The availability of nitrate and ammonium significantly affects plant growth. Co-provision of both nutrients is generally the best nutritional condition, due to metabolic interactions not yet fully elucidated. In this study, maize grown in hydroponics was exposed to different nitrogen (N) availabilities, consisting of nitrate, ammonium and co-provision. Roots and leaves were analyzed after 6, 30, and 54 h by biochemical evaluations and proteomics. The ammonium-fed plants showed the lowest biomass accumulation and the lowest ratio of inorganic to organic N content, suggesting a metabolic need to assimilate ammonium that was not evident in plants grown in co-provision. The N sources differently affected the root proteome, inducing changes in abundance of proteins involved in N and carbon (C) metabolisms, cell water homeostasis, and cell wall metabolism. Notable among these changes was that some root enzymes, such as asparagine synthetase, phosphoenolpyruvate (PEP) carboxylase, and formate dehydrogenase showed a relevant upsurge only under the sole ammonium nutrition. However, the leaf proteome appeared mainly influenced by total N availability, showing changes in the abundance of several proteins involved in photosynthesis and in energy metabolism. Overall, the study provides novel information about the biochemical determinants involved in plant adaptation to different N mineral forms. In several plant species, some members of the AS gene family are upregulated by increases in the levels of amino acids and/or of internal NHnditions . In our n n, na, , confirm4+ nutrition [4+ content in roots never reached levels associated with toxicity in maize [4+ inputs or during longer exposures.In this context, it is important to note that it was proposed that glutamate dehydrogenase (GDH) could contribute to the assimilative process when plants are exposed to an excessive NHutrition . Our proin maize ,27, but 3− to change the balance between the quota of NH4+ drained by assimilation and the quota of NH4+ delivered to vacuole storage.On the whole, the proteomic analysis provided evidence that co-provision could also have positive effects on plants’ growth because of the ability of NO4+, both during (a) and (na) treatment  plants at 54 h could indicate that a massive allocation of photoassimilates in this organ occurred when NH4+ was provided as the sole N nutrient  at 54 h, several events occurred at once, along with the increase in NH content . Firstly4 plants . Finally4 plants , of two 4 plants , as wellstresses , it is pnutrient C. This rnutrient B.3−, as indicated in maize root plastids [3− than by NH4+ [In roots, C metabolism was instead more specifically affected by the kind of N source . Seeing plastids  and by tplastids , it is i by NH4+ . 3− in (n) and (na) roots  roots . Althouga) roots . With th authors ,10,45. TOverall, this proteomic study highlights the different metabolic roles for roots and leaves and it allows us to propose novel molecular determinants involved in the adaptation of plants to different N sources.+ channel  and tonoplast (TIP) . In partionality ). Howevereatment . It seem tissues A could h vacuole . This pr3− might promote a higher accumulation of K+ channels and aquaporins in roots, maybe acting as an osmolyte. Considering that both protein families are involved in the NH4+/NH3 transport [3− and the highest NH4+ content in roots in co-provision  and (a) plants  plants A. This tThe “protein synthesis and folding” was the major functional class both in roots and leaves, accounting for 27% and 46% of the DPs, respectively . In both3− in N-starved Arabidopsis seedlings induces changes in the expression of more than 100 genes encoding ribosomal proteins [Ribosomal proteins accounted for 36% and 59% of the category in roots and leaves, respectively, and, in particular, in leaves they were identified as both chloroplastic (30%) and cytosolic (70%) members . In geneproteins . Howeverproteins , encodedproteins ,56. To uSeveral peptidyl-prolyl cis-trans isomerases (PPIases) of the cyclophilin-type (Cyp), namely, two in the roots  plants were characterized by a relevant upsurge in abundance of formate dehydrogenase (R105) in roots and of lipoxygenase (L76) in leaves  and (na) plants, which are therefore probably related to the presence of NOn leaves . Both inn leaves . + dependent oxidation of formate to CO2 [4+ nutrition, a trait that was less pronounced in co-provision  [4+, all together these results support the hypothesis that LOX accumulation in the leaf could be a way to store N, a new and intriguing role of LOX in cereal crops. Plant lipoxygenases (LOX) are involved in polyunsaturated fatty acid and membrane metabolisms, but it has also been proposed that these enzymes could be accumulated as vegetative storage proteins in leaves, as observed in soybean (Glycine max L.) in response to sink limitation . In one −, 30 h) . AlthougIn addition, three root peroxidases of the class III (Prxs) showed significant changes in abundance  seeds of the line PR33A46  were germinated in the dark at 26 °C for 72 h. Seedlings were then grown by a hydroponic system in a growth chamber with a 16/8 h day/night regime, at 26/22 °C, constant relative humidity of 65%, and PPFD of 500 μmol·m−2·s−1. After 48 h of incubation in 4 mM CaSO4, the plants were transferred into a growth solution with low N input 2SO4, 60 μM Fe-EDTA, 25 μM KCl, 12.5 μM H3BO3, 1 μM MnSO4, 0.25 μM CuSO4, 0.25 μM ZnSO4, 0.25 μM Na2MoO4, pH = 6.1). After six days, at the beginning of the day (t0), the plants were transferred into new growth solutions in which N availability was changed according to the following treatments (abbreviated by letters in brackets): (i) 5 mM NO3− (n); (ii) 5 mM NH4+ (a); (iii) 2.5 mM NO3− + 2.5 mM NH4+ (na). All the solutions were balanced with K2SO4 and continuously aerated by electric pumps. The plants were sampled at t0 and after 6, 30, and 54 h of treatment. Roots were rinsed with water and blotted with paper towels. Roots and leaves were separately collected, weighed and immediately frozen in liquid N2. Each biological sample was composed of roots or leaves collected from four plants. Samples were stored at −80 °C. The significance of the changes in the biomass accumulation in roots and in leaves (n = 8) was assessed by the ANOVA test .Maize  method, as described by Coskun and coworkers [2, homogenized in 5 volumes of ice-cold 10 mM formic acid (FA), and centrifuged at 14,000 g for 10 min at 4 °C. The supernatants were filtered by Millipore Millex HC cartridges (0.45 μm). An aliquot of the extract was added to 3 mL of OPA reagent , pH = 7). After incubation for 30 min in the dark, the sample absorbance was determined at 410 nm. The contents of NHoworkers . Briefly3− and NH4+) plus the organic N  and compared by the ANOVA test .Amino acids, reducing sugars and sucrose were extracted from leaf and root samples as previously described . Amino a in corn ). All of2 using a mortar and pestle and an aliquot of 200 mg was suspended in 3 volumes of extraction buffer  glycerol, 2% (w/v) sodium dodecyl sulfate (SDS), 2% (v/v) 2-ME, 0.1 mg·mL−1 Pefablock (Fluka)). Samples were vortexed for 10 min at room temperature and incubated for 30 min at 90 °C. After centrifugation at 14,000× g for 15 min, the collected supernatants were recentrifuged at 14,000× g for 5 min. The supernatants were stored at −80 °C until further use. Protein concentration was measured by 2-D Quant Kit .Frozen samples of roots or leaves were powdered in liquid Nw/v) polyacrylamide gel [Mr 12 000-225 000) . Briefly, the run was conducted applying 60 mV for 30 min until protein samples completely entered in the running gel. The gels were then incubated for 1 h in fixing solution (10% (v/v) acetic acid, 50% (v/v) methanol), stained for 1 h with Coomassie Brilliant Blue (CBB) (0.1% (w/v) CBB R-250, 10% (v/v) acetic acid), and destained in 10% (v/v) acetic acid. Protein samples were colored with traces of bromophenol blue and aliquots of 15 µg were purified by partial 1D SDS-PAGE  conducted on 16%  formic acid (FA). All of the procedures were replicated on three independent biological samples (n = 3).The portion of gel containing proteins was excised and subjected to tryptic digestion. In-gel digestion was performed as previously described  with the−1 in 0.1% (v/v) FA. The peptides were then eluted during a 100-min non-linear gradient of acetonitrile (from 3% to 50% v/v) in 0.1% (v/v) FA at 0.4 µL·min−1. The mass spectrometer ran in positive ion mode and MS scans were acquired over a range from 300 to 3000 mass-to-charge ratio (m/z) at 4 spectra·s−1. Precursor ions were selected by auto-MS/MS with a maximum of 4 precursors per cycle and active exclusion set at 2 spectra for 0.1 min. All mass spectrometry experiments were conducted on an Agilent 6520 Q-TOF mass spectrometer equipped with an HPLC Chip Cube source driven by a 1200 series nano/capillary LC system ). Both systems were controlled by a MassHunter Workstation . Chromatography was performed into Polaris-HR-Chip-3C18 (Agilent Technologies), consisted of a 360-nL trap column and a 75 μm × 150-mm analytical column . An aliquot of sample was loaded onto the trap column at 2 µL·minhttp://www.uniprot.org/), and concatenated with the reverse one. The threshold used for protein identification was false discovery rate (FDR) ≤ 1%, number of unique peptides (NUP) ≥ 2, protein amino acid coverage ≥ 5% if NUP < 4. Peptide quantification was obtained as the spectrum intensity (SI) of the precursor (MH+). Protein quantification was obtained summing the SI of all the identified peptides in the protein. Protein abundance was normalized as the % with respect to the abundance of all validated proteins in the sample (%SI). Proteins showing at least a two-fold change in their %SI among at least 2 of all the experimental conditions were analysed according to the two-way ANOVA test to ascertain the source of variations and then by one-way ANOVA to assess the significance of the differences .Analysis of MS/MS spectra were performed by Spectrum Mill MS Proteomics Workbench . Cysteine carbamidomethylation and methionine oxidation were used as fixed and variable modifications, admitting 2 tryptic missed cleavages per peptide. The search was conducted against the database of Zea mays (ID 4577) protein sequences  downloaded from UniProtKB/Swiss-Prot (


Functional BAT was detected in 4 out of 11 individuals with T1D with a prevalence of 36%. The glucose utilization rate in the supraclavicular BAT regions ranged from 0.75–38.7 µmol × min−1 × 100 g−1. The glucose utilization per gram tissue was higher in BAT when compared with both WAT (p = 0.049) and skeletal muscle (p = 0.039). However, no correlation between BAT activity and metabolic control or insulin requirements was found. In conclusion, for the first time, cold-induced BAT was detected in patients with T1D with a wide range in metabolic activity. Contrary to findings in animal models, the metabolic activity of BAT had negligible impact on insulin requirements or metabolic control in T1D under normal physiological conditions.Type 1 diabetes (T1D) is characterized by the loss of insulin-producing cells and hence insulin secretion and metabolic control. In addition to insulin, there are a number of hormones and cytokines that influence metabolism, and many of these can be secreted from brown adipose tissue (BAT). However, the presence and activity of BAT in T1D have not been studied, despite the fact that preclinical studies have shown that transplantation of BAT in mouse models of T1D can restore metabolic control. The metabolic activity of BAT, white adipose tissue (WAT), and skeletal muscle was investigated in patients with T1D ( Type 1 diabetes (T1D) is characterized by a progressive loss of beta cells and thereby insulin secretion, resulting in hyperglycaemia. Since the discovery of insulin by Banting and Best in 1921, exogenous insulin has been the cornerstone of T1D treatment. In fact, since then, no fundamentally new drug treatments have been clinically established for the treatment of T1D. However, apart from insulin there are a number of hormones and cytokines that influence glucose homeostasis. Cytokines have traditionally been studied for their inflammatory effects, but have in recent years also attracted increasing interest due to their effects on metabolism. For instance, IL-6 has a complex role in metabolism and has been shown to increase insulin sensitivity . Many of18F)fluoro-D-glucose (18F-FDG). Interestingly, the metabolic activity of BAT is blunted in patients with obesity and type 2 diabetes [Functional BAT was only recently confirmed in adult humans through hybrid positron emission tomography (PET) and computed tomography (CT) studies ,8. The mdiabetes ,10. ThusThere is also an increasing interest in the hormones and cytokines secreted by BAT (“batokines”) and their beneficial impact on whole body metabolism . Great eDespite these interesting preclinical and clinical data on the role of BAT in metabolism in metabolic disease, there have been no reports on the activity and occurrence of BAT in T1D.18F-FDG PET/CT in response to cold stimulation in 11 individuals with long-standing T1D. The primary endpoint was to evaluate the correlation between the glucose utilization in BAT and insulin requirements in individuals with established T1D. All participants were characterized regarding metabolic control, insulin requirements, body composition, and hormonal status in order to study potential correlations to metabolic activity in different tissue compartments, with emphasis on BAT.In the present study, we have investigated metabolic activity, measured as glucose utilization rate, in three different tissue compartments, BAT, WAT, and skeletal muscle by n = 11 subjects were successful. Descriptive data of all participants can be found in Glu) varied substantially between individuals, ranging from 0.75 µmol × min−1 × 100 g−1 (indicating negligible BAT activity) to 38.7 µmol × min−1 × 100 g−1 , weight adjusted (p = 0.64), or adjusted for lean body mass (p = 0.67) (p = 0.91) or HbA1c (p = 0.99). BAT activity was not correlated to blood lipid profiles: total cholesterol (p = 0.72), HDL-cholesterol (p = 0.86), LDL-cholesterol (p = 0.58) or triglycerides (p = 0.78), nor did the metabolic activity of BAT correlate to body composition expressed as body mass (p = 0.71), BMI , fat mass (p = 0.93), or lean body mass (p = 0.71).We found no correlation between metabolic activity of BAT and insulin requirements, whether expressed as total daily insulin doses ( = 0.67) A. The mer = 0.44, p = 0.027) (max)  (mean) .However, the metabolic activity of BAT correlated to the cold-induced levels of IL-6 (= 0.027) B. Cold-i 0.0005) C or meanr = 0.80, p = 0.003) and the delta IL-6 levels  correlated positively to HbA1c levels but not insulin requirements or fasting glucose. There was a tendency towards a positive correlation to IGF-1 levels both prior to cooling  and cold-induced . No correlation was observed for FGF-21 (p = 0.47), leptin (p = 0.70), or adiponectin (p = 0.60).The cold-induced levels of IL-6 were not, however correlated to fasting glucose, HbA1c, or insulin requirements (data not shown), whereas the IL-6 levels prior to cooling  but not with HbA1c (p = 0.49). However, metabolic activity of WAT did not correlate to insulin requirements, markers of body composition, or investigated hormones/cytokines. The metabolic activity of WAT and skeletal muscle were correlated .The metabolic activity of WAT was found to correlate with fasting glucose levels  and body volume , but not with fat mass (p = 0.27) or lean body mass (p = 0.16).As for BAT, we did not observe any correlation between the metabolic activity in skeletal muscle and insulin needs, fasting glucose, HbA1c, or investigated hormones/cytokines. However, there was a negative correlation with body mass . These four individuals did not differ when compared to the individuals with low BAT activity regarding markers of metabolic control or body composition (data not shown). However, there was a tendency towards increased insulin requirements in these four patients . In addition, the cold-induced levels of IL-6 were significantly higher in the patients with high BAT activity . Both the pre-cooling and cold-induced levels of FGF-21 were increased in the patients with high BAT activity .Notably, four participants had markedly increased BAT activity (23.94 ± 6.0 vs. 3.32 ± 1.03 µmol × minp = 0.049) and to skeletal muscle (p = 0.039) (p < 0.0001) and WAT (p = 0.0001) when compared with BAT (The glucose utilization per gram tissue was higher in BAT when compared both to WAT (= 0.039) A. Howevewith BAT B.−1 × 100 g−1) to negligible in others. Using SUVmean > 1.5 as defining cut-off for the presence of functional BAT [mean between 1.2 and 1.5, just below the threshold. SUVmax is usually not used for assessing BAT prevalence as it potentially is less reproducible than SUVmean in this specific setting [max > 2.5 as threshold.We demonstrated, for the first time, the presence of BAT in individuals with T1D. The metabolic activity of BAT ranged from considerable in some individuals  in patients with T1D. There was no difference in the metabolic activity of BAT in participants with low insulin requirements compared to those with high insulin requirements. Nor were insulin requirements lower in the four participants with the highest metabolic activity of BAT. In fact, there was a tendency towards increased insulin requirements in the individuals with the highest metabolic activity of BAT.However, the metabolic activity of BAT correlated with the levels of IL-6, a known batokine, and was found to be higher in participants with the highest BAT activity. This is suggestive of a systemic effect of BAT in T1D. Traditionally, IL-6 is viewed as a pro-inflammatory cytokine, but it can also exert endocrine effects and has been described to increase insulin sensitivity in both skeletal muscle and WAT . In addiIn the publication by Gunawardana et al., diabetic mice were cured by a transplantation of embryonic BAT ,5. The lFGF-21 was recently shown to correlate to the metabolic activity of BAT in healthy individuals . FGF-21 mean > 1.5 [Glu ranging from 11.4 to 38.7 (average 23.94 ± 6.0) µmol × min−1 × 100 g−1. Despite that, we did not observe any signs of improved metabolic control in those individuals. It is tempting, based on preclinical literature, to speculate that BAT could serve as a back-up mechanism in order to clear glucose from circulation and thereby protect against severe hyperglycaemia in T1D under hypoinsulinemic conditions. However, we found no evidence for such a mechanism under these experimental conditions.We identified four individuals with functional BAT, defined as SUVan > 1.5 . These in = 11). The original primary endpoint was to evaluate whether BAT glucose utilization correlated with insulin requirement in subjects with T1D. At the interim analysis at n = 11 individuals, no such tendency was observed, as outlined above. Further enrollment was thus deemed unnecessary. The remaining correlations observed in this material should therefore be considered exploratory, as the study was not specifically designed or powered otherwise. We hope, however, that the exploratory observations will generate novel hypotheses on the role of BAT in T1D and motivate future clinical studies. One such research question is the prevalence of functional BAT during the progress of T1D, which could be examined by a longitudinal study design following individuals from T1D debut.The major limitation of this study was the low number of enrolled individuals , the total body contribution to glucose clearance was negligible compared to that of the whole-body mass, especially of skeletal muscle, but also of WAT. It remains to be investigated whether induced browning of adipose tissue might increase the total beige/brown adipose tissue to meaningful levels, which might be able to help correct hyperglycemia in humans.2, HbA1c 45–60 mmol/mol (DCCT 6.3–7.6%). Exclusion criteria were: other metabolic disorders, treatment with glucose-lowering drugs other than insulin, drug abuse, extreme physical activity, and pregnancy. In total, 11 individuals with T1D were included. Participants were sub-grouped based on their average insulin requirements: low (<0.65 U × kg−1 × 24 h−1) or high insulin needs (>0.65 U × kg−1 × 24 h−1), see The study was approved by the regional ethical board of Uppsala County  and conducted in accordance with the declaration of Helsinki as revised in 2000. Participants were recruited from the Diabetes and Endocrinology Department at Uppsala University Hospital. All study participants were given oral and written information and gave their written consent prior to participation in the study. Inclusion criteria were: clinically established diagnosis of T1D, age 18–35 years, BMI 19–25 kg/mDF2100, DLP00 and DRP300 respectively, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol. All other blood samples were analyzed at the central laboratory of Uppsala University Hospital according to clinical routine.Fibroblast growth factor-21 (FGF-21), leptin, and adiponectin were analyzed using commercially available ELISA kits (Art. No All examinations were conducted in the morning under fasting conditions. Body weight and volume was determined by air displacement plethysmography (Bod Pod)  and body density was calculated based on these two measurements. Body fat mass and lean body mass were determined by a two-compartment model. Measurements of waist, hip, and upper arm circumference were taken by the same examiner for all patients according to standardized techniques.All PET/CT examinations were conducted under fasting conditions. Blood samples were collected prior to cooling and after cooling just prior to the PET/CT examination. To induce BAT activation, participants rested in light clothing in a room with an ambient temperature of 19 °C for 60 min while wearing a cooling vest .18F-FDG was administered intravenously and each participant was examined by dynamic PET for 40 min.Participants were positioned in a Discovery ST PET/CT scanner  centred at the clavicular level. A localization dose CT examination  was performed for attenuation and anatomical co-registration of PET images. A target dose of 2 MBq/kg The PET list data was sorted into 11 frames  and reconstructed using OSEM VUEPOINT .Regions of interest (ROIs) were placed in the supraclavicular regions where BAT is normally located, guided by CT (−190 to −10 Houndsfield units) according to standard practise . ROIs weGlu, µmol × min−1 × 100 g−1) in BAT (Lumped Constant (LC) 1.15), WAT (LC 1.0) and skeletal muscle (LC 1.2) was calculated from dynamic PET data by Patlak graphical analysis using the signal from the aortic arch as arterial blood input and the average B-glucose over the scan for normalization . The mean standardized uptake values (SUVmean) and maximal standardized uptake values (SUVmax) at the 40 min time-point were also calculated for each region.The metabolic rate of glucose per grams of tissue (MR−1) in each tissue was approximated by multiplying the MRGlu per grams of tissue (µmol × min−1 × 100 g−1) by the estimated total mass of BAT (by direct segmentation on PET/CT images), WAT (BodPod), and skeletal muscle  and females (25%).The total glucose utilization . Two-tailed Student’s Cold-induced BAT was detected in patients with T1D with a varying degree of metabolic activity. The metabolic activity of BAT was correlated to the cold-induced levels of IL-6. However, the normal physiological range of metabolic activity in BAT had no impact on insulin requirements and metabolic control in T1D.


Thin films of CZTS were then prepared by spin coating, followed by thermal annealing. The effects of the fabrication parameters, such as ink concentration, spinning rate, and thermal treatment temperatures on the morphology and structural, optical, and electrical properties of the films were investigated. As expected, very thin films, for which the level of transmittance and band-gap values increase, can be obtained either by reducing the concentration of the inks or by increasing the rate of spinning. Moreover, the thermal treatment affects the phase formation and crystallinity of the film, as well as the electrical conductivity, which decreases at a higher temperature.Cu As a semiconducting compound, kesterite has p-type conductivity [2 (CIGS), whose elements are expensive and scarcely available. Indeed, CZTS exhibits good light-harvesting performance, with an optimal band gap energy around 1.5–1.6 eV [Kesterite, or CZTS, is a quaternary sulfide containing copper (Cu), zinc (Zn), tin (Sn), and sulfur (S) with the formula (I)uctivity  and can 5–1.6 eV ,4, given5–1.6 eV  radiatioNowadays, fabrication of films from CZTS suspension through the ink-based approach has attracted considerable research interest as a simple, economic, and industrially scalable route, compared to the more expensive vacuum deposition methods. This method allows better material utilization with high throughput. Moreover, an ink-based approach lets simple deposition methods, such as spin coating, dip coating, spray coating, screen printing and ink-jet printing, be used ,9,10. InWhere the electrical properties of binary and ternary semiconductors have been studied for years ,18,19, tSo far, properties of micrometric and sub-micrometric (>0.5 μm) CZTS thin films fabricated using several different methods have been extensively studied ,24,25,26The first parameter studied was ink concentration. SEM observations of the cross-section of the thin films with different ink concentrations  allowed Tauc’s plot of CZTS thin films with different ink concentrations are shown in the insert of Although the band gaps obtained were still in accordance with the band gap range of CZTS reported in the literature ,5,6, theThe effect of TT temperature on CZTS thin film samples, obtained from 0.2 g/mL ink spread at a 6000 rpm spin rate, was studied in terms of its surface morphology, grain growth, optical behavior, and electrical properties. In The crystallization process, and especially the grain growth, as demonstrated by the sharpness of peaks, was strongly promoted at 500 °C, while the formation of a secondary phase (SnS) might be due to unbalanced stoichiometry . The Williamson–Hall (W-H) analysis ,44 was pFrom the UV–vis spectroscopy data, the band gaps of the thin films were determined to be at ~1.62 eV for TT 200 °C, ~1.57 eV for TT 350 °C, and ~1.73 eV for TT 500 °C, respectively, which is in good agreement with those reported in the CZTS literature . The preElectrical properties of CZTS thin films were studied at different TT temperatures  by Hall effect measurements. Results shown in While resistivity and carrier concentration obtained at 500 °C were better than literature values ,55, mobipc) was determined by the difference between the maximum peak current density and the baseline of charging current density [3− + 2e− → 3I−, where the current density can be related to the rate of reduction of I3– to I–, occurring at the CZTS electrode. As shown in The better electrical properties of the film made at 500 °C was also demonstrated by cyclic voltammetry (CV) measurements. The cathodic current density , zinc chloride , and tin (II) chloride  were purchased by Sigma-Aldrich Inc. and dehydrated in vacuum at 200 °C. Sulfur (S), oleylamine , toluene (99.9%), and ethanol (>99%) were also provided by Sigma-Aldrich Inc. and used without further purification.Copper (II) chloride dihydrate  was rapidly injected in the hot solution at 270 °C under fast stirring and nitrogen (N2) flux. The mixture was kept at 270 °C for 30 min after the injection and then cooled slowly to room temperature. The obtained suspension of nanoparticles was washed with a mixture of 5 mL toluene and 25 mL ethanol and centrifuged for 10 min at 4000 rpm to separate the solvent from the nanoparticles. Finally, the CZTS nanoparticles were extracted, dispersed in toluene, and sonicated to obtain an ink with the desired concentration.CZTS nanoparticles ink was prepared according to the method described by Ataollahi et al. : 0.538 g2 stream. The cleaning process of the FTO glass was similar, except for the treatment with KOH, where 20 μL of ink was dropped on the substrate. After spin coating, the residual toluene was removed at 150 °C for 15 min. The TT below 350 °C was carried out on a hot-plate under N2 atmosphere for 15 min. High-temperature TT (500 °C) was performed in a tubular furnace at a heating rate of 3 °C/min for 1 h under N2 atmosphere. Samples were labelled according to ink concentration (0.2 and 0.5 g/mL), spin rate (2000–6000 rpm), and TT temperature (150–500°C). The CZTS nanoparticles ink was deposited on SLG substrates by spin coating. Before deposition, the substrates were cleaned with soap, treated with concentrated potassium hydroxide (KOH) in ethanol, rinsed with distilled water and ethanol, then dried in NThe morphology of CZTS thin film samples was observed either by optical microscopy or by scanning electron microscopy (SEM). Optical microscopy was made on a REMET HX-1000  at 400× magnification, whereas for SEM we used a JEOL JSM-7001F FEG-SEM  equipped with an energy-dispersive X-ray spectroscopy detector . Observations were made at 10.00–15.00 keV electron beam energy with a working distance between 5–10 mm. Surface morphology images were acquired in top-down modes, whereas a cross-sectional analysis was performed by putting the cross-section of the film on a 90° stub.α = absorption coefficient,d = film thickness,= normal incidence transmittance, and= normal incidence reflectance.UV–vis spectroscopy of CZTS thin films was made using a Perkin-Elmer LAMBDA 950 spectrophotometer  equipped with a 150 mm integrating sphere. By measuring normal incidence transmittance and reflectance, the absorption coefficient was calculated using the equationFrom UV–vis spectroscopy data, the bandgap energy was also obtained by a linear fit of a Tauc plot.Structural information on CZTS thin films were obtained by X-ray diffraction (XRD), using a Panalytical X’Pert MRD instrument equipped with CoKα sealed tube operated at 40 kV and 40 mA. Phase identification was supported by JADE Software .−10–10−2 A, with maximum resolution of 2.5 × 10−12 A. The maximum output voltage was ± 20 V with the output resistance using a typical 1013 Ω resistor. Besides that, the voltage measurement range was 10−6–101 V, with maximum resolution of 3 × 10−7 V. The instrument had a sensitivity of <1 mV and an input resistance of >1015 Ω. The magnetic field was provided by a standard magnet with magnetic field (B) range of 0.05–0.8 T and an achievable maximum resolution of 0.001 T. The pole diameter and gap were 45 and 0–75 mm, respectively. The resistivity value was calculated by using the formulaρ = sample resistivity (Ω∙cm),−1∙cm−1),σ = sample conductivity (Ω−19 C),= charge constant (1.6 × 102/Vs), andμ = carrier mobility (cm−3).= carrier concentration  with a current source range of 10−/I3−) electrolytes using a methanol solution containing 0.5 M potassium iodide (KI) and 0.5 mM I2. Scanning was done at a rate of 50 mV s−1.Cyclic voltammetry (CV) was conducted on Princeton Applied Research (PAR) model no. 4000 A with a three-electrodes system. Silver/silver chloride (Ag/AgCl) was the reference electrode, platinum (Pt) was the counter electrode, and CZTS thin film samples were deposited on the FTO glass as working electrodes with iodide/triiodide ion  affect more than other parameters on the overall quality, surface morphology, crystal structure, transmittance behavior, band gap energy, and electrical properties of the CZTS thin film. The increase in TT temperature induces significant phase changes  in addition to an improvement in the crystallinity degree and electrical properties. While high-temperature TTs are required to get rid of organic residuals and increase conductivity and carrier concentration, they can also lead to formation of secondary phases like SnS, which is detrimental to the mobility of the carriers. A correct balance between different requirements in thin film production still has yet to be found. The method is confirmed to be very versatile for preparing CZTS sub-micrometric thin films (< 0.5 µm) and semiconductors suitable for a transport layer in many applications such as in solar cells, diodes, and thermoelectric devices.


Results showed that Si supplied in the form of silicic acid (12 kg ha−1) has no effect on the agronomic performance of plants cultivated with the lower N input. In contrast, in plants fertilized with 160 kg N ha−1, Si supply promotes the preservation of green leaves (until the flowering stage) and at harvest stage, increases biomass, yield, and seed micronutrient concentrations . The agronomic indexes show that the increase in seed yield is related to a better uptake of N from the soil by Si-treated plants, but is not an improvement in N mobilization towards the seeds. This study showed that Si supply combined with high N inputs (160 kg ha−1) improves usage of N fertilizer and yield. The possibility that a Si supply could allow for a reduction in N input without altering the yield of rapeseed is discussed.To limit the environmental pollution associated with intensive nitrogen (N) fertilizer usage, alternative cultural practices must be considered for crops requiring high N inputs such as rapeseed. In this context, the effects of silicon (Si) supply on the agronomic performance of rapeseed cultivated under field conditions with two N fertilizer levels (60 and 160 kg ha Si is 0.6 mM) ,6.Vascul 0.6 mM) ,8,9,10.  0.6 mM) . For exa 0.6 mM)  and othe 0.6 mM) ,14. More 0.6 mM) ,16 and s 0.6 mM) . Further 0.6 mM) , promote 0.6 mM)  reported−1) and supplied or not with a form of Si available to plants (12 kg Si ha−1 in the silicic acid form). For this, the effects of Si have been monitored in the whole plant biomass and during leaf senescence at the flowering stage , but alThe field experiments were conducted in a lysimeter system located at Lieury  from 17 September, 2016 to 7 July, 2017. The lysimeter device comprised of 15 polyester resin lysimeter boxes with an area of 1.96 m² (1.4 m × 1.4 m), and a depth of 1.10 m. Rainfall and air temperature were measured continuously at the meteorological station at the Lycée Le Robillard in Lieury  were sown  as previously described by Génard et al. [2 of area), with a density of 60 plants per lysimeter. At stage C1 , the 15 lysimeter boxes were randomly separated into five batches and each batch was composed of three lysimeter boxes. As usually recommended [−1 (as P2O5), 50 kg K ha−1 (as K2O), and 30 kg S ha–1 (as MgSO4). Among these 15 lysimeter boxes, three of them did not receive N fertilization  and the others received 160 kg N ha−1 (160 N). Nitrogen was supplied as ammonium nitrate fractioned into two N inputs  and March 31, 2017 (stage E) for plants fertilized with 60 and 160 kg N ha−1 (−1 were supplied with 12 kg Si ha−1 (60 N + Si and 160 N + Si) in the form of Si(OH)4, which was fractioned into four inputs: 1 kg ha−1 at stage C1 , 1 kg ha−1 at stage E , and two inputs of 5 kg ha−1 during stage F . In contrast, the remaining 60 N and 160 N lysimeters were considered as controls of Si supply (60 N and 160 N) and did not receive Si inputs  and its concentration was determined using the colorimetric method previously described by Haddad et al. [4 solution at 57 mg L−1 was applied, and for the last two fertilizations with Si, a 20 L of Si(OH)4 solution (pH 6) at 171.5 mg L−1 was applied uniformly to each set of lysimeter boxes with a watering can with sprinkler system. At each date of Si supply, the same volumes (12 or 20 L) of water were applied to the lysimeter boxes without Si. Seeds of rapeseed  were harvested at the G1 stage  and the G5 stage . At each harvest date, plant organs ) were separated, weighed, and oven dried for 48 h at 60 °C for DW determination and then ground to fine powder for further chemical analysis. For all treatments, plants cultivated on half of the respective lysimeter area  were placed into tin capsules using a microbalance and the total N concentration was determined with a continuous flow isotope ratio mass spectrometer  linked to a C/N/S analyzer . TM). Dry matter of each of the samples (40 mg) was suspended with 800 µL of concentrated HNO3, 200 µL of H2O2, and 1 mL of Milli-Q water. All samples were then spiked with three internal standard solutions containing gallium, rhodium, and iridium with final concentrations of 5, 1, and 1 µg L−1, respectively. After microwave acidic digestion , all samples were diluted with 50 mL of Milli-Q water to obtain solutions containing 2.0% (v/v) nitric acid. Before HR ICP-MS analysis, samples were filtered at 0.45 µm using a Teflon filtration system . Quantification of each element was performed using external standard calibration curves and concentrations were expressed in mg g−1 and µg g−1 of seed DW for macronutrients and microelements, respectively.In mature seeds, macronutrients  and microelements  were quantified by high-resolution inductively coupled plasma mass spectrometry , the nitrogen harvest index (NHI), and agronomic nitrogen recovery (ANR) were calculated to characterize the nitrogen use efficiency of Agronomic efficiency (AE), which represents the efficiency of the crop to convert the applied nitrogen to seed yield, was calculated as follows: ANR, defined as the efficiency of nitrogen capture from soil was calculated as follows: rtilizer  have bee−1 and supplied (60 N + Si or 160 N + Si) or not (60 N or 160 N) with Si (12 kg ha−1), respectively. NHI, which represents the efficiency of nitrogen mobilization towards the seeds, was calculated using the following equation: p ≤ 0.05).The experiment was performed with three replicates for each crop . The resulting variations in data are expressed as the mean ± standard error (SE). Data were analyzed using analysis of variance (ANOVA), after verifying compliance of normality, and significantly different means between treatments were separated with the Tukey’s multiple range test (−1) at the G1 developmental stage  in Brassica napus, Si supply (12 kg Si ha−1) had no significant effect on the biomass of different compartments or on the total biomass of plants. Thus, the cumulative biomasses of plants were 18.91.0 ± 1.74, 18.47 ± 0.95, 24.18 ± 1.24 and 25.46.0 ± 1.33 g for 60 N, 60 N + Si, 160 N and 160 N + Si plants, respectively  and senescent leaves . In contrast, for plants cultivated with 160 kg N ha−1, the distribution of green and senescent leaves was significantly different in plants treated with Si (160 N + Si) compared to the control (160 N). Thus, in plants cultivated with 160 N, Si supply increased the percentage of green leaves  and decreased the percentage of senescent leaves  (For a given level of nitrogen fertilization (60 or 160 kg N haectively A. Moreovctively) B.−1 in 160 N + Si and 160 N plants, respectively) and whole plant biomass  , Si supply had no significant effect on the biomass of different compartments or on the total biomass of plants cultivated with 60 N. In plants cultivated with 160 N, Si supply led to an increase in taproot biomass (2.94 ± 0.03 vs. 2.67 ± 0.09 g plantctively) A. Moreovely) (−1 B. Howeveely) (−1 C but rately) (−1 B. −1 (60 N), whereas it led to a significant increase in yield in plants cultivated with 160 kg N ha−1 (160 N). Thus, the yield of 160 N + Si plants reached 4247.20 ± 75.23 vs. only 3644.37 ± 182.48 kg ha−1 in the 160 N plants. Since the thousand-seed weight is similar between the Si treatments , the Co and Fe concentrations in seeds of plants supplied with Si (60N + Si and 160N + Si) increased compared to their respective controls (60N and 160N)  and nine microelements  were analyzed in seeds from plants cultivated with 60 or 160 N and supplied with or without Si. For a given N fertilization, Si supply had no effect on either the N concentration A or on tnd 160N) B,C. Ther60N) (−1 D. For th60N) (−1 . −1 (60 N), Si supply had no significant effect on agronomic efficiency of N fertilizer , Si supply led to significant increases in AE and ANR of 1.5- and 1.3-fold, respectively. In addition, neither the NHI nor the residual N concentrations in fallen leaves were modified by the Si supply. Moreover, it was notable that the residual N concentrations in fallen leaves of plants cultivated with 60 kg N ha−1 (60 N and 60 N + Si) was significantly lower than those in fallen leaves from plants cultivated with 160 kg N ha−1 (160 N and 160 N + Si) and reached about 0.9% and 1.2%, respectively  with the lower nitrogen input (60 kg N ha−1) had no significant effect on the growth, yield, or seed quality of rapeseed cultivated under field conditions (−1) was applied over a long period, which has mainly limited the growth of plants cultivated under field conditions. Indeed, Good et al. [The developmental stages studied  showed that Si supply  increased the percentage of green leaves on plants until at least the flowering stage (84.85%) compared to control plants (75.73%), while the biomass of total leaves was not significantly different between either of the Si treatments , which would have allowed an improvement in N uptake, especially in plants cultivated with the higher N input (160 kg ha−1). Another assumption is that Si is able to increase N availability in the soil. This is reinforced by several works demonstrating that Si is able to modify physicochemical (such as soil exchange capacity) [−1) may have increased availability of N in the soil, improved N uptake, and led to an accumulation of N in the plants cultivated with the higher dose of N fertilizer (160 kg ha−1). Furthermore, our experiments showed that Si supply resulted in a significant increase in seed yield (4.2 T ha−1) of 16.6% under 160 N + Si compared to the 160 N plants (3.6 T ha−1). This result agrees with recent observations by Kuai et al. [Brassica napus cultivated under field conditions showed that Si supplied via spraying resulted in yield improvement. Interestingly, in our study, the increase in yield in 160 N + Si plants was associated with a preservation of seed yield quality components such as thousand-seed weight or the oil, glucosinolate, and protein concentrations, and the linoleic acid (ω-6): linolenic acid (ω-3) ratio was also not significantly different between seeds from plants cultivated with 160 kg N ha−1 and supplied or not with Si  ,27 and bapacity)  and to iapacity) ,31,32,33apacity)  reportedi et al.  in which with Si . Moreove with Si C. The in with Si A, an ind−1; −1) there was an increase in iron (Fe) and cobalt (Co) concentrations in the seeds  concentration, but only in seeds from plants cultivated under the lower N fertilization regime (60 kg N hahe seeds B,C. Althcusta L. ,25. Thescusta L.  but alsocusta L. . Indeed,custa L. ,39 and Ccusta L. , Fe is ocusta L. . Brassica napus when the crop is cultivated with a 160 kg N ha−1 input enabling good growth and development. In our conditions of N inputs, this improvement in N efficiency is mainly due to better N uptake, but not better N remobilization, and increases yield while preserving seed quality. In addition, this study indicates the potential for combining N and Si fertilizers to promote mineral biofortification in Brassica napus seeds. In conclusion, this study has established that supplying Si under field conditions leads to better N efficiency in


Exosomes play a crucial role in the progression of infectious diseases, as exosome release and biogenesis are affected by external factors, such as pathogenic infections. Pyrogens may aide in the progression of diseases by triggering inflammation, endothelial cell injury, and arterial plaque rupture, all of which can lead to acute coronary disease, resulting in cardiac tissue death and the onset of a cardiac event (CE). To better understand the effects of Gram-negative bacterial infections on exosome composition and biogenesis, we examined exosome characteristics after treatment of AC16 human cardiomyocytes with lipopolysaccharide (LPS), which served as a model system for Gram-negative bacterial infection. Using increasing doses  of LPS, we showed that treatment with LPS substantially altered the composition of AC16-derived exosomes. Both the relative size and the quantity (particles/mL) of exosomes were decreased significantly at all tested concentrations of LPS treatment compared to the untreated group. In addition, LPS administration reduced the expression of exosomal proteins that are related to exosomal biogenesis. Conversely, we observed an increase in immunomodulators present after LPS administration. This evaluation of the impact of LPS on cardiac cell death and exosome composition will yield new insight into the importance of exosomes in a variety of physiological and pathological processes as it relates to disease progression, diagnosis, and treatment. Gram-negative bacteria are responsible for millions of nosocomial infections and are of particular interest . These mEscherichia coli (E. Coli) and are shared by the majority of Gram-negative bacteria [E. coli and Salmonella sp., in investigations of the essential components of biogenesis, cell viability, and a myriad of other biological processes [Pseudomonas aeruginosa, has not been thorough, despite the urgent need to understand the role of these bacteria in cardiac cell death. Although the precise molecular structure of LPS differs from one bacterium to another, most enzymes and genes related to LPS formulation and transportation were identified in bacteria ,7. LPS srocesses ,6,8. TheCardiovascular disease is the primary cause of roughly one-third of deaths worldwide ,10. GiveExosomes are formed from the plasma membrane and the fusion of multi-vesicular bodies and are found in various bodily fluids ,11. Exos2 until the cells reached 70–80% confluency. Cells were then washed with 1 × phosphate-buffered saline (PBS) and removed with 0.05% trypsin, 0.053 mM EDTA, and 1X-sodium bicarbonate. Cells (5 × 105 cells per flask) were then plated in new a T-25 flasks (Corning) in 3 mL of DMEM/F-12 medium. The following day, media were removed, and AC16 cells were washed with sterile 1 × PBS. Following removal of the PBS, exosome-free DMEM/F-12, which was prepared as described above with supplemental 15% exosome-depleted FBS (prepared by System Biosciences), was added.AC16 human cardiomyocytes (Millipore Sigma) were cultured in Dulbecco’s Modified Eagle’s Medium/Hams F-12 50/50 mix 1 × with L-glutamine  supplemented with 15% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 4% amphotericin. Cells were maintained at 37 °C with an atmosphere of 5% COAfter replacement of original media with exosome-free DMEM/F-12 the day after plating, cells were treated with 0.1, 1, or 10 µg LPS. For control cells, exosome-free DMEM/F-12 only was used. The cells and LPS were incubated for 48 hours (h) without any further manipulation ,19,20. TAfter harvest, AC16 cells were stained with 0.4% trypan blue solution (Cellgro), and viability was measured using a Countess automated cell counter (Invitrogen).Exosomes were collected following 48 h of LPS treatment via filtration and ultracentrifugation. Media was filtered through a 3 mL syringe with a 25 mm syringe filter with a pore size of 0.22 µm. Then, 1 × PBS was added to the media and centrifuged at 32,000 revolutions per minute (rpm) for 70 min in a swinging bucket rotor SW41T1 at 4 °C using a Beckman Coulter Optima L-70K Ultracentrifuge. The supernatant was discarded, and 400–500 µL resuspended exosomes were collected from each sample. Exosome quantitation was performed using the Bradford-Lowry protein quantitation procedure (BioRad).A Nanosight Tracking Analysis (NTA) NS300 Submicron Particle Imaging System was used to visualize and quantify exosomes from cells treated with LPS. Exosomes were diluted 1:1000, and five independent experiments were analyzed. The mean values for exosome quantity and size measurements were determined. Exosomes (40 µg) were plated in wells of a 96-well plate. In addition, 50 µL buffer was plated as a control. Then, 100 µL bicarbonate buffer with a pH of 9.5 was added to each well, and plates were incubated at 4 °C overnight. The next day, plates were washed with 1 × PBS with 0.05% Tween 20 three times. After washing, plates were blocked with 5% skim milk in PBS with 0.09% Tween 20 at 4 °C for 1 h. After blocking, primary antibodies were added to each well, and plates were incubated at room temperature (RT) for 2 h or at 4 °C overnight. After incubation, the plates were rewashed as described above, and horseradish peroxidase-conjugated secondary antibody  was added to each well for incubation at RT for 2 h or at 4 °C overnight. After incubation, the ELISAs were developed with SigmaFast™ OPD peroxidase substrate (Sigma Aldrich) in the dark at RT for 30 min. Intensities were determined at 405 nm using a Genemate UniRead 800 microplate reader.t-test and ANOVA. The error of probability was indicated as * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.Statistical analyses were conducted using the standard functions of Excel and GraphPad Prism . Mean values were compared using an unpaired two-tailed To examine the effect of LPS treatment on human cardiomyocytes, AC16 cells were treated with increasing concentrations of LPS, which represent varying levels of bacterial infection ,19,20. CThe mean exosome size and concentration were determined using NTA , which i8 particles/mL, while cells treated with 0.1 and 1 µg LPS yielded decreased numbers of exosome particles (5.54 × 107/mL). Furthermore, treatment with 10 µg LPS decreased the number of exosomes to 4.73 × 107 particles/mL. These findings indicate AC16 cell exposure to LPS significantly decreases the number of exosomes (particles/mL) at all LPS concentrations tested compared to untreated cells.Further Nanosight Tracking Analysis (NTA) analysis revealed a significant decrease in the number of exosome particles/mL after LPS treatment B. In the−2 (0.1 μg group), +/−7.3 × 10−2 (1 μg group), and +/− 5.2 × 10−2 (10 μg group) , tumor necrosis factor-alpha (TNFα), cleaved caspase 3, and cleaved caspase 9 were evaluated to investigate any changes in expression following LPS treatment. CD81 and actin were both expressed in exosomes, as expected A,B. We ag group) C. Intereg group) C.−2). Additionally, Rab5 was also decreased in exosomes from cells treated with 0.1 μg LPS compared to those treated with 1 μg LPS (+/− 5.8 × 10−2) and was significantly increased in exosomes from cells treated with 10 μg LPS compared to those treated with 1 μg LPS (+/− 9.4 × 10−2) (−2) of LPS compared to exosomes from untreated controls (−2) and the 0.1 µg (+/− 1.6 × 10−2) LPS-treated cells  A. Rab27Acontrols B. TLR4 acontrols C,D. The ed cells C. Furtheed cells D. The cled cells A,B. Whiled cells A, actin ed cells B, TNFα (ed cells D, caspased cells A, and caed cells B, the exGram-negative bacterial infections are responsible for millions of infections and deaths worldwide . With anThe primary causative events of Gram-negative bacterial infections are bacteremia, and septic shock, which are caused by the release of LPS . We seleCD81 is a small membrane tetraspanin that is characteristically found on exosomes and is involved in a wide variety of biological functions, such as cell migration, cell proliferation, immune response, and thrombosis . CD81 haThe cytoskeleton of cardiac myocytes consists of actin, the intermediate filament, and alpha- and beta-tubulin, which form the microtubules . The cytRab5 and Rab27A belong to a family of GTPases that play an essential role in intracellular vesicle transport via control of membrane trafficking between intracellular compartments . Rab5 isA previous study conducted by Davidson et al. showed that plasma exosomes protect the hearts of rats from injury both in vitro and in vivo . These sCaspases are a family of proteases that play a role in programmed cell death and inflammation. A study conducted by Park et al. concluded that the release of exosomes is dependent upon caspases, particularly caspase 3 and caspase 9 . In our In conclusion, we confirmed the presence of exosomes from AC16 cardiac cells and described the relative exosomal changes after the treatment of these cells with LPS. LPS administration significantly decreased AC16 cell viability, exosome quantity, and exosome size. Exosomal proteins were altered by LPS treatment, with a significant decrease in structural and vesicle trafficking proteins. These data illuminate the facilitatory role that exosomes and their constituents play in cardiac cell death after bacterial infection and, therefore, these findings could have clinical implications. In particular, the non- invasive surveillance of exosomes numbers in the form of a blood test could provide information regarding Gram-negative bacterial infections. In future studies in AC16 cells, we will compare the effects of LPS treatment with those induced by infection by some common opportunistic Gram-negative bacteria. Furthermore, these studies shed light on the impact of LPS on cardiac cell death and exosome biogenesis and offer insight into the importance of exosomes in a variety of processes related to cardiovascular disease. Additional investigations will allow us to identify potential exosomal markers for the further identification of pathogen-specific contributions to cardiovascular disease.


This study is a retrospective analysis of the prenatal genetic diagnosis results of fetuses with high risk of major thalassemia to provide information for clinical genetic counseling and to better control the birth of major thalassemia child in Hakka population. Totally, 467 fetuses in at-risk pregnancies were collected from Meizhou people's hospital from January 2014 to December 2017. Genomic DNAs were extracted from peripheral blood of the couples and villus, amniotic fluid or cord blood of the fetuses. DNA-based diagnosis was performed using polymerase chain reaction (PCR) and flow-through hybridization technique. Follow-up visits were done half a year after the fetuses were born. Around 467 fetus at-risk pregnancies were performed prenatal diagnosis. We detected 88 CVS samples, 375 amniocentesis fluid samples and, 4 cord blood samples. The 356 fetuses in α-thalassemia families consisted of 69 (19.38%) with Bart's hydrops syndrome, 20 (5.62%) fetuses with Hb H disease, and 184 (51.68%) fetuses with heterozygote. And the 111 fetuses in β-thalassemia families consisted of 31 (27.93%) thalassemia major, 51 (45.95%) fetuses with heterozygote. There are 13 fetuses with α+β-thalassemia, including 2 cases with severe β-thalassemia. DNA-based testing prenatal diagnosis of thalassemia was found to be highly reliable. Our findings provide key information for clinical genetic counseling of prenatal diagnosis for major thalassemia in Hakka pregnant women. Our work plays an important role in the prevention and control of thalassemia in Hakka population. We will also combine other techniques to further improve our molecular prenatal diagnostic capabilities, including the next-generation sequencing (NGS), Sanger sequencing and MLPA. Thalassemia is classified into 2 major types, including α- and β-thalassemia.,8Thalassemia is considered to be one of the most common genetic diseases in the world, with a high frequency in tropical and subtropical regions such as Mediterranean countries, Indian subcontinent, Middle East, North Africa, and South Ocean.,10 The normal genotype can be represented as αα/αα. Alpha-thalassemia is characterized caused by deletion or nondeletional mutation of one  or both (--/αα or -α/-α) α-globin genes. Severe alpha-thalassemia can cause fetal death at the time of birth, represented as Hb Bart's (γ4) disease (--/--). People with loss or inactivation of 3 α-globin genes (--/-α or --/αTα or --/ααT) which in adult life excess β globin also form active tetramer with the structure β4 (HbH). HbH disease is highly heterogeneous and there are many different rules about their pathogenesis.–13Alpha-thalassemia is a hereditary hematological disorder caused by the deletion or dysfunction of alpha-globin genes in chromosome 16 contributing to the absence or inadequate synthesis of alpha-globin peptide chains.+) or absence (β0) in beta chains of hemoglobin synthesis, resulting in variable phenotypes ranging from severe anemia to clinically asymptomatic level.,15Beta-thalassemia is a group of hereditary blood disorder characterized by point mutations or, more rarely, deletions in the beta globin gene on chromosome 11, leading to reduction  in Guangdong Province between January 2014 and December 2017. All of them come from 7 areas within Meizhou City, Guangdong Province and their ancestral home is Meizhou. Before the prenatal diagnosis, the genotypes of both father and mother were tested. This study was carried out in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Meizhou People's Hospital , Meizhou Hospital Affiliated to Sun Yat-sen University.2.2Samples were obtained via venipuncture of an antecubital vein using Ethylenediaminetetraacetic acid (EDTA) anticoagulant tube collection, and 2 mL peripheral blood were collected for relative detection. Erytrocyte correlation indices were determined followed the standard operating procedures provided by Sysmex XE-2100 blood analyzer . Subjects with low mean corpuscular volume values (MCV) (<82 fl) or Mean corpuscular hemoglobin values (<27 pg) were considered possible thalassemia carriers.2 (<2.5%) were considered possibly α-thalassemia carriers, with high HbA2 (>3.5%) were considered possibly β-thalassemia carriers, respectively.Hemoglobin electrophoresis analysis was detected according to standard laboratory procedures by Sebia capillary electrophoresis system . Subjects with low HbA2.3There are 3 main ways to obtain fetal samples: chorionic villus sampling (CVS) was conducted at 10 to 12 weeks of gestation; amniotic fluid (10 mL) was collected at 15 to 22 weeks; cord blood (1.0–2.0 mL) was sampled at 18 to 28 weeks. All procedures were performed under ultrasonography guidance. Meanwhile, 2 ml peripheral blood of the mother was sampled for short tandem repeats (STR) analysis.Villi were washed twice with normal saline. Villus tissue was removed from 15 mL centrifuge tubes with hook tweezers, placed in Petri dishes with saline. Parts of villi were removed from stripped maternal decidua with hooks at low magnification. Some of the tissues were used for the extraction of fetal DNA. The remaining villi were soaked in saline and frozen at −80°C. Amniocentesis fluid cells were isolated from 10 ml amniocentesis fluid using a centrifuge at 1000 g for 10 minutes. Pipette off supernatant and add 800 ml sterile saline to resuspend the cell pellets. Then take 400 μL of pellet for the extraction of DNA. Take 200 μl umbilical cord blood directly extracted the group DNA, similar to extraction of DNA from peripheral blood.2.4,20 The STR analysis with the common markers including D19S433, D5S818, D21S11, D18S51, D6S1043, AMEL, D3S1358, D13S317, D7S820, D16S539, CSF1PO, Penta D, D2S441, vWA, D8S1179, TPOX, Penta E, TH01, D12S391, D2S1338 and FGA  using ABI 3500xl Genetic Analyzer . When all the polymorphic alleles of the mother were absent from fetal sample, the fetal sample was considered free from maternal contamination.Genomic DNAs were extracted from peripheral blood of the couples and villus, amniotic fluid or cord blood of the fetuses using the Tiangen DNA extract kit . Maternal cell contamination (MCC) was tested by STR analysis. Fetal samples are always confused by maternal tissue, so STR analysis was conducted before detecting of the fetal samples.2.5SEA, -α3.7, -α4.2 and nondeletion of Hb Constant Spring (αCSα) , Hb Quong Sze (αQSα) and Hb Westmead (αWSα) . Polymerase chain reaction for detection α-thalassemia mutations was performed according to the following protocol: denaturation at 95°C for 15 minutes, and then 35 cycles of amplification, with 40 seconds at 98°C for denaturation, 1 minute and 10 seconds at 64°C for annealing, and 2 minutes and 30 seconds at 72°C for elongation.Gap-polymerase chain reaction (gap-PCR) and flow-through hybridization technology  were used to detect the α-thalassemia mutations, including deletion of --Mutation analysis for the β-globin gene were performed using the polymerase chain reaction (PCR) and flow-through hybridization technology , including 16 common nondeletion β-globin gene mutations: CD41-42(-TCTT), CD43(G→T), IVS-II-654(C→T), CD17(A→T), CD14-15(+G), -28(A→G), -29(A→G), CD71-72(+A), βE(G→A), IVS-I-1(G→T), IVS-I-1(G→A), CD27-28(+C), IVS-I-5(G→C), Cap+40-43(-AAAC), initiation condon (T→G) and CD31(-C). Polymerase chain reaction for detection β-thalassemia mutations was performed according to the following protocol: 37 °C for 5 minutes, initial denaturation at 94°C for 4 minutes, and then 40 cycles of amplification, with 30 seconds at 94 °C for denaturation, 30 seconds at 55 °C for annealing, and 30 seconds at 72 °C for elongation. Flow-through hybridization was operated according to the manufacturer's instructions.2.6We consulted the clinical records of the subjects when performed molecular prenatal diagnosis. Fetuses with nonsevere thalassemia by genetic diagnosis and were chosen to continue pregnancy were followed up 6 to 8 months after birth. SPSS statistical software version 19.0 was used for data analysis. Data were reported with the descriptive statistics method and showed as the means ± SD.32 was showed less than 3.5% or between 2.5% and 3.5% in the parents with α-thalassaemia. The parents with β-thalassaemia showed that Hb A2 was beyond 3.5% and mean corpuscular volume (MCV) was <80fl and/or mean corpuscular hemoglobin (MCH) was <27 pg. And the results of 467 pregnant women of hematological screening showed in Table Between January 2014 and December 2017, prenatal diagnosis was performed in 467 fetuses of 465 mothers. The age group for fathers and mothers ranged from 18 to 50 years (30.55 ± 5.81) and 16 to 42 years (27.72 ± 5.34), respectively. The mean ages for mothers carried α, β and α compound β globin mutation gene were 27.69 ± 5.19, 27.69 ± 5.83 and 28.13 ± 5.58, respectively. The mean gestational age carrying out the invasive procedure was 16 to 42 weeks (18.47 ± 4.93). Hb ASEA), in which included 281 couples. According to previous researches, most of regions indicated a similar feature for higher prevalence genotype of --SEA deletion.,21–24 Such couples have 25% chance to produce fetus/fetuses with Hb Bart's. In this study, we identified 69 (19.38%) fetuses with Bart's hydrops syndrome.In the 465 couples, there were 642 subjects (69.03%) that carry Southeast Asian type deletion , 33 fetuses with severe β-thalassemia and accepted the termination of pregnancy under informed consent. In addition, we detected 20 cases with HbH disease, ten fetuses with deletion HbH disease (8 with --SEA/-α3.7 and 2 with --SEA/-α4.2), 4 fetuses with --SEA/α WSα, 4 fetuses with --SEA/αCSα and 2 fetuses with --SEA/αQSα. The pregnancy of 4 fetuses with --SEA/ αCSα, 2 fetuses with --SEA/αQSα and one fetus with fetal malformation (--SEA/-α3.7 at the same time) were terminated in time. The remaining parents were determined chose to continue pregnancy, and the infants showed no severe anemia phenotype according follow-up within the first half year after birth.After molecular prenatal diagnosis completed, 69 fetuses were diagnosed as Bart's hydrops syndrome , a karyotype of 46,XX,der,+21 , 2 karyotype of 46,XY,inv(9)(p13q13) , a karyotype of 46,XY,21pstk+ , a karyotype of 47,XY,+18  and a karyotype of 46,XY,14pstk+ karyotype  we found. The pregnancy of fetuses with 46,XX,der,+21; 47,XY,+18 and 46,XY,14pstk+ karyotype  were terminated the pregnancy. The results of karyotype analysis showed in Figure According to the results of karyotype analysis of fetal samples, a karyotype of 46,XY,dup1)(q11q12) (--(q11q12) All the mothers with fetal severe thalassemia or other abnormalities chose to terminate pregnancy with informed consent, at which time fetal tissues were examined by molecular diagnosis and the results were confirmed. Fetuses with nonsevere thalassemia by genetic diagnosis and were chosen to continue pregnancy were followed up 6 to 8 months after birth, asked about their growth and development, anemia symptoms and so on. Because most they did not return to the hospital after birth for thalassemia gene testing, have not the comparison between the results of prenatal and post natal genetic tests. However, according to the growth and development and other phenotypes, it is consistent with our prenatal genetic diagnosis, which confirmed the reliability of molecular prenatal diagnosis. The comparison between the results of prenatal and post natal genetic tests is the next step we need to accomplish.4 Meizhou is located in eastern of Guangdong province in which resident population was up to 5.28 million and annual birth rate reached 12.45‰ . Meizhou is a mountainous city, and the vast majority of the permanent residents are Hakka people. Due to geography, culture, customs, and other reasons, the proportion of intermarriage between Hakka people in Meizhou and other regions is relatively small, which consequently triggered higher genetic frequency of thalassemia and that will cause a public health burden in this region. It has been reported that the incidence of thalassemia in Meizhou, Guangdong Province, higher than the average level of the Guangdong Province. The spectrum of both alpha- and beta-thalassaemia mutations is similar to that previously described in southern China, such as Shenzhen, Guangzhou and Hong Kong. Prenatal diagnosis is one of the most effective and direct method to prevent from inherited disease, including thalassemia.Thalassemia serves as an endemic disease that mainly populars in Guangdong, Guangxi and Hainan province, resulting in numerous health expenditure. According to the epidemiological results, it showed high prevalence of carriers of α thalassaemia (8.53%), β thalassaemia (2.54%), and both α and β thalassaemia (0.26%) in Guangdong province of China. Overall, 11.07% of the population in this area was heterozygous carriers of α and β thalassaemia.SEA), accounting for 69.03%. Showing that the --SEA deletion is the most common type of thalassemia in this area. Such couples have a 25% chance to give birth to fetus with Hb Bart's syndrome. In this study, we identified 69 fetuses with Hb Bart's hydrops syndrome, accounting for 14.78%, and 33 with severe β-thalassemia . And all accepted the termination of pregnancy according to selection principle after informed consent. Especially, we prevented the 33 newborn births with β-thalassemia major syndrome by prenatal diagnosis, which would cost expensive treatment if pregnancies had continued and given birth.In this study, there are 642 subjects in which 281 couples were detected as heterozygous of the Southeast Asian type deletion  were terminated, the remaining parents continue to pregnancy. They showed no severe anemia phenotype within the first half year after birth and showed good growth according the follow-up visit.HbH disease can be divided into 2 types according to genotype, deletion HbH disease and nondeletion HbH disease. The vast majority of nondeletion mutations are in the strong function of α2 gene, so the clinical manifestation of nondeletion HbH disease more serious than deletion HbH disease, except --In this study, we identified 2 fetuses with Hb Bart's disease and 1 fetus with deletion Hb H disease, in whom one of his/her parent was α-thalassemia carrier and the other was α+β-thalassemia carrier. These results indicate the existence of at-risk of α-thalassmia homozygous in their fetuses. We suggest that it should be necessary to detect for α-thalassemia mutations in β-thalassemia couple when the other company of the couple is α-thalassemia carrier, for there is 25% chance to produce a fetus with Hb Bart's or Hb H disease.N/βN and --SEA/αα, βN/βN, respectively. However, amniotic fluid culture and karyotype analysis showed that the number of fetal chromosomes was 47, XY, +18 and 46, XX, der  , +21, respectively. One fetus with nuchal translucency (NT) was thickening, and karyotype analysis showed 46, XX, inv (9) (p13q13). These finally end up with terminate pregnancy after informed consent. These cases suggest that, while prenatal diagnosis and genetic counseling of thalassemia are able to carry out in families, attention also could be paid in the prenatal diagnosis indications of other genetic diseases to avoid missed diagnosis of other genetic diseases.In addition, 2 pregnant women with fetal abnormalities were detected with ultrasonography. Molecular prenatal genetic diagnosis of thalassemia in these 2 fetuses were αα/αα, β,28 We also retrospectively analyzed the data of prenatal diagnosis of α- and β- thalassemia from 2014 to 2016. Summarizing and analyzing our data are helpful to provide reference and prenatal diagnosis ability for our future work.In this study, to eliminate the risk of maternal cell contamination, we performed DNA analysis twice. The first analysis was done after amniocentesis or CVS, and the second analysis was carried out after fetal DNA was re-extracted from amniocytes or CVS after culture. When we used STR detection technology to rule out maternal tissue contamination, we performed DNA analysis twice after confirmed no maternal cell contamination. With these methods, we successfully minimized the risk of maternal cell contamination to guarantee the accuracy and reliability of results.5In this study, DNA-based diagnosis was performed on the tissues of fetuses who have risk with severe thalassemia using polymerase chain reaction (PCR) and flow-through hybridization technique. We identified 69 fetuses with Hb Bart's hydrops syndrome and 33 cases with severe β-thalassemia, were terminated the pregnancy. Follow-up visits were performed in 6 to 8 months after the fetuses with nonsevere thalassemia were born. Our findings provide key information for clinical genetic counseling of prenatal diagnosis for thalassemia major in Hakka pregnant women. Our work plays an important role in the prevention and control of thalassemia in Hakka population. We will also try to combine with other techniques to further improve our molecular prenatal diagnostic capabilities, including next-generation sequencing (NGS), Sanger sequencing and MLPA.6Pingsen Zhao conceived and designed the experiments. Heming Wu conducted the laboratory testing. Huaxian Wang, Wei Guo, Zhiyuan Zheng, Huichao Zhu and Jie Wu helped to collect data. Liubing Lan and Mei Zeng recruited subjects, collected clinical data. Pingsen Zhao and Heming Wu prepare the manuscript. Pingsen Zhao reviewed the manuscript.The author would like to thank other colleagues whom were not listed in the authorship of Clinical Core Laboratory and Center for Precision Medicine, Meizhou People's Hospital , Meizhou Hospital Affiliated to Sun Yat-sen University for their helpful comments on the manuscript.Conceptualization: Pingsen Zhao.Data curation: Heming Wu, Pingsen Zhao.Formal analysis: Heming Wu, Huaxian Wang, Zhiyuan Zheng, Pingsen Zhao.Funding acquisition: Pingsen Zhao.Investigation: Heming Wu, Huaxian Wang, Zhiyuan Zheng, Jie Wu, Pingsen Zhao.Methodology: Heming Wu, Huaxian Wang, Liubing Lan, Mei Zeng, Wei Guo, Zhiyuan Zheng, Huichao Zhu, Jie Wu, Pingsen Zhao.Project administration: Pingsen Zhao.Resources: Heming Wu, Huaxian Wang, Liubing Lan, Mei Zeng, Wei Guo, Huichao Zhu, Pingsen Zhao.Software: Heming Wu, Huaxian Wang, Wei Guo, Huichao Zhu, Jie Wu, Pingsen Zhao.Supervision: Pingsen Zhao.Validation: Heming Wu, Huaxian Wang, Pingsen Zhao.Visualization: Heming Wu, Pingsen Zhao.Writing – original draft: Heming Wu, Pingsen Zhao.Writing – review & editing: Pingsen Zhao.


Collectively, DUXAP10 plays a critical role in regulating HCC development, potentially by regulating EMT and cell proliferation through the PI3K/Akt and Wnt/β-catenin signaling. Inhibition of DUXAP10 in HCC HepG2 cells could attenuate the EMT and cell proliferation and invasion. Therefore, DUXAP10 might be a promising therapy target to inhibit the growth of HCC.The long non-coding RNA DUXAP10 has been involved in the development, progression, and metastasis in several human cancers, but its biological function and underlying mechanism in hepatocellular carcinoma (HCC) still undetermined. The present study was proposed to explore the effect of DUXAP10 on the growth and metastasis of HCC cells and the potential mechanisms involved. The results showed that DUXAP10 is dramatically elevated in HCC tumor tissues and cell lines. Knockdown of DUXAP10 by DUXAP10 si-RNA significantly inhibited the cell viability, proliferation and induce the apoptosis of HCC cell line. Meanwhile, inhibition of DUXAP10 attenuates the cell migration, invasion, and epithelial–mesenchymal transition (EMT) process. No significant change of JNK MAPK pathway was detected in DUXAP10 siRNA transfected HCC cell lines. The β-catenin and pAkt levels were decreased in the Hep G2+DUXAP10 siRNA and SMMC7721+DUXAP10 siRNA groups, while the activation of Wnt/β-catenin or PI Hepatocellular carcinoma (HCC) is one of the most aggressive cancers due to its high rate of recurrence ,2. It isIn the past decade, the significance of long non-coding RNA (lncRNA) has been identified . These tWe obtained tumor tissues and adjacent non-tumor tissues from 32 advanced HCC patients, from the Xi’an Central Hospital. The diagnosis standard for advanced HCC was from the current management of HCC: an eastern perspective, etc. ,10. The 2 supply.HCC cell lines, including Hep G2, SMMC7721, and normal HCC cell line LO2, were all purchased from Chinese Academy of Science . Cells were cultured in DMEM culture medium , 100 U/ml penicillin and streptomycin 10% fetal bovine serum (Gibco) were added . All theC−ΔΔT method was used for calculation, and each sample was examined in triplicate.Trizol was used for the isolation of total RNA from the tumor tissues and adjacent non-tumor tissues. The quantitative real-time PCR was performed using a PrimeScript™ RT-PCR Kit  on a Roche LightCycler480-II real-time thermal cycler (Roche). In brief, 45 μl reaction solutions containing 50 ng cDNA, 25 μl of FastStart Universal SYBR Green Master, 0.5 μl of forward primer and reverse primer, and 19 μl PCR-grade water were subjected to 95°C for 10 min and then 40 cycles of 95°C for 15 s, 56°C for 60 s, and 68°C for 45 s. The primers used were as follows: DUXAP10 Forward primer 5′-GGTTCAACAGTATGGCTCCAAAG-3′ Reverse primer 5′-GACTGCCCATCCACAGATGAAG-3′; GAPDH was used as the reference gene. RT-PCR was conducted at 95°C for 3 min, 95°C for 3 s, 60°C for 30 s, 40 cycles. 24/well in a six-well plate, and transfected with DUXAP10-siRNA and negative control-RNA . The transfection was performed according to the manufacturer’s protocol of lipofectamine 2000 . Total 6 h after transfection, the lipofectamine 2000-coated siRNA were all washed out with culture medium. And 48 h after transfection, cells were used for other experiments [Hep G2, SMMC7721 cells were cultured 6 × 10eriments .The MTT assay was used for cell viability evaluation. Cells were seeded to 96-well plates with 2000 cells/well and incubated for 1, 2, 3, 4, and 5 d. In the MTT assay, in each group, 20 μl 5 mg/ml, MTT  solution was added to each well and then the cells were continue to incubate at 37°C for 4 h. After that, the MTT solution was removed, and 200 µl of DMSO was added for each well. After 15 min incubating in 37°C, the plate was measured by a microplate reader at 490 nm .For BrdU binding, remove the existing culture medium from the cells and replace with 10 µM BrdU labeling solution . Incubate the cells in the BrdU labeling solution at 37°C for 12 h and then immunofluorescence staining was used. Fix cells using 4% paraformaldehyde in 0.M PBS for 15 min at room temperature. For sample permeabilization, using 0.3% Triton X-100 for 15 min. For DNA hydrolysis, incubate cells in 1 M HCL for 10 min at 37°C. Then incubate cells with 3% BSA for 20 min to block the possible unspecific binding of the antibodies. Incubate cells with diluted BrdU-antibody  in 1% BSA overnight at 4°C. Then incubate cells with secondary antibody Cy3-goat anti-mouse . Images were collected from at least three independent cultures per group were used for statistical analysis . For CCK5 cells/well) in the top chamber of the inserts cultured in the serum-free DMEM. Then, in the lower chamber of the well, 500 μl of 10% serum-containing DMEM were added. The cells were allowed to migrate at 37°C for 24 h and then removed from the upper chamber using cotton swabs. For invasion experiment, cells were seeded on Matrigel-coated  membrane inserts (1 × 104/well). Cells that successfully migrated to the underside of the membrane were stained with 0.1% crystal violet and counted in four randomly selected fields by using a light microscope.Transwell membranes  were used to detect cell migration and invasion. Before the experiment, SMMC7721 and Hep G2 were suspended separately and cultured in the serum-free DMEM culture medium for 24 h. After that, they were plated (1 × 10The Annexin V and FITC-propidium iodide (PI) double staining assay  were used to estimate the apoptotic cells. Briefly, 48 h of post-DUXAP10 siRNA transfection cells were harvested and stained with 5 µl of FITC-Annexin V and 5 µg/ml of PI. After incubation for 15 min in the dark, the cells were detected by flow cytometry. The Annexin V positive and PI negative cells are in the apoptotic stage; the double negative cells are considered alive; the double positive cells are dead cells.Total protein was extracted using RIPA , and the concentrations were determined by BCA kit . Total 10 μg of samples were loaded and separated by 10–12% SDS-PAGE gels. Proteins on the gel were transferred to PVDF. After blocking membrane in 10% skim milk, the membrane was incubated in primary antibody overnight at 4°C. Following by incubating with the HRP-conjugated goat anti-rabbit  secondary antibodies for 2 h at room temperature. The membranes were visualized with ECL systems . GAPDH was used to normalize the data. The primary antibodies used: E-cadherin , N-cadeherin , Vimentin , pAkt , Akt , pJNK , JNK , GAPDH , and β-catenin  .t test. A P<0.05 was considered as statistically significant.Data were processed using SPSS13.0 software and expressed as means ± S.D. Differences amongst groups were assessed by one-way ANOVA; differences between groups were analyzed by The expression level of DUXAP10 in advanced HCC, 32 HCC tissues and normal adjacent tissues were dissected from HCC patients, and the mRNA expression of DUXAP10 was detected using RT-PCR analysis. Amongst these tissues, there are 25 normal adjacent tissues that express DUXAP10, and 26 tumor tissues that express DUXAP10. Our result showed that DUXAP10 expression level was significantly higher in HCC than in the adjacent tissues A. In ordMTT and CCK assay were used for the cell viability study. The results showed that the HCC cell line viability is higher than the normal hepatic cell LO2. However, by suppressing the DUXAP10, the cell viability was decreased significantly compare with the control group A. BesideNext, the cell migration and invasion status of HCC cells were evaluated. HepG2 and SMMC7721 cells have higher migration and invasion ability compare with DUXAP10 siRNA treated group A,B. Next3K/Akt signaling pathway were involved in the HCC cell migration and invasion. In the present study, we evaluated the phosphorylate-JNK (pJNK)/JNK, and Wnt/β-catenin, and phosphorylate-Akt/Akt level in the Hep G2, Hep G2+DUXAP10 siRNA, SMMC7721, SMMC7721+DUXAP10 siRNA groups. Western blotting data illustrate that there is no significant change of pJNK/JNK in Hep G2+DUXAP10 siRNA and SMMC7721+DUXAP10 siRNA groups. Indicating the DUXAP10 regulates the HCC cell migration and invasion through other pathways , combined with DUXAP10 siRNA could suppress the inhibition of DUXAP10 siRNA on cell proliferation and migration (It was suggested that the JNK MAPK, Wnt/β-catenin, and PIpathways A,B. The pathways A,C,D. Weecreased E,F. In aigration E,F.LncRNAs have diverse cellular functions such as cell proliferation and differentiation, and it has demonstrated that they are crucial regulators for a variety of tumor growth ,15,16. Iin vivo study showed that knockdown DUXAP10 could impair non-small cell lung cancer growth [It has been reported that DUXAP10 is significantly increased in non-small cell lung cancer tissues and knockdown of DUXAP10 could inhibit the inhibited non-small cell lung cancer cell migration and invasion. Moreover, the cell proliferation, migration, and invasion in non-small cell lung cancer cells are significantly up-regulated in the DUXAP10 overexpression cell model. The r growth . We illu3K/Akt signaling pathway [3K/Akt signaling has also attenuated. This suggested that PI3K/Akt is involved in the DUXAP10 related HCC cell proliferation and invasion. Moreover, Wnt/β-catenin is activated in the tissue of HCC patients, which indicates that Wnt/β-catenin is involved in HCC tumor growth. This is in parallel with our result. More importantly, we have proved that Wnt/β-catenin is taken part in the DUXAP10 related HCC cell proliferation [The JNK signal pathway plays an important role in the advance of HCC ,19. It w pathway ,21. Combferation .3K/Akt and Wnt/β-catenin signaling. Inhibition of DUXAP10 in HCC SMMC-7721 and Hep G2 cells could attenuate the EMT and cell proliferation and invasion. Therefore, DUXAP10 might be a potential target for overcoming therapy for HCC in the future.In summary, our present study showed that DUXAP10 plays a critical role in regulating HCC development, potentially by regulating EMT and cell proliferation through the PI