TAR DNA-binding protein 43 (TDP-43) is a protein that is involved in the pathology of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). In patients with these neurodegenerative diseases, TDP-43 does not remain in its normal nuclear location, but instead forms insoluble aggregates in both the nucleus and cytoplasm of affected neurons.We used high density peptide array analysis to identify regions in TDP-43 that are bound by TDP-43 itself and designed candidate peptides that might be able to reduce TDP-43 aggregation. We found that two of the synthetic peptides identified with this approach could effectively inhibit the formation of TDP-43 protein aggregates in a concentration-dependent manner in HeLa cells in which a mutated human TDP-43 gene was overexpressed. However, despite reducing aggregation, these peptides did not reduce or prevent cell death. Similar results were observed in HeLa cells treated with arsenite. Again we found reduced aggregation, in this case of wild type TDP-43, but no difference in cell death.Our results suggest that TDP-43 aggregation is associated with the cell death process rather than being a direct cause. Recent evidence links TDP-43 pathology to at least two forms of neurodegeneration that had heretofore been thought to be quite separate. FTLD is the second most common type of early-onset neurodegenerative dementia after Alzheimer’s disease, and ALS is the most common adult-onset progressive motor neuron disease (MND). The TAR DNA binding protein 43 (TDP-43) has been found to be the major protein constituent of the intracellular aggregated inclusions in both FTLD with ubiquitin-positive inclusions (FTLD-U) and ALS ,2.TDP-43 is a 414 amino acid protein encoded by the TARDBP gene on chromosome 1. It was originally identified as a transcriptional repressor of the human immunodeficiency virus type 1 (HIV-1) gene . TDP-43 The mechanism through which TDP-43 is involved in neuronal death and degeneration remains unclear. One of the best characterized pathological features of TDP-43 proteinopathies is the cytoplasmic inclusions of TDP-43 aggregates. As with other protein misfolding diseases, TDP-43 mediated toxicity may result from a toxic gain of function associated with its aggregation. Johnson and colleagues have established a yeast model involving overexpressed full-length human TDP-43 or various TDP-43 truncation products . They foTo further elucidate the links between TDP-43 aggregation and disease mechanisms, we used two different models. The first was an aggregation model pioneered by Nonaka and colleagues . They foThe protein array described in the Methods section was used to identify candidate binding regions at which TDP-43 might interact with itself. Given the nature of the overlapping sequences associated with each neighboring peptide (frame shift of 2 aa), only positive spots in a series were considered as possible binding sites. Five separate and robust binding regions were found on the TDP-43 peptide array after incubating the membrane with recombinant TDP-43 protein followed by visualization of an antibody against TDP-43 Figure . This reTo verify whether the five regions of TDP-43 identified through the array assay are actually involved in self binding of the protein, the five peptides indicated in Figure To examine whether the peptides blocking interaction of recombinant TDP-43 protein with the peptide array could also inhibit TDP-43 aggregation in cells, a construct expressing a mutant TDP-43 lacking residues 187–192 [To investigate whether the synthetic peptides could inhibit the aggregation of mutant TDP-43, N-terminally TAT conjugated peptides A, B, C, D, E and/or TC were added to the cell culture medium after transfection. The peptides were added either individually or combined together in various ways . The TAT peptide has been used to deliver large molecules and small particles across both the plasma membrane and the nuclear membrane . The numTo investigate whether our peptides could inhibit aggregation of wild-type TDP-43 under stress conditions, HeLa cells were treated with arsenite with or without addition of synthetic peptides. After exposure to 0.5 μM arsenite with or without 10 μM of the synthetic peptide mixture (ABCDE) for 4 hours, the cell lysates were sequentially extracted and separated into soluble and insoluble proteins. The relative ratio of insoluble to soluble TDP-43 was used as a measure of TDP-43 aggregation propensity. After the treatment with arsenite, soluble endogenous TDP-43 was transformed into predominantly insoluble protein and showed a significant increased aggregation propensity (243.23 ± 22.97%) compared with control cells without treatment (41.02 ± 24.02%)  compared to untreated cells. Treatment with Peptide B and C, which significantly reduced the aggregation, led to similar levels of cell viability  as was found in untreated and scrambled peptide treated cells   compared to the basal condition. Our synthetic peptides decreased insoluble TDP-43 but did not prevent cell death  Figure . These rTo further elucidate the relationship between the cell death and TDP-43 aggregation, the cells were triple-labeled with TDP-43, GFP and the cell death marker, TUNEL. The numbers of transfected cells, aggregation-positive cells, or TUNEL-positive cells were counted by a blinded independent investigator. We measured four fields and 1000–1200 cells per microscope slide. We found that almost all the cells with TDP-43 aggregation that were untreated, Peptide B and C treated, or scrambled peptide (TC) treated showed TUNEL positive signals. In addition, even though Peptides B and C combined reduced TDP-43 aggregation, a much higher percentage of dead cells were observed in Peptides B and C treated cells without aggregation (12.22% ± 0.75%) than in scrambled peptide treated cells (5.95% ± 0.95%). Finally, we also measured cell death in cells without mutant TDP-43 overexpression but treated with the peptides. Cell death percentage in the untransfected cells of wild-type TDP-43, mutant TDP-43, mutant TDP-43 plus peptides BC and mutant TDP-43 plus scrambled peptide groups were 3.14% ± 0.56%, 2.28% ± 0.36%, 3.22% ± 0.35%, and 3.57% ± 0.39%, respectively  can induce TDP-43 aggregation and cell death in HeLa cells 7-192 can. Accordi2O2 and NMDA. Despite this increased neuronal vulnerability, we did not observe either nuclear or cytoplasmic TDP-43 aggregation. These results suggesting that reduced levels of TDP-43 in the nucleus may be critical for cytoxicity are consistent with evidence reviewed by Z.S. Xu in a recent paper [Our results indicating that aggregation may not be the causative factor for cytotoxicity in cells expressing mutant TDP-43, appear consistent with other emerging evidence that aggregation is not necessarily the cause of cellular toxicity. Previous work from our lab  showed tnt paper . These rnt paper . Recent nt paper . Moreovent paper ,32. Overnt paper . Togethein vivo. Furthermore, the same approach as the one taken here may be used to study the role of aggregation of other proteins, e.g. Huntingtin, α-synuclein, β-amyloid, in other neurodegenerative diseases.Protein aggregation has been observed in a number of neurodegenerative diseases. While aggregation is generally to be associated with cytotoxicity, it can also be a phenomenon associated with excessive ER stress, which is caused by an unusually large amount of misfolded proteins in the cell -38. In fIn this study, we are the first group to identify the regions in TDP-43 protein that involve in its self-aggregation. Peptides derived from those regions can effectively reduce the aggregation in cells transfected with an aggregation prone mutant TDP-43 or treated with arsenite. We then showed that reducing TDP-43 aggregation did not protect cells from cell death caused by either the expression of mutant TDP-43 or arsentie treatment. Thus, our study is the first to separate aggregation of TDP-43 from other factors involved in TDP-43 proteinopathy and demonstrate that TPD-43 aggregation may not be the cause of cytotoxicity.The peptides were synthesized on derivatized cellulose-based membranes  by the UBC Peptide Synthesis facility using a previously described protocol . The pepCell-penetrating peptides consisting of the truncated TAT domain at the N-terminal were synthesized by GL biochem Ltd. (Shanghai) and purified by HPLC. TAT, the trans-acting activator of transcription of the human immunodeficiency virus (HIV-1), has been used as an efficient way of delivering proteins or peptides into cells . The resMembranes were incubated with the mixture of TDP-43 protein (5 μg/ml) and/or single synthetic peptides (100 μg/ml) overnight at 4°C. Then the membranes were incubated sequentially with primary antibody and secondary antibody and washed with TBST. The protein interaction was visualized using an enhanced chemiluminescence reaction assay (PerkinElmer Life Sciences). Optical densities of each peptide array were measured using NIH ImageJ software.2. Wild type TDP-43 plasmid and mutant TDP-43 plasmid lacking residues 187–192 were obtained from Dr. Nonaka and Dr. Arai. The plasmids were transfected into the HeLa cells using the Lipofectamine 2000 system  according to the manufacturer’s instructions. After incubating the cells with the transfection mixture for 5 hours, the medium was replaced with fresh DMEM  with 2.5–20 μM synthetic peptides. After 24 hours, the medium was replaced with fresh peptides. The cells were returned to the incubator for an additional 24 hours before study.HeLa cells were obtained from the American Type Culture Collection (ATCC) and grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and 1% antibiotics . Cultures were maintained at 37°C in a humidified incubator  with 5% COFor studies using arsenite, arsenite  was added to the cell culture medium to a final concentration of 0.5 μM with or without the presence of synthetic peptides. The cells were then returned to the incubator for 4 hours before harvesting and analysis.Cells were fixed with pre-warmed 4% Paraformaldehyde  containing 4% sucrose  in PBS for 10 minutes at room temperature and permeabilized with 0.1% Trition X-100  in PBS for 2–3 minutes at room temperature. Then the reaction was blocked with 10% Bovine serum albumin  in PBS for 1 hour at room temperature and incubated with a primary antibody against TDP-43  overnight at 4°C. Reactions were visualized with anti-rabbit antibody conjugated with Alexa 488 . Cell nuclei were labeled with DAPI  for 2–3 minutes at room temperature. Images were obtained with an Olympus Fluoview FV1000 Confocal scanning microscope.The viability of cultured cells was assessed using the MTT assay. MTT -2,5-diphenyltetrazolium bromide)  was added to each well . After 4 h incubation, cells were lysed overnight. The optical density of test and control samples was measured at 590 nm absorbance with a reference filter of 750 nm using a “uQuant” microplate spectrophotometer . The absorbance was translated into cell viability ratios for comparison: cell viability ratio = (test sample absorbance/control sample absorbance) × 100%.in situ cell death detection kit . Cells were seeded on poly-D-lysine coated cover slips at a density of 2.5 × 104 cells per well in 24 well plate. After treatment, cells were fixed and stained with anti-TDP-43 , anti-GFP , Alexa 647 anti-rabbit  and Alexa 488 anti-chicken . The enzyme solution  and label solution were mixed in a volume ratio of 1:9 to obtain the TUNEL reaction mixture. The cells were incubated with the 50 μl TUNEL reaction mixture at 37°C for 1 hour and then washed three times with PBS. Then the cover slips were mounted on glass slides with antifade reagent . Images were obtained with an Olympus Fluoview FV1000 Confocal scanning microscope.Deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL) staining was performed using an To examine the effect of arsenite on solubility of endogenous TDP-43, preparation of soluble/insoluble protein were performed. HeLa cells were washed twice with cold PBS, lysed in cold lysis buffer . Cell lysates were rotated for 15 min and then centrifuged at 55,000 rpm for 15 min at 4°C. The supernatants were collected as soluble proteins. To prevent contamination caused by carrying over, the pellets were re-centrifuged at 55,000 rpm for 15 min at 4°C and washed once with lysis buffer. Then the pellets were lysed in PBS with 1% SDS and sonicated. The supernatants were collected as insoluble protein. Soluble and insoluble proteins were analyzed by immunoblotting.Protein concentrations of cell lysate and medium were measured using a standard BCA assay (Bio-Rad) as per the manufacturer’s instructions. Proteins were separated in SDS–PAGE (10%) and transferred to nitrocellulose membranes (Bio-Rad) at 4°C. Membranes were blocked with 5% nonfat dry milk in TBST for 1 hour, then incubated in 5% nonfat dry milk/TBST overnight at 4°C with antibodies for TDP-43 or β-actin . After washing with TBST, membranes were incubated with anti-rabbit secondary antibody conjugated with horseradish peroxidase for 1 hour at room temperature. Blots were developed using an enhanced chemiluminescence reaction assay.All data were expressed as mean ± SD and analyzed by one-way ANOVA. A value of p < 0.05 was considered statistically significant.The authors declare that they have no competing interests.RL, WJ and MSC conceived and designed the experiments. RL performed the experiments and analyzed the data. GY provided assistance in application of immunofluorescence methods. TN and TA were involved in plasmid construction. RL, WJ and MSC drafted and edited the manuscript. All Authors read and approved the final manuscript.


The tbx5 mutation in human causes Holt-Oram syndrome, an autosomal dominant condition characterized by a familial history of congenital heart defects and preaxial radial upper-limb defects. We report aberrant apoptosis and dormant cell growth over head, heart, trunk, fin, and tail of zebrafish embryos with tbx5 deficiency correspond to the dysmorphogenesis of tbx5 morphants.Wild-type zebrafish embryos at the 1-cell stage were injected with 4.3 nl of 19.4 ng of tbx5 morpholino or mismatch-tbx5-MO respectively in tbx5 morphants and mismatched control group. Semi-quantitative RT-PCR was used to for expression analysis of apoptosis and cell cycle-related genes. TUNEL and immunohistochemical assay showed the apoptosis spots within the local tissues. Ultra-structure of cardiac myocardium was examined by transmission electron microscope.Apoptosis-related genes , and cell cycle-related genes  showed remarkable increases in transcriptional level by RT-PCR. Using a TUNEL and immnuohistochemical assay, apoptosis was observed in the organs including the head, heart, pectoral fins, trunk, and tail of tbx5 knockdown embryos. Under transmission electron microscopic examination, mitochondria in cardiomyocytes became swollen and the myocardium was largely disorganized with a disarrayed appearance, compatible with reduced enhancement of myosin in the cardiac wall. The ATP level was reduced, and the ADP/ATP ratio as an apoptotic index significantly increased in the tbx5 deficient embryos.Our study highlighted that tbx5 deficiency evoked apoptosis, distributed on multiple organs corresponding to dysmorphogenesis with the shortage of promising maturation, in tbx5 knockdown zebrafish embryos. We hypothesized that mesenchymal cell apoptosis associated with altered TBX5 level may subsequently interfered with organogenesis and contributed to dysmorphogenesis in tbx5 deficiency zebrafish embryos. Tbx5 belongs to the T-box family of transcription factors and is required for the embryonic development of the heart and forelimbs [Tbx5 mutations in humans cause Holt-Oram syndrome (HOS), an autosomal dominant condition characterized by a familial history of congenital heart defects and preaxial radial ray upper-limb defects [tbx5 deficiency in different vertebrates are quite similar. Tbx5 deficiency in zebrafish causes multiple organ defects during organogenesis, including a shortened trunk, failure of cardiac looping formation, and hypogenesis or agenesis of the pectoral fins [tbx5 genotype and phenotype.orelimbs ,2. Tbx5  defects ,4. The pral fins . Either tbx5 encodes a transcription factor, tbx5 deficiency is presumed to disrupt development by altering the transcriptional function of the TBX5 protein, thereby affecting downstream target gene expressions, particularly affiliation binding proteins or transcription factors [Since  factors -9. Thougtbx5 leading to cardiac cell cycle arrest have been proved to elicit defect in cardiac sacromere formation and decreased cardiac cell number [The syndrome caused by mutations in T-box transcription factor 5 (TBX5) was described as "premature stops" or "developmental delay" in embryogenesis, though the mechanism how to delay or stop the development of the embryo, especially the cardiogenesis, remained indistinct . Some rel number .Apoptosis is a key event in many biological processes including embryogenesis and occurs in a variety of circumstance in specific time window in embryos. Zebrafish undergoes different levels of developmental cell death throughout embryonic stages precisely, including brain, spinal cord, ear, eyes, germ cells, tail bud and excretory system . Apoptostbx5 deficiency; meanwhile, the excessive apoptosis is in coincidence with the dysmorphogenesis in individuals with tbx5 deficiency.In this study, we reported that aberrant apoptosis and cell cycle arrest are increasing in zebrafish embryos with The approval of this study was granted by the National Taiwan Ocean University Aquaculture Animal ethics review board with annual evaluation.Zebrafish were maintained in 45-liter aquaria heated to 28.5°C with 25 fish per tank. The water was filtered, and about half of the water was replaced at least once a week. Adult zebrafish were fed 1 or 2 times per day with a variety of food, and the tank was cleaned by siphoning off any excess food after the second daily feeding. The day-night cycle was controlled with an automatic timer (14 h light/10 h dark).Zebrafish reach sexual maturity in 10~12 weeks, but breeding fish should be between 7 and 18 months of age for maximum embryo production. The day before breeding, 1/3 of the water was replaced and the tank was cleaned after feeding (1~2 h before the end of the light period). Finally, a collection box was placed at the bottom of the tank and preparation was made to collect the embryos the next day.When the light came on, we removed the collection box and placed the collected embryos into an incubator maintained at a temperature of 28.5°C.Total RNA was isolated from 50 embryos using the guanidine isothiocyanate-based TRIzol solution. RNA samples were resuspended in DEPC-treated water and quantified spectrophotometrically at 260 nm. The RNA quality was then checked by 1.2% agarose gel electrophoresis, after staining with 1 μg/ml ethidium bromide. The RNA stock was stored at -80°C.bcl2 , bad , and bax , p27 , p57 , and pcna , cdk2 . PCR conditions were as follows: denaturation at 95°C for 3 min, followed by 50 cycles of amplification . Quantification of PCR product was performed on an electrophoresis agarose gel using Kodak image 3.5 software. All measurements were performed in triplicate (n = 3).Total RNA was prepared from 50 morpholino (MO) injected or normal embryos . Three microliters of 1st-strand cDNA was amplified. Amplification primers for each specific mRNA were deduced from published sequences and included tbx5-MO (5 GAAAGGTGTCTTCACTGTCCGCCAT-3), was designed against the tbx5 translational start site  and a mismatch tbx5-MO (5'-GTCTCTTGACTCTCCGCGATCTCGG-3') was designed for control . Wild-type embryos primarily at the 1-cell stage with the chorion intact were injected with 19.4 ng/4.3 nl of stock morpholino diluted in Danieau's solution. The injected embryos were raised at 28.5°C. Embryos used for analyzing the expression of various markers were fixed with 4% paraformaldehyde. Otherwise, embryos were scored after 2 days of development for late effects. In our previous study, three control groups, including 3' end of tbx5-MO(2) (5'-GCCTGTACGATGTCTACCGTGAGGC-3') and embryos with blank microinjection and wild-type ones without microinjection, were included to identify the specific blockage of tbx5 mRNA translation effect of tbx5-MO.6 In order to examine the knockdown effectiveness of the tbx5-MO, a tbx5-GFP construct was generated by fusing a 468-bp fragment of tbx5, which included the morpholino target side into a GFP producing vector. Tbx5-MO at 19.4 ng and tbx5-GFP DNA at 150 μg were co-injected into 1-cell stage embryos. In control group, only 150 μg of tbx5-GFP was microinjected into 1-cell stage embryos [The morpholino antisense oligonucleotide, Zebrafish embryos were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS). Deparaffinized sections (3 μm) of zebrafish embryo tissues were placed on slides and processed for immunohistochemistry. After blocking with a biotin blocking system  for 30 min, sections were incubated with target-purified rabbit primary antibodies, including BCL2 , BAD , CASPASE-3 , CASPASE-8 ,CDK2 , P27  and MF20 , washed in PBS, and then incubated with a rhodamine-conjugated secondary antibody: goat anti-rabbit immunoglobulin G (IgG). After washing in PBS, sections were incubated with mounting medium and kept at 4°C.4 in Sorenson's phosphate buffer followed by dehydration through a graded series of ethanol washes, and embedded in Spurr's EPON. Blocks were heated in an oven for 8 h at 70°C. Semithin (1-μm) sections were cut and stained with toluidine blue for adequate preview under a microscope. Ultrathin sections (900 Å) were cut with a diamond knife, stained with uranyl acetate and lead citrate, and examined with an electron microscope.Embryos were fixed at 48 h post-fertilization (hpf) with 2.5% glutaraldehyde in Sorenson's phosphate buffer, postfixed with 1% OsOBoth whole mount and sectioned TdT-UTP nick end labeling (TUNEL) assays were performed using an ApopTag kit . Briefly, zebrafish embryos were fixed with 4% paraformaldehyde in PBS. Proteinase K-treated whole embryos or deparaffinized sections (5 μm) of embryos were incubated with the TdT enzyme followed by anti-digoxigenin. Finally, embryos or slides were stained with DAB for 5 min.Embryos were homogenized on ice in lysis buffer . Then, cellular debris was pelleted by centrifugation at 12,000 rpm for 20 min, and the supernatant was collected and measured. Proteins were mixed with sample buffer before separating in 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. The SDS-PAGE was then transferred onto nitrocellulose membranes at 100 V for 1 h. Membranes were blocked with blocking buffer ) at room temperature for 1 h. The TBX5 primary antibody  was incubated overnight at 4°C using the appropriate dilution (1: 1000). The nitrocellulose membranes were washed with PBST followed by incubating with a horseradish peroxidase (HRP)-conjugated secondary antibody (1: 5000) for 1 h at room temperature before the images were digitized.n = 10 in each subgroup) were separately placed in 96-well round-bottomed, flat, white plates for detection. The RLU (relative luminescence unit) value was detected using the PARADIGM™ Detection Platform .ATP and ADP/ATP ratio assay were performed using an ApoSENSOR™ ADP/ATP Ratio Assay Kit . Ten embryos  directly into embryos at the 1-cell stage resulted in retardation of the looping process, and the most prominent cardiac finding in tbx5 morphants was a string-like heart  embryos at 48 hpf, meanwhile the ventricle overlapped with the atria during the diastolic phase Figure . Injectit Figure . At 96 ht Figure ; howevers Figure . The tais Figure  and truns Figure  of knocks Figure  and truns Figure , both oftbx5 knockdown embryos  construct [us study . The effg Figure  and tbx5onstruct .bad, bax, and bcl2, showed remarkable increases in tbx5 morphants in all studied periods but not in embryos injected with mismatch-tbx5-MO, which has no significant different with the wild-type and control mismatch   Figure .tbx5 knockdown embryos. In addition, myofibrils appeared to be disarrayed  was remarkably decreased  or pro-apoptosis related genes (bad and bax) were all transcriptionally activated, and so were the cell proliferation markers (cdk2 and pcna) and cyclin kinase inhibitors (p27 and p57). In protein level, remarkably plentiful production of apoptosis-related proteins including CASPASE-3, CASPASE-8, and BAD were proved by immunohistochemistry These data disclose that a deficiency of tbx5, a transcription factor, in zebrafish embryos may trigger multiple signal pathways including promoting or inhibiting molecules and eventually come to the end sequel of apoptosis and cease of cell growth.Our data revealed activation of apoptosis and inhibition of cell growth in embryonic tbx5 in the embryos might be associated with the dysmorphogenesis, especially engendering the string-like heart, short fins, and malformed trunk during zebrafish embryonic development.Besides, the distribution of aberrant apoptosis and deviant cell cycle caused by a deficiency of tbx5-deficient embryos at the late stage of organogenesis [There are two main apoptosis pathways including cell-intrinsic  pathway and cell-extrinsic (death receptor) pathway; the intrinsic pathway is largely regulated by mitochondria, whereas the extrinsic pathway interacts with death receptors . Mitochoogenesis ,26.tbx5 knockdown embryos, rather than small and dense in WT embryos. This phenomenon is compatible with the morphological changes of mitochondria during apoptosis, which culminates in permeabilization of the mitochondrial outer membrane and release of soluble proteins from the mitochondrial intermembrane space. Once the mitochondrial permeability transition pore is opened, proapoptotic proteins are released, and the membrane potential collapses, which results in swelling of mitochondria. The number of mitochondria in tbx5 deficiency embryos also increased in addition to their size. Although a similar phenomenon was described during apoptosis induced by anticancer drugs in colon cancer cells and apoptosis in mitochondrial myopathy, mitochondrial proliferation accompanied by mitochondrial swelling was first reported in apoptosis of zebrafish embryos with tbx5 deficiency [The mitochondrial apoptotic process causes the outer membrane of mitochondria to release proapoptotic proteins and eventually gives rise to swollen mitochondria ,28. Our ficiency . Until ntbx5 knockdown embryos appeared eminently disorganized. The myofibrils were quantitatively diminished at both the atrial and ventricular levels. We used MF20, an anti-myosin antibody, to prove a decrease in cardiac myosin. This result is in accordance with conclusion of our previous study that cardiomyogenesis-related genes were perturbed in tbx5 knockdown embryos [Under TEM, the ultrastructure of the myocardium in  embryos .tbx5 activated aberrant apoptosis and dormant cell growth in the head, heart, fins, and trunk. Although abnormal apoptosis-related genes were expressed throughout the entire process of organogenesis in tbx5 knockdown embryos, the degree and distribution of apoptosis varied in different locations at 48 hpf. Such aberrant apoptosis may contribute to consequent morphologic anomalies including a string-like heart, a curled trunk, and hypogenesis of the pectoral fins, but it did not interfere with the migration and differentiation of progenitor cells during early development. For example, no ectopic fin was found in embryos, although hypogenesis or agenesis of the pectoral fin was frequently observed in tbx5 morphants, as was the string-like heart. We hypothesized that the aberrant apoptosis may play a role in the interruption of cardiac looping, chamber formation, and growth of pectoral fins in zebrafish. Nevertheless, mutations in tbx5 gene underlie Holt-Oram syndrome, the majority of which is assumed as sequel "premature stops" or "developmental delay", may induce inappropriate apoptotic processes during embryogenesis and lead to the dysmorphogenesis including cardiac and limb malformation.The TUNEL assay showed that a shortage of From a genomic analysis, dozens of genes were related to the underlying causes of isolated congenital heart disease and genetic syndrome associated with cardiac anomalies. However, genotype-phenotype distinctions exist. The discrepancy was frequently related to multiple genetic pathways, the size of the deletion, different allele/loci, point mutations, or a haploinsufficiency. Our observation presumed that the genotype-phenotype distinction might be a consequence of variable degrees of apoptosis in progenitor cells.tbx5 insufficiency ultimately resulted in activation of apoptosis and inhibition of cell growth in whole individual of zebrafish embryo, though knockdown of TBX5 leads to overexpression of both anti-apoptotic and pro-apoptotic gene than wild type embryos and the controls. Those data implied that abnormal tbx5 level play a role in aberrant apoptosis and cell growth contributing to dysmorphogenesis.Some study declared that exogenous TBX5 could induce apoptosis and inhibit cell proliferation in vitro . Our restbx5 deficiency including string-like heart, curled trunk, curled tail, and pectoral fins might be a consequence of aberrant apoptosis and dormant cell growth in early embryonic development, which affected late differentiation and growth of organs as well as maturation.In summary, abnormal appearances in zebrafish embryos with The authors declare that they have no competing interests.JHL and JKL conceived of the study, participated in its design, coordination and drafted the manuscript. TT participated in its design and drafted the manuscript, SC, SY and AY carried out the molecular genetic studies. RT and HL participated in its design and coordination. All authors read and approved the final manuscrpt.


Bacillus cereus group.Group II introns are widespread genetic elements endowed with a dual functionality. They are catalytic RNAs (ribozymes) that are able of self-splicing and they are also mobile retroelements that can invade genomic DNA. The group II intron RNA secondary structure is typically made up of six domains. However, a number of unusual group II introns carrying a unique extension of 53-56 nucleotides at the 3' end have been identified previously in bacteria of the In the present study, we conducted combined sequence comparisons and phylogenetic analyses of introns, host gene, plasmid and chromosome of host strains in order to gain insights into mobility, dispersal, and evolution of the unusual introns and their extension. We also performed in vitro mutational and kinetic experiments to investigate possible functional features related to the extension.B. cereus group, Bacillus pseudofirmus OF4 and Bacillus sp. 2_A_57_CT2, an uncharacterized species phylogenetically close to B. firmus. Interestingly, the B. pseudofirmus intron has a longer extension of 70 bases. From sequence comparisons and phylogenetic analyses, several possible separate events of mobility involving the atypical introns could be identified, including both retrohoming and retrotransposition events. In addition, identical extensions were found in introns that otherwise exhibit little sequence conservation in the rest of their structures, with the exception of the conserved and catalytically critical domains V and VI, suggesting either separate acquisition of the extra segment by different group II introns or a strong selection pressure acting on the extension. Furthermore, we show by in vitro splicing experiments that the 3' extension affects the splicing properties differently in introns belonging to separate evolutionary branches.We report the identification of novel copies of group II introns carrying a 3' extension including the first two copies in bacteria not belonging to the Altogether this study provides additional insights into the structural and functional evolution of unusual introns harboring a 3' extension and lends further evidence that these introns are mobile with their extension. Group II introns are genetic elements that are widespread in bacteria and in the organelles of eukaryotes. They are self-splicing catalytic RNAs (ribozymes) that remove themselves from precursor mRNA transcripts and ligate their flanking sequences (exons). Group II introns are also mobile retroelements which can invade genomic DNA sites -5. SplicBacillus cereus group, including B. cereus, B. thuringiensis, B. mycoides, and B. pseudomycoides. These bacterial species are genetically closely related and are known to harbor a range of mobile elements such as plasmids and introns [B. cereus group. Using the complete and diverse set of introns with an extra segment we conducted a detailed sequence and phylogenetic analysis of the introns together with their host genes and strains in order to gain insight into mobility, dispersal, and evolution of these elements and their domain VII. Functional studies were also carried out to investigate possible features related to the extension.The secondary structure of the group II intron RNA typically consists of six domains (numbered I to VI) that are linked by a network of tertiary interactions, and introns are classified based on structural features and IEP phylogeny ,5,14-16. introns ,20-23. F introns ,18,24. P introns ,18. In tB. cereus group, two of the newly discovered elements are encoded by strains of unrelated species, namely Bacillus pseudofirmus OF4  and theB.th.I6 intron from B. thuringiensis kurstaki BGSC 4D1/HD1 (Table B.th.I6-like introns (B.th.I6 and B.c.I16) are all inserted in the same homing site within the pXO1-42 plasmid gene, and the two intron copies (B.th.I6a and b) found in B. thuringiensis kurstaki BGSC 4D1/HD1 were suggested to be the result of intron mobility [With respect to the phylogeny of the introns themselves, many of the introns with a 3' extension belong to the β subgroup within the bacterial B class ,18, and mobility ,18. HereB. cereus group. Indeed, large plasmids are the main vectors of group II intron spread in this bacterial group [B.th.I6-like introns are distributed in both groups. Identical copies are present in distantly related pXO1-42 sequences, as exemplified by B.th.I6b and c/d and by B.c.I16a and b/c. This strongly suggests mobility of the individual introns rather than transfer of the whole locus containing host gene and intron  belonging to the "pXO1-like" family . Phylogeal group . Secondln Figure . A diffee Figure . The lat Figures  and 2A. B.th.I7a and b copies indicated that the B.th.I7 intron as well must be mobile with its extension. These two intron copies are inserted in different genes in B. thuringiensis kurstaki BGSC 4D1/HD1 . While the nucleotide sequences of the two intron copies are identical, the insertion sites exhibit little similarity, with the exception of the IBS2 motif  is identical to that of B.th.I5 from B. thuringiensis BGSC 4D1/HD1 and CT-43  has adapted to function with the 3' extra domain, the extension was not essential for splicing since the intron could splice nearly as efficiently as wildtype (WT) when the entire extension was deleted [B.c.I4_dS1S2) appeared to produce somewhat more linear form of the intron, suggesting more hydrolytic splicing without the 3' extension [B.c.I4 WT and dS1S2 constructs. These analyses confirmed that the linear form is not the major product of the splicing reaction of WT B.c.I4, even in buffers containing KCl, which is known to promote the hydrolytic splicing pathway [B.c.I4_dS1S2 construct produced a linear and lariat fraction of ~40% and ~45%, respectively, after the same time period . Even though splicing of the B.th.I6a construct deleted of the full 54-nt 3' extension (B.th.I6a_dS1S2) also appeared to give an increased amount of free linear intron in KCl-containing buffer 2SO4 and KCl buffers showed a dramatic inhibition of the second splicing step. This was revealed by the accumulation of the first step intermediate "lariat + 3' exon" product compared to the B.th.I6a WT construct containing the extension  was searched as well. Strain HD1 corresponds to strain BGSC 4D1, whose genome has also been sequenced by our laboratory and the Norwegian High-Throughput Sequencing Centre, University of Oslo, Norway . Introns were named following the nomenclature used in the Group II Intron Database [The nucleotide sequences of the previously identified xtension -19 were xtension  using BLxtension  for addiDatabase .B. cereus group strains publicly available in Genbank and Microgen at the time of analysis were screened for the B.th.I6 intron's host gene, pXO1-42, using BLAST. The BLAST search was conducted using the pXO1-42 sequence of the pXO1 plasmid of B. anthracis Ames Ancestor strain (locus tag GBAA_pXO1_0064) as query, and was performed both at the amino acid and nucleotide levels .The 86 completely sequenced B. cereus group were selected for screening, including strains closely related to those harboring B.th.I6 and the B.th.I6-like B.c.I16 intron, based on the multiple locus sequence typing (MLST) supertree of chromosomal housekeeping genes available in the SuperCAT database at the University of Oslo's typing website ([http://mlstoslo.uio.no/).An additional 36 isolates were screened by PCR, and positive products were sequenced. PCR was performed as previously described using the B.th.I6a_exon_left/right and B.th.I6b_exon_left/right oligonucleotide primer pairs . Strainswebsite  were computationally predicted by constrained folding using the MFOLD 3.1 package [The secondary structures of the  package ,35 follo package ,36. ThatB.th.I6-like introns was available for the strains that were screened by PCR. Thus, sites with gaps were removed in a pairwise manner when computing distances. Statistical support for branches in the tree was assessed by 1000 bootstrap replicates [Homologous pXO1-42 nucleotide sequences were aligned using CLUSTALW 2 ,38, follplicates . Phylogeplicates .B. cereus group strains encoding the pXO1-42 gene was reconstructed using the MLST data available in the SuperCAT database ([http://mlstoslo.uio.no/). The data included the nucleotide sequences of 7 to 26 chromosomal housekeeping genes, depending on the strain. According to information in culture collections, B. thuringiensis kurstaki BGSC 4D1 and ATCC 33679 should be the same strain and both correspond to strain HD1. However, typing studies revealed that the former two strains exhibit genotypic differences [B. cereus group isolates was reconstructed using the matrix representation by parsimony (MRP) technique as done in SuperCAT  according to the manufacturer's instructions using two complementary oligonucleotides (of ~40 bases) containing the desired mutation(s) with either B.c.I4 or B.th.I6a ΔORF constructs as templates [B.th.I6a (B.th.I6a_dS1S2 construct) was performed in the same manner as done previously for B.c.I4  according to the manufacturer's instructions. Transcription and gel-purification of radiolabelled and unlabelled RNA were conducted as previously described .2, and either 500 mM (NH4)2SO4 or 500 mM KCl at 47°C. Reactions were initiated by adding pre-warmed splicing buffer to the transcript RNA giving a total reaction volume of 40 μl. At each time point of the time-course analysis, 2 μl were taken out, quenched with loading buffer (Ambion) and storing samples on dry ice. Samples were then heated to 95°C and cooled on ice, before being separated on a 7.5 M Urea 4% polyacrylamide gel. Gels were then vacuum dried, exposed, and analyzed using a Molecular Dynamics Storm 860 Phosphorimager.In vitro generated transcripts were denatured and refolded using a GenAmp 2700 PCR machine (Applied Biosystems), by incubating the transcripts in 10 mM MOPS, pH 7.5 at 90°C for 1 min, 75°C for 5 min, and then slow cooling to the splicing temperature of 47°C. Intron transcripts were spliced with 70000 cpm RNA or ~0.1 μg unlabelled transcripts in 40 mM MOPS, pH 7.5, 100 mM MgClFor subsequent RT-PCR and sequencing of these splicing products, either unlabeled spliced transcripts, purified with Nucleotide purification kit (Qiagen), or labeled spliced transcript species, excised from gels, were used as templates.For kinetic analysis, the intensities of the radioactive bands were quantified using the ImageQuant 5.0 software. The relative fractions of unspliced precursor and free lariat RNA were computed from the intensities of the radioactive bands of all intron-containing products.The authors declare that they have no competing interests.NJT and FBS jointly conceived the study and wrote the manuscript. NJT conducted the bioinformatic analyses. FBS performed the laboratory experiments. ABK supervised the study and contributed to the interpretation of the data. All authors have read and approved the current manuscript.Figure S1-S3 and Table S4. The file includes three supplementary figures (S1-S3) along with the corresponding legends and associated references. The figures show respectively a phylogenetic tree of Bacillus species , comparisons of sequence homology between large plasmids , and drawings of the secondary structure of unusual group II introns . The file also includes a supplementary table (S4) listing the oligonucleotide primers used for the in vitro splicing experiments.Click here for file


Most of our time is spent indoors where we are exposed to a wide array of different microorganisms living on surfaces and in the air of our homes. Despite their ubiquity and abundance, we have a limited understanding of the microbial diversity found within homes and how the composition and diversity of microbial communities change across different locations within the home. Here we examined the diversity of bacterial communities found in nine distinct locations within each of forty homes in the Raleigh-Durham area of North Carolina, USA, using high-throughput sequencing of the bacterial 16S rRNA gene. We found that each of the sampled locations harbored bacterial communities that were distinct from one another with surfaces that are regularly cleaned typically harboring lower levels of diversity than surfaces that are cleaned infrequently. These location-specific differences in bacterial communities could be directly related to usage patterns and differences in the likely sources of bacteria dispersed onto these locations. Finally, we examined whether the variability across homes in bacterial diversity could be attributed to outdoor environmental factors, indoor habitat structure, or the occupants of the home. We found that the presence of dogs had a significant effect on bacterial community composition in multiple locations within homes as the homes occupied by dogs harbored more diverse communities and higher relative abundances of dog-associated bacterial taxa. Furthermore, we found a significant correlation between the types of bacteria deposited on surfaces outside the home and those found inside the home, highlighting that microbes from outside the home can have a direct effect on the microbial communities living on surfaces within our homes. Together this work provides the first comprehensive analysis of the microbial communities found in the home and the factors that shape the structure of these communities both within and between homes. Globally, homes vary from simple stick structures similar in their climate to the outdoor environment to climate-invariant mansions. But even within regions, house conditions can vary in subtler but important ways, and it is likely that microbial communities also vary among such houses. Such variation could be stochastic–a function of the complex dynamics influencing the fate of the many species and their populations among which we live. Alternately, composition might be influenced deterministically by differences between homes in outdoor or indoor environmental conditions, surface materials, and the inhabitants (both human and otherwise) of the homes. Both indoor humidity and temperature appear to influence the diversity and composition of building-associated bacterial communities To understand the differences in bacterial communities found across specific house locations (habitats) within homes and the variation that exists within habitats between homes, we sampled bacteria in nine standardized locations within each of forty homes in the Raleigh-Durham area of North Carolina. The sampled homes spanned a range of designs, sizes, numbers and types of occupants , degrees of urbanization, and socioeconomic status. They were identified as part of a volunteer-based citizen science project in which participants sampled their own homes. We compared the diversity and composition of the bacterial communities in the 360 collected samples using high-throughput sequencing of the bacterial 16S rRNA gene.http://www.yourwildlife.org/projects/wild-life-of-our-homes/).Historically, most efforts to engage the public in scientific research have rallied participation around charismatic organisms or easily observable environmental phenomena Here we present the results from the first forty households we recruited to participate in our study in North Carolina in autumn 2011. Each participant was provided with a written Informed Consent form approved by the North Carolina State University’s Human Research Committee  as well as instructions for sampling their home and a home microbe sampling kit.Each home sampling kit contained nine dual-tipped sterile BBL™ CultureSwabs™. Participants were instructed to sample nine standardized locations within their home: kitchen cutting board, kitchen counter, a shelf inside a refrigerator, toilet seat, pillowcase, exterior handle of the main door into the house, television screen, the upper door trim on the outside surface of an exterior door, and the upper door trim on an interior door . These lWe used the direct PCR approach described in Flores et al. Singletons and phylotypes classified as chloroplasts were removed from further analyses. Phylotypes observed in extraction controls were removed as per Flores et al. http://dx.doi.org/10.6084/m9.figshare.674588).Data have now been deposited online at figshare  in R In order to identify the relative importance of different potential source environments to the bacterial communities found on the sampled surfaces we used a source-tracking approach similar to that described previously Across all samples, a total of 7726 unique phylotypes were identified with nearly all individual samples having at least 100 unique OTUs (out of 1000 reads per sample) . We obseP<0.05 for all pairwise comparisons). In general, samples clustered into the following general groups: depositional environments , kitchen-associated , and frequently touched surfaces   were Proteobacteria, Firmicutes, and Actinobacteria which represented 34%, 32%, and 20%, respectively, of all 16S rRNA gene sequences. The bacterial families with the highest relative abundances across all of the collected samples were the Streptococcaceae (8.9%), Corynebacteriaceae (5.6%), and Lactobacillaceae (5.1%). Bacterial community composition differed significantly between each sampling site across the 40 homes ( handle) . The rel handle) .As we have previously analyzed a large number of samples from possible bacterial source environments inside and outside the home, including soil P = 0.001). The between-home differences accounted for one-quarter to one-half as much variation as the location-specific differences within homes. This was particularly evident for the communities collected from the kitchen environments that were highly variable in composition across the homes (As evident from he homes .P = 0.001). In particular, the presence of dogs was associated with a different community composition on pillowcases and TV screens  and 57% (P = 0.004) more OTUs on pillowcases and TV screens, respectively, compared to homes without pets.Although we had limited data on the specific environmental conditions found within the homes, we could use collected data on house occupancy, location, and design to ascertain which of these factors contributed to the inter-home variability in community composition for the individual sampling locations. We found that this variation could be attributed, in part, to the presence of dogs in homes , suggesting that information on the types of bacteria found in the air outside the home can be used to predict, in part, the composition of the bacterial communities found within homes. We tested other factors, including number of human occupants, presence of cats, presence of children, use of pesticides, presence of carpet, and the presence of and types of allergies, but none of these factors had a significant influence on the bacterial communities at any of the sampling locations . Either these factors were not important in structuring house-associated bacterial communities across the sampled locations or our sampling effort was insufficient to quantify the importance of those factors. For example, we could not detect an effect of cats on house-associated microbial communities; however, with only three homes having cats and no dogs, we may have lacked sufficient replication to detect such an effect. Likewise, we could not test the influence of other factors that may influence the structure of house-associated bacterial communities, including house environmental conditions , cleaning frequency, or specific surface materials, as we did not collect sufficient information on these factors from the homes sampled.We also found a significant relationship between exterior and interior microbial community composition  to have higher diversity  common on other types of moist surfaces Compared to the samples collected from inside homes, the exterior door trim had far lower abundances of human-associated bacteria and appeared to be dominated by microbes commonly found in soil and on leaf surfaces . Both so2 so any potential differences in airborne microbial taxa may be minimal if they are regionally driven. Future work would benefit from a broader sampling of homes across larger geographic regions to determine the extent to which the outside environment may shape the composition of those bacterial communities found within our homes.While strong differences across locations within houses exist and were consistent from one house to the next, some of the locations harbored bacterial communities that were highly variable in composition across the homes. A number of factors could account for this inter-home variability, including factors that we did not include in our analyses because they were either unmeasured  a phenomenon that has also been observed in other studies e.g., . The preOur work highlights that our homes are composed of many distinct habitats that harbor distinct bacterial communities. The composition of these communities is likely driven by numerous factors, including the differential sources of bacteria dispersed onto the surfaces, and many of the community assembly patterns appear to be predictable. The microbial communities that live within homes clearly provide unique opportunities for testing ecological theories of community assembly, theories that have been derived almost exclusively from research on plant and animal communities. Practically speaking, however, the more significant result is our observation of strong differences in bacterial communities among houses, even for particular habitats. In our dataset, a single feature of the home environment (the presence of a dog) explained a large portion of the variability in bacterial community composition and diversity in some of the habitats sampled – but clearly there are other unmeasured or undetected factors that are likely important in structuring communities across homes. To the extent that the differences in household microbial communities are associated with differences in health and well-being, understanding these missing factors seems important. However, given the large number of potential explanatory variables, such work will require much larger sample sizes than were possible here.Figure S1Diagram of the nine locations sampled within each of the 40 homes. Insets emphasize the habitat characteristics of each sampling site.(TIFF)Click here for additional data file.Figure S2Principle coordinates plots showing effect of dogs on bacterial community composition on (A) pillowcases and (B) TV screens. Points closer together are more similar in terms of their bacterial composition.(TIF)Click here for additional data file.Figure S3Presence of dogs in home influences relative abundance of bacterial taxa. Differences in relative abundance of selected taxa between homes with dogs and those without pets on pillowcases and TV screens (A) and between dog fur and human skin Click here for additional data file.Table S1OTUs considered to be contaminants and removed prior to downstream analyses. OTU IDs and taxonomy labels are from the 97% similarity OTUs of the Greengenes February 2011 release.(DOCX)Click here for additional data file.Table S2Indicator taxa used for source tracking analysis.(DOCX)Click here for additional data file.


Abnormal distribution, modification and aggregation of transactivation response DNA-binding protein 43 (TDP-43) are the hallmarks of multiple neurodegenerative diseases, especially frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS).M337V). We found hTDP-43M337V was expressed primarily in the nuclei of neurons in the brain and spinal cord, intranuclear and cytoplasmic phosphorylated TDP-43 aggregates were frequently detected, and the levels of TDP-43 LMW products of ~25 kDa and ~35 kDa species were also increased. Overexpression of hTDP-43M337V dramatically down regulated the levels of mouse TDP-43 (mTDP-43) protein and RNA, indicating TDP-43 levels are tightly controlled in mammalian systems. TDP-43M337V mice displayed reactive gliosis, widespread ubiquitination, chromatolysis, gait abnormalities, and early lethality. Abnormal cytoplasmic mitochondrial aggregates and abnormal phosphorylated tau were also detected in the mice.To explore the pathogenic properties of mutant forms of TDP-43, we generated and characterized two mouse lines expressing human TDP-43 carrying the M337V mutation (hTDP-43M337V mouse models produces similar phenotypes, our results suggest that overexpression of the hTDP-43M337V can cause neuronal dysfunction due to its effect on a number of cell organelles and proteins, such as mitochondria and TDP-43, that are critical for neuronal activity.While overexpression of hTDP-43 in wild-type TDP-43 and TDP-43


Due to unique properties and unlimited possible applications, graphene has attracted abundant interest in the areas of nanobiotechnology. Recently, much work has focused on the synthesis and properties of graphene. Here we show that a successful reduction of graphene oxide (GO) using spinach leaf extract (SLE) as a simultaneous reducing and stabilizing agent. The as-prepared SLE-reduced graphene oxide (S-rGO) was characterized by ultraviolet–visible spectroscopy and Fourier transform infrared spectroscopy. Dynamic light scattering technique was used to determine the average size of GO and S-rGO. Scanning electron microscopy and atomic force microscopy images provide clear surface morphological evidence for the formation of graphene. The resulting S-rGO has a mostly single-layer structure, is stable, and has significant water solubility. In addition, the biocompatibility of graphene was investigated using cell viability, leakage of lactate dehydrogenase and alkaline phosphatase activity in primary mouse embryonic fibroblast (PMEFs) cells. The results suggest that the biologically synthesized graphene has significant biocompatibility with PMEF cells, even at a higher concentration of 100 μg/mL. This method uses a ‘green’, natural reductant and is free of additional stabilizing reagents; therefore, it is an environmentally friendly, simple, and cost-effective method for the fabrication of soluble graphene. This study could open up a promising view for substitution of hydrazine by a safe, biocompatible, and powerful reduction for the efficient deoxygenation of GO, especially in large-scale production and potential biomedical applications. Recently, carbon-based nanomaterials such as carbon nanotubes, graphene oxide, and graphene have been explored extensively by researchers as well as the industry. Graphene is an emerging nanomaterial which has greater scientific and commercial advantages. Recently, single-layer and few-layer graphenes received great interest due to its exceptional characteristics including high surface area as well as strong electronic, mechanical, thermal, and chemical properties in various fields such as materials science, physics, chemistry, biotechnology, and nanomedicine -3. PartiBiocompatibility is an essential factor for tissue engineering applications. Recent studies suggest that the biocompatibility of carbon-based nanomaterials depends strongly on mass, purity, ratio, and surface functional groups. A variety of biological applications depend on the functionalization of graphene. The ability of the functionalization of graphene and its derivatives brought the attention of nanomaterials in various applications including biosensors and tissue engineering. Several studies have reported the biocompatibility of graphene derivatives in proximity of mammalian cells. Biris et al.  demonstrRegarding synthesis of nanomaterials, various phytochemicals have been used from different natural sources like leaves, peels, roots, seeds, and other parts of plants as reducing agents for the synthesis of different metal nanoparticles like silver and gold -47. Here4, anhydrous ethanol, 98% H2SO4, 36% HCl, and 30% hydrogen peroxide aqueous solution were purchased from Sigma-Aldrich (USA) and used directly without further purification. All aqueous solutions were prepared with deionized (DI) water. All other chemicals were purchased from Sigma-Aldrich  unless stated otherwise.Graphite powder was purchased from Sigma-Aldrich . NaOH, KMnO2. The finely cut spinach leaves were mixed in 100 mL of sterile distilled water and then boiled for 2 min. After boiling, it was filtered through Whatman filter paper no. 1. Further, the extracts were used for synthesis of graphene. The extracts were stored at 4°C until further use.Spinach leaves were obtained from the local market and stored at 4°C until needed. Twenty grams of spinach leaves was washed thoroughly with double distilled water and was then sliced with a sharp stainless steel knife into fine pieces, about 1 to 5 cmNatural graphite powder was utilized as the raw material to prepare graphite oxide by suspension through a modified Hummers’ method  and accoReduced graphene oxide was obtained from the reaction of a plant extract with graphene oxide. In the typical reduction experiment, 10 mL of spinach leaf extract was added to 40 mL of 0.5 mg/mL aqueous GO solution and then the mixture was kept in a tightly sealed glass bottle and stirred at 30°C for 24 h. Then, using a magneto-stirrer heater, reduced graphene oxide suspension was stirred at 400 rpm at a temperature of 30°C for 30 min. A homogeneous S-rGO suspension was obtained without aggregation. Then, the functionalized S-rGO was filtered and washed with DI water. Finally, a black S-rGO dispersion was obtained.λ = 1.5406 A at 40 kV and 40 mA was recorded in the range of 2θ = 5° − 80°. The changes in the surface chemical bonding and surface composition were characterized using a Fourier transform infrared spectroscopy (FTIR) instrument . A JSM-6700F semi-in-lens FE-SEM operating at 10 kV was used to acquire SEM images. The solid samples were transferred to a carbon tape held by an SEM sample holder for analyses. The analyses of the samples were carried out at an average working distance of 6 mm. Raman spectra of graphene oxide and reduced graphene oxide were measured by WITec Alpha300  with a 532-nm laser. The calibration was initially made using an internal silicon reference at 500 cm−1 and gave a peak position resolution of less than 1 cm−1. The spectra were measured from 500 to 4,500 cm−1. All samples were deposited on glass slides in powder form without using any solvent. Surface images were measured using tapping-mode atomic force microscopy  operating at room temperature. Height and phase images were recorded simultaneously using nanoprobe cantilevers .Ultraviolet–visible (UV–vis) spectra were obtained using a WPA . The aqueous suspension of GO and S-rGO was used as UV–vis samples, and deionized water was used as the reference. The particle size of dispersions was measured by Zetasizer Nano ZS90 . X-ray diffraction (XRD) analyses were carried out on an X-ray diffractometer . The high-resolution XRD patterns were measured at 3 kW with Cu target using a scintillation counter, and v/v) fetal bovine serum, plus 2 mM of l-glutamine, and 1% (v/v) penicillin-streptomycin (10 U mL−1 penicillin and 0.1 mg mL−1 streptomycin) and grown at 37°C in a 5% CO2 humidified environment. When the cells had reached 70% confluence, they were trypsinized  and passaged (1:3). Cells within three passages were used for experiments. GO or S-rGO suspensions were freshly prepared before the cells were exposed and diluted to appropriate concentrations from 20 to 100 μg mL−1 with the culture medium; they were then immediately applied to the cells. DMEM without GO and S-rGO supplements served as a negative control in each experiment.PMEF cells were routinely cultured in Dulbecco’s modified Eagle’s low-glucose medium  supplemented with 10%  was added to a 96-well plate and 10 μL of WST-8 reagent solution was added to each well; the mixture solution was incubated at 37°C under 5% CO2 for 1 h. After incubation, GO or S-rGO was centrifuged and 50 μL of the supernatant was transferred to another 96-well plate. The optical density was measured at 450 nm.WST-8 assay was followed as described earlier by Liao et al. . Typicalin vitro toxicology assay kit, TOX7, Sigma-Aldrich). The LDH assay is based on the release of the cytosolic enzyme, LDH, from cells with damaged cellular membranes. Thus, in cell culture, the course of GO- and S-rGO-induced cytotoxicity was followed quantitatively by measuring the activity of LDH in the supernatant. Briefly, cells were exposed to various concentrations of GO and S-rGO for 24 h, and then 100 μL per well of each cell-free supernatant was transferred in triplicates into wells in a 96-well plate, and 100 μL of LDH assay reaction mixture was added to each well. After 3 h of incubation under standard conditions, the optical density of the color generated was determined at a wavelength of 490 nm using a microplate reader.Cell membrane integrity of PMEF cells was evaluated by determining the activity of lactate dehydrogenase (LDH) leaking out of the cell according to manufacturer’s instructions . The plates were incubated at 37°C for 30 min. The amount of released p-nitrophenol was measured at 405 nm in a 96-well microplate reader. Enzyme activity was evaluated as the amount of nitrophenol released through the enzymatic reaction, and absorbance was recorded using a microplate reader  at 405 nm. For normalization, the total protein content was measured using a bicinchoninic acid protein assay kit. Thus, the alkaline phosphatase (ALP) activity was expressed and normalized by the total protein content (U/mg).PMEF cells were cultured in a 48-well culture dish at a density of 5 × 10π-π* transitions of the aromatic C-C bonds and a weak shoulder at 300 nm due to n-π* transitions of C=O bonds. After complete reduction, a red shift of this characteristic peak was observed at 265 nm for S-rGO  reflection peak shifts to a lower angle at 2θ = 11.7, (d spacing = 0.76 nm). The increase in d spacing is due to the intercalation of water molecules and the formation of oxygen-containing functional groups between the layers of the graphite [θ = 26.4° corresponding to a d spacing of 0.36 nm which may be due to the restacking of graphene layers. The disappearance of 002 reflection peak of graphite oxide and the appearance of a broad band at 2θ = 26.4° in the S-rGOs indicate the formation of few-layer graphene, which are close to that of pristine graphene nanosheets (26.6°), revealing the reduction of graphene oxide by spinach leaf extract. These XRD results suggest that spinach leaf extracts are capable in reducing GO and in removing intercalated water molecules and oxide groups in GO.XRD is an effective technique to investigate the interlayer changes and the crystalline nature of the synthesized material. XRD patterns of GO and S-rGO are shown in Figure graphite . In contWe employed dynamic light scattering (DLS) technique to elucidate the status of GO and S-rGO sheets in aqueous solution. DLS measurement was performed in aqueous solution to elucidate the size of reduced graphene oxide after reaction with GO. It was found that the average hydrodynamic diameter (AHD) of GO was 2,000 ± 50 nm Figure . However−1, C-O stretching at 1,720 cm−1, skeletal vibration of unoxidized graphitic domains at 1,620 cm−1, O-H deformation at 1,400 cm−1, C-OH stretching at 1,220 cm−1, and C-O stretching at 1,030 cm−1, were clearly observed in the FTIR spectrum of GO [−1 (O-H) attributed to the water molecules. For the S-rGO sample, the intensities of the bands associated with the oxygen functional groups strongly decreased in relation to those of GO. The results indicate that graphite is successfully oxidized and probably cleaved in the form of GO. GO has two new peaks at 1,720 cm−1 (C=O) from carbonyl and carboxylic groups and at 1,050-cm−1 (C-O) peak from carbonyl, carboxylic, and epoxy groups, which confirms the presence of oxygen-containing functional groups. The peak at 1,625 cm−1 indicates the restoration of sp2. The peak at 1,720 cm−1 almost disappeared in S-rGO because of the removal of C=O. While being reduced by the extract of leaf, the peaks for oxygen functional groups at 3,400 cm−1 significantly decreased. These observations confirmed that most oxygen functionalities in the GO were removed [FTIR is a valuable technique to prove the degree of GO reduction. Figure um of GO ,50,51. A removed ,50,51. T removed , tea pol removed ,35, and 2O molecules and hydroxyl/carboxyl groups attached to GO [The dispersions of GO and S-rGO were further analyzed using SEM. Images were taken randomly from each sample. GO sheets were prepared from natural Gt flakes and had significant solubility in water because of their plentiful oxygen-containing functional groups -58. In g−1. The G vibration mode, owing to the first-order scattering of E2g phonons by sp2 carbon of GO and S-rGO, were at 1,611 and 1,603 cm−1, respectively, while the D vibration band obtained from a breathing mode of k-point photons of A1g symmetry of GO and S-rGO appeared at 1,359 and 1,342 cm−1, respectively  was increased significantly, which indicates the introduction of sp3 defects after functionalization and incomplete recovery of the structure of graphene [2 carbon cluster, a higher intensity of D band suggested the presence of a more isolated graphene domain in S-rGO compare to GO and that SLE is able to remove oxygen moieties from GO. Wang et al. [−1, and increasing the intensity of the D band at 1,350 cm−1 could be attributed to the significant decrease of the size of the in-plane sp2 domains due to oxidation and ultrasonic exfoliation and partially ordered graphite crystal structure of graphene nanosheets. The Raman spectra of graphene-based materials also show a two-dimensional (2D) band which is sensitive to the stacking of graphene sheets. It is well known that the two-phonon (2D) Raman scattering of graphene-based materials is a valuable band to differentiate the monolayer graphene from multilayer graphene as it is highly perceptive to the stacking of graphene layers [−1, whereas this peak is broadened and shifted to a higher wave number in the case of multilayer graphene [−1 for GO and S-rGO, respectively. The results of the Raman spectrum are in good agreement with those of previous studies in which using aqueous leaf extracts of Colocasia esculenta and M. ferrea Linn, an aqueous peel extract of orange [−1, while the D band is shifted to a lower region  and becomes more prominent, indicating the destruction of the sp2 character and the formation of defects in the sheets due to extensive oxidation [Raman spectroscopy is an effective tool to characterize graphite and graphene materials, which strongly depend on the electronic structure. As shown in Figure e layers -29. Genegraphene -29. In txidation . This obxidation .Atomic force microscopy (AFM) is an important technique for the morphological characterization of GO and graphene materials and is also capable of imaging and evaluating the surface morphology and properties -58. Figu2 is the safest graphene derivative with potential for biomedical applications due to its lack of thrombotic and hemolytic activities. Biocompatibility of graphene films was compared with carbon nanotubes using a mouse fibroblast cell line (L-929) to assess the cytotoxicity; the results suggested that the cells adhered and proliferated on graphene film well than carbon nanotubes, which indicated that the material is biocompatible [Measuring the biocompatibility of graphene is complex and depends on the techniques used for synthesis and the selection of the biological model system for study. In order to evaluate the biocompatibility of as-prepared S-rGO, the cytotoxic effect of GO and S-rGO against PMEF cells was investigated. As shown in Figure mpatible ,68. Akhampatible  demonstrmpatible  reportedThe reactive oxygen species (ROS) generated in a concentration-dependent graphene is known as one of the important mechanisms describing the cytotoxicity of graphene . TherefoALP activity is an important and quantitative marker of osteogenesis. Furthermore, ALP is an important marker for functional activity of cells such as cell proliferation. Cell numbers and ALP activity were used as measures of cell proliferation, self-renewal, and pluripotency. ALP is a membrane-bound enzyme that exhibits biphasic behavior. It is expressed on the surface of pluripotent undifferentiated ES cells and disappears as cells begin to differentiate. To examine cell differentiation, the ALP was measured as a marker of differentiation. The ALP activity was measured in GO- and S-RGO-treated cells, and the results are represented in Figure We demonstrated a simple and green approach for the synthesis of water-soluble graphene using spinach leaf extracts. The transition of GO to graphene was confirmed by various analytical techniques such as UV–vis spectroscopy, DLS, FTIR, SEM, and AFM. Raman spectroscopy studies confirmed that the removal of oxygen-containing functional groups from the surface of GO led to the formation of graphene with defects. The obtained results suggest that this approach could provide an easy technique to produce graphene in bulk quantity for generating graphene-based materials. In addition, SLE can be used as an alternative reducing agent compared to the widely used and highly toxic reducing agent called hydrazine. Further, the cells treated with S-rGO show a significant compatibility with PMEF cells in various assays such as cell viability, LDH leakage, and ALP activity. The significance of our findings is due to the harmless and effective reagent, SLE, which could replace hydrazine in the large-scale preparation of graphene. The biocompatible properties of SLE-mediated graphene in PMEFs could be an efficient platform for various biomedical applications such as the delivery of anti-inflammatory and water-insoluble anticancer drugs, and also it can be used for efficient stem cell growth and differentiation purposes.The authors declare that they have no competing interests.SG participated in the preparation and characterization of GOs and S-rGO. JWH, VE, AAD, DNK participated in culturing, cell viability, LDH assay, and ALP assay. SG and JHK participated in the design and coordination of this study. All authors read and approved the final manuscript.


The composite was characterized by optical microscopy, scanning electron microscopy, Raman spectroscopy, energy dispersive spectroscopy, and X-ray photoelectron spectroscopy (XPS). The experimental results indicate that Ga2O3 flakes grew on the surface of graphene film during the cooling process. In particular, tenfold enhancement of graphene Raman scattering signal was detected on Ga2O3 flakes, and XPS indicates the C-O bonding between graphene and Ga2O3. The mechanism of Raman enhancement was discussed. The 2D Ga2O3-2D graphene structure may possess potential applications.2D β-Ga Considerable investigation has been carried out on the assembly of graphene powder with functional materials, such as reduced graphene oxide-TiOactivity -3, graphreaction ,5, and g battery -9. Two t battery -5,7. The battery ,8,9. In  battery . ZnO was battery . A graph battery .2O3 is a deep ultraviolet transparent semiconductor [2O3[2O3 is the most stable form with a wide bandgap of 4.9 eV [2O3 has a useful application in phosphors. The hybrid structure of graphene and Ga2O3 is promising for flexible display devices by exploiting high conductivity and flexibility of graphene and the good luminescence of Ga2O3. Herein, we report a simple and one-step CVD process to assemble β-Ga2O3 flakes on a continuous graphene film. The morphology of the composite was characterized by optical microscopy (OM), field emission scanning electron microscopy (FESEM), Raman spectroscopy, and energy dispersive spectroscopy (EDS) mapping. The assembly mechanism was discussed. Importantly, it was found that the as-grown β-Ga2O3 flakes enhanced the intensity of the graphene Raman signal ten times. The possible Raman enhancement mechanism is proposed.Gaonductor ,14, whicctor [2O3. Among t2. The sample was annealed at 1,000°C for 1 h to remove the surface oxide. The graphene film was synthesized through CVD for only 3 min under 200 sccm Ar flow with 1.5 sccm CH4. After the growth of the graphene, the carbon source was turned off and the temperature was kept at 1,000°C for 30 min. Then, the furnace cover was opened for fast cooling down to room temperature either immediately at 1,000°C or after controllably cooling (approximately 10°C/min) down first to 800°C, 600°C, and 400°C, respectively. The samples were placed in a refrigerator for several hours for solidification before characterization since the melting temperature of gallium is about 29.8°C and has strong supercooling effects, causing its liquid state at room temperature.A 0.2 g Ga with 7 N purity from UMC was laid on a designed quartz bowl, loaded into the quartz tube, and heated to 1,000°C under the protection of 200 sccm Ar and 2 sccm H+ laser , FESEM , and energy dispersive spectroscopy (EDS) analysis  were employed to characterize the samples. X-ray photoelectron spectroscopy  with a monochromatized Al Kα X-ray source  was used to study the bonding between graphene and Ga2O3. A Shirley background was removed from the atomic spectra prior to deconvolution. We tried to conduct an atomic force microscopy and transmission electrical microscopy in order to directly characterize the thickness and the interface between the layers, but the graphene film decorated by Ga2O3 flakes curled up after removing the Ga substrate, rendering high-quality sample impossible.OM  was used for the preliminary exploration. Raman microprobe spectroscopy  with an Ar2O3 flakes could grow on graphene films by controlling the cooling step after graphene-film growth. When the tube furnace cover was opened immediately at 1,000°C for fast cooling after cutting off CH4 gas and keeping Ar flow, no Ga2O3 flakes were observed on the sample. In contrast, the Ga2O3 flakes could be observed by OM on the samples, which were cooled down to 800°C with a rate of approximately 10°C/min in Ar and then fastly cooled down to room temperature by opening the furnace cover, as shown in Figure The CVD graphene growth on liquids, including Ga, Sn, and In, has been reported in our previous work . Liquid 2O3 flakes. Figure −1, more than ten additional peaks appear. These characteristic peaks correspond to β-Ga2O3[2O3, as well as the comparison of their peak positions with bulk Ga2O3 powders, are presented in Additional file 2O3 flakes. A Raman mapping on the area marked as a white square in Figure 2O3 flakes do not change the ratio of I2D/IG of graphene, i.e., the formation of the β-Ga2O3 sheets seems to have no effect on the continuity and thickness of graphene.Raman measurements confirmed the formation of continuous graphene film and Gao β-Ga2O3,17. The 2. The flat and clear area at the left is the bare Ga surface, which is exposed due to volume expansion during the solidification of liquid Ga and/or due to the different thermal expansion coefficients between Ga and graphene [2O3 flakes, the carbon signal does not decrease. This result indicates that the β-Ga2O3 flakes do not hinder the formation of the continuous graphene film and is consistent with the above Raman analysis. The red dots in Figure 2O3 flakes, since the shape and position of Ga and O distribution is consistent with the polygons in Figure During the FESEM characterization, EDS mapping was employed to further confirm the components and element distribution. Figure graphene . The EDS2O3-graphene composite on Ga. This structure may have potential applications due to the 2D-2D assembly. More importantly, we found the interesting property of the graphene Raman enhancement by the β-Ga2O3 flakes, as shown in Figure IG/I2D ratio does not change. Figure 2O3. Through comparing two images of Figure 2O3 flakes have same efficiency to enhance the graphene Raman signal. This phenomenon indicates that the graphene Raman enhancement may be related to the thickness of Ga2O3.The above results from OM, FESEM, Raman, and EDS mapping confirm the formation of a special β-Ga2O3 flakes, the charge transfer is confirmed by the Raman and XPS data. In the Raman spectra of Figure −1  at the locations of the β-Ga2O3 sheets, which indicates the charge transfer between the graphene and β-Ga2O3, and β-Ga2O3 as an electron donor to the graphene [2O3 (4.11 ± 0.05 eV) [2O3. The two peaks of Ga2p for the Ga-O bondings are also clearly observed in Figure sp2 C, and the 285.7 and 286.2 eV peaks are attributed to sp3 carbon and C-O bonds [2O3-graphene structure, and XPS also presents the evidence of the C-O bands, which confirms the negative-charge doping effect from the Ga2O3 sheets on the graphene film. Due to chemical doping, polarizability of graphene is increased, leading to an increase in the Raman scattering cross-section [The surface-enhanced Raman scattering (SERS) has been extensively investigated -20. The graphene . In addigraphene  and Ga2O0.05 eV)  is proxi-O bonds ,28. The -section .2O3-graphene. Ga itself is very reactive and can react with most materials under high temperature. In the periodic table of elements, Ga and Al are in the same main group and have similar characteristics. Analogously, Ga can form a continuous and compact oxidized layer in air, which impedes further oxidation of Ga. Therefore, we need to remove the very thin surface oxide before graphene growth through pre-annealing in Ar/H2 atmosphere. During the CVD graphene growth, hydrogen is hazardous for the graphene formation [2 was not applied during the growth stage. We proposed that the graphene grows on the surface of the liquid Ga at first and then the Ga2O3 sheets come into being on the graphene during the cooling process, as shown in the schematic illustration of Additional file 2O3 on graphene because they will act as nucleation points of Ga2O3 since Ga atoms in the vapor will obviously prefer O as a bonding target, not the carbon atoms.It is necessary to discuss the formation mechanism of Gaormation , and H2 2O3-2D graphene into dilute hydrochloric acid for 1 h at room temperature, the Ga2O3 sheets will disappear. If the graphene covers and protects the Ga2O3 sheets, it is hard to remove Ga2O3 in hydrochloric acid. Another evidence is related to the cooling process. We chose different rapid cooling starting points of 800°C, 600°C, and 400°C. More polygon sheets or granules deposited on the graphene surface when the sample underwent longer cooling durations. According to the illustration depicted in Additional file 2O3 sheets on the graphene surface to form the special 2D Ga2O3 nanosheet-2D graphene sheet structure through a one-step CVD process. Compared to the general Raman enhancement by metals, such as silver and gold, the Ga2O3 nanosheets have remarkable thermal stability. Conversely, silver will oxidize excessively and becomes quenched within 36 h in the air [2O3 film can be transferred onto other targets and may be used as bio-substrate through SERS. The stability of the Ga2O3 nanosheets and the structure stability need to be further investigated.This mechanism is supported by two evidences. The first one is that after immersing the samples of 2D Ga the air . The 2D 2O3 nanosheets, with a lateral size of 1 to 10 μm, on continuous 2D graphene film were synthesized by a one-step CVD process on liquid gallium substrate. The Raman and EDS mapping confirm the formation of the β-Ga2O3 sheets on the graphene surface. The formation mechanism was proposed as a β-Ga2O3 sheet formation after graphene synthesis during the cooling process. The graphene Raman enhancement over ten times was detected on the β-Ga2O3 sheets due to the charge transfer. The 2D-2D structure may have potential application in optical and electronic devices.In summary, separated 2D thin GaThe authors declare that they have no competing interests.YZ, X-GX, and G-QD carried on the experimental parts. Q-KY, G-QD, T-RW, and QG analyzed and interpreted the data. Q-KY and G-QD wrote the manuscript. N-YY, J-ND, S-MW, X-MX, and M-HJ were involved in the discussions and revision of the manuscript. All authors read and approved the final manuscript.Raman data, XPS analysis, and proposed growth mode.Click here for file


The response time of the chip was also experimentally verified to be within 5 seconds from RH 36% to RH 64%. As a consequence, the implemented humidity SSoC paves the way toward the an ultra-small sensor system for various applications.A fully integrated humidity sensor chip was designed, implemented, and tested. Utilizing the micro-stamping technology, the pseudo-3D sensor system-on-chip (SSoC) architecture can be implemented by stacking sensing materials directly on the top of a CMOS-fabricated chip. The fabricated sensor system-on-chip (2.28 mm × 2.48 mm) integrated a humidity sensor, an interface circuit, a digital controller, and an On-Off Keying (OOK) wireless transceiver. With low power consumption,  To build ubiquitous sensor networks, moreover, the small system form factor should be addressed to seamlessly integrate the sensor system into environments. To match the above requirements, monolithic sensor architecture has been proposed and implemented by micro-electro-mechanical system (MEMS) technology for years [i.e., sensing area is much larger than the circuit area. As a consequence, novel sensor chip architectures need to be developed.With the advancement of communication and information technologies, sensor networks have become an emerging technology to be developed for the next generation . This teor years ,4. Howev4, CO and CO2 environment, and good temperature stability of 7.85 × 10−10 A/°C [Among the various sensing quantities sought in different applications, humidity is one of the most important factors –7. As a −10 A/°C . In thisWith the circuits fabricated by a CMOS process, specifically the Fe-Al-polyaniline humidity sensing material is micro-stamped on the top of CMOS circuits to form the pseudo three-dimensional (3D) sensor chip architecture. 3D integration is one of the methods to integrate heterogeneous layers with interconnecting metal lines to improve performance or create new functions of chips . With th2.2.1.3+ and Al3+ with specific concentration, can affect the selectivity and resistance of the blends. Following stirring within 200 mL 10% NaOH solution and precipitation, polyaniline blends can be obtained. Subsequently, the precipitate is washed with distilled water to reduce the acidity [To fabricate the sensing material, a solution-based process has been developed . In brie acidity ,15. The 2+ +H2O → NH + H3O+, is stimulated as the sensing film is exposed to humidity. As a result, the resistance of the polyaniline blends decrease. This reaction can be triggered in room temperature. The resistance change of polyaniline blends introduced by the reaction between low and high relative humidity (35% to 70% RH) is three orders [The fundamental sensing mechanism of the polyaniline film has been investigated in previous works . In briee orders . The int2.2.6 to 108 [To the analog front-end (AFE) circuit, the humidity sensing device plays as a variable resistance in the voltage dividing circuit, as shown in 6 to 108 , the vol6 to 108 .i.e., a cell phone or laptop, for further data analysis and saving. In addition, the wireless circuit cannot only transmit data but also receive command codes to wake up this SSoC from standby mode to active operation mode. Moreover, this chip also includes a built-in junction-based temperature sensor with good linearity. This temperature sensor shares the same chopper DDA with the humidity sensor by a multiplexer to reduce the circuit power consumption and complexity. Although the developed polyaniline blends have good temperature stability (7.85 × 10−10 A/°C) [After the sensing is amplified, the sensing signal is then translated into digital data by a 10-bit successive approximation register analog-to-digital converter SAR ADC with low power consumption. With the help of a built-in digital signal processor (8051-based), this digital data is packaged into RS232 format to be sent by OOK wireless circuits (TX/RX) to an external host, 10 A/°C) , this te2.3.To implement the proposed system architecture, the Taiwan Semiconductor Manufacturing Company (TSMC) 0.35 μm two-polysilicon-four-metal (2P4M) CMOS process is employed to implement the humidity sensor system. Previously designed analog and digital circuits are implemented following a standard CMOS process flow. The top metal layer  of the chip is used as the interdigitated electrodes. These interdigitated electrodes are directly connected to the AFE input by vias. It should be noted that the position of the intedigitated electrodes is on the top of the chopper DDA and OOK TX/RX to form the proposed pseudo-3D architecture. On the top of interdigitated electrodes, a layer of gold is e-beam evaporated to form a good contact to the sensing material . Then th2.4.The humidity sensing experiment was designed and used to determine the performance of the developed heterogeneous humidity sensing SoC. The testing board module can be seen in 3.2. The performance table shows the power consumption of the circuit system is around 730 μW without RF operation. By considering the sensing device and the off-chip resistor operation, the total power consumption is around 750 μW. The region marked by a blue line is the place for stamping the sensing material. After stamping the sensing material, the chip is wire-bonded and ready for test.The fabricated sensor chip photo and electrical performance are shown in i.e., from 32% to 66% in controlled chamber. The experimental results are shown in To examine if the sensing material is functional on the top of the interdigitated electrodes on the fabricated chip, the humidity SSoC was placed into a controlled chamber. With gating the injection of different relative humidity into the chamber, the voltage signal at the output of AFE was measured by Keithley 2400 source-meter. The experimental date of a single humidity injection can be shown in 4.2). With low power consumption, i.e., 750 μW without RF operation, experimental results have shown that the humidity sensing SoC had a fast response time of about 5 seconds and high sensitivity of about 30 mV/% at room temperature. In addition, the SSoC integrates a polymer-based humidity sensing device, AFE, SAR ADC, 8051-based microcontroller, and OOK wireless TX/RX on a single chip. Therefore, this work demonstrates not only a practical humidity sensor system-on-chip, but also a novel method to implement sensor systems for applications in sensor network technologies.In this work, a fully-integrated humidity sensor system-on-chip (SSoC) is demonstrated. The SSoC is fabricated by the TSMC 0.35 2P4M CMOS process. Implemented by a micro-stamping technique to stack the sensing device on the top of CMOS circuits, the SSoC can be formed in a pseudo 3D architecture to obtain an ultra-small form factor (5.65 mm


LINE-1 (L1) retrotransposons are mobile genetic elements whose extensive proliferation resulted in the generation of ∼34% of the human genome. They have been shown to be a cause of single-gene diseases. Moreover, L1-encoded endonuclease can elicit double-strand breaks that may lead to genomic instability. Mammalian cells adopted strategies restricting mobility and deleterious consequences of uncontrolled retrotransposition. The human APOBEC3 protein family of polynucleotide cytidine deaminases contributes to intracellular defense against retroelements. APOBEC3 members inhibit L1 retrotransposition by 35–99%. However, genomic L1 retrotransposition events that occurred in the presence of L1-restricting APOBEC3 proteins are devoid of detectable G-to-A hypermutations, suggesting one or multiple deaminase-independent L1 restricting mechanisms. We set out to uncover the mechanism of APOBEC3C (A3C)-mediated L1 inhibition and found that it is deaminase independent, requires an intact dimerization site and the RNA-binding pocket mutation R122A abolishes L1 restriction by A3C. Density gradient centrifugation of L1 ribonucleoprotein particles, subcellular co-localization of L1-ORF1p and A3C and co-immunoprecipitation experiments indicate that an RNA-dependent physical interaction between L1 ORF1p and A3C dimers is essential for L1 restriction. Furthermore, we demonstrate that the amount of L1 complementary DNA synthesized by L1 reverse transcriptase is reduced by ∼50% if overexpressed A3C is present. Apolipoprotein B mRNA Editing Enzyme Catalytic Polypeptide 3, A3) protein family of cytidine deaminases . Since iewed in ].23-28-P-C-X2-4-C  or two  cytidine deaminase (CDA) domains containing a conserved zinc-binding motif (C/H)-X-E-XC-X2-4-C . MultiplC-X2-4-C . A3 protC-X2-4-C ,23. All C-X2-4-C . All A3 C-X2-4-C ]. HoweveC-X2-4-C . For insC-X2-4-C .Alu with varying degrees of efficiency , washed in 1× PBS-T and incubated overnight with the respective primary antibody at 4°C. HA-tagged A3 proteins and L1 ORF1p were detected using an anti-HA antibody  in a 1:5000 dilution and the polyclonal rabbit-anti-L1 ORF1p antibody #984 , for 1 h at room temperature. Anti-rabbit HRP  was used as secondary antibody. Immunocomplexes were visualized using lumino-based ECL immunoblot reagent (Amersham Biosciences Europe GmbH).To analyze expression of WT and mutant A3 proteins, and of the tagged L1 reporter element, co-transfected HeLa cells were lysed 48 h after transfection using triple lysis buffer ; 1% Triton X-100; 1% deoxycholate; 1× complete protease inhibitor cocktail [Roche]), and lysates were cleared by centrifugation. Twenty micrograms of each protein lysate were boiled in Laemmli buffer, loaded on 12% polyacrylamide gels, subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and electroblotted onto nitrocellulose membranes. After protein transfer, membranes were blocked for 2 h at room temperature in a 10% solution of nonfat milk powder in 1× PBS-T  and posTo analyze for potential co-localization of L1 ORF1p-T7 or A3C-HA fusion proteins with the stress granule marker G3BP, the following primary and secondary antibodies and dilutions were applied: anti-T7 antibody , anti-HA antibody . As secondary antibodies, anti-mouse Alexa488  or anti-mouse Alexa594  and anti-goat Alexa546  were used, respectively. To detect the stress granule marker G3BP, anti-G3BP antibody  and secondary antibody anti-rabbit Alexa488  were used.To evaluate whether the P-body–specific marker rck/p54 co-localizes with L1 ORF1p-T7 or A3C-HA fusion proteins, the following antibodies and dilutions were used: anti-T7 antibody , anti-HA  and anti-rck/p54 .6 HeLa cells per co-transfection were seeded in a T-75 cell culture flask. Twenty-four hours later, cells were co-transfected with 10 µg of the L1 reporter plasmid pJM101/L1RP and 10 µg of the A3C-WT or mutant expression plasmid or the empty expression vector pcDNA 3.1/Zeo(+), respectively, using FuGENE HD. Three days later, cells were washed twice with cold PBS, detached with Trypsin EDTA pelleted for 5 min at 3000g and 4°C and incubated on ice for 5 min in lysis buffer . The lyf et al. . Briefly (Roche) ] in ultr6 cells per T-175 cell culture flask. Cells were transfected 24 h later with 20 µg L1 reporter plasmid pDK101 . After 4 days of selection, cells were transfected with 8 µg of the A3 expression plasmid using FuGENE 6 or FuGENE HD transfection reagent. Three days later, cells were washed twice with cold phosphate-buffered saline/5% PMSF (PBSP), detached with a cell scraper in 10 ml PBSP and pelleted for 5 min at 3000g and 4°C. Cells were incubated on ice for 5 min in lysis buffer . To precipitate HA-tagged A3 proteins, the cleared lysates were incubated with 50 µl of anti-HA Affinity Matrix Beads (Roche) overnight or 1 h at 4°C under gentle agitation. For RNase treatment, the cleared lysate was incubated with 50 µl of anti-HA Affinity Matrix Beads and RNase A (Qiagen) at a final concentration of 50 µg/ml. The beads were washed five times with ice-cold lysis buffer. Subsequently, the pelleted beads were boiled in SDS loading buffer for 8 min at 95°C before separation by SDS-PAGE.For co-immunoprecipitation, 1.6 × 10RNA isolation, reverse transcriptase-polymerase chain reaction (RT-PCR) and L1 element amplification protocol (LEAP) reaction procedures were adopted from  with the2, 10 mM DTT, 0.4 mM LEAP primer, 20 U RNaseOut (Invitrogen), 0.2 mM dNTPs and 0.05% Tween 20 in a final volume of 50 µl for 1 h at 37°C. Reactions with RNPs from untransfected HeLa cells (HeLa) or in the absence of RNPs (No RNP/RNA) were used as negative controls. After incubation, 1 µl of each LEAP reaction was used in a standard 30 µl of PCR using AmpliTaq DNA polymerase (Applied Biosystems) and 0.4 mM of each linker PCR primer and L1.3 end primer (The LEAP reaction was performed as described previously  with thed primer  accordin27-P-C-X2-C) is involved in the inhibition of L1 retrotransposition, we introduced mutations that were reported to abolish the editing activity in the context of A3G  did not affect A3C-mediated L1 restriction significantly , and mutating both Cys residues separately even slightly increased the L1 inhibition from 54 to 87% (P = 0.0023) or 71% (P = 0.0349), respectively (Supplementary Table S1). These findings clearly provide evidence that deaminase activity is not required for A3C-mediated L1 restriction. Replacing Glu68 by Gln (E68Q) still resulted in a significant inhibition of L1 by ∼40% . Since our Escherichia coli mutation assays demonstrate that the A3C CDA mutants H66R, E68Q, C97S, C100S and C97S/C100S are devoid of any meaningful DNA-editing activity relative to A3C-WT , the retrotransposition reporter assays . Mutating these residues was also reported to abolish the capability of A3C to introduce G-to-A hypermutations in an E. coli mutation assay , indicating that A3C oligomerization is required for A3C-mediated L1 restriction. To evaluate the possibility that the numbers of L1 retrotransposition events in A3C-WT and F55A or W74A-transfected cells is a consequence of discrepancies in WT and mutant A3C protein levels, we assessed expression levels of WT and mutant A3C proteins in a parallel set of co-transfected HeLa cells. Immunoblot analysis of cell extracts isolated 2 days after co-transfection showed that comparable amounts of A3C proteins were expressed . However, the K22A mutation reduced the L1 restricting effect to only ∼29% relative to mock-transfected cells . Initial retrotransposition reporter assays also suggested that the R122A mutation in A3C not only fully restored the retrotransposition activity of the L1 reporter to 100% (defined as the number of L1 retrotransposition events in the absence of overexpressed A3C in HeLa cells)  A, but acn levels B, which Supplementary Table S1). Expression of comparable amounts of R122A and A3C-WT protein  and an ∼7-fold decrease (P = 0.0003) of L1 retrotransposition, respectively (P = 0.1022), whereas A3C-WT inhibited L1 by ∼4-fold (P = 0.0035). These data confirmed that the restoration of the relative L1 retrotransposition frequency of 100%, observed after co-expression of the R122A mutant (P = 0.5866) nor between L1-restricting effects of increasing A3C-WT protein levels resulting from transfection of 0.25, 0.5, 1 or 1.5µg A3C-WT plasmid DNA (P = 0.4190). The 1.7- to 2-fold increase of L1 retrotransposition resulting from co-transfection of 0.5–1.5 µg of the R122A expression construct  showed that the incidence of co-localization drops significantly to ∼5.6–15.9% for the A3C mutant proteins tested. Interestingly, the most prominent drop (by 83 and 78%) was observed for the R122A and W74A mutations, respectively, which were both shown earlier to abolish the L1-restricting effect of A3C entirely . Three days later, the cleared cell lysate was centrifuged through a sucrose cushion and the resultant L1 RNP–containing pellet was resuspended , and were then hygromycin selected for the presence of the respective plasmid for 11 days A. Then, uspended . Whole-cuspended B. Immunouspended B.FigureTM III RT  and were therefore not able to evaluate any potential effect of the A3C mutants on L1 RNP–associated L1 RT activity. The absence of R122A and W74A from the RNP fractions is consistent with their inability to interact with L1 ORF1p  to only ∼29% or even restored and enhanced L1 retrotransposition activity, respectively, the N177A mutation did not affect L1 inhibition by A3C significantly (pN177A = 1). The L1-restricting effect of A3C-WT and its mutant proteins K22A and N177A, as well as the absence of any L1-inhibiting effect of R122A, correlates well with the antiretroviral effects of K22A and N177A and the loss of any inhibitory effect of R122A against SIVΔvif reported previously ; Stiftung zur Erforschung infektiös-immunologischer Erkrankungen‘ (to H.H.). Funding for open access charge: Paul-Ehrlich-Institut.Conflict of interest statement. None declared.