GUCY2D), which encodes guanylate cyclase 1 (ROS-GC1), are associated with LCA1 and account for 6%–21% of all LCA cases. In this study, one family with LCA1 was recruited from China. A combination of next generation sequencing and Sanger sequencing was used to screen for disease-causing mutations. We found three novel mutations  in the GUCY2D gene. Proband III-2 carries mutations c.139delC and c.2783G>A, which are inherited from the heterozygous mutation carriers, II-2 (c.139delC) and II-3 (c.2783G>A) that possess c.139delC and c.2783G>A. Additionally, II-8 carries heterozygous mutation c.835G>A. Sanger sequencing was used to confirm the presence of the three novel mutations in other family members. Mutation c.139delC results in a truncated protein. Mutations c.835G>A and c.2783G>A significantly reduce the catalytic activity of ROS-GC1. Our findings highlight the gene variants range of LCA. Moreover, HPLC-coupled tandem mass spectrometry (HPLC-MS/MS) was used to analyze the concentration of 3',5'-cyclic guanosine monophosphate (cGMP), suggesting that HPLC-MS/MS is an effective alternative method to evaluate the catalytic activity of wild-type and mutant ROS-GC1.Leber congenital amaurosis (LCA) is a group of severe congenital retinal diseases. Variants in the guanylate cyclase 2D gene ( GUCY2D) is a prevalent LCA gene because mutations to GUCY2D account for 6%-12% of all LCA (LCA1) cases [Leber congenital amaurosis (LCA), accounts for at least 5% of all inherited retinal dystrophies, and is the earliest and most severe form of all inherited retinal dystrophies . LCA is 1) cases , 5.GUCY2D, is expressed in rod and cone cells of vertebrae retina and catalyzes the synthesis of 3',5'-cyclic guanosine monophosphate (cGMP). ROS-GC1 is negatively controlled by a Ca2+ feedback loop because a reduction in Ca2+ concentration causes an increase in ROS-GC1 activity. However, the effects of the Ca2+ concentration on ROS-GC1 activity are indirect because this feedback loop is regulated by small Ca2+-binding guanylate cyclase-activating proteins (GCAPs), which interact with overlapping binding sites within the juxtamembrane domain (JMD), kinase homology domain (KHD) and dimerization domain (DD) of ROS-GC1. In addition to these latter domains, ROS-GC1 also possesses a leader sequence (LS), an extracellular domain (ECD), a transmembrane domain (TM) and a cyclase catalytic domain (CCD) [GUCY2D point mutations in different domains of ROS-GC1 cause protein dysfunction and subsequently result in LCA1 [Guanylate cyclase 1 (ROS-GC1), encoded by in (CCD) . CurrentGUCY2D gene were found, c.139delC (p.Ala49Profs*36), c.835G>A (p.Asp279Asn) and c.2783G>A (p.Gly928Glu). Mutations c.139delC and c.2783G>A were carried by proband III-2, and heterozygous mutation c.835G>A was carried by II-2. This study probes the pathogenesis of LCA1 and addresses the effects of mutations on ROS-GC1 activity. The three novel mutations may be suitable for LCA1 screening, and HPLC-MS/MS represents a convenient and effective method for cGMP quantification.In the present study, targeted-next generation sequencing (NGS) was performed to screen 222 genes that are responsible for 24 kinds of ophthalmic genetic diseases in the proband. After confirming the results by Sanger sequencing, the identity of three novel variants of the All procedures performed were in accordance with the Declaration of Helsinki and approved by the ethical standards of the Institutional Review Board, Zhejiang University. Written informed consent was obtained from all individuals who participated in this study and the parent of children under 16.A family with a 5-year-old proband (III-2) and two suspected patients (III-1 and III-3) was recruited for this study. The proband was diagnosed with LCA at Guangdong Zhongshan Hospital, Shanghai General Hospital and the Second Affiliated Hospital of the School of Medicine, Zhejiang University. The pedigree was constructed for the proband based on information provided by the guardians.Total genomic DNA was extracted using the Relax Gene Blood DNA System  following the manufacturer’s instructions. All DNA was dissolved in sterilized double-distilled water and kept at –20 °C until assayed.260/A280 ratio) and reagent contamination (indicated by the A260/A230 ratio) with a NanoDrop ND 1000 spectrophotometer .One percent agarose gels were used to monitor DNA degradation and contamination. All DNA samples were examined for protein contamination  was designed to capture 3093 exons (including 100 bp regions that flanked the exons) from 222 genes known to be associated with common genetic diseases, including retinitis pigmentosa, Waardenburg syndrome, X-linked juvenile retinoschisis, crystalline retinitis pigmentosa, albinism, LCA, Bardet Biedl syndrome and cone-rod dystrophy  used in Sanger sequencing were designed by Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) and synthesized by Sangon Biotech   . All ampXhoI and AgeI restriction sites were used to amplify ROS-GC1  to give the recombinant plasmid pEGFP-GC1.The cDNA of human ROS-GC1 was obtained from Gene Copoeia (EX-Z0715-M98). Primers F and R containing  ROS-GC1 . The pEGSite-directed mutagenesis PCR was used to construct the ROS-GC1 mutants. Primers used in site-directed mutagenesis PCR are shown in Recombinant plasmids pEGFP-GC1, pEGFP-Asp279Asn and pEGFP-Gly928Glu were transformed into Escherichia coli DH5α cells. DNA was prepared by using a plasmid DNA purification kit from Macherey-Nagel following the manufacturer’s instructions. Sanger sequencing was used to verify sequences.+/K+-ATPase  was used to identify the plasma membrane of HeLa cells. Details about the process have been described previously [pEGFP-N1, pEGFP-GC1, pEGFP-Asp279Asn and pEGFP-Gly928Glu were transfected into HeLa cells using the PolyJet Reagent . After 36 h, cells were washed and fixed in 4% paraformaldehyde. The antibody anti-NaProteins from transfected HeLa cells were electrophoresed on a 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes , and then incubated with the primary antibodies, anti-GFP  or anti-glyceraldehyde 3 phosphate dehydrogenase (GAPDH)  overnight at 4 °C. The PVDF membranes were washed with PBS and incubated with fluorescent secondary antibodies  for 2 h at room temperature. The protein bands were visualized using an Odyssey Imager . GAPDH was used as the standard.g at 4 °C for 10 min. The supernatant was transferred into a new tube and the non-dissolved residue was extracted two more times with 400-μL extraction medium. After evaporation, residues were collected and dissolved in water for further analysis.HeLa cells were transfected with pEGFP-N1, pEGFP-GC1, pEGFP-Asp279Asn and pEGFP-Gly928Glu. After 36 h, cells were collected from 100 mm plates and washed three times with PBS. The supernatant was removed carefully and 300 μL of ice-cold extraction medium  was added to each tube. Twenty-five ng/mL Tenofovir (TNF) was added as the internal standard. After dissolving, the sample was frozen immediately in liquid nitrogen for 30 s to terminate cGMP metabolism, and this was followed by incubation in a 37 °C water bath for 60 s. After repeating 6 times, samples were heated at 98 °C for 20 min. Samples were cooled on ice and centrifuged at 20,000 × P-values were calculated by means of the independent sample T-test.The cGMP concentrations were analyzed via HPLC-MS/MS. The samples were applied to an HPLC utilizing an ACQUITY CSH-C18 column . The binary pump system supplied two eluents for chromatographic analysis, eluent A (10 mM formic acid) and eluent B (acetonitrile). The flow rate was 0.3 mL/min. Analyte detection was conducted on a sensitive triple quadrupole mass spectrometer . Nitrogen was used as the collision gas. One-hundred ng/mL cGMP  was used as the standard. All processes were referenced to the method described previously [GUCY2D (NG_009092.1). The pathogenicity of mutations was evaluated using the in silico predictors SIFT (http://sift.jcvi.org/), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) and Mutation Taster (http://www.mutationtaster.org/). Computational modeling of the mutant ROS-GC1 by Chimera (PDB ID: 1AWL) was carried out to study the effect of the p.Gly928Glu mutation on the three-dimensional (3D) structure of ROS-GC1.All sequences were analyzed by Mutation Surveyor software and aligned to the NCBI nucleotide sequence of The proband (III-2) was a five-year-old girl who showed the disorder visually but could perceive light in a dark room. The proband displayed the classic signs of oculo-digital symptoms. Ophthalmological examination revealed pendular nystagmus, roving eye movements, macular “colobomas” and optic disc abnormalities. Additionally, dim retina and salt-and-pepper pigmentation were found in the fundus. The results of flash visual evoked potential (VEP) showed that the latent period of binocular P-waves was severely prolonged and the amplitudes were severely reduced. The electroretinogram (ERG) results showed that the latent period and the amplitude of binocular scotopia rod response b-wave, mixed response a-wave and mixed response b-wave were flat. With an attenuated photopic 30-Hz flicker, the full-field ERG (ffERG) showed a worsening delay of the cone-dependent response. The latent period and amplitude of the binocular photopic cone response a-wave and b-wave were flat. Clinical examination of the parents confirmed that neither was affected.GUCY2D gene were identified in the proband (III-2). Sanger sequencing confirmed the presence of c.139delC and c.2783G>A in other family members. The sequencing results showed that II-2 and II-3 are heterozygous mutation carriers that possess c.139delC and c.2783G>A, respectively  and parents (II-7 and II-8). The results showed that II-7 is a c.2783G>A mutation carrier, and II-8 possesses another novel mutation c.835G>A , which contains 158 amino acids from 871 to 1028 was used to study the impact of c.2783G>A (p.Gly928Glu) on the 3D structure of ROS-GC1. 3D modeling analysis showed that Glu928 directly interacts with GTP  and this+/K+-ATPase antibody was used as a specific marker of the plasma membrane  carried by II-1 and putatively by II-2 and III-3 generates a truncated protein (83 amino acids), containing only a part of the LS . As all Multiple sequence alignments indicated that aspartic acid and glycine at positions 279 and 928 are highly conserved across species, indicating that these residues are important for protein function. This is consistent with previous observations that mutations located in the ECD decrease cGMP production and result in LCA1 . The p.AGUCY2D mutations, such as HEK293 cells expressed mutant ROS-GC1 (A710V and P873R) showed undetectable cGMP concentrations, indicating that endogenous cGMP concentration exerted no contribution to cGMP levels. Although GUCY2D gene encodes a retina-specific protein, use other cell lines to investigate the localization and enzymatic activity of ROS-GC1 is feasible, for example, HEK293 cells were widely used to study the cellular localization of ROS-GC1 [In this study, the endogenous concentration of cGMP in HeLa cells, which were transfected with pEGFP-N1, is too low to be measured. This phenomenon can be observed in other studies of  ROS-GC1 ,17,18. I ROS-GC1 . However ROS-GC1 . Thus, iGUCY2D gene. Mutation c.139delC results in a truncated protein that lacks all functional domains of ROS-GC1. Although mutations c.835G>A and c.2783G>A showed normal protein expression levels and subcellular localization, both mutations significantly reduced the catalytic activity of ROS-GC1. As an alternative to radioisotope labeling assays, HPLC-MS/MS was used herein to analyze cGMP concentrations. Compared with traditional methods, HPLC-MS/MS is more convenient and is an effective alternative to evaluate the cGMP concentration in cells.In this study, we found three novel mutations  in the S1 FigResults from multiple sequence alignment reveal that codon 279 and 928 where the mutations Asp279Asn and Gly928Glu occurred are located within a highly conserved region.(TIF)Click here for additional data file.S2 FigSanger sequencing was used to verify the sequences of the recombinant plasmids pEGFP-GC1, pEGFP-Ala49Profs*36, pEGFP-Asp279Asn and pEGFP-Gly928Glu.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 File(RAR)Click here for additional data file.S2 File(RAR)Click here for additional data file.S3 File(RAR)Click here for additional data file.S4 File(RAR)Click here for additional data file.S5 File(RAR)Click here for additional data file.S6 File(RAR)Click here for additional data file.S7 File(RAR)Click here for additional data file.S8 File(RAR)Click here for additional data file.S9 File(RAR)Click here for additional data file.S10 File(RAR)Click here for additional data file.S11 File(RAR)Click here for additional data file.


Activation of P2X7 signaling, due to high glucose levels, leads to blood retinal barrier (BRB) breakdown, which is a hallmark of diabetic retinopathy (DR). Furthermore, several studies report that high glucose (HG) conditions and the related activation of the P2X7 receptor (P2X7R) lead to the over-expression of pro-inflammatory markers. In order to identify novel P2X7R antagonists, we carried out virtual screening on a focused compound dataset, including indole derivatives and natural compounds such as caffeic acid phenethyl ester derivatives, flavonoids, and diterpenoids. Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) rescoring and structural fingerprint clustering of docking poses from virtual screening highlighted that the diterpenoid dihydrotanshinone (DHTS) clustered with the well-known P2X7R antagonist JNJ47965567. A human-based in vitro BRB model made of retinal pericytes, astrocytes, and endothelial cells was used to assess the potential protective effect of DHTS against HG and 2′(3′)-O-(4-Benzoylbenzoyl)adenosine-5′-triphosphate (BzATP), a P2X7R agonist, insult. We found that HG/BzATP exposure generated BRB breakdown by enhancing barrier permeability ) and reducing the levels of ZO-1 and VE-cadherin junction proteins as well as of the Cx-43 mRNA expression levels. Furthermore, HG levels and P2X7R agonist treatment led to increased expression of pro-inflammatory mediators  and other molecular markers , along with enhanced production of reactive oxygen species. Treatment with DHTS preserved the BRB integrity from HG/BzATP damage. The protective effects of DHTS were also compared to the validated P2X7R antagonist, JNJ47965567. In conclusion, we provided new findings pointing out the therapeutic potential of DHTS, which is an inhibitor of P2X7R, in terms of preventing and/or counteracting the BRB dysfunctions elicited by HG conditions. Diabetic retinopathy (DR) is one of the microvascular complications of diabetes. DR is a leading cause of vision impairment among the working-age population . It has Zonula occludens-1, -2, and -3 , occludins, and claudins are tight junctions essential for BRB structural stability . A decreDuring the last three decades, several studies have demonstrated the pivotal role played by ROS in retinal microvascular complications, such as DR . Since tInside the retina, the combination between hyperglycemic and hypoxic conditions represents a strong stimulus for both astrocytes and endothelial cells, which, therefore, enhance the expression of a vascular endothelial growth factor (VEGF) . When hyEndothelial and pericyte dysfunction represents a key factor for DR progression and increased extracellular ATP (eATP) that has been observed at the site of inflammation as a consequence of endothelial cell injury . MoreoveSalvia miltiorrhiza, against the retinal damage elicited by HG and a selective P2X7R agonist [2′(3′)-O-(4-Benzoylbenzoyl)adenosine-5′-triphosphate (BzATP)].In the present work, we aimed to explore the role played by the P2X7R in a recently established in vitro primary culture based on triple co-culture BRB model entirely based on human cells  . Along wAccording to the last solved structure of a full-length rat P2X7R , we builA series of compounds  have bee® task of Schrodinger Maestro. According to our computational analysis  calculations of DHTS binding at P2X7R allosteric, orthosteric pockets. We included in our analysis a pocket in the cytosolic domain of P2X7R, as identified by the SiteMapanalysis , DHTS caiBRB integrity was evaluated by performing trans-endothelial electrical resistance (TEER) measurements and the paracellular permeability assay, following an exposure for 24 or 48 h to HG levels and BzATP (200 µM) in the absence or in the presence of JNJ47965567 (100 nM) or DHTS (500 nM) (2 h pre-treatment). Co-stimulus with HG and BzATP led to significant, albeit small, decreased TEER values, −18.5%, at 24 h, that further increased to −25.3% at 48 h, i.e., an increased permeability of our triple co-culture model of iBRB . DHTS anThese data were also confirmed by measuring the apical-to-basolateral permeability of sodium fluorescein (Na-F). Na-F permeability accounts to paracellular permeability across the endothelial cells/perycites monolayers under our experimental conditions  Figure .p < 0.05 vs. NG), confirming the TEER measurements. The inhibition of P2X7R by pre-treatment with JNJ47965567 significantly decreased the Na-F permeability, counteracting the effects of HG + BzATP. According to virtual screening predictions, DHTS (putative P2X7R antagonist) also decreased the monolayer Na-F permeability, preventing and/or counteracting the damage induced by both HG and P2X7R agonist (BzATP) as already shown in TEER measurements. Therefore, JNJ47965567 and DHTS exerted protective effects on iBRB integrity challenged with the co-stimuli HG and BzATP.HG/BzATP stimulus significantly increased the Na-F fluorescence, indicating that HG and activation of P2X7R led to higher permeability of endothelial cells/perycites monolayers  and adherens junctions, we carried out immunocytochemistry experiments to assess the expression of ZO-1 and VE-cadherin proteins in endothelial cells’ monolayer, which is part of our iBRB model. As shown in A similar trend was observed for expression of the adherens junction VE-cadherin. p < 0.001) increase in ROS formation compared to control conditions. JNJ47965567 or DHTS pre-treatment significantly prevented the increase in ROS production elicited by HG + BzATP . After stimulation for 48 h with HG + BzATP, there was a significant .As shown in p < 0.05), TNF-α (p < 0.05), and IL-8 (p < 0.01), counteracting the inflammatory stimuli exerted by HG + BzATP. The “predicted” P2X7R antagonist DHTS significantly decreased the expression levels of the analyzed pro-inflammatory markers (p < 0.05) to values of control cells. Furthermore, HG + BzATP stimulation led to a significant increased expression of TLR-4 expression compared to NG conditions (p < 0.05) (p < 0.05 vs. HG + BzATP). As observed for IL-6, JNJ47965567 pre-treatment diminished, although not significantly, the mRNA levels of TLR-4 in endothelial cells challenged with HG + BzATP. The expression at the mRNA level of matrix metallopeptidase 9 (MMP-9), which is an additional pro-inflammatory marker, NADPH oxidase 2, which is a pro-oxidant enzyme, and transforming growth factor beta-1 (TGF-β1), which is an anti-inflammatory cytokine, was measured. We did not detect any significant changes for these three targets under our experimental conditions (data not shown).Inhibition of P2X7R, through JNJ47965567 pre-treatment, significantly decreased cytokines levels such as IL-1β ( < 0.05) E. DHTS pp < 0.05), while the opposite effect was observed for Cx-43 (p < 0.001). JNJ47965567 or DHTS pre-treatment significantly counteracted the over-expression of P2X7R, VEGF-A, and ICAM-1 induced by HG + BzATP treatment, and rescued Cx-43 expression levels to values of control cells (NG condition).In order to assess the effects of HG and P2X7R activation on the expression of connexin-43 (Cx-43) and P2X7R, we carried out qRT-PCR experiments. Furthermore, accordingly to pathophysiology markers of DR, we included the study of VEGF-A and ICAM-1 mRNA levels under our experimental conditions in our analysis assessment . The treBRB dysfunction represents a well-known hallmark of DR . HoweverDuring the last decade, the signaling pathway of P2X7R, which is an ATP-gated purinergic channel, has been associated with inflammatory phenomena at a retinal level . Accordi2 receptor [Along with inflammation, another factor that contributes to DR progression is oxidative stress ,67. As sreceptor . We inclWe also focused on the intracellular production of total ROS in endothelial cells, showing that their levels, significantly increased as a consequence of HG/BzATP treatment, were significantly decreased by the presence of DHTS or JNJ47965567, that antagonized the BzATP damage . We alsoAs mentioned before, it has been shown that P2X7R activation leads to increased ROS production in retinal micro-vessels  and VEGF2 polystyrene culture flasks, 12-well and 96-well plates, and rat-tail collagen type I were obtained from Corning Inc. . 2′,7′-dichlorofluorescin diacetate (DCFDA)—cellular ROS assay kit was purchased from Abcam . Anti-VE-cadherin primary antibody was supplied by Cell Signaling . Na-F was obtained from Santa Cruz Biotechnology, Inc. . The materials necessary to perform qRT-PCR experiments  were purchased from Qiagen , while 18S rRNA, TLR-4, P2X7R, Cx-43, and ICAM-1 primers were all purchased by Eurofins MWG Synthesis GmbH .All materials and reagents were of an analytical grade and purchased from Thermo Fisher Scientific  or Sigma-Aldrich  unless specified otherwise. The three human retinal cell lines  along with astrocyte cell medium (AM), pericyte cell medium (PM), endothelial cell medium (ECM), endothelial cell growth supplement (ECGS), fetal bovine serum (FBS), pericyte supplement factor (PGS), astrocytes supplement factor (AGS), polylysine (PLL), and penicillin–streptomycin were purchased from INNOPROT . JNJ47965567 was purchased by Tocris Bioscience . Cell culture inserts, 75-cm® Schrodinger Maestro task, using implicit solvation and the membrane. The protein model quality check has been carried out and the Ramachandran plot was calculated. SitMap® identified three druggable pockets, the allosteric, orthosteric, and cytosolic cavities at a hP2X7 full-length model. Glide® grids were built and grid origin was fixed at the center of mass of the SitMap pockets. The previously published compounds database [® docking was carried out for DHTS at allosteric, orthosteric, and cytosolic pockets. MM/GBSA calculation, using implicit solvation and the membrane, was carried out according to a previous published protocol [The human full-length model of P2X7R has been built with the advanced homology modeling task of Schrodinger Maestro, using the following input: primary sequence (uniprot accession# Q99572) and the template full-length rat P2X7 apo structure (PDB:6U9V). The hP2X7 model was minimized with the Primedatabase  was modidatabase . SIFT stdatabase  was aimeprotocol .A previous human triple co-culture BRB model  was usedThe activity of DHTS and JNJ47965567 as antagonists of HG + BzATP challenge was evaluated by measurements of TEER by using a Millicel-Electrical Resistance System (ERS2)  at different time points: 0 (T0), 1 (T24), and 2 (T48) days after treatment as previously described ,85.To evaluate the modification of paracellular permeability under the above-mentioned conditions, the luminal-to-abluminal movements of Na-F across endothelial cell monolayers after 48 h of treatment were measured by using a Varioskan Flash microplate reader  as previously described .Astrocytes, pericytes, and endothelial cells were characterized as previously described . To evalThe semi-quantitative evaluation of ZO-1 and VE-cadherin expression levels was carried out as previously described ,33. CoveThe ability of JNJ47965567 and DHTS in counteracting the changes in intracellular ROS due to HG + BzATP treatment for 48 h was carried out in endothelial cells, which is part of the in vitro tri-culture model, using a 2′,7′-dichlorofluorescin diacetate (DCFDA) cellular ROS assay kit, according to the manufacturer’s recommendations. To determine total ROS formation, the fluorescence (485 nm excitation/535 nm emission) was measured by using a Varioskan Flash microplate reader and normalized to the fluorescent intensity of control conditions (NG).® ND-1000 (Thermo Fisher Scientific) and the RNA quality was tested by a Qubit® 3.0 Fluorometer ). For reverse transcription, sample amplification, fluorescence data collection, and sample quantification, the same protocol as previously described was used [The procedure to extract the RNA by a TRIzol reagent from the endothelial cells, part of the in vitro BRB model, is the same as recently described by us . RNA conwas used ,88. The –∆∆Ct) method.The sequences of the primers purchased by Eurofins MWG Synthesis GmbH  are the following: 18S rRNA , TLR-4 , P2X7R , Cx-43 , ICAM-1 , and MMP-9 . One-way or two-way ANOVA, followed by Tukey The effects of the combined treatment with HG and the selective P2X7R agonist 2BzATP were investigated in our recently developed in vitro BRB model, more closely mimicking the inner retinal barrier compared to the cell monolayer, entirely based on human retinal cells. This stimulation led to significant BRB breakdown (decreased TEER and increased Na-F permeability), along with a significant reduction of both ZO-1 and VE-cadherin in endothelial cells. An increased oxidative stress, measured as a total intracellular ROS, and an up-regulation of the genes responsible for the formation of P2X7R and pro-inflammatory mediators was also observed in endothelial cells, which is part of the BRB model. The predicted P2X7R allosteric antagonist DHTS and the validated P2X7R antagonist JNJ47965567 significantly antagonized HG/BzATP-induced damage in our BRB model. In particular, DHTS maintained the integrity of the BRB and preserved the expression levels of endothelial junction proteins, ZO-1 and VE-cadherin. The increase of the expression of the TLR-4 gene as well as of IL-1β, IL-6, TNF-α, and IL-8 pro-inflammatory genes was also significantly counteracted by both compounds with the DHTS showing a greater protective effect against the HG/BzATP insult. The anti-inflammatory features along with the antioxidant action of DHTS were also demonstrated by the down-regulation of VEGF-A and ICAM-1 mRNA expression levels, the rescue of Cx-43, and the decrease in total ROS.In conclusion, we provided new findings pointing out the therapeutic potential of DHTS in preventing and/or counteracting the blood retinal barrier dysfunctions elicited by high glucose and P2X7R activation.


Furthermore, pharmacological profiles of ACE inhibitors and their impurities were determined via an in silico approach. The above data were used to develop comprehensive classification, which may be useful in further drug design studies. Proline is a unique, endogenous amino acid, prevalent in proteins and essential for living organisms. It is appreciated as a tecton for the rational design of new bio-active substances. Herein, we present a short overview of the subject. We analyzed 2366 proline-derived structures deposited in the Cambridge Structure Database, with emphasis on the angiotensin-converting enzyme inhibitors. The latter are the first-line antihypertensive and cardiological drugs. Their side effects prompt a search for improved pharmaceuticals. Characterization of tectons (molecular building blocks) and the resulting supramolecular synthons (patterns of intermolecular interactions) involving proline derivatives, as presented in this study, may be useful for in silico molecular docking and macromolecular modeling studies. The DFT, Hirshfeld surface and energy framework methods gave considerable insight into the nature of close inter-contacts and supramolecular topology. Substituents of proline entity are important for the formation and cooperation of synthons. Tectonic subunits contain proline moieties characterized by diverse ionization states: -N and -COOH(-COO Proline was isolated for the first time by Richard Willstätter in 1900  who deriN-atom. Its ring, constrained by the tertiary amide bond, is a remarkably rigid structure in the world of peptides , perindopril structures and also EDALEC, GERXEI, GERWUX01, TUHMOY, TUHMUE.HThe enrichment ratios (ER) of the interactions available in the analysed crystal structures were based on the HS methodology, see ..H, C…H and S…H (1.61) in perindoprilat, followed by O…O (1.25) in perindopril and N…H (1.15) in DKP polymorph, among the structures of the perindopril family. The H…H contacts are the most abundant interactions (~70%), but they are rather moderately enriched with the ER close to unity (0.9).The highest value of ER is calculated for the contacts OIn the case of other proline-based ACEI, the most enriched contact is between oxygen and sodium atoms, in TUHMOE and TUHMUE, although it represents ~5% of the contact surfaces. …O, C…O, Na…O, O…H/C…H, S…O, Cl…H, C…C, N…H have ER values of 2.03 (in DIVHOF01), 1.88 (in EDALEC), 1.41 (in TUHMUE), 1.38 (in CIYNIH), 1.33 (in RUWBAM), 1.29 (in RUWBAM), 1.25 (in DIVHOF01) and 1.2 (in QOQWAU), respectively.The O…H (0.86 in TUHMOY), C…O (0.8 in FIFGEG), S…H (0.77 in MCPRL01), S…O (0.63 in NELSEC and 0.5 in TUHMUE), Na…H (0.23 in TUHMOY) and C…O (0.21 in DIVHOF01) are disfavoured in the crystal packing of proline-based ACEI. The two latter contacts are impoverished.On the other hand, weak contacts O…O, O-H…N, N-H…O, C-H…O, S-H…O and O-H…Cl− synthons are formed. More specifically, COOH and COO− groups of proline participate in either single or bifurcated H-bonding interactions. The latter play an important role in supramolecular architectures of ACEI crystals. The COO− group is involved in the N-H…O…H-N, C-H…O…H-C, N-H…O…H-C, while the COOH group builds two types of H-bonds systems: N-H…O…H-C, C-H…O…H-C, C-H…O…H-S and O…H-O…H-C, N…H-O…H-C. Moreover, the pyrrolidine ring of proline participates in the formation of (-CcyclPRO)C-H…O(COOH/COO-/SH/Cl-) interactions. Synthons formed by weak inter-contacts only are marked in yellow frames in − groups of pyrrolidine, aliphatic groups COOH/COO− and NH  have relevance in the ACE binding. Therefore, from the point of view of the bio-complexes of inhibitors with ACE, in the −) of proline (−) in the chain of proline (2+) in the proline chain (C22(6) based on the N-H…O hydrogen bonds and the cyclic ring R22(12) based on the C-H…O and N-H…O bonds. The same patterns exist most commonly in perindopril-derived structures.The thorough investigation revealed a wide variety of supramolecular interactions (synthons) resulting from three types of proline-based tectons . General proline , COOH, R22(9), D22(5), D22(6), D22(9), D22(10), D33(16), D33(20), while in the IQISAE: R22(13), C12(16), C(11), C22(13), C22(15), C22(19), C22(20) graphs are observed. The sulphur atom participates in the formation of synthons via O–H…S H-bonding with water molecules. In EDALEC, intramolecular synthon S(6) is observed. A Tert-butyl moiety in perindopril crystals is also involved in the synthons  plane and constitute Long-Range Synthon Aufbau Modules (LSAM)  interwoven by N-H…O zigzags along the (COOH/COO-/SH/Cl–) interactions. The Hirshfeld surface analysis revealed that C…C, C…O/O…C, O…O and Na…O(H)/(H)O…Na inter-contacts cannot be neglected. Energy framework calculations identified the most important inter-contacts in ACEI. In particular, proline, perindoprilat, and lisinopril structures are governed by the classic, electrostatic forces while, in DKP, perindopril polymorphs, ramipril, trandolapril, dispersion effects are preferred. The tiny balance between electrostatic and dispersion was observed in captopril and its disulfide. ADMET pharmacological profiles are similar for all ACEI and their impurities. Prodrugs are mostly hepatotoxic with the captopril and perindopril impurities, with G, K and L being the exception. Libraries of structural, conformational, and pharmacological data for ACE inhibitors may be used in studies on smart ligands containing proline and acting via inhibition of either ACE or similar targets. A comprehensive characterization of related synthonic interactions can help to design model ligands for efficient enzyme binding. Proline is a unique amino acid that is attracting increasing attention in contemporary medicinal chemistry and supramolecular pharmacology with special emphasis on its specific topological properties. It is frequently localized in turns and other active sites of protein chains and contrains conformation of short peptides. In this work, we thoroughly analysed supramolecular features of prolines and further applied this approach to angiotensine converting enzyme inhibitors. The latter are frontline therapeutics applied in hypertension and cardiovascular diseases. Their supramolecular structures have not been precisely systematically analysed by contemporary approaches to date. The rational drug design involves multidisciplinary studies. A detailed characterization of synthons and tectons involving proline derivatives may be useful for in silico molecular docking and macromolecular modelling studies. The comprehensive search of the Cambridge Structural Database yielded an impressive number of entries (2366) which were used to create the libraries of conformational and supramolecular features. Twenty proline-based ACEI were subjected to detailed DFT, Hirshfield surface and energy framework studies for the comprehensive description of tectons and synthons. Tectonic subunits contain proline moieties characterized by diverse ionization states: –N and –COOH(–COOThe comprehensive summary of weak intermolecular interactions in proline-based ACEI enhances knowledge on the supramolecular recognition in bio-complexes of ACE with potential new effective inhibitors. As highlighted in this paper, the synthon concept can also be successfully applied in the protein environment. Structural data determined by X-ray crystallography gave insight into the supramolecular forces. Additional methods, like Hirshfeld surface analysis and energy frameworks help to explain how tiny preferences of H-bonds donors and acceptors affect the overall supramolecular topology. Those results allow the identification of binding predispositions of conformationally flexible ACEI. The advanced bio-structural summary sheds light on new supramolecular strategies of rational design of modern inhibitors with better biological activity and reduced side effects combined with a high affinity and specificity. In this respect, we proved that not only the macromolecular, but also small-molecular, approaches have relevance. Future work will focus on proline substituents as building blocks for the formation of synthons. Special attention will be paid to cooperative effects, which are frequently neglected in the crystal structure interpretations. They affect the entropy of supramolecular entities and are important for their overall characterization.


Seriola fasciata) from the Madeira archipelago . This reference material was used to implement a liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) for the detection of C-CTX1, acquisition of full-scan as well as collision-induced mass spectra of this particular analogue. Fragmentation pathways were proposed based on fragments obtained. The optimized LC-HRMS method was then applied to confirm C-CTX1 in fish (Bodianus scrofa) caught in the Selvagens Islands .Ciguatera poisoning (CP) is a common seafood intoxication mainly caused by the consumption of fish contaminated by ciguatoxins. Recent studies showed that Caribbean ciguatoxin-1 (C-CTX1) is the main toxin causing CP through fish caught in the Northeast Atlantic, e.g., Canary Islands (Spain) and Madeira . The use of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) combined with neuroblastoma cell assay (N2a) allowed the initial confirmation of the presence of C-CTX1 in contaminated fish samples from the abovementioned areas, nevertheless the lack of commercially available reference materials for these particular ciguatoxin (CTX) analogues has been a major limitation to progress research. The EuroCigua project allowed the preparation of C-CTX1 laboratory reference material (LRM) from fish species ( Ciguatera poisoning (CP) is a food intoxication mainly related to the consumption of fish contaminated with ciguatoxins (CTXs) . CiguatoThe potential for confirmation provided by high-resolution mass spectrometry (HRMS) makes this approach necessary, in particular when the availability of reference materials is limited. Nevertheless, the lack of sensitivity is a challenge that compromises its applicability as concentrations of CTXs are typically low. The complexity of the matrix and the difficulty in the ionization of CTXs also jeopardizes the applicability of LC-HRMS for the detection of CTXs. Despite this, the applicability of HRMS has been demonstrated in particular for CTX1B and CTX3C as the main toxins responsible of CP in the Pacific Ocean, the fragmentation pathways have been identified, increasing the knowledge on how these complex toxins fragment in the mass spectrometer ,18,19. RThis study responds to one of the objectives included in the EUROCIGUA project, which focused on the risk characterization of ciguatera food poisoning in Europe, and it is partially funded by the European Food Safety Authority (EFSA). One of the objectives of the project is the confirmation by HRMS of the CTX analogues previously detected by LC-MS/MS in fish samples from selected areas in the EU. To accomplish this objective, an LC-HRMS method was developed to obtain C-CTX1 fragmentation pathways, which will allow the characterization of the C-CTX1 molecule. The method was subsequently applied to confirm the presence of C-CTX1 in a contaminated fish sample from the Northeast Atlantic , previously identified by LC-MS/MS.Seriola fasciata, was analyzed by LC-HRMS. The use of aqueous methanol as mobile phase was dismissed due to the formation of highly stable [M + Na]+ and the difficulty in its fragmentation. For this reason, the LC and ion source conditions proposed by + followed by the sodium adduct m/z 1163.6106 (−1.6 ppm) [M + Na]+. The protonated molecule [M + H]+ as well as ammonium [M + NH4]+ and potassium adducts [M + K]+ were present but with a lower intensity and with higher mass differences (>10 ppm) (LC-HRMS in full-scan MS acquisition mode detected C-CTX1 at a retention time of 7.8 min with an ion pattern where the predominant ion was the first water loss >10 ppm) A. The as+ was not the predominant ion cluster; so due to its high intensity, the first water loss m/z 1123.6200 [M + H - H2O]+ was selected as precursor ion to be fragmented. C-CTX1 fragmentation pathways were proposed based on HRMS/MS spectra in positive targeted MS/MS mode with a range of six collision energies (from 20 to 70 eV). In HRMS spectra A, the prm/z 1123.6200 [M+H-H2O]+ were characterized by the detection of successive water losses at m/z 1105.6094 [M+H−2H2O]+, m/z 1087.5989 [M + H - 3H2O]+, and m/z 1069.5883 [M + H - 4H2O]+  or MRM (multiple reaction monitoring) mode [These results confirmed that the C-CTX1 molecular ion with theoretical ng) mode . Bodianus scrofa) from Selvagens Islands  was extracted and purified following the conditions described by [m/z 1123.6214 (+1.2 ppm) [M + H - H2O]+ and the sodium adduct at m/z 1163.6138 (+1.1 ppm) [M + Na]+  was prepared at the University of Vigo, in the framework of the Specific Grant 4 of the project EuroCigua . C-CTX1 in the LRM was identified by comparison with authentic C-CTX1 kindly provided by Robert W. Dickey  via Ronald Manger .C-CTX1 LRM (estimated to 123 ng/mL) purified from amberjack B. scrofa) was captured in the marine protected area of Selvagens Islands  under the EUROCIGUA framework agreement in September 2018. A portion of flesh tissue was dissected and stored raw at −20 °C until use. Barred hogfish .Water, acetonitrile, formic acid, and ammonium formate used to prepare mobile phases were of LC-MS grade. All these chemicals were purchased from Sigma Aldrich .Barred hogfish was extracted and purified following the conditions proposed by . LC-HRMS analyses were carried out using a UHPLC system 1290 Infinity II  coupled to a HRMS quadrupole/time-of-flight (Q-TOF) mass spectrometer, i.e., Q-TOF 6550 iFunnel . Chromatographic separation was performed using a Kinetex C18 column  at 40 °C with 5 mM ammonium formate and 0.1% formic acid in water (A) and acetonitrile (B). The flow rate was set at 0.4 mL/min and the injection volume was 5 µL. Gradient of mobile phase was carried out as follows: 35% B was kept for 1 min increasing to 80% B in 15 min, 95% of B in 16 min keeping for 5 min, and returning to the initial conditions in 0.1 min equilibrating the column for 4.9 min prior to the next injection.m/z) ranging from 800 to 1200 with a scan rate of 1 spectrum/s. Targeted MS/MS was performed in a collision induced dissociation (CID) cell at 45,000 FWHM over the scan rage from m/z 50 to 1200 with a scan rate of 10 spectra/s and a scan rate of 3 spectra/s applying three different collision energies in order to have a good fragmentation pathway. A reference mass of m/z 922.0099 (hexakisphosphazine) was continuously monitored during the entire run. Acquisition was controlled by MassHunter software . Raw data were processed with Agilent MassHunter Qualitative Analysis software  using the Find by Formula (FbF) algorithm screening with a Personal Compound Database and Library (PDCL) created by the Phycotoxins Laboratory .LC-HRMS analyses were carried out in full-scan and targeted MS/MS mode in positive ionization. Full-scan analysis operated at a mass resolution of 40,000 full width at half maximum (FWHM) over a mass-to-charge ratio (Source conditions were as follows: gas temperature, 250 °C; gas flow, 16 L/min; nebulizer, 15 psi; sheath gas temperature, 400 °C; sheath gas flow, 12 L/min; capillary voltage, 5000 V, and nozzle voltage, 1000 V. The instrument was calibrated, using the Agilent tuning mix, in positive ionization mode before each analysis.


Enterococcus hirae or Lactobacillus johnsonii, transferred bacterial species and host mouse strain were critical determinants of the anti-tumour efficacy of Tim-3 blockade. Bacterial gavage did not increase the alpha diversity of gut microbiota in antibiotic-treated mice but did alter the microbiome composition, which was associated with the restoration of the anti-tumour efficacy of Tim-3 blockade. Conclusively, our results indicate that gut microbiota modulation may improve the therapeutic efficacy of Tim-3 blockade during concomitant antibiotic treatment. The administered bacterial species and host factors should be considered in order to achieve therapeutically beneficial modulation of the microbiota.T cell immunoglobulin and mucin domain-containing protein-3 (Tim-3) is an immune checkpoint molecule and a target for anti-cancer therapy. In this study, we examined whether gut microbiota manipulation altered the anti-tumour efficacy of Tim-3 blockade. The gut microbiota of mice was manipulated through the administration of antibiotics and oral gavage of bacteria. Alterations in the gut microbiome were analysed by 16S rRNA gene sequencing. Gut dysbiosis triggered by antibiotics attenuated the anti-tumour efficacy of Tim-3 blockade in both C57BL/6 and BALB/c mice. Anti-tumour efficacy was restored following oral gavage of faecal bacteria even as antibiotic administration continued. In the case of oral gavage of  Havcr2) gene and is an emerging target for cancer immunotherapy. It was discovered as a molecule that distinguished type 1 helper T (TH1) cells from type 2 helper T (TH2) cells [T cell immunoglobulin and mucin domain-containing protein-3 (Tim-3) is an immunoregulatory protein encoded by the Hepatitis A virus cellular receptor 2 (2) cells . In T ce2) cells . Tim-3 i2) cells . Tim-3 i2) cells . In addi2) cells . Increas2) cells ,10,11. B2) cells ,13. Furt2) cells ,15,16. S2) cells ,18,19.Escherichia coli enhanced fludarabine activity [Enterococcus hirae enhanced the efficacy of cyclophosphamide in mice [The efficacy of anti-cancer therapies is influenced by gut microbiota composition . Certainactivity . In contactivity . Manipul in mice . Further in mice ,25,26. M in mice . HoweverIn this study, we examined whether gut microbiota modulation influences the efficacy of Tim-3 blockade in mouse tumour models. In particular, the efficacy of gut microbiota modulation for enhancing the tumour-suppressive effects of Tim-3 blockade was evaluated in mice receiving antibiotics. We observed that oral gavage of bacteria altered the gut microbiota of mice despite continuous antibiotic administration. Further, the anti-tumour efficacy of Tim-3 blockade depended on the particular bacterial species administered through oral gavage and the mouse strain.B16 melanoma cells (American Type Culture Collection (ATCC), Manassas, VA, USA) and Chinese hamster ovary-K1  cells were cultured in Dulbecco’s modified Eagle’s medium  containing 10% foetal bovine serum , 100 U/mL penicillin, and 100 µg/mL streptomycin . CT-26 BALB/c colon carcinoma cells   were culThe IgV domain of Tim-3 was amplified by PCR using primers (forward primer: 5′-CGG GGT ACC GAT TGG AAA ATG CTT ATG TGT TTG AG and reverse primer: 5′-GAA TTC TGC TTT GAT GTC TAA TTT CAG TTC) and the pIRES2-EGFP-Tim3SVMhIg plasmid . The Tim® Laboratories, Invitrogen, Carlsbad, CA, USA) and then developed using an enhanced chemiluminescence kit .CHO cells and HEK-293T cells were transfected with a Tim-3 expression vector using Polyethylenimine . After 2 days, the culture supernatant was collected and loaded on sodium dodecyl sulfate polyacrylamide gel for electrophoresis (SDS-PAGE) or non-denatured PAGE followed by transfer onto a polyvinylidene difluoride (PVDF) membrane . The membrane was incubated with an anti-mouse IgG antibody conjugated to horseradish peroxidase (ZYMED6/mL) were transfected with pSecTag2C-Tim-3VdIg using PEI. After 7 days, supernatant was harvested from the culture, and the Tim3VdIg protein was purified using Protein A beads .HEK-293T cells (2 × 106 cells/mL): B16 cells for B6 mice and CT-26 for BALB/c mice. Mice were intraperitoneally injected with Tim-3VdIg (60 µg/mouse) every second day for 12 days after tumour challenge. Tumour progression was assessed every second day by determining tumour volume using the formula: tumour volume = 0.523 × tumour length × (tumour width)2.Tumour growth experiments in mice were approved by the Institutional Animal Care and Use Committee, Ajou University Medical Center (IACUC protocol #2016-0003). Six-week-old male C57BL/6 (B6) and BALB/c mice were purchased from OrientBio . Mice were maintained in specific-pathogen-free conditions and in separate cages based on strain and treatment. An antibiotic mixture containing 900 mg/L ampicillin , 900 mg/L neomycin , 900 mg/L metronidazole , and 300 mg/L vancomycin  was administered to mice via drinking water. Drinking water was replaced every three days. After three weeks, all mice were subcutaneously injected with 100 µL of tumour cells  and Enterococcus hirae (Korean Culture Center of Microorganisms) were cultured at 37 °C in Brain Heart Infusion media and De Man, Rogosa, and Sharpe (MRS) agar, respectively. The bacteria were cultured to an OD of 1.8 measured at 600 nm (corresponding to 109 CFU/mL) and then aliquoted and cryopreserved in 15% glycerol. Each bacterial suspension (100 µL/head) was administered to a mouse seven times in total, on every third day.A faecal bacteria stock was prepared by collecting faeces from the large intestine of eight-week-old BALB/c and B6 mice under anaerobic conditions. Faeces were suspended in PBS at a concentration of 60 mg/mL followed by centrifugation at 800× Mice were sacrificed on the eighth day after tumour cell challenge to collect caecal content, which was immediately frozen at −70 °C for microbiome analysis. DNA was extracted from caecal samples using the DNeasyPowerSoil Kit  according to the manufacturer’s instructions. DNA quality was assessed by gel electrophoresis and fluorometry. Sequencing libraries were constructed according to the Illumina 16S Metagenomic Sequencing Library protocols using Herculase II fusion DNA polymerase  and the universal primer pair specific for the V3–V4 region of the 16S rRNA gene with Illumina adapter overhang sequences. The purified PCR product was quantified according to the qPCR Quantification Protocol Guide (KAPA Library Quantification kits for Illumina Sequencing platforms) and qualified using the TapeStation D1000 ScreenTape . Paired-end sequencing was performed with Macrogen  using the MiSeq™ platform .The FLASH  program was employed to merge paired-end reads . Open ret-test or ANOVA with Bonferroni correction for multiple comparisons. p < 0.05 indicated statistical significance. Significant differences in alpha diversity were computed using the Kruskal–Wallis test with Dunn’s multiple comparison test. Significant differences in beta diversity were computed using PERMANOVA and ANOSIM. Significant differences in dispersion were determined by permDISP.The statistical significance of differences in tumour growth between groups was analysed using the Student’s p < 0.001).We previously reported the tumour-suppressive effects of Tim-3 blockade using a Tim-3hIg fusion protein comprised of the Tim-3 variable domain (V) and mucin domain linked to the Fc region of human IgG . The Timp < 0.001). However, significant suppression of tumour growth by Tim3VdIg was not observed in mice treated with antibiotics except for in 1-year-old B6 mice on day 14. These results indicate that Tim3VdIg may exert tumour-suppressive effects in subjects with different genetic backgrounds, tumour types, and age, but not in subjects treated with antibiotics.To investigate the impact of gut microbiota modulation on the tumour-suppressive efficacy of Tim3VdIg, we examined tumour growth in mice administered Tim3VdIg while receiving or not receiving antibiotics, based on a previous report of gut microbiota disturbance by antibiotic treatment . A mixtuEnterococcus hirae, or Lactobacillus johnsonii once every three days. While the tumour-suppressive effect of Tim3VdIg was consistently suppressed in antibiotic-treated mice, it was partially but significantly restored by the oral transfer of faecal bacteria or E. hirae . However, tumour suppression was not maintained thereafter   b. In BALohnsonii c,d. ThesTo verify changes in the gut microbiota of mice receiving antibiotics and bacteria, we analysed the microbiome of the cecum harvested on the eighth day after tumour challenge. At that time point, antibiotic treatment was ongoing, and oral gavage of bacteria had been performed five times in total. Read count ranged from 69,012 to 106,016 per sample. Rarefaction measurement of 12,311 reads per sample revealed a sufficiently covered diverse microbiome a. We comR = 0.813, p = 0.001 via ANOSIM). B_T and B_T/A were separated along PC2, whereas C_T and C_T/A were separated along PC1. The B6 groups administered Tim3VdIg, antibiotics, and bacteria  were separated from B_T and B_T/A along PC1 as well as along PC2. The samples from BALB/c mice treated with Tim3VdIg, antibiotics, and faecal bacteria (indicated as C_T/A/F) were separated from C_T and C_T/A along PC1, as well as from the samples from mice treated with Tim3VdIg, antibiotics, and E. hirae (indicated as C_T/A/E) along PC1 and PC2. Notably, B_T/A/F, B_T/A/E, and C_T/A/F, the groups exhibiting restoration of Tim-3VdIg anti-tumour efficacy, were clustered. Furthermore, C_T/A/E, in which tumour suppression was not restored, was proximal to B_T/A. These results demonstrated clustering of experimental groups based on tumour suppression.We then analysed beta diversity using Bray–Curtis distance . PrincipFirmicutes (phylum) Clostridia (class) Clostridiales (order) was observed in the B_T group, whereas in the C_T group, two major populations belonging to Bacteroidetes (phylum) Bacteroidia (class) Bacteroidales (order) and Firmicutes Clostridia Clostridiales, respectively, were observed. In the B_T/A group of B6 mice and in all groups of BALB/c mice , Proteobacteria Gammaproteobacteria Enterobacterales were predominant . In B_T/A/F and B_T/A/E, there were two dominant populations, namely, Proteobacteria Gammaproteobacteria Enterobacterales and Firmicutes Bacilli Lactobacillales. These results indicated that antibiotic treatment drastically altered the microbiome, and feeding antibiotic-treated mice with bacteria did not fully reconstitute gut microbiota even though it restored the tumour-suppressive effect of Tim3VdIg.We then compared microbiome composition at the phylum, class, and order levels . A majorBeduini massiliensis and Propionispira paucivorans, were potentially associated with the suppression of anti-tumour efficacy of Tim3VdIg in B6 mice, as they were observed in the T/A group but not in the T, T/A/F, and T/A/E groups. The increased abundance of Proteus alimentorum and Akkermansia muciniphila in T/A/E or T/A/F relative to T/A was indicative of their potential role in enhancing the anti-tumour effects of Tim3VdIg in B6 mice. We could not identify any bacterial species whose modulation may be associated with the tumour-suppressive efficacy of Tim3VdIg in BALB/c mice. Finally, we compared the microbiota composition of Tim3VdIg-treated B6 and BALB/c mice, as oral gavage of E. hirae enhanced the tumour-suppressive efficacy of Tim3VdIg in B6 mice but not in BALB/c mice . The cause of this difference remains unknown. It may be attributed to variations in the intestinal response to transferred bacteria, differences in the immune response, and differences in gut microbiota composition between B6 and BALB/c mice. In agreement with this possibility, differences in intestinal gene expression between the BALB/c and C57BL/6 mice after probiotic treatment have been reported [Recovery of Tim3VdIg efficacy through the oral administration of bacteria depended on the transferred bacterial species and the host strain. The transfer of ibiotics . Howevery humans ,36,37. E tissues . Additio in mice . Furtherpatients . Contrarreported ,42. Our Our results emphasize the critical role of gut microbiota in cancer immunotherapy via Tim-3 blockade and reveal the beneficial effect of gut microbiota modulation on tumour suppression even during the continuous administration of antibiotics. Further research will help ascertain the appropriate modulation of gut microbiota via the selection of appropriate bacteria and the careful consideration of relevant host factors.


PfSNP-LAMP-LFD) was recently developed to detect single nucleotide polymorphism (AAT → ATT), corresponding to substitution of asparagine to isoleucine at amino acid position 51 in the P. falciparumdhfr-ts gene associated with antifolate resistance. In this present study, the PfSNP-LAMP-LFD was validated on 128 clinical malaria samples of broad ranged parasite densities . The results showed 100% accuracy for the detection of single nucleotide polymorphism for N51I mutation. Indeed, the high prevalence of N51I in the Pfdhfr-ts gene detected in the clinical samples is in line with reports of widespread antifolate resistant P. falciparum in Thailand. The relationship between enzyme choice and reaction time was observed to have an effect on PfSNP-LAMP-LFD specificity; however, the method yielded consistent results once the conditions have been optimized. The results demonstrate that PfSNP-LAMP-LFD is a simple method with sufficient sensitivity and specificity to be deployed in routine surveillance of antifolate resistance molecular marker and inform antimalarial management policy.The loop-mediated isothermal amplification coupled with lateral flow dipstick ( Plasmodium falciparum [P. falciparum, the dihydropteroate synthetase (DHPS), and the dihydrofolate reductase (DHFR), respectively [The global malaria morbidity and mortality have decreased substantially over the past decades, yet malaria still remains a significant global health threat. More than 228 million malaria cases and 405,000 deaths were reported in 2019 [lciparum . SP blocectively ,11,12.PfSNP-LAMP-LFD). While S108N is the most common SNP identified; however, single mutation is rarely found in pyrimethamine resistant parasites [Pfdhfr has been described with an initial S108N mutation and sequential additional mutations at N51I and/or C59R, and I164L [P. falciparum genome represented technical challenges in the design of a SNP-LAMP primer set. The position of N51I SNP allowed for the best primer design and development of a robust PfSNP-LAMP-LFD that could capture most of the circulating pyrimethamine-resistant parasite population including double mutant (N51I + S108N), triple mutant (N51I + C59R + S108N), and quadruple mutant (N51I + C59R + S108N + I164L).The surveillance of molecular markers associated with antimalarial resistance can be informative for evidence-based policy decision on antimalarial management. The prevalence of known single nucleotide polymorphisms (SNPs) attributed to pyrimethamine resistance was considered during the development of SNP-loop-mediated isothermal amplification coupled with lateral flow dipstick . The use of gel electrophoresis can distinguish specific amplification from non-specific products but it is more time consuming, while the visualization of SYBR green dye intercalated LAMP products is simple but can be subjective. Real-time turbidity resulting from precipitation of magnesium-pyrophosphate can be used; however, it requires the purchase and maintenance of a turbidimeter. The LAMP method does not require specialized equipment, so it has the potential to be used in molecular diagnostic at point-of-care or in surveillance programs. Indeed, LAMP test kits are available for the detection variety of pathogens such as COVID-19, E. coli O157, and tuberculosis [Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method performed under single temperature that is relatively simple, cost-effective, and time-efficient . Key fearculosis .PfSNP-LAMP-LFD used the same read out format as most commercially available rapid diagnostic tests to detect N51I mutation in the Pfdhfr-ts gene [PfSNP-LAMP-LFD was further optimized and evaluated on clinical malaria samples, in order to validate its application as an alternative test for point-of-care diagnostics and for the molecular surveillance of malaria drug-resistant biomarkers.There have been numerous reports demonstrating comparable or even improved performances of LAMP-based protocols compared to PCR-based protocols for species detection of malaria parasites ,19,20,21-ts gene . In thisP. falciparum in this area was approximately 3.7%, as determined by PCR [P. falciparum infection were invited to participate in the study after providing informed consent, following an approved protocol by the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University . Briefly, blood sample was collected from each patient by finger prick using heparinized capillary tubes. A volume of 100 µL of the packed red blood cells was stored in the freezer. Genomic DNA extraction was performed on the frozen blood samples using ®QIAamp DNA Blood minikit , as described by the manufacturer’s protocol. A total of 2 µL of eluted DNA was used directly for PfSNP-LAMP-LFD to detect N51I SNP on the dhfr-ts gene associated with pyrimethamine resistance and for nested PCR to confirm malaria species based on the detection of the 18S ribosomal RNA gene [Blood samples were collected from malaria patients presented at the malaria clinics in Thailand between 2013 and 2018. The prevalence of d by PCR . PatientRNA gene .Pfdhfr-ts gene products of P. falciparum clinical samples were obtained by PCR using specific primer pairs . PCR reactions were conducted in a 25 µL reaction mixture using the following condition: initial denaturation at 95 °C for 2 min, followed by 35 cycles of 95 °C for 1 min, 51 °C for 1 min, and 72 °C for 1 min, with a final extension at 72 °C for 5 min. A PCR purification kit  was used to obtain purified PCR products for DNA sequencing. The sequencing reactions were conducted with the BigDye Terminator Version 3.1 Cycle-Sequencing Kit , using the same primers as above. The DNA sequences were analyzed with the Bioedit program.Briefly, the amplified Pfdhfr-TM4/8.2 containing the wild-type SNP (AAT) of the Pfdhfr-ts gene was used as a negative control. The pUC18-Pfdhfr-V1/S containing the mutant SNP (ATT) corresponding to the N51I mutation was used as positive control. The V1/S strain of P. falciparum is considered to be highly resistant to pyrimethamine, with 4 reported mutations (N51I + C59R + S108N + I164L) in the Pfdhfr-ts gene. These two plasmids were constructed as previously described [The pUC18-escribed .PfSNP-LAMP reaction mixture contained the following components: 2 µM each of Pf-snp-FIP and Pf-snp-BIP primers, 0.2 µM each of Pf-snp-F3 and Pf-snp-B3 primers, 1X isothermal amplification buffer 2SO4, 10 mM KCl2, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8), 0.4 M betaine , 8 mM MgSO4 , 1.4 mM dNTP mix , 8 units of Bst DNA polymerase, a large fragment or Bst 2.0 DNA polymerase or Bst 2.0 WarmStart DNA polymerase , and 2 µL of DNA sample. The design of the PfSNP-LAMP primer set was also previously described [Pfdhfr-TM4/8.2 and pUC18-Pfdhfr-V1/S were used, respectively. For optimization, LAMP reactions were performed at 60–63 °C and observed for 60–90 min and LAMP product signals were monitored using Loopamp Realtime Turbidimeter at 650 nm wavelength . For the validation of clinical samples, PfSNP-LAMP reactions were performed using Bst 2.0 WarmStart DNA polymerase  at 63 °C for 75 min. Then, 20 pmol Pf-FITC-probes was directly added to the reaction and allowed to hybridize with products at 63°C for 5 min. Subsequently, 8 µL of the hybridized PfSNP-LAMP-probe were transferred into a new Eppendorf tube containing 120 µL of room-temperature assay buffer . The LFD strip was dipped into the assay solution for 5 min to allow the solution to migrate by chromatography effect. Two bands were observed on the control line and the test line of the LFD strip for samples with N51I mutation. One band was observed on the control line, representing a negative result for negative sample (no DNA template) or wild-type SNP sample. If no signal appeared on the control line, then the result was invalid.The 25 µL-volume of escribed . For negBst 2.0 DNA polymerase and Bst 2.0 WarmStart DNA polymerase are homologues of Bst DNA polymerase large fragment that have been designed for improvements in amplification speed, thermostability, and salt tolerance, in order to increase the quantity of product, according to the manufacturer’s product descriptions. Here, we compared the effect of enzyme choice on PfSNP-LAMP reactions. The PfSNP-LAMP reactions were evaluated under varying temperatures from 60 to 63 °C for up to 90 min using Loopamp Realtime Turbidimeter.PfSNP-LAMP. Signals represented amplified products using Bst, Bst 2.0, and Bst 2.0 WarmStart DNA polymerases in the reactions with either pUC18-Pfdhfr-V1/S  or pUC18-Pfdhfr-TM4/8.2  as DNA templates.Bst DNA polymerases could distinguish the V1/S SNP  from TM4/8.2 SNP  when performed at the optimal temperature of 63 °C. Signals began to appear around the 60-min mark and reached the optimal time around the 75-min mark. The appearance time of peak signal was dependent on the amount of the starting DNA templates in the reaction. The signal intensities were similar for all reaction conditions and concentrations of templates tested.The overall observation suggested that all three versions of Pfdhfr-TM4/8.2 when using Bst DNA polymerase (Line 2) for all temperatures. Non-specific signals clustered closer to the true positive signals  when performed at the lower range of temperature at 60 °C . These results were consistent with the detection limit of pUC18-Pfdhfr-V1/S observed in previous report [PfSNP-LAMP reaction products were resolved on gel electrophoresis where the characteristic ladder pattern was observed for reactions amplified from pUC18-Pfdhfr-V1/S but not from pUC18-Pfdhfr-TM4/8.2 (data not shown). The amplified PfSNP-LAMP products were visualized on LFD strips, as shown in To further explore the detection limit of 0–75 min A. The Pfproducts B, as exps report . FurtherPfSNP-LAMP-LFD. There were 55 P. falciparum samples and 73 P. vivax samples, the latter served as additional negative control samples. A recent investigation of P. falciparum samples collected in Thailand between 2008–2016 showed high prevalence of N51I among the surveyed parasites, where up to 11% were triple mutants (N51I + C59R + S108N) and 83% were quadruple mutants (N51I + C59R + S108N + I164L) [Pfdhfr-ts in Thailand, we included 20 ng of genomic DNA from two different lab strains, with wild-type Pfdhfr-ts (TM4/8.2 and NF54) as negative controls for this validation study.A total of 128 clinical blood samples from malaria clinics in Thailand were used to validate + I164L) ,31. Due PfSNP-LAMP-LFD reactions correctly detected SNP associated with pyrimethamine resistance  in 55 out of 55 P. falciparum samples, with parasite density from 10 to 87,634 parasites per microliter (P/µL) of blood (100% accuracy), while no signal was observed in all 73 P. vivax samples with parasite density from 9 to 12,632 P/µL and P. falciparum strain NF54 sample (100% specificity). The range of parasite density used in this study was sufficiently broad to allow insights into the performance of PfSNP-LAMP-LFD in actual clinical settings. The performance observed for PfSNP-LAMP-LFD was comparable to the typical performance range seen with expert level malaria microscopy (50–500 P/µL), commercially available rapid diagnostic test , and other nucleic acid-based detection methods (1–5 P/µL) [P. falciparum samples contained the mutant SNP in the Pfdhfr-ts gene. In addition, C59R and S108N mutations were also observed in these samples; however, it is noted that SNP associated with I164L in the Pfdhfr-ts gene was not examined. Nonetheless, these observations were in agreement with other studies that also reported high prevalence of N51I, C59R, S108N, and I164L mutations in the Pfdhfr-ts gene, among circulating parasites in the Greater Mekong Subregion [PfSNP-LAMP-LFD validation on clinical malaria samples tested are shown in The validation results are summarized in –5 P/µL) ,34. DNA ubregion ,31. ReprPfSNP-LAMP-LFD using clinical samples while exploring the robustness of the method when performed under different conditions. Our results demonstrated that the PfSNP-LAMP-LFD could accurately distinguish SNP  associated with pyrimethamine resistance on 128 clinical samples with a broad range of parasite density from as low as 10 parasites/µL of blood. The PfSNP-LAMP-LFD performance was comparable to typical LAMP-based protocols and other PCR-based protocols for distinguishing Plasmodium species [Pfdhfr-ts gene with the detection limit of seven parasites/µL at 60 °C for 45 min [The primary objective of this study was to validate the  species . For a mr 45 min .PfSNP-LAMP-LFD method, once optimized, was sufficiently robust to withstand some variations to the protocol, such as batch-to-batch variation of reagents, equipment and laboratory environment, and the experience level of laboratory technicians. Nonetheless, we observed that the efficiency of PfSNP-LAMP-LFD to distinguish mutant SNP from wild-type SNP was affected by the relationship between enzyme choice and the reaction cut-off time. In our observations at 63 °C, amplified signals from mutant samples could be detected within the 60-min mark for all versions of Bst DNA polymerases, with Bst DNA polymerase large fragment showing the earlier peak signal, followed by Bst 2.0 DNA polymerase and Bst 2.0 WarmStart DNA polymerase. However, non-specific signals were detected in wild-type samples when the reactions extended well beyond the 60-min mark. We observed an approximate delay of 25 min between the appearance of peak signals from mutant samples and wild-type samples for all three versions of the Bst DNA polymerases used in the study; this delay represented the window cut-off which PfSNP-LAMP-LFD could distinguish between the mutant and wild-type Pfdhfr-ts gene at codon 51. In previous PfLAMP and PfSNP-LAMP development efforts, the amplification reactions were optimized within the 60-min mark, in favor of a faster turnaround of reliable results; therefore, we did not observe the non-specific signals in prior experiments [In general, we noted that eriments ,26.PfSNP-LAMP-LFD, and efforts were made to not exceed more than two freeze-thaw cycles of DNA samples and reagents. The design of the PfSNP-LAMP primer set relies on a single nucleotide base difference to distinguish between mutant from wild-type genotypes; the efficiency and stability of primers binding to the mutant sequences would be more favorable than binding to the wild-type sequences. This preferential binding can be leveraged by optimizing the relationship between enzyme and reaction time to promote favorable amplification from mutant sequences over wild-type sequences, thereby improving the sensitivity and specificity of PfSNP-LAMP. Similar observation patterns were made on the effects of enzyme choice and reaction temperature on the sensitivity and specificity of SNP-LAMP by Mohon et al. in their development of SNP-LAMP to detect the C580Y mutation in the Kelch13 gene corresponding to artemisinin-resistance in P. falciparum [Several factors can be attributed to non-specific amplification in LAMP products, including sample quality and partial hybridization of one or more LAMP primers to the fragmented DNA of target organisms or of host DNA. To ensure sample quality, purified genomic DNA samples were used for lciparum . These oPfSNP-LAMP-LFD protocol includes a genomic DNA purification step, and thus requires the laboratories to be equipped with basic equipment, including microcentrifuge, temperature-control heating block or water bath, and pipetting instruments. Simplified sample preparation can improve the applicability of nucleic acid-based detection method as point-of-care diagnostics, particularly for use in more remote settings where malaria is endemic. However, this convenience in sample preparation can affect the quality of starting DNA templates, and consequently the limit of detection. Previous attempts to simplify DNA preparation showed a 10-fold difference in the limit of detection of PfSNP-LAMP-LFD, for example, when purified genomic DNA (LOD = 0.2 ng of genomic DNA) was compared to non-purified genomic DNA (LOD = 2 ng of genomic DNA in the presence of red blood cell lysate) [PfSNP-LAMP-LFD can provide consistent results for the detection of SNP associated with pyrimethamine resistance, which can be a proxy for treatment efficacy, particularly in areas where a sulfadoxine-pyrimethamine regimen is still in use. With increasing threats of multidrug-resistant malaria, PfSNP-LAMP-LFD can be used as part of a toolkit for a comprehensive molecular surveillance program to monitor known biomarkers for drug resistance to support evidence-based malaria treatment policies.The current  lysate) . The ove


N-alkyl drugs is crucial in drug-development as over 50% of the top-selling drugs contain N-alkyl groups, in which it is very challenging to selectively replace protons with deuterium atoms. With the goal of achieving controllable isotope-labeling in N-alkylated amines, we herein rationally design photocatalytic water-splitting to furnish [H] or [D] and isotope alkanol-oxidation by photoexcited electron-hole pairs on a polymeric semiconductor. The controlled installation of N-CH3, -CDH2, -CD2H, -CD3, and -13CH3 groups into pharmaceutical amines thus has been demonstrated by tuning isotopic water and methanol. More than 50 examples with a wide range of functionalities are presented, demonstrating the universal applicability and mildness of this strategy. Gram-scale production has been realized, paving the way for the practical photosynthesis of pharmaceuticals.Precisely controlled deuterium labeling at specific sites of  Controllable deuteration of N-alkyl amines is crucial in drug-development as they constitute over 50% of the top-selling drugs. Here, the authors report a polymeric semiconductor acting as photocatalyst in water-splitting and in isotope alkanol-oxidation by photoexcited electron-hole pairs, and furnishing deuterated amines. The higher stability of C–D bonds than C–H bonds because of the deuterium kinetic isotope effect (DKIE) motivates the need for a “deuterium switch” in drug synthesis to improve biological properties, such as pharmacokinetics, pharmacodynamics (PK/PD), and metabolic stability11. In 2017, the first deuterium-labeled drug, deutetrabenazine, was approved by the FDA and initiated a new era of deuterated clinical drug development12. Among the myriad of commercial drugs, over 50% of the top sellers contain N-alkyl amine units13, and the N-dealkylation metabolized cytochrome P450 (CYP450) are commonly found in such N-alkyl drugs and other bioactive molecules19. Thus, deuterium substitution of N-alkyl groups in N-alkyl drug molecules could contribute to slow down the N–C bond cleavage, and impacts their pharmacodynamic properties and improve pesticide effects24. In this regard, the precision synthesis of drug analogs with deuterated N-alkyl amine units holds great promise and has been attracting increasing interest  catalysis mediated the abstraction of deuterium from D2O or T2O to furnish α-deuterated or tritiated amine product. Multi-deuterium atoms incorporation at all α-position of pharmaceutical amines (more than 4.0 deuteriums per molecule) with a wide range of D-incorporation ratio (from 1 to 91%) is generally occurred. Still, the development of a general and mild method for the substitution of the traditional deuterated alkylation from toxic deuterated reagents like CD3I is in high demand to effectively and selectively functionalize pharmaceutical amines43. Further, the precise control of deuterium atoms number at the α-position of N-alkyl drugs with high deuterium incorporation currently remain unexplored, while it is particularly attractive for their potential use in mechanistic and metabolic studies45.Traditional approaches to 2O/D2O to furnish reductive [H]/[D] and simultaneously oxidize organics by the photoexited electron–hole pairs49. Synergistic utilization of those reductive [H]/[D] and reactive organic species holds great potential for production of deuterated chemicals and pharmaceuticals, e.g. D-labeling N-alkyl pharmaceuticals, from isotopic water and organics. Polymeric carbon nitride (PCN) is a nontoxic, highly stable, low-cost, and scalable polymer semiconductor with a suitable redox window [from approximately +1.2 V to −1.5 V vs. saturated calomel electrode (SCE)]51. These characteristics define PCN as an ideal semiconductor photoredox catalyst for effective water splitting coupled with controlled oxidation by photoexcited electron–hole pairs. Herein, we utilize highly crystalline PCN as a semiconductor photocatalyst for the sustainable synthesis of N-alkyl chemicals and drugs with well-controlled isotope labeling52. Upon visible-light irradiation, electron–hole pairs are generated on crystalline PCN. Photogenerated electrons are transferred to the anchored Pd nanoparticles and utilized to reduce water to furnish absorbed [H]/[D] species. Meanwhile, photogenerated holes with appropriate oxidative ability are designed to selectively oxidize isotopic alkanols, furnishing isotopic aldehydes for aldehyde-amine condensation to produce imine intermediates. These imines are subsequently reduced by [H]/[D] from water splitting, producing corresponding N-alkyl chemicals and drugs  at the metabolic position of N-Me drugs is enabled by simply tuning the isotopic water and methanol  excN-methylation of amines using highly crystalline PCN (CPCN) as the semiconductor photocatalyst53, water and methanol as the green methylating reagents, and p-toluidine as the amine. The optimized conditions are summarized in Supplementary Table N-methylation product of p-toluidine, N,N-(CH3)2p-toluidine, was obtained in 94% yield. Using the optimized conditions, isotopic water and methanol were used to investigate the synthesis of deuterated compounds and the reaction pathway. Generally, multiple reaction processes are required to achieve high deuteration content in the production of deuterated chemicals and pharmaceuticals. Here, the use of D2O and CD3OD afforded N,N-(CD3)2p-toluidine in 89% yield with high D incorporation (97%). To trace the deuterium source, H2O/CD3OD was used, which afforded N,N-(CD2H)2-p-toluidine in 91% yield, with nearly quantitative D-incorporation (>99%). The obtained partially deuterium-labeled product suggests that CD3OH/CD3OD are probably oxidized to [D2C=O] by photogenerated holes, which is consistent with the mechanism of photocatalytic water splitting using methanol as the sacrificial agent50. Aldehyde-amine condensation of [D2C=O] and p-toluidine occurs to furnish imine intermediates for sequential hydrogenation by reductive [H] from water splitting 2-p-toluidine product. Consistent with the aforementioned reaction pathway, using the D2O/CH3OD system could introduce N-CDH2 groups (91%) with high D content (>99%). The controllable D-labeled N-alkylation of secondary amines was also examined, affording N-CH3, -CD3, -CD2H, and -CDH2 diphenylamines in high yields (74–94% yields) with excellent D incorporation (>97%) 2 units with excellent D incorporation (97-99%) . The use of aniline substrates bearing both electron-donating groups  and electron-withdrawing groups  produced the corresponding N,N-(CD3)2-anilines in 69–89% yields (3aa-3ad). Sensitive substrates with alkyl chiral centers (3ae and 3af) were compatible and unperturbed. Since most N-alkyl drugs are fabricated from secondary amines via N-alkylation reactions, the N-trideuteromethylation of secondary amines was investigated with great interest. To our delight, this protocol with secondary amines exhibits a broad reaction scope, good functional group tolerance and excellent D incorporation. N-alkyl anilines, including substituted N-Me anilines and N-Bn anilines, furnished the corresponding products with high D incorporation (91–98%) and in excellent yields (84–94%). N-trideuteromethylation of ethyl phenylglycinate (3bg), a representative amino acid derivative, as well as estrone derivate (3bi) was achieved, attesting to the ability to deuterate bioactive molecules. For the diamine substrate 3bh, di-CD3 was simultaneously introduced in 71% yield. This protocol was also applicable to a wide range of diary amines bearing substituted phenyl (3ca-3ce), naphthyl (3cf), and pharmaceutical units such as chlorambucil (3ci), oxaprozin (3cj), and (R)-naproxen (3ck). A steric effect-controlled highly chemoselective N-trideuteromethylation of diary amines is observed (3cl). The N-CD3 incorporation of heterocyclic amines such as indoline and iminodibenzyl was achieved successfully. These heterocyclic skeletons are widespread in natural products, pharmaceuticals and key intermediates. Aliphatic amines were also found to be competent substrates, providing the desired products (3da–3db) in good yields. Finally, the strategy could be extended to the N-deuterated alkylation of amines by replacing d4-methanol with other deuterated alkanols, such as d5-ethanol for N-CD2CD3 incorporation (3e). This protocol exhibits highly efficient in production of deuterated N-alkyl chemicals with excellent D-incorporation, thus holding great potential application towards the synthesis of stable isotope-labeled compounds for synthetic mechanism study as well as LC/MS quantification38.Next, the generality of the water-splitting-based N-Me amine units are present in many of the 200 top-selling drugs produced in 2018 and are often required for their intended pharmacological functionality58. Deuterium substitution of the N-Me groups of these drugs is highly desired. We tested the protocol developed above for the synthesis of N-CD3-based pharmaceuticals and bioactive molecules  without affecting the amide and sulfamine functionalities. A variety of commercially available pharmaceuticals with secondary amine units, namely, fluoxetine, tetracaine, atomoxetine, sertraline, paroxetine and vortioxetine, smoothly underwent N-trideuteromethylation , reconfirming the universality of our strategy. More importantly, this mild and general process enables access to site-specifically labeled drugs in a single step. Deuterium-labeled analogs of butenafine could be obtained in 67% yield (4k). Trideuteromethylation of monomethylated desipramine and amoxapine gave imipramine-d3  and loxapine-d3 , respectively. The use of C2D5OD/D2O as an alkylation reagent successfully afforded alverine-d5 (4l) in high yield (84%). In addition, synthesis of dofetilide-d3 was achieved in four steps with 32% overall yields from low-cost and commercially available starting materials. Gram-scale syntheses of both loxapine-d3 and dofetilide-d3 with high yields were demonstrated, highlighting the practical utility of this protocol. Again, all D-labeled pharmaceuticals and their analogs gave excellent deuterium incorporation.les Fig. . Here, t61. In particular, the controllable incorporation of partially deuterium-labeled N-methyl groups  slows drug metabolism to improve the pesticide effect62. However, their synthesis remains a great challenge. Our controllable deuterium-labeling strategy was successfully applied for the facile synthesis of N-CD3, N-CD2H and N-CDH2 nimesulide derivatives , butenafines-d3, d2 and d1 , loxapines-d3, d2 and d1  and imipramines-d3, d2 and d1  with high yields and uniformly high D incorporation (>95%) . In all these drugs, only the target N-alkyl units were specifically labeled with deuterium. 13C-labeled drugs are of significant importance in medical biology for tracking metabolites and quantitative analysis by mass spectrometry and 13C NMR spectroscopy63. This protocol can also be applied for the sustainable synthesis of 13C-labeled drugs by replacing methanol with 13CH3OH. As expected, 13C-labeled nimesulide derivative (4q), butenafine (4t), loxapine (4w) and imipramine (4z) were readily obtained with comparable yields64.Isotope-labeled bioactive compounds are extensively used to study interactions with lipid membranes, proteins, nucleic acids, etcration >9%  at the metabolic position of pharmaceuticals, which is critically important for deuterated drug discovery. Finally, the present results reveal a new horizon of photosynthesis for direct pharmaceutical production.In summary, a powerful semiconductor photocatalytic system for the sustainable and scalable construction of deuterated pharmaceuticals and chemicals has been discovered. This strategy is characterized by high yields, excellent D incorporation in a single step, the use of low-cost and sustainable deuterated methylating reagents (isotopic water and methanol), excellent functional group tolerance including a range of pharmaceutically relevant functionalities, and mild conditions. Significantly, the unique controllable D-labeling protocol provides the ability to precisely control the number of deuterium atoms  was ground with KBr . Then, the resultant mixture was heated to 550 °C for 3 h in a tube furnace. After cooling to room temperature, the bright yellow-green product was washed with boiling deionized water several times and collected by filtration, followed by drying at 60 °C under vacuum. As-prepared sample is denoted as CPCN. Pd/CPCN photocatalyst was prepared by photodeposition process. In brief, as-synthesized CPCN (0.3 g) was dispersed in a mix solution with 80 mL deionized water and 20 mL glycol. After untrasonication treatment for 2 h, 84 μL of 1.0 M H3 (0.3 mmol) were dispersed in a mixture solution with Acetonitrile/D2O/CD3OD = 2 ml/1.5 ml/1.0 ml, and then sonicated for 1 min. The reaction mixture was then irradiated with a LED lamp  for 4-24 h under Argon at 25oC by using a flow of cooling water during the reaction. After reaction, the mixture was centrifuged to remove photocatalyst. The supernatant was extracted by adding 5 mL of CH2Cl2. The reaction mixture was concentrated under reduced pressure and the residue was purified by column chromatography on silica gel to furnish the corresponding product. The isolated yield was calculated by dividing the amount of the obtained desired product. Deuterium incorporation were checked and calculated by NMR.Typically, 25 mg of Pd/CPCN and 0.4 mmol of substrate and AlClo min−1 and 0.01o, respectively. Transmission electron microscope (TEM) images were obtained using a HT7700 TEM (Hitach). The solid diffuse reflectance spectra (DRS) were collected on a UV–Vis–NIR spectrophotometer . NMR tests were conducted on Bruker AVANCE III NMR spectrometer (500 and 600 MHz). The high-performance mass spectrometry was conducted by a Q Exactive GC Orbitrap GC-MS/MS (Thermo Scientific).The crystal structure of catalyst was characterized by X-ray diffraction (XRD)  at 40 kV and 40 mA (Cu Kα X-ray radiation source) with a scanning speed and step interval of 4Supplementary informationPeer Review File