O-sulfotransferases 1 and 2 in vertebrates. These two enzymes showed about 50% amino acid sequence identity, and appeared to be complementary to each other in acceptor substrate preferences. Gene targeting experiments showed that the deficiency of these genes was lethal for medaka fish during young fry development and accompanied by different phenotypes. Thus, the sulfation of sialic acids is essential for the vertebrate development.Glycosylation of proteins and lipids occurs in vertebrates, usually terminating with sialylation, which regulates the physicochemical and biological properties of these glycoconjugates. Although less commonly known, sialic acid residues also undergo various modifications, such as acetylation, methylation, and sulfation. However, except for acetylation, the enzymes or functions of the other modification processes are unknown. To the best of our knowledge, this study is the first to demonstrate the ubiquitous occurrence of sulfated sialic acids and two genes encoding the sialate:  Sias have been demonstrated to play an essential role for embryo survival in vertebrates, because loss of critical enzymes in the Sia metabolic pathway leads to the embryonic death. Mice deficient in the UDP-N-acetylglucosamine (GlcNAc) epimerase/N-acetylmannosamine (ManNAc) kinase (GNE) gene or the CMP-sialic acid synthetase (CMAS) gene are lethal during early development5. Medaka fish with a point-mutated CMAS gene are also lethal in young fry6. Sias mediate and regulate various cellular recognition and signaling events2. For example, sialylation of podocalyxin in the mouse kidney is essential for the formation of glomerular filtration7. Polysialylation of the neural cell adhesion molecule (NCAM) in the mouse embryonic brain not only negatively regulates the NCAM homophilic binding9, but also retain neurotrophins, fibroblast growth factor 2, and dopamine to control their signal transductions13.Sialic acids (Sias) are a group of nine-carbon carboxylated sugars that modify the termini of glycans of proteins and lipids on the cell surface and the extracellular matrix14. However, the metabolism and biological significance of the modified forms of Sia is unclear, except for O-acetylated Sia (SiaAc)15. O-Acetylation of Sia occurs in glycans in proteins and lipids of a wide range of organisms from bacteria to vertebrates16. SiaAc is involved in murine development at the 2-cell stage17, regulation of apoptosis19 and immune recognition24. SiaAc-containing gangliosides are prominently expressed in various cancer, and can be a target for cancer immunotherapy25. The cell surface SiaAcs are also utilized as targets for bacterial and viral infection28. Thus, a myriad of information on occurrence and functions of SiaAc have been accumulated. On the other hand, a single mammalian sialic acid O-acetyltransferase (SOAT), or CASD1  has been so far identified as a biosynthetic enzyme30.One of unique features of Sia absent from other monosaccharides is that Sia displays a marked structural diversity owing to modifications such as acetylation, methylation, and sulfationO-Sulfation of Sia has not been studied well, compared with the Sia O-acetylation. Sulfated Sia 46. SiaS was detected in all the examined tissue sections from mice and humans . Based on the two conserved motifs of the 3′-phosphoadenosine-5′-phosphosulfate (PAPS)-binding domain among sulfotransferases, 61 genes that were already annotated as sulfotransferases in mice were selected  and Wscd2 . Wscd1 and Wscd2 full-length cDNA was cloned by RT-PCR using total RNA from the E14.5 mouse embryonic brain  cells expressing only Wscd1 -Neu5Ac, ganglioside GM1, and transferrin (TF). For Neu5Ac and CMP-Neu5Ac, no SiaS derivative was detected by the fluorometric HPLC analysis of the reaction mixture with Wscd1 or Wscd2 (data not shown). The findings indicated that sulfation of free Sia or donor substrate of sialyltransferases did not occur.To test the in vitro sulfotransferase activity of mWscd1 and mWscd2, the recombinant enzyme fractions prepared from authentic) was detected in Wscd1 , but not in the Mock fractions . The co-injection experiment with authentic Neu5Ac8S also confirmed that the peak was Neu5Ac8S . These results indicate that mWscd1 shows SulT-Sia activity on Neu5Ac residue on GM1, while mWscd2 has no activity against GM1.For the glycolipid substrate, GM1 was incubated with the recombinant mWscd1, mWscd2, and Mock-derived enzyme fraction (Mock), and the reaction products were analyzed by thin-layer chromatography (TLC) Fig. a. The baN-glycan chains terminated with α2,6-Neu5Ac residues, was incubated with mWscd1, mWscd2, and Mock, and subjected to western blotting with 3G9 ; however, only when incubated with mWscd2 , but not mWscd1  or Mock , the 3G9 epitope intensity was greatly increased in the TF band at 78 kDa. The amount of TF analyzed in each lane was the same based on the Coomassie Brilliant Blue (CBB) staining intensity of the same gel . In addition, this intensity increase did not occur in the absence of PAPS  or TF . These results indicate that Wscd2 shows SulT-Sia activity on TF, whereas Wscd1 has no effect on TF. The reaction product of Wscd2 was also analyzed to determine the increase in SiaS by fluorometric HPLC  but not in Mock . The co-injection experiment also confirmed that the peak was Neu5Ac8S . Thus, Wscd2 shows SulT-Sia activity on the Neu5Ac residue of the TF N-glycan.The TF glycoprotein substrate, containing two Oryzias latipes, as a vertebrate model. Medaka has a single copy of each Wscd1 and Wscd2 orthologs genes. The Wscd1 and Wscd2 cDNAs were cloned from the fry at 6 days post-fertilization (dpf) and 7 dpf, respectively. Their deduced amino acid sequences showed 72% and 75% identity with those from mice, respectively , had a 19-bp deletion in exon 2 , had a 1-bp insertion/32-bp deletion in exon 1  and Wscd2(+/−)] were established, and subcultured. The survival rate in the crossed offspring was investigated. Wscd1(−/−) fry showed lethality at 10 ~ 33 dpf with a dpf value of 50% death (D50) of 19.5 dpf  fry showed lethality at 9 ~ 60 dpf with a D50 of 18 dpf  fry lived a little longer than the Wscd1(−/−) fry, and 10% of Wscd2(−/−) fry could live until 60 dpf.We generated Wscd1- and Wscd2-deficient medaka using the CRISPR-Cas9 system. The  Supp_Figa and the Supp_Figb. Since dpf Fig. a. Wscd2(48 was observed in Wscd1(−/−) fry at 8 dpf and beyond . Consistent with this result, the western blotting with an anti-myosin heavy chain (MHC) antibody (MF20)49 showed that the amount of cardiac MHC was significantly reduced in Wscd1(−/−) fry compared to the WT and Wscd1(+/−) fry . In contrast, Wscd2(−/−) fry did not develop cardiac arrhythmia  fry  fry died by 18 dpf, 8% lived as long as 60 dpf. Notably, Wscd2(−/−) fry at 50 dpf were smaller than the WT by 49% and 17% in weight and length, respectively  fry did not  fry, partly due to impaired muscle, eyes and brain development.Although 50% of the  Supp_Fig. These oWscd1(+/−) and Wscd2(+/−) showed high lethality rates  fry compared to the WT or Wscd1(+/+) fry and was even higher in Wscd1(−/−) fry . The same or even more prominent results were obtained for Wscd2(+/−) and Wscd2(−/−) fry . Since CRP increases in response to inflammation51, the homozygous and heterozygous fry of both types are suggested to be in an inflammatory state, which is more severe in the homozygous than heterozygous fry. The findings further suggest that the loss of Wscd1 or Wscd2 increases the severity of inflammation in the fry, probably through the dysfunction of various tissues during the growth stages. Further studies are necessary to understand the linkage between the deficiency of these SulT-Sias and the inflamed state. The collective findings indicate the critical roles of Wscd1 and Wscd2 in the survival of medaka.Interestingly, even heterozygous fry of  Supp_Fig. The CRPO-sulfotransferases, Wscd1 and Wscd2, responsible for the sulfation of Sia residues on glycoproteins and glycolipids are constitutively expressed during embryogenesis and in various adult organs. Wscd1 and Wscd2 are structurally and phylogenetically close to each other, and this gene pair widely distributes in the deuterostomes from echinoderms to vertebrates. Interestingly, mWscd1 and mWscd2 may have complementary substrate preferences to each other, because mWscd1, but not Wscd2, is active for glycolipid GM1, while mWscd2, but not mWscd1, for glycoprotein transferrin. This feature might explain constitutive co-expression profiles of these two genes in embryos and adult organs of medaka. Both enzymes are Golgi-localized, type II transmembrane proteins with a short N-terminal cytosolic tail and a C-terminal luminal catalytic domain, and share the common structural and topological features with sialyltransferases53. Thus, sequential reactions of sialylation and sulfation may effectively proceed in the Golgi compartment.In conclusion, SiaS occurs in various cells and tissues in vertebrates including fish and mammals, and the sialate: O-acetyltransferase CASD130. CASD1 converts CMP-Sia into CMP-Sia9Ac using acetyl-coenzyme A as a donor substrate30. Although both Sia modification enzymes are localized in the Golgi, Wscd1 and Wscd2 transfer sulfate group on sialoglycans on proteins and lipids, but not CMP-Sia, using PAPS as a donor substrate , but not Wscd2(−/−), fry at 8 dpf suffer from cardiac arrhythmia48, suggesting that Wscd1 is essential for heart development. Wscd2(−/−), but not Wscd1(−/−), fry show growth retardation, accompanying impaired muscle, eyes, and brain development. Wscd2 might be involved in survival of cells in muscle, eye, and brain. Furthermore, the loss of Wscd1 or Wscd2 increases the severity of inflammation in the fry, which may be related to the lethality of fry. Although further studies are necessary to gain in-depth insight into pathophysiology of the lethality, the Sia: O-sulfation must have multiple functions such as embryonic development, organogenesis, and immune recognition. Finally, the present data emphasize the importance of modified Sias that have been uncovered for a long time.Examination of gene knockout medaka revealed that SiaS is essential at the organism level. 54. Since CHO cells did not express Wscd1 or Wscd2 before and after the G418 treatment (unpublished results), the presence of other SulT-Sia than Wscd1 and Wscd2 is suggested in CHO cells. Thus, more sulfotransferases with the SulT-Sia activity might occur in mammalian cells. We can also seek for substrate specificity of the SulT-Sias in detail. This report shows that Wscd1 and Wscd2 can synthesize Neu5Ac8S-residues from Neu5Ac-residues; however, Sia species-specificity of the enzymes to Neu5Ac, Neu5Gc, and Kdn, which must be dependent on organism-species, and the linkage-specificity of sulfation not only to 8-O-position, but also to 4-O-, 7-O-, and 9-O-positions of Sia remains to be investigated. Last but not least, we should pursue a regulatory feature of SiaS expression through O-sulfation and de-O-sulfation like that of SiaAc expression through O-acetylation and de-O-acetylation23. To the best of our knowledge, no de-O-sulfating activity of SiaS has been identified so far, and this important aspect still remain an open question.Now that two different genes for SulT-Sia are evident, many questions would immediately emerge in various aspects of biochemistry and biology of SiaS. We can ask if there are still other genes for SulT-Sia. Recently, we reported that the surface expression of SiaS reversibly induced by treatment of CHO cells with the antibiotic G41855, and Keith Joung (Addgene 42250)56, respectively. Restriction enzymes, Ex Taq polymerase, and an In-Fusion HD Cloning Kit were purchased from TaKaRa Bio Inc. . KOD SYBR qPCR mix (QKD-201), KOD-Plus-Neo polymerase and Bgl II were purchased from TOYOBO . Polyvinylidene difluoride (PVDF) membranes and enhanced chemiluminescence (ECL) reagents were purchased from . Molecular weight markers and bicinchoninic acid (BCA) assay kits were purchased from Bio-Rad . PEI-Max Transfection Reagent was a product of Polysciences Inc. . Anti-β-actin antibody was purchased from Santa Cruz Biotechnology . Peroxidase-conjugated goat anti-mouse (IgG + IgM) was purchased from American Qualex . The monoclonal anti-myosin 4 antibody (MF20), Alexa Fluor 488-labeled goat anti-mouse IgM, and Alexa Fluor 488-labeled goat anti-mouse (IgG + IgM) antibodies were obtained from Thermo Fisher Scientific KK. Alexa Fluor 488-conjugated anti-chicken IgY, anti-GM130, and anti-KDEL antibodies were products of Abcam . Alexa Fluor 555-conjugated anti-rabbit IgG was purchased from Invitrogen . Chicken anti-V5 antibody was purchased from Bethyl laboratories . 4-Methylumbelliferyl ⍺-glycoside of 8-O-sulfated Neu5Ac (4MU-Neu5Ac8S) was prepared as previously described45. The monoclonal IgM antibody 3G9 (3G9), which specifically recognizes the α-glycosides of Neu5Ac8S, was previously prepared using sea urchin sperm as an immunogen46. mAb.2G9 (10 μg/mL) was obtained as an IgM clone, which did not react with Neu5Ac8S-containing gangliosides, during the 3G9 preparation and was used as the isotype control. Unless otherwise stated, primers for PCR amplification were purchased from RIKAKEN .N-acetylneuraminic acid (Neu5Ac), cytidine-5′-monophosphateNeu5Ac (CMP-Neu5Ac), bovine serum albumin (BSA), and trifluoroacetic acid (TFA) were purchased from Nacalai Tesque . Ganglioside GM1  Galβ1 → 4Glcβ1-Cer) was purchased from AdipoGen . 1,2-Diamino-4,5-methylenedioxy-benzene (DMB) was purchased from Dojindo Molecular Technologies, Inc. . 3′-Phosphoadenosine 5’-phosphosulfate (PAPS) and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich . Transferrin (TF) from humans was purchased from Wako . TRI REAGENT LS was a product of Molecular Research Center, Inc. . pGEM-T Easy plasmid was purchased from Promega . The pcDNA3.1(neo) plasmid, pcDNA4-V5/His, and the mMessage mMachine SP6 transcription kits were purchased from Thermo Fisher Scientific KK . pSUPER.neo vector was a product of Oligoengine . AmpliScribe T7-Flash Transcription kit was a product of Lucigen . ProtoScript II and BsaI were products of New England Biolabs . The Cas9 expression vector with SP6 promoter, pCS2 + hSpCas9, and the sgRNA expression vector with a T7 promoter, pDR274, were gifts from Masato Kinoshita (Addgene 51815)All procedures for the use of animals were approved by the Animal Care and Use Committee of Nagoya University , and performed under the relevant Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and regulations, which are set by the same committee. All methods were performed in accordance with the relevant guidelines and regulations. Every effort was made to minimize the number of animals used and their suffering.Oryzias latipes, was used as the wild type (WT). Wild type medaka strain was supplied by the National Bioresource Project (NBRP) Medaka. Fish stocks were maintained in 16-L tanks with a circulating water system maintained at 26 °C and a 14 h light/10 h dark cycle. The development and phenotype of medaka fish were observed using an SZX12 DP80 microscope .Mice (C57/BL6J) were obtained from Japan SLC  and brain samples were prepared at different stages: embryo 14.5 days (E14.5), postnatal day 1 (P1), P15, P30, and P45. The Nagoya strain of medaka fish, 2O2 for 6 min. After washed three times, they were blocked with 2% BSA in 10 mM sodium phosphate buffer, pH 7.2, 0.15 M NaCl (PBS) for 30 min, and incubated with 1 µg/mL 3G9 at 4 °C for overnight. After washed, they were incubated with Alexa Fluor 488-labeled goat anti-mouse IgM (1:500 dilution) for 30 min. Nucleus was stained with 1 µg/mL DAPI for 15 min. The immunofluorescence was observed under the fluorescent microscopy BX51 45.Mouse tissue section slides  were purchased from Super Bio Chips  and human tissue section slides  were obtained from Gentaur Molecular Products . According to the manufacturer’s instruction, these sections were 4% formaldehyde-fixed paraffin-embedded, 4 mm-thick section on the glass slide. The tissue sections on the slide were rehydrated, proteinase K-digested  at room temperature (rt) for 10 min, followed by treatment with 3% Ho-vanadate) in PBS and incubated on ice for 1 h. Homogenates were centrifuged at 15,000 × g at 4 °C for 15 min. The total protein concentration of the supernatants was quantified using the BCA assay. Each lysate was denatured with 5% (v/v) β-mercaptoethanol at 60 °C for 20 min to detect SiaS and then subjected to 6% SDS-PAGE, followed by blotting onto a PVDF membrane using a semi-dry blotting apparatus45. The PVDF membrane was blocked with 2.5% dry milk in PBS containing 0.05% Tween 20 (PBST) at 37 °C for 1 h. Incubation with the primary antibody (2 µg/mL 3G9) was performed in PBST containing 2.5% dry milk at the 4 °C overnight. After washing three times with PBST, the membrane was incubated with peroxidase-conjugated anti-mouse IgG + M as a secondary antibody (1: 5000 dilution) at 37 °C for 1 h. After washing three times with PBST, the ECL reagents were used to visualize the blotted components.Cells and tissues were homogenized with the lysis buffer containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), protease inhibitor cocktail , phosphatase inhibitor cocktail (50 mM sodium fluoride (NAF), 10 mM β-glycerophosphate, and 1 mM sodium 57, E14.5 mouse brain homogenates (100 µg) were applied to 10% SDS-PAGE followed by blotting on a PVDF membrane. The left edge of the membrane was removed and used for western blotting with 3G9. The rest of the membrane was equally cut into nine pieces according to the molecular size. Each slit was cut into small pieces and subjected to mild acid hydrolysis in 0.4 mL of 0.1 N TFA at 80 °C for 2 h. The hydrolysates were dried using SpeedVac vacuum concentrator . Twenty microliters each of 0.01 M TFA and DMB solution45 were added and incubated at 50 °C for 2 h. The DMB-derivatized samples were directly applied to an octadecylsilyl (ODS) column  and eluted with acetonitrile/methanol/0.05% TFA  at 1.0 mL/min for 120 min on a JASCO HPLC system  as previously described45. 4MU-Neu5Ac8S was used as a positive control. For identification of DMB-derivatives of Neu5Ac and Neu5Ac8S, the identical retention times to the authentic sialic acid species were confirmed. In addition, co-injection experiments of the samples and CMP-Neu5Ac or CMP-Neu5Ac8S were also performed.To assess the amount of SiaS components of the glycoproteins blotted onto PVDF membranesmWscd1) and mWscd2 genes  were obtained by amplifying the coding regions by PCR using specific primers (Table Wscd1 (mdkWscd1) and mdkWscd2 genes  were obtained by amplifying the coding regions through PCR using the primary cDNA prepared from 6-dpf and 7-dpf fry, respectively, primers  domain containing 1 . Multiple sequence alignment of all the sequences was performed by ClustalW 2.1  and the phylogenic tree was obtained by the Neighbor-joining method (Kimura method) on GENETYX software Ver.14.Nucleotide sequences of genes that are annotated as mouse sulfotransferases or Wscd1/Wscd2 of various animals were obtained from the National Center for Biotechnology Information (NCBI) gene database  were designed using the siDirect tool (http://sidirect2.rnai.jp), and purchased from Eurofins Japan  . The annealed oligonucleotides were then ligated into the BsaI-digested pDR274 vector. The obtained plasmids were denoted pDR274-sgWscd1 and pDR274-sgWscd, respectively.(a) Mammalian expression plasmids for Wscd1 and Wscd2. The cDNA fragments were amplified by a two-step cycle PCR  with KOD-Plus-Neo polymerase from the id Table , and subrs Table , and pcDrs Table . Four crs) Table . They wees Table  were syn2 and 95% air-humidified atmosphere at 37 °C. HEK cells were cultured under the same conditions except that Dulbecco's modified Eagle's medium was used instead of MEM-α.Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells were purchased from Riken Cell Bank . The human neuroblastoma (SK-N-SH) cell line was purchased from the Japanese Collection of Research Bioresources Cell Bank . CHO and SK-N-SH cells were cultured in MEM-α  supplemented with 100 U/mL penicillin G and 100 μg/mL streptomycin sulfate, and 10% fetal bovine serum in a 5% CO6 cells) were cultured in a 6-well plate overnight at 37 °C, and transiently transfected with 3 μg of each pcDNA3.1 plasmid for mWscd1 and mWscd2, and pcDNA4 plasmids for mdkWscd1 and mdkWscd2 using the PEI-Max Transfection Reagent. At 48 h post-transfection, the cells were collected and subjected to flow cytometry and fluorometric HPLC analyses to evaluate the SiaS epitope. Transfection efficiency was evaluated by observing the transfected cells with a plasmid encoding the green fluorescent protein (GFP) gene by fluorescent microscopy. For RNA interference with shRNA plasmids, transfection of HEK and SK-N-SH cells was performed with the shWscd1 and shWscd2 plasmids, respectively, according to the above-mentioned method.CHO cells  at 4 °C for 1 h, and washed twice with PBS. mAb.2G9 (10 μg/mL) was used as an isotype control. The cells were incubated with Alexa Fluor 488-labeled goat anti-mouse (IgG + IgM) (2 µg/mL) at 4 °C for 30 min. After washing twice with PBS, the cell surface fluorescence was analyzed using a Galios flow cytometer . The collected data were analyzed using the Kaluza software (Beckman Coulter). The proportion of the 3G9 epitope-positive to negative cells was analyzed.6) were cultured on glass coverslips in 6-well plates until 60% confluency. The cells were then transfected with 3 μg of pcDNA-mWscd1, pcDNA-mWscd2, and pcDNA3.1-Mock (empty) plasmids. All cells were washed once with PBS and fixed by incubation with 4% paraformaldehyde in PBS at room temperature for 8 min. The membrane was permeabilized with 0.1% Triton-X-100 in PBS at room temperature for 15 min. The cells were blocked with 2% BSA in PBS for 1 h. V5-tagged Wscd1 and Wscd2 were stained by incubation with anti-V5 chicken mAb (1:500 dilution) at 37 °C for 1 h, followed by incubation with a 1:1000 dilution of Alexa Fluor 488-conjugated anti-chicken IgY at room temperature for 30 min. Golgi apparatus and endoplasmic reticulum were stained with GM130 mAb and KDEL mAb at 37 °C for 1 h, followed by incubation with Alexa Fluor 555-conjugated anti-rabbit IgG (1:1000 dilution). DAPI was used for nuclear staining at 37 °C for 15 min. The stained slides were examined using a KEYENCE BZ-X810 florescent microscope.CHO cells (0.5 × 106 cells) were inoculated in a 10-cm dish overnight, transfected with 3 μg of pcDNA-mWscd1, -mWscd2, or -Mock and incubated for 2 days. The transfected CHO cell lysate was used as the crude enzyme fraction for the in vitro activity. The reaction mixtures (50 μL) containing 50 mM Tris–HCl, pH 7.2, 0.375 mM 1,4-dithiothreitol, 2 mM ATP, 5 mM NAF, 10 mM MnCl2, 2 mM PAPS, Sia-containing acceptor substrates , and 25 µg of the enzyme fraction were incubated at 20 °C for 18 h. (b) GM1 substrate: The reaction mixtures with GM1 were mixed with 1 mL of chloroform/methanol/water  (CMW), and applied to a DEAE-Sephadex A-25 column . The column was washed with 1 mL of CMW, and eluted with 12.5 mL of chloroform/methanol/1 M CH3COONa  to obtain the acidic glycolipid fraction. After desalting with the SepPak C18 cartridge , the acidic lipid fraction was spotted on a thin-layer chromatography (TLC) plate , and developed with chloroform/methanol/0.2% CaCl2 . The TLC sheet was cut into two parts. One part was visualized for detecting the acidic glycolipid products (P) by the orcinol/sulfuric acid method59. The other part was used to collect the product P by scratching the silica gels at the corresponding position of P on the visualized plate. The product P was extracted from the collected silica gels by CMW, and the supernatant was subjected to fluorometric HPLC after hydrolyzed in 0.02 mL of 0.1 N TFA at 80 °C for 2 h. (c) TF substrate: The reaction mixtures with and without TF were subjected to SDS-PAGE/western blotting with 3G9 as described above. CBB staining was performed to check for equal substrate loading in the western blotting. The reaction products were also analyzed by the fluorometric HPLC, after hydrolyzed in 0.1 N TFA.(a) In vitro enzyme reactions: CHO cells  was subjected to reverse transcription using ProtoScript II with a random hexamer primer. qRT-PCR was performed using a pair of oligonucleotides Table  and SYBRhttps://shigen.nig.ac.jp/medaka/)58. After pCS2 + hSpCas9 was linearized by NotI digestion, the vector was used as a template for the synthesis of capped Cas9 mRNA with an mMessage mMachine SP6 kit and then purified using the RNeasy Mini Kit. For the synthesis of sgRNAs, the pDR274 vector containing sgWscd1 or sgWscd2 was first digested by DraI. This was use as the template for synthesizing sgRNA using the AmpliScribe T7-Flash Transcription kit. The sgRNAs were purified using the RNeasy Mini kit. Approximately 2–4 nL of a mixture containing 100 ng/µL of Cas9 mRNA and 25 ng/µL of sgRNA of Wscd1 or Wscd2 was injected into embryos at the one-cell stage.All the procedures followed the instruction protocol by the NIBB55 . It was then incubated at 55 °C for 3 h, followed by denaturation at 95 °C for 15 min to inactivate the protease K. After centrifugation, the supernatant of each sample was used as genomic DNA. To detect CRISPR/Cas9-induced mutations, a heteroduplex mobility assay (HMA) was performed as described previouslyet Table .The G0 medaka were out-crossed with the wild-type medaka to obtain the F1 hetero-mutant medaka. The F1 hetero-mutants were subjected to genotyping to find Wscd1(+/−) and Wscd2(+/−) medaka, which contained a knockout allele arisen from frameshifts. The Wscd1(+/−) or Wscd2(+/−) female and male medaka of the same genotype were in-crossed with each other to obtain Wscd1(−/−) or Wscd2(−/−) offspring at F2. The Wscd1(+/−) or Wscd2(+/−) female and male medaka were carefully maintained as the stable strain containing the knockout allele. Their offspring that should contain all the (−/−), (+/−) and (+/+) genotypes were daily collected and maintained in a separate plastic rearing tank under a 14 h-day/10 h-night cycle at 26 °C. For each group, at least 5 small tanks were established to assess the life span. To understand the life span, the medaka were observed every day for a certain period. When some of them died, the dead fish were immediately collected for genotyping.Young fry from 3 to 9 dpf (hatching day) was immobilized in a hole made by 1.5% agarose. The heart contractions were recorded using an SZX12 DP80 microscope (Olympus). Digital pictures were captured at maximum frame rate at a resolution of 1360–1024 pixels for up to 3 min and recorded in a PC using CellSens Standard software. Movies of heart movements were processed using ImageJ software. Contraction rhythms were measured based on alterations in the intensity of blood cell flow into and out of ventricle. Regions of interest (ROIs) in the ventricle were selected. The pixel intensities of the ROIs were digitized throughout the entire time series examined using ImageJ software.64. Briefly, after anesthetized, the fish were weighed, and the body length was measured using the ruler. Five Wscd2(−/−) fish and 15 WT fish were used.Young fry of Wscd2(−/−) that were alive at 60 dpf and WT fry were measured for the body weight and body length as described previouslyCRP in WT, Wscd1(−/−), and Wscd2(−/−) fry at 15 dpf was determined by qRT-PCR using the primary cDNA as a template and the primers for CRP and β-actin , (+/−), and (−/−) genotypes of Wscd1 and Wscd2.Expression level of in Table . β-actinAll values were expressed as the mean SE (n is three) and p-values were evaluated by the Student’s t-test.Supplementary Information.


The understanding of biomolecular recognition of posttranslationally modified histone proteins is centrally important to the histone code hypothesis. Despite extensive binding and structural studies on the readout of histones, the molecular language by which posttranslational modifications on histone proteins are read remains poorly understood. Here we report physical-organic chemistry studies on the recognition of the positively charged trimethyllysine by the electron-rich aromatic cage containing PHD3 finger of KDM5A. The aromatic character of two tryptophan residues that solely constitute the aromatic cage of KDM5A was fine-tuned by the incorporation of fluorine substituents. Our thermodynamic analyses reveal that the wild-type and fluorinated KDM5A PHD3 fingers associate equally well with trimethyllysine. This work demonstrates that the biomolecular recognition of trimethyllysine by fluorinated aromatic cages is associated with weaker cation–π interactions that are compensated by the energetically more favourable trimethyllysine-mediated release of high-energy water molecules that occupy the aromatic cage. Lysine trimethylation is a key post-translational modification, which some epigenetic reader proteins detect through binding to aromatic cages involving cation-π interactions. Here systematic modification of the aromatic cage reveals that weaker cation-π interactions do not correlate with weaker binding owing to a compensating release of bound water molecules. Many covalent modifications have been identified and characterized on histone tails and core histones; methylation, acetylation, phosphorylation and ubiquitination have been known for some time, whereas crotonylation and succinylation, among others, have been discovered more recently7. Methylated lysine residues can exist in the form of monomethyllysine (Kme), dimethyllysine (Kme2) or trimethyllysine (Kme3), and can lead to gene activation or repression, depending on the histone site and methylation state8. Histone lysine methylation is dynamically regulated by three classes of functionally related proteins9. The installation of the methyl group(s) from S-adenosylmethionine (SAM) onto lysine is catalyzed by histone lysine methyltransferases (KMTs)10. The opposite reaction, i.e., the removal of methyl group(s) from methylated lysine, is catalyzed either by flavin-dependent lysine specific demethylases or a larger family of non-heme Fe(II) and 2-oxoglutarate (2OG)-dependent histone lysine demethylases (KDMs)11. Recent structural and functional studies have revealed that histones that possess unmethylated and methylated lysine residues can be specifically recognized by a large number of reader domain proteins that differ in the composition of the lysine recognition site13. Electrostatic interactions and H-bonding appear to be of central importance in the recognition of unmethylated lysines by interacting reader domains 13. Similarly, electrostatic interactions and H-bonding play important roles in the readout of the lower methylation states Kme and Kme2 via a cavity-insertion binding mode 13. Numerous epigenetic reader proteins, including plant homeodomain (PHD) zinc fingers and members of the Royal superfamily , have been involved in the recognition of trimethyllysine via the so-called surface-groove binding mode13. Despite different folding patterns, these reader proteins have a common feature as they all possess electron-rich aromatic cages, most often comprised of 1–4 side chains of Phe, Tyr and Trp, although some cages also include the negatively charged Asp or Glu residues14. Comparative binding studies between trimethyllysine and its neutral carba analogue led to the conclusion that the recognition of the positively charged trimethyllysine by the aromatic cage containing readers is predominantly driven by a combination of favourable cation–π interactions and the release of high-energy water molecules located inside the aromatic cages17.Posttranslational modifications on histone proteins have a profound effect on the structure and function of human chromatinTo provide a deeper understanding of the origin of molecular recognition of trimethyllysine by aromatic cages, we report here a complementary physical-organic chemistry approach in which the two electron-rich tryptophans that solely constitute the aromatic cage of KDM5A reader are substituted by fluorinated tryptophans, thus resulting in electron-poorer π systems. Our experimental and computational investigations reveal that despite weaker aromatic character of fluorinated cages, the association between trimethyllysine and fluorinated KDM5A is comparable to that of the wild-type KDM5A. The underlying molecular mechanism for such observation is a well-balanced compensation between energetically less favourable cation–π interactions and more favourable release of high-energy water molecules that occupy fluorinated aromatic cages.25. We envisioned that this elegant chemical approach could be employed for probing the involvement of cation–π interactions in the readout of trimethyllysine-containing histones by epigenetic reader domains. We chose the PHD3 finger of KDM5A reader protein as a model system, because its specific recognition of H3K4me3 is required for leukemogenesis and, importantly, its aromatic cage is composed of only two tryptophan residues (Trp18 and Trp28) , position 6 (6F-Trp), and at positions 5 and 6   Fig. 26. Sincerp) Fig. . FluorinE. coli Castellani and Chalmers strain in a method similar to the procedure described by Budisa and coworkers28. We successfully produced the protein variants with the three tryptophan analogues: 5F-Trp, 6F-Trp and 5,6diF-Trp in the KDM5A PHD3 reader domain. Additional attempts to incorporate 4,5,6,7tetraF-Trp, the most electron-poor tryptophan analogue, did not lead to production of detectable amounts of fluorinated KDM5A. Interestingly, the wild-type (WT) construct expressed significantly less well than its fluorinated counterparts. Wild-type and fluorinated KDM5A PHD3 fingers were purified using standard biochemical techniques to obtain proteins of high purity in reasonable yields pLysS (hereafter referred to as BL21) and E. coli Castellani and Chalmers (hereafter referred to as AUX) showed that both expression strains produced proteins with identical masses and tertiary structures , enthalpy of binding (ΔH°) and entropy of binding (ΔS°) , ΔH°  and −TΔS°  were also observed to be virtually indistinguishable  was examined by isothermal titration calorimetry (ITC), which provided values of the Gibbs free energy of binding , thus supporting the presence of two fluorine atoms located at 6F-Trp18 and 6F-Trp28 of the PHD3 finger of KDM5A. Upon binding of H3K4me3 to 6F-KDM5A, we observed down-field shifts of approximately +1 ppm (−121.1 ppm and −121.2 ppm), consistent with the magnitude of shifts found in other ligand binding studies utilizing 6F-Trp labelled proteins30. A down-field shift was also observed upon binding of H3K4me3 to 5F-KDM5A  simulations to examine the behaviour over time of the reader–ligand complex and effects of fluorination on key interactions for binding. Four variations of the PHD3 finger of KDM5A were simulated, including the wild-type (PDB: 2KGI) and three variants containing F-substituted Trp18-Trp28 aromatic cages: 5F-Trp18/5F-Trp28, 6F-Trp18/6F-Trp28, and 5,6diF-Trp18/5,6diF-Trp28. Adopting a recently described molecular mechanics-based approachIn all cases of fluorinated KDM5A, the trimethyllysine side chain of H3K4me3 occupies the aromatic cage throughout the simulation Fig. . Flexibi33. To quantify the strength of these energetically favourable cation–π interactions, average ΔEele values were calculated between the quaternary ammonium cations of H3K4me3 to each aromatic side chain of Trp18-Trp28 . Binding of H3K4me3 to 6F-KDM5A leads to differences in ΔEele values when comparing 6F-Trp18 and 6F-Trp28 int of −10.3 kcal mol−1, due to an almost negligible deformation strain energy (ΔE(aq)strain = 0.1 kcal mol−1) associated with the subtle variation of geometry upon complexation. The interaction energy between Kme3 and TRP2 without water is stronger , because the system presents an unfavourable desolvation energy of 17.3 kcal mol−1. The interaction energy in the absence of water, ∆Eint, can be decomposed into Pauli repulsion , electrostatic attraction , orbital interaction  and dispersion  terms.Next, we aimed to elucidate the nature of the non-covalent interactions between the Kme3 side-chain of the histone peptide and the aromatic cage that consists of two fluorinated tryptophan residues of the KDM5A PHD3 finger (hereafter designated as TRP2 fragment) to understand the underlying origin of the recognition. We characterized quantum chemically the energetics and bonding mechanism in the four model complexes, using dispersion-corrected density functional theory at BLYP-D3BJ/TZ2P and COSMO for simulating aqueous solution, as implemented in the ADF program (Supplementary Table E(aq) = −10.3–−10.4 kcal mol−1). The same happens for both the deformation strain and interaction energies, with a maximum change of 0.1 kcal mol−1. So, even with the presence of fluorine on TRP2, complexation only very slightly changes the geometry of Kme3 side chain. However, changes appear when these interactions are analysed without water. Fluorination of TRP2 causes a weakening of the interaction in the absence of water, ∆Eint, between Kme3 and TRP2 of 2.4 and 3.0 kcal mol−1 for 5F-TRP2–Kme3 and 6F-TRP2–Kme3, respectively, and of 5.3 kcal mol−1 for 5,6diF-TRP2–Kme3 int values.Furthermore, we performed an analogous series of analyses as the one described above, but this time for di- and tetra-fluorinated TRP2 as the aromatic cage with Kme3. First, bond energies hardly change for difluorinated 5F-TRP2–Kme3 and 6F-TRP2–Kme3, or tetrafluorinated 5,6diF-TRP2–Kme3 systems , with a maximum difference of 1.0 kcal mol−1 compared with unfluorinated TRP2 system. This constant Pauli term is in agreement with the minor geometrical changes among the different systems, which can be also followed from distances enclosed in Table 3+ H atom and a C atom of a tryptophan in TRP2–Kme3 is 2.78 Å, while the same H atom is 3.38 Å away from the closest C atom of the other tryptophan . Thus overall, we can conclude that the five-membered rings of TRP2 contribute more to binding to Kme3, and even more so in case of fluorinated aromatic cages.Further insight into the effect of fluorination of the aromatic cage on the interaction with Kme3 can be gained by estimating the interaction of the cationic nitrogen of Kme3 with either exclusively the five-membered ring or exclusively the six-membered ring of TRP2. We achieved this by introducing tailor-made, for this purpose, modifications into our model systems. Thus, we have constructed modifications of our TRP2–Kme3 system by just keeping one five-membered ring of one TRP unit and one six-membered ring of the other TRP unit, whereas Kme3 has been simplified to NMeng Table . Further16. We conceived that fluorination of tryptophan residues that constitute the aromatic cage of the KDM5A PHD3 finger presumably leads to altered energetics of high-energy water molecules in their proximity. Therefore, water thermodynamic analyses were carried out to compute thermodynamic parameters for water molecules located in wild-type and fluorinated KDM5A  Solute–solute interactions, i.e., cation–π interactions between the positively charged trimethyllysine and the electron-rich aromatic cage of the KDM5A PHD3 finger; (ii) Ligand desolvation, i.e., partial desolvation of trimethyllysine upon the KDM5A–Kme3 complex formation; and (iii) Protein desolvation, i.e., desolvation of the aromatic cage of KDM5A upon Kme3 binding. A strategy in which the aromatic character of tryptophan residues is perturbed by the introduction of fluorine substituents, while keeping all other parameters of the KDM5A–H3K4me3 system unaltered, eliminates the contribution from trimethyllysine desolvation in our comparative analyses . Our thermodynamic results  that are compensated by energetically more favourable desolvation of aromatic cages (when compared with the wild-type aromatic cage) upon trimethyllysine binding. More detailed examinations of biomolecular recognition of histones will greatly contribute to our basic understanding of the histone code48, which postulates that the molecular landscape of posttranslational modifications on histone proteins is tightly associated with interactions with chromatin-associated proteins, thus altering the chromatin structure and function.Understanding the molecular origin of biomolecular recognition processes that play essential roles in human health and disease is important from a basic molecular perspective as well as from a biomedical perspective. Despite extensive examinations of non-covalent interactions in various chemical and biological systems in the past two decades52 will importantly contribute to a better understanding of underlying molecular mechanisms that govern the specific recognition of other types of posttranslational modifications found on histones and other proteins.This work demonstrates that a holistic physical-organic chemistry approach, based on synergistic experimental and computational tools, enables a more advanced understanding of biomolecular recognition of trimethyllysine-containing histones by epigenetic reader proteins. It is envisioned that compelling physical-organic chemistry approaches, which collectively examine non-covalent interactions and desolvation effects, along with modern chemical biology approaches2O  was heated to 70 °C under Ar atmosphere in microwave vial. After 16 hours of stirring the solvent was coevaporated with toluene. Crude brown oil was purified by column chromatography (MeOH in CH2Cl2 (0–5%) and with AcOH (0.1%)), affording N-acetyl-5,6-difluorotryptophan  as a yellowish oil. 1H NMR  δ: 7.36–7.29 , 7.17–7.11 , 7.11 , 4.65 , 3.23 , 3.08 , 1.89 . 13C NMR  δ: 173.5, 171.7, 131.5 , 124.7 , 122.8 , 110.2 , 104.3 , 98.4 , 53.2, 20.9. ESI-MS calcd for C13H13F2O3N2 [M + 1]+ 283.0894, found 283.0888. A solution of N-acetyl-5,6-difluorotryptophan  was dissolved in aqueous NaOH  and heated at 100 °C for 16 h. The reaction mixture was then cooled and acidified to pH 1. Solvent was evaporated and the crude product was purified by preparative HPLC, affording racemic 5,6-difluorotryptophan as a TFA salt  as a yellowish oil. 1H NMR  δ: 7.44–7.36 , 7.26–7.17 , 4.21 , 3.39 , 3.30 . 13C NMR  δ: 170.2, 148.1 , 145.7 , 131.9 , 126.0 , 122.3 , 106.9 , 104.3 , 98.8 , 52.9, 25.9. 19F NMR  δ −78.2 , −148.3 , −151.6 . ESI-MS calcd for C11H11F2O2N2 [M + 1]+ 241.0789, found 241.0797.Supplementary Fig. E. coli containing the KDM5A-GST construct in TB medium supplemented with the appropriate antibiotics. At OD600 ~0.6, expression was induced with 0.4 mM IPTG and 0.1 mM ZnCl2  and cultured overnight at 16 °C. Cells were then harvested, lysed and purified using GST affinity. The GST tag was cleaved off with TEV-protease under reducing conditions (10 mM dithiothreitol), and the KDM5A PHD3 finger was subsequently purified by size exclusion chromatography on a Superdex 75 column using 20 mM TRIS-HCl pH 7.5, 50 mM NaCl, 1 mM DTT as running buffer. Protein concentration was measured spectrophotometrically using a Denovix DS-11 spectrophotometer and protein masses were confirmed by ESI-MS analyses. Wild-type and fluorinated PHD3 finger of KDM5A-GST  expressed in the auxotrophic E. coli (Migula) Castellani and Chalmers strain were cultured in either New Minimal Medium (NMM) or in Unnatural amino acid New Minimal Medium (UNMM) supplemented with appropriate antibiotics, respectively. NMM was prepared as described by Budisa and coworkers28. In brief, NMM contained 100 mM K2HPO4, 55 mM KH2PO4, 20 mM D-glucose, 8.5 mM NaCl, 7.5 mM (NH4)2SO4, 1 mM MgSO4, 10 mg l−1 biotin, 10 mg l−1 Thiamine-HCl, 1 mg l−1 CaCl2 and FeCl3, 1 μg l−1 CuSO4, MnCl2, ZnCl2, NaMoO4 and 50 mg l−1 of each individual amino acid. UNMM was prepared similarly except that tryptophan was substituted by the desired fluorinated tryptophan analogue, at a final concentration of 25 mg l−1. E. coli (Migula) Castellani and Chalmers containing the wild-type KMD5A construct was cultured in NMM at 37 °C. At OD600 ~0.6, the NMM medium was refreshed by harvesting the cells, after which they were resuspended in fresh NMM. Expression was then induced with 0.1 mM IPTG and 0.1 mM ZnCl2 . The cells were subsequently cultured for 3 h at 37 °C, after which the culture was harvested, lysed and purified as described above. Fluorinated tryptophan analogues were introduced into KDM5A as follows: E. coli (Migula) Castellani and Chalmers containing the KDM5A construct was initially cultured in NMM at 37 °C. At OD600 ~0.6, the cells were harvested and subsequently washed three times with 0.9% NaCl at room temperature. Following the washing steps, the cells were resuspended in fresh UNMM. Expression was then induced with 1.0 mM IPTG and 0.1 mM ZnCl2 . The cells were subsequently cultured for 3 h at 37 °C after which the culture was harvested, lysed and purified as described above.The wild-type KDM5A PHD3 finger  fused to GST was expressed in Rosetta BL21 (DE3)pLysS −1 in 10 mM phosphate buffer, pH 7.5, on a J-815 circular dichroism spectropolarimeter. The samples were measured over a range of 180–260 nm with normal sensitivity and a bandwidth of 1 nm. Scanning was performed at 50 nm per minute, a data integration time (D.I.T.) of 0.5 s and a data pitch of 0.5 nm. The spectra for each protein are a result of 10 accumulations.CD experiments were carried out at a protein concentration of 0.1 mg ml53. SYPRO-Orange protein gel stain (Invitrogen) was used as a reporter dye, emitting fluorescence in the FAM channel. Total reaction volume was 25 μl:20 μl buffer , 2.5 μl of 25 μM protein and 2.5 μl SYPRO-Orange dye (diluted 1:100 in ddH2O). Melt curve data were obtained in triplicate, in a temperature range of 20–95 °C at a stepwise temperature increment of 1 °C min−1. Obtained data were analyzed using DSF Analysis v3.0.2 software, designed by Niesen et al. (available via ftp://ftp.sgc.ox.ac.uk/pub/biophysics/)54.Protein melt curves were obtained as described by Reinhard et al. using a StepOne-Plus Real-Time PCR system (Applied Biosystems) and MicroAmp fast optical 96-well reaction plates (Applied Biosystems)The same batch of H3K4me3 histone peptide  was titrated to all KDM5A PHD3 fingers (28 μM). Due to lower expression of the auxotrophic WT-KDM5A, H3K4me3 (190 μM) and AUX WT-KDM5A (21 μM) were used. The buffer used for ITC experiments was the same as the elution buffer used for SEC; 20 mM TRIS-HCl pH 7.5, 50 mM NaCl, 1 mM DTT. Each ITC titration consisted of 19 injections. ITC experiments were performed on the fully automated Microcal Auto-iTC200 . Heats of dilution for histone peptides were determined in control experiments, and were subtracted from the titration binding data before curve fitting. Curve fitting was performed by Origin 6.0  using one set of sites binding model. With the exception of the auxotrophic WT-KDM5A–H3K4me3 (replicate), 7–9 independent ITC experiments were carried out for other four reader–histone systems.19F nucleus detection with 1H decoupling. Samples were prepared using 5 mm Shigemi tubes matched to D2O to minimize solvent volume required. 19F NMR experiments were performed 10 mM H2KPO4 pH 7.5, at a concentration of 450 μM of 5F-KDM5A/6F-KDM5A and 1 mM of H3K4me3 peptide (ARTKme3QTARKS). All measurements were performed at 288 K. After samples were inserted into the magnet, the sample was shimmed using the lock nucleus in D2O and a 1H spectrum was acquired to assess the quality of the shims. The probe was then manually tuned and matched to 19F to optimize 19F detection. A 15 μs @ 23 Watts 90-degree pulse was used. 19F{1H} spectra were then acquired with the following parameters: NS = 1.5 k–28 k, d1 = 1, aq = 1.09 s, sw = 20.1 ppm and o1p near −120 ppm. 19F NMR spectra were externally referenced to CFCl3 using the frequency of residual solvent signal in the 1H spectrum and the ratio between the 1H and 19F gyromagnetic ratios.Measurements were obtained on a Bruker AVANCE III 400 MHz system equipped with a BBFO probe capable of 32 water and neutralized explicitly with either sodium or chloride counterions. Non-bonding parameters of Zn(II) previously established from studies of KDM4A35 were employed. Atomic partial charges for 5F-Trp, 6F-Trp, and 5,6diF-Trp correspond to the Restrained Electrostatic Potential (RESP)55 charges, as shown in Supplementary Tables 29. The final systems were minimized for 1000 cycles of steepest-descent minimization followed by 1000 cycles of conjugate-gradient minimization to remove close van der Waals contacts using the sander program in AMBER12. Equilibration was achieved using PMEMD to heat the systems to 310 K followed by independent MD simulations performed with a periodic boundary condition at a constant pressure of 1 atm with isotropic molecule-based scaling at a time step of 2.0 fs. All simulations used a dielectric constant of 1.0, Particle Mesh Ewald summation56 to calculate long-range electrostatic interactions and bond-length constraints applied to all bonds to H atoms. Trajectories were saved at 20 ps intervals and visualized using VMD57. Electrostatic energies between the terminal modified Kme3 side chain (e)-N atom and the π-system of surrounding aromatic cages were calculated with the NAMDEnergy Plugin57. The π-system was defined for tryptophan and fluorinated tryptophans as the side chain indole ring (non-H) atoms. Energy values were measured every 20 ps and averaged over 100 ns.Four MD simulations were carried out for 100 ns each using the Amberff12SB force field. A PDB structure for the model representing KDM5A PHD3 (PDB: 2KGI) was used as a template for building the readerKme3 systems. KDM5A residues Trp18 and Trp28 were manually modified to generate the 5F-Trp18/5F-Trp28, 6F-Trp18/6F-Trp28, and 5,6diF-Trp18/5,6diF-Trp28 complexes. Hydrogen atom addition was performed with LEaP. Systems were solvated in a 10 Å truncated octahedral box of TIP3P58. The effect of solvation was simulated by means of the Conductor-like Screening Model (COSMO) of solvation as implemented in ADF. The approach has been benchmarked against highly correlated post-Hartree-Fock methods and experimental data and was found to work reliably62.All calculations were carried out with the Amsterdam Density Functional (ADF) program using dispersion-corrected density functional theory at the BLYP-D3BJ/TZ2P level of theory64. The bond energy in aqueous solution ∆E(aq) consists of two major components, namely, the strain energy ∆E(aq)strain associated with deforming the Kme3 and the reader from their own equilibrium structure to the geometry they adopt in the complex, plus the interaction energy ∆E(aq)int between these deformed solutes in the complex  molecular orbital (MO) theory in combination with an energy decomposition analysis (EDA)plex Eq. :1\documeE(aq)int into the effect caused by the change in solvation ∆E(desolv) and the remaining intrinsic interaction ∆Eint between the unsolvated fragments in vacuum ∆Eint:To arrive at an understanding of the importance of desolvation phenomena during the complexation process, we separate the solute–solute interaction ∆Eint can be further decomposed as shown in Eq. In the EDA, the intrinsic interaction energy ΔVelstat corresponds to the classical electrostatic interaction between the unperturbed charge distributions of the deformed fragments which is usually attractive. The Pauli repulsion ∆EPauli comprises the destabilizing interactions between occupied orbitals and is responsible for the steric repulsions. The orbital interaction ∆Eoi accounts for charge transfer  and polarization . Finally, the ∆Edisp term accounts for the dispersion interactions based on Grimme’s DFT-D3BJ correction. Furthermore, the charge distribution has been analysed using the Voronoi deformation density (VDD) method65.Here, ∆67. All calculations were run in with default settings. In brief, a 2 ns molecular dynamic (MD) simulation of the KDM5A PHD3 finger with the histone peptide removed, was performed using the Desmond molecular dynamic engine with the OPLS2.1 force field43. Protein atoms were constrained throughout the simulation. Water molecules from the simulation were then clustered into hydration sites for thermodynamic analysis. Enthalpy values for each hydration site were obtained by computing the average non-bonded interaction for each water molecule in the cluster over the course of the MD simulation. Entropy values were calculated using a numerical integration of a local expansion of the entropy in terms of spatial and orientational correlation functions69. The contribution of water-free energy to the binding free energy of the peptide was approximated by the sum of the free energies of hydration sites displaced by the ligand upon binding.Water thermodynamic calculations were performed with the program WaterMap, as described in previous reportsFurther information on research design is available in the Supplementary InformationReporting Summary


The complement system is part of the innate immune system that clears pathogens and cellular debris. In the healthy brain, complement influences neurodevelopment and neurogenesis, synaptic pruning, clearance of neuronal blebs, recruitment of phagocytes, and protects from pathogens. However, excessive downstream complement activation that leads to generation of C5a, and C5a engagement with its receptor C5aR1, instigates a feed-forward loop of inflammation, injury, and neuronal death, making C5aR1 a potential therapeutic target for neuroinflammatory disorders. C5aR1 ablation in the Arctic (Arc) model of Alzheimer’s disease protects against cognitive decline and neuronal injury without altering amyloid plaque accumulation.To elucidate the effects of C5a–C5aR1 signaling on AD pathology, we crossed Arc mice with a C5a-overexpressing mouse (ArcC5a+) and tested hippocampal memory. RNA-seq was performed on hippocampus and cortex from Arc, ArcC5aR1KO, and ArcC5a+ mice at 2.7–10 months and age-matched controls to assess mechanisms involved in each system. Immunohistochemistry was used to probe for protein markers of microglia and astrocytes activation states.ArcC5a+ mice had accelerated cognitive decline compared to Arc. Deletion of C5ar1 delayed or prevented the expression of some, but not all, AD-associated genes in the hippocampus and a subset of pan-reactive and A1 reactive astrocyte genes, indicating a separation between genes induced by amyloid plaques alone and those influenced by C5a–C5aR1 signaling. Biological processes associated with AD and AD mouse models, including inflammatory signaling, microglial cell activation, and astrocyte migration, were delayed in the ArcC5aR1KO hippocampus. Interestingly, C5a overexpression also delayed the increase of some AD-, complement-, and astrocyte-associated genes, suggesting the possible involvement of neuroprotective C5aR2. However, these pathways were enhanced in older ArcC5a+ mice compared to Arc. Immunohistochemistry confirmed that C5a–C5aR1 modulation in Arc mice delayed the increase in CD11c-positive microglia, while not affecting other pan-reactive microglial or astrocyte markers.C5a–C5aR1 signaling in AD largely exerts its effects by enhancing microglial activation pathways that accelerate disease progression. While C5a may have neuroprotective effects via C5aR2, engagement of C5a with C5aR1 is detrimental in AD models. These data support specific pharmacological inhibition of C5aR1 as a potential therapeutic strategy to treat AD.The online version contains supplementary material available at 10.1186/s12974-022-02539-2. Alzheimer’s disease (AD) is the most common form of dementia and is characterized by the accumulation of extracellular amyloid beta (Aβ) plaques, hyperphosphorylated tau, synaptic loss, neuronal death, and ultimately cognitive decline . EvidencThe complement system is a powerful effector of the innate immune system that is activated via three distinct pathways, classical, lectin, and alternative, all of which converge on proteolytic cleavage of C3 into the chemoattractant C3a and opsonin C3b. C3b also forms part of the C5 convertase, which cleaves C5 into C5a, a potent pro-inflammatory chemoattractant, and C5b, the initiating molecule of the lytic membrane attack complex (MAC) . In addiIn two mouse models of AD, pharmacologic inhibition of the C5a receptor (C5aR1) with PMX205 reduced amyloid plaque accumulation, attenuated activation of microglia and astrocytes, and protected mice against hippocampal synaptic loss and cognitive decline . InhibitC5aR1 expression is highly induced in AD mouse models . To furt. The Arctic48 mouse model of AD (hereafter referred to as Arc), which carries the human APP transgene with three mutations—the Indiana (V717F), the Swedish (K670 N + M671 L), and the Arctic (E22G), was generated on a C57BL6/J background and originally provided by Dr. Lennart Mucke . This hemizygous mouse model produces fibrillar plaques as early as 2 to 4 months of age [The Institutional Animal Care and Use Committee of University of California at Irvine approved all animal procedures, and experiments were performed according to the NIH Guide for the Care and Use of laboratory animals. Mice were grouped housed in ambient temperature and given access to food and water ad libitum Arctic EG, was ges of age , were crs of age  (originaObject location memory (OLM) as previously described , 21 took2HPO4, 1.47 mM KH2PO4, pH 7.4). Half brains were quickly dissected and fixed in 4% paraformaldehyde for 24 h, then stored in PBS with 0.02% sodium azide at 4 °C. 30–40 µm coronal sections were generated using a vibratome and stored in PBS with 0.02% sodium azide at 4 °C until ready for use.Mice were deeply anesthetized with isoflurane and perfused transcardially with cold phosphate buffered saline . Low magnification images (10×) were acquired using ZEISS Axio Scan.Z1 Digital Slide Scanner. The mean areas of C3, GFAP, CD11b, CD11c, CD68, Iba1, and plaques were quantified in the hippocampal regions CA1, CA3, and DG, as well as the entire hippocampus and cortex using the Surfaces feature of Imaris ×64 (version 9.5.0). Confocal images were acquired at 20× using a Leica SP8 confocal microscope. Z stacks of 1-µm-step interval within a depth of 30 µm were obtained per area of interest, and volume was analyzed with Imaris. Quantitative comparisons between groups were carried out on comparable sections of each animal processed at the same time with same batches of solutions, unless otherwise noted.Sections were incubated in blocking solution  for 1 h on a shaker at RT. Tissues were then incubated with primary antibody diluted in blocking solution overnight on a shaker at 4 °C. Primary antibodies used were rabbit anti-Iba1 , rat anti-CD68 , rat anti-CD11b , hamster anti-CD11c , rat anti-Lamp1 , rat anti-mouse C3 , rabbit anti-GFAP . Alexa Fluor secondary antibodies were diluted 1:500 in blocking solution and included 568 goat anti-Armenian hamster , 555 goat anti-rat (Invitrogen #A21434), 488 goat anti-rat (Invitrogen #A21212), 488 goat anti-rabbit (Invitrogen #A-11070), and 647 goat anti-rabbit (Invitrogen #A21244). To counterstain fibrillar plaques with Thioflavin-S (Thio-S) sections were incubated in 0.1% or 0.5% Thio-S for 10 min after secondary antibody. To counterstain fibrillar plaques with Amylo-Glo , tissues were incubated for 10 min before the first blocking step, washed in PBS for 5 min, and quickly rinsed with MilliQ HTo confirm that mice expressing the C5aGFAP+/− transgene produce higher quantities of C5a protein, we performed enzyme-linked immunosorbent assay (ELISA) on pulverized hippocampus and cortex and on plasma taken from WT, Arc, C5a+, and ArcC5a+ mice at 5, 7, and 10 months. Pulverized cortex and hippocampal samples were homogenized on ice using a pestle pellet motor in 10× volume of homogenization buffer containing PhosphoSTOP (Roche) and cOmplete Mini Protease Inhibitor Cocktail (Roche) in TPER. Samples were then centrifuged at 14,000 rpm for 30 min at 4 °C and supernatant was collected and stored at − 80 °C. C5a concentration was determined with the R&D Systems Mouse Complement Component C5a DuoSet ELISA (DY2150) according to the manufacturer’s instructions, in triplicate. Optical density was read on SpectraMax Plus 384 using and SoftMax Pro 7.1 . Standard curve was fitted to 4-parameter logistic expression. The total protein concentration was assessed using the Pierce BCA Protein Assay Kit (Thermo Scientific). Any plates lacking a full standard curve were normalized to the previous plate while C5a levels were averaged to generate one value per animal for samples repeated in more than one ELISA experiment. To assess whether the C5aGFAP transgene resulted in peripheral effects, plasma levels of C5a were measured. Blood collected via cardiac puncture was mixed immediately with 10 mM EDTA on ice. After centrifugation , plasma was collected and diluted 1:80 in reagent diluent for ELISA. When plasma from FVB/NJ C5-deficient mice  was assaMice were perfused with PBS and dissected cortex and hippocampus were stored at − 80 °C prior to RNA extraction. Hippocampus and cortex were lysed separately in RLT (Qiagen # 80204) buffer with 1% β-mercaptoethanol by the QIAGEN TissueLyser. Total RNA of each tissue was extracted using the QIAGEN RNeasy mini kit and QIAcube (Qiagen # 80204 and # 9002864) and quantified using the NanoDrop ND-1000 spectrophotometer. The RNA integrity number (RIN) was assessed using the Agilent 2100 Bioanalyzer and the samples with RIN > 8.0 were used for library preparation.Bulk sequencing experiments were conducted utilizing 5–10 mice per genotype per age . RNA-seq libraries were built following the Smart-seq2 protocol using the Nextera library preparation kit . In brieM-values (TMM) function [http://crick.bio.uci.edu:3838/ArcticMonday/).Paired-end RNA-seq reads were aligned to mm10 reference genome and annotated with Gencode v21 transcriptome using STAR v.5.1 . Gene exfunction . TPM wasfunction . A totalk-means clustering of genes that present similar patterns of expression during the time-course [The R package maSigPro (version 1.58.0) was used to identify gene expression changes over time allowing for e-course . In addie-course  using a p-values smaller than 0.05 [Gene ontology enrichment analysis was performed using Metascape and DAVID online tools computing gene-set overlaps between pathways and biological processes, that were selected based on han 0.05 , 34.Unless otherwise stated, all statistical analyses were performed with GraphPad Prism (V9.3.1). When appropriate, comparisons were performed with two-way ANOVA and Tukey’s post hoc test.We previously reported that ablation of C5aR1 in Arc mice prevents memory deficits assessed with the OLM test at 10 months of age , suggestNeuronal loss and cognitive decline are common features of Alzheimer’s disease . TherefoTo explore molecular basis of C5a–C5aR1 signaling, we dissected 372 cortices and hippocampi of mice representing six distinct genotypes  throughout disease progression at 2.7, 5, 7, and 10 months of age in order to build RNA-seq libraries and thus identify transcriptome changes in different cohorts  , 40, andAdditional changes in expression of complement genes were driven by transgene overproduction of C5a. Complement genes Masp1 and C1ra, regulators Cfh and Serping1, and the α chains Itgam (CD11b) and Itgax (CD11c) of CR3 and CR4, respectively, were upregulated early by the presence of the Arctic APP transgene but showed increased expression in the ArcC5a+ hippocampus at 10 months Fig. D. Thus, To understand the effects of C5a–C5aR1 signaling on gene expression in AD, changes in expression of genes found in the current bulk RNA analysis were compared to our previous reports of microglia isolated from combined cortex and hippocampus from Arc and ArcC5aR1KO at the same ages , to thosWhile many of the above genes are considered predominantly microglial genes, given the evidence for altered astrocyte states in AD , 49, 50,We performed gene ontology analysis to identify relevant biological processes and pathways associated with differentially expressed genes present in each maSigPro cluster. Expression of genes in Rc6 was higher in Arc mice and peaked in the hippocampus of the Arc cohort at 7 months. However, in the ArcC5a+ cohort, expression did not increase until 10 months of age. Rc6 was enriched for DAM genes, such as Trem2, Tyrobp, Cybb (gp91 of the NADPH oxidase), Cst7, Ctss, and Spp1 Fig. A and C. Rc7 mainly consisted of genes whose expression was increased in the Arc peaking at 7 months. While expression in the ArcC5aR1KO remained higher than C5aR1KO controls, it did not increase uniformly with age. Interestingly, genes in Rc7 were highly upregulated in the ArcC5a+ mice at 10 months compared to earlier ages and Arc and ArcC5aR1KO Fig. A. Rc7 alC5a has been shown to promote neuronal damage in vitro , althougAmyloid pathology in humans and animal models can activate microglia to an inflammatory and disease-enhancing state . We obseCD11b is part of the integrin receptor, CR3, with multiple ligands including iC3b/C3b. It is expressed exclusively on microglia in the CNS , and preCD11c-positive microglia appear early in response to plaque deposition and continue to increase with disease progression around plaques in APP/PS1 mice . A time-We further assessed pan-reactive and lysosomal markers Iba1 and CD68  decrease in C3 volume in CA1 in ArcC5aR1KO relative to Arc at 7 months of age  that drives rapid fibril formation and resilience to degradation, resulting in aggressive fibrillar plaque accumulation . Using tInflammatory stimuli such as lipopolysaccharide or IFN-γ, or chronic inflammatory states, promote polarization of astrocytes towards a pro-inflammatory state and the subsequent expression and secretion of Lcn2 , 85. FurAlthough overexpression of C5a under the GFAP promoter resulted in acceleration of behavioral deficits in the Arctic mouse, RNA-seq data revealed that overexpression of C5a in the Arctic mice delayed the rise of many inflammatory genes until later in disease progression (10 months). Interestingly, genes associated with synaptic transmission were upregulated in both C5a+ and ArcticC5a+ hippocampi. While these data appear to be opposed to the C5a-induced neuronal damage previously reported, the difference may be due to the in vitro cell cultures and/or the specific in vivo injury , 8. FurtWe observed peak hippocampal expression of most complement pathway genes at 7–10 months in the Arc and ArcC5aR1KO mice. Surprisingly, peak complement expression in the ArcC5a+ mice was delayed to 10 months. Consistent with the findings here, others have reported that stimulation with C5a increases gene expression of C5ar1 and C3ar1 in human-derived dendritic cells . FurtherC5a overexpression also induced genes involved in lipids and cholesterol metabolic processes, some of which were reduced upon C5ar1 deletion in the Arctic mouse and further enhanced (at later ages) by the transgenic expression of C5a. Although cholesterol cannot cross the blood–brain barrier, high levels of circulating cholesterol have been correlated with increased risk of dementia and drugs that reduced levels of cholesterol have been shown to reduce prevalence of AD , 102. OuThe clusters detected in our study have a partial overlap 94 genes) with the AD subtype signatures described by [ genes wiConsistent with RNA-seq gene expression, immunohistochemistry showed markedly induced levels of CD11b and CD68 in all Arctic groups at 5–10 months in hippocampus and cortex. Microglia surround Aβ plaques and promote elimination of deleterious proteins , and thuCD11c was also increased in all Arctic mice compared to WT, but at 7 months, the increase was not as pronounced in the ArcC5aR1KO or ArcC5a+ hippocampi compared to Arctic. By 10 months, CD11c levels were comparable between all Arctic groups, suggesting a delay, but not a complete attenuation of differentiation/polarization of this microglial state or subset activation. CD11c+ microglia are sparse in the healthy brain, but in models of AD, their proliferation is tightly associated with plaque pathology , 104. InEliminating C5aR1 from the Arctic mice delayed or reduced expression of genes enriched for inflammatory signaling pathways and microglial and astrocyte cell activation. Given the lack of altered amyloid plaque accumulation in the ArcC5aR1KO mice, these results demonstrate a separation between genes whose expression change in response to amyloid plaque deposition and genes whose expression change in response to C5a–C5aR1 signaling and thus enhanced inflammation. Ablation of C5ar1 in Arctic mice also delayed upregulation of some complement components, including C4b and C3ar1, suggesting that targeted inhibition of C5aR1 may directly or indirectly (via reduced inflammatory and neurotoxic environment) limit the supply of upstream complement components, such as C4b, thereby controlling excessive synaptic pruning as well as microglial activation. Taken together, these data are consistent with a neuroprotective effect of inhibition of the potent anaphylatoxin C5a–C5aR1 interaction, while leaving C1q and C3 immunoprotection intact, making C5a–C5aR1 signaling a promising therapeutic target for AD.Additional file 1: Fig S1. Presence of the C5aGFAP transgene increases C5a protein levels in brain. Fig S2. Locomotion and exploration during open field and object location memory training at 7 months. Fig S3. C5aR1 ablation reduces dystrophic neurites and reduces toxicity of amyloid plaques. Fig S4. Differential expression analysis show lack of sex-specific changes in gene expression in the Arctic mice. Fig S5. C5ar1 knockout in Arctic mice delays expression of characteristic AD and astrocyte-associated genes. Fig S6. Plaque accumulation is faster in hippocampus compared to cortex and is unaltered by C5a–C5aR1 modulation. Fig S7. Iba1 and CD68 not altered by C5a-C5aR1 modulation in Arctic mice, mirroring gene expression. Fig S8. Astrocyte polarization state in Arctic, ArcticC5aR1KO, and ArcticC5a+ mice.


I–V) characteristics of the n-GaN NRs/p-Si heterojunction were measured in the temperature range of 300–475 K. The ideality factor (n) and zero-bias barrier height (ϕB0) are found to be strongly temperature-dependent. The calculated values of ϕB0 are 0.95 and 0.99 eV according to Gaussian distributions (GD) and modified Richardson for GD, respectively, which are in good agreement with the band offset of GaN/Si (0.95 eV). A Richardson constant of 37 cm−2 K−2 was obtained from the modified Richardson plot, which is close to the theoretical value for p-Si (32 cm−2 K−2). The Gaussian distributions (GD) of inhomogeneous barrier height (BHs) and modified Richardson for GD of BHs with TE have also been used to explain the obtained transport properties.We report on the structural, electrical, and transport properties of high quality CVD-fabricated n-GaN nanorods (NRs)/p-Si heterojunction diodes. The X-ray diffraction (XRD) studies reveal the growth of hexagonal wurtzite GaN structure. The current–voltage /p-Si heterojunction diodes. Xu and co-workers investigated the I–V–T characteristics of n-GaN/n-Si heterojunctions at different temperatures (18–400 K) and they reported an ideality factor of 10.23 Recently, Tuan et al.24 studied the electrical properties of p-GaN/n-Si at a testing temperature of (300–450 K) and they estimated a Schottky barrier height in the range 0.5 to 0.62 eV. However, to the best of our knowledge, there is no report on the I–V–T characteristics of n-GaN/p-Si diodes in the absence of buffer layers.Therefore, it is of great importance to understand the nature of electrical transport through GaN/Si heterojunctions. In order to improve the performance of devices, it is neccessary to study the electrical characteristics of GaN/Si Schottky interfaces at different temperatures. A rigorous analysis of the current–voltage  directly on Si substrate and to examine the temperature-dependent electrical properties of the fabricated GaN NRs/p-Si heterojunctions. The obtained parameters, including ideality factor diode, barrier height (BHs), flat-band barrier height and Richardson constant, have been extracted from the thermionic emission and discussed.2.3 on Si substrate using chemical vapour deposition (CVD) system at 1050 °C for one hour. An alumina boat containing 0.2 g of Ga (99.99%) metal was placed at the center of a three-zone horizontal tube furnace (HTF) and Si (111) substrate was placed at 0.2 m away downstream from the Ga metal. The temperature of the center zone  was kept at 1000 °C during the evaporation process. The condensation products were deposited onto the Si substrate placed in a temperature zone of 1050 °C. An aqueous NH3 solution (120 mL) with a weight concentration of 25% solution was used as the nitrogen source and was kept at room temperature (RT). The N2 flow rate was maintained at 2 L min−1 under atmospheric furnace pressure during the growth process. The NH3 flow rate was controlled by the N2 flow-rate. When the temperature reached to growth temperature, NH3 gas was introduced into the HTF. After the reaction, the furnace was allowed to cool to RT naturally under an N2 flow rate of 2 L min−1. For further details, the reader may refer to  with a step size of 0.05°. The scanning range was between 2θ = 20° and 75°. The carrier type and concentration of the Si substrate were examined by Hall measurements using the HL5500PC system. After the contact formation, the sample was characterized by I–V in the dark condition. The electrical measurements were carried out by using a computer-controlled integrated source meter (Keithley 2400) at different temperatures (300–475 K). The operation temperature of the diode was measured using a calibrated K-type thermocouple mounted on the device.The morphological characterization of the sample was performed by field emission scanning electron microscopy (FE-SEM). The phase and purity of the GaN NRs were examined by X-ray diffraction (XRD) using Cu K3.2,25 The sharp and intense peaks indicate the good crystalline quality of the grown GaN NRs.I–V curves of the heterojunction device measured at different temperatures, indicating temperature-dependent behavior, which can be ascribed to the inhomogeneities at the interface.16,18 The current increases with increasing temperature due to the increase of the thermally-activated carriers and the reduction of the internal resistance. At high temperature, the rectifying nature deteriorates, possibly owing to the thermally-generated carrier tunneling.17,18 A slight decrease in the forward current at a higher temperature of 475 K was also observed. This behavior can be ascribed to the pronounced diffusion current at high bias voltage arising from the linear reduction in the barrier for electron and hole diffusion current. According to Einstein's relation, the diffusion current is strongly dependent on the carrier mobility.18 However, at higher temperatures, the phonon population increases resulting in decreased mobility. However, at a certain bias, an increase in temperature results in an increase in the forward current, an indicator that the current could be caused by the thermionic emission (TE). Thus, the current transport through a heterojunction at a forward bias can be understood based on the TE theory.26 The forward bias I–V measurements of the GaN/p-Si heterojunction can be given by 26,27q is the electronic charge, V is the applied forward bias voltage, k is the Boltzmann's constant, T is the absolute temperature, n is the diode ideality factor, A is the contact area, Js is the reverse-bias saturation density current, ϕB0 is the zero-bias barrier heights (BHs) of device, and A* is the effective Richardson constant (A* = 26.4 A cm−2 K−2 for n-GaN and 32 A cm−2 K−2 for p-Si).16,28 The values of BHs and the ideality factor (n) for the fabricated heterojunction were calculated as a function of temperature as extracted from the forward bias I–V curves. Both the saturation current density (Js) and n were estimated from the linear regions of the semi-log forward bias I–V curve according to 26J) against voltage, the slope gives the n and the intercept gives the Js. The obtained value of Js is used to determine the zero bias BHs (ϕB0):26The electrical characteristics of the grown n-GaN NRs/p-Si heterojunction were monitored as a function of forward and reverse bias voltage conditions at 300–475 K. n of the fabricated n-GaN/p-Si heterojunction diode were estimated at each temperature, as shown in 29 It was also observed that n deviates from unity and decreases with increasing temperature, which suggests the presence of an inhomogeneous barrier. The high values of n can be attributed to a wide distribution of low-Schottky barrier height (SBH) areas, tunneling through the interfacial layer, and a barrier inhomogeneities.20 Note that the dependence of ϕB and n on the temperature can be attributed to the heterogeneous and potential fluctuation at the interface.16,18,30 The BHs are possibly a function of the atomic inhomogeneities at the GaN/Si interfaces and the interface atomic structure, which are caused by nonuniformity of the interfacial charges, defects, and interfacial oxide layer composition.16 Consequently, the inhomogeneous BH may be varied as a result of the different types of defects that might exist at the GaN/Si interface. These observations are consistent with the TE theory and reported studies InGaN/Si barrier height17,21 and GaN/Si heterojunctions.11,20,23 However, in cases where the ideality factor is larger than unity, the TE would not be the only factor that promotes the transfer of current. As the BH depends on the applied voltage and, consequently, on the current flow across the interface, it is requisite to consider the standard field conditions. The electric field is zero across the interface under flat band (FB) conditions.The zero bias BHs and J–V graphs, which is derived from the zero-bias barrier height (ϕB0) via26ϕBF is the flat-band barrier height, EF is the Fermi energy, EV is the valence band energy, NV the effective density of states of the valence band, and NA is the carrier concentration. Hall measurements revealed that the carrier density of the Si substrate was ∼6 × 1019 cm−3 (p-Si). Notably, the deposition of the GaN layer formation causes the surface of the Si substrate to become heavily p-type doped through Ga in-diffusion.5 The GaN NRs layer is thought to have n-type behavior because of the N vacancies or/and oxygen contamination during the growth process.5,31 The FBBH was derived based on the assumption that the electron effective mass and NA  do not vary with temperature. While the effective density of states in the valence band (NV) will change with the temperature and is given by mh is the effective mass of hole (mh/mo = 0.56 for p-Si), T is the absolute temperature, and h is the Planck's constant. The relation can be written as NV (cm−3) = 4.3 × 1014(T)3/2 for p-type Si.32ϕBF slightly decreases with increasing temperature, whereas the zero-bias (ZB) BHs increase with increasing temperature. The difference between the ZB and the FBH values suggests low inhomogeneity and potential fluctuation at the interface.18,19 Futhermore, the temperature dependence of the ϕBF can be defined by ϕBF (T = 0) is the barrier height extrapolated to zero-temperature, and α is the temperature cofficient of ϕBF(T). The values of ϕBF(T = 0) and α have been extracted fom the intercept and slope according to ϕBF(T = 0) and α were ∼2.5 eV and 0.0023 eV K−1, respectively. The value of ϕBF(T = 0) is larger, which can be ascribed to the series resistance.33 Also, the value of average barrier height  PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.phvgrm.i" format="PBM" resolution="300" x-size="9" y-size="12" xml:space="preserve">001111100000000000000101110001001010010010010010010010010010110010100100001111000000100000001000000001000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="12.500000pt" height="16.000000pt" viewBox="0 0 12.500000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M160 920 l0 -40 200 0 200 0 0 40 0 40 -200 0 -200 0 0 -40z M240 760 l0 -40 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 -160 0 -160 40 0 40 0 0 -40 0 -40 40 0 40 0 0 -40 0 -40 -40 0 -40 0 0 -80 0 -80 40 0 40 0 0 80 0 80 40 0 40 0 0 40 0 40 80 0 80 0 0 40 0 40 40 0 40 0 0 40 0 40 40 0 40 0 0 200 0 200 -120 0 -120 0 0 -80 0 -80 -40 0 -40 0 0 -120 0 -120 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 160 0 160 40 0 40 0 0 40 0 40 40 0 40 0 0 40 0 40 -40 0 -40 0 0 -40z m320 -160 l0 -120 -40 0 -40 0 0 -80 0 -80 -80 0 -80 0 0 40 0 40 40 0 40 0 0 120 0 120 40 0 40 0 0 40 0 40 40 0 40 0 0 -120z"/></g></svg>B is lower than the ϕBF(T = 0), and can be explained by lateral inhomogeneities of barrier heights.To extract more details on the barrier height, the flat band barrier height (FBBH) can be obtained from the Js) were calculated at each temperature, and a “conventional” Richardson's plot, ln Is/T2versus 1/kT, is plotted as shown in 26,27Another method to extract the BH is through the application of a Richardson plot. From the experimental data measured in −8 A cm−2 K−2, respectively. The obtained Richardson's constant is eight orders of magnitude smaller than the theoretical value of A* for p-Si.32 This deviation in A* is generally explained by the barrier inhomogeneity of the contacts, which means that it consists of high and low barrier areas at the interface.34 The inhomogeneous BH probably arises due to several types of defects present at the GaN NRs/Si hetero-interfaces.35 In addition, the effective area for current conduction (Aeff) is significantly lower than the geometric contact area (Ageom) due to the preferential current flow through the lower barrier height regions.18 In order to account for the temperature dependent n and BH, the modified Richardson's plot of ln(Is/T2) versus 1/nkT as proposed by Hackam and Harrop36 is shown in ϕB0) and modified Richardson's constant were determined from the slope and intercept, respectively. The values of effective ϕB0 and the Richardson's constant A* were 1.475 eV and 37.48 A cm−2 K−2, respectively. The value of A* was eight orders of magnitude higher than that obtained from the conventional Richardson's plot. However, the value of A* obtained from the modified Richardson plot was found to be closer to the theoretical value for p-Si.11,32From this linear fit, the BH and the conventional Richardson's constant were 0.366 eV and 8.76 × 1037 This method uses GS to relate experimental values of BH extracted from I–V measurements. Using ϕB allows the values of BH calculated from the I–V data to be plotted against the inverse thermal energy to extractthe standard deviation (σs) and mean BH B, which can be given by 26 is the normalization constant of the Gaussian barrier height distribution. The total current I(V) across a heterojunction diode containing barrier inhomogeneities is expressed by 37I is the current at a bias V for a barrier of height based on the ideal thermionic emission diffusion (TED) theory and P(ϕB) is the normalized distribution function giving the probability of accuracy for barrier height. Solving the 26,27,38The other method is to use the Gaussian statistics (GS) to modify the conventional Richardson plot to determine the BHs, taking into account SBH lowering due to an inhomogeneous contact.I(V) can be derived through a Schottky barrier at a forward bias but with a modified barrier as:38ϕap and nap are the apparent barrier height at zero bias and apparent ideality factor, respectively, and can be resolved by 37,38B) and standard deviation (σs) are assumed to be linearly bias-dependent on Gaussian parameters. The values of B and σs can be expressed as: ϕB = ϕB0 (T = 0) + ρ2V and σs = σs0 + ρ3V, where ρ2 and ρ3 are voltage coefficients, which may depend on temperature. The voltage coefficients quantify the voltage deformation of the barrier height distribution.38 The value of σs is usually very small therefore, can be ignored.39 Verification of σs0 and ϕB0 values can be carried out using a modified Richardson plot. I–V analysis plotted against the q/2kT and ideality factor versus q/2kT according to the Gaussian distribution (GD) of the BHs. The plot of ϕBversus q/2kT should be a straight line. The values of σs0, and B were extracted from the slope and intercept and found to be 0.0091 V and 0.993 eV, respectively. However, the voltage coefficients ρ2 and ρ3, which can be extracted from (1/n) − 1 versus q/2kT, were also reported in ρ2 of 0.123 and ρ3 of 0.025 eV were obtained. The linear behavior plot shows that the ideality factor expresses the voltage deformation of the GD of the SB contact. The structure with the best rectifying performance presents the best barrier homogeneity with the lower value of the standard deviation.Applying the above integration , the curσs0 and ϕB0 values can be carried out using a modified Richardson plot. The Richardson plot can be modified by combining Verification of Is/T2) − σs02q2/2(kT)2versus 1/kT. From the slope and the intercept of the modified plot, the mean B0 and modified Richardson's were extracted. The value of mean B0 obtained is 0.95 eV. Also, using AA*) at the ordinate determined A* for a given diode area (i.e. A ∼ 1 cm2) as 39.25 A cm−2 K−2. From the modified Richardson's plot and according to ϕB0 = 0.95 eV is consistant with that of ϕB0 = 0.99 eV extracted from the plot in ϕB0 = 1.475 eV from 20 The preceding results confirm that predominant current transport is not the only method for ascertaining barrier height inhomogeneities (BHI) in GaN/Si heterojunction sample. The results also establish that Gaussian distributions (GD) of BHs and modified Richardson for GD of BHs are also applicable. Eg) and electron affinities (χ) used are Eg(Si) = 1.12 eV, χ(Si) = 4.05 eV, Eg(GaN) = 3.4 eV, and χ(GaN) = 3.1 eV, respectively, hence the conduction band offset (ΔEC = χ(GaN) − χ(Si)) is 0.95 eV, which is much smaller than the valence band offset, i.e., ΔEV = ΔEg − ΔEC = (1.33 eV).35,394.I–V characteristics of the fabricated heterojunction has been discussed. Using the TE model, the ideality factor (n) and barrier height (BH) of the heterojunction device were determined from the forward I–V curves under dark conditions. It is found that the values of ideality factor decreases with increasing the temperature, which suggests the presence of an inhomogeneous barrier. Deviations of n from unity reveal that TE will not be the only way that promotes the current transfer. It is observed that the ϕBF slightly decreases with increasing temperature, whereas the zero-bias (ZB) BHs increase with temperature. We have obtained a temperature coefficient BH of (α = 2.3 meV K−1) for this heterojunction. The measured value of the effective barrier height (∼1.48 eV) via the modified Richardson's plot is larger than that of BHs of 0.99 eV and 0.95 eV obtained by GD and modified Richardson GD, respectively. The calculated values of BHs and Richardson constant, which were extracted from the modified Richardson's GD of BHs, are reasonably acceptable.In conclusion, a high-quality n-GaN NRs/p-Si heterojunction device has been successfully fabricated. The relatively sharp diffraction peaks in the XRD spectra indicated the growth of highly crystalline GaN nanorods with wurtzite structure. The temperature dependence of the The data that supports the findings of this study are available upon request.There are no conflicts to declare.