In summary, the N-terminal region of Jaw1 inhibits the formation of OSER as an IDR to maintain the homeostatic localization of interactors on the ER membrane.Jaw1/LRMP is a type II integral membrane protein that is localized at the endoplasmic reticulum (ER) and outer nuclear membrane. We previously reported that a function of Jaw1 is to maintain the nuclear shape as a KASH protein via its carboxyl terminal region, a component of linker of nucleoskeleton and cytoskeleton complex in the oligomeric state. Although the oligomerization of some KASH proteins via the cytosolic regions serves to stabilize protein-protein interactions, the issue of how the oligomerization of Jaw1 is regulated is not completely understood. Therefore, we focused on three distinct regions on the cytosolic face of Jaw1: the N-terminal region, the coiled-coil domain and the stem region, in terms of oligomerization. A co-immunoprecipitation assay showed that its coiled-coil domain is a candidate for the oligomerization site. Furthermore, our data indicated that the N-terminal region prevents the aberrant oligomerization of Jaw1 as an intrinsically disordered region (IDR). Importantly, the ectopic expression of an N-terminal region deleted mutant caused the formation of organized smooth ER (OSER), structures such as nuclear karmellae and whorls, in B16F10 cells. Furthermore, this OSER interfered with the localization of the oligomer and interactors such as the type III inositol 1,4,5-triphosphate receptor (IP In addition, in the NE, a continuous region of the ER membrane, i.e. an inner nuclear membrane (INM) and an outer nuclear membrane (ONM), a linker of nucleoskeleton and cytoskeleton (LINC) complex consisting of Klarsicht/ANC-1/Syne/homology (KASH) proteins in the ONM and Sad-1/UNC84 (SUN) proteins in the INM functions as a physical linkage between cytoskeletons and nuclear lamina for the maintenance of the nuclear network such as shaping, positioning and movement5. To date, several proteins implicated in ER morphogenesis have been reported to be involved in hereditary spastic paraplegia (HSP) and hereditary sensory and autonomic neuropathy (HSAN), neurodegenerative diseases7. In addition, dysfunctions of nuclear physiology due to defects in the LINC complex have been reported to be causes of Emery–Dreifuss Muscular Dystrophy9, infertility11, cerebellar ataxia13 and the Hutchinson-Gilford Progeria syndrome (HGPS)14. It has been also reported that the expression levels of some components of the LINC complex are altered in cancer tissues15. Thus, the integrated morphological homeostasis of the ER including the nucleus is crucial for regulating many cellular events and physiology.The endoplasmic reticulum (ER) is a reticular membranous network consisting of a nuclear envelope (NE), sheets surrounding the nucleus and tubules in the periphery. The ER is involved in many cellular events such as protein synthesis, lipid synthesis, calcium transfer, membrane trafficking, stress responses as well as related actions. ER morphogenesis is regulated by several proteins, including Climp-63, kinectin and p180 on the sheets25. For example, previous investigations have reported that the ectopic expression of 3-Hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase16, the lamina-associated polypeptide 2 beta (Lap2β)17 starch-binding domain-containing protein 1 (Stbd1)18 causes the OSER formation. The oligomeric fluorescence protein tagged cytochrome b(5)19 and Sec61γ19, calnexin21 and Langerin22 has also been reported to form the OSER, indicating that a tightly stacked membranous environment is formed between the apposed membranes due to the irregular or excessive oligomerization of ER resident membrane proteins. In addition, OSER formation that results from the expression of mutants such as TorsinA23, TorsinB24 and the vesicle-associated membrane protein-associated protein B/C (VAPB)25 has also been reported to be relevant to diseases such as dystonia and amyotrophic lateral sclerosis, which suggests the existence of OSER structures under conditions of a physiological disease. Nevertheless, the details of the OSER, such as morphogenesis, plasticity and the effects on the cellular events remain to be unveiled.It has been reported that the overexpression of several ER resident membrane proteins results in the formation of a stacked ER with highly dense structures, such as cisternae, whorls, nuclear karmellae which are grouped as organized smooth ER (OSER)28. Current information indicates that Jaw1 is expressed in taste buds and lymphoid organs such as the spleen, thymus, bone marrow and tonsils30. We recently reported that Jaw1 has a role in maintaining the nuclear shape at ONM as a KASH protein28. Trimeric KASH proteins interact with trimeric SUN proteins in the perinuclear space (PNS) via the KASH domain and the SUN domain, respectively, resulting in the formation of a hetero-hexamer complex33. Intriguingly, some KASH proteins such as Nesprin3α and KASH5 have been reported to be oligomerized via their cytosolic regions to stabilize the interaction with proteins34. As we previously reported, Jaw1 also forms an oligomer, but the issue of whether or not the oligomerization is mediated by its cytosolic region has not been revealed28. Furthermore, Jaw1 has a predicted coiled-coil domain in the middle region on its cytosolic face which interacts with the type III inositol 1,4,5-triphosphate receptor (IP3R3), a calcium-gate channel on the ER membrane30. However, the biological significance of the interaction between these molecules is not well understood. Furthermore, the structure and function of the rest of cytosolic regions except the coiled-coil domain have also not been determined. In this study, the cytosolic face of Jaw1 was defined as follows: the N-terminal region (44-183) as an uncharacterized N-terminal region of the coiled-coil domain, the predicted coiled-coil domain (194-333), and stem region (334-469) as an uncharacterized intermediate region between coiled-coil domain and single trans-membrane domain. According to these definitions, we attempted to clarify the function of the cytosolic region of Jaw1.Jaw1 is a type II integral membrane protein that is localized on the ER and ONMThe findings reported herein show that Jaw1 probably forms an oligomer via its coiled-coil domain. We also show that the N-terminal region has a role in inhibiting OSER formation, preventing the irregular oligomerization as an intrinsically disordered region (IDR), a structurally unstable region within the protein. The OSER derived from the expression of a mutant (Jaw1 ΔN) lacking the N-terminal region recruits the interactors to this structure, but does not affect the conventional ER resident proteins, calnexin and calreticulin. Finally, based on our findings, we propose that the precise oligomerization of Jaw1 regulated by the N-terminal region is crucial for the normal localization of Jaw1 at the ER and the precise localization of interactors.28. However, the oligomerization site has not been identified. Furthermore, the possibility that the oligomerization is due to the interaction between the KASH domain and the SUN domain of the PNS has not been excluded. Therefore, we first investigated the issue of whether the cytosolic region is involved in the oligomerization or not. To accomplish this, we focused on three distinct cytosolic regions: the N-terminal region of the coiled-coil domain , the coiled-coil domain and the C-terminal region of the coiled-coil domain (stem region). For a co-immunoprecipitation (Co-IP) assay, we prepared plasmids encoding GFP-fused mouse Jaw1 long form (GFP Ms LJaw1) and GFP-fused mutants encoding entire cytosolic regions (GFP Ms LJaw1 ΔTM) and single regions   tagged protein (MBP Ms LJaw1 N). The MBP Ms LJaw1 N was purified by affinity chromatography using an amylose resin and the MBP tag was digested with TEV protease. After the digestion, MBP and Ms LJaw1 N were separated by anion exchange chromatography and affinity chromatography using amylose resin , LaminB1 and SUN224. In order to characterize the OSER derived from Jaw1 ΔN, the effects on the localization of ER resident proteins were first evaluated. Here, in cells expressing Ms LJaw1, Ms SJaw1 or Ms Jaw1 ΔN, the localization of the ER markers; calnexin or calreticulin were observed. The confocal images and line plot profiles showed that both Ms LJaw1 and Ms SJaw1 are co-localized with calnexin nearly and calreticulin strongly within the conventional ER network  or SUN2 (FLAG SUN2) were co-expressed with Ms LJaw1, Ms SJaw1 or Ms Jaw1 ΔN in B16F10 cells and co-immunostaining using an anti-FLAG antibody and an anti-Jaw1 antibody was carried out. The confocal images and line plot profiles showed that FLAG IP3R3 was nearly co-localized with Jaw1 in the cells expressing Ms LJaw1 and Ms SJaw1  compared to remainder of the region (71.9–92.9%)  and a non-diabetic model  provided some candidate mutations of mouse Jaw1 that are related to type I diabetes such as G6D, L145P, duplication of three amino acids [STL (452-455)]44. Intriguingly, all of the mutated positions are located in the N-terminal region or stem region. Furthermore, some of the residues are also conserved in humans  was observed. However, interestingly, the national center for biotechnology information (NCBI) database shows the existence of a transcript variant NM_001361633) coding for the amino acid sequence corresponding to Ms Jaw1 ΔN1, which would be a variant with a shorter N-terminal region and would form the OSER at slightly higher percentage than LJaw1 and SJaw1, as shown in Fig. 61633 cod28. From the same library, a DNA fragment coding Ms SJaw1 (NM_001368864.1) was amplified by PCR with primer set 1 and the PCR product was subcloned into the pcDNA5 FRT/TO HA FLAG vector (digested with KpnI/BamHI) using an In-Fusion HD cloning Kit , resulting in pcDNA5/FRT/TO HA FLAG Ms SJaw1. These plasmids were digested with KpnI/BamHI and the fragment was ligated into the pTagGFP2-C vector (Evrogen) (digested with the same enzymes), resulting in pTagGFP2-C Ms LJaw1 and SJaw1, respectively. To produce pTagGFP2-C Ms LJaw1 ΔTM coding for entire cytosolic regions, the PCR product amplified from pcDNA5FRT/TO HA FLAG Ms LJaw1 with primer set 2 was inserted into the pTagGFP2-C vector  using an In-Fusion HD cloning Kit. Furthermore, for the generation of the plasmids encoding Jaw1 each cytosolic region, the PCR products amplified from pTagGFP2-C Ms LJaw1 with primer sets 3, 4 and 5 were inserted into the pcDNA3.1(+) vector (Invitrogen) (digested with BamHI/XhoI) using an In-Fusion HD cloning Kit. These plasmids were digested with KpnI/ApaI and each fragment was then ligated into the pTagGFP2-C vector (digested with the same enzymes), resulting in pTagGFP2-C Ms LJaw1 N, Jaw1 Coil and Jaw1 Stem, respectively. For the generation of deletion mutants: pcDNA5 FRT/TO HA FLAG Ms Jaw1 ΔN and ΔN Coil, the PCR products amplified from pTagGFP2-C Ms LJaw1 using primer sets 6 and 7, respectively, were inserted into the pcDNA5/FRT/TO HA FLAG vector  using an In-Fusion HD cloning Kit. Furthermore, pcDNA5 FRT/TO HA FLAG Ms Jaw1 ΔN was digested with KpnI/BamHI and the fragment was ligated into the pTagGFP2-C vector, resulting in pTagGFP2-C Ms Jaw1 ΔN. To produce pcDNA5 FRT/TO HA FLAG Ms LJaw1 ΔCoil and ΔStem, pcDNA5 FRT/TO HA FLAG Ms LJaw1 was performed by inverse PCR to remove the region coding for the coiled-coil domain or stem region using 5′-phosphorylated primer sets 8 and 9, respectively, and self-ligated. To remove the possible effects of the N-terminal tag, pcDNA5 FRT/TO HA FLAG Ms LJaw1 and pcDNA5 FRT/TO HA FLAG Ms SJaw1 was digested with KpnI/BamHI and each fragment was ligated into the pcDNA3.1(+) vector (digested with the same enzymes), resulting in the pcDNA3.1(+) Ms LJaw1 and SJaw1, respectively. In addition, for the generation of pcDNA3.1(+) Ms Jaw1 ΔN, the PCR product amplified from pTagGFP2-C Ms LJaw1 with primer set 10 was inserted into the pcDNA3.1(+) vector (digested with BamHI/XhoI) using an In-Fusion HD cloning Kit. To produce the limited N-terminal region-truncated mutants, pcDNA3.1(+) Ms LJaw1 was performed by inverse PCR to remove the region coding for each limited N-terminal region using primer sets 11, 12 and 13, resulting into pcDNA3.1(+) Ms LJaw1 ΔsN1, ΔsN2 and ΔsN3. For the generation of pcDNA3.1(+) Ms LJaw1 ΔN1 and ΔN2, the PCR products were first amplified from pTagGFP2-C Ms LJaw1 using primer sets 14 and 15, respectively, and inserted into the pcDNA5 FRT/TO HA FLAG vector (digested with BamHI/XhoI) using an In-Fusion HD cloning Kit. These plasmids were then digested with BamHI/XhoI and each fragment was ligated into the pcDNA3.1(+) vector (digested with the same enzymes). To generate pcDNA3.1(+) PA Ms LJaw1 and Jaw1 ΔN, pcDNA3.1(+) PA vector was first prepared by the insertion of DNA cassette encoding PA tag (primer set 16) into the pcDNA3.1(+) vector (digested with NheI/HindIII). pcDNA3.1(+) Ms LJaw1 and Jaw1 ΔN were then digested with KpnI/BamHI and KpnI/XhoI, respectively, and each fragment was ligated into the pcDNA3.1(+) PA vector. To produce the pMALcRI TEV Ms LJaw1 N, LJaw1 N Coil and the Jaw1 Coil, the PCR products amplified from pTagGFP2-C Ms LJaw1 with primer sets 3, 17 and 4, respectively, were inserted into the pcDNA3.1(+) vector (digested with BamHI/XhoI) using an In-Fusion HD cloning Kit. These plasmids were digested with BamHI/XhoI and each fragment was then ligated into the pMALcRI TEV vector (previously described28) , resulting in pMALcRI TEV Ms LJaw1 N, LJaw1 N Coil, Jaw1 Coil, respectively. For the generation of the pcDNA5 FRT/TO HA FLAG Hu LJaw1, SJaw1 and Jaw1 ΔN, the PCR products were amplified from a total cDNA derived from Burkitt’s lymphoma cell line (Raji) with primer sets 18, 19 and 20 and inserted into the pcDNA3.1 (+) vector (digested with BamHI/XhoI) using an In-Fusion HD cloning Kit. pcDNA5 FRT/TO HA FLAG Ms SUN2 was produced from EGFP-C1 SUN2  as previously described28. pCMV6 Myc-DDK-tagged Mouse IP3R3 (NM_08053) was purchased from Ori Gene (MR225699). The identity of cloned sequences was verified by the DNA sequence contract service (eurofin). After confirmation, each plasmid was prepared using the Plasmid Mini Kit  and used for following experiments.The primer sets used to produce the plasmids below are shown in Supplementary Table L-glutamine and in DMEM containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin (Sigma) and 5.84 mg/mL l-glutamine, respectively, and maintained at 37 °C in 5% CO2.B16F10 and HEK293 cells were incubated in MEM supplemented with 10% fetal bovine serum (FBS) and 5.84 mg/mL of FectTMA plus  according to the manufacturer’s instructions. After the treatment for 24 h, the cells were used for the following assays.Plasmids were introduced into B16F10 or HEK293 cells using ScreenB16F10 cells were grown on 8 well chamber slides . After the transfection, the cells were washed with PBS and fixed in 4% paraformaldehyde (PFA)/PBS for 10 min. After washing with PBS, the cells were permeabilized by treatment with 0.1% Triton X-100/PBS for 5 min. After washing with PBS, the cells were blocked with 3% bovine serum albumin (BSA) diluted with PBS for 1 h and reacted with the following primary antibodies diluted with 1% BSA/PBS for 1 h; anti-FLAG mouse monoclonal antibody (1:500) , anti-FLAG rabbit antibody (1:500) , anti-PA rat antibody (1:500) , anti-GM130 mouse antibody (1:250) , anti-calnexin rabbit antibody (1:500) , anti-Calreticulin rabbit antibody (1:200)  and anti-Jaw1 rat antibody (1:200) (produced in our laboratory as previously described (28)). After washing with 0.1% BSA/PBS for 5 min three times, the cells were incubated with the following secondary antibodies diluted into 1:500 with 0.1% BSA/PBS containing Hoechst33342 for 1 h; Alexa Fluor™ 488 goat anti-mouse IgG (H + L) , Alexa Fluor™ 488 goat anti-rabbit IgG (H + L) , Alexa Fluor™ 488 goat anti-rat IgG (H + L) , Alexa Fluor™ 568 goat anti-mouse IgG (H + L)  and Alexa Fluor™ 568 goat anti-rabbit IgG (H + L) . After washing with 0.1% BSA/PBS for 5 min three times, the cells were mounted with VECTOR SHIELD (Vector laboratories). For the staining of mitochondria, Mito Tracker Orange CMTMRos  was used before the fixation according to manufacturer’s instructions. The images for statistics were acquired using fluorescence microscopy  . The confocal images were acquired using confocal microscopy  .g for 10 min at 4 °C. The pellets were lysed in lysis buffer  containing 1 µL of a protease inhibitor cocktail . The lysates were sonicated for 10 min on ice and centrifuged at 12,000×g for 20 min at 4 °C. A portion of the supernatants was used as an input and the remainder was added to Anti DYDDDDK tag Antibody Beads  equilibrated with incubation buffer  at 4 °C for 2 h. The beads were then collected by brief centrifugation and washed four times with washing buffer . For elution, the beads were mixed with SDS-PAGE buffer and heat blocked at 95 °C for 5 min. After a brief centrifugation, the supernatants were subjected to western blotting.HEK293 Cells were cultured on a 6 well plate . After the transfection, the cells were peeled off by gentle pipetting and centrifuged at 500×The samples were subjected to western blotting following SDS-PAGE. Polyvinylidene Fluoride membranes were blocked in 3% skim milk  diluted with Tris-Buffered Saline (TBS) (20 mM Tris–HCl pH7.6 and 137 mM NaCl) containing 0.1% Tween-20 (TBS-T) for 1 h. After washing the membrane with TBS-T, it was reacted with the following primary antibodies diluted with 1% skim milk/TBS-T for overnight; anti-FLAG rabbit antibody (1:1000)  and anti-GFP rabbit polyclonal antibody (1:1000) . After washing the membrane with TBS-T, it was incubated with a secondary antibody ) diluted into 1:5000 with TBS-T for 1 h. After washing the membrane with TBS-T, Immunostar Zeta  was used as substrates and the bands were detected using LAS3000.47. B16F10 cells grown in a 35 mm dish and transfected with pcDNA5 FRT/TO HA FLAG Ms Jaw1 ΔN were prefixed by treatment with 2% PFA and 2% glutaraldehyde in 30 mM HEPES buffer (pH 7.4) for 4 h at room temperature. After washing with 30 mM HEPES containing 5% sucrose gently twice, post-fixation was performed in a 1% OsO4 mixture containing 0.8% K3[Fe(CN)6] in 30 mM HEPES buffer (pH 7.4) for 1 h at room temperature. After washing with hyperpure water, the cells were stained en bloc with EM stainer (Nisshin EM) and dehydrated in ethanol, and then embedded in Quetol 812 (Nisshin EM). The blocks were sectioned using an ultramicrotome  and were then observed by electron microscopy .For observing OSER structures using transmission electron microscopy, the samples were prepared as previously describedd-1-thiogalactopyranoside for 4 h at 25 °C. 9 mL of culture media was centrifuged at 8000 rpm for 10 min and the pellets were resuspended in washing buffer (10 mM Tris–HCl pH 7.4 and 200 mM NaCl). The suspensions were sonicated on ice and centrifuged at 12,000 rpm for 30 min. The supernatants were performed for purification using an amylose resin . MBP tag was removed by the treatment of TEV proteases (0.58 mg/mL) for 16 h at 4 °C. The purified recombinant proteins were mixed with SDS-PAGE sample buffer, heat blocked at 95 °C for 5 min and subjected to SDS-PAGE. The resulting purified proteins were mixed with Native-PAGE sample buffer and subjected to Native-PAGE. The gels were then subjected to CBB staining. For the preparation of the samples for the circular dichroism spectrometry, BL21(DE3) transformed with pMALcRI TEV Ms LJaw1 N was cultured in 1 L LB medium and the expression of the MBP Ms LJaw1 N was induced by treatment with 1 mM isopropyl β-d-1-thiogalactopyranoside for 4 h at 25 °C. The culture media was centrifuged at 8000 rpm for 10 min and the pellet was resuspended in washing buffer. The suspension was sonicated on ice and centrifuged at 12,000 rpm for 30 min. The supernatant was purified using an amylose resin. Purified MBP Ms LJaw1 N was treated with TEV proteases (0.58 mg/mL) for 16 h at 30 °C to remove the MBP tag, resulting to MBP tag and Ms LJaw1 N. After digestion, the His tagged TEV proteases was removed by the affinity chromatography using Ni–NTA Agarose . The solution containing MBP tag and Ms LJaw1 N was concentrated with Amicon Ultra-15 Centrifugal Filter , desalted in 20 mM Tris–HCl pH 8.0 and each protein was separated by anion exchange chromatography [HiTrap Q FF ] on FPLC ÄKTAPurifier system . A gradient of 0 to 50% of buffer B (20 mM Tris–HCl pH 8.0 and 1 M NaCl) was applied. The fractions containing Ms LJaw1 N were concentrated with Amicon Ultra-15 Centrifugal Filter, desalted in 20 mM Tris–HCl pH 8.0 and subjected to anion exchange chromatography followed by affinity chromatography using amylose resin to remove the MBP tag or MBP tagged proteins. Each solution containing MBP or Ms LJaw1 N was concentrated with Amicon Ultra-15 Centrifugal Filter and desalted in 20 mM Tris–HCl pH 8.0. After that, the samples were mixed with SDS-PAGE sample buffer, heat blocked at 95 °C for 5 min and subjected to SDS-PAGE followed by CBB staining.BL21(DE3) transformed with pMALcRI TEV Ms LJaw1 N Coil and Ms Jaw1 Coil were cultured in three test tubes containing 3 mL LB medium and the expression of the MBP Ms LJaw1 N Coil or the Ms Jaw1 Coil were induced by treatment with 1 mM isopropyl β-48. Briefly, purified MBP or Ms LJaw1 N were dissolved in 20 mM Tris–HCl pH7.4, 200 mM NaCl at a concentration of 0.1 mg/mL. CD spectra were recorded on a J-720WI spectrophotometer (Jasco GmbH) using at a 0.1 cm optical path, 0.1 nm interval, 1.0 nm bandwidth and 50 nm/min scanning speed. The spectra were recorded five times followed by averaging and background subtraction. The HT voltage was below 700 V over the entire range of recording (200–250 nm).CD spectroscopy was performed as previously described2P2 platform to explore the intrinsically disordered region within the amino acid sequence coding for Jaw1 were used in the online page below (http://210.137.243.169/dichot/) and (http://d2p2.pro), respectively57. The entire amino acid sequences of human and mouse Jaw1 were analyzed.The DICHOT algorithm and Dt-test.The collected data were analyzed and graphically presented using GraphPad Prism7 (GraphPad). Statistical significance was determined by one-way ANOVA followed by Tukey Kramer’s Supplementary Information.Supplementary Table S4.


Graves’ disease(GD) has a tendency for familial aggregation, but it is uncommon to occur in more than two generations. However, little is known about susceptibility genes for GD in the three-generation family.DNA were extracted from three-generation familial GD patient with a strong genetic background in a Chinese Han population. The Whole Exome Sequencing (WES) was utilized to screen the genome for SNVs associated with GD and the Sanger Sequencing was used to confirm the potential disease-causing genes.MAP7D2(c. 452C > T: p. A151V), SLC1A7(c. 1204C > T: p. R402C), TRAF3IP3(c. 209A > T: p. N70I), PTPRB(c. 3472A > G: p. S1158G), PIK3R3(c. 121C > T: p. P41S), DISC1(c. 1591G > C: p. G531R) were found to be associated with the familial GD and the Sanger sequencing had confirmed these variations. Furthermore, PolyPhen-2 score showed that the variants in TRAF3IP3, PTPRB, PIK3R3 are more likely to change protein functions.In the case study, there were five patients with Graves’ disease(GD) from a three-generation family. The SNVs of MAP7D2, SLC1A7, TRAF3IP3, PTPRB, PIK3R3, DISC1 may be the candidate susceptibility genes for familial GD from a three generations family.The  Graves’ disease is a thyroid-related autoimmune disorder which is caused by a complex interaction between susceptibility genes and multiple environmental factors. Previous familial and twin studies had shown that there was an association of genetic factors with Graves’ disease in 79% cases of Graves’ Disease which influenced the familial clustering in GD . People HLA gene and CTLA-4 gene. However, linkage analysis has not addressed the need for fine gene mapping in complex diseases and linkage analysis also requires a large familial sample size to localize the pathogenic genes by observing whether the genetic markers are co-segregated with the disease. In contrast, Whole Exome Sequencing can explore the susceptibility genes with a small sample size. Whole Exome Sequencing is an efficient strategy for fine mapping to determine the exact location of variant [In addition to twins study, the familial GD is the ideal object of study on contribution of genetic heritability to complex disease. Findings from family linkage analysis indicated that the 5q31-q33, 6p, 7q, 8q, 10q, 12q, 14q and 20q regions were related to GD susceptibility [131 for anti-thyroid treatment. As of the month before the article was submitted, it was confirmed that the control group was still free of Graves' disease. Table A three-generation family from Zhanjiang, Guangdong Province had been targeted for the case study. There were 25 people in the family tree including 13 males and 12 females  was used and the reference range of indicators are: FT3: 2.3–6.8 pmol/L; FT4: 10–23.5 pmol/L; TSH: 0.34 ~ 4.0mIU/L; TRAb: 0 ~ 1.75 IU/L. For preparation of DNA, genomic DNA from EDTA-treated peripheral blood was extracted according to DNA extraction kit manual MAP7D2, SLC1A7, TRAF3IP3, PTPRB, PIK3R3, DISC1) that were in all the participants.The Agilent SureSelect Human All Exome Kit(Agilent) was used for exon capture. The sequencing processes were exon capture, hybrid library cleaning and purification, PCR amplification of exon DNA library, library quality detection, sequencing, and data analysis. The PCR reaction conditions of PCR Amplification was initially 30 s at 98 °C, 10 cycles of 98 °C for 10 s, 10 cycles of 60 °C for 30 s, 10 cycles of 72 °C for 30 s, 10 cycles of 72 °C for 5 min at 72 °C, followed by a final extension of 4 °C. The exome region was sequenced by illumine hiseq2500, and GATK standard procedure was adopted to calibrate the initial data. Quality control of raw reads was performed with fastqc. Transition and Transversion (SNV) and Insertion and Deletion (InDel) of each sample were detected though VarScan and GATK HaplotypeCaller. In addition, SNV stands for single nucleotide variants and SNP stands for single nucleotide polymorphism. Both concepts refer to single nucleotide changes, but SNPs are generally two-state and SNV has no such restriction. In addition, if the frequency of the single-base variation in a species reaches a certain level, it is called SNP, and if the frequency is unknown , it is called SNV. Sanger sequencing was used to confirm the genotype variant of 6 genes , SLC1A7(c. 1204C > T: p. R402C), TRAF3IP3(c. 209A > T: p. N70I), PTPRB(c. 3472A > G: p. S1158G), PIK3R3(c. 121C > T: p. P41S), DISC1(c. 1591G > C: p. G531R). All SNV/InDel sites in the case study were rare variants according to several known population databases . In addition, Chinese Millionome Database  were applied for the comparative analysis. CMDB contain considerable variation and their allele frequency information came from 141,431 unrelated healthy Chinese individuals (Phase I results). SNV function analysis indicated that all the SNV of MAP7D2, SLC1A7, TRAF3IP3, PTPRB, PIK3R3, DISC1 were of nonsynonymous variations. The genotype of the 6 genetic variations from the 7 samples by Sanger sequencing was the same with the results from the whole exome sequencing  is located on X chromosome. MAP7D2 is specifically expressed in human brain tissue which has impact on the behavioral traits and cognition in human. MAP7D2 is also associated with sex-biased mental illnesses [MAP7D2 study is likely to provide important general information about the reason why women are more vulnerable to GD. SLC1A7 and DISC1 are also susceptibility genes for mental illnesses. In one research, Keith A. Young et al. discovered that DISC1 gene played a vital role in post-traumatic stress disorder (PTSD) severity of US military veterans [SLC1A7 gene expression in peripheral blood leukocytes was responsible for the association between socioeconomic status and depressive mood in healthy adults [SLC1A7 and DISC1 are involved in regulating the symptoms of GD such as nervousness and irritabilityllnesses . Previoullnesses . Thus, tveterans . In anoty adults . We specPTPRB belongs to the family of protein tyrosine phosphatases (PTP). The activation of PTK (protein tyrosine kinase) was regulated through the binding of SH2 domains from PI3K(PIK3R3 gene encode). The balance of tyrosine protein phosphorylation was regulated by PTP/PTK which participated in cell signal transduction, cell growth regulation, differentiation, metabolism, transcription, immune responses, etc. Researches have demonstrated that the significant role of PTKs and PTPs were to modulate the tyrosine phosphorylation-dependent signaling pathways which were critical for the effector of NK cell and Neutrophil cell [TRAF3IP3 is also highly expressed in CD34 + CD38 + CD7 + common lymphoid progenitors (CLPs) Furthermore, CD34 + CD38 + CD7 + cells have the capacity to differentiate into B/NK/T cell which implies that TRAF3IP3 possibly may play a role in lymphoid development [CTLA-4 and CD40 gene variants which has been confirmed in the pathogenesis of autoimmune diseases including GD. Therefore, further studies are necessary to figure out whether the variations of PTPRB, PIK3R3 and TRAF3IP3 are involved in the dysfunction of thyroid autoimmune.The protein encoded by hil cell , 17. TRAelopment . The oveThe case study is about the susceptibility genes of a single three-generation family of Graves disease, but there are insufficient samples of similar families to verify the results of this study. Also, the frequency of susceptibility genes screened in this study has not been further verified in the population of patients with sporadic Graves disease and the relationship between these gene mutations and sporadic Graves disease is uncertain. Finally, the susceptibility genes screened this time need to be further studied at the protein molecular level to further determine the biological significance of these mutations.MAP7D2, SLC1A7, TRAF3IP3, PTPRB, PIK3R3, DISC1 genes and the familial GD of a three-generation family. The findings in this cast studies are clues for further study and more verification and function researches are needed to explore these genes related to GD susceptibility.To summarize, the WES was applied to establish an association between Additional file 1. Gene function analysis.


In HSCs, CDs induced higher cytotoxicity correlated with lower cell viability than CHR–CD. The 1:1 CHR–RAMEB pretreatment avoided p65 translocation. The 1:2 CHR–RAMEB complex increased ORAC values, improved SOD activity and produced the highest stimulation of GPx activity. CHR–RAMEB reduced α-sma expression at lower concentration than CHR–HPBCD, proving to be more efficient. In conclusion, both CHR–CD complexes proved to be biocompatible, but CHR–RAMEB showed improved anti-inflammatory, antioxidant and anti-fibrotic effects that could recommend its further use in liver fibrosis treatment. Liver fibrosis results from chronic liver injury and is characterized by the accumulation of extracellular matrix in excess driven by hepatic stellate cells (HSCs) activation. Chrysin (CHR) is a natural flavonoid that is limited by its low solubility to exert its anti-inflammatory, antioxidant and anti-fibrotic properties. The aim of this study was to investigate the biocompatibility of CHR complexes with two cyclodextrins (CDs)-(2-hydroxypropyl)-β-cyclodextrin (HPBCD) and random methyl-β-cyclodextrin (RAMEB), and their potential to induce anti-inflammatory, antioxidant and anti-fibrotic effects. Biocompatibility of the complexes was evaluated on Huh7 and LX2 cell lines: MTT and Live/Dead tests indicated the cell viability and an LDH test showed the cytotoxicity. Immunohistochemical staining of Nuclear Factor Kappa B (NF-κB) nuclear translocation was performed to evaluate the anti-inflammatory effect of the complexes. Oxygen Radical Absorbance assay, Superoxide Dismutase activity and Glutathione Peroxidase (GPx) assays indicated the antioxidant properties of the chrysin complexes. Finally, the complexes’ anti-fibrotic potential was evaluated at the protein and gene level of  These cells are responsive to multiple growth factors, such as transforming growth factor β (TGF-β) and platelet-derived growth factor (PDGF) that induce their proliferation and fibrogenic behavior. Furthermore, activated HSCs also produce TGF-β themselves, creating a positive activating loop [Liver fibrosis is generated in the presence of a chronic liver injury that induces hepatic stellate cells (HSCs) activation and excessive extracellular matrix (ECM) accumulation ,2. If uning loop ,4. α-sma and TGF-b1/Smad 2 and 3 expression.As liver fibrosis is a reversible stage in liver disease progression, many treatments target it to prevent or attenuate its progress. Some of them target activated HSCs to either reverse to a latent state, or to undergo apoptosis. To date, there is no standard treatment to induce liver fibrosis reversion, and new potential therapies are currently searched and tested. Natural compounds such as helioxanthin, silymarin, silybinin, curcumin were evaluated for their anti-fibrotic potential ,6,7. SucHowever, the efficiency of this potential treatment is limited by its low solubility and reduced absorption . Therefo(2-hydroxypropyl)-β-cyclodextrin (HPBCD) (degree of substitution (DS) ~ 4.5; MW = 1396) and random methyl-β-cyclodextrin (RAMEB)  were the product of Cyclolab Ltd. . CHR–RAMEB and CHR–HPBCD complexes with 1:1 and 1:2 molar ratios were synthesized as previously described . ComplexSaturated solutions of CHR–cyclodextrin complexes were prepared with PBS by mixing excess amounts of complexes for 24 h at room temperature. The solutions were centrifuged at 10,000 rpm for 6 min and the clear supernatants were used for the measurements applying Zetasizer Nano ZSP .2 with humidity. HSCs were activated with 10 ng/mL TGF-β1 for 24 h . Human Caco-2 intestinal epithelial cells were obtained from the European Collection of Cell Cultures  and grown routinely in Dulbecco’s Minimum Essential Medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acid and 1% penicillin–streptomycin solution.Cells derived from human hepatocellular carcinoma (Huh7) were propagated in culture with Dulbecco’s Modified Eagle Medium (DMEM)  media supplemented with 10% fetal bovine serum  and 1% antibiotic antimycotic . In addition, hepatic stellate cells from LX2 cell line were kindly provided by Dr. Pau Sancho-Bru with permission from Dr. Scott Friedman . HSCs were maintained in DMEM media supplemented with 2% SFB and 1% ABAM. Cell cultures were incubated at 37 °C, 5% CO4 cells/cm2 (Huh7) and 1.5 × 104 cells/cm2 (LX2). After 24 h, cells were put in contact with the complexes at concentrations of 10–300 µM for another 48 h. Biocompatibility of the complexes was evaluated by quantitative  and qualitative tests . By MTT, cell viability was evaluated in the presence of the complexes. Cells were washed with phosphate buffered saline , and maintained with 3--2,5-diphenyltetrazolium bromide 1 mg/mL for 4 h. Metabolic active cells turned the compound to formazan crystals, soluble in isopropanol and the absorbance was measured at 550 nm by spectrophotometry. The values for the absorbance were proportional to the number of live cells in the wells. LDH assay indicated the cytotoxicity levels induced by the complexes on the cells they were exposed to. Cell media was collected and following the kit instructions, the mix was added for 15 min RT in a dark room. The absorbance was measured at 490 nm by spectrophotometry. The absorbance values correspond to the number of dead cells in the wells. For the LiveDead Assay, live cells were stained with calcein AM (green) and nuclei of the dead cells with ethidium homodimer (red). Cells were visualized by fluorescence microscopy .Cells were cultured in 48 well plates at 4 × 104 cells/slide and cultured for 6 days. Caco-2 cultures were divided into groups according to the stimulant(s) used. Following 6 days of culture in DMEM, the medium was renewed, and cells were pretreated with different CHR–CD complexes at 25 µM concentration or with saturated chrysin aqueous solution for 60 min. After that, cells were treated with 50 ng/mL Tumor Necrosis Factor-α (TNF-α) for 30 min at 37 °C. Then, each slide was fixed with ice-cold methanol-acetone (1:1) and non-specific antibody binding sites were blocked with FBS for 15 min. Primary labelling of the NF-κB p65 subunit was conducted using 2 μg/mL rabbit anti-human p65 antibody (Santa Cruz Biotechnology) for 60 min at 37 °C. The samples were washed three times with Hank’s Balanced Salt Solution (HBSS) and secondary staining with Alexa Fluor 488-conjugated goat-anti-rabbit IgG was applied . Samples were washed four times with HBSS and cell nuclei were stained with Hoechst 33342 (0.1 µg/mL) for 15 min at room temperature. After washing with HBSS, the cover slides were mounted on glass microscopy slides and observed by a Zeiss Axio Scope A1 fluorescent microscope (HBO 100 lamp) . Images were analyzed with ZEN software, and the ratio of nuclear and perinuclear fluorescence intensities of the cells was calculated.Caco-2 cells were seeded onto sterile uncoated microscope cover slides at a density of 5 × 105 cells per well and then pretreated with 25 µM CHR–CD complexes for 1 h. After that, samples were treated with 50 ng/mL TNF-α for the induction of O2− or 1 h and then the wells were washed with PBS. Caco-2 cells were collected by adding 500 µL trypsin to each well and incubating at 37 °C for 10 min. Cells were placed into Eppendorf tubes on ice, the volumes were completed with 4 mL PBS and then they were centrifuged at 500× g for 10 min at 4 °C. The supernatants were discarded. Cell pellets were re-suspended in 1.2 mL ice-cold PBS, centrifuged at 250× g for 10 min at 4 °C and the supernatant was discarded. Cell pellet was homogenized in 0.5–1 mL PBS per 100 mg of cells and then centrifuged at 10,000× g for 15 min at 4 °C. The supernatant was collected for experiments. The preparation of standard solutions and reagents as well as the procedure for assay were done by the protocol provided with the kit. The antioxidant enzyme activity was measured by inhibiting of the dye reduction as the result of oxygen generation by xanthine oxidase. The samples were measured at 450 nm with a FLUOstar OPTIMA microplate reader , and the results were expressed as U/mL.The SOD Assay kit  was used for measuring SOD enzymes. Caco-2 cells were cultured in 12-well plates at the density of 4 × 105 cells per well were treated with 25 µM Chrysin–CD complexes for 1 h. Cell lysates were produced by removing medium then washing cells with 5 mL Hank’s solution for two times. After that, cells were collected, centrifuged (1000–2000× g for 10 min at 4 °C) and homogenized in cold buffer then centrifuged again . The supernatant was removed for assay and stored on ice. Experimental reagents such as GPx assay and GPx sample buffer, GPx (control), GPx Co-substrate mixture, GPx cumene hyperoxide and NADPH were prepared according to protocol of Glutathione Peroxidase assay kit .The seeded Caco-2 cells at the density of 4 × 10−1.The depletion of NADPH at 340 nm was recorded with a FLUOstar OPTIMA microplate reader  once every minute for 7 cycles at 25 °C. The enzyme activity then was calculated as nmol/min/mg protein using the NADPH extinction coefficient of 0.00373 µM2PO4-K2PO4  to obtain an 18.75 mM concentration solution. The fluorescein solution was applied at 0.6 µM final concentration. Cyclodextrins and CHR–CD complexes were dissolved at a concentration of 25 µM; chrysin aqueous solution was prepared by saturation in water.The free-radical generator 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH) was prepared by mixing with phosphate buffer KHThe reaction was initiated by immediately adding AAPH to samples and the signal was measured for 1 h at every 2 min with a FLUOstar OPTIMA microplate reader . The free-radical generator produced peroxyl radicals, resulting in loss of fluorescence due to the damage of fluorescein molecules. The intensity of fluorescence vs. time was plotted and the area under the curve of chrysin and complexes were determined. The ORAC fluorescence indexes were calculated for different C–CD and cyclodextrin derivates. The difference of ORAC indexes for C–CD and same cyclodextrin derivate was compared to the ORAC index of chrysin.4 cells/cm2. Following TGF-β activation, cells were put in contact with the complexes for a further 48 h. The level of activation marker α-sma was evaluated both at the gene (qPCR) and protein  levels. For the gene expression study, total RNA was extracted with TRIzol Reagent . RNA concentration and purity were determined on a NanoDrop 8000 Spectrophotometer  and RNA integrity was assessed on a BioAnalyzer 2100 (Agilent Technologies). Using the iScript cDNA synthesis kit (BioRad), cDNA was obtained and amplified on a Veriti 96-Well Thermal Cycler (Applied Biosystems). For Real Time PCR, the SYB Select Master Mix  and ViiA 7 Real-Time PCR System  were used. Samples were tested in triplicate and the glyceraldehyde 3-phosphate dehydrogenase (gapdh) gene was used as reference. Primers used were: α-sma F 5′ TTCGCATCAAGGCCCAAGAA 3′, α-sma R 5′ GTCCCGGGGATAGGCAAAG 3′, gapdh F 5′ GAGTCAACGGGGTCGT 3′, gapdh R 5′ TTGATTTTGGATCTCG 3′.To evaluate the antifibrotic potential of the complexes, HSCs were cultured in 48 well plates at 1.5 × 10α-sma expression, cells were fixed with 4% paraformaldehyde solution for 20 min, followed by permeabilization for 1 h with 2% bovine serum albumin (BSA) solution with 0.1% Triton X100. Overnight at 4 °C, samples were incubated with antibody anti-α-sma , and then incubated for 1 h with antibody anti-mouse conjugated with fluorescein isothiocyanate (FITC) (Santa Cruz Biotechnology). To visualize the nuclei, cells were stained with Hoechst 33342 (Sigma-Aldrich). Cells were visualized by fluorescence microscopy (IX-73 Olympus).For the evaluation of p-values < 0.05 were considered statistically significant in accordance with the 95% confidence interval established for the analysis.All results were statistically analyzed with GraphPad Prism 6.0, one-way ANOVA and Bonferroni correction; The characteristics of CHR–RAMEB and CHR–HPBCD complexes were measured by dynamic light scattering and the results are presented in p < 0.05), suggesting the efficient release of CHR from the complexes. As the concentration was increased, the cell viability was statistically significantly reduced starting from 30 µM (p < 0.05) 1:1 and 1:2 CHR–HPBCD and only 1:2 CHR–RAMEB. Both CHR–HPBCD complexes (1:1 and 1:2) induced similar responses in Huh7 viability all throughout the five concentrations tested (p < 0.01). As for RAMEB complexes 60 and 100 µM, the same behavior was observed as they induced similar cell viability levels for each concentration, but the cell viability in contact with 1:1 complexes, even though was reduced compared to 30 µM, was still higher than for 1:2 complexes. The most reduced Huh7 viability compared to control was in contact with CHR–HPBCD 300 µM (p < 0.01) (cell viability reduced ~50%) and also for CHR–RAMEB 300 µM, but only the 1:2 complex (cell viability reduced ~80%), suggesting the higher efficiency of 1:2 RAMEB complex in releasing CHR.Cell viability was evaluated by MTT test both on Huh7 and LX2 exposed to CHR complexes (1:1 and 1:2), as well as CDs alone . Both CDs tested a. In conp < 0.01) (~35%) and to 100 µM RAMEB complexes (p < 0.001) (~45%). Cell viability levels in the presence of 300 µM of both types of complexes were significantly affected compared to control and not recommended for use (p < 0.001).HSCs were also exposed to CDs and CHR complexes and cell viability was measured c,d. In cp > 0.01) of cytotoxicity at 300 µM concentration compared to control, suggesting the harmful effect the complexes could induce in such high concentrations.The LDH test showed cytotoxicity levels induced by exposure to CDs and CHR complexes on Huh7 and LX2 cells . The cytThe LiveDead assay showed the live cells (green) and the nuclei of the dead cells (red) in both Huh7 and LX2 cells  exposed HPBCD and RAMEB showed no significant difference in the number of Huh7 live cells between the three concentrations used. In contrast, even though 30 µM 1:2 CD–CHR did not strongly affect the cells, starting with 60 µM, the number of live cells was significantly reduced and the number of dead cells was highest when exposed to 100 µM of complexes, especially for 1:2 CHR–RAMEB. These results are in accordance with the quantitative MTT and LDH results, confirming the release of CHR from the complexes.As for HSCs, 30 µM complexed CHR with RAMEB and HPBCD, did not strongly affect the cells. However, when exposed to 60 µM CHR–CD, they showed reduced number of live cells compared to 30 µM CHR–CD. In addition, their normal cellular distribution was also affected by the presence of the complexes, and they showed a tendency to form aggregates. This tendency was best observed in the case of exposure to 100 µM CHR–CD, in which cells tended to form aggregates and detached from the culture surface, causing their further death. As determined by quantitative tests, this qualitative assay confirmed the previous results and indicated that increasing the concentration by more than 100 µM of complexes could affect the normal cells too much.p < 0.0001). After CHR pretreatment the p65 translocation to the cell nucleus remained significantly high, compared to the control (p < 0.0001), however 1:1 CHR–RAMEB pretreatment avoided p65 translocation, there was no significant difference compared to the untreated cells. Similar enhanced effect could be observed in the case of 1:1 CHR–HPBCD pretreatment, while 1:2 CHR–CD complexes were less effective. It should be noted, that each pretreatment significantly decreased the p65 translocation, compared to TNF-α stimulation (p < 0.01).The translocation of NF-κB p65 subunit from the cytosol to cell nuclei after TNF-α stimulation with or without CHR and CHR–CD complex pretreatments was detected by antibody labelling in Caco-2 cells . In contp < 0.05) (Each CHR–CD complex showed significant differences in ORAC assay compared to chrysin ( < 0.05) . RAMEB cα-sma was evaluated at the gene and protein levels in TGF-β activated HSCs exposed for 48 h to CHR–RAMEB/-HPBCD 10, 30 and 60 µM  higher levels (~10×) of α-sma, indicating that HSCs responded to TGF-β treatment and induced α-sma production. Exposure to CD–CHR complexes, even at the smallest 10 µM concentration, reduced the α-sma relative expression in HSCs by ~50%, suggesting the efficiency of CDs to release CHR, and also the anti-fibrotic potential of CHR, even in small quantities. CHR–RAMEB 30 and 60 µM statistically significantly reduced (p < 0.001) the expression levels of α-sma in HSCs compared to 1:2 CHR–RAMEB 10 µM, suggesting the efficiency of RAMEB to release CHR at smaller concentrations. CHR–HPBCD 30 µM induced statistically significant (p < 0.01) lower α-sma expression compared to 10 µM, suggesting the potential of this complex in increasing the bioavailability of CHR that regulates α-sma expression. Furthermore, CHR–HPBCD 60 µM reduced α-sma expression even more, with similar levels to control, indicating the potential of these complexes in antifibrotic treatments. All tested complexes, at 60 µM concentrations, reduced the expression of α-sma to control levels, but only 1:2 CHR–RAMEB induced the same response in 30 µM, indicating its potential future use at this concentration of complexed CHR.α-sma gene expression were confirmed at protein level as well, by immunofluorescence staining  similar to the effect induced by 60 µM, suggesting RAMEB to be a better CD than HPBCD in releasing CHR. The favorable release of a flavonoid (silymarin) from RAMEB and HPBCD complexes was demonstrated in another study as well, on CCl4-liver fibrosis induced mice [4-liver fibrosis induced mice, by assessing the impact on TGF-β/Smad pathway [Based on biocompatibility results, the anti-fibrotic potential of CHR complexes was evaluated at three concentrations  by assessing the expression level of the HSC activation marker progress . Both coced mice . The hepced mice ,12,29,30 pathway .In conclusion, the complexes CHR–RAMEB and CHR–HPBCD proved to be biocompatible, and showed anti-inflammatory, antioxidant and anti-fibrotic effects. The 1:2 CHR–CD complexes were more efficient in releasing CHR that further exerted its beneficial properties, and the favorable results were obtained for RAMEB complexes. To confirm its potential use in liver fibrosis therapies, 1:2 CHR–RAMEB could be used for further in vivo studies.


BCL2 expression has been observed with many diseases and is associated with cellular exposure to reactive oxygen species. A region upstream of the P1 promoter in the human BCL2 gene plays a major role in regulating transcription. This G/C-rich region is highly polymorphic and capable of forming G-quadruplex structures. Herein we report that an oxidative event simulated with an 8-oxo-7,8-dihydroguanine (oxoG) substitution within a long G-tract results in a reduction of structural polymorphism. Surprisingly, oxoG within a 25-nt construct boosts thermal stability of the resulting G-quadruplex. This is achieved by distinct hydrogen bonding properties of oxoG, which facilitate formation of an antiparallel basket-type G-quadruplex with a three G-quartet core and a G·oxoG·C base triad. While oxoG has previously been considered detrimental for G-quadruplex formation, its stabilizing effect within a promoter described in this study suggests a potential novel regulatory role of oxidative stress in general and specifically in BCL2 gene transcription.Misregulation of   BCL2 gene was found to be aberrantly overexpressed in a wide range of human tumours, including B-cell and T-cell lymphomas, breast, lung, prostate, cervical and colorectal cancers as well as gliomas, melanomas and neuroplastomas  is commonly used as a biomarker for oxidative stress  is a part of normal cellular metabolism and exerts a constant strain on DNA. Among the four DNA nucleobases, guanine has the lowest redox potential and is therefore the easiest to oxidize ,14. Basee stress ,17. oxoGhe sugar . Furtherelements . In codistalling . In promplex DNA ,22. It itructure . oxoG waruplexes .BCL2 gene with the highest G/C frequency was designated as Pu39 stabilizing effect of oxoG substitutions within the G5-tract and establish potential changes in which nucleotides are involved in G-quartet formation and stacking. Only individual substitutions were tested as any successive oxidative events would take place on the initially oxidized oxoG nucleotide due to its lowest redox potential  oligonucleotide adopts an antiparallel basket-type G-quadruplex structure, which expands the array of non-B-DNA structures associated with the BCL2 promoter. Both bcl2ex and its analogue containing oxoG exhibit novel structural elements, which deepen our understanding of G-quadruplex folding in general.A 39-nt region within the P1 promoter of the 9 Figure  27). It. ItBCL2 morphism . We desimorphism . All, exmorphism ,30. Howere tire’ ,31. On totential . Our ini13C,15N-enriched guanine, adenine and cytosine nucleotides. Oligonucleotides were deprotected with AMA (1:1 mixture of aqueous ammonium hydroxide and methylamine) at 65°C for 30 min. Samples were purified using reverse-phase HPLC chromatography, followed by removal of the DMT group with 80% AcOH for 30 min. Then the oligonucleotides were transferred to a pure water phase by ether extraction and desalted using FPLC and a Sephadex G25 column. DNA solutions were dried on a vacuum centrifuge and redissolved in 9:1 H2O/2H2O, 20 mM potassium phosphate buffer, pH 7, and 70 mM KCl. Then the samples were concentrated using Amicon Ultra – 0.5 ml Centrifuge Filters. Final NMR solutions collected from Amicon tubes were heated to 95°C for 10 min and left to cool on the bench. DNA concentrations were determined by measuring UV absorption at 260 nm. Extinction coefficients of 231 300, 251, 500 and 251 300 M−1cm−1 were calculated with the nearest-neighbour method for nonsubstituted bcl2MidG4, bcl2ex and bcl2exT, respectively. Also, the same extinction coefficients were used for oligonucleotides contained oxoG substitution. The final oligonucleotide concentrations were in the range from 0.5 to 1.2 mM. bcl2ex and bcl2ex-oxoG19 samples for 1H–2H exchange experiment, were dried on a vacuum centrifuge and redissolved in 2H2O.All oligonucleotides were synthesized on a K&A Laborgeraete DNA/RNA Synthesizer H-8 using standard phosphoramidite chemistry. Isotopically labelled samples contained 8% Tm were determined from the first derivative of A295 versus temperature plot.UV melting experiments were performed on an Agilent Cary 3500 UV–Vis spectrophotometer using 1 cm path length cells. Samples were heated/cooled at a rate of 0.5°C/min in the range of 10–95°C and absorbance at 295 nm was monitored. CD spectra were acquired on Chirascan CD spectrometer at 25°C using 2.0 mm light path quartz cuvettes. All samples for CD were prepared by dilution of NMR samples to final 50 μM DNA concentrations in 20 mM potassium phosphate buffer, pH 7, and 70 mM KCl solution. CD spectra were acquired in the wavelength range from 220 to 320 nm.τm = 80, 150, 200, 250, 300, 350 ms) were utilized for 1H resonance assignments. Identification of guanine imino proton resonances was aided by 1D 15N-edited HSQC experiments, while aromatic of adenine, guanine and cytosine proton resonances were assigned with the help of 13C-edited HSQC experiments using 8% 13C,15N-labeled oligonucleotides. 2D TOCSY (τm = 80 ms) and DQF-COSY experiments gave information on sugar puckering and cytosine H5 protons resonances. Also, 2D 1H–13C HSQC spectra were used to help to assign H8/H2 aromatic resonances of adenines, guanines and to confirm syn conformation of nucleotides. Data were analysed with VNMRJ, NMRPipe, CcpNMR and Origin . Restraint bounds for cross-peaks in aromatic and anomeric regions were set at ±0.5 Å for dij ≤ 3 Å, ±0.6 Å for 3 Å < dij ≤ 4 Å, ±0.7 Å for 4 Å < dij ≤ 4.5 Å, and ±0.8 Å for dij > 4.5 Å. For some restraints corresponding to overlapping or broader cross-peaks, larger distance ranges were used (±0.9–1.5 Å). All cross-peaks in imino-aromatic, imino-imino, amino-imino, and amino-aromatic regions were classified as strong (2.5 Å), medium (3.5 Å) or weak (4.5 Å) with ±1–1.5 Å restraint ranges.Interproton distances were calculated from 200 ms and 250 ms NOESY spectra of bcl2ex-χ was restrained to a range between -90° and 90° for syn and between 90° and 270° for anti nucleotides, and oxoG19 was left unrestrained. Restraints for backbone dihedral angles included α (–120–120°), β (150–210°), γ (30–90°), δ (130–190°), ϵ (170–300°), and ζ (–120–120°). All sugar puckers were determined to be of S-type (C2′-endo) based on 2D TOCSY and DQF-COSY NMR spectra, expect oxoG19, which was determined to adopt a predominate N-type (C3′-endo) sugar pucker.Dihedral angle ϵ/ζOL1 and χOL4 modifications (oxoG nucleotide were derived from the RESP ESP charge derive (RED) Server  Server .−1Å−2 for hydrogen bonds, 20 kcal·mol−1Å−2 for NOE distance restrains, 200 kcal·mol−1rad−2 for backbone, and 50 kcal·mol−1rad−2 for the χ torsion angle. Twelve structures were selected based on the lowest energy and subjected to energy minimization with a maximum of 10000 steps.Calculations were started from initial linear structure of the oligonucleotide, created with the LEAP module of AMBER 18. A total of 100 structures were obtained in 100 ps restrained SA simulations using Born implicit solvent model with random starting velocities. In the first 20 ps of SA, the temperature was kept at 1000 K, followed by slow cooling in the next 60 ps down to 300 and to 0 K in the last 20 ps. Force constants were 50 kcal·mol1H NMR spectrum with narrow imino resonances in the Hoogsteen chemical shift range . Following this interesting observation we probed the ability of the bcl2ex structure to tolerate oxidative lesions by individually substituting guanine nucleotides in the G5-tract with oxoG and examined G-quadruplex forming ability of bcl2ex analogues with 1H NMR and UV melting experiments  containing the central four G-tracts of Pu39 with 5′ and 3′ single nucleotide overhangs C1 and A25) exhibits a  and A25 eriments . oxoG20 . Figure . Interese Figure . SurprisoxoG19 were acquired for an initial assessment of their structural properties. While the CD spectrum of bcl2ex exhibits a strong positive signal at 265 nm and a less intense one at 295 nm, spectral properties of bcl2ex-oxoG19 differ considerably with positive signals at 250, 270 and 295 nm . The remaining intense inter-nucleotide H6–H1’ NOESY cross-peak was assigned to C1. The syn conformation of C1 was confirmed by a downfield shift (δ 147.5 ppm) of its C6 resonance  and last (G20–C21) are of the edgewise type, while the longer second loop (A11–G12–G13–A14–A15–G16) is diagonal. Intra-nucleotide G12H8-H1’ and G13H8-H1’ cross-peaks could not be observed most likely due to the dynamic character of this part of loop region with its orientation changing on the intermediate NMR timescale. These structural features are consistent with the antiparallel basket-type G-quadruplex topology  and last (oxoG19–G20–C21) are of the edgewise type, while the second one (A11–G12–G13–A14–A15) is a diagonal loop. The structure of bcl2ex-oxoG19 is consistent with an antiparallel basket-type G-quadruplex topology  between G2–G4 and G8–G10 tracts, two medium grooves (∼15 Å) between G2–G4 and G16–G18, as well as between G8–G10 and G22–G24, and a narrow groove (∼9 Å) between G16–G18 and G22–G24  whereas the remaining nucleotides adopt the south (C2'-endo) pucker. Interestingly, C5 is bulged out of the G-quadruplex core and positioned in the wide groove, which facilitates efficient stacking of the G6·oxoG19·C21 base triad with the adjacent G-quartet  to reduce structural polymorphism and solve a hybrid type G-quadruplex structure. Interestingly, we found that bcl2ex and bcl2MidG4 exhibit similar CD spectra . Non-B-DNA secondary structures within promoters are attractive targets for small molecule binding. Studies showed that stabilization of  effects ,39. G-quharacter . Many cug et al. . bcl2Mid spectra , which c spectra . The par5-tract is a potential source of structural polymorphism. The G-tract regions with connecting loops are formally extended in 5′ and 3′ directions by a single nucleotide (C1 and A25). These play an instrumental role in determination of the G-quadruplex structure of bcl2ex by NMR. C1 forms a Watson–Crick base pair with G16 and both are adjoined by A11 and A25 to form a planar CGAA-quartet. This structural element apparently stabilizes only a single basket-type G-quadruplex as no strand slippage derived polymorphism or other competing folds were observed. Interestingly, contribution of the CGAA-quartet to the overall thermal stability of the bcl2ex G-quadruplex is negligible since its melting temperature is comparable to that of bcl2MidG4 (69°C). An unusual structural feature of the CGAA-quartet is the syn conformation of C1. Antiparallel and hybrid type G-quadruplexes often start with a 5′ guanine in syn conformation  while a single longer Gormation . Howeverulations . C1 in soxoG appears in long loop regions. However, oxoG increases binding of transcription factors to the G-quadruplex, which destabilizes the structure. DNA reverts to the duplex form and oxoG can be efficiently repaired by base excision repair (BER) where the nucleobase is excised by the oxoG glycosylase 1 (OGG1). This creates an abasic site (AP), which is cleaved by the apurinic/apyrimidinic endonuclease 1 (APE1). Lesion free promoter DNA has increased affinity towards nuclear factors, which activates KRAS transcription. Regulation of BCL2 expression, which sensitizes cells to apoptosis, was also shown to be related to exposure to ROS  contains additional G-tracts of different lengths, which offers a level of redundancy. Burrows et al. have shown that redundant G-tracts in VEGF and NEIL3 promoters play a pivotal role in regulating gene expression (oxoG in duplex DNA is normally removed by BER. This creates an AP site, which is highly destabilizing to the duplex and thus promotes formation of a G-quadruplex structure. The AP site is excluded from the G-quadruplex core by utilizing redundant G-tracts. Interestingly, AP sites were suggested to decreases the number of possible structures and slightly stabilizes the fold. Subsequently, APE1 is recruited to the excluded AP sites, but does not cleave the DNA strand. Instead, APE1 recruits transcription factors, which generally upregulate gene expression. However, depending on location, strand, and promoter the reverse effect of gene suppression can also be observed and may involve i-motif structures (oxoG19 with APE1. While oxoG19 is not expected to cause a major decrease in duplex stability, the change in structural equilibrium would be driven by increased stability of the oxidized G-quadruplex. Furthermore, once the bcl2ex-oxoG19 structure is formed, repair could be less efficient due to high thermal stability of the G-quadruplex. OGG1 initiates excision by hydrogen bonding with the protonated N7 of oxoG (oxoG19, H7 of oxoG19 is hydrogen bonded to G6, which could negatively affect removal of oxoG19 by OGG1. Additional structural transformations to partially or completely unfold the structure could be required. On the other hand, the structure of bcl2ex-oxoG19 could hinder ROS from accessing oxoG19 and prevent secondary oxidation reactions despite the low redox potential of oxoG. The inability to remove oxoG19 could lead to a stable lesion, which would act as an epigenetic modification that might regulate gene expression by altering the binding of transcription factors to the DNA.The G-rich strand of the BCL2 promoter exists in equilibrium between duplex and G-quadruplex forms. This equilibrium is likely shifted towards the duplex form where G-tracts are likely targets for oxidation, either direct or via electron hole migration . Due to  systems ,30, othe systems . Analogopression ,30. Oxidructures . The samructures . Normall of oxoG ,62. In tBCL2 and suggests that multiple structures are relevant for regulation of transcription. Diversity of BCL2 G-quadruplex structures is further expanded by the action of reactive oxygen species. While oxidative lesions were mostly found to reduce G-quadruplex stability, we report a significant stabilizing effect on a biologically relevant G-quadruplex. Unique structural features presented in this study could prove significant for detection and mitigation of oxidative lesions in the human BCL2 gene and related diseases.In summary, identification of new non-B-DNA secondary structures adds additional levels of complexity to the folding landscape of the G-rich region of the P1 promoter of The coordinates for the 12 lowest energy structures of bcl2ex and bcl2ex-oxoG19 have been deposited in the Protein Data Bank with accession codes 6ZX7 and 6ZX6, respectively. Chemical shifts have been deposited in the Biological Magnetic Resonance Data Bank as entries 34543 and 34542.gkab057_Supplemental_FileClick here for additional data file.


The initial radiolabeling is achieved in a short reaction time (10 min) and purified through solid-phase extraction (SPE) with modest radiochemical yields  in excellent radiochemical purity . The de-protection of the tert-butyloxycarbonyl (Boc) and trityl group was achieved with mild heating under acidic conditions to provide 18F-tagged dipeptide 1. Preliminary analysis of 18F-dipeptide 1 was performed to confirm uptake by peptide transporters (PepTs) in human pancreatic carcinoma cell lines Panc1, BxPC3, and ASpc1, which are reported to express the peptide transporter 1 (PepT1). Furthermore, we confirmed in vivo uptake of 18F-dipeptide tracer 1 using microPET/CT in mice harboring subcutaneous flank Panc1, BxPC3, and Aspc1 tumors. In conclusion, we have established the radiolabeling of dipeptide 1 with fluoride-18, and demonstrated its potential as an imaging agent which may have clinical applications for the diagnosis of pancreatic carcinomas. We have developed a novel fluorine-18 radiotracer, dipeptide  The proton-coupled oligopeptide transport (POT), also called the peptide transport (PTR), comprises a family of transporters which are found in animal, plant, yeast, archaea, and both Gram-negative and Gram-positive bacterial cells ,2,3. In +/Peptide transporters have been shown to be responsible for peptide transport activity in cancer cells, including pancreatic carcinoma glycylsarcosine 2 (Gly-Sar) [18F]FDG FDG ,11. The [18F]FDG , in whic[18F]FDG  lists cr18F-dipeptide 1, (S)-N-(2-amino-3-(4-(2-(fluoro-18F)ethoxy)phenyl)propanoyl)-N-methylglycine ([18F]FEPPG), that meets the criteria as a suitable PepT binding substrate. We also demonstrate preliminary data confirming cell uptake of the radiolabeled dipeptide by several human pancreatic carcinoma cell lines and uptake of the [18F]FEPPG 1 by μ-PET imaging on subcutaneous human xenograft Panc1, BxPC3, and ASpc1 tumor models in mice. This suggests that [18F]FEPPG 1 is suitable for further study to explore its specificity as a PET tracer.Herein we report the synthesis of a novel 11C]Gly-Sar , high resolution with a low positron energy (0.64 MeV), and a half-life of 109.8 min that allows for easier commercial distribution and longer scan times as compared to carbon-11. FEPPG 1 with the minimal side products [To obtain the de-protection of both the Boc and products . The use18F]FEPPG 1 was achieved with the use of reverse phase HPLC  followed by the formulation process, less than 10% of radioligand was secured in >95% radiochemical purity. Current work is underway with the use of different SPE cartridges. Purification of [ase HPLC . The fra18F]FEPPG 1, starting from a raw solution of aqueous fluoride-18/[18O]-enriched water (98%), was 19 ± 9% (n = 5) with an average total synthesis time of 117 min. The chemical purity of the final product, determined by HPLC analysis, was 33 ± 5% (n = 5) with a range of 0.5–4.8 μg/mL of the fluorine-19 isotope of FEPPG. The radiochemical purity determined by HPLC analysis of [18F]FEPPG was determined to be 89 ± 5% (n = 5).The overall decay corrected yield for the formation of [18F]FEPPG 1, we performed initial cell and tumor uptake studies to ensure that newly introduced fluorine-18 moiety did not negate cell and tumor binding previously reported in the carbon-11 analog. We therefore performed initial cell uptake studies on Panc1, BxPC3, and AsPC1 cells, which have been reported to express the PepTs, and found that all tested cell lines had uptake of [18F]FEPPG 1  of [18F]FEPPG 1  and could be easily purified by way of solid-phase extraction (t-C18 plus cartridge), eluted with acetonitrile, dried, and used directly in the next synthetic sequence without any further purification. The desired final product was formulated into an isotonic injectable solution. Of significance, the final injectable product demonstrated excellent radiochemical purity. We also performed preliminary cell and tumor uptake studies to demonstrate retained in vitro and in vivo uptake of our new molecular entity that has an additional fluorine-18 moiety compared to the originally reported carbon-11 tracer, justifying its further study to confirm PEPT specificity. We have reported a new fluorine-18 labeled dipeptide 3, intermediate 4, and final product standard 1 were purchased from Syngene International Ltd. . Hydrogen chloride solution was purchased from Sigma-Aldrich  and used without further purification. Oxygen-18 enriched water (min. 98%) was purchased from Advanced Accelerator Applications  and used for the cyclotron bombardments (2.4 mL per bombardment). The cell lines  and culture media were obtained from American Type Culture Collection  Company. Fetal bovine serum came from Atlanta Biologicals . DMEM was obtained from Fisher Scientific . Fetal bovine serum (FBS) was obtained from Atlanta Biologicals . All other reagents not listed above were of the highest grade available from Sigma-Aldrich  and Fisher Scientific . Radiochemical yields (rcy) are reported as decay corrected to the end of cyclotron target bombardment. Mesylate precursor CrTac:NCr-Foxn1nu mice  referred to in text as NCr were maintained on a normal mouse diet. All animal experiments were conducted according to protocols approved by the Institutional Animal Care and Use Committee of Columbia University Medical Center.4 is as follows: [18O]-Enriched water (98%) aqueous solution of fluoride-18, produced from a Siemens 111 RDS cyclotron , was trapped on a pre-conditioned QMA light  cartridge. A solution of 1.5 mL  containing cryptand 222  and 3.0 mg potassium carbonate, was passed through the QMA light cartridge into a reaction vessel to provide K[222]18F complex. This complex was dried in a heat block at 110 °C with a flow of argon until most of the solvent was evaporated. The solution was further azeotroped with the use of acetonitrile (3 × 1 mL) at 110 °C over a stream of argon gas. A solution of mesylate 3  in 0.5 mL of anhydrous dimethyl sulfoxide was added to the “dry” K[222]18F complex at 110 °C. After 10 min, the reaction mixture was removed from the heat and allowed to cool. The reaction mixture was then taken up in 20 mL of water and passed through a t-C18 plus (Waters Corporation) cartridge (activated by flushing with 10 mL ethanol followed by 10 mL water). The cartridge was then washed with another 10 mL of water and eluted into a test tube with 2.0 mL of acetonitrile to provide radiolabeled intermediate 4 . Radiochemical purity of intermediate 4 was determined by HPLC. HPLC conditions: Luna 5 µ C18(2) 100 Å column, 250 × 4.60 mm, 5 µm; acetonitrile/0.1 M ammonium formate (aq) in 0.5% acetic acid solution 50/50 ramped to 80/20 over 20 min with a flow rate of 2.0 mL/min; rt = 10.05 min. The newly formed intermediate was dried at 90 °C over a stream of argon. The dried compound was taken to the next step.A typical procedure for the formation of radioactive intermediate Method A: A typical procedure for the formation dipeptide 1 is as follows: Tetrahydrofuran (167 µL) followed by hydrogen chloride solution  was added to dry intermediate 4. The reaction mixture was placed in a heat block set to 60 °C. After 20 min, remove from heat and allowed to cool. The reaction mixture was diluted with 9/1 (v/v) 0.1% trifluoroacetic acid (aq)/acetonitrile (1 mL) solution and purified by HPLC (Luna 10 µ C18(2) 100 Å column, new column 250 × 10 mm; acetonitrile/0.1% trifluoroacetic acid (aq) solution 10/90 ramped to 90/10 over 30 min as mobile phase with a flow rate of 4.0 mL/min; fractions collected from 10.00 to 10.80 min). The fractions were pooled together and diluted with 40 mL of sterile water for injection. The reaction media were then passed through a t-C18 light  cartridge (activated by flushing with 10 mL ethanol followed by 10 mL water). The t-C18 light cartridge was then washed with sterile water for injection (10 mL), eluted into sterile 10 mL final product vial with ethanol (0.45 mL) followed by 0.9% saline (2.55 mL). An aliquot was removed for quality control testing.Method B: A typical procedure for the formation of dipeptide 1 is as follows:4. The reaction mixture was placed in a heat block set to 80 °C. After 20 min, was removed from heat and allowed to cool. The reaction mixture was then taken up in 50 mL of 10% sodium ascorbate solution (pH = 7.5). An aliquot was removed for quality control testing.Dimethyl sulfoxide (20 µL) followed by 6 N hydrogen chloride (200 µL) was added to intermediate 18F]FEPPG 1 passed all required quality control tests, which included radionuclidic purity (511 keV), chemical  and radiochemical purity , excipients present , pH (range 4–8), and half-life (110.0 ± 5.0 min). Radiochemical purity/identity of 1 was determined by analytical HPLC. Analytical HPLC conditions: Luna 5 µ C18(2) 100 Å column, 250 × 4.60 mm, 5 µm; acetonitrile/0.1% trifluoroacetic acid (aq) solution 10/90 ramped to 90/10 over 20 min with a flow rate of 1.5 mL/min; rt = 8.68 min. Fluorine-19 derivative of 1 varied from 0.5–4.8 µg/mL, based off calibration curve  at 280 nm; molar activity (Am) = 17,000 ± 7000 MBq/µmol (460 ± 190 mCi/µmol), where n = 5.Each batch of FEPPG 1, at ~1.5 MBq per well (~40 μCi), was added to cells for 30 min and washed four times with PBS. Cells were lysed with 0.1 M sodium hydroxide and the resulting gamma-activity was measured on Hidex gamma counter . For protein concentration, cells were harvested using 100 μL of RIPA buffer; protein concentration was measured using colorimetric assay.For cell-uptake studies, Panc1, BxPC3, and AsPC1 human pancreas carcinoma cells were plated at 1 × 106 of Panc1, BxPC3 or AsPC1 human pancreatic carcinoma cells (ATCC) in 200 μL of Matrigel®. After tumors reached 1 cm3, mice were injected IV with 3.7–5.5 MBq (100–150 μCi) of [18F]FEPPG 1. Static 60 min PET was acquired 30 min after [18F]FEPPG 1 injection using Inveon micro PET scanner . PET images were reconstructed using the 3D-OSEM algorithm with three iterations in a 256 × 256 matrix, without attenuation correction  and analyzed using VivoQuant ver 4 .CrTac:NCr-Foxn1nu mice  were injected sc with 1 × 10p-values were calculated using two-tailed Student’s t-test for unpaired samples.Statistical analysis was performed using Prism 8.0 . All data are represented as mean ± standard error. Statistical


Cytochrome P450 (CYP) enzymes are involved in the metabolism of many important endogenous substrates , drugs and toxic xenobiotics. Their induction accelerates drug metabolism and elimination. The present study aimed at examining the inducing abilities of two antipsychotic drugs levomepromazine and clozapine for the main CYPs.N-demethylation (CYP1A1/2), diclofenac 4′-hydroxylation (CYP2C9), perazine N-demethylation (CYP2C19) and testosterone 6β-hydroxylation (CYP3A4). In parallel, CYP mRNA levels were measured in neuroleptic-treated hepatocytes.The experiments were performed using cryopreserved human hepatocytes. The hepatotoxicity of levomepromazine and clozapine was assessed after exposure to the neuroleptics (LDH test). CYP activities were measured in the incubation medium using the CYP-specific reactions: caffeine 3-The results indicate that levomepromazine and clozapine induce the expression of main CYP enzyme CYP3A4 in human hepatocytes. Levomepromazine and clozapine at concentrations of 2.5 and 10 µM, respectively, caused a significant increase in the mRNA level and activity of CYP3A4. Both neuroleptics did not produce any changes in CYP1A1/2, CYP2C9 and CYP2C19.Levomepromazine and clozapine induce CYP3A4 in human hepatocytes in vitro. Further in vivo studies are advisable to confirm the CYP3A4 induction by levomepromazine and clozapine in the liver, and to assess the effect of these drugs on their own metabolism and on the biotransformation of other co-administered drugs which are the CYP3A4 substrates. Cytochrome P450 (CYP) is one of the largest drug-metabolizing enzyme systems whose expression has significant substrate and tissue specificity. The CYP enzymes are involved in the biotransformation of most drugs and other xenobiotics, and help to eliminate them out of the body . MoreoveLevomepromazine is a typical phenothiazine antipsychotic with sedative/hypnotic, anxiolytic, antiemetic, analgesic and antipsychotic activities. It is used for treating schizophrenia, paranoia, mania, toxic psychosis and mental organic syndromes associated with delirium . ClozapiN-demethylation [N-demethylation and by CYP3A4 via N-oxidation [The knowledge of the ability of levomepromazine and clozapine to induce CYP enzymes would be of pharmacological and clinical importance, since these drugs are administered to patients for months or years, and very often in combination with other drugs, which are substrates of the CYP enzymes. Although there are some experimental data suggesting a possibility of induction of CYP isoforms by typical and atypical neuroleptics , the abihylation , while cxidation .The aim of this work was to ascertain whether levomepromazine and clozapine, the neuroleptics with different chemical structures and pharmacological profiles, may induce CYP1A1/2, CYP2C9, CYP2C19 and CYP3A4 enzymes in human liver.N-desmethylperazine were purchased from Sigma-Aldrich . Perazine was obtained from Labor . Testosterone and its metabolites were provided by Steraloids . All of the organic solvents with HPLC purity were supplied by Merck .All media for culture of human hepatocytes, PBS Buffer, LDH Cytotoxicity Detection Kit were purchased from ThermoFisher Scientific . Levomepromazine was obtained from Egyt , clozapine from Anpharm . Trypan Blue, dimethyl sulfoxide (DMSO), rifampicin, β-naphthoflavone, TRI Reagent, caffeine, paraxanthine, diclofenac, 4-hydroxydiclofenac, and 5 viable cells in 0.25 ml of plating medium for each well in Williams Medium E. The plates were incubated at 37 °C in 5% CO2 with saturating humidity to allow cells to adhere to the plate. The neuroleptics and positive inducers were added to the culture medium in 0.1% DMSO at a concentration of 0.25, 0.75, 2.5 µM for levomepromazine, 1, 2.5, 10 µM for clozapine, 25 µM for rifampicin , and 20 µM for β-naphthoflavone (CYP1A1/2 inducer). 0.1% DMSO was used as a vehicle control. The above procedure simulates the conditions of long-term treatment in the clinic. For each lot of hepatocytes, five separate wells per treatment conditions were used. The hepatocytes were incubated for 72 h with a daily replacement of the culture medium containing one neuroleptic or one inducer (a positive control).Experiments were performed in vitro using inducible-qualified human cryopreserved hepatocytes from three different lots. Cryopreserved human hepatocytes constitute a universally accepted experimental in vitro system for the evaluation of drug metabolic properties in human liver. Human hepatocyte donors  were thawed in hepatocyte recovery medium, viability was determined using Trypan Blue, according to the manufacturer’s protocols. The viability of the cell suspensions was > 90%. Hepatocytes were seeded in collagen type-I-coated 48-well plates from Corning , at a density of 1.35 × 10Thawed human hepatocytes were seeded in collagen type-I-coated 96-well plates in 100 µl of incubation medium at conditions described above. After 24 h, the hepatocytes were treated with 0.01, 0.1, 0.5, 1, 2.5, 5, 10, 25, 50, 100 and 200 µM of levomepromazine or clozapine in 0.1% DMSO (0.1% DMSO served as a control). At the end of the treatment, 50 µl of medium solution from all wells was transferred to a 96-well plate and the LDH reaction was performed using Pierce LDH Cytotoxicity Assay Kit following the manufacturer’s instructions.N-demethylation (CYP1A1/2), diclofenac 4′-hydroxylation (CYP2C9), perazine N-demethylation (CYP2C19) and testosterone 6β-hydroxylation (CYP3A4). The concentrations of CYP-specific substrates and their metabolites formed in the culture medium were measured by the HPLC method with UV detection, as described previously [After 72-h incubation of hepatocytes with the tested neuroleptics or inducers, the culture media were changed to a medium without the neuroleptics or inducers, but containing the CYP enzyme-specific substrates to assess specific CYP activities. After the incubation for 60 min, 100 µl of the culture medium from each well was transferred into Eppendorf tubes containing 10 µl of acetonitrile. CYP enzyme activities were determined in the culture medium using the following CYP-specific reactions: caffeine 3-g). Total cellular RNA was prepared using TRI Reagent method, according to the manufacturer’s instructions. cDNA was synthesized from total RNA using a Transcriptor High Fidelity cDNA Synthesis Kit (Roche) according to the manufacturer’s instructions. The cDNA was used as a template for qPCR using TaqMan Gene Expression Assays: CYP1A1 (Hs01054797_g1), CYP1A2 (Hs00167927_m1), CYP2C9 (Hs02383631_s1), CYP2C19 (Hs0042638_m1), CYP3A4 (Hs00604506_m1), β-actin (Hs99999903_m1). The expression levels of CYP1A1, CYP1A2, CYP2C9, CYP2C19 and CYP3A4 were normalized to β-actin and calculated using 2-delta Ct method as described previously [After incubation with the tested compounds, the cells were washed with ice-cold PBS buffer, scraped and collected by centrifugation  followed by Dunnett’s post hoc test (GraphPad Prism 7.0). The results were regarded as statistically significant when The hepatotoxicity of levomepromazine and clozapine on cryopreserved human hepatocytes was routinely assessed by estimating cellular damage by measuring lactate dehydrogenase (LDH) release into the culture medium. Cytotoxicity of levomepromazine and clozapine was expressed as the percent of control (0.1% DMSO-treated hepatocytes)  and inducers on CYP enzyme activities were evaluated in three human hepatocyte donors. 0.1% DMSO did not affect CYP enzyme activities (data not shown). Rifampicin potently raised the CYP3A4 activity in all three donors Fig. a, but loMoreover, β-naphthoflavone potently enhanced the activity of CYP1A1/2 and rifampicin that of CYP2C9 and CYP2C19, while levomepromazine and clozapine did not significantly affect the activity of those enzymes at any concentration tested in any of the evaluated human hepatocyte donors (data not shown).CYP3A4 mRNA level in all the three donors .Besides, β-naphthoflavone potently enhanced the level of The results of the statistical analysis are summarized in Table CYP3A4 and CYP2C genes [The present study indicates that levomepromazine and clozapine induce the expression of CYP3A4 enzyme in human hepatocytes. Levomepromazine at a concentration of 2.5 µM and clozapine at a concentration of 10 µM caused significant increase in the CYP3A4 mRNA and activity level. Both neuroleptics at the tested concentrations did not produce any changes in the expression and activity of CYP1A1/2, CYP2C9 and CYP2C19 enzymes. It suggests that the neuroleptics tested do not act as direct ligands of the nuclear receptors PXR or CAR, which are engaged in the transcriptional regulation of both CYP induction did not cause any cytotoxic effect on the cryopreserved human hepatocytes. But the concentrations of levomepromazine above 50 µM and of clozapine above 25 µM were toxic for hepatocytes. Similar level of clozapine hepatotoxicity was observed by Magliaroa and Saldanha [CYP1A1/2 or CYP2C9, CYP2C19 and CYP3A4, respectively) in all the tested hepatocyte donors, suggesting that the hepatocytes from different donors used in the present study retained the regulation mechanisms of CYP expression.It is worth noting that levomepromazine and clozapine at the concentrations tested for Saldanha  and Lu aSaldanha . On the Saldanha , 13 sign, and will lead to pharmacokinetic interactions.As mentioned above, levomepromazine and clozapine increased the expression and activity of CYP3A4 at the highest concentrations tested . These concentrations are higher than the therapeutic ones in the blood plasma  , 15. HowKi = 34 μM) [CYP gene expression, such as enzyme induction.Our previous experiment performed in vitro using the human cDNA-expressed CYP3A4 enzyme showed that levomepromazine mildly inhibited the activity of CYP3A4 (testosterone 6β-hydroxylation) via a mixed mechanism (= 34 μM) . Thus, l= 34 μM)  and clasA few clinical reports suggest that levomepromazine can induce cytochrome P450. A significant decrease in the plasma levels of clozapine or quetiapine was indeed observed in psychiatric patients simultaneously treated with levomepromazine , 18. SinThe obtained results show that levomepromazine and clozapine at the concentrations of 2.5 µM and 10 µM, respectively, increase the expression and activity of the main drug-metabolizing enzyme CYP3A4 in human hepatocytes. Since these neuroleptics can reach such concentrations in the liver during a long-term therapy of psychiatric patients, they may increase the CYP3A4 activity in clinical conditions. Further in vivo studies are advisable to confirm the CYP3A4 induction by levomepromazine and clozapine in the liver, and to assess the effect of these drugs on their own metabolism and on the biotransformation of other co-administered drugs which are the CYP3A4 substrates.


Mechanochemistry is an emerging and reliable alternative to conventional solution (batch) synthesis of complex molecules under green and solvent-free conditions. In this regard, we report here on the conjugation of a dextran polysaccharide with a fluorescent probe, a phenylboronic acid (PBA)-functionalized boron dipyrromethene (BODIPY) applying the ball milling approach. The ball milling formation of boron esters between PBA BODIPY and dextran proved to be more efficient in terms of reaction time, amount of reactants, and labelling degree compared to the corresponding solution-based synthetic route. PBA-BODIPY dextran assembles into nanoparticles of around 200 nm by hydrophobic interactions. The resulting PBA-BODIPY dextran nanoparticles retain an apolar interior as proved by pyrene fluorescence, suitable for the encapsulation of hydrophobic drugs with high biocompatibility while remaining fluorescent. In the last few decades, mechanochemistry has gathered a great deal of attention and a lot of efforts have been focused on its use in organic synthesis, catalysis, biotransformation reactions, and even in nanomaterials preparation –5. Indeecis-1,2 and 1,3-diols has been successfully exploited in many applications including the selective sensing of saccharides, the separation and purification of glycoproteins, and the immobilization of antibodies in microarrays  = 4.4 × 10−8 M in water. Pyrene fluorescence was measured using a stock solution of pyrene prepared in methanol due to its poor solubility in water (with a concentration of 4.45 × 10−6 M). Ten µL of this solution were dissolved in 1.0 mL of water to obtain a final concentration of 4.4 × 10−8 M.All reagents, whose synthesis is not described, were commercially available and were used without any further purification, if not specified otherwise. Ball milling was carried out with a Retch MM 400 mixer mill. Filtration was carried out using a glass vacuum filter with 0.2 µm sartorius. UV–vis spectra were recorded on a Varian Cary 4000 UV–vis spectrophotometer using a 1 cm cell or a BioTeK Synergy H4 microplate reader. Fluorescence spectra were registered on a Jasco FP750 spectrofluorometer using a 1 cm cell. NMR spectra were recorded on an Inova 400 instrument. Fourier transform infra red absorption spectroscopy (FTIR) was carried out with a Shimadzu IRAffinity-1S spectrometer. Potassium bromide (KBr) pellets were prepared using 100 mg of potassium bromide and 1.0 mg of sample. The spectra were recorded under inert atmosphere and a pure potassium bromide pellet was used as background. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was used to determine the boron (B) concentrations and was performed in triplicate using a Varian 720-ES Inductively Coupled Plasma Atomic Emission Spectrometer (ICP-AES). An accurately weighted amount of each sample was treated with a microwave-assisted digestion (CEM MARS Xpress) using 1 mL of suprapure HNO2 in a humidified chamber.Human lung adenocarcinoma (A549) cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) antibiotic solution . The cells were maintained at 37 °C, with 5% CODex-1b at different concentrations. After 24 h the cells were washed and 135 μL fresh medium with 15 μL of MTT (at 5 mg/mL in PBS) were added to each well. Culture plates were then incubated at 37 °C for 2 h, after which the medium-containing MTT was discarded and formazan crystals were dissolved in 150 µL DMSO. The absorbance at 550 nm (with automatic discount of reference wavelength 630 nm) of the resulting solution was measured in a 96-well spectrophotometer microplate reader. The percentage of cell mitochondrial activity was determined by the following formula: (Absorbance of treated cells/ Absorbance of control cells) × 100 (%).Cell mitochondrial activity was tested using the 3--2,5-diphenyltetrazolium bromide (MTT) assay, which is based on the mitochondrial conversion of the tetrazolium salt into a formazan dye with absorption characteristics in the visible region. Cells were incubated with 1) was prepared following the synthetic strategy reported in ref [Supporting Information File 1PBA-BODIPY , and 6.6 mL of dry dimethyl sulfoxide. The mixture was stirred for 6 h, then the product was precipitated in cold ethanol (50 mL), and filtered over a 0.2 µm nylon membrane. The powder was recovered and dispersed (6.8 mg/mL) in ethanol and sonicated for 5 min at 59 kHz. The dispersion was filtered over a 0.2 µm nylon membrane recovering both solid and solution. This protocol was repeated and then the solid was washed over the filter with ethanol (2 × 30 mL) and dried under vacuum to obtain 250 mg of Dex-1a as orange solid. FTIR data are shown in Figure S7 of In a dried sealed tube under a nitrogen atmosphere were added 4 Å molecular sieves (100 mg), 400 mg of 9–11 kDa dextran (at least 0.044 mmol), 17 mg of PBA-BODIPY , and milled for 90 min at 25 Hz. The powder was recovered, dissolved in DMSO (6.6 mL) and the product precipitated from cold ethanol (50 mL) and filtered over a 0.2 µm nylon membrane. The powder was recovered and dispersed (6.8 mg/mL) in ethanol and sonicated for 5 min at 59 kHz. The dispersion was filtered over a 0.2 µm nylon membrane recovering both solid and solution. The last protocol was repeated and then the solid was washed over the filter with ethanol (2 × 30 mL) and dried under vacuum to obtain 380 mg of Dex-1b as dark orange-solid. Boron content measured by ICP-AES 0.98 mg/g. FTIR data are given in Figure S8 of A 10 mL stainless-steel mixing mill equipped with a 1 cm diameter ss ball was charged with 400 mg of 9–11 kDa dextran (at least 0.044 mmol), 17 mg of PBA-BODIPY , and milled for 90 min at 25 Hz. The powder was recovered, dissolved in DMSO (6.6 mL) and the product precipitated from cold ethanol (50 mL) and filtered over a 0.2 µm nylon membrane. The powder was recovered and dispersed (6.8 mg/mL) in ethanol and sonicated for 5 min at 59 kHz. The dispersion was filtered over a 0.2 µm nylon membrane recovering both solid and solution. The last protocol was repeated and then the solid was washed over the filter with ethanol (2 × 30 mL) and dried under vacuum to obtain 336 mg of Dex-1c as orange-solid. FTIR data are given in Figure S9 of A 10 mL stainless-steel mixing mill equipped with a 1 cm diameter ss ball was charged with 400 mg of 9–11 kDa dextran (at least 0.044 mmol), 8.5 mg of PBA-BODIPY , and milled for 90 min at 25 Hz. The powder was recovered, dissolved in DMSO (6.6 mL) and the product was precipitated from cold ethanol (50 mL) and filtered over a 0.2 µm nylon membrane. The powder was recovered and dispersed (6.8 mg/mL) in ethanol and sonicated for 5 min at 59 kHz. The dispersion was filtered over a 0.2 µm nylon membrane recovering both solid and solution. The last protocol was repeated and then, the solid was washed over the filter with ethanol (2 × 30 mL) and dried under vacuum to obtain 360 mg of Dex-1d as light orange-solid FTIR data are given in Figure S10 of A 10 mL stainless-steel mixing mill equipped with a 1 cm diameter ss ball was charged with 400 mg of 9–11 kDa Dextran (at least 0.044 mmol), 1.7 mg of PBA-BODIPY (File 1Dex-1b, Dex-1c, and 1 in DMSO, FTIR spectra of PBA-BODIPY 1, dextran and Dex-1a–d; MTT assay and correlation coefficient of Dex-1b.Methods for UV–vis and fluorescence measurements, UV–vis and fluorescence spectra of