-of-function phenotype55.The verification that CRY2olig-mediated opTDP-43 oligomerization is toxic to the spinal motor neurons instead made it difficult to evaluate the toxicity derived from opTDP-43 aggregates alone, as the oligomeric and aggregate forms of opTDP-43 coexists during illumination. In this regard, it is noteworthy that the opTDP-43hnts Fig. , providi58. As a result, the surviving motor units are enlarged to preserve maximal force-generating capacity by compensatory collateral reinnervation59. ALS has also been characterized by an elevated number of muscle fibers innervated by a single subterminal axon60, which is likely a remnant of such compensatory collateral reinnervation events. In the present study, we found by live imaging of axon collateral that an innervation territory of healthy spinal motor neurons is determined by a balance between assembly and disassembly of neuromuscular synapses in zebrafish. We further discovered that optogenetic opTDP-43 oligomerization could tip the balance toward disassembly and decrease the total collateral length. Therefore, our results predict that, once a cellular concentration of IDR-mediated TDP-43 oligomers reaches a critical level, a spinal motor neuron would begin to reduce its motor unit size through repetition of incomplete denervation/reinnervation cycles. Such neurons would also be defective in complementing damaged neighboring motor units through collateral reinnervation, which would accelerate the manifestation of motor decline. We envision that opTDP-43 allows for approaching the mechanisms underlying such dynamic innervation/reinnervation balancing of spinal motor axons in health and TDP-43-associated pathology, as well as for interrogating how not only motor neurons but also diverse types of surrounding cells, including muscle, glial and endothelial cells, respond to and modify TDP-43 toxicity. Moreover, combined with the feasibility of high-throughput, whole organism chemical screening in zebrafish, opTDP-43-mediated motor neuron pathogenesis should be extended for exploring small molecules that restore a normal denervation/reinnervation balance for spinal motor neurons, which might serve as drugs for ALS and other TDP-43 proteinopathy.It has been estimated that, even during healthy aging, the spinal motor neurons are substantially lostThis study was carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the Institutional Animal Care and Use Committee  of the National Institute of Genetics , which has an Animal Welfare Assurance on file (assurance number A5561-01) at the Office of Laboratory Animal Welfare of the National Institutes of Health . Fish were raised under 12:12 light-dark (L/D) cycles during the first 5 days after birth, unless otherwise stated.Tol2 transposon-based cassette (UAS:mRFP1-TDP-43z) carrying the zebrafish tardbp (Genbank accession # NM_2014476) that was tagged with mRFP1 (Genbank accession # AF506027.1) at the N-terminus without linker and placed downstream of x5 upstream activation sequence (UAS)61 . For the construction of Tg[UAS:mRFP1-CRY2olig], the zebrafish codon-optimized photolyase homology region (PHR) of Arabidopsis thaliana CRY2 carrying the E490G mutation (CRY2olig)34 was synthesized (Biomatik) and N-terminally tagged with mRFP1 with a linker peptide TRDISIE encoded by ACG CGT GAT ATC TCG ATC GAG (mRFP1-CRY2olig). The mRFP1-CRY2olig fragment was fused to 5xUAS, cloned into the Tol2-transposon cassette. For the construction of Tg[UAS:opTDP-43z], the mRFP1-TDP-43z fragment was C-terminally fused to CRY2olig with the linker peptide TRDISIE (opTDP-43z). The opTDP-43z fragment was fused to 5xUAS, cloned into the Tol2-transposon cassette. NEBuilder HiFi DNA Assembly Master Mix was used for the vector construction. For the generation of Tg[mnr2b-hs:EGFP-TDP-43z], EGFP was directly fused to zebrafish tardbp gene (EGFP-TDP-43z) and the resulting EGFP-TDP-43z was linked to the hsp70l promoter (650 bp) and introduced into downstream of the mnr2b 5′UTR in the mnr2b-BAC DNA  via homologous recombination using a Kmr-resistance as essentially described62. Tg[mnr2b-hs:opTDP-43h] and Tg[mnr2b-hs:opTDP-43hA315T] lines were generated by the same procedure except that opTDP-43h and opTDP-43h A315T were used, respectively, instead of EGFP-TDP-43z. The opTDP-43h consists of the zebrafish-codon-optimized human TDP-43 that is fused directly to the zebrafish-codon-optimized mRFP1 at the N-terminus and indirectly to CRY2olig via the linker peptide TRDISIE. opTDP-43hA315T is identical to opTDP-43h except the A315T mutation (GCT>ACT). All transgenic lines were created via Tol2-mediated transgenesis.Tg[UAS:mRFP1-TDP-43z] was generated by synthesizing a h2afva encoding a histone H2A variant (NM_153644.1) was C-terminally tagged with EGFP or mRFP1 with a linker nucleotide (GGA GGC TCG AAT CTC GAG) and cloned into the UAS vector pT2MUASMCS33 to generate pUAS:h2afva-EGFP and pUAS:h2afva-mRFP1, respectively. Twenty-five pg of these plasmids were injected into the embryos at the one-cell stage.The open reading frame of tardbp and tardbpl knockout fish, target sequences for Cas9-mediated cleavage were searched by CRISPRscan63. The target sequences CAAGACTTAAAAGACTACTTcgg and CAAGACTTAAAAGACTACTTcgg, where the protospacer adjacent motifs (PAMs) are indicated by lower cases, were chosen for the generation of tardbp-n115 and tardbpl-n94 alleles, respectively. hSpCas9 was in vitro-transcribed with mMESSAGE mMACHINE Kit  by using pCS2 + hSpCas9 plasmid as a template . Wild type embryos were injected with 25 pg of sgRNA and 300 pg of hSpCas9 mRNA at the one-cell stage.For the generation of tardbp (TDP-43z), zebrafish-codon optimized human TDP-43 (TDP-43h), mRFP1-tagged zebrafish tardbp (mRFP1-TDP-43z) and opTDP-43z was cloned into pCS2+ vector in vitro-transcribed with mMESSAGE mMACHINE Kit. First, we injected varied amount of TDP-43z mRNA into the offspring obtained from incrosses of parental zebrafish carrying homozygous tardbp-n115 and heterozygous tardbpl-n94 mutation or heterozygous tardbp-n115 and homozygous tardbpl-n94 mutation at the one cell stage. After investigating the presence or absence of blood flow at 36–48 hpf, all fish were subjected individually to genotyping for tardbp-n115 and tardbpl-n94 alleles. The uninjected tardbp-n115 tardbpl-n94 double homozygotes displayed a swollen heart that was beating, but the blood cells were completely stacked on the yolk surface and cannot reach the heart. An injection of 300 ng of TDP-43z mRNA was the most effect effective in restoring blood flow (up to 40% of the double homozygotes) of the double homozygotes with a minimum developmental abnormality due to overexpression. Throughout the assay, we scored that the blood flow was “rescued” when any blood cell flowing through the beating heart was observed. The function of TDP-43h and TDP-43 derivatives were tested by the microinjection of 300 ng mRNA each. The tardbpl-n115 allele was identified by performing Heteroduplex Mobility Assay (HMA) against PCR product obtained with a primer pair: tardbp-6F3 (5′-gcc aga taa taa gag gaa gat gga-3′) and tardbpl-6R3 (5′-tga cag tac aaa gac aaa cac cac-3′). The tardbpl-n94 allele was similarity identified by using a primer pair: tardbpl-4F2 (5′-caa tca ctg aat gaa tgc act ttt-3′) and tardbpl-4R2 (5′-gtt tgc tta tac taa cct gca cca-3′).For the expression of human and zebrafish TDP-43 and its derivatives via mRNA injection, the open reading frames of zebrafish 2. For longer-term illumination, fish were raised in 6-well dish  with 8 ml E3 buffer, and the dish was placed on a blue LED panel. The intensity of blue LED light that reached the E3 buffer was ~0.69 mW per cm2 and its wavelength peaked at 456 nm.Short-term (<4 h) light stimulations of mRFP1-CRY2olig and opTDP-43z were carried out by embedding fish in the 0.8–1% low-melting agarose  and conducting confocal scanning with the laser with 473 nm wave length using an Olympus FV1200 microscope. The average optical power of the confocal laser was ~44.66 µW per cmZebrafish larvae were fixed for overnight in PBT  containing 4% paraformaldehyde . After wash with PBT, fish were incubated at 70 °C in 150 mM Tris-HCl, pH 9) for 15 min, permeabilized in PBT with 0.025% trypsin and 0.01% EDTA for an hour on ice and treated with antibodies after an hour of blocking with PBT with 1% bovine serum albumin, 2% normal goat serum and 1% dimethyl sulfoxide. For the mono- and poly-ubiquitinated protein staining, Tg[SAGFF73A] Tg[UAS:opTDP-43z] fish at 31.5 hpf that had illuminated with a blue light were taken out from the agarose and immediately subjected to immunofluorescence. The mouse monoclonal antibody for mono- and polyubiquitinate conjugates  and goat anti-mouse IgG Alexa Fluor 488  were used as primary and secondary antibodies, respectively. For the detection of opTDP-43z and opTDP-43h, the rabbit anti-RFP polyclonal antibody  and goat anti-rabbit IgG Alexa Fluor 633  were used as primary and secondary antibodies, respectively. Mouse anti-phospho TDP-43 (pS409/410)  and mouse anti-human G3BP antibody  were detcted by goat anti-mouse IgG Alexa Fluor 488 . Rabbit anti-TIAL1 antibody  was detected by goat anti-rabbit IgG Alexa Fluor 488 .z-axis and analyzed with Olympus Fluoview Ver2.1b Viewer and Image J, and processed for presentation with Adobe Photoshop CS6. The axon length and branching frequency were measured by Imaris Filament Tracer. Morphological analyses of CaPs were restricted to the spinal segment 14–17 before 50 hpf and to 13–17 during 56–72 hpf. A neurite with more than 5 µm of length was counted as branch.A fluorescence stereomicroscope MZ16FA, Leica) equipped with a CCD camera  was used to observe the whole fish . Photobleaching of opTDP-43h was conducted by scanning of a region of interest (ROI) set in the nucleus or cytoplasm, which was determined using EGFP-TDP-43z signal as a reference, with 599-nm laser at 100% intensity for 5 s. Fluorescence recovery was monitored at 10 min intervals for 30 min. For nuclear opTDP-43h bleaching (Dark), the shape of the nucleus was determined based on EGFP-TDP-43z signal and the opTDP-43h signal included in the nuclear ROI was used to estimate the photobleaching due to the post-bleach imaging for fluorescence recovery. Similarly, for cytoplasmic opTDP-43h foci bleaching (BL), cells with at least two distant cytoplasmic foci were chosen, and the one that was not beached was used to estimate the photobleaching during the post-bleach imaging for fluorescence recovery. The recovery of opTDP-43h signal was determined by subtracting the signal reduction caused by the post-bleaching imaging.A315T] (A315T) larvae at 72 hpf (17 larvae each) by homogenizing in 1 ml of Trizol Reagent (Life Technologies). Three µg of the total RNA is used for cDNA synthesis using oligo dT . opTDP-43h and opTDP-43hA315T were detected by a primer pair against the zebrafish codon-optimized mRFP1: zmRFP1-123f (5′-TCA GAC AGC TAA ACT GAA GGT CAC-3′) and zmRFP1-633r (5′-GAC GAT GGT ATA GTC TTC GTT GTG-3′). EGFP-TDP-43z was detected by a primer: EGFP-f2s (5′-CAC ATG AAG CAG CAC GAC TTC T-3′) and EGFP-r5s (5′-ACG TTG TGG CTG TTG TAG TTG T-3′). zfand5b expression was detected by a primer pair: zfand5b–133f (5′-ATA GTA CAC ACC GAA ACG GAC AC-3′) and zfand5b-772r (5′-TTA TAT TCT CTG GAT TTT ATC GGC-3′)The total RNA was prepared from Tg[mnr2b-hs:EGFP-TDP43z] (none), Tg[mnr2b-hs:EGFP-TDP43z] Tg[mnr2b-hs:opTDP-43h] (opTDP-43), and Tg[mnr2b-hs:EGFP-TDP43z] Tg[mnr2b-hs:opTDP-43hA315T, and EGFP-TDP-43z in the individual ROI were measured by Image J software.The z-stack images of the spinal motor column were first created by Sum Slices function of Image J. Then, the ROIs covering the somas of individual spinal motor neurons were determined based on the contour of weak cytosolic fluorescence of EGFP-TDP-43z that became clearly visible by signal enhancement. Fluorescence intensities of opTDP-43h, opTDP-43hStatistical analyses were performed using GraphPad Prism Software. Micrographs shown are the representative of results from at least three independent animals (except in Fig. Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Reporting summary