A combined proteomics and metabolomics analysis revealed that P2RY12 deficiency causes redox imbalance and leads to reduced aerobic glycolysis by downregulating the expression of hexokinase 2 (HK2), a rate-limiting enzyme of the glycolytic pathway, indicating that HK2 might be a promising candidate for the treatment of diseases associated with T cell activation. Further analysis showed that P2RY12 prevents HK2 degradation by activating the PI3K/Akt pathway and inhibiting lysosomal degradation. Our findings highlight the importance of the function of P2RY12 for HK2 stability and metabolism in the regulation of T cell activation and suggest that P2RY12 might be a pivotal regulator of T cell metabolism in ConA-induced immune hepatitis.Increasing evidence suggests that immunometabolism has started to unveil the role of metabolism in shaping immune function and autoimmune diseases. In this study, our data show that purinergic receptor P2Y12 (P2RY12) is highly expressed in concanavalin A (ConA)-induced immune hepatitis mouse model and serves as a potential metabolic regulator in promoting metabolic reprogramming from oxidative phosphorylation to glycolysis in T cells. P2RY12 deficiency or inhibition of P2RY12 with P2RY12 inhibitors (clopidogrel and ticagrelor) are proved to reduce the expression of inflammatory mediators, cause CD4 A combined proteomics and metabolomic analysis were performed to determine the role of P2RY12 and the detailed underlying mechanism. Additionally, we found that P2RY12 functions post-translationally by mediating the stabilization of hexokinase 2 (HK2), a rate-limiting enzyme of the glycolytic pathway. P2RY12 prevents the degradation of HK2 by controlling lysosomal degradation and activating Akt phosphorylation. These findings suggest that P2RY12 function as a pivotal regulator of T cell metabolism and identify a novel link between glycolysis and AIH pathogenesis.In this study, we hypothesized that P2RY12 is of unique significance to T lymphocytes in AIH disease progression. To confirm the validity of this hypothesis, we systematically evaluated the effects of P2RY12 on T lymphocytes in ConA-induced immune hepatitis-/- mice on a C57BL/6 background were described previously-/- mice on C57BL/6 background were obtained from The Jackson Laboratory . All mice were housed under specific pathogen-free conditions and fed with standard laboratory chow and water ad libitum. All animal experiments were performed in accordance with the guidelines of Tongji University Animal Care Committee and the Animal Research Ethics Committee.C57BL/6 mice were purchased from GemPharmatech . P2RY12A single dose of ConA  was dissolved in normal saline solution at a concentration of 10 mg/ml. At the age of 8-10 weeks, mice were intravenously injected with ConA at 7.5, 10, 12.5, 15 or 20 mg/kg body weight, through the tail vein and their survival rate was monitored continuously for 7 days. The ConA dose of 12.5 mg/kg body weight injected intravenously was selected to induce immune hepatitis in mice. Clopidogrel   or ticagrelor   was administered orally once daily for 7 days before ConA treatment. Clopidogrel (15 mg/kg body weight) or ticagrelor (15 mg/kg body weight) was administered orally once after ConA treatment 30 min, 1 hr or 12 hr. A 0.9% saline solution was used as vehicle.Blood was collected from the post-orbital venous plexus vein at different times after the ConA injection, and the serum was separated by centrifugation at 4,000×g at 4 °C for 10 min. Serum levels of alanine aminotransferase test (ALT) and aspartate aminotransferase test (AST) were measured using reagent kits  following the manufacturer's specifications. For histological staining, mice were anesthetized and perfused with phosphate-buffered saline (PBS), followed by fixation with 4% (w/v) paraformaldehyde. The isolated livers tissues were then fixed in 4% (w/v) paraformaldehyde for 24 hr, embedded in paraffin, sectioned into 4-µm-thick sections, stained with hematoxylin and eosin (H&E), and examined for the presence of inflammation by light microscopy using an inverted Olympus IX51 microscope .The samples of human blood were obtained from healthy volunteers and AIH patients following the procedures and guidelines approved by the Institutional Review Board of Tongji University School of Medicine. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all subjects for being included in the study.TM density gradient solution , and centrifuged at 800×g for 30 min at room temperature. Cells were collected from the interphase layer, and the collected fraction was diluted with PBS and centrifuged at 1,000×g for 5 min. Collected cells were transferred to RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin-streptomycin (100 ×) and glutamine (2 mM) and maintained at 37 °C in a humidified atmosphere containing 5% CO2. The obtained PBMCs were stimulated with 2 μg/ml of ConA in the culture medium except for the negative control group, other groups were treated with different appropriate treatments. After 24 hr, cells and supernatants were collected for flow cytometry, RT-qPCR and enzyme-linked immunosorbent assay (ELISA) analysis.Peripheral blood mononuclear cells (PBMCs) were isolated as previously describedHepatic lymphocytes were prepared as previously describedTM Universal PCR Master Mix  and TaqManTM Gene Expression Assays (Applied Biosystems) following the manufacturers' instructions. QRT-PCR analysis was conducted in a Light Cycler quantitative PCR apparatus  using the SYBR Green JumpStart Taq ReadyMix kit (Sigma-Aldrich). The expression value was normalized to β-actin in the same sample. The primers used in the PCR reactions, including those for mouse Il6, Il12a, Il10, Tnf, Ifng, and Actb and for human IL-6, IL-12A, IL10, TNF, IFNG and ACTB . After synthesizing cDNA from the RNA samples, cDNA samples were subjected to quantitative real-time polymerase chain reaction (qRT-PCR) using the TaqManand ACTB  were syn-/- mice were isolated and cultured in 96-well plates at a density of 1×106 cells per well, then stimulated with ConA (2 μg/ml) (Sigma-Aldrich) for 12 hr or 24 hr and co-cultured with the P2RY12 inhibitors clopidogrel  or ticagrelor , HK2 inhibitor 3-bromopyruvate , proteasome inhibitor MG-132 , lysosomal inhibitor bafilomycin A1 (BafA-1) (Med Chem Express LLC) or Akt activator SC79 (Med-Chem-Express LLC). The cells were analyzed by flow cytometry and supernatants were collected for measurement of cytokines.Splenocytes from wild type (WT) mice and P2RY12TM Flow Cytometer (BD Bioscience) and the analysis was used by The Guava Soft software.Freshly isolated hepatic lymphocytes separated from liver tissue sections or human PBMCs were stained with various antibodies in the dark at 4°C for 30 min, then washed twice with PBS. After fixation and permeabilization with a Cytofix/Cytoperm kit  and staining with various antibodies, including anti-mouse CD3-PE , anti-mouse NK1.1-FITC , anti-mouse CD8a-FITC , anti-mouse CD4-PE-Cy7 , anti-mouse IFN-γ-APC , anti-human CD4-PE-Cy7 , anti-human CD8a-FITC , anti-human IFN-γ-APC . The cells were analyzed by flow cytometry on a BD FACSVerseTo evaluate the expression levels of different proteins in liver, mouse livers were homogenized in radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail  and centrifuged at 10,000×g at 4 °C for 15 min. The proteins in the supernatants were solubilized by boiling for 5 min in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample-loading buffer. Then, 50 μg of solubilized proteins were separated on SDS-PAGE gels and subsequently electro-transferred onto a nitrocellulose membrane . After blocking the membrane with 5% nonfat milk to prevent nonspecific binding, immunoblotting was performed using the following antibodies against mouse proteins: GPI, PFK2, MCT1, TPI; JNK, p-JNK (Thr183/Tyr185), cleaved caspase3 and β-actin ; P2RY12 ; HK2, P62 and LC3 ; Akt, P-Akt (Ser473) ; P-Akt (Thr308) .Whole proteins of liver tissue were extracted and measured protein concentrations using the bicinchoninic acid (BCA) protein assay. Part of the supernatant was used as input control and the rest was immunoprecipitated overnight at 4ºC by gently rocking with anti-HK2 antibody .Approximately 5 μl antibodies were used for 500 μg total protein. Then protein A/G agarose beads  were added to bind with the immunoprecipitates for 2 hr with gentle shaking at room temperature. Precipitated proteins were washed 3 times with PBS and boiled with 5×loading buffer, then immunoblotting was performed as previously described. Rabbit normal IgG (Santa Cruz Biotechnology Inc.) served as negative control.Individual mouse serum and culture supernatants were collected, and the levels of inflammatory cytokines were measured using an ELISA kit . according to the manufacturer's instructions.-/- mice with ConA-induced immune hepatitis and T cells were sorted by the MagnisortTM mouse CD3 positive selection kit . The sorted T cells were lysed in 1,000 µl RIPA buffer at 4 °C. The BCA protein assay was used to determine the total protein content of each sample. Samples were analyzed on Orbitrap Fusion Lumos, and Q Exactive Plus mass spectrometers  coupled with an EASY-nLC 1000 nanoflow liquid chromatograph system (Thermo Fisher Scientific) connected to an UltiMate 3000 RSLCnano system (Thermo Fisher Scientific).Hepatic lymphocytes were isolated from the liver of WT mice and P2RY12Hepatic lymphocytes isolated from ConA-induced immune hepatitis and sorted T cells. T cells were washed twice with ammonium carbonate (75 mM) at pH 7.4 and snap frozen in liquid nitrogen. Metabolomics analysis was performed using a 1260 infinity high-performance liquid chromatography (HPLC) system . Before LC/MS analysis, samples were prepared by different procedures and stored at -80°CR statistical software (http://www.r-project.org).For LC/MS, samples were separated on an amide column, using water mixed with 25 mM ammonium acetate and 25 mM Ammonium hydroxide as mobile phase A and ACN as mobile phase B. MS analysis was performed on a UHPLC-Q-Exactive MS/MS system (Thermo Fisher Scientific Inc.) in both positive and negative ion modes. Different data-dependent analysis (DDA) methods and the full scan method were performed, and the results were processed using untargeted metabolomics workflow with minor modification to find and identify the differences between samples. The exact mass of each feature was submitted to selected ChemSpider with 4 databases  and then analyzed with -/- mice and age- and sex-matched WT control mice (8-10 weeks) were used to establish the ConA-induced immune hepatitis model by injecting ConA into tail vein at the dose of 12.5 mg/ kg of body weight for 12 hr. Hepatic lymphocytes isolated and further sorted T cells with MagnisortTM mouse CD3 positive selection kit . OCR was evaluated under basal conditions and in response to 1 μM oligomycin, 0.6 μM FCCP, 100 nM rotenone, plus 1 μM antimycin A. ECAR was measured under basal conditions and in response to 100 mM glucose, 1 μM oligomycin, and 5 mM 2-d-glucose (2-DG). Activated T cells were cultured in XF-medium ; Agilent Technologies Inc.) containing 10 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate. Assays were analyzed with the Wave Desktop software (Agilent Technologies Inc.).The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined using a Seahorse XF96 Extracellular Flux Analyzer (Agilent Technologies Inc.) following established protocolsATP production level was quantified by a luminescent available assay kit . Briefly, T cells isolated from liver tissues were lysed to release ATP into medium containing luciferase and luciferin, which produces light in the presence of ATP. The light emission, which is proportional to the amount of ATP present was detected.t test. For three or more groups, a one-way analysis of variance (ANOVA) was used, followed by Tukey post-hoc test. All the experiments were repeated at least three times. P < 0.05 was considered to be statistically difference.Data are expressed as the means ± SEM and were analyzed using the Graphpad Prism software . Differences between groups were assessed by means of a two-tailed unpaired Student P2ry12 was up-regulated in hepatic lymphocytes and down-regulated in hepatic cells, suggesting that hepatic lymphocytes are the main contributor to the increased P2RY12 level in the liver of mice with ConA injection  and aspartate aminotransferase (AST) levels, and increased rate of death in a dose-dependent manner 37. A signjection C-E.-/-) mice to determine the role of P2RY12 in ConA-induced immune hepatitis , CD8+ T cells (CD3+ CD8+), NK cells (CD3- NK1.1+), and NKT cells (CD3+ NK1.1+)  in the liver has been shown to play a key role in ConA-induced immune hepatitis) Figure A. The nus Figure A-C.-/- mice and WT mice were transferred into Rag1-/- mice for 48 hr, followed by ConA injection  were also affected by P2RY12 inactivation  for 12 hr or 24 hr. The results indicate that ticagrelor and clopidogrel significantly reduce the proportions of CD4+IFN-γ+ and CD8+IFN-γ+  cut-off of 5% and a 1.5-fold change threshold were used, and 146 proteins were found differentially expressed between two groups  indicated that P2RY12” Figure A-C. Next” Figure D, 5E. Moin vitro A-D. Thesin vitro , indicatin vitro and in vivo. The proportions of CD4+ IFN-γ+ and CD8+ IFN-γ+ as well as the level of IFN-γ in supernatant were rapidly decreased in splenocytes treated with 3-BrPA for 4 hr  and cultured for 8 hr. Then cells were transferred into Rag1-/- mice via tail vein injection, followed by challenging these mice with ConA -induced colitis by decreasing inflammation-induced epithelial cell deathIn conclusion, our study reveals that P2RY12 is involved in the pathogenesis of ConA-induced immune hepatitis. We also propose that intracellular metabolic reprogramming driven by the HK2/Akt axis is the driving mechanism by which P2RY12 orchestrates ConA-induced immune hepatitis disease. These findings will have implications for the development of future therapeutic approaches involving inhibition of P2RY12 or synergism with therapy target HK2 as a therapeutic tool for the treatment of autoimmune hepatitis.Supplementary figures and table.Click here for additional data file.


Little is known about U.S. outpatient prescribing trends for type 2 diabetes (T2DM) in adolescents and young adults.To determine (a) trends in the outpatient prescribing of pharmacological and nonpharmacological therapies and (b) factors influencing prescribing trends for adolescents and young adults with T2DM.A retrospective, cross-sectional analysis was conducted on office visits of adolescents (12-17 years) and young adults (18-39 years) with T2DM or impaired glucose tolerance (IGT), using the National Ambulatory Medical Care Survey (NAMCS) from 1996-2005. Logistic regression was used to test for prescribing trends over time.There were an estimated 1.6 million  and 22.2 million  office visits for adolescents  and young adults  associated with T2DM based on ICD-9-CM codes, respectively. In young adults, diabetes drug mentions increased significantly from 39% of visits with T2DM to 61% in 2004-2005 (P = 0.04). Oral diabetes medication mentions increased from 20% to 49% (P = 0.001). However, reports of nonpharmacological therapy decreased from 53% in 1996-1997 to 37% in 2004-2005 (P = 0.14).The prescribing of pharmacological treatment for T2DM increased with emphasis on oral agents, while reports of nonpharmacological therapy for T2DM decreased over the 9-year study period with increased use of oral medications in both adolescents and young adults. Health care providers should consistently consider both treatment approaches when prescribing patient care as recommended by treatment guidelines.


Divalent lanthanide(Ln) compounds are excellent reducingagentswith unique reactivity profiles. These reagents are typically usedin superstoichiometric amounts, often in combination with harmfuladditives. Reactions catalytic in Ln(II) reagents that retain thereactivity and selectivity of the stoichiometric transformations arecurrently lacking due to the absence of effective and selective methodsto form reactive Ln(II) species from stable precursors. Here, activeLn(II) is generated from a Ln(III) precursor through reduction bya photoexcited coumarin or carbostyril chromophore, which, in turn,is regenerated by a sacrificial reductant. The reductant can be metallic(Zn) or organic (amines) and can be used in strictly stoichiometricamounts. A broad range of reactions, including C–halogen, C=C,C=X , P=O, and N=N reductions, as wellas C–C, C–X , and N–N couplingswere readily carried out in yields and selectivities comparable toor better than those afforded by the analogous stoichiometric transformations.The reaction outcomes could be altered by changing the ligand or thelanthanide or through the addition of environmentally benign additives. EPR spectroscopy supported the formation of both Ln(II)and oxidized chromophore intermediates. Taken together, these resultsestablish photochemical Ln(II) generation as a powerful strategy forrendering Ln(II)-mediated reactions catalytic. Lanthanides, like many transition metals, are expensive, andtheir mining causes significant environmental damage.14 Thus, there is an urgent need to develop protocols that are catalyticin Ln(II).15 A handful of examples havedemonstrated the possibility of Ln(II) catalysis by regenerating Ln(II)photochemically with rhodamine 6G and an amine,16 with Zn,17 or electrochemically;18 sacrificial reductants were used in large excess.18 Crucially, existing examples have transformedonly a narrow range of substrates lacking potentially sensitive functionalgroups and have thus not demonstrated the retention of the most attractivefeatures of the stoichiometric reactions: high functional group tolerance,tunability of reducing power, and versatility.The unique physical and chemical propertiesof lanthanides (Ln)make them indispensable for applications ranging from renewable energyproduction to bioimaging and catalysis.22 Catalysts are often based on organic dyes19 or metal complexes, mainly transition metals;24 they have short (ns) excited-state lifetimes, and their reducingpower is limited to a narrow range. Ln(II)-based reductants have oxidationpotentials that cover a broad range from −0.4 to −3.9V; however, lanthanides have been underutilized as photocatalysts.Most Ln(III) ions can luminesce when sensitized by a light-harvestingchromophore (25 Thephotoexcited chromophore is a powerful reductant. In the case of themost reducible Yb(III)26 (E1/2(Yb(III)/Yb(II), YbCl3) = −1.1 Vvs NHE) and Eu(III)27 (E1/2(Eu(III)/Eu(II), EuCl3) = −0.42 Vvs NHE), Ln(III) reduction competes with Ln(III) luminescence sensitizationvia energy transfer  from a Ln(III) precursor followed by reoxidation of theLn(II) by a substrate and the reduction of the oxidized chromophorewith a sacrificial reductant enables the use of catalytic amountsof lanthanide reagent. While such a lanthanide turnover has been demonstrated,the reported procedure relies on strongly reducing Dy(II), Nd(II),or Tm(II) (E1/2(Ln(III)/Ln(II) = −2.5,−2.6, – 2.3 V vs NHE, respectively29));16 the authors noted thatusing the much more accessible Ln(III) precursors instead of the Ln(II)ones afforded the product in lower yields.16 We hypothesized that a practical protocol for the in situ generationof Ln(II) from Ln(III) could be obtained by combining the chromophorewith a multidentate ligand.In photoredoxcatalysis, excited-state photosensitizers initiatechemical transformations.2. Reductionswere successfully coupled to a variety of C–C, C–N,C–P, C–S, and N–N bond formations, as well asto carbocycle and heterocycle syntheses. Taken together, these practicalcatalytic processes reproduce and even surpass stoichiometric Ln(II)-mediatedreductions in scope, functional group tolerance, and versatility andare thus viable alternatives to the attractive, but resource-intensivestoichiometric transformations.Here, we show that insitu-generated Ln(II)  speciescan reduce benzylic, allylic, and aryl halides efficiently and affecta broad range of functional group interconversions previously achievedonly by (super)stoichiometric amounts of SmILnL1–LnL3)  with different coordination environments (L1 and L2 were expected to strongly bind Ln(III) and Ln(II)and thus yield stable, long-lived catalysts. L3 relieson weak interactions between Eu(III) and 7-aminocarbostyril, and mostlanthanide coordination sites are occupied by labile solvent molecules. L1–L3 incorporate a light-harvesting heterocycle,either a 6,7-oxycoumarin , or a7-aminocarbostyril (L3), which allows for excitationwith UV and visible light. Structurally L1 is relatedto the Allen cryptands.30L2 isa flexible and synthetically more accessible open-chain analogue of L1. L1 and L2 were expected to stabilizeLn(II) compared to the solvated ion. The excited-state reduction potentialsof the chromophores are < –1.88 V , which is sufficiently negative to reduce Eu(III) and Sm(III)and thus yield the reactive Ln(II) species. The catalyst design describedhere is highly modular. Catalyst reactivity can be tuned through theligand that regulates Ln(II) stability and substrate access, throughthe chromophore excited state and the lanthanide.We designedthree catalysts (ronments 1. L1 and17 Irradiation with 365 nm light or with a blueLED (λem = 463 nm) of a solution of benzyl chloride(1a) containing 0.1 equiv of Eu(III)L  yieldedbibenzyl 1b using only 1 equiv of Zn . A range of nonmetallic sacrificialdonors were then screened to reduce the amount of metal in the reactionsusing 1a as the substrate (Table S1). These studies allowed optimized conditions to be identifiedfor the use of EuL1 and EuL2, as well asconditions for using either metallic (Zn) or organic sacrificial reductants. Conditions B (Eu(III)L1 (0.1 equiv), N,N-diisopropyl ethylamine ,HCO2H (0.5 equiv), 365 nm or blue LED) and C (Eu(III)L2 (0.1 equiv), DIPEA (10 equiv), LiCl (10equiv), H2O (20%), blue LED). Conditions B and C are tailored to EuL1 and EuL2, respectively, and differ in the excitation wavelength and the amountof sacrificial donor. The shorter excitation wavelength is incompatiblewith several iodinated and brominated substrates that absorb in thisregion. Water addition  shifts EuL2 absorption above 400 nm  = 3094 M–1 cm–1 with watervs 714 M–1 cm–1 without water),which allows for excitation with a blue LED, and thus increases thesubstrate scope and the selectivity toward bibenzyl formation. A catalystloading of 10% was suitable for both small- and large-scale reactionsand could be lowered to 5 mol % for reactions with >0.1 mmol substrate. EuL can be generated in situ from the ligand and an Eu(III)salt without impacting the reaction outcome (Table S4). Control experiments established that light was necessaryfor catalysis, as was the presence of EuL or SmL. The replacement of Eu/SmL either with the uncomplexedligand  or withredox-inactive Gd(III)L gave only a trace amount of product.Initialinvestigations focused on the mildest divalent lanthanidereductant Eu(II) to ensure a high functional group tolerance for thereaction and Zn as the sacrificial donor due to its ability to reducecryptand-encapsulated Eu(III) to Eu(II) when used in large excess.1b or 1c could be obtained selectively from 1a in excellent yield , ketone (11a), nitrile (2a), aryl bromide (7a), ether , thioether (10a) and silylether (12a), and heteroaryl groups (13a–17a).Under the optimized conditions, either and EuL2 2a. Benzy1c–17c) or bibenzyl (1b–18b) products, including biologically activeBrittonin A (18b). While the reaction outcome was substrate-dependent,in several cases, either product could be selectively obtained bya simple change in the catalyst or reaction conditions. Such ligand-basedselectivity is unprecedented for Ln(II)-mediated reductions. The productdistribution likely depends on the rate at which a benzyl radicalis generated and its stability, as is the case for Ir and Ru catalysts33  were efficiently dehalogenated, as were electron-richbromides  and iodide (20a). The functional group tolerance of C(Ar)-X reductionswas remarkable: aldehydes (21a) and esters  were left intact. In contrast, the stoichiometrictransformations either did not afford the desired product (21b) or gave mixtures of dehalogenated products and byproducts formedby ketone and ester reduction, while requiring 2–8 equiv. ofSmI2, and either HMPA (>20 equiv) or a proton source.36 Deuterium labeling experiments (24c) reveal the DMFsolvent as a proton source under conditions A and C. However, no deuterium incorporation was seen under condition B when conducting reactions either in DMF-d7 or with added formic acid-d2. These results are consistent withDIPEA being the proton source in condition B (Table S18).Aryl halides were successfully reducedby using catalytic amountsof lanthanide 2b. Elect29a–37a proceed without side product formationin yields comparable to or better than that of the stoichiometricprocess while requiring up to 99% less lanthanide. Diazo compound 30a was reduced to hydrazine 30b, while azidein 32a yielded diazo compound 32b in 69%yield without over-reduction presumably due to the presence of theelectron-donating p-amino groups. Pinacol couplingof 35a proceeded in yields comparable to that obtainedpreviously using 2–10 equiv of SmI2.38 The reaction of 35a was catalyzed by EuL3 and not by EuL1 or EuL2. Substrate bindingwas ligand- and substrate-dependent, as indicated by the differencesin the changes of Eu(III) luminescence spectral shapes and luminescencelifetimes (τEu) upon the addition of various substratesto EuL1–3 . The open Eu(III) coordination sphere of EuL3 is likelybeneficial for binding a bulky substrate such as 35a.Additionally, the coordination of the H-donor (XH) to Ln(II) is morelikely for EuL3 than for ligand-encapsulated EuL1 and EuL2. Such XH-coordination has been shown to generatea strong proton-coupled electron transfer reagent, capable of simultaneouslytransferring an electron and a proton to the substrates that are difficultto reduce.39 The reduction of pentavalentphosphorus compounds is an industrially important reaction, oftenrequiring silyl or metal trapping reagents.40 Trimethyl phosphate was reduced in moderate yield to phosphite 34b using EuL2 as the catalyst and 1 equiv ofZn.A range of catalytic functionalgroup transformations that havepreviously been performed using superstoichiometric amounts of Ln(II)were then carried out under photocatalytic conditions 3. The re36b or 36c could be obtainedselectively from 36a by performing the reaction in thepresence of strictly10 equiv. or a large excess of H2O (20% v/v), respectively.Catalyst reactivity was also lanthanide-dependent. Imine (31a), aldehyde (49a), and oxime (50a) werenot efficiently reduced with EuL2, but the more powerful SmL2 afforded the products in yields comparable to what wasobserved using stoichiometric Sm(II) reagents.42 A similar improvement in the yield was seen upon reduction of 24a with SmL2 rather than EuL2,presumably also because of the higher reducing power of SmL2. Conversely, the more reducible substrates 1a and 2a underwent unselective reactions in the presence of SmL, while with the milder reductant EuL productswere obtained with excellent selectivity (38a–41a), aldehydes , heteroarenes , and arenes(47a) enabling C–C bond formation in good yields(38a–41a) was possible in a 1,4-fashion to afford 38b–41b in good yields. Allylic bromides 42b and 43b reacted with aldehydes 42a and 43a, respectively; the 1,2-addition products wereobtained in moderate yields. Quinoline (44a) alkylationproduced a mixture of C2/C4 alkylated isomers  (45a gave tricyclic fluorene (45b), albeit inlow yield. A similar addition of 46b to isoquinoline 46a enabled the synthesis of a natural product, papaverine,in a good yield. The aryl radical formed from 24a couldalso be harnessed to enable C–S and C–P bond formations 4c, whilermations4d.47b and 47c, respectively. Such synthetic modificationsof the isoquinoline pharmacophore43 couldopen up new possibilities for medicinal chemists.Reactions with triethyl phosphite and DMSO gavethe correspondingphosphate and methyl sulfide-substituted isoquinolines 49a to trans-1,2-diol 49b, astructural motif central to the pradimicin and benanomicin antibioticclasses,44 was stereoselective. Preparationsof formamide analogue 48b and tetrasubstituted pyrazines 50b from simple precursors  couldopen up new avenues for the synthesis of various drugs incorporatingthese important pharmacophores.46Some productsare of particular interest. Pinacol cyclization of Figure S7 presents a range of aryl halides that were not reduced underconditions A, B, or C. Thesesubstrates may require more powerful reductants, e.g., Ln(II) specieswith more negative reduction potentials.47 Attempted cross-reactions between benzyl chloride and aryl, alkynyl,or carbonyl acceptors and unsuccessful pinacol coupling reactionsare shown in Tables S48–S51 and Figure S7, respectively. The success of these cross-couplings likelyrequires an adjustment of the ligand.Limitations tothe protocol remain. LnL absorb at theexcitation wavelengths used in the reactions; absorption is throughthe chromophore  andlifetimes (τL) of the chromophores  hasa similar ionic radius and heavy atom effect to Eu(III) and Sm(III)but is not photoactive.25 The reductionpotential required to access Gd(II) is much more negative than thatrequired for accessing Eu(II).48 Thus, Gd(III)L provides a model for Eu(III)L that recreates the coordinationand electronic properties of the complexes but does not allow forLn(II) formation. The Gd complexes had higher ΦL andlonger τL, which is consistent with the presenceof additional processes (energy or electron transfer) quenching thefirst singlet excited state of Chr in EuL and SmL. Electron transfer from Chr toLn(III)  was calculated to be thermodynamically feasible(Tables S55 and S56).ore Chr, 5a. To inmophores 1 in GdL150 which may be intercepted by asubstrate either before or after Chr regeneration bythe sacrificial reductant (Eu/SmL that yields the active reagent (Table S2). Neither the uncomplexed ligand  nor Gd(III)L gave trace amounts of product. These experiments show that Ln(II)formed from the reducible Ln(III) rather than the excited chromophorereduces the substrate. An Eu(III) complex of the ligand lacking thesensitizing antenna  can be reduced by the photoexcited Chr and inturn can reduce the substrate; the role of the sacrificial reductantis to regenerate Chr and enable the use of catalyticamounts of EuL. Eu(II) is a more powerful reductant inits excited state than in its ground state.51Eu(II)L1 was inactive in the absence of light (Scheme S2). Thus, either the photoinduced electrontransfer (PeT) yields Eu(II) in its excited state or ground-stateEu(II) is initially formed and gets excited.There are several conceivable mechanistic pathwaysfor the catalyticprocesses to follow. Ln(III) reduction by the photoexcited chromophoreyields Ln(II),eductant 1. Contronna L2m, 1 did nottable S6). LCMS analysis of an irradiated sample containing EuL1 and TEMPO showed the protonated molecule ion of the L1-TEMPO adduct . To gather further evidence of either •+Chr or Eu2+ formation, twoEPR experiments were carried out. First, solutions of EuL/GdL containing equimolar amounts of the radical quenchersTEMPO or PBN (N-tert-butyl-α-phenylnitrone)were irradiated. TEMPO, but not PBN, is an EPR-active free radical.PBN can instead form stable radical species after reaction with anorganic radical. EPR analysis of the irradiated TEMPO-containing solutionsindicated 20–50% reduction of the TEMPO signal in the presenceof EuL . The irradiation of a mixture of EuL2 and PBN showed the emergence of an EPR signal correspondingto a N-based radical from a PBN adduct .52 EPR analysis at cryo temperatures(10 K) of an irradiated  solution of EuL2 showed features at g = 5.9, 3.13, 2.8 and a broadfeature at g = 2, in addition to a sharp radical-likesignal visible at g = 2.01 L1 and isoelectronic GdL2 . These results are consistent with the formation of aradical cation and Eu2+ in EuL under illuminationby electron transfer from Chr. To the best of our knowledge,the Eu(II) EPR spectrum is the first spectroscopic evidence of suchphotochemical Ln(II) formation.In the presenceof the radical quencher 2,2,6,6-tetramethylpiperidinyloxy(TEMPO), the reaction does not proceed (2O). Ln(II) catalysis hasbeen demonstrated on a broad substrate scope with the yield and selectivitycomparable to or better than the corresponding stoichiometric processeson a synthetically useful scale. The scope of the Ln(II) catalysisincludes synthetically important C–C, C–N, C–P,C–S, and N–N bond formation reactions, C–halogen,P–O, C–C, C–S, and N–N bond cleavage reactions,and the synthesis of biologically important carbocyclic and heterocyclicstructural motifs. Eu(II) formation via the reduction of Eu(III) bya photoexcited nearby chromophore was supported by EPR spectroscopicfindings. These photocatalytic reactions are substantially more benignthan their stoichiometric analogues, as they can be performed withouttoxic HMPA, and with up to 99% less lanthanide.Stronglyreducing Ln(II) species were accessedfrom Ln(III) precursorsthrough photochemical reduction by an excited-state organic chromophoreand used as catalysts. Excitation could be performed with both UVand visible light. The Ln(II) could reduce a broad range of substrateswith excellent functional group tolerance. The catalytic cycle wasclosed by reduction of the oxidized chromophore with Zn or nonmetallicsacrificial reductants. The selectivity of the reaction and the reducingpower of the catalyst could be independently tuned by the ligand,the lanthanide, and additives (H


Coturnix c. japonica). Consensus HA1 sequences were generated to capture antigenic diversity among isolates. We constructed chimeric H9N2 viruses containing the HA1 of each consensus sequence on a constant isogenic backbone. The resulting viruses were used to generate antisera from quail, a common and significant minor poultry species whose anti-HA response profiles remain poorly defined. Antigenic maps were generated by plotting the cross-hemagglutination inhibition (HI) data from the panel of quail sera against the chimeric constructs and 51 H9 field isolates. The chimeric antigens were divided into four different antigenic profiles . Site-directed mutagenesis analysis showed nine amino acid positions of antigenic relevance. Substitutions at amino acid positions 149, 150, and 180 (H9 HA numbering) had relatively significant impact on HI activity using quail sera. Substitutions E180A and R131K/E180A led to the most significant antigenic change transitions. This study provides insights into the antigenic profile of H9 FLUAVs, with important implications for understanding antigenic drift and improving vaccine development for use in minor poultry species.Influenza A viruses (FLUAV) of the H9N2 subtype are zoonotic pathogens that cause significant economic damage to the poultry industry. Vaccination to prevent and control H9N2 infections in poultry is widely employed in the Middle East and Asia. We used phylogenetics and antigenic analysis to study the antigenic properties of the H9 hemagglutinin (HA) using sera produced in Japanese quail (Determining the relevant amino acids involved in antigenic drift on the surface protein hemagglutinin (HA) is critical to understand influenza virus evolution and efficient assessment of vaccine strains relative to current circulating strains. We used antigenic cartography to generate an antigenic map of the H9 hemagglutinin (HA) using sera produced in one of the most relevant minor poultry species, Japanese quail. Key antigenic positions were identified and tested to confirm their impact on the antigenic profile. This work provides a better understanding of the antigenic diversity of the H9 HA as it relates to reactivity to quail sera and will facilitate a rational approach for selecting more efficacious vaccines against poultry-origin H9 influenza viruses in minor poultry species. Influenza A virus (FLUAV) of the H9N2 subtype are enzootic in poultry in Asia, the Middle East, and parts of Africa, where they cause significant economic losses due to high morbidity and mortality of poultry flocks , 2. MoreTo prevent and control H9N2 virus infections in poultry, several countries in Asia and Middle East have resorted to vaccination programs –10. AnTo broaden the understanding of the antigenic diversity of HAs of H9 FLUAVs, we included strains from the American and Eurasian lineages. Starting from an initial phylogenetic analysis of nucleotide sequences corresponding to the HA1 region of the HA, we identified 18 clades utilizing sequence information of strains from 1966 to 2020. Analyses of these clades led to the selection of 10 consensus sequences that largely embodied the amino acid diversity within each H9 lineage/sub-lineage/sub-sub-lineage. The 10 HA1 sequences were used to generate chimeric H9 HA gene segments carrying a constant HA2 portion derived from the prototypic strain gf/HK/WF10/1999 (H9N2) (WF10) , 31. Then = 10 . Consensus sequences were generated for each clade, n = 10 . The pern = 10 . Chimerin = 10 . For WF1in vitro in Madin-Darby canine kidney (MDCK) cells showed that all chimeric H9 viruses (10TCID50eq/mL except the h9.1.1 consensus virus (5 log10TCID50eq/mL at 96 h). Replication and transmission of consensus chimeric of H9 viruses were evaluated (n = 6 per group) that were directly inoculated (DI) with the H9N2 chimeric viruses (one chimeric virus per group) or the WF10 wild-type virus. At 24 h post-inoculation, naive quail were introduced in direct contact  with the corresponding DI group. We chose Japanese quail (Coturnix c. japonica) as a relevant minor poultry host of H9 FLUAVs  were used to generate antisera against the chimeric HA consensus viruses . Dimensional analysis of the HI data set led to lower error yield in the 3D maps, though 2D maps were selected for better visualization, given that the relationship between consensus antigens remained unvaried. The antigens were grouped into four different clusters . We usedTo better define whether the consensus chimeric H9 HA viruses captured the antigenic profile of prototypic strains within each clade, the quail sera was used in HI assays using a subset of closest prototypical field strains available ; Table 2We expanded these analyses to 48 additional field strains , bringinTo better define the amino acid signatures involved in the antigenic profile of H9 HA antigens, the differences among the prototypic WF10 h9.4.1 (cyan), the h9.4.2 (blue), and the h9.3.9 consensus viruses were further analyzed. The antigenic units (AU) distance between WF10 h9.4.1 and h9.4.2 are lower (3.4 AU) than the distance between WF10 h9.4.1 and h9.3.9 (4.1 AU). Amino acid substitution differences between WF10 h9.4.1 (cyan) and the h9.4.2 (blue) include E72G, G135D, E180A, and I186T, which have been previously reported as antigenically relevant for H9 , 16, 33.The HA plays a pivotal role in the antigenicity of FLUAV as it is the major target of neutralizing antibodies and subject to positive selection. Phylogenetics combined with antigenic analysis is the basis for human, avian, and equine influenza vaccine selection . AntigenFor antigenic characterization, boost immunizations with inactivated-whole virus adjuvant formulations were performed in quail at 14 dpi to increase HI titer levels of poorly immunogenic antigens. Quail antibody responses to H9 FLUAV mimicked what was previously reported in the literature for chicken sera , 16. TheLevels of HI titers were consistent with the virus’ ability to replicate in quail. Perhaps, not surprisingly, the poultry-adapted chimeric viruses and particularly the wild-type WF10 virus were the most efficient in replication and transmission in quail. In contrast, limited replication and transmission were observed with the h9.2.2 wild bird Eurasian-origin chimeric virus, whereas no replication was observed with the h9.1.1 wild bird American-origin virus. It can be speculated that the replication and transmission of the recombinant consensus viruses were affected by the chimeric nature of the HA segment. However, these observations are in agreement with previous observations on the limited capacity for replication and transmission in quail of non-poultry-adapted wild bird-origin FLUAVs , 21.Most wild bird isolates from Europe and North America clustered with the h9.1.1 and h9.2.2 consensus antigens (red cluster) , as predThe generation of a humoral response that interferes with the interaction of HA with its receptor is key to achieving sterilizing immunity against FLUAV. Seven residues  near the receptor binding site (RBS) were identified as the major determinants of antigenic drift in human and swine H3N2 FLUAVs , 38. SimThe impact of single or double amino acid substitutions was less clear . The E18Other single or double substitutions showed changes in the level of antigenicity; however, none were enough on their own to produce complete antigenic cluster transitions . ReducedDespite the remarkable plasticity of the H9 HA of WF10, reversions were observed in 5 out of 19 mutants, suggesting that tolerability of changes in antigenically relevant amino acids may be context dependent and likely encompass compensatory substitutions , 53. In Most studies of H9N2 antigenicity in poultry involve the use of chicken sera but not sera from minor land-based poultry species, such as quail. Japanese quail have been suggested as key players in the genesis of influenza viruses with respiratory tract tropism , 21. QuaIn conclusion, phylogenetics was used to generate consensus on H9 viruses encompassing their natural diversity. We demonstrated that these consensus H9 viruses were biologically active, capable of triggering an immune response associated with the generation of neutralizing antibodies, and manifested important distinctive biologic characteristics driven only by their differences in the HA1 domains. Using this system, we explored antigenicity and modulation of HI profiles using antisera obtained from quail. The sera obtained allowed us to narrow down antigenically relevant amino acids, as many as nine for h9.4.2  and six for h9.3.9  to as few as one (E180A), to produce antigenic cluster transitions. The results are relevant to pave the way for a better understanding of the molecular signatures of antigenicity in H9 viruses, facilitating a rational approach for selecting more efficacious vaccines against poultry-origin H9 influenza viruses.2.MDCK cells were a kind gift from Robert Webster . Human embryonic kidney 293T cells were obtained from the American Type Culture Collection . Cells were maintained in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum (Sigma-Aldrich), 1% antibiotic-antimycotic (Sigma- Aldrich), and 1% L-glutamine (Sigma- Aldrich). Cells were cultured at 37°C in a humidified incubator under 5% COAY206676  and then sub-cloned into the plasmid pDP-BsmbI-WF10_HA2 encoding the HA2 portion of WF10. All chimeric constructs contained the identical cleavage site motif (PARSSR) of the WT HA of WF10.50) as described . Next-generation sequencing was performed on all consensus viruses’ whole genomes to exclude unwanted substitutions. For whole-genome sequencing, amplicon sequence libraries were prepared using the Nextera XT DNA library preparation kit  according to the manufacturer’s protocol. Barcoded libraries were multiplexed and sequenced on a high-throughput Illumina MiSeq sequencing platform in a paired-end 150-nucleotide run format. eviously Growth kinetics were performed as previously described . MDCK ceVirus RNA was extracted from cell supernatant or quail oropharyngeal swabs and quantitated . Viral RCoturnix c. japonica) were purchased from Plantation Quail . The seronegative status against FLUAV was determined via IDEXX AI MultiS-Screen Ab Test ELISA  before infection. Quail (n = 6 per group) were inoculated by the oculo-nasal-tracheal route with 106 TCID50 per quail of the following WF10-chimeric HA (H9N2) viruses: h9.1.1, h9.2.2, h9.3.3, h9.3.4, h9.3.5, h9.3.7, h9.3.9, h9.4.1 (WF10), and h9.4.2. A negative control  was included. At 24 hpi, naive quail (n = 6 per group) were brought into direct contact with inoculated quail to monitor for virus transmission. Active infections were monitored by Flu DETECT  on tracheal swabs collected from days 1–7 post-inoculation. HI titers against homologous viruses in each case were tested from each individual quail  as previously described  generated single and double amino acid substitutions in the WF10 HA gene segment following manufacturer conditions. Plasmid sequences were confirmed by Sanger sequencing.


O-methyl-nordihydroguaiaretic acid , a global transcription inhibitor, in combination with a second anticancer drug induces strong tumoricidal activity and has the ability to suppress energy metabolism in cultured cancer cells. In this study, we showed that after continuous oral consumption of high-fat (HF) diets containing M4N, the M4N concentration in most of the organs in mice reached ~1 μM (the M4N concentration in intestines and fat pads was as high as 20–40 μM) and treatment with the combination of M4N with temozolomide (TMZ) suppressed glycolysis and the tricarboxylic acid cycle in LN229 human glioblastoma implanted in xenograft mice. Combination treatment of M4N with TMZ also reduced the levels of lactate dehydrogenase A (LDHA), a key enzyme for glycolysis; lactate, a product of LDHA-mediated enzymatic activity; nicotinamide phosphoribosyltransferase, a rate-limiting enzyme for nicotinamide adenine dinucleotide plus hydrogen (NADH)/NAD+ salvage pathway; and NAD+, a redox electron carrier essential for energy metabolism. It was also shown that M4N suppressed oxygen consumption in cultured LN229 cells, indicating that M4N inhibited oxidative phosphorylation. Treatment with M4N and TMZ also decreased the level of hypoxia-inducible factor 1A, a major regulator of LDHA, under hypoxic conditions. The ability of M4N to suppress energy metabolism resulted in induction of the stress-related proteins activating transcription factor 4 and cation transport regulator-like protein 1, and an increase in reactive oxygen species production. In addition, the combination treatment of M4N with TMZ reduced the levels of oncometabolites such as 2-hydroxyglutarate as well as the aforementioned lactate. M4N also induced methylidenesuccinic acid (itaconate), a macrophage-specific metabolite with anti-inflammatory activity, in tumor microenvironments. Meanwhile, the ability of M4N to suppress energy metabolism prevented obesity in mice consuming HF diets, indicating that M4N has beneficial effects on normal tissues. The dual ability of combination treatment with M4N to suppress both energy metabolism and oncometabolites shows that it is potentially an effective therapy for cancer.Tetra- Larrea divaricata), a desert medicinal plant . In. In4N+TMreatment .4N-mediated induction of multiple stress-related genes are not clearly understood  in LN229 tumors Click here for additional data file.S2 Table(TIF)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


The GsGd lineage (A/goose/Guangdong/1/1996) H5N1 virus was introduced to Canada in 2021/2022 through the Atlantic and East Asia-Australasia/Pacific flyways by migratory birds. This was followed by unprecedented outbreaks affecting domestic and wild birds, with spillover into other animals. Here, we report sporadic cases of H5N1 in 40 free-living mesocarnivore species such as red foxes, striped skunks, and mink in Canada. The clinical presentations of the disease in mesocarnivores were consistent with central nervous system infection. This was supported by the presence of microscopic lesions and the presence of abundant IAV antigen by immunohistochemistry. Some red foxes that survived clinical infection developed anti-H5N1 antibodies. Phylogenetically, the H5N1 viruses from the mesocarnivore species belonged to clade 2.3.4.4b and had four different genome constellation patterns. The first group of viruses had wholly Eurasian (EA) genome segments. The other three groups were reassortant viruses containing genome segments derived from both North American (NAm) and EA influenza A viruses. Almost 17 percent of the H5N1 viruses had mammalian adaptive mutations  in the polymerase basic protein 2 (PB2) subunit of the RNA polymerase complex. Other mutations that may favour adaptation to mammalian hosts were also present in other internal gene segments. The detection of these critical mutations in a large number of mammals within short duration after virus introduction inevitably highlights the need for continually monitoring and assessing mammalian-origin H5N1 clade 2.3.4.4b viruses for adaptive mutations, which potentially can facilitate virus replication, horizontal transmission and posing pandemic risks for humans. Influenza A viruses (IAVs) are segmented and enveloped RNA viruses ,2. Each H5Nx viruses belonging to clade 2.3.4.4 viruses and their different reassortant variants are the major causes of large-scale influenza outbreaks in poultry and wild birds worldwide ,12. In EIsolated cases of influenza infection associated with GsGd lineage H5Nx viruses were also documented in terrestrial mammals such as domestic dogs and cats ,19, tigeThis study describes the presence of wholly Eurasian and three novel reassortant H5N1 clade 2.3.4.4b viruses with various genome constellations in free-living mesocarnivore species  in Canada in 2022. The viruses characterized from the mesocarnivore species had similar genetic compositions with H5N1 viruses circulating in avian species in the same geographic areas. The spillover of these viruses from wild birds to mammals could cause a potentially devastating pandemic if the H5N1 viruses mutate into forms that can spread efficiently among the mammalian species.Various samples including oropharyngeal swabs, lung, brain, and other tissues were collected between 12 April 2022 and 29 July 2022 from mesocarnivore species that were found dead or euthanized at various rehabilitation centres or at the wildlife clinics by Canadian Wildlife Health Cooperative regional centres. Clinical samples collected from animals with neurological disease were first tested for rabies and canine distemper at regional Animal Health Laboratories or provincial Canadian Animal Health Network Labs. Samples that tested negative for rabies and canine distemper were further tested for the presence of IAVs. All IAV H5-positive samples were submitted to the National Centre for Foreign Animal Disease (NCFAD) laboratory in Winnipeg for confirmatory testing. Clinical histories of all the cases involving mesocarnivore species used in this study are summarized in Total RNA was extracted from clinical specimens (swabs and tissues) and virus isolates using the MagMax 1836 Nucleic Acid Isolation Kit using the KingFisher Duo prime platform . The presence of IAV genomic material was verified using the matrix gene-specific real-time RT–PCR followed by H5-specific real-time RT–PCR as described previously ,28.n = 959,847) (https://www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select.cgi?go) and influenza virus sequences from the 2021/2022 H5N1 outbreaks.The full genome segments of IAVs were amplified directly from clinical specimens and isolates using RT–PCR as described . Nanoporn = 26), Asia (n = 124) and Europe (n = 1168) were downloaded from the Global Initiative on Sharing All Influenza Data . Sequences downloaded from GISAID and generated in our study, 40 from mesocarnivores and 41 from birds, were aligned using MUSCLE, and alignments were trimmed to contain major open reading frames for PB2 (nucleotides [nt] = 2280), PB1 (nt = 2274), PA (nt = 2151), HA (nt = 1704), NP (nt = 1497), and NA (nt = 1410), whereas M and NS segments were trimmed as 982 and 838 nt, respectively. Before selecting representative strains, all sequences of eight segments were separately pooled with sequences generated in this study, and the alignments were subject to maximum likelihood (ML) tree inference using IQ-TREE web server (http://iqtree.cibiv.unvie.ac.at). The best-fitting substitution model for each segment dataset was determined by IQ-TREE and the ultrafast bootstrap with 1000 bootstraps was used for branch support analysis. For the 81 H5N1 full genome sequences generated in this study, a Bayesian phylogenetic analysis was used for concatenated sequences from the coding region of PB2, PB1, PA, HA, NP, NA, M1, M2, NS1, and NS2 using a Bayesian Markov Chain Monte Carlo (BMCMC) method [http://tree.bio.ed.ac.uk/software/tracer). All parameter estimates for each run showed ESS values > 200. A maximum clade credibility (MCC) phylogenetic tree was generated to summarize all 10,000 trees after a 10% burn-in using TreeAnnotator in BEAST 2 [http://tree.bio.ed.ac.uk/software/figtree).A total of 2364 full genome sequences of IAVs of various subtypes collected between 1 January 2015 and 19 July 2022 from North America and a total of 1318 full genome sequences of highly pathogenic H5N1 clade 2.3.4.4b avian influenza viruses were collected between October 2020 and 19 July 2022 from Africa  method  as impleBlood samples were collected from red foxes in Ontario that survived clinical disease 3–4 weeks after infection. IAV antibodies in the convalescent sera were detected using an in-house developed IAV nucleoprotein (NP) specific competitive ELISA (cELISA)  or H5 HA+ system (anti-mouse) , and reacted with the chromogen 3,3′-Diaminobenzidine. The sections were counter-stained with Gill’s hematoxylin.Brain, lung, and various other tissues collected from red foxes in Ontario, Manitoba, Nova Scotia, and Prince Edward Island (PEI) were processed for hematoxylin and eosin staining for microscopic analysis. Immunohistochemical detection of IAV antigen in tissue sections was conducted as described previously. A mouse monoclonal antibody specific for influenza A nucleoprotein (F26NP9)  was appln = 40) and wild birds (n = 41) showed closer relationships to HPAI H5N1 viruses from Ireland (isolated on 19 November 2021) and Iceland (isolated on 25 October 2021). The same genetic relationships were observed for NA and M segments in all 81 Canadian H5N1 viruses (n = 81) generated in this study were deposited at the GISAID. ORG database and accession numbers were indicated in To confirm the origin of each EA genome segment of the 40 H5N1 viruses obtained from the mesocarnivore species, phylogenetic tree analysis was conducted for each genome segment individually in comparison with HPAI H5N1 virus sequences deposited at GISAID between October 2020 and 19 July 2022 from Africa, Asia, and Europe. In addition, to confirm the origin of NAm lineage genome segments, IAVs of various subtypes collected from NAm between 1 January 2015 and 19 July 2022 were used. Phylogenetic analyses of the HA gene of viruses originated from Canadian mesocarnivore species  demonstrated to provide H5 subtype IAVs with strong binding capability to α-2,6 human-like receptors. Significant amino acid substitutions in all the putative antigenic sites of the HA protein were present in all viruses. These substitutions were present in the antigenic site B , antigenic site C (E268G), and antigen site D (V210A) (H5 numbering) when compared to the A/Gyrfalcon/Washington/41088-6/2014 H5N8 (clade 2.3.4.4c) virus that was previously reported in North America for the first time in 2014/2015.The full genome sequences of H5N1 viruses obtained from foxes, skunks, and mink were assessed for previously known adaptive mutations that may enhance the replication and transmission to other mammalian hosts. All viruses isolated from the mesocarnivores retained some amino acid residues in their hemagglutinin (HA) protein that might enable them to bind to the α-2,6-linked sialic acids. These residues include the substitutions of S137A and T160A that were demonstrated to moderately enhance the binding activity of IAVs to α-2,6-linked sialic acids under The majority of H5N1 viruses obtained from mesocarnivores had glutamic acid in their PB2 protein at amino acid position 627 (627E). A couple of red foxes from Atlantic Canada (FAV-0544 and FAV-0592-01) and Ontario (FAV-0558-01) had a substitution of the avian signature glutamic acid (E) to a human signature lysine (K) at position 627 (E627 K). In one of the red foxes (FAV-0592-01) from Prince Edward Island (PEI), a virus originating from the brain sample contained a mixture of the mammalian 627 K and avian 627E signatures, however, the proportion of the mammalian signature was about 66.7%. In contrast, a virus from the lung of the same fox had more of avian signature PB2-627E (87.9%) than the mammalian signature PB2-627 K (10.8%) indicating the occurrence of an adaptive mutation in this specific animal. The PB2 sequence information of a virus originating from the oropharyngeal swabs of this kit’s littermate identified as FAV-0544 had 100% 627 K. Three red foxes from Ontario  had valine in PB2-627 (E627 V). Another red fox (FAV-0824-096) from Saskachewan had a substitution of D701N in PB2. The other important mutations in the PB2, PB1, PA, and NP segments that have been described to enhance either the polymerase activity or virus replication in mammalian cells are summarized in Three serum samples collected from red foxes in Ontario that survived H5N1 infection tested positive for the presence of anti-IAV NP antibodies as well as H5 HA-specific antibodies in cELISA tests. On the HI assay using A/Fancy Chicken/NL/FAV-0033/2021 (H5N1 – 2.3.4.4b) antigen, the serum samples obtained from two foxes (FAV-0300-1 and FAV-0300-6) had HI titers of 320, whereas a serum sample from another red fox (FAV-0300-5) had relatively higher HI titer of 640 units.Histological lesions in the cerebral cortex of red foxes naturally infected with a reassortant H5N1 virus  include multifocal necrotic neurons and glial cells, neutrophilic infiltrates into the cortex and perivascular cuffing A and C. Influenza A viruses continuously evolve and as a result accumulate adaptive mutations, which facilitate virus replication, interspecies transmission, and pose pandemic risks for human and animals. In the current study, we examined 40 HPAI H5N1 viruses from clade 2.3.4.4b virus with various genome constellations in multiple mammalian species, notably in mesocarnivore species in Canada. Infections with HPAI H5N1 viruses and mortality in these free-living red foxes, striped skunks, and mink reaffirm that these viruses can overcome interspecies barriers and infect new hosts. The clinical presentation of the disease in red foxes, skunks, and mink was mostly neurologic, resembling clinical and pathologic features described in seals and foxes that were naturally infected with HPAI H5N8 viruses . OropharTo date, few cases of HPAI H5N1 and H5N8 clade 2.3.4.4b viruses were found in free-living mesocarnivores  comparedin vitro studies [The full genome sequence analysis of H5N1 viruses derived from red foxes, skunks and mink revealed critical mutations that can affect receptor binding and adaptation to a mammalian host. The mammalian viruses demonstrate significant antigenic drift in their HA protein compared to A/Gyrfalcon/Washington/41088-6/2014 (H5N8) – a zoonotic candidate vaccine virus selected after the 2014/2015 outbreak in North America. The mammalian and avian viruses in the 2021/2022 HPAI virus outbreaks in Canada have substitutions at S137A and T160A in their hemagglutinin that may provide a degree of binding preference to the α-2,6-linked sialic acids as indicated under  studies . However studies .n = 15) with the E627 K mutation in their PB2 gene. E627 K mutation was also observed by deep sequencing of H7N9 viruses from oropharyngeal swabs originated from a number of human patients [in vitro in mammalian cells and in mammalian hosts [The replication of H5N1 viruses in mammalian cells is influenced by the polymerase complex activity; among which PB2 plays a main role in virulence . The majpatients  and in tpatients . E627 V patients . E627 K an hosts  and havean hosts . A cladean hosts . The mecan hosts . As wellan hosts ,54 of inan hosts . Virusesan hosts . A mutatan hosts . The polan hosts . Influenan hosts . A HPAI an hosts  are presan hosts ,60 and wan hosts . PB2 seqan hosts . The virClick here for additional data file.


SLC13A3 gene, and the X-linked neurodevelopmental condition Christianson Syndrome is caused by mutations in the SLC9A6 gene, which encodes the recycling endosomal alkali cation/proton exchanger NHE6, also called sodium-hydrogen exchanger-6. As a result, there are severe impairments in the patient’s mental capacity, physical skills, and adaptive behavior.Intellectual disability (ID) is a condition that varies widely in both its clinical presentation and its genetic underpinnings. It significantly impacts patients’ learning capacities and lowers their IQ below 70. The solute carrier (SLC) family is the most abundant class of transmembrane transporters and is responsible for the translocation of various substances across cell membranes, including nutrients, ions, metabolites, and medicines. The SLC13A3 gene encodes a plasma membrane-localized Na+/dicarboxylate cotransporter 3 (NaDC3) primarily expressed in the kidney, astrocytes, and the choroid plexus. In addition to three Na + ions, it brings four to six carbon dicarboxylates into the cytosol. Recently, it was discovered that patients with acute reversible leukoencephalopathy and a-ketoglutarate accumulation (ARLIAK) carry pathogenic mutations in the SLC13A3 gene (NM 022829.5) and the SLC9A6 gene (NM 001042537.2) were identified using whole exome sequencing. Family-A segregated a novel homozygous missense variant  in the exon-11 of the SLC13A3 gene. At the same time, family-B segregated a novel missense variant  in the exon-10 of the SLC9A6 gene. By integrating computational approaches, our findings provided insights into the molecular mechanisms underlying the development of ID in individuals with SLC13A3 and SLC9A6 mutations.Two Pakistani families (A and B) with autosomal recessive and X-linked intellectual disorders were clinically evaluated, and two novel disease-causing variants in the We have utilized in-silico tools in the current study to examine the deleterious effects of the identified variants, which carry the potential to understand the genotype-phenotype relationships in neurodevelopmental disorders.The online version contains supplementary material available at 10.1186/s12883-023-03397-y. Intellectual disability (ID) refers to a range of neurodevelopmental abnormalities, and about 2% of children or young people have ID, which is described as having significant deficits in intellectual functioning and adaptive behavior and is associated with an IQ below 70 . ChromosNaDC3) gene encodes the Na+/dicarboxylate cotransporter 3 (SLC13A3), found on the plasma membrane and carries necessary metabolic intermediates into cells ) to generate BAM files. BAM files were processed using GATK best practices  for single nucleotide variants (SNV) and small insertions/deletions (indel) variant calling to generate VCF files [https://3billion.io/resources) are used. The Homozygosity (ROH) regions were mapped from the VCF file using AutoMap v1.2 [Genomic DNA was extracted from the patient’s blood samples of both families. First, the exonic regions of all 22,000 human genes were captured by the xGen Exome Research Panel v2 . After capture, Novaseq 600 was used to sequence all captured regions . We acquired ≥ 20X coverage in > 98.9% and ≥ 10X coverage in > 99.4% of target sequences. Following the sequencing, the data was analyzed using open-source bioinformatics tools and proprietary software. bcl2fastq v2.20.0.422  and a 3-billion genome database were utilized in the first step. According to ACMG guidelines, gene variations with more than 5% allele frequency were filtered out. Next, the VarSome [https://www.ncbi.nlm.nih.gov/clinvar/) were utilized for the evaluation of variants. Then, each variant concerning disease phenotype was assessed using the ACMG guidelines [https://hpo.jax.org/) and retrieved to determine the degree of similarity [https://omim.org/ and https://www.orpha.net/consor/cgi-bin). According to the ACMG guideline, the similarity score between each patient’s phenotype and symptoms related to that disease caused by priority variations varied from 0 to 10. Medical geneticists and physicians then manually evaluate probable alterations and related disorders. Bidirectional Sanger sequencing is used to confirm single nucleotide variants and all indels.One of the in-house tools, EVIDENCE, was designed to select variants based on ACMG guidelines and each patient’s phenotype. Variant filtration, categorization, and similarity score for the patient’s phenotype are three significant steps in this approach. For allele frequency estimation, a genome aggregation database . Two sets of primers were designed:SLC13A3_Forward 5ˊCACACATGCATGGGACTC 3ˊ,SLC13A3_Reverse 5ˊCACTGTGCAGAGAGTGCAG 3ˊ.SLC9A6_Forward 5ˊ GAAGCTGTTAGGGGAAAT 3ˊ,SLC9A6_ Reverse 5ˊ CACTTATCTTTTGGGGTTGG 3ˊ,SLC13A3 [We utilized homology modeling to generate the protein structures without available crystal structures for the proteins of interest. The SWISS-MODEL server was employed for this purpose . For SLCSLC13A3 . Both wiSLC13A3 , and a sSLC13A3 , 40, whiSLC13A3 .To investigate the effect of mutations on the overall structural dynamics of the proteins in comparison to their wild-type counterparts, we conducted MD simulations in two steps: a 100 ns MD simulation for refining and optimizing the models (wt and mut), followed by another 100 ns MD simulation to analyze the residual fluctuations with or without the reported mutations. All simulations were executed using AMBER 20  followinV:2) was born to first cousins in the fifth generation of the presented family pedigree  were born to first-cousin parents, making this the fifth generation in the family’s pedigree. Before our first visit for sampling, patient IV:7 appeared in the fourth generation and had already passed away for unknown reasons. No history of a genetic disorder had been detected in the family . The segregation of this variant with disease phenotype was confirmed by Sanger sequencing. Zygosity analysis found both unaffected parents (IV:1 as well as IV:2) heterozygous carriers (c.1478 C/T), unaffected sibling (V:1) to be homozygous wild-type (c.1478 C/C), and the affected individuals (V:2) to be homozygous affected (c.1478T/T) for missense variant as mentioned earlier  from family-A revealed a novel homozygous missense variant  in the exon-11 of SLC9A6 gene (NM_001042537.2). Sanger sequencing validated and confirmed the segregation of the variant with the disease phenotype in the family. Furthermore, the zygosity analysis determined that both the affected individuals (V:3 and V:4) were hemizygous affected (c.1342 A), mother and sister (IV:2 and V:1) were heterozygous carriers (c.1342G/A). At the same time, the father and phenotypically unaffected brother (IV:1 and V:2) were hemizygous wild-types (c.1342G) for the identified missense variant , consistent with an X-linked mode of inheritance.In family-B whole exome sequencing identified a novel missense variant  in the exon-10 of SLC9A6, denoted as ‘SLC9A6_G448R mutant’, and Pro493Leu in SLC13A3, denoted as ‘SLC13A3_P493L mutant’. To investigate the structural implications of these mutations, homology models of SLC9A6 and SLC13A3 were generated using the SWISS-MODEL server. The SLC9A6 model exhibited 68.47% sequence identity and 0.66 query coverage with the cryo-electron microscopy (cryo-EM) structure of NHE isoform 9 (SLC9A9) from Equus caballus (PDB ID: 6Z3Y). Both proteins share a conserved domain architecture, including a core ion-transport domain that is open toward the intracellular side. Near the base of the cavity lies the strictly conserved aspartate residue, Asp244 in SLC9A6 (Asp292 in SLC9A9), which is crucial for ion-binding and transport [In our study, we identified the mutations Gly448Arg in + binding sites. The Na1 is coordinated by conserved residues such as Ser139, Trp141, Gly252, and Asn144. Whereas, Na2 is surrounded by Thr479, Ala482 (Thr463 in SLC13A5), Ser526 (Ala507 in SLC13A5), and Asn484. The proximity of the Pro493Leu mutation to the sodium-binding site suggests a possible impact on the transporter’s function . The Pro493Leu mutation is positioned close to the substrate and NaSLC13A3 and SLC9A6 models, respectively, indicative of reliable structural models.Subsequently, the homology models were refined and optimized through 100 ns molecular dynamics (MD) simulations. The evaluation of the final models showed that 90.30% and 90.54% of the residues were in Ramachandran favoured regions for vib of -1.523 for SLC9A6_G448R and − 0.583 for SLC13A3_P493L), leading to contrasting effects on thermal stability . The SLC9A6_G448R mutation lies within the core ion-binding site, while the SLC13A3_P493L mutation is located close to the sodium binding pocket. These locations and the observed contrasting effects on protein stability and flexibility might potentially influence ion transport efficiency, thereby affecting the protein’s function and ultimately contributing to the development of intellectual disability. These preliminary findings were further investigated and validated through Molecular Dynamics (MD) simulations by analyzing the impact of these mutations on the overall structural dynamics and residual fluctuations.The protein stability predictions for the mutated models of SLC9A6_G448R and SLC13A3_P493L, derived from the combined computational approach of mCSM and SDM, provided insights into the possible structural consequences of these mutations and their potential impact on the development of intellectual disability. For the SLC9A6_G448R mutation, the consensus prediction revealed a destabilizing effect on the protein structure, with a ΔΔG value of -0.138 kcal/mol. Conversely, in the case of the SLC13A3_P493L mutation, the consensus prediction from mCSM and SDM indicated a stabilizing effect on the protein structure, with a ΔΔG value of 0.446 kcal/mol. However, the Elastic Network Contact Model (ENCoM) server predicted a decrease in molecular flexibility for both mutations (ΔΔSA comprehensive 300 ns molecular dynamics (MD) simulations were conducted on the wild-type and mutant (SLC9A6_G448R and SLC13A3_P493L) protein models. Snapshots were taken every 30 ns throughout the simulation period to monitor the evolution of the protein structure and dynamics Fig. . The iniDetailed structural insights revealed that substituting glycine for arginine at position 448 in SLC9A6_G448R led to more interactions due to a longer and more flexible side chain in arginine Figure . The posSLC13A3 variants. After a bout of fever caused by a respiratory tract infection, one patient exhibited symptoms of acute neurological deterioration, including drowsiness, ataxia, and dysarthria. The other patient had febrile tonsillitis and relapsed six years later due to a febrile respiratory tract infection, presenting with drowsiness, poor contact, dysarthria, peripheral motor abnormalities, and global hypotonia. After a short supportive treatment, they returned to nearly complete health. It is the first study to link a-ketoglutarate accumulation and reversible leukoencephalopathy to biallelic variants of SLC13A3 [SLC13A3 variants significantly reduced the capacity of NaDC3 to transport the three substrates due to loss of function that may lead to physiological conditions [Solute carriers, or SLCs, are the most prominent family of transmembrane transporters responsible for the diffusion of nutrients, ions, metabolites, and drugs across cell membranes , 46 Abou SLC13A3 . Proxima SLC13A3 . Previounditions .SLC9A6, is primarily found in early and recycling endosomes, where it participates in endosomal trafficking, signaling, and the regulation of luminal pH [Christianson syndrome (CS) is a neurodevelopmental and degenerative X-linked intellectual disability disorder with a growing number of confirmed cases. The enzyme NHE6 controls the pH balance and trafficking of recycled endosomes . Since tminal pH , 55–57. minal pH , 59. WesSLC13A3 gene (NM_022829.5), while in family-B, a novel missense variant  in the exon-10 of the SLC9A6 gene (NM_001042537.2) was identified.Our current genetic study enrolled two consanguineous Pakistani families with Pashtun ethnicity segregating intellectual disability. Genetic analysis in family-A revealed a novel homozygous missense variant  in the exon-11 of SLC13A3 gene and found in the kidney, brain, liver, placenta, and eye [SLC13A3 are known to reduce the transport capacity for a-ketoglutarate, succinate, and NAA, suggesting a loss-of-function mechanism [NaDC3 is a plasma membrane cotransporter encoded by the  and eye , 61. It  and eye . Abnormaechanism . NaDC3 iechanism .SLC9A6 mutations mirror developmental and progressive pathophysiology [SLC9A6 knockout mouse models revealed abnormalities in endosomal-lysosomal function and cholesterol accumulation in specific neuronal populations, similar to those in primary lysosomal storage diseases [SLC9A6/ mutant female mice and SLC9A6−/0 mutant male mice [SLC9A6 mutations [SLC9A6 mutations in CS patients [Clinical manifestations attributable to ysiology . SLC9A6 diseases . In addiale mice . Failureutations . Putativpatients .Through state-of-the-art in silico studies, we explored the impact of these mutations, G448R in SLC9A6 and P493L in SLC13A3, on protein stability and function and their potential link to the development of intellectual disability. Protein flexibility plays a crucial role in determining the functional properties of a protein, as it is often associated with conformational changes and dynamic interactions required for proper function, ligand binding, and protein-protein interactions , 65. AltThese observed stability and molecular flexibility changes could be linked to either gain or loss of function in the proteins. In intellectual disability, both gain and loss of function can potentially contribute to the disease’s development by disrupting normal cellular processes or signaling pathways . TherefoBelow is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3