Plasmodium falciparum histidine-rich protein 2 (pfhpr2) gene deficiency has been found to be the major reason of RDTs failure in many countries. This article analysed the deletion of pfhpr2 gene of falciparum malaria cases isolated in Yunnan Province, China.Failed diagnoses of some falciparum malaria cases by RDTs are constantly reported in recent years. Plasmodium genomic DNA was extracted and the pfhrp2 gene exon2 region was amplified via nested PCR. The haplotype of the DNA sequence, the nucleic acid diversity index (PI) and expected heterozygosity (He) were analyzed. Count PfHRP2 amino acid peptide sequence repeat and its times, and predict the properties of PfHRP2 peptide chain reaction to RDTs testing.Blood samples from falciparum malaria cases diagnosed in Yunnan Province were collected. pfhrp2 PCR amplification products (gene exon2) were obtained, while the remaining 47 samples were false amplification. The length of the 250 DNA sequences ranged from 345 - 927 bp, with 151 haplotypes, with PI and He values of 0.169 and 0.983, respectively. The length of the PfHRP2 peptide chain translated from 250 DNA sequences ranged from 115 to 309 aa. All peptide chains had more than an amino acid codon deletion. All 250 PfHRP2 strands ended with a type 12 amino acid repeat, 98.0% (245/250) started with a type 1 repetition and 2.0% (5/250) with a type 2 repetition. The detection rate for type 2 duplicates was 100% (250/250). Prediction of RDT sensitivity of PfHRP2 peptide chains based on type 2 and type 7 repeats showed that 9.60% (24/250), 50.0% (125/250), 13.20% (33/250) and 27.20.5% (68/250) of the 250 peptide chains were very sensitive, sensitive, borderline and non-sensitive, respectively.A total of 306 blood samples were collected, 84.9% (259/306) from which pfhrp2 gene deletion from different infection sources in the Yunnan province are extremely complex. The cause of the failure of pfhrp2 exon2 amplification is still to be investigated. The results of this study appeal to Yunnan Province for a timely evaluation of the effectiveness and applicability of RDTs in the diagnosis of malaria.The diversified polymorphism of the  Plasmodium falciparum in 2008 [P. falciparum infections despite the tendency to overestimate the sensitivity and specificity [P. falciparum histidine-rich protein 2 (PfHRP2) gene deletion may be the mainly biological factor accounting for failed diagnoses of falciparum malaria. Meanwhile, the WHO appeal to the regions with falciparum malaria epidemic worldwide for promptly clarification and validation of the PfHRP2 (genes) deletion as well as its influence on the accuracy of RDTs diagnosis of malaria [Yunnan was one of the only two provinces in China where malaria was indigenous endemic cases as of 2013 –7. A spe in 2008 , 7, 9. Acificity . The Wor malaria .PfHRP2, P. falciparum lactate dehydrogenase (pLDH) and P. falciparum aldolase (ALD). The first two are the detection targets of most malaria RDT products [PfHRP2 is a specific diagnosis target protein for falciparum malaria. Unfortunately, since the existence of P. falciparum strains with pfhrp2 gene deletion was first reported in the 1980s [pfhrp2 gene deletion has been found in Peru [pfhrp2 gene was as high as 40%. In contrast, in areas with a false negative rate of 2%, only 22% of pfhrp2 genes were undetected [P. falciparum without the pfhrp2 gene had increased the incidence of errors in RDT diagnosis of falciparum malaria in Ghana. The studies conducted by Akinyi et al. and Wurtz et al. indicated these phenomena of a combination of RDT diagnosis failures for falciparum malaria, and the pfhrp2 gene entire deletion in P. falciparum isolates from Peru and Senegal [pfhrp2 gene exon2 regions reached 9.5%, 10%, and 23%, respectively, indicating that these countries may have a higher risk of misdiagnosis with the pfhrp2 (gene)-deficient strains when using PfHRP2-target protein RDTs for falciparum malaria diagnosis. In 2014, Yang et al. detected the polymorphisms of pfhrp2 gene in 20 P. falciparum isolates collected from the China-Myanmar border [pfhrp2 gene deletion in falciparum malaria case isolates from Yunnan Province [pfhrp2 gene in P. falciparum [pfhrp2 gene deletion and its changes need to be further verified. More importantly, in response to the WHO’s appeal for the selection and improvement of RDTs, it is essential to understand the prevalence of pfhrp2 gene deficiency, PfHRP2 protein deficiency, the extent of antigenic variation, and to provide evidence for Yunnan Province to improve the quality of falciparum malaria RDT diagnoses [pfhrp2 gene deletion in Yunnan Province.The target proteins of malaria RDTs diagnosis mainly include products , 12, andhe 1980s –15, the  in Peru , Mali [1 in Peru , India [ in Peru , 19, Phi in Peru , Senegal in Peru , Brazil  in Peru , Yemen [ in Peru , Hondura in Peru . Amoah edetected ; hence,  Senegal , 26. In  Senegal , Angola  Senegal , and Indr border . In 2015Province . Certainlciparum , 30. Howiagnoses , 30. ThiThe study was approved by Yunnan Institute of Parasitic Diseases and by the Ethical Committee. Genetic testing experiment, etc. were performed on stored blood samples obtained as part of routine diagnostic work-up from patients with fever suspected of malaria. Although the absence of risk and the anonymous data processing are ensured among the study, consent from potential malaria patients need to be obtained during collecting blood samples.P. falciparum infection was parasitological confirmed by both microscopic examination [pfhrp2 gene sources of falciparum malaria infection were confirmed by epidemiological investigation.Blood samples from falciparum malaria cases were continuously collected diagnosed and reported by Yunnan Province, and officially registered and approved by the “China Information System for Disease Control Prevention from January 2013 to December 2018. mination  and genemination . The filPlasmodium genomic DNA was extracted according to the instructions of QIAgen Mini Kit , and extracted genomic DNA liquid stored at − 20 °C for later use. The pfcrt gene exon2 region was amplified to verify whether the DNA extraction of the Plasmodium genome was normal [Three filter paper blood drops, each with a diameter of 5 mm, was taken, and s normal .https://www.ncbi.nlm.nih.gov/gene/) reference sequence (ID: nc_004329.3) was used to design primers and reaction conditions for PCR amplification of pfhrp2 gene exon2 [pfhrp2 gene exon2 region, and the product size was expected to be about 824 bp. Both first and second PCR reactions contained 2.6 µL DNA template, 14 µL of 2× Taq PCR mixed system (containing Taq enzyme that was purchased from Qiagen Biotech (Shanghai), 0.7 µL each forward and reverse primers (20 µmol/L), and ddH2O for a volume of 25 µL. Both the conditions of first and second PCR reactions were as follows:95 °C for 5 min; 35 cycles of 95 °C for 30 s, 55 °C for 40 s, and 72 °C for 90 s; and 72 °C for 7 min. The amplified product of the second PCR was detected by 1.5% agarose, observed via 1.5% agarose gel electrophoresis . Then the positive products were sent to Shanghai Meiji Biomedical Technology Co., Ltd. for sequencing using the dideoxy chain-termination method. Sequenced data were analysed using Chromas Pro 2.33 software.The GenBank  using BLAST module of NCBI after splicing or transformation in the DNA Star Lasergene 7.1 software [pfhrp2 gene exon2 region was analysed by Arlequin 3.5.2.2 software, and the haplotypes, the nucleic acid diversity index (PI) and expected heterozygosity (He) were calculated [PfHRP2 peptide chain were counted, and the identification of the amino acid repeat types followed the principle of “long rather than short”. Such as, the “ AHHAHHAAD “ amino acid sequence was identified only as a type 2 repeat, not as a linkage between type 4 (AHH) and type 7 (AHHAAD), and the “AHHAHHATD” amino acid sequence was identified only as a type 14 repeat, not as a linkage between type 4 (AHH) and type 6 (AHHATD) [F test was performed on the amino acid residues and sequence repeats in pfhrp2 gene exon2 region using the SPSS16.0 software, and the inspection level was 0.05.DNA sequences from PCR product sequencing were aligned with the reference sequence of software –36. The software –36. The lculated , 36. Jal(AHHATD) , 30. F tPfHRP2 repeat types 2 and 7 (type 2 × type 7) was used to distinguish the sensitivity of PfHRP2 peptide chain reaction to RDTs. Assay products with sensitivity responses > 100, 50–100, 43, 44–49 and ˂43 to the RDTs were classified as very sensitive (group A) and sensitive (group B), borderline (group I) and non-sensitive (group C) peptides, respectively [PfHRP2 peptide chain in geographical populations of P. falciparum was evaluated via the T test using SPSS16.0 software, and the inspection level was 0.05. Arc Gis10.1 was used to map the distribution of samples including normal sequencing pfhrp2 gene exon2 and failed cases [The product of the number of ectively . The resed cases .pfhrp2 gene exon2 region was amplified, with a fragment length of about 345 (not shown in electrophoresis)–927 bp. 47 blood samples was not amplified to cover the target pfhrp2 gene exon2 region because its pfcrt gene exon2 fragment (150 bp) was amplified and identified as normal DNA extracted from the Plasmodium genome.A total of 306 blood samples were collected from falciparum malaria cases, 84.9% (259/306) from which the target phhrp2 gene exon2 region, collected from prefecture falciparum malaria cases including Dehong (103 cases), Kunming (43 cases), Baoshan (41 cases), Dali (16 cases), Lincang (15 cases), Qujing (7 cases), Zhaotong (7 cases), Puer (7 cases), Wenshan (5 cases), Nujiang (4 cases), Xishuangbanna (4 cases), Lijiang (3 cases), Honghe (2 cases), Yuxi (1 case), Chuxiong (1 cases) , 23.4% (11/47) and 14.9% (7/47), respectively. Yunnan indigenous infection cases blood samples with failed amplification of pfhrp2 exon2 region were only limited to three prefectures including Dehong, Baoshan and Lincang. On the contrary, the blood samples with failed amplification of that gene fragment which were collected from Kunming and Qujing prefectures involved a large number of cases imported from Africa counties infection , 23.4% 1/47 and pfhrp2 exon2 region were obtained by sequencing 259 PCR amplification products, with lengths ranging from 345 to 927 bp. Polymorphic analyses showed that there were 151 haplotypes, Hap_001–Hap_151, and that PI and He were 0.169 and 0.983, respectively. There were 87, 77 and 10 haplotypes in 146 Myanmar geographical populations, 89 African geographical populations and 15 Yunnan geographical populations which were traced by epidemiological investigation, respectively. The PI and He of the three geographical populations were 0.167 and 0.974, 0.219 and 0.997, 0.249 and 0.981, respectively.A total of 250 DNA sequences of the Hap_016, with a DNA sequence length of 771 bp, was present in the largest proportion of the 151 haplotypes, accounting for 16.0% 40/250). The remaining haplotypes were Hap_004 , Hap_014 , Hap_008 and Hap_058 , Hap_105 , and Hap_030    and the largest score  in the X-axis direction were two sequences from Myanmar geographical isolates. The haplotypes with the smallest score (− 13.685)  and the largest score (14.593)  in the Y-axis direction included one Myanmar isolate and one African isolate. Haplotypes with the smallest score (− 12.873)  and the largest score (34.599)  in the z-axis direction were also from two Myanmar isolates.Eleven high frequency haplotypes, including “Hap_014″, “Hap_016″, “Hap_105”, “Hap_058”, “Hap_030”, “Hap_038”, “Hap_044”, “Hap_004”, “Hap_008”, “Hap_096”, and “Hap_064”, with the PCA locations, X: 4.134, Y: 2.598, Z: 1.951; X: − 3.927, Y: 8.212, Z: 2.775; X: − 6.644, Y:− 8.952, Z: − 2.350; X: 2.480, Y: 11.215, Z: − 3.569; X: − 4.944, Y: 0.798, Z: 0.178; X: 9.492, Y: − 0.388, Z: − 8.409; X: − 0.427, Y: 0.694,Z: − 0.463; X: 10.719, Y: − 12.883, Z: − 6.025; X: 8.430, Y: 1.204, Z: 2.151; X: 6.962, Y: 2.430, Z: 0.230; and X: 13.543, Y: − 4.160, Z: 34.599, respectively, were discrete and non-overlapping. Also, the haplotype which only composed of the sequences from Myanmar isolates, Africa isolates or Yunnan isolates, did not show any aggregation tendency .Following the translation of 250 DNA sequences into amino acid sequences, the longest was 309 aa of haplotype Hap_114, and the shortest was 115 aa of Hap_064 . Moreover, the three amino acid repetitions of “AHHAHHAAH”, “AHHAHHAPD” and “AHHAHHAPH” similar to type 2, the “AHHAHHAPY” similar to type 3, the “AHHAPD” and “AHHAPH” similar to type 7, and the “AHHAPY” similar to type 8 only appeared in the sequences of Myanmar population and African population  were between 52 and 99 in group B accounted for most PfHRP2 peptide chains . The shared advantages in Myanmar, Africa, and Yunnan was also reflected, but the separation plant taxa of group B peptides in Myanmar were lower than African and Yunnan isolates  , and the proportion of group A peptide chains isolated from Myanmar was significantly lower than those from African and Yunnan isolates   and Yunnan  isolates (Table The sensitivity testing of RDTs for 5) Table . The propfhrp2 gene (1064 bp) is located in the chromosome 8 telomere area [pfhrp2 gene region is prone to lead to increased mutations and a more complex diversity of amino acid tandem repeats [pfhrp2 gene exon2 [PfHRP2 not only affect the accuracy of RDTs diagnosis falciparum malaria against PfHRP2 target protein, but also lead to the potential harm of changing the habits of Plasmodium and even changing the prevalence intensity of malaria [ere area , and con repeats , 37–40. ne exon2  and the  malaria .PfHRP2 is not only involved in transforming the toxicity for pigment to poison Plasmodium parasites, but also plays a role in reshaping the structure of the erythrocyte membrane to enhance the immune escape of malaria parasites [arasites –44.pfhrp2 gene in the 57–301 aa coding region, which is also a dense repeat region of alanine and histidine in the exon2 region, of P. falciparum isolates in Yunnan province in the past 6 years was analyzed. Of the 306 blood samples, 250 samples had obtained the target DNA sequence (345–927 bp), which are similar in length from the geographical isolates in Senegal [pfhrp2 gene in the sample isolates was greater than that previously reported in African isolates [pfhrp2 gene from the Yunnan isolates was as high as 0.249, which is the highest among Myanmar, African and Yunnan groups.In this study, the polymorphism of  Senegal , Nigeria Senegal , Yemen [ Senegal , and Mozequence 3–927 bp,  Senegal . There w Senegal , and lowequence 3–927 bp,  Senegal  and Bake Senegal  in theirisolates , 29, whiPfHRP2 peptide chains derived from the DNA sequence were between 115 and 309 aa in length, with an average of 245 aa, from total cases isolates. The length of 242 aa from the Myanmar isolates was consistent with the study conducted by Baker et al. [pfhrp2 gene exon2 region in Southeast Asia. It is interesting to note that for the same infection across the same period, when we compared the pfhrp2 gene exon2 area and DNA sequences, and found that the peptides and amino acids of pfhrp2 with the same chain length as other infection isolates have different sequence structures. A prompt investigation of pfhrp2 gene mutation and gene exon2 region diversity may have potential gene traceability between different P. falciparum isolates.The 250 r et al. , but shoPfHRP2 peptide amino acid repeat feature recognition, all 250 peptide chains showed 19 kinds of repeats, including type 1, type 2 and its three variation types, type 3 and its one variation type, type 4, type 5, type 6, type 7 and its two variation types, type 8 and its one variation type 10, type 12 and type 13 , but it was still higher than that of the samples from Senegal  [PfHRP2 peptide chains belonging to group B, accounting for 50.0% (125/250), which was lower than 71.3% (87/122) [PfHRP2 peptide chain have not been implemented. Therefore, it is not possible to infer whether PfHRP2 peptide chain variation affects falciparum malaria diagnosis in Yunnan province.Studies have demonstrated that antigen variation is a risk factor for the unstable quality of falciparum malaria diagnosed by RDTs based on immune response , 49, andl region . Therefo a trend , 29, 30. a trend  and Wurt a trend  found th, 9/122)  and in K(87/122)  and 75.8pfhrp2 gene was not amplified in 15.36% (47/306) of the samples when extracting P. falciparum genomic DNA was confirmed to be normal from these samples. Still, the author could not ensure that the P. falciparum isolates in these samples can be considered entire deficiency of pfhrp2 gene. First, pfhrp2 gene deletion was not verified through Cheng’s method [pfhrp2 gene with higher amplification efficiency than Kumar et al. [pfhrp2 gene, and the sensitivity to confirm pfhrp2 (gene) deletion may not have been high enough. Parr et al. [pfhrp2 gene extension [pfhrp2 gene deletion was not found or rarely found. However, more phenomenon of pfhrp2 gene deletion were found only in amplifying exon2 region alone [pfhrp2 gene alone may be less sensitive than whole-genome or upstream and downstream extension assays for confirmation of pfhrp2 gene entire deletion. Third, the failure to amplify the pfhrp2 gene above 47 samples does not rule out the effect of low Plasmodium density. Gupta et al. indicated that the analysis on pfhrp2 gene deletion was unreliable when Plasmodium density is only 3 ~ 330 parasites/µl. Therefore, the study conducted by Beshir et al. excluded samples with Plasmodium densities < 5 parasites/µl during validation of pfhrp2 gene deletion. In conclusion, the certification of pfhrp2 gene entire deletion must be rigorous, as the WHO requires that programme must adjust their RDTs product when the proportion of pfhrp2 gene entire deletion of P. falciparum achieve to 5% in a population within an area [In this study, s method . Second,r et al.  and Baker et al. , the ampr et al.  and Trour et al.  adopted r et al.  and Ranar et al.  amplifiextension , while ton alone , 36, 55, an area .pfhrp2 gene polymorphism from falciparum malaria cases isolates from January 2013 to December 2018 in Yunnan province. The current pfhrp2 gene polymorphism situation from falciparum malaria case isolates in Yunnan province was reflected due to good continuity and systemic characteristics of these samples. The results of the study will be the basis for further verification of pfhrp2 gene entire deletion and screening of optimal epitopes of PfHRP2 peptide chain. However, this study include several limitations: (1) due to the inability to determine the Plasmodium density in some blood samples, the effect of low protozoan density on the confirmation of pfhrp2 gene deletion cannot be ruled out; (2) polymorphic and deletion of pfhrp3 gene, which are considered as early signs of pfhrp2 gene deletion [PfHRP2 peptide chains exists or not in blood samples was not examined in parallel with RDT products in the study.This study comprehensively analysed the deletion , were nopfhrp2-deficient plasmodium [pfhrp2-deficient Plasmodium deserves attention. Further rapid verification of the entire deletion of pfhrp2 gene from P. falciparum in China deserve higher priority. Fortunately, a more sensitive and specific sequencing analysis method for pfhrp2 gene deletion has developed.The study conducted by Watson et al. indicated that low prevalence of malaria and the high frequency for treatment could increase the risk for inappropriate use of antimalarial drugs because malaria might be misdiagnosed by RDTs, which will also lead to transitionally screening on asmodium . Currentpfhrp2 gene diversity information of isolates from falciparum malaria cases diagnosed and reported by Yunnan Province, China. Although the presence of pfhrp2 gene entire deletion is uncertain, the results are a general reflection of the presence of pfhrp2 gene polymorphism in Yunnan Province since the study samples are from official cases reported over the years. Therefore, it is necessary that the effectiveness and applicability of RDTs in diagnosis falciparum malaria in Yunnan province should be evaluated in time and it is important that the pfhrp2 gene-deficient P. falciparum isolates should not be excessively screened by blindly extending the use of RDTs.In conclusion, this study firstly, provides the


Glycine contributed 43% to total N uptake of Pinus, but only 20, 11, and 21% to N uptake by Larix, Quercus, and Juglans, respectively (whole plant), whereas nitrate uptake was 24, 74, 88, and 68% of total uptake for these four species, respectively. Retention of glycine carbon versus nitrogen in Pinus roots indicated that 36% of glycine uptake was retained intact. Nitrate was preferentially used by Larix, Quercus, and Juglans, with nitrate uptake constituting 68∼88% of total N use by these three species. These results demonstrated that these dominant tree species in secondary forests in northeast China partitioned limited N resource by varying uptake of glycine, ammonium and nitrate, with all species, except Pinus, using nitrate that are most abundant within these soils. Such N use pattern may also provide potential underlying mechanisms for the higher retention of deposited nitrate than ammonium into aboveground biomass in these secondary forests.Fundamental questions of how plant species within secondary forests and plantations in northeast China partition limited nitrogen (N) resource remain unclear. Here we conducted a  In Northeast China, natural secondary forests (NSFs) account for as much as 70% of the regional forests . They arA complete understanding of forest N cycling requires that we understand how much N plants take up, and which form of inorganic or organic N they assimilate. Soil N occurs in a variety of organic and inorganic forms that are differentially available to plant roots. Complex organic polymers in litter are depolymerized into bioavailable, N-containing monomers  weakly preferring specific N forms  methods and a 15N tracer experiment with ammonium, nitrate and glycine to determine if these tree species show preference or flexibility in N use. Glycine is the most frequently used amino acid in field studies of plant uptake of amino acids  the two coniferous species Pinus koraiensis and Larix keampferi will prefer ammonium or glycine to nitrate  thest sites ; and (3)Pinus koraiensis and Larix keampferi, and two broadleaf species, Quercus mongolica and Juglans mandshurica, which make up 5, 30, 8, and 27% of the mature tree composition in this forest, respectively . These four species are known to be associated with different mycorrhizal types, with Juglans mandshurica associated with arbuscular mycorrhizal fungi, with the three other species, Pinus koraiensis, Larix keampferi, and Quercus mongolica all being ectomycorrhizal type , and the ratio of ammonium to nitrate is 2:1 . These species include two coniferous species, zal type . The cone is 2:1 . The soiPinus koraiensis, Ulmus laciniata, and were classified as brown forest soils, with pH of 6. The seedlings were transplanted individually to each pot (15.3 L). All pots received natural light levels and were watered every 3 days with tap water to keep the soil surface moist. The average high temperature in the greenhouse during the experiment was 26.5°C and the average low temperature was 18.3°C. Pots of all seedlings were arranged randomly and were moved within the greenhouse regularly to reduce the effects of environmental variation. These seedlings were acclimated to greenhouse conditions for 1 year prior to the 15N labeling experiment. No nutrients were added to the pots during this 1 year acclimation.One-year old seedlings of these four species were purchased from a local nursery and transplanted to the greenhouse in Yunong Campus, Institute of Applied Ecology, Chinese Academy of Sciences . Soils for transplanting these seedlings were topsoils from low hills adjacent to the greenhouse, which are dominated by 15N tracers in July 2018. For 15N labeling, we randomly allocated four individuals of each species to one of the three N species treatments . Seedlings received 10.17 mg 15N per individual  dissolved in 20 mL deionized water containing equimolar concentrations of nitrate, ammonium, and glycine, with only one N form labeled. The total amount of N (including 15N) added to each plant individual was 1.02 μg N g-1soil, with 1/3 15N, representing a roughly 16∼37% increase in DIN (ammonium+nitrate) and a 8∼14% increase in total N. The organic N source was provided as U-13C2, 15N-glycine (both 13C and 15N at 99 atom%), while ammonium and nitrate were supplied as 15NH4Cl (99.08 atom%) and Na15NO3 (99.26 atom%). We could then test for uptake preferences of each of the labeled N forms by individual plant species and also test the proportion of glycine taken up directly by plants, as evidenced by the relative enrichment of plant tissue with 13C and 15N, or as mineral N after microbial mineralization . A control group with no added N provided background 15N values for 15N uptake rates calculations.Most leaves reached full expansion by middle May. We labeled each individual seedling with lization . The nit15N-label zone) was collected for analysis. Seedlings were immediately rinsed, separated into coarse roots (>2 mm), fine roots (<2 mm), stems, and leaves, dried at 60°C for 48 h, weighed, and ground for 15N and 13C isotope analyses by elemental analyzer-isotope ratio mass spectrometry . Four compounds were used as references: L-histidine, D-glutamic acid, glycine, and acetanilide. The analytical precision for δ15N and δ13C were both 0.2‰. Whole plant δ15N values were calculated from mass-weighted δ15N values of each plant organ.We removed any leaf litter within the surface of the pots and injected nitrogen tracer solutions into the soil at four locations (5 ml for each) equally spaced around, and approximately 3 cm from, the stem of each seedling to a depth of 10 cm using a syringe and needle with four lateral holes. After 24 and 48 h, seedlings and intact root systems were harvested and bulk soil surrounding each seedling (∼3 cm radius or within the 15N atom% excess (APE) was calculated as the difference in atom% 15N between 15N-labeled plant organs and unlabeled (control) organs. 15N uptake rates (μg 15N g-1 dry mass h-1) were calculated by multiplying plant organ N content (μg g-1 plant organ biomass) by the corresponding (APE/100), divided by labeling time in hours and (atom% 15N/100) of the applied 15N-labeled N form  as follows: (N content (μg g-1 plant organ biomass) × APE/100)/(time (h) × (at% 15N tracer/100)) (15N enrichment in plant leaves (data not shown).er/100)) . Uptake Total dissolved N (TDN), ammonium, and nitrate were immediately extracted within 4 h of sampling in 50 mL of 2 mol/L KCl on a shaker table for 1 h, filtered through Whatman GF/A filters , and frozen at -36°C until colorimetric determination of nitrate+nitrite and ammonium using a continuous chemical analyzer . Gravimetric soil water content was 66 ± 9.7% on average at the time of harvest. Total dissolved N in KCl extracts was determined by alkaline persulfate digestion  and nitr15N measurements of actual and derived nitrate samples were then made using the modified azide method  was calculated as the mass-weighted difference between the concentrations of KCl-extracted TDN, ammonium, and nitrate, and the δ15N values obtained from the modified azide method  or diffusion (ammonium) methods using the following equation: δ15NDON = (δ15NTDN × [TDN] - (δ15Nammonium × [ammonium] + δ15Nnitrate × [nitrate]))/[DON].The δe method . Methodoe method . The δ15cted TDN . The δ1515N values of soil N  in control pots and δ15N values of the leaves in a Bayesian isotope-mixing model [Stable Isotope Analysis in R (SIAR)] to estimate the proportional contributions  of the three N forms  to total N of each species. The SIAR model uses a Bayesian framework to establish a logical prior distribution (60) for estimating f values, and then determines the probability distribution for the f values of each soil N source  to the mixture . The mixing model considers isotope effects during plant N uptake . We used the normalized 15𝜀 values for worldwide plants associated with ectomycorrhizae (ECM) and arbuscular mycorrhizae (AM) ;∗δ15Nleaves = δ15Nleaves + 2‰ (for AM species Juglans);∗δ15Nleaves = δ15NDON × fDON + δ15Nammonium × fammonium + δ15Nnitrate ×fnitrate.15N uptake rates among the three labeled N forms for each species were tested through one-way analysis of variance. P-values < 0.05 were considered to indicate a significant difference among means. Analyses were followed by Tukey’s b post hoc test. All statistical analyses were conducted using SPSS 19.0 .The differences in 15N tracer addition, plant organs of the four species had δ15N values ranging from -3.1 to -0.5‰, with N concentrations ranging from 0.4 to 3.6%  and ammonium (1.2 ± 0.1 to 2.5 ± 0.4 μg g-1). The nitrate : ammonium ratios varied between 1.3 ± 0.2 and 2.1 ± 0.4 (Quercus had the lowest N availability (DON + ammonium + nitrate) after 1 year growth  in control soils and δ15N values of the leaves after correction of the fractionation factor (15𝜀) showed that soil nitrate was the major N source for all species, with nitratef values ranging from 43 to 76%  and ammonium , and the lowest being the assimilation of ammonium to Quercus stems  and 8 ∼ 85 times that of ammonium (0.001∼0.04 μg 15N g-1h-1). Quercus had significantly higher uptake and assimilation rates for nitrate than for glycine or ammonium , followed by ammonium and nitrate , the coarse and fine roots of Pinus were associated with significantly higher δ13C values  and the 13C excess (x) under U-13C2, 15N-glycine treatment had a slope of 0.71  at 48 h after 15N tracer addition, indicating that 36% of glycine was derived from intact uptake by Pinus roots  and the13C excess of coarse and fine roots of this species were not significantly correlated (data not shown).In contrast to the other three species, us roots . In addireatment –D, but tP < 0.05) for Pinus, and nitrate > glycine > ammonium (P < 0.001) for Larix, Quercus, and Juglans   . In cont15N-labeled glycine, ammonium, or nitrate by four tree species common in secondary forests and larch plantations in northeast China. These species exhibited different preferences for different N forms, with Pinus predominantly accumulating glycine into fine roots and coarse roots, followed by ammonium, while the other three species primarily used nitrate, followed by glycine. These results partly supported our hypothesis, suggesting that tree species within these secondary forests and larch plantations partition N through preferential uptake of different N forms. Nitrate was the most available N form in these sites and was preferred by all species except Pinus.Nutrient acquisition strategies incorporate nutrient uptake rates, mycorrhizal associations, nutrient requirements, and preferences for chemical forms of N, which are analogous to above-ground economic strategies . In thisNevertheless, given that this experiment was done in pots and trees were kept in a greenhouse for a year prior to the study, these results may not reflect what would occur in the field. Being a pot study in greenhouses, the soil within pots might be warmer than field soils, which may enhance both N mineralization and nitrification processes, with the net effects on soil ammonium and nitrate availability not straightforward. Despite this, all species were exposed to the same conditions, so the differences among species are noteworthy.15N uptake rates ranged from 0.001∼0.8 μg 15N g-1h-1. These uptake rates, however, were low comparing with those by plant species from temperate forests in China . The overall uptake pattern in Pinus was glycine > ammonium > nitrate. Our results agree with studies on Pinus sylvestris L. and Picea abies L. seedlings growing in pots (peat as medium) showing similar uptake rates of glycine, arginine and ammonium, being 7 to 8 times higher than uptake rates for nitrate  was observed for each Pinus individual . Plants with mycorrhizal associations are generally favored over non-mycorrhizal plants in N-limited ecosystems, where most soil N cycling will largely occur in organic forms. Due to the wider enzymatic capabilities of mycorrhizal fungi compared to plant roots , and three of the species took up more nitrate than other forms. Considering the dominance of nitrate over ammonium in the ambient soils , corresponding well to the observed different sink strength for ammonium and nitrate by these plants over relative longer terms.Distinguishing the different fates of ammonium and nitrate is a prerequisite to better understand the consequences of increasing N deposition. Deposited ammonium and nitrate are processed differently in a mixed forest in Changbai Moutain  and in NPinus assimilated predominantly glycine into fine roots and coarse roots, in contrast to primarily nitrate assimilation by the other three species, Larix, Quercus, and Juglans, implied that some coniferous trees may compete less well than other species, particularly if they are unable to shift root preference toward increased nitrate availability. Therefore, increasing N deposition may influence the coexistence of plant species and lead to community composition change in these forest stands.Recently increased proportions of nitrate in N deposition has decreased the mean ammonium : nitrate ratio from 6 in 1980 to 2 in 2010 . This maThe raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.YF and FZ conceived and designed the experiments. FZ and LD performed the data acquisition. FZ analyzed the data and wrote the manuscript. YF, EH, KK, XL, GG, SHu, SL, YL, and SHa commented and edited the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


LTα), located in the Major Histocompatibility Complex (MHC) class III region on chromosome 6, encodes a cytotoxic protein that mediates a variety of antiviral responses among other biological functions. Furthermore, several genotypes at this gene have been implicated in the onset of a number of complex diseases, including myocardial infarction, autoimmunity, and various types of cancer. However, little is known about levels of nucleotide variation and linkage disequilibrium (LD) in and near LTα, which could also influence phenotypic variance. To address this gap in knowledge, we examined sequence variation across ~ 10 kilobases (kbs), encompassing LTα and the upstream region, in 2039 individuals from the 1000 Genomes Project originating from 21 global populations.Lymphotoxin-α , in global populations. While these results are generally consistent with a model of balancing selection, we also uncovered a signature of positive selection in the form of long-range LD on chromosomes with derived alleles primarily in Eurasian populations. To reconcile these findings, which appear to support different models of selection, we argue that selective sweeps  of multiple derived alleles in and/or near Overall, this study identified candidate functional alleles in a biologically-relevant genomic region, and offers new insights into the evolutionary origins of these loci in modern human populations. LTα) in the MHC class III region encodes a potent cytotoxic polypeptide that plays a key role in regulating a number of biological processes, including lipid metabolism, coagulation, neurotransmission, and immunological response ) and the adjacent 5′ non-coding region (7411 bps) on chromosome 6, in 21 global populations from the 1000 Genomes Project. Of the 183 polymorphisms, 52 SNPs were located within LTα, while the remaining 131 SNPs were found in the 5′ region  occurred at relatively high frequency in all populations. More specifically, the minor allele frequency (MAF) at rs2229094  and rs1041981  ranged from 27.3 to 45.8% and from 20.2 to 33.8%, respectively, in South Asian populations. In Europeans, the rs2229094 minor C-allele and rs1041981 minor A-allele varied from 22.9 to 38.3% and from 24.8 to 30.9%, respectively, while the MAF at these loci ranged from 14.4 to 22.8% and from 38.4 to 54.8%, respectively, in East Asian populations. Likewise, the minor C-allele at rs2229094 and the minor A-allele at rs1041981 varied from 22.4 to 30.8% and from 36.3 to 61.5%, respectively, in populations of African descent . Further comparative analysis also showed that allelic variation at rs2229094 was present in the genomes of two closely related archaic species to modern humans—Neanderthals and Denisovans  across ~ 10 kbs of sequence, encompassing rs538402044, rs562333039, rs538877791, rs566451995 occurred at < 2.5% frequency in non-Africans, while these alleles were absent in populations of African descent. Furthermore, the minor C-allele at rs2229092 was common in Europeans varying from 5.1 to 7.0%, while it occurred at lower frequency in South Asians (from 1.7 to 5.5%) and in East Asians (from < 1 to 2.4%). In contrast, the rs2229092 C-allele was absent in indigenous Africans, but was observed at relatively low frequency (~ 1–2.5%) in African American and African Caribbean populations. Additionally, we found that synonymous variants were either absent or occurred at very low frequency (< 1.0%) in all populations  occurred at relatively high frequency in global populations  have previously been implicated in the onset of complex diseases  Africa includes 99 Esan , 113 Mandinka , 54 Mende , 107 Yoruba , 99 Luhya , 60 African Americans , 96 Barbadians ; [2] South Asia encompasses 84 Bengali , 103 Gurjarati , 99 Indian Telugu , 93 Punjabi , 99 Tamil ; [3] East Asia consists of 93 Dai , 103 Han , 105 Han , 104 Japanese , 99 Kinh ; [4] Europe comprise 90 Great British , 99 Finnish , 107 Iberian , 104 Toscani Italians  [We analyzed the 9637 bps, encompassing the , Italy) . Althougregion 741 bps on LTα gene and the adjacent 5′ region in the 1000 Genomes sequencing dataset using vcftools [LTα and 5′ region, in Neandertal and Denisovan samples from the UCSC genome browser [We extracted variant calls  from the vcftools . The stavcftools . The min browser .D (DT) [H (H) [LTα and the adjacent 5′ region, separately, in each population. Significance of the test statistics was assessed by comparing the observed values to expected values generated from 10,000 neutral coalescent simulations incorporating different models of growth using the ms software [DT and H statistics under a 2-, 4-, 6-, 8-, and 10-fold increase in population size  beginning 70,000 ya until the present [DT and H values under a range of demographic scenarios that included a population bottleneck at 60,000 ya , followed by 10-, 20-, and 40- fold growth beginning at 50,000 ya until the present [LTα and the 5′ region are different sizes, we also incorporated sequence length as a parameter in these simulations.We calculated the Tajima’s D (DT)  and Fay ) [H (H)  statistisoftware . If a sa present , 143–145 present , 145. FoLTα coding region using DnaSP [N/DS) is expected to equal the ratio of replacement to silent polymorphisms within species (PN/PS).In addition, we applied the McDonald–Kreitman (M-K) test to the ng DnaSP . The M-Kh-diversity) was also calculated for each population using the DnaSP software [We extracted fully phased haplotype data from the 1000 Genomes Project for 2039 individuals using vcftools , and thesoftware .LTα region spanning ~ 10 kbs, and separately 2) in an ~ 35.5-kb genomic region, encompassing neighboring genes , using the Haploview software [D′ statistic [We examined pairwise LD: 1) in the software . LD was tatistic , which itatistic . The boliHS statistic [iHS| > 2 represent the most extreme 5% of scores. We also estimated the length of haplotype homozygosity with another haplotype-based statistic, nSL, using selscan [iHS statistic, making it robust to recombination rate variation [iHS. To complement these scans for selection, we quantified the decay of identity of haplotypes with distance by calculating the EHH statistic using loci with extreme iHS and/or nSL scores as core SNPs [We characterized long-range LD on chromosome 6 for each population using the tatistic , which itatistic . SNPs wi selscan . This stariation . The outore SNPs .FST derived from genomic data using the Weir and Cockerham method implemented in vcftools [FST at individual polymorphic sites across the ~ 10-kb region of interest and at ~ 7.5 million randomly selected SNPs from across the genome. The observed FST estimates for SNPs in and/or near LTα were then compared with the empirical distribution of FST values derived from genome-wide SNPs in order to identify outlier values (≤ 2.5th percentile or ≥ 95th percentile of the distribution). FST values at individual sites in or near LTα, and FST values at the ~ 7.5 million genome-wide polymorphisms were calculated using the same 21 populations.To measure the degree of genetic divergence among global populations, we calculated average vcftools , 148. Weθ over 1,000,000 runs [θ and our estimate of μ, we calculated the effective population size (Ne) parameter, which was determined to be 18,367 based on the formula Ne = θ/4μ. Then, the TMRCA of the gene tree and the ages of individual polymorphisms were estimated from the weighted average of simulated ages over 100,000 independent runs [We used a coalescent-based approach to estimate: 1) the expected TMRCA of the gene tree and 2) the expected ages of individual polymorphisms. This method requires an outgroup that provides the ancestral state at each polymorphic site. We applied the GENETREE software  to our s000 runs . Using tent runs . GENETREent runs . We remoLTα on protein function were inferred using the SIFT algorithm implemented in the Ensembl Variant Effect Predictor toolset. SIFT predicts the functional impact of amino acid substitutions  based on sequence homology, the physical properties of amino acids and multiple alignment information [G is the difference in the Gibbs free energy for folding between the final state (the mutant) and the reference state (the wild-type). For each polymorphic site, ΔΔG was computed from the free energy of the wild type protein  minus the Gibbs free energy of the mutated protein . In other words, ΔΔG = ΔG WT – ΔG MUT. Given this definition, mutations resulting in negative ΔΔG values were classified as “destabilizing”, while mutations resulting in positive ΔΔG values were categorized as “stabilizing”. We constructed the protein structure of LTα, which served as the input for FoldX, with the homology modelling server SWISS-MODEL [rs2229092 (H51P) and rs1041981 (T60N), but excluded rs2229094 (C13R). Therefore, the ΔΔG could not be calculated for rs2229094 using the FoldX tool.The effects of nonsynonymous SNPs at ormation . In addiormation , phyloP ormation  and phasormation  to evaluormation . We alsoormation  and I-Muormation  tools. ΔSS-MODEL . The resLTα gene using the SNP2TFBS tool [We also analyzed polymorphisms in and near the FBS tool , which pFBS tool , 56. In FBS tool , we searAdditional file 1 : Figure S1. contains pairwise LD plots (spanning ~9.64 kbs) for African and non-African populations that were not included in Fig. Figure S2. contains pairwise LD plots for loci located across a larger genomic region (spanning ~ 35.5 kbs) for each population. Figure S3. displays Manhattan plots of standardized |iHS| values in populations that were not included in Fig. Figure S4. shows Manhattan plots of standardized |nSL| scores for all populations. Figure S5. presents the remaining EHH plots for populations that were not included in Fig. Additional file 2 : Table S1. contains the frequency of alleles at polymorphic sites across LTα and 5′ regulatory region in global populations, and estimates of per site FST. Tables S2. gives summary statistics for the 5′ regulatory region only. Table S3. lists the observed Tajima’s D (DT) statistics for the LTα gene and their corresponding P-values under different scenarios of population growth. Table S4. shows the observed Fay and Wu’s H (H)  statistics for the LTα gene and their corresponding P-values under different scenarios of population growth. Table S5. lists the observed DT statistics for the 5′ regulatory region and their corresponding P-values for different scenarios of population growth. Table S6. contains the observed H statistics for the 5′ regulatory region and their corresponding P-values under different scenarios of population growth. Table S7A. presents haplotype frequencies in global populations. Table S7B. lists the polymorphic sites where the highest-frequency haplotypes in the network differ from one another and also indicates the alleles (on these haplotypes) that are shared with Neandertal and Denisovan genomes. Table S8. presents the frequencies of common haplotypes in non-African populations. Table S9. shows the frequencies of common haplotypes in African populations. Table S10. gives the genomic coordinates for markers in the LD plots (spanning ~9.64 kbs) in Fig. Table S11. gives the genomic coordinates for markers in the LD plots (spanning ~35.5 kbs) in Additional file Table S12. lists outlier standardized |iHS| and |nSL| scores for SNPs in each population. Table S13. presents the inferred ages of mutations across LTα and the 5′ regulatory region.


The influence of materials, heterostructure, and orientation for nanohybrids on photocatalytic activity is discussed for the first time. The heterojunction structures show remarkable enhancement compared to the bare semiconductor. The morphology of nanostructure has mainly an influence on the photocatalytic activity. NiO has the highest catalytic activity among the four pristine semiconductor nanostructures of ZnO, Cu2O, CuSCN, and NiO. The greatest enhancement of the photocatalytic activity is obtained using a ZnO/NiO (1 min) heterostructure attributed to the heterojunction structure and extremely higher specific surface area, which can degrade MO (20 mg/L) into colorless within 20 min with the fastest photocatalytic speed among homogeneous heterojunction structures. Meanwhile, the methodology and data analysis described herein will serve as an effective approach for the design of hybrid nanostructures for solar energy application, and the appropriate nanohybrids will have significant potential to solve the environment and energy issues.In this work, different structures based on electrodeposited n-type ZnO nanorods and p-type CuThe online version of this article (10.1186/s11671-019-2851-z) contains supplementary material, which is available to authorized users. So far, ZnO nanomaterials obtained the most widely investigation among various semiconductors, which could be attributed to their efficient electron transfer performance [2O-ZnO heterostructures on photocatalysis were reported by Wang et al. [2O, CuSCN, and NiO nanostructures are prepared by a low-cost, simple electrodeposition method at room temperature. Heterojunction structures of different materials and different orientation are fabricated based on n-type ZnO nanorods and p-type Cu2O, CuSCN, and NiO nanostructures. The heterostructures exhibit much better photocatalytic performance for photocatalytic degradation of MO than pristine n-type material or p-type material. The influence on the orientation of the heterojunction is dependent on the crystal quality of the upper material of the heterojunction. The influence on the material of different reaction conditions is dependent on the morphology and quality of the nanostructures. Among the three p-type materials used in our work, NiO has the most excellent photocatalytic performance. The ZnO/NiO (1 min) can decompose the MO (20 mg/L) aqueous solution from orange to colorless within 20 min. It reveals that the material and the orientation can both give an effect on the photocatalytic performance, which has great importance meaning for the decomposition of organic pollutants; moreover, this study is the first thorough study of the influence of materials, orientation, and heterostructure on photocatalytic activity and may promote further investigations on more nanohybrids to obtain higher photocatalytic efficiency.Hybrid nanomaterials with outstanding optical, electronic, and magnetic properties have attracted much interest in recent years due to their widespread applications in environment remediation , 2 and smposites , 3D-prinmposites , and Ru/mposites . Among tmposites –11 and tmposites –14 propemposites , bioinormposites , and gremposites . Due to mposites , 19. Thumposites –22, espeformance , providiformance , 25. Howformance , 27. In formance , composiformance , 30 and formance ; and comformance , ZnSe [3formance , CdSe [3formance , 35, andformance , 37. Obtg et al.  and Yu eg et al. . Luo andg et al. . Liu et g et al. . ZnO/CdSg et al. . These r3)·6H2O), hexamethylenetetramine (HMT), copper (II) sulfate pentahydrate, sodium hydroxide, lactic acid, potassium thiocyanate, ethylenediaminetetraacetic acid, triethanolamine, and nickel nitrate hexahydrate are all purchased from Sinopharm Chemical Reagent Co., Ltd. All these materials are of analytical grade and used as received without further purification.Indium tin oxide (ITO)-coated glass , zinc nitrate  at room temperature. The scan rate is 10°/min. The voltage and current are 40 kV and 40 mA. Surface and cross-sectioned structure of the samples are characterized by scanning electron microscopy (SEM) using a Philips-FEI XL 30-SFEG at room temperature without any surface coating. The accelerating voltage is 10–20 kV. The optical properties of the samples are investigated by UV-vis diffuse reflectance spectrometry (UV-vis DRS) using a Shimadzu UV-3101PC UV-vis spectrophotometer at room temperature.X-ray diffraction (XRD) patterns are measured with a Rigaku D/Max-2500 diffractometer using Cu Kα radiation  aqueous solution. Before irradiation, the aqueous solution is kept in the dark for 60 min to reach the adsorption equilibrium of MO. After a specific irradiation time, the photocatalytic decomposition performance is analyzed by measuring the absorbance of MO solution at its characteristic wavelength (465 nm) with a UV-vis spectrophotometer. All of the samples are performed in independent experiments and carried out at room temperature.2O, CuSCN, and NiO nanostructures are synthesized by the cost-effective electrodeposition method at room temperature. The electrodeposition is performed in a standard three-electrode electrochemical cell in the potentiostatic mode. The crystal textures of ZnO, Cu2O, CuSCN, and NiO nanostructures are characterized via XRD profiles. The XRD images of ZnO, Cu2O, CuSCN, and NiO nanostructures prepared by the electrodeposition method are shown in Fig. θ of ca. 34.36°, 36.12°, and 47.48° for the ZnO nanorods, which are assigned to the (002), (101), and (102) of ZnO crystals, respectively. All the peaks in the ZnO nanorods can be indexed to the hexagonal wurtzite structure of ZnO, and no other detectable phases exist in the ZnO nanostructures, which are similar as XRD profiles in Ref. [θ of ca. 29.78°, 36.81°, and 42.89° are observed for the electrodeposited Cu2O film on ITO substrate, which are assigned to the (110), (111), and (200) of Cu2O crystals, respectively, indicating that Cu2O has the pure cupric cubic structure with a (111) preferred orientation, which is the same as XRD profiles in Ref. [θ of ca.16.21°, 27.20°, and 32.69° and can be assigned to the (003), (101), and (006) planes of CuSCN crystals, respectively, which can be indexed to a rhombohedral structure β-CuSCN [2O, CuSCN, and NiO nanostructures prepared by the electrodeposition method. As shown in Fig. 2O, as shown in Fig. 2O (2.1 eV). As shown in Fig. 2O, CuSCN, and NiO nanostructures is in the ultraviolet and visible range, and this will guarantee the absorption of the ultraviolet light under Xe lamp irradiation in the photocatalytic decomposition experiments and the consequent generating of electron-hole pairs.All the ZnO, Cu in Ref. . Moreove in Ref. . The dif β-CuSCN . The XRD β-CuSCN . All the2O, CuSCN, and NiO nanostructures with different orientation are fabricated. Firstly, the ZnO, Cu2O, ZnO/Cu2O, and Cu2O/ZnO are prepared for photocatalytic degradation of MO. Figure 2O  heterojunction (b), Cu2O  (c), and Cu2O /ZnO heterojunction (d). Additional file 2O  heterojunction (a), ZnO/Cu2O  heterojunction (b), Cu2O /ZnO heterojunction (c), Cu2O /ZnO heterojunction (d), Cu2O  (e), and Cu2O  (f). Additional file 2O crystals grow from cubes to octahedras when the pH value of the electrodeposition solution alters from 10 to 12. The crystals obtained at pH ~ 10 with the reaction time of 20 and 40 min are not all the perfect cubes due to the different dissolution of the crystal in the reaction solution [2O crystals grow bigger and denser according to longer time, and the Cu2O crystals will crowd and aggregate together when the reaction time is longer. In the aggregating process, the Cu2O crystals will be crowded out of shape due to the great density. As shown in Figs. 2O crystals grown on the ZnO nanorods are as compact as the Cu2O crystals grown on the bare ITO glass substrate and are some smaller than the Cu2O crystals grown on ITO glass due to the different nucleating point. When the pH value of the reaction solution is 10, the ZnO nanorods grown on the Cu2O crystals are more compact than the ZnO nanorods grown on ITO glass, and the diameter and the length of the ZnO nanorods grown on the Cu2O crystals are almost the same with the ZnO nanorods grown on ITO glass, as shown in Fig. 2O crystals  are a little denser than the ZnO nanorods grown on the Cu2O crystals , and much larger ZnO rods will appear on the layer of the ZnO nanorods. As shown in Fig. 2O crystals  are much sparser than the ZnO nanorods grown on the ITO glass. The diameter and the length of the ZnO nanorods grown on the Cu2O crystals  are not homogeneous in a large range.Different heterojunctions based on n-type ZnO nanorods and p-type Cure 2O pH 2, 20 minre 2O pH 2, 20 minre 2O pH 2, 20 minsolution . It can Secondly, ZnO, CuSCN, ZnO/CuSCN, and CuSCN/ZnO are prepared for photocatalytic degradation of MO. Two different CuSCN nanostructures, hexagonal prism-like (3D) and nanowire (NW) structures, are prepared by the electrodepositon method. Figure Finally, the ZnO, NiO, ZnO/NiO, and NiO/ZnO are prepared for photocatalytic degradation of MO. Figure 2O, ZnO/CuSCN, and ZnO/NiO are discussed including pristine ZnO, pristine Cu2O (or CuSCN or NiO), ZnO/Cu2O (or CuSCN or NiO), and Cu2O (or CuSCN or NiO) with four structures in every system. Although the electrodeposition method used in this work for the preparation of the nanostructures is green and environmental, the preparation method may require a considerable amount of undesirable chemicals which undermines the greenness of the methods. The problem such as the sustainable membrane recovery of chemicals can be solved by a continuous hybrid process comprising of a flow reactor and a subsequent nanofiltration unit for in situ solvent and reagent recycle which was developed by Szekely et al. [2O , Cu2O , Cu2O , ZnO/Cu2O , ZnO/Cu2O , ZnO/Cu2O , Cu2O /ZnO, Cu2O/ZnO, and Cu2O /ZnO as the catalysts are shown in Additional file 2O, Cu2O , Cu2O , and Cu2O , is similar due to the similar morphology and the specific surface area, as shown in Figs. 2O is poorer than that of pristine ZnO nanorods due to bigger crystal boundary, lower carrier mobility, smaller specific surface area, and easier recombination of electrons and holes. The photocatalytic performance of ZnO/Cu2O , ZnO/Cu2O , and ZnO/Cu2O  is almost the same owing to the similar morphology and the specific surface area of the upper layer Cu2O, as shown in Figs. 2O is lower than that of pristine ZnO nanorods due to the smaller specific surface area of the upper layer Cu2O as a direct connect to the MO solution but is higher than that of pristine Cu2O owing to the effect of heterojunction of ZnO and Cu2O. The photocatalytic performance of Cu2O/ZnO architecture is the highest in the ZnO/Cu2O system due to their heterojunction structure and larger specific surface area of the upper layer ZnO. Compared to the sparse ZnO nanorods on Cu2O  and too much large ZnO nanorods on Cu2O , Cu2O /ZnO has the best photocatalytic performance among three Cu2O/ZnO architectures as a result of perfect ZnO nanostructures grown on Cu2O , as shown in Figs. 2O system. In summary, in the ZnO/Cu2O system, Cu2O /ZnO has the best photocatalytic performance.The photocatalytic properties are investigated via the degradation of MO, a common organic pollutant , 47. Thry et al. . Additioy et al. –51. The The concentration changes of MO with ZnO, CuSCN (3D), CuSCN (NWs), ZnO/CuSCN (3D), ZnO/CuSCN (NWs), CuSCN (3D)/ZnO, and CuSCN (NWs)/ZnO as the catalysts are shown in Additional file Additional file 2O , CuSCN (3D), NiO (1 min), Cu2O /ZnO, ZnO/CuSCN (3D), and ZnO/NiO (1 min) as the catalysts. Among the four semiconductor nanostructures ZnO, Cu2O , CuSCN (3D), and NiO (1 min), NiO has the most excellent photocatalytic performance owing to the multi-layer interspersed meshwork uniformly distributed and the consequent extremely higher specific surface area. ZnO has the bigger mobility and bigger specific surface area than Cu2O and CuSCN so that ZnO has the better photocatalytic performance. Cu2O has the better photocatalytic performance than CuSCN due to the bigger specific surface area. ZnO/NiO (1 min) heterostructure has the most excellent photocatalytic performance among all the heterostructures based on n-type ZnO and p-type Cu2O, CuSCN, and NiO. It is owing to more charge transfer caused by heterojunction structure, more photo-generated carrier as a result of higher specific surface area caused by the meshwork nanostructure of the upper NiO directly contacting to MO, and less carrier recombination caused by more compact contact of NiO/ZnO than Cu2O/ZnO and CuSCN/ZnO, as shown in Additional file 2O, CuSCN, and NiO. The photocatalytic performance of the semiconductor can be affected both by the mobility and the specific surface caused by the nanostructure. ZnO/NiO (1 min) heterostructure has the most excellent photocatalytic performance among all the architectures based on ZnO, Cu2O, CuSCN, and NiO. Influencing factor on the photocatalytic performance of all these architectures can be summarized as the inherent mobility of the material, the heterojunction architecture, and the morphology of nanostructure. The scheme of the photocatalysis mechanism using heterostructure photocatalyst is shown in Additional file Figure 2O pH 0, 20 min2O pH 0, 20 min2O pH 0, 20 min2O, CuSCN, and NiO nanostructures with different orientations are fabricated. All these structures exhibit certain photocatalytic activity for the degradation of MO. Several conclusions can be summarized with analysis of these photocatalytic data as follows: the morphology of nanostructure has significant influence on photocatalytic activity; the photocatalytic activity of heterojunction structure is better than pristine semiconductor except consideration of the influence of the nanostructure morphology; the orientation of the heterojunction has no remarkable influence on photocatalytic activity; NiO has the best photocatalytic activity among the four pristine semiconductor nanostructures ZnO, Cu2O, CuSCN, and NiO; and ZnO/NiO (1 min) heterostructure has the most excellent photocatalytic performance among all the architectures. The great enhancement of the photocatalytic activity is obtained using ZnO/NiO (1 min) heterostructure attributed to the heterojunction structure and extremely higher specific surface area. The study on the influence of materials, nanostructure morphology, and orientation in heterostructure on photocatalytic activity can provide a theoretical direction for the photocatalyst research with application in the energy and environment fields, and it can be concluded with a perspective on the future photocatalyst and a bright prospect of these controllable nanohybrid materials.In summary, different heterojunctions based on n-type ZnO nanorods and p-type CuAdditional file 1:Figure S1. The cross-sectional SEM image of ZnO nanorods (a), ZnO/Cu2O  heterojunction (b), Cu2O  (c), and Cu2O /ZnO heterojunction (d). Figure S2. The cross-sectional SEM image of ZnO/Cu2O  heterojunction (a), ZnO/Cu2O  heterojunction (b), Cu2O /ZnO heterojunction (c), Cu2O /ZnO heterojunction (d), Cu2O  (e), and Cu2O  (f). Figure S3. The cross-sectional SEM image of ZnO/CuSCN (3D) heterojunction (a), ZnO/CuSCN (NWs) heterojunction (b), CuSCN (3D)/ZnO heterojunction (c), CuSCN (NWs)/ZnO heterojunction (d), CuSCN (3D) (e), and CuSCN (NWs) (f). Figure S4. The cross-sectional SEM image of ZnO/NiO (1 min) heterojunction (a), ZnO/NiO (10 min) heterojunction (b), NiO (1 min)/ZnO heterojunction (c), NiO (10 min)/ZnO heterojunction (d), NiO (1 min) (e), and NiO (10 min) (f). Figure S5. The UV-vis absorption spectra of MO aqueous solution with different photocatalysts: (a) MO degradation in the absence of catalysts; (b) ZnO, Cu2O , Cu2O , Cu2O , ZnO/Cu2O , ZnO/Cu2O , ZnO/Cu2O , Cu2O /ZnO, Cu2O /ZnO, and Cu2O /ZnO; (c) ZnO, CuSCN (3D), CuSCN (NWs), ZnO/CuSCN (3D), ZnO/CuSCN (NWs), CuSCN (3D)/ ZnO, and CuSCN (NWs)/ZnO; (d) ZnO, NiO(1 min), NiO (10 min), ZnO/NiO (1 min), ZnO/NiO (10 min), NiO (1 min)/ZnO, and NiO(10 min)/ZnO. Figure S6. The relative concentration (Ct/C0) of MO versus time under light irradiation in the absence and presence of various photocatalysts: (a) ZnO, Cu2O , Cu2O , Cu2O , ZnO/Cu2O , ZnO/Cu2O , ZnO/Cu2O , Cu2O /ZnO, Cu2O /ZnO, and Cu2O /ZnO; (b) ZnO, CuSCN (3D), CuSCN (NWs), ZnO/CuSCN (3D), ZnO/CuSCN (NWs), CuSCN (3D)/ZnO, and CuSCN (NWs)/ZnO; (c) ZnO, NiO (1 min), NiO (10 min), ZnO/NiO (1 min), ZnO/NiO (10 min), NiO (1 min)/ZnO, and NiO (10 min)/ZnO. Figure S7. Scheme of the photocatalysis mechanism using heterostructure photocatalyst. 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Proper neuronal circuit function relies on precise dendritic projection, which is established through activity-dependent refinement during early postnatal development. Here we revealed dynamics of dendritic refinement in the mammalian brain by conducting long-term imaging of the neonatal mouse barrel cortex. By “retrospective” analyses, we identified “prospective” barrel-edge spiny stellate (SS) neurons in early neonates, which had an apical dendrite and primitive basal dendrites (BDs). These neurons retracted the apical dendrite gradually and established strong BD orientation bias through continuous “dendritic tree” turnover. A greater chance of survival was given to BD trees emerged in the barrel-center side, where thalamocortical axons (TCAs) cluster. When the spatial bias of TCA inputs to SS neurons was lost, BD tree turnover was suppressed, and most BD trees became stable and elaborated mildly. Thus, barrel-edge SS neurons could establish the characteristic BD projection pattern through differential dynamics of dendritic trees induced by spatially biased inputs. Layer 4 stellate neurons in barrel cortex have a characteristic dendritic pattern. Here, the authors conduct long-term imaging from postnatal day 3–6 to show that an orientation bias is established through dendritic tree turnover and selective elaboration, which may be induced by biased thalamocortical inputs. Dendritic projection patterns of neurons define the types of information the neuron receives and processes; thus, it is critical to understand how specific dendritic projection patterns are established during development5. However, the dynamic nature of developmental dendritic refinement, in which cortical neurons extend their dendrites toward specific sets of presynaptic axons, has remained largely unexplored.A central question in neuroscience is how cortical circuits are established during postnatal development, wherein initial primitive circuits are refined by periphery-derived neural activity7. For studies of structural plasticity in the developing mammalian brain, two-photon in vivo imaging has been used to monitor morphological dynamics of dendritic spines and filopodia of cortical pyramidal neurons9, climbing fiber axons in the cerebellum10, thalamocortical and Cajal–Retzius axons in cortical layer 111, and so on. In contrast, technical difficulties have hindered the use of in vivo time-lapse (TL) imaging in studies on the dynamics of dendritic development in mammals, which occurs during early postnatal development. Major difficulties include (1) sparse yet intense in vivo labeling of neurons, which is necessary for visualization of detailed dendritic morphology, (2) in vivo labeling of specific axons that are presynaptic to dendrites of an identified neuron, and (3) use and maintenance of fragile newborn mice during in vivo imaging sessions. For these reasons, instead of in vivo imaging, acute or chronic slice culture has been predominantly used for two-photon or confocal TL imaging of dendritic development  in the mammalian cortex12, hippocampus14, and cerebellum15. Alternatively, transparent vertebrates, such as Xenopus tadpoles17 and zebrafish larvae18, have been used for in vivo TL imaging of dendritic development in the brain and/or retina.Two-photon microscopy has been widely applied for in vivo imaging of structural plasticity in adult neuronal circuits, such as spine formation/elimination associated with learning and memory22. This area contains “barrels”, which are morphological and functional modules arranged to correspond with facial whiskers23  spiny stellate (SS) neurons in the mouse primary somatosensory cortex (barrel cortex) as a model30, and (2) TCA-GFP transgenic (Tg) mice, which allows in vivo labeling of TCAs30. These innovations have allowed us to conduct short-term (18-h) in vivo imaging of SS neuron BDs starting at postnatal day 5 (P5)30. This study provided the first in vivo observation of dendritic dynamics in the neonatal mammalian brain, in which small-scale dynamics  were analyzed. However, this study was far from sufficient, because dendritic refinement was already nearly completed at P530. Therefore, to fully understand dynamic mechanisms of BD refinement of SS neurons, long-term (over days) imaging starting at earlier neonatal stages was awaited.As a first step toward in vivo imaging of dendritic refinement in the mammalian brain, we recently developed two methods: (1) the “Supernova” system, which allows sparse yet bright in vivo labeling of cortical L4 neurons when used in combination with in utero electroporation-based gene transfectionIn the current study, we have succeeded in vivo visualization of SS neuron dendrites in mouse pups as early as P3. We also developed a system by which neonatal mice as early as P3 grow up with adequate maternal care during imaging sessions over days. Based on these preparations, we here achieved long-term (3-d-long) imaging of the SS neuron dendrites starting at P3 and characterized large-scale dendritic dynamics . Our long-term imaging in neonatal mice revealed the selection dynamics by which only a fraction of inner BD trees become “winners”, exhibiting high stability and extensive elaboration. Also, we revealed the role of spatial patterns of periphery-derived inputs in the selection dynamics of BD refinement.31. Our system can use information obtained at later developmental stages  to identify prospective SS neurons at earlier stages, which enabled us for the first time to characterize features of SS neurons during early neonatal stages. Our retrospective analyses also revealed the presence of two phases in eSS neuron BD refinement during neonatal stages. Thus, our novel in vivo imaging system contributes to our understanding of developmental mechanisms of cortical maturation in neonates.In addition, it is also important that our long-term imaging of the same neurons over time allowed for “retrospective” identification of prospective SS neurons during early neonatal stages, such as P3. In these stages, most SS neurons in mammals show similar morphological features as another type of L4 excitatory neurons, star pyramid (SP)30, and eSS neurons acquire their characteristic BDs, which are asymmetrically extended within single barrels, primarily by P632. Therefore, in the present study, to characterize the detailed time course of L4 SS neuron BD refinement, we sought to perform long-term in vivo TL imaging starting at P3 or earlier and ending at P6  were neglected, killed, and/or injured by mothers within a day after surgery. To solve this problem, we first designed an extremely small titanium bar to help minimize discomfort of mothers during breastfeeding  to late P5 (P5L) and at late P6 (P6L) . Because L4 neurons were sparsely labeled and the relative positions of neurons were roughly conserved, it was easy to identify the same neurons in images taken at different time points (imaging sessions)  Fig. . In a fens) Fig. . All neuL to P6L, as did control pups, and there was no significant difference between TL and control pups even at P6L  had no AD. Analysis of daily changes in AD length by comparing images of the same L4 neurons taken at P3L, P4L, P5L, and P6L  allowed us to classify individual L4 neurons into two groups (Groups 1 and 2). Neurons which shortened AD during imaging sessions were classified as Group 1  Fig. . We thenigh Fig. , indicatigh Fig. , indicat36. As described above, 76% of L4 neurons were Group 1 and 24% were Group 2. Group 1 neurons possessed no (or short) AD and their BDs exhibited strong orientation bias at P6L, while Group 2 neurons possessed a long AD and their BDs exhibited no orientation bias. These characteristics of Group 1 and Group 2 neurons were consistent with those of SS and SP neurons, respectively. Therefore, hereafter, we refer to Group 1 neurons as SS neurons and Group 2 neurons as SP neurons, although we could not exclude the possibility that Group 2 may contain a few SS neurons whose maturation was slower than others.In the mature mouse barrel cortex, the majority (65–80%) of L4 neurons are SS neurons with no AD and multiple BDs projecting specifically within a single barrel. The other 20–35% are SP neurons with an AD and multiple BDs showing no orientation bias31. This was confirmed in the current study in the mouse barrel cortex. We also found that prospective SS neurons that exhibit a pyramidal shape lose their AD during development, providing direct evidence for developmental sculpting of SS neuron AD, as proposed by Callaway and Borrell31. In addition, we report three new findings. First, the AD was lost from SS neurons by gradual retraction  by total BD length . In this analysis, only neurons located at the barrel edge at P6L (eSS and barrel-edge SP (eSP) neurons) were included.To elucidate whether SS and SP neurons exhibit morphological differences in early neonatal stages, it is useful to compare the BD orientation bias of SS and SP neurons at P3ent Fig.  and possL . We then repeated OBI quantification using only eSS neurons that had an intact AD at P3L (see Methods for details). Again, the OBI of these neurons was significantly larger than that of eSP neurons at P3L  during the first postnatal week Fig. . By P3  as we did in our previous workL and P5L over 8-h intervals , 82% (18/22) disappeared by the next imaging session (one time frame), and only a small portion of outer trees survived longer than one imaging interval , and eliminated trees were those that disappeared during imaging sessions. The lengths of surviving inner trees increased in proportion to survival time frames  Fig. . The cSSL  , and shorter and longer trees were rare at both ages  growth were similar between eSS and cSS neurons, in which the data of inner and outer BD trees from eSS neurons were pooled  Fig. , while aµm) Fig. . Insteadµm) Fig. . On the ges Fig. , j. It iL and P5L  morphology of SS neurons in ION-cut mice (iSS neurons) was similar to that of eSS and cSS neurons  Fig. . At P3L,ons Fig. . At P6L,ons Fig. . These r39. Importantly, this activity is not affected by ION cut at P4 or P539.We recently report unique features of spontaneous activity in neonatal barrel cortex L4, which shows a “patchwork”-type pattern corresponding to the barrel map. This patchwork activity is delivered to L4 via TCAs39 and conspecifics receiving ION cut at P0 (Early-ION-cut mice)  Fig. . In contce) Fig. . In addice) Fig. . To furtce) Fig. . Color-cce) Fig.  clearly ce) Fig. . In contce) Fig. . The frece) Fig. , althougIn normal mice, SS neurons located at the barrel edge  receive spatially biased TCA inputs only from one direction . On the contrary, like cSS neurons in normal mice, SS neurons of ION-cut mice  should not have any specific direction for proper TCA inputs. It is likely that this disruption of polarized TCA inputs results in altered dynamics of iSS neurons.To our knowledge, the present study is the first to report long-term (days) in vivo imaging analysis of dendritic dynamics in developing mammals. Herein, we have successfully accomplished in vivo imaging of the mouse barrel cortex starting at P3 and ending at P6. We revealed dynamics of SS neuron BD trees associated with circuit refinement in the neonatal barrel cortex, as well as possible involvement of spatial patterns of periphery-derived inputs in these dendritic tree dynamics as summarized in Fig. 40 but not the spontaneous activity, which is most likely derived from the trigeminal ganglion or further downstream42. Instead, the patchwork-type pattern39 of spontaneous activity is disrupted by ION-cut in early neonates (such as at P0)  Fig. . In the P0) Fig. . In both30. The Rim1/Rim2 double knockout mice, which lack thalamocortical synaptic transmission, also show overtly normal TCA clustering but impaired BD refinement28. These results clearly demonstrate that the presence of physical TCA clusters are not sufficient and synaptic inputs from TCAs to SS neurons are necessary for BD refinement.It appears that spatially biased TCA inputs to SS neurons facilitate cell-wide BD tree turnover, because turnover rate is higher in eSS neurons than in cSS and iSS neurons. Another scenario is that biased distributions of physical structures of TCA clusters and/or unidentified molecules derived from TCAs could induce high levels of BD dynamics in eSS neurons. Although we cannot exclude these possibilities, previous studies oppose them. For example, mice in which the gene encoding the NMDA receptor NR1 subunit is knocked out in single SS neurons showed normal TCA clusters but impaired BD refinement18. Our previous short-term (hours) in vivo imaging of mouse barrel cortex eSS neurons also revealed an important role for NMDA receptors in stabilization of BD branches30. It is likely that postsynaptic signaling induced by NMDA and/or AMPA receptors stabilizes those BD branches that make synaptic contact with appropriate TCAs, which in turn stabilizes the tree .Previous in vivo imaging studies using the tectum of Xenopus tadpole or zebrafish larvae suggested that dendritic branches forming synapses with axons are stabilized and elaborated, and that glutamate receptor (NMDA and AMPA receptor)-mediated synaptic transmission is important for this stabilization processSS neurons may have homeostatic mechanisms to maintain BD tree numbers, because these numbers did not change substantially between P3 and P6 Fig. . If so, 31. Then by P3, L4 eSS neurons create nascent orientation bias by forming more BD trees inside than outside. At this stage, both inner and outer BDs are similarly primitive. It is possible that TCAs secrete molecules that produce more BD trees on the side of the barrel center, where TCA termini form a cluster by P3, although neural activity transmitted through TCAs could also be involved. In contrast, Phase II starts approximately at P3 and ends at P6 or later. In this phase, the ratio of inner to total BD trees does not change . In the mammalian brain, developmental dendritic refinement is found not only in barrel cortex SS neurons, but also in various types of neurons in other brain areas, such as SS neurons in L4 of the visual cortex 47, Purkinje cells in the cerebellum48, and mitral cells in the olfactory bulb49. Long-term in vivo imaging will be a powerful method for uncovering dynamic mechanisms of dendritic refinement in these neurons and the cellular and molecular mechanisms regulating these dynamics.Gene knockout and knockdown approaches have identified many molecules involved in SS neuron BD refinement, including the NMDA receptor, metabotropic glutamate receptor 5, adenylyl cyclase 1, protein kinase A, BTBD3, and Lhx230 which were backcrossed from B6 to ICR more than four times or male TCA-GCaMP Tg mice39 which were backcrossed from B6 to ICR more than one time. The day at which the vaginal plug was detected was designated as embryonic day 0 (E0) and E19 was defined as postnatal day 0 (P0). For histological analysis of ION-cut mice  and were approved by the animal experimentation committee of the NIG. To obtain pups, ICR female mice were mated with male TCA-GFP Tg mice50. Pregnant mice at 14 postcoitus days were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg) in saline. Isoflurane was used to control anesthesia level. A midline laparotomy was performed to expose the uterus. DNA solution mixed with trypan blue  was injected into the right lateral ventricle of embryos via a pulled glass capillary (Drummond), and square electric pulses  were delivered five times at the rate of one pulse per second by a CUY21SC electroporator (NepaGene). After electroporation, the uterus was repositioned, and the abdominal wall and skin were sutured. After surgery, pregnant mice were kept on a 37 °C heater until they recovered from anesthesia.In utero electroporation was conducted as described38 was performed as follows  and small (7 × 2 × 0.5 mm) (T and I) and used the round cover glass whose diameter is 2.5 mm (Matsunami). In the morning of P2 or P3, mice were anesthetized with isoflurane. Skin covering the right hemisphere was removed using scissors to expose skull followed by applying of Vetbond (3 M) to fix the margin and to stop bleeding. Barrel area was detected by TCA-GFP signal and a small piece of bone covering the Supernova-labeled barrel area was removed with a sterilized razor blade, keeping the dura intact. Gelfoam (Pfizer) was used to stop bleeding, as necessity. To keep the brain moist, cortex buffer51  was applied during surgery. After that, the window was covered with 1% low melting point agarose (Sigma) in cortex buffer and 2.5 mm diameter round cover glass. The custom-made titanium bar was glued to the skull near the window to attach mouse to two-photon microscope stage  that secured with dental cement. A titanium bar30 (~30 mg) was attached to the area adjacent to the cranial window. After surgery, pups were kept on a heater for recovery.Craniotomy for in vivo calcium imaging was performed as described30 pups, in which L4 neurons were sparsely labeled by in utero electroporation-based Supernova-RFP29 [pK036.TRE-flpe-WPRE (10–15 ng/µl) and pK037.CAG-FRT-STOP-FRT-RFP-ires-tTA-WPRE (1000 ng/µl)], were anesthetized with 0.8–1.2% isoflurane and fixed to the microscope stage using the titanium bar. Heating pad was used to keep pups warm. Images were acquired using an LSM 7MP two-photon microscope (Zeiss) with a W Plan-Apochromat 20×/1.0 DIC objective lens (Zeiss) and an LSM BiG detector (Zeiss). HighQ-2 laser (Spectra-Physics) at 1045 nm was used in most experiments. GFP and RFP were simultaneously excited and emitted fluorescence was filtered (500−550 nm for GFP and 575−620 nm for RFP). In an experiment ; at P2L, P3E, P3M, P3L, P4E, P4M, P4L, P5L, and P6L ; at P3L, P4L, P5L, and P6L (#205); at P3L, P4E, P4M, P4L, P5L, and P6L (#227); at P2L, P3E, P3M, P3L, P4E, P4M, P4L, P5E, P5M, and P5L (#313); at P2L, P3E, P4L, P5L, and P6L (#314). PXE, PXM, and PXM indicate around 4 a.m., noon, and 8 p.m. at postnatal day X (PX) as shown in Fig. Images were taken at P3L imaging session. Mice were decapitated, and brains were fixed with 4% paraformaldehyde (PFA) in 0.1 M PB at 4 °C for 1–3 days. For tangential sectioning, right hemispheres were flattened and transferred to 2% PFA/30% sucrose in 0.1 M PB. Flattened cortex was kept at 4 °C for 1–2 days and tangential slices (100 µm thick) were obtained with a ROM-380 freezing microtome (YAMATO). Slices were mounted with Anti-fade Mounting Medium52. Images were acquired by a TCS SP5 confocal microscope (Leica). The layout of the barrel map was identified by TCA-GFP signals.Histological analyses after the end of in vivo imaging confirmed that all analyzed neurons of both normal and ION-cut mice were located within the large-barrel field of the primary somatosensory cortex  was used to reduce the noise from respiratory movements. For tracing and quantification, Imaris Filament Tracer 7.0 and 8.3 (Bitplane) were used. Only neurons of which all BD and AD terminals were clearly visible were used for BD and AD analyses, respectively. Dendrite traces were generated semi-automatically and validated manually. Any dendritic processes greater than 5 µm in length was designated as a dendritic segment. Dendritic trees of which origins were in the same position between time-sequential images were considered as the same tree. AD and BDs were distinguished by their shape at the initial imaging session. Dendrites that had the same orientation as neighboring neuron ADs were judged as ADs. At early neonatal stages, neurons usually had single long thick AD toward pial surface. Axon was distinguished from BDs by the following features: (1) Axon was thinner than BD. 2) Axon emerged from the bottom of the soma. (3) Axon projected toward deep brain regions. All imaged neurons were categorized either Group 1 (SS) or Group 2 (SP) neurons as follows: Group 1 neuron was the neuron that shortened AD during imaging sessions or the neuron which had no AD at P3 Axon emeL Fig.  was GrouBecause mice were detached from the two-photon microscope stage after each imaging session, orientations of acquired images were slightly different among imaging sessions. Prior to dendritic orientation analyses, these orientation artifacts among images were adjusted as follows: for each Z-stack two-photon image, neuronal coordinates were measured by Fiji/ImageJ as centroids of binary images. The central point of each image was determined by the centroid of neuronal coordinates. The central points of images were laid over and images were rotated around the central point. Angle difference between two images was calculated to minimize the error (determined by the least squares method) between coordinates of same neurons in two images. The orientation artifact was corrected by applying this angle difference to the image . The barrel edge was determined by the contrasting difference of TCA signal intensities in the area lying between barrel center and septa. Neurons whose cell-body center was located within 12.5 µm from the barrel edge were classified as barrel-edge neurons. Other neurons located within the barrel were classified as barrel-center neurons. The simple version of OBI, which was used in the current study, was defined as the ratio of BD segment length in the barrel-side half to the total BD length. Inside–Outside boundary, which separates barrel-side half and the other half, was determined as shown in Supplementary Fig. The layout of the barrel map was identified by TCA-GFP signals of in vivo images and/or tangential section images taken at P639 at P5 or P6 under an unanesthetized condition. In these mice, L4 neurons were sparsely labeled by in utero electroporation-based Supernova-nlsRFP [pK031.TRE-Cre30 (10 ng/µl) and pK263.CAG-loxP-STOP-loxP-nlsRFP-ires-tTA-WPRE39 (1000 ng/µl)] as markers of the in vivo imaged areas. Images were acquired at 1 Hz (512 × 512 pixels) using an LSM 7MP two-photon microscope (Zeiss) with a W Plan-Apochromat 20×/1.0 DIC objective lens (Zeiss) and an LSM BiG detector (Zeiss). Mai Tai eHP DeepSee titanium–sapphire laser at 940 nm was used. Emitted fluorescence was filtered 500−550 nm for GCaMP6s and 575−620 nm for nlsRFP. During imaging, the body temperature of the pup was maintained using a heating pad. When the pup head moved during imaging, these frames were excluded from the analyses.In vivo calcium imaging was performed for the large-barrel field of the primary somatosensory cortex L4 of TCA-GCaMP pupsF/F = (F−F0)/F0 was calculated in each pixel at each time. F0 of each pixel was obtained by averaging more than 50 images in which calcium transients were obviously absent such as under anesthesia. ΔF/F >100% were considered as activated pixels. With these criteria, boundaries of activated zones were well matched with the barrel edges in control mice. The zones that were less than 2500 µm2 were excluded as noise.The boundaries of the activated zones Fig. b−f were F of ROI was obtained by averaging intensities of pixels inside the ROI. F0 of each ROI was obtained by averaging more than 50 images in which calcium transients were obviously absent. For the raster plots, the threshold ΔF/F > 50% was used, because with this condition calcium transients in the control mice were most accurately detected. With the threshold ΔF/F > 100%, although the barrel boundaries in control mice were most sharply visible, some apparent calcium transients  were failed to be detected . Supplementary Movies F/F > 50%. Correlation matrices were sorted by principal component analysis with a few exceptions  in 0.1 M PB. Rabbit anti-VGluT2  and Alexa 488-conjugated goat anti-rabbit IgG  antibodies were used. Although, in the ION-cut mouse cortex, barrel map is impaired, still identification of the large-barrel field was possible.29 [pK036.TRE-flpe-WPRE (10–15 ng/µl) and pK037.CAG-FRT-STOP-FRT-RFP-ires-tTA-WPRE (1000 ng/µl)]. P16 mice in which IONs were cut at P0 (or uncut as control) were perfused by saline and 4% PFA in 0.1 M PB before decapitated. Brains were fixed with 4% PFA in 0.1 M PB at 4 °C for 3 days. Then, brains were transferred to 30% sucrose in 0.1 M PB and kept at 4 °C for 1–2 days. Coronal slices (100 µm thick for most experiments and 50 µm thick for cytochrome oxidase (CO) staining) were obtained with a ROM-380 freezing microtome. DAPI staining  was used to determine L4 barrel field and to confirm whether IONs were cut properly. After DAPI staining, slices were mounted with Anti-fade Mounting Medium47, and fluorescent images were acquired by a TCS SP5 confocal microscope. CO staining was also used to confirm whether IONs were cut properly. Coronal sections were incubated with CO stain solution (0.05% Cytochrome C (Sigma)/0.08% 3–3′-diaminobenzidine tetrahydrochloride /30% sucrose in 0.1 M PB) for 4 h at 37 °C. After visual detection of stain, sections were washed three times with 0.1 M PB and mounted with EUKITT (Kindler).L4 neurons were labeled by in utero electroporation-based Supernovap < 0.05, **p < 0.01, and ***p < 0.001. p < 0.05 was considered statistically significant. g and r indicate the effect size for parametric and for non-parametric tests, respectively55. Error bars in bar graphs and line graphs represent SEM. In box plots, upper and lower limits of box represent 75th and 25th percentile, crosses represent mean, horizontal lines represent median, upper and lower whiskers represent maximum and minimum within 1.5 interquartile range, and observations beyond the whisker range were marked with open circles as outliers. Brunner–Munzel test was performed by R 3.2.5 and its additional package lawstat 3.0. All other analyses and visualizations were performed using Fiji/ImageJ 1.51p53 and custom-written scripts in Python 3.5.2 with its additional packages Numpy 1.11.3, Scipy 0.18.1, Matplotlib 1.5.1, Pandas 0.19.2, Lifelines 0.9.3.2, PIL 4.2.1, OpenCV 3.3.1, Scikit-learn 0.19.1, Glob 0.6, and their later versions. Blinding was not performed. Pre-determination of sample sizes by statistical methods were not performed. At least two mice were analyzed per condition in each experiment. Exact sample sizes were provided in figure legends or following paragraphs in this section. See also Supplementary Table Two-tailed parametric and non-parametric tests were used to show the differences among means and medians, respectively (see figure legends). The asterisks in the figures indicate the following: *n = 35, 39, 38, and 33 neurons from 7, 8, 8, and 6 mice for P3, P4, P5, P6, respectively); Group 2 (SP) . Figure n = 30, 38, 36, and 29 from 7, 8, 8, and 6 mice for P3, P4, P5, P6, respectively); Group 2 (SP) .Figure n = 28, 12, 36, and 18 BDs for P3L inside, P3L outside, P6L inside, and P6L outside, respectively. Data were collected from eight eSS neurons of four mice.Figure n = 2 mice) were analyzed every 8 h between P3L and P5L, and the other four neurons (n = 2 mice) were analyzed between P4E and P5L because at P3L cell morphology was not very clear due to clouding of the window. Figure n = 9, 9, 7, 6, 4, and 1 trees for 1, 2, 3, 4, 5, and 6 surviving time frames, respectively); Surviving outer BD trees ; Eliminated inner BD trees ; Eliminated outer BD trees .Figure n = 15 eSS neurons from seven mice, 15 cSS neurons from five mice, and 7 iSS neurons from two mice at P3L. n = 13 eSS neurons from five mice, 13 cSS neurons from four mice, and 9 iSS neurons from three mice at P6L. Supplementary Fig. n = 8 eSS neurons from four mice, 7 cSS neurons from two mice, and 7 iSS neurons from two mice at P3L. n = 8 eSS neurons from four mice, 7 cSS neurons from two mice, 9 iSS neurons from three mice at P6L.Supplementary Fig. L analyses because the mouse died during the imaging session at P5L by an anesthesia problem. One neuron (neuron ID #314-1) was excluded from P3L analyses because the P3L imaging of the mouse was skipped.Sample sizes were often different among time points for following reasons. Some neurons at some time points were excluded from the analyses because cranial windows were cloudy, and terminals of AD and/or BDs were not clearly visible. One neuron (neuron ID #231-9) was excluded from AD length analysis because its AD terminals were out of imaging range. Two neurons (neuron ID #313-1 and 2) were excluded from P6All data that support the conclusions in the study and computational code used in the study are available from the authors on reasonable request.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2