Structural studies showed that TAF8 forms a histone fold pair with TAF10 in lobe B and participates in connecting lobe B to lobe C. To better understand the role of TAF8 in TFIID, we have investigated the requirement of the different regions of TAF8 for the in vitro assembly of lobe B and C and the importance of certain TAF8 regions for mouse embryonic stem cell (ESC) viability. We have identified a region of TAF8 distinct from the histone fold domain important for assembling with the 5TAF core complex in lobe B. We also delineated four more regions of TAF8 each individually required for interacting with TAF2 in lobe C. Moreover, CRISPR/Cas9-mediated gene editing indicated that the 5TAF core-interacting TAF8 domain and the proline-rich domain of TAF8 that interacts with TAF2 are both required for mouse embryonic stem cell survival. Thus, our study defines distinct TAF8 regions involved in connecting TFIID lobe B to lobe C that appear crucial for TFIID function and consequent ESC survival.The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID is thought to nucleate RNA polymerase II (Pol II) preinitiation complex formation on all protein coding gene promoters and thus, be crucial for Pol II transcription. TFIID is composed of three lobes, named A, B, and C. A 5TAF core complex can be assembled  TAFs formed . In addis formed . Early es formed , 17. Recin TFIID , 14, 18.in TFIID A  results in early embryonic lethality around E4.0 (Taf8 in mouse embryonic stem cells (ESCs)   or Taf10inocytes , 25 or dic liver  or ablatymocytes  compromiTAF8 TAF8c.781–1G>A splice site mutation in a human patient causes intellectual disability , the interaction partner of TAF8, which are also associated with intellectual disability syndrome . In all of the analyzed endogenous hTFIID complexes, TAF8 cross-linked extensively to TAF2, TAF6, TAF9/9b and less frequently to TAF1, TAF3, TAF5, and TAF12 with cross-linking hotspots in TAF8 at K:20, K:112, K:132, K:178, and K:205 . The K:20 hotspot of TAF8 cross-links to the TAF9/TAF6 HFD, to the TAF6 middle (TAF6M) and TAF6C domains, containing five HEAT repeats (A and B); however, K20 is absent from the published human TFIID structure , suggesting that either the C-terminal end of TAF8 does not produce peptides that are amenable to MS analysis, it is buried in a non-cross-linkable structure or positioned away from the TFIID surface. Note that the visible path of hTAF8 in the cryo-EM structure of the human TFIID is from amino acids 28 to 229 . Nevertheless, these cross-linking results in agreement with cryo-EM structures further indicate that TAF8 plays a triple role in TFIID: (i) participates in the assembly of the six HFD-containing 7TAF complex in lobe B via its N-terminus and HFD regions; (ii) connects lobes B and C by interacting with the HEAT repeat regions of two TAF6 subunits, the TAF1 region between amino acid (aa) 378 and 427, and TAF2 via its PRD; and (iii) interacts with TAF2 in lobe C via its C-terminal part.To gain more insights into the structure/function relation of human TAF8, first we carried out a multiple sequence alignment of TAF8 proteins from several eukaryotic species to better understand the potential domain conservation of TAF8 in addition to its HFD . FollowiFigs. 1 repeats , and to B: 6MZM, ), suggesB: 6MZM, ) encompaA). To evaluate the impact of these deletions on the assembly of recombinant 3TAF (TAF10-TAF8-TAF2), 7TAF (TAF5-TAF6-9/9b-TAF4-12-TAF8-TAF10), and 8TAF (7TAF complex+TAF2) complexes, we used the Baculovirus expression system.To further delineate the regions of TAF8 that are important for lobe B and/or lobe C assembly/interactions in TFIID, based on the above determined domain organization  and S1 aFig. 2B–D), to test whether TAF8 deletion mutants would affect interaction(s) of the TAF8-TAF10 HF pair with TAF2. Our anti-TAF10 coimmunoprecipitation (co-IP) experiments indicated that deletion of PRD (aa 141–184), T2R1 (aa 185–224), T2R2 (aa 225–271), or T2R3 (aa 272-Stop) of TAF8 all abrogated TAF8-TAF10 interactions with TAF2 (C and D). Deletions of N-terminal (aa 1–23) and ID (aa 117–140) regions of TAF8 did not influence the formation of the 3TAF (TAF10-TAF8-TAF2) complex (C and D). In our negative control experiments, when TAF10 was absent from the coinfections, TAF8 or TAF2 did not copurify by the anti-TAF10 IP, or when TAF8 was absent from the coinfections, TAF10 did not interact with TAF2 .First, we coexpressed TAF10-TAF2 with wild-type (WT) TAF8, or with the six deletion mutants, and carried out TAF10 immunoprecipitations (IPs) , B–D, toith TAF2 , C and D complex , C and DA) followed by anti-TAF10 IPs. When analyzing the 7TAF complex formation, we found that only the deletion of the ID region of TAF8 (Δ117–140) severely reduced the interactions with subunits of the 5TAF core complex, while the TAF8-TAF10 HF pair could still form (A and B and S2A). All other tested TAF8 deletion mutants, including the delta N-terminus mutant deleting the K:20 cross-linking hotspot, which cross-linked extensively to subunits of the 5TAF complex (B), formed the 7TAF complex (A and B). As described previously (A and B).Next, subunits of the 7TAF complex were coexpressed with either WT TAF8 or with one of the six deletion mutants A followe complex B, formed complex , A and Bof TAF8 Δ7–140 seveviously , A and BS. cerevisiae Taf8 sequences from amino acids 156 to 179. TAF10 co-IP results showed that the amino acid substitution in this region could not functionally replace the human sequences, as the 7TAF complex did not form efficiently under these conditions (C and D). The results show that the amino acid sequence of the ID domain is required for efficient formation of the 7TAF complex and that the ID is not functionally conserved between S. cerevisiae and humans.To test whether the amino acid sequence of the ID is required for formation of 7TAF complex, we substituted the human TAF8 117–140 amino acid sequences with the nditions , C and DA and S3B) and anti-TAF10 IPs carried out, the results recapitulated the observations made individually with either the 3TAF complex or with the 7TAF complex (B–D and A and B), and deletion of PRD (Δ141–184), T2R1 (Δ185–224), T2R2 (Δ225–271), or T2R3 (Δ272-Stop) of TAF8 all severely abrogated the TAF8–10 HF pair interactions with TAF2, but did not influence the interactions with the 5TAF core complex  (A and B).When subunits of the 8TAF complex were coexpressed with either WT TAF8 or with one of the six deletion mutants A and S3B B–D and , A and B, 4–12,) , A and Bin vitro TFIID subassembly results show that (i) deletion of the nonconserved N-terminal region of TAF8, which cross-links to many core TAF subunits in TFIID (B), is not required for formation of the 7TAF core complex, or to interact with TAF2, (ii) the ID region of TAF8 (aa 117–140) is required to interact with the 5TAF core complex, and it cannot be replaced by the ID region from S. cerevisiae Taf8, and (iii) PRD (aa 141–184), T2R1 (aa 185–224), T2R2 (aa 225–271), or T2R3 (aa 272-Stop) of TAF8 are all required for stable interactions between TAF8-TAF10 and TAF2.Thus, together our recombinant in TFIID B, is notin vitro identified regions of TAF8 play a role in TFIID assembly/function in a cellular context, we created mouse ESCs expressing Flag-WT TAF8, Flag-TAF8Δ117–140 (ID), or Flag-TAF8Δ141–184 (PRD) mutants. To generate a Dox-inducible expression system, first we integrated an expression cassette encoding the reverse tetracycline-controlled trans-activator . Barely any Flag-tagged protein could be detected in cells grown in the absence of Dox, whereas after Dox treatment Flag-TAF8 WT, Flag-TAF8Δ117–140, or Flag-TAF8Δ141–184 mutant proteins were clearly detectable in the corresponding ESC populations (A–C). From these ESC populations individual clonal ESC lines, originating from a single cell, were isolated, amplified in the presence of Dox, and tested for the expression of the Flag tagged TAF8 variants versus endogenous TAF8 (D–F). By selecting the positive ESC clones for further experiments, clones were chosen in which the expression level of each exogenously expressed Flag-TAF8 variants was close to endogenous TAF8 (D–F). When we compared the expression levels of the exogenously expressed Flag-WT-TAF8, Flag-TAF8Δ117–140, or Flag-TAF8Δ141–184 proteins in the selected ESC clones, the expression levels of the Flag tagged TAF8 variants (full-length and deletions) were comparable among themselves and also comparable to endogenous TAF8 (A).To test whether the r  in E14 r . This observation is in agreement with our earlier data, showing that mass spectrometry analysis of an anti-TAF10 IP from WT ESC whole cell extracts does not identify all holo-TFIID subunits, but only a “7TAF-type” complex (B). Surprisingly, under the same IP conditions the Flag-TAF8Δ141–184 (PRD) did not interact with any endogenous TFIID subunits (B). Importantly, none of the exogenously expressed Flag-tagged TAF8 proteins (full-length or mutants) impaired endogenous TFIID assembly when nuclear extracts from these cell lines were analyzed by an anti-TAF7 IP (C). Thus, these results suggest that the ID or the PRD regions of TAF8 are required for efficient assembly of 7TAF-type complexes (or TFIID subcomplexes) in mESCs.Next, we set out to analyze whether Flag tagged TAF8 variants (full length and deletions) would incorporate in TFIID, TFIID building blocks and/or would influence endogenous TFIID assembly in mESCs. To this end, we prepared whole cell or nuclear extracts from Dox-treated R:Flag-TAF8 WT, R:Flag-TAF8Δ117–140, or R:Flag-TAF8Δ141–184 expressing cell lines, carried out an anti-Flag (on whole cell extracts) or anti-TAF7 IPs (on nuclear extracts), and subjected the IP-ed complexes to mass spectrometric (MS) analyses. Analyses of the MS data from the Flag IP-ed mESC complexes showed that exogenously expressed WT Flag-TAF8 incorporated in a 7TAF-like complex, indicating that the exogenously expressed TAF8 can form a TFIID assembly intermediate, and that this intermediate resembles the 7TAF complex, containing TAF6–9, TAF4b–12, TAF5, as well as TAF8–10 B. This o complex . In cont complex B. Surprisubunits B. Import-TAF7 IP C. Thus, in vivo TFIID assembly and consequent mESC survival, we set out to inactivate the endogenous Taf8 gene using CRISPR/Cas9 genome editing in the ESC lines expressing R:Flag-TAF8 WT, R:Flag-TAF8Δ117–140 (ID), or R:Flag-TAF8Δ141–184 (PRD) (A). Using this strategy in the presence of Dox in the R:Flag-TAF8 WT mESC expressing line, we obtained several viable homozygous Taf8 deletion ESC lines (B), indicating that the CRISPR/Cas9 worked efficiently. By using the same gene editing strategy in the presence of Dox in R:Flag-TAF8Δ117–140 or R:Flag-TAF8Δ141–184 expressing cell lines, we obtained heterozygous clones in the same proportion as for the Flag-TAF8 WT expressing lines, indicating that the CRISPR/Cas9 worked with comparable efficiency at the Taf8 genomic locus in these heterozygous mESC lines, as in the R:Flag-TAF8 WT mESC expressing line (B). In contrast, we could not isolate any homozygous knockout clones when inactivating the Taf8 locus in the R:Flag-TAF8Δ117–140 or in the R:Flag-TAF8Δ141–184 mESC lines, suggesting that the deleted ID or PRD regions of TAF8 have essential roles in endogenous TFIID assembly and/or function, and consequently for mESC survival, and that these functions cannot be compensated by other TAFs or TAF assemblies.To test whether the above identified ID or PRD regions of TAF8 are required for 84 (PRD) A. Using SC lines B, indicaing line B. In conCharacterization of the structure–function relationship of multisubunit transcription complexes is crucial to understand gene regulation. Whereas three-dimensional models of fully assembled multiprotein complexes derived mainly from single-particle cryo-electron microscopy provide a wealth of information on the architecture of such complexes, the requirement of the precise subunit–subunit and domain–domain interactions to maintain the architecture and function of these gene regulatory complexes, such as TFIID, remains largely elusive.A). Out of these seven regions, the HFD of TAF8 has already been characterized (in vitro assembly of TAF8-containing TFIID building blocks. We identified three types of regions: (i) the N-terminus of TAF8 that did not disrupt the tested interactions with either the 5TAFcore complex or with TAF2, (ii) the ID region that is required for interactions with the 5TAF core complex, but does not influence the TAF2 interactions, and (iii) four successive TAF2 regions, which are all required for normal TAF8-TAF2 interactions, but do not influence the interactions with the 5TAF core complex (C). Our TAF8-TAF2 interaction results agree with previous surface plasmon resonance (SPR) experiments using immobilized full-length TAF2 as ligand and maltose-binding protein (MBP) fusions of TAF8 fragments (C). These interactions could happen through several TAF8-TAF2 contact surfaces, which would be all synergistically required for connecting lobes B and C. In the cryo-EM structures of TFIID, the structured TAF8 path stops at amino acid 229 (A)  would also cause cell death.Based on several functional and structural studies , 19, 32,Fig. 2cterized ; thus we complex C. Our TAragments . Thus, oragments , suggestragments . The facragments , 14. Furragments C. These acid 229 A (14), l 229 (A) . In addi 229 (A) , 28. It ethality , 22, 23.adiation . Also, mcription . Thus, iC). TAF8 plays a critical role in the genesis of TFIID and mutations in TAF8 can lead to human diseases such as ID. As such, defining the role of TAF8 regions in TFIID assembly and function provides key information about how TFIID assembly occurs and how mutations in a particular domain can lead to human diseases. Altogether, we show the significance of precise interactions of TAF8 with other TAFs, establishing TAF8 as a functional bridge between lobes B and C in TFIID (C). Moreover, our experiments indicate that these interactions in TFIID are required for mESCs to function properly.Here we have identified regions of TAF8 that are required for (1) anchoring TAF8-TAF10 to the core 5TAF complex and (2) for connecting the 7TAF complex in Lobe B to Lobe C C. TAF8 pin TFIID C. MoreovA). The deleted Flag-TAF8 cassettes were inserted into the pVL1393 Baculovirus expression vector. The constructs were verified by sequencing. All the other Baculovirus expression vectors have been described previously (The Baculovirus expression vector for FLAG-tagged human (h) TAF8 WT was PCR amplified from the pPBAC MultiBac vector, expressing TAF8 and TAF10 together, described in A. The deeviously , 33.Mouse (m) WT Flag-TAF8 cDNA and its deletions, corresponding to the hTAF8 deletions , were cloned into the pUHD10-3 vector  in whichTaf8 guide (g) RNAs and coexpressing high-fidelity Cas9  and rabbit polyclonal (pAb) antibodies raised against the following proteins have been described previously or were purchased commercially: anti-TBP (mAb 3G3)  and mAb, anti-TA2. For cell collection and amplification, ESCs were trypsinized for 2 to 3 min with trypsin-EDTA  and the digestion was stopped by the addition of prewarmed 15% FCS. Where indicated doxycycline (Dox) was added to the medium at a final concentration of 1 μg/ml (Sigma Cat#D9891).Mouse E14 ESCs were cultured on plates coated with 0.1% gelatin solution in 1× PBS  using DMEM (4.5 g/l glucose) with 2 mM Glutamax-I medium supplemented with 15% fetal calf serum (FCS) , 0.1% β-mercaptoethanol , 40 μg/ml gentamicin , 0.1 mM nonessential amino acids . 1.500 U/ml leukemia inhibitory factor (LIF) (home-made), 3 μM CHIR99021  together with 1 μM PD0325901  were added freshly to the medium. Cells were grown at 37 °C with 5% COR RNA mini isolation kit , and RT-qPCR was carried out using HA273 and HA274 primers for detecting rtTA expression, and HA817 and HA818 primers for Rplp0 housekeeping gene amplification , together with 1/10 of pKJ1B plasmid  as described previously , 44, usification , which wfication .Taf8 transgenes for 3 days with 1 μg/ml doxycycline , positive ESC clones were selected and analyzed by western blot assays using an anti-Flag mouse monoclonal antibody or purified anti-TAF8 rabbit polyclonal antibody (pAb3478).Next the rtTA expressing ESC line was transfected using Lipofectamine2000 , with pUHD-Flag-mTAF8, pUHD-Flag-mTAF8Δ117–140, or pUHD-Flag-mTAF8 Δ141–184 plasmids (each Hind III linearized), in which the expression of full-length mTAF8 and the two mTAF8 deletion mutants was under the control of tet-operator, together with 1/10 of pGK Hygro plasmid . Hygromycin (Sigma Cat#HO654) selection (250 μg/ml) was started 2 days after transfection . Single 6 cells on a 10 cm Petri dish) were transfected with the pUES-3 plasmid (24 μg), expressing the two Taf8 gRNAs  following manufacturer’s instructions. Then the cDNA samples were amplified using LightCycler 480 SYBR Green 2× PCR Master Mix I  and 0.6 μM of forward and reverse primers with a LightCycler 480 (Roche). The primer pairs used for different RT-qPCR reactions are listed in g 4 °C for 2 min, and washed once with 1 ml 1× PBS. Pellets were resuspended in one packed cell volume (PCV) extraction buffer , incubated 5 to 10 min on ice, and mixed with three PCV of “IP0 buffer” . After further 10 min incubation on ice, tubes were centrifuged at 14,000g at 4 °C for 10 min. The supernatant fractions, called whole cell extracts (WCEs), were stored at −80 °C.The required number of cells were trypsinized, transferred to 1.5 ml Eppendorf tubes, centrifuged at 600Sf9 insect cell culture. Infected insect cells were harvested 48 h post cell infection by centrifugation and stored at −80 °C until further use. Pellets of Baculovirus-infected Sf9 insect cells, coexpressing the different TAFs and distinct TAF8 proteins . Starting input protein extracts were either ESC nuclear extracts (0.5–2 mg), ESC WCEs (0.5–2 mg), or Baculovirus-infected Sf9 cell extracts (2–5 mg). If needed, protein extracts were diluted with “0” buffer  to reach a final 100 mM KCl concentration in the extracts. Protein inputs were then precleared by the addition of 1/10 volume of 100% protein A or G beads for 1 h at 4 °C with overhead agitation. During this time beads were coupled to the different antibodies (as indicated in the figure legends). Approximately, 1 mg of indicated antibody per ml of protein A or G bead was bound. Beads were incubated with the antibodies for 1 h at room temperature with agitation, unbound antibody was removed by washing the beads twice with IP500 buffer (“0” buffer containing 500 mM KCl) and twice with IP100 buffer before addition of the precleared protein extracts, and further incubated overnight at 4 °C with overhead agitation. The following day the beads were collected, and subjected to two rounds of washing for 10 min each with ten volumes of IP500 buffer, followed by 2× IP100 buffer washes. Proteins IP-ed with an anti-TBP mAb were eluted by adding one bead volume of 2 mg/ml competing epitope peptide for 4 h, repeated again for 2 h  or Protein-A  beads were washed twice with 1× PBS and twice with IP100 buffer  were cross-linked by addition of BS3  to 2 mM for 2 h at 25 °C. The reaction was quenched by addition of 10 μl of 1 M ammonium bicarbonate. Then 20 μl SP3 beads (20 μg/μl) were added, as described , followehttps://www.dropbox.com/sh/o7z1h12sf3nu89f/AAD5tR_iEXaf8IUDcYZjSj3ja?dl=0. For Nexus searches, the same databases were used with the following parameter settings: (a) up to three miscleavages; (b) static modification on Cysteines (+57.0215 Da); (c) differential oxidation modification on Methionines (+15.9949 Da); (d) differential modification on the peptide N-terminal Glutamic acid residues (−18.0106 Da) or N-terminal Glutamine residues (−17.0265 Da); (e) differential mono-BS3 modification on Lysine residue (+156.0806 Da). A 5% of FDR cutoff was used for both pLink and Nexus. After performing the pLink and Nexus analyses, the search results were combined and each spectrum was manually evaluated for the quality of the match to each peptide using the COMET/Lorikeet Spectrum Viewer (TPP). Cross-linked peptides are considered confidently identified if at least four consecutive b or y ions for each peptide are observed and the majority of the observed ions are accounted for. Search results that did not meet these criteria were removed. Intralinks involving a cross-link between identical residues were only kept if the spectral evidence strongly supported the identification; that is, the major fragment ions correspond to the intralinked peptide sequence and no/few other fragment ions were observed. All the cross-linked spectra and cross-linking maps can be viewed at: https://www.yeastrc.org/proxl_public/viewProject.do?project_id=153.The RAW files were converted to mzXML files by Rawconverter . For crog for 30 min at 4 °C. Pellets were washed twice with 1 ml cold acetone and centrifuged at 14,000g for 10 min at 4 °C. Washed pellet were then urea-denatured with 8 M urea  in Tris-HCl 0.1 mM, reduced with 5 mM TCEP for 30 min, and then alkylated with 10 mM iodoacetamide  for 30 min in the dark. Both reduction and alkylation were performed at room temperature and under agitation (850 rpm). Double digestion was performed with endoproteinase Lys-C  at a ratio 1/100 (enzyme/proteins) in 8 M urea for 4 h, followed by an overnight modified trypsin digestion  at a ratio 1/100 (enzyme/proteins) in 2 M urea for 12 h.Protein mixtures were precipitated with TCA  overnight at 4 °C. Samples were then centrifuged at 14,000via a nano-electrospray ionization source (Thermo Fisher Scientific). Peptide mixtures were loaded on a C18 Acclaim PepMap100 trap-column  for 3.5 min at 5 μl/min with 2% ACN , 0.1% formic acid  in water and then separated on a C18 Accucore nano-column  with a 90 min linear gradient from 5% to 35% buffer B , then a 20 min linear gradient from 35% to 80% buffer B, followed with 5 min at 99% B and 5 min of regeneration at 5% B. The total duration was set to 120 min at a flow rate of 200 nl/min. The oven temperature was kept constant at 38 °C. The mass spectrometer was operated in positive ionization mode, in data-dependent mode with survey scans from m/z 350 to 1500 acquired in the Orbitrap at a resolution of 120,000 at m/z 400. The 20 most intense peaks (TOP20) from survey scans were selected for further fragmentation in the Linear Ion Trap with an isolation window of 2.0 Da and were fragmented by CID with normalized collision energy of 35%. Unassigned and single charged states were rejected. The Ion Target Value for the survey scans (in the Orbitrap) and the MS2 mode (in the Linear Ion Trap) were set to 1E6 and 5E3, respectively, and the maximum injection time was set to 100 ms for both scan modes. Dynamic exclusion was used. Exclusion duration was set to 20 s, repeat count was set to 1, and exclusion mass width was ±10 ppm. TFIID subunit identification details are shown in Samples were analyzed using an Ultimate 3000 nano-RSLC (Thermo Fisher Scientific) coupled in line with a LTQ-Orbitrap ELITE mass spectrometer Mus musculus database . Precursor and fragment mass tolerances were set at 7 ppm and 0.6 Da, respectively. Trypsin [KR/P specificity] was set as enzyme and up to two missed cleavages were allowed. Oxidation was set as variable modification, and carbamidomethylation as fixed modification. Peptides were filtered with a false discovery rate  at 1%, rank 1, and proteins were identified with 5% FDR and one unique peptide. For the Label-Free Quantification, the protein abundancies were calculated from the average of the peptide abundancies using the TOP N , and only the unique peptide was used for the quantification. Data were analyzed by calculation of NSAF (Proteins were identified by database searching using SequestHT (Thermo Fisher Scientific) with Proteome Discoverer 2.4 software  on  of NSAF , 49.via the PRIDE (The proteomics data have been deposited to the ProteomeXchange Consortium he PRIDE  partner This article contains The authors declare that they have no conflicts of interest with the contents of this article.


These specific modulators are powerful probes for investigating the structural and functional features of Nav channels. Here, we report the isolation and characterization of δ-theraphotoxin-Gr4b (Gr4b), a novel peptide toxin from the venom of the spider Grammostola rosea. Gr4b contains 37-amino acid residues with six cysteines forming three disulfide bonds. Patch-clamp analysis confirmed that Gr4b markedly slows the fast inactivation of Nav1.9 and inhibits the currents of Nav1.4 and Nav1.7, but does not affect Nav1.8. It was also found that Gr4b significantly shifts the steady-state activation and inactivation curves of Nav1.9 to the depolarization direction and increases the window current, which is consistent with the change in the ramp current. Furthermore, analysis of Nav1.9/Nav1.8 chimeric channels revealed that Gr4b preferentially binds to the voltage-sensor of domain III (DIII VSD) and has additional interactions with the DIV VSD. The site-directed mutagenesis analysis indicated that N1139 and L1143 in DIII S3-S4 linker participate in toxin binding. In sum, this study reports a novel spider peptide toxin that may slow the fast inactivation of Nav1.9 by binding to the new neurotoxin receptor site-DIII VSD. Taken together, these findings provide insight into the functional role of the Nav channel DIII VSD in fast inactivation and activation.Venomous animals have evolved to produce peptide toxins that modulate the activity of voltage-gated sodium (Na Notably, these subtypes have different tissue-specific localization and functions. The Nav1.1–Nav1.3 subtypes are expressed primarily in the central nervous system (CNS); the Nav1.6 subtypes are expressed in the central and peripheral nervo us system; the Nav1.7–Nav1.9 subtypes are mainly expressed in the peripheral nervous system (PNS); Nav1.4 is present in skeletal muscle; and Nav1.5 is mainly expressed in cardiac muscle  or altering gating kinetics (gating modifier toxins). At least three distinct binding sites of neuropeptide toxins have been identified  have evolved the ability to produce peptide toxins with high affinity to target Naredators . These pentified . Peptideentified . Site 3 entified . Site 4 entified . It is kGrammostola rosea, which is a gating modifier and significantly inhibits fast inactivation of the Nav1.9 channel. Like the previously described Nav1.9 peptide toxin HpTx1  in double-distilled water to a final concentration of 10 mg/ml immediately before being subjected to reversed-phase high-performance liquid chromatography (RP-HPLC) purification. First, reverse-phase HPLC purification was performed using a water HPLC system  with an Ultimate® XB-C18 column  with a flow rate of 3 ml/min and a gradient of 10–55% A for more than 45 min . The absorbance was measured at 215 nm. The fractions were collected, lyophilized, and then stored at –20°C until the next subdivision. Next, the target fraction containing Gr4b was subjected to a second round of RP-HPLC  using an analytic XB-C18 column,  with a linear increasing acetonitrile gradient (acetonitrile at an increasing rate of 0.5% per minute and a flow rate of 1 ml/min) to obtain the purified Gr4b. The molecular weight of the peptide was confirmed by matrix assisted laser desorption/ionization-time of-flight mass-spectrometry (MALDI-TOF-TOF MS) spectrometry . The N-terminal amino acid sequence of the peptide was determined by automated Edman degradation in a PPSQ-53A protein sequencer .As described in previous studies, the venom of the v1.4, human Nav1.7 and rat Nav1.8 cDNA clones were kindly gift from Dr. Theodore Cummins  and were subcloned into the pCMV or pCDNA3.1-blank vectors. Human Nav1.9 was subcloned into the pEGFP-N1 vector. The C-terminal of hNav1.9 was linked to GFP to construct a fusion protein channel (hNav1.9-eGFP), which was as described in our previous studies (Rat Na studies . Mutatio2 incubator in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin, 100 U/ml penicillin, and 2 mM L-glutamine. The cells were trypsinized, diluted with 1 ml of culture medium, and seeded at a 1:5 ratio in 35 mm Petri dishes for culture. When grown to 80–90% confluence, the ND7/23 cells were transfected with hNav1.9-GFP or hNav1.9-GFP mutants using the X-tremeGENE HP DNA Transfection Reagent  according to the manufacturer’s instructions. ND7/23 cells were used for hNav1.9-GFP chimeric channel expression and the conditions were as previously described (v1.9 (v channel plasmid (4 µg) plus 0.5 µg pEGFP-N1 (except for hNav1.9) plasmid were co-transfected into HEK293T or ND7/23 (rNav1.8 and hNav1.9 only) cells. The cells were seeded onto several 3.5 cm dishes at a 1:10 ratio at 4–6 h after transfection. Cells with green fluorescent protein (GFP) were selected for whole-cell patch-clamp analysis at 24–36 h post-transfection.ND7/23 and HEK293T cells were maintained at 37°C in a humidified 5% COescribed , the beted : 150 NaCl, 2 KCl, 1.5 CaCl2, 1 MgCl2 and 10 HEPES ; the pipette solution contained (in mM): 35 NaCl, 105 CsF, 10 EGTA, 10 HEPES . The osmotic pressure of the intracellular fluids and extracellular fluids is adjusted to 300–320 mOsm with sucrose. Before use, Gr4b was dissolved in ddH2O to make a 250 μM stock solution at −20°C. TTX was dissolved in DMSO to make a 1 mM stock solution. TTX was added to bath solution to a final concentration of 1 µM when used to inhibit TTX-sensitive (TTX-S) Nav channels. Unless otherwise indicated, all chemicals were products of Sigma-Aldrich . For electrophysiology experiments, the stock solution of Gr4b was diluted with fresh bath solution to a concentration of tenfold of the interested concentration, 30 µl of the concentrated peptide was diluted into the recording chamber (containing 270 µl bath solution) far from the recording pipet (the recording cell), and was mixed by repeatedly pipetting to achieve the specified final concentration.Whole-cell current recordings were performed using an EPC-10 USB patch-clamp amplifier operated by Patch Master software . The recording pipettes were fabricated from borosilicate glass capillaries using a two-step vertical microelectrode PC-10 puller , and the pipette resistance was controlled to be 2.0–3.0 MΩ. Voltage-clamp recordings were acquired with Patch Master software 2 × 73 (HEKA Elektronik) 4 min after establishing whole-cell configuration, and the currents elicited were sampled at 20 kHz and filtered at 5 kHz. After breaking in, the serial resistance was controlled below 5 MΩ, the voltage error was minimized by using 80% serial resistance compensation, and the compensation speed value was 10 µs. For recording Nap < 0.05.Data were analyzed using the PatchMaster v2x73 , Igor Pro 6 , Office Excel 2010 , and GraphPad Prism 7 . All data points are shown as mean ± standard error of the mean (SEM), and n was presented as the number of separate experimental cells. The Boltzmann function was used to fit steady-state activation and deactivation curves. Concentration-response curves were fitted using the Hill equation. One-way ANOVA was used to assess the difference between multiple groups. Significant levels were set at Grammostola rosea contains several classes of peptide toxins that target ion channels , which comprise 42–44 residues and contained six residues and form a conserved cysteine pattern-inhibitor cystine knot (ICK) motif  motif   motif . NaSpTx channels . Interestivation .v1.9 current was evoked to −40 mV by a 100-ms depolarization potential from a holding potential of −120 mV in ND7/23 cells. A concentration of 250 nM Gr4b slowed the fast inactivation of Nav1.9, leading to a large sustained current (50) of Gr4b was determined to be 25 ± 1.0 nM  value of 27 ± 3.0 nM. It had substantially weaker effects on Nav1.4, with an IC50 value of 411 ± 184 nM  to analyze the effects of Gr4b on the voltage dependence of activation and inactivation properties of the Nav1.9 channel. As shown in v1.9 current (p < 0.0001) (p < 0.0001), indicating that toxin binding might affect the cooperativity of the four voltage sensors of the Nav1.9 channel. As shown in p < 0.0001), whereas the slope of the curve was not changed . We also found that Gr4b introduced a non-inactivated component in the steady-state inactivation curve around the test potential. The β1 subunit is known to modulate the kinetics of fast inactivation  (p < 0.001), potentially increasing Na+ influx (v1.9 channel.A major effect of gating modifier toxins on Na current . However current . In addi 0.0001) . Furthertivation . Because nM Gr4b . Indeed, < 0.05) . Consist+ influx . Based ov1.9 channel. As shown in v1.9 was characterized by a slow onset of action, with a τon value of 42.8 ± 3.5 s (n = 3). Inhibition of fast inactivation by Gr4b slowed the reversible recovery upon perfusion bath solution washing, with a recovery of approximately 54.39% of the control current within 2.5 min  being the hotspot for spider peptide toxins to inhibit fast inactivation  responsible for the toxin’s ability to reduce fast inactivation of Nav1.9. Firstly, we made the chimera Nav1.9/1.8 DIV VSD, in which the DIV VSD (DIV S1–S4) of Nav1.9 was replaced with the corresponding domain of Nav1.8. As shown in v1.9/1.8 DIV VSD chimeric channel (p < 0.05) and the slope of the curve was also significantly changed   , that cov channels have been also shown to modulate channel inactivation (v1.9. Thus we made the two chimeras Nav1.9/1.8 DIII VSD and Nav1.9/1.8 DIII & DIV VSD. The results showed that replacing these regions of Nav1.9 abolished the effects of Gr4b on the channel. Even at high concentrations (2.5 μM), the chimeric channels were almost unaffected by Gr4b  enhanced sensitivity to Gr4b  the effect of Gr4b on the Nav1.9 channel is voltage-dependent and the relationships of the current-voltage curve and voltage-dependent steady-state activation curve both shift in the direction of depolarization rization . Moreovetivation ; some muaralysis . Togethev channel might be a neurotoxin binding site. To date, at least six different neurotoxin receptor sites have been identified on Nav channels  in the DIII S3-S4 linker of Nav1.9 might be involved in the interaction with Gr4b (v1.9 at voltages of −60 to −40 mV (v1.9 exists (v1.9 DIII VSD affects fast inactivation of the channel as well as activation.The DIII VSD of the Nachannels ; howeverith Gr4b . Previouith Gr4b . In conto −40 mV . These e9 exists . The chiv1.9 channel, as well as a novel Nav channel neurotoxin binding to the site DIII VSD. The toxin binding to DIII VSD of Nav1.9 affects both activation and fast inactivation, thereby providing pharmacological insight into the role of the DIII VSD in Nav channel activation and fast inactivation.In summary, our study has revealed a novel spider peptide toxin that specifically interacts with the Na


Gastric cancer (GC) is the second leading cause of cancer-related deaths. Because it is hard to diagnose at early stage, the overall 5 years survival rate is lower than 25%. High migration is the main hallmark of malignant cells at advanced stage of GC. Thus, it is urgent to find biomarkers for early diagnosis and more effective therapy of GC.In this study, lentivirus-mediated silencing and overexpression lentiviruses targeting the ubiquitin-conjugating enzyme E2 D1 (UBE2D1), transwell, wound healing, and pulmonary metastasis mouse model were applied to analyze the function of UBE2D1 in vitro and in vivo. Real-time PCR and immunohistochemistry were used to elucidate the level of UBE2D1 in GC samples.Silencing of UBE2D1 inhibited cell migration and the levels of epithelial-mesenchymal transition makers (MMP2 and MMP9) in AGS and MKN45 cells. Silencing of UBE2D1 inhibited cell metastasis in mouse model. On the contrary, UBE2D1 overexpression increased cell migration and the levels of MMP2 and MMP9 in MGC-803 cells. Further, silencing of UBE2D1 decreased the ubiquitination level of mothers against decapentaplegic homolog 4 (SMAD4), and the increase of cell migration induced by UBE2D1 overexpression could be reversed by SMAD4.Silencing of UBE2D1 inhibited cell migration through transforming growth factor β (TGF-β)/SMAD4 signaling pathway in GC.The online version contains supplementary material available at 10.1186/s13027-021-00402-2. Helicobacter pylori and Epstein-Barr virus, and genetic factors including somatic mutations, mismatch repair gene deficiency, gene amplifications, and deletions are widely accept as the main causes of GC [Gastric cancer (GC), a common malignant tumor of the digestive system, is the second leading cause of cancer-related deaths, with an overall 5 years survival rate lower than 25% . The infes of GC . As mostes of GC . AlthougTransforming growth factor β (TGF-β) signaling pathway is of great importance in regulating a large number of biological processes, including cell proliferation, apoptosis, differentiation, migration, and the initiation and progression of cancer . MothersThe ubiquitin-conjugating enzyme E2 D1 (UBE2D1) is a member of UBE2D family which has been found to play a role in some important pathways of carcinogenesis , 10. UBEIn the current study, we revealed that silencing of UBE2D1 suppressed cell migration in vitro and in vivo. In addition, TGF-β/SMAD4 signaling pathway was involved in the function of UBE2D1 in GC.Human GC cell lines, including AGS, BGC-823, MGC-803, MKN45, SGC-7901, and human normal gastric epithelial cell line GES-1 were purchased from iCell Bioscience Inc. . AGS, BGC-823, and MGC-803 were cultured in DMEM medium, and MKN45, SGC-7901, and GES-1 were cultured in RPMI-1640 medium. All the mediums were purchased from Gibco  and 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin sulfate (Gibco) were added. 10 μM of MG132 , a proteasome inhibitor, or 100 μg/ml of cycloheximide (Selleckchem), a protein synthesis inhibitor were used to treat cells.−ΔΔCT method and normalized to β-actin. The primer sequences are presented below.TRIzol reagent  was used to extract cellular RNA and PrimerScript RT reagent Kit  was used to synthesize cDNA. SYBR Green kit was used to perform the PCR reactions in a 7500 Fast Real-time PCR System (Thermo Fisher Scientific). Relative gene expression levels were calculated using the 2The treated cells were lysed in RIPA buffer  on ice. After 12,000 rpm centrifugation at 4 °C, a BCA kit  was used to measure protein concentration. Proteins (30 μg) from different samples were separated by 10% SDS-PAGE and blotted onto PVDF membranes . After blocking in 5% non-fat milk, PVDF strips were incubated overnight with primary antibodies, followed by incubation with secondary antibodies. Primary antibodies were listed as following: UBE2D1 , SMAD4 , MMP2 , MMP9 , and β-actin . At last, the membranes were scanned using a Bio-Rad imaging system .All these lentiviruses were constructed by Genechem company , including three interfering  and one overexpression lentiviruses targeting UBE2D1, and one SMAD4 overexpression lentivirus. Briefly, shRNA oligos targeting UBE2D1 were annealed and cloned into AgeI- and EcoRI-digested pLKO.1 . The full-length of UBE2D1 and SMAD4 were separately cloned into pLVX-puro . The lentivirus was produced in 293 T cells along with packaging plasmids, psPAX2 and pMD2.G.4 cells per well (about 200 μl). Next, the culture medium containing 10% FBS was added to the lower chamber and cultured for 24 h. After removing the non-migratory cells, the chambers were stained with 1% crystal violet  for 15 min, followed by acquiring images (200 ×) using a microscope .The transwell chambers  were set into 24-well plates and incubated with culture medium overnight. Treated cells were harvested with FBS-free culture medium and seeded into the upper chamber at 2 × 105 treated cells were planted into each well of a 6-well plate and cultured to 80% confluence. Then, a 200 µL pipette tip was used to create wounds in the middle of each well. After washing with PBS, the cells were incubated with culture medium containing 1% FBS. Images (200 ×) of wounds were acquired at 0 and 24 h. Each experiment was performed in triplicate.A total of 1 × 106 cells were subcutaneously injected into the tail vein of nude mice. After 4 weeks of feeding, all these mice were euthanized. The image of lung profile was acquired, and the metastatic nodules were counted. Then, the lung samples were fixed in 4% paraformaldehyde (Beyotime) for the hematoxylin–eosin staining. All the animal protocols were approved by the Institutional Animal Ethics Care and Use Committee of The First People’s Hospital of Changzhou. This study was performed in line with the principles of the Declaration of Helsinki.A total number of 12 BALB/c nude mice  were purchased from Shanghai SLAC Laboratory Animal Co.,Ltd . Mice were randomly divided into two groups: AGS-shNC and AGS-shUBE2D1. The cells transduced with shNC or shUBE2D1 lentivirus were harvested and suspended with PBS, then 1 × 10http://www.broad.mit.edu/gsea). GSEA was used to generate an ordered list of genes according to their correlation to UBE2D1 expression. The phenotype marker was UBE2D1-high versus UBE2D1-low.GSEA is used to analyze gene expression data using biological knowledge . Lots ofAfter fixing in 4% paraformaldehyde, the lung samples were paraffin-embedded, sliced (4 µm), baked, and stained with Hexion and E staining kit (Bio-Rad Laboratories) according to the manufacturer’s protocol. Images were acquired by light microscopy .Treated cells were lysed as previously described . Whole c2O2 in methanol, blocked, and incubated with anti-UBE2D1 antibody at 4 °C overnight. After washing twice with PBS-T for 10 min each, the slice was incubated with second antibody 1 h and DAB buffer for detection. Images were acquired by light microscopy .Human gastric tissue microarray  was deparaffinized, rehydrated, antigen retrieved with 3% HP < 0.05 was considered significant.All data are reported as means ± standard deviation (SD). Student’s t-test and one-way ANOVA were used in data analysis. Survival curves were calculated using the Kaplan–Meier method and P = 0.029).To determine the significance of UBE2D1 in GC, we analyzed the expression level of UBE2D1 in TCGA-STAD and found that UBE2D1 was upregulated in primary tumor (n = 415) compared to the normal tissue (n = 14, Fig. To explore the function of UBE2D1, we firstly measured its expression in human normal gastric epithelial cell line GES-1 and GC cell lines AGS, BGC-823, MGC-803, MKN45, and SGC-7901. As shown in Fig. Based on the result of transwell and wound healing assays Fig. A, B, celTo further confirm the function of UBE2D1, overexpressing lentivirus targeting UBE2D1 was used to treat MGC-803 cells. As shown in Fig. GSEA, a method of interpreting gene expression data using biological knowledge , has beeSMAD4 is the central mediator of TGF-β signaling, thus we detected whether there was a direct interaction between UBE2D1 and SMAD4 using Co-Ip assay. As we expected, UBE2D1 could interacted with SMAD4 directly and vice versa Fig. B. Next, To further evaluate the function of UBE2D1 and SMAD4, SMAD4 overexpressing lentivirus was constructed to treat MGC-803 cells. As shown in Fig. GC, the second leading cause of cancer-related deaths, has a low overall 5 years survival rate and a high recurrence rate . Due to Previous studies reported that upregulated UBE2D1 is found in several cancers including non-small-cell lung cancer , osteosaThe TGF-β/SMAD4 signaling pathway has a crosstalk with many other pathways and is famous in a plenty area, including cancer, EMT, microRNA, DNA damage response and DNA damage repair . In cancIn conclusion, silencing of UBE2D1 decreased the ubiquitination and degradation of SMAD4, resulting the inhibition of cell migration in GC.Additional file 1. The protein levels of E-cadherin and N-cadherin were measure after transduction of shUBE2D1 lentiviruses in AGS and MKN45 cells.


Hyperuricemia has a strong association with diabetes mellitus. Hyperuricemia can lead to cardiovascular and renal complications in patients with diabetes. The goal of this study was to compare the effect of sodium-glucose cotransporter-2 (SGLT-2) inhibitors dapagliflozin and empagliflozin on serum uric acid (SUA) levels in patients with type 2 diabetes against traditional oral antihyperglycemic drugs (OADs).  In this double-blind randomized controlled trial, 70 patients with type 2 diabetes and elevated SUA levels were assigned to two treatment groups. Patients in group A received SGLT-2 inhibitors tablet dapagliflozin 5 mg to 10 mg and empagliflozin 10 mg to 25 mg. Group B patients received OADs such as glimepiride, metformin, sitagliptin, gliclazide, and glibenclamide as monotherapy or combination therapy. The changes in SUA level were primary end points while changes in body weight and body mass index (BMI) from baseline to end point were secondary end points. p = 0.001). Mean body weight was significantly reduced in the SGLT-2 group from 82 ± 10.4 to 78 ± 12.5 kg versus comparator group from 78 ± 13.2 to 79.2 ± 9.7 kg (p = 0.001). Similarly, the mean BMI of patients in the SGLT-2 group was significantly reduced from 25.7 ± 3.2 to 24.2 ± 3.2 kg/m2 versus comparator group from 27.5 ± 4.2 to 28 ± 3.6 kg/m2 (p = 0.002).  After four weeks of treatment, we noted a significant reduction of mean SUA levels in the SGLT-2 inhibitor group from 7.5 ± 2.5 to 6.3 ± 0.8 mg/dl versus comparator group from 7.1 ± 1.8 to 6.8 ± 2.2 mg/dl ( SGLT-2 inhibitors have a strong potential to decrease SUA levels in patients with type 2 diabetes. The prevalence of diabetes is growing worldwide and continues to represent a global health challenge. In 2015, the estimated prevalence of diabetes was 8.8%, but projections estimate this will rise to 10.4% by 2040  <7%), and blood pressure within the reference range (<140/90 mmHg). Patients with a history of smoking, alcohol use, and cardiovascular and renal disease were excluded from the study. Also, a complete history was taken to screen for drugs that can affect uric acid levels, such as thiazide diuretics, aspirin, niacin, statins, anticancer agents, immunosuppressants, and pyrazinamide. Patients taking any antigout drug were also excluded from the study. Patients were also asked to avoid all fructose-containing beverages and foods for the duration of the study.We conducted this four-week double-blind randomized controlled trial at a government clinic and five private clinics in the District of Rahim Yar Khan, South Punjab, Pakistan, from February to March 2020. Three hundred eighty overweight patients with type 2 diabetes on OAD agents were screened for elevated SUA levels. Of this group, 70 patients were enrolled in the study according to our inclusion and exclusion criteria. To be included in the study, patients must have a body mass index (BMI) < 28 kg/mn = 35) were treated with daily empagliflozin tablet 10 mg to 25 mg and dapagliflozin 5 mg to 10 mg, and patients in group B (n = 35) continue their routine OADs either alone or in combination as the standard treatment of diabetes. These OADs were glimepiride, metformin, sitagliptin, gliclazide, and glibenclamide. Dosages of the drugs were adjusted according to patient fasting blood glucose and random blood glucose level testing. Patients, investigators, and laboratory staff were blinded to the study plan throughout clinical trial.Patients were randomly assigned to one of two groups by simple random number generation via computer software. Patients in group A (The changes in SUA level were primary end points while changes in body weight and body mass index (BMI) from baseline to end point were secondary end points.All clinical and laboratory parameters were measured by following standard guidelines and methods. The study design was approved by the institutional review board of Sheikh Zayed Medical College/Hospital Rahim Yar Khan ( 26/IRB/SZMC/SZH). All study participants provided written informed consent.t-test was used to compare the changes between the test groups. A paired t-test was applied to compare each group's changes, while the Mann-Whitney U-test and t-test were applied to compare changes between groups from baseline to four weeks. A p value <0.05 was considered statistically significant.Values were expressed as mean ± standard deviation and percentages. Numeric data were analyzed using SPSS Statistics for Windows, Version 18.0. (Chicago: SPSS Inc.). A All patients completed the study with none lost to follow-up. In group A, 20 received empagliflozin while 15 patients received dapagliflozin. In group B, 35 patients received their routine OADs. The safety and tolerability profile of empagliflozin and dapagliflozin were quite good, and no adverse effects were recorded during the study.p = 0.001; The baseline demographic characteristics of study participants are presented in p = 0.001). Similarly, BMI was reduced significantly from 25.7 ± 3.2 kg/m2 to 24.2 ± 3.2 kg/m2 in the SGLT-2 inhibitor group while the control group's mean BMI at baseline was 27.5 ± 4.2 kg/m2 and 28 ± 3.6 kg/m2 by the end of the study (p = 0.002).Mean body weight was significantly reduced in the SGLT-2 group from 82 ± 10.4 kg to 78 ± 12.5 kg compared to the comparator group, which had a mean body weight of 78 ± 13.2 kg at baseline and 79.2 ± 9.7 kg at the end of the study period  on SUA in patients with type 2 diabetes. Both drugs significantly reduced SUA as compared to control with good safety and tolerability profile.SGLT-2 inhibitors are widely prescribed for patients with diabetes as monotherapy and combination therapy. Their mechanism of action is unique among conventional OADs. They have an insulin-independent effect and increase the excretion of glucose in the urine. These drugs also have beneficial effects on conventional risk factors of cardiovascular disease , 15.SGLT-2 inhibitors also lower SUA levels in various clinical studies , 13. TheOur results align with a meta-analysis that showed dapagliflozin significantly reduced SUA by 1.14 mg/dl at a dose of 10 mg daily. However, in our study, both dapagliflozin and empagliflozin reduced uric acid by 1.21 mg/dl at different doses. We did not analyze the dose-dependent effects of the SGLT-2 inhibitors. Moreover, in these studies, the status regarding drugs affecting SUA was lacking. Our study excluded all patients receiving drugs that would affect SUA levels , 18.A meta-analysis of 62 studies by Zhao et al.  concludeA meta-analysis of 31 studies on 13,650 diabetic patients demonstrated that SGLT-2 inhibitors significantly decreased SUA levels compared with placebo, canagliflozin 0.62 mg/dl, dapagliflozin 0.64 mg/dl, and empagliflozin 0.71 mg/dl . Our stuThe proposed mechanisms by which SGLT-2 inhibitors decrease SUA include the expression of glucose transporter 9 isoform 2 in the kidney tubules that causes the excretion of D-glucose and uric acid in urine . Other sOur study's main strength was the exclusion of risk factors that can affect SUA levels, such as obesity, hypertension, chronic kidney disease, and uncontrolled diabetes. We also excluded patients taking any drugs that can affect SUA levels. However, our study was limited in that we did not check urine for a complete examination for uric acid, and we did not compare empagliflozin and dapagliflozin for effects on SUA levels.SGLT-2 inhibitors have a strong potential to decrease SUA levels in patients with type 2 diabetes. SGLT-2 inhibitors can be administered in patients with diabetes with abnormal SUA levels. Hyperuricemia is one of the potential risk factors for cardiovascular and kidney diseases. Therefore, lowering SUA levels is crucial to prevent morbidity and mortality in patients with type 2 diabetes.Further studies with larger sample sizes and longer durations should be conducted to illuminate the uric acid-lowering effects of SGLT-2 inhibitors.


The post-synthesis transformation of HKUST-1 into Cu(BTC)·3H2O is further demonstrated. Single crystals of both structures are studied by multi-laser Raman and luminescence spectroscopy. It is found that both crystals exhibit photoluminescence in the range of 700–900 cm−1 within the optical gap of the bulk materials, which can be associated with crystallographic defects. This work gives impetus for the synthesis of large metal–organic crystals based on which optical properties can be studied in depth.Synthesis of crystalline materials is elemental in the field of coordination chemistry towards optical applications. In the present work, coordination between copper and benzene-1,3,5-tricarboxylic acid (BTC) is controlled by adjusting the pH scale of the reaction mixture at room temperature to synthesize two crystalline structures: metal–organic framework HKUST-1 and coordination polymer Cu(BTC)·3H Synthesis of crystalline materials is elemental in the field of coordination chemistry towards optical applications. Coordination polymers are solid-state structures consisting of repeating coordination units to form 1D extended chains, 2D sheets or 3D frameworks.5–8 Coordination polymers having a porous structure are known as metal–organic frameworks (MOFs).9–16 Synthesizing large and quality crystals of MOFs benefits in a wide range of applications such as separation, heterogeneous catalysis,17–19 gas-sorption,20,21 chromatography,22,23 optoelectronics, magnetism and sensing applications.24,25Organic coordination compounds consist of organic ligands (Lewis base) bound to a central metal ion (Lewis acid).26–28 The pH modulating technique was previously utilized to synthesize MOFs.29–34Controlling the process of crystallization by pH adjustment is a challenging, yet fascinating process in understanding the chemistry of materials.3(BTC)2(H2O)3  and Cu(BTC)·3H2O  acid is controlled by adjusting the pH of the reaction mixture to obtain crystals of two different structures: Cu3H2O see .2O is obtained as a by-product. This by-product remains in the pores of the framework and reduces the specific surface area thereby causing hinderance in gas absorption.36 Thus, room-temperature synthesis is beneficial. Our pH-controlled synthesis procedure will help the community to synthesize quality and large crystals of HKUST-1 at room temperature.HKUST-1 is typically synthesized using a hydrothermal/solvothermal method. The high-temperature synthesis yields larger crystals, but the problem is that a significant amount of Cuii) based coordination polymers are some of the well studied compounds in the field of natural science and medical research. Owing to the antibacterial activity, low toxicity and potential as biomaterials, these compounds are gaining interest among researchers.37 In the present work, large crystals of Cu(BTC)·3H2O are synthesized at room temperature under weak acid conditions i.e. pH greater than 2.1. Single crystal X-ray diffraction (SXRD) reveals that the structure is composed of 1D zig-zag chains interconnected by hydrogen bonding which makes the structure stable and generates a layered coordination network.38In turn, copper·3H2O are examined on their single crystals with multiple visible lasers. It is found that both crystals luminesce in the range 700–900 cm−1 with a red excitation laser, that can be attributed to crystallographic defects.Finally, luminescent properties of both the HKUST-1 framework and Cu(BTC)·3H22O can be synthesized in the pH range 2.2–3.2 as shown in All synthesis demonstrated in this work is done at room temperature. It is found that crystals of HKUST-1 can be synthesized by adjusting the pH of the reaction mixture in the pH range 0.7–1.6 as shown in 2.13·3H2O (1 mmol) and BTC (1 mmol) is dissolved in a mixture of three solvents  i.e. in a volume ratio (V.R.) of 1 : 1 : 1 (pH = 1.8–2.0). After 16–20 hours, crystals of HKUST-1 are formed at the bottom of the vial (pH = 2.1). A clear depiction of the morphology of the as-synthesized HKUST-1 crystal is shown in First, we present room temperature synthesis of HKUST-1  without additional pH adjustment. Copper nitrate trihydrate CuNO3) in the aforementioned 12 ml reaction mixture of V.R. = 1 : 1 : 1. The resulting pH scale is in the range 0.7–0.9. After 8–10 days, crystals of HKUST-1 can get as large as ∼1–1.2 mm, as shown in 3 and copper(ii) chloride (CuCl2) instead of CuNO3·3H2O. Their crystal size and morphology are shown in Fig. S3 in the ESI.†Much larger crystals can be obtained in more acidic conditions prepared by adding 0.1 ml of nitric acid ·3Hules see . The sta2.32O packing structure along the [100], [001] and [010] crystallographic axes are shown in 2O, see Section S2 in the ESI.†The crystallographic representations of the Cu(BTC)·3HP21/n and lattice parameters a = 6.7654, b = 18.8135, c = 8.5144; α = γ = 90°, β = 92.439°. The empirical formula of the structure is C9H10CuO9. This structure was previously reported.37,38,40–4238,42 BTC consists of three carboxyl groups attached to the benzene ring. Two of the three carboxyl groups of the ligand are coordinated to the metal ion via one oxygen to form the zig-zag Cu–BTC polymeric chain. The other oxygen atoms contribute to the hydrogen bonding (dotted line) between the Cu–BTC chains. The Cu–BTC layers are held together by a combination of hydrogen bonding (dotted line) and π–π stacking of the BTC ligand ·3H2O  is displaced away from the framework. Ultimately, the metal hydroxide remains along with the protonated linker.42One of the major concerns in MOF research is that the porous framework can absorb harmful gases. These harmful gases trapped in the pores of the framework can cause threats to the environment. Our results strongly suggest that post-synthesis transformations between HKUST-1 and Cu(BTC)·3H3H2O see , the HKU2.52O measured with a green laser (wavelength λ = 514.5 nm) focused on the single crystals in the insets. For HKUST-1, the bands appearing in the low frequency region i.e. below 600 cm−1 is due to the copper ions in the framework. The doublet at 177.54 cm−1 and 197.63 cm−1 indicates that the MOF is hydrated and correspondingly Cu–Cu stretching modes appear.43–45 The band at 275.49 cm−1 is indicative of the Cu–Ow stretching vibration, where Ow represents the oxygen of the water molecule adsorbed on Cu2+ ions.−1 are mostly due to vibrational modes of the BTC ligand. The C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C stretching modes of the benzene ring are found at 1008.9 and 1615.4 cm−1 for HKUST-1,43,44,46–48 and at 1003.5 and 1614.4 cm−1 for Cu(BTC)·3H2O. The peaks at 745.15 and 829.29 cm−1 for HKUST-1 can be attributed to the C–H bending modes.43,44 The corresponding peak for Cu(BTC)·3H2O is located at 811.13 cm−1. The peaks assigned to the C–O–O symmetric and C–O–O asymmetric stretching modes are centered at 1464.7 and 1552.6 cm−1, respectively, for HKUST-1,48,49 and at 1459.8 and 1567.9 cm−1 for Cu(BTC)·3H2O.The bands in the range 700–1800 cm2.650–52 The luminescence from HKUST-1 and Cu(BTC)·3H2O single crystals has been examined using seven different excitation lasers. The photoluminescence spectra of HKUST-1 and Cu(BTC)·3H2O crystals measured at laser wavelengths λ = 458, 488, 514.5, 531, 568, 633 and 647 nm are shown in λ = 633 and 647 nm ·3H2O ,58 This indicates the presence of defects either in the bulk or on the surface of HKUST-1. The luminescence in the wavelength range 675–1000 nm is excited at laser wavelengths of 633 nm (1.96 eV) and 647 nm (1.92 eV) that is in resonance with the optical band gap of defects.58,702O crystals. The photoluminescence spectra of Cu(BTC)·3H2O measured at λ = 458, 633 and 647 nm are superposed onto the UV-Vis absorption spectrum. To the best of our knowledge, there have been no reports of UV-Vis absorption and luminescence spectroscopy on Cu(BTC)·3H2O. The UV-Vis spectrum shows the strong UV absorption at wavelengths below 400 nm and the broad absorption above 600 nm. The long-wavelength tail of the UV absorption coincides with the luminescence excited at λ = 458 nm. The broad band above 600 nm leads to the luminescence observed at λ = 647 nm. The much weaker luminescence at 633 nm as compared with HKUST-1 can be attributed to the lower-wavelength absorption onset.32O. Crystals of HKUST-1 can be synthesized in the pH range 0.7–0.9, whereas Cu(BTC)·3H2O in the pH range 2.8–3.2. The post-synthesis transformation from HKUST-1 to Cu(BTC)·3H2O is possible, which can be useful as means for environmentally-friendly disposal of the MOF. Both species are found to be luminescent. HKUST-1 exhibits luminescence in the range 675–1000 nm that can be associated with crystallographic defects. Cu(BTC)·3H2O in turn is luminescent in the ranges 458–600 nm and 675–1000 nm, that are in-line with the UV-Vis absorption spectrum. The present work demonstrates band-gap engineering by the coordination chemistry and defect-induced luminescence in bulky crystals, that in unison will pave the way for optical applications of coordination polymers and MOFs.The present work has demonstrated that the reaction between copper and benzene-1,3,5-tricarboxylic (BTC) acid can be controlled by adjusting the pH of the precursor solution to obtain millimeter-sized crystals of two different coordination compounds, namely, metal–organic framework HKUST-1 and coordination polymer Cu(BTC)·3HThere are no conflicts to declare.MA-003-D1MA00866H-s001MA-003-D1MA00866H-s002


Rilpivirine (RPV) was approved by the U.S. FDA (Food and Drug Administration) in 2011 to treat individuals infected with human immunodeficiency virus 1 (HIV-1). Significantly, rilpivirine is three fold more potent than etravirine. Once-daily, it is used with a low oral dose (25 mg/tablet), decreasing the drug administration and bringing a better choice to the patients. However, there are many shortcomings in the existing synthesis route of RPV, such as the high cost, prolonged reaction time and low yield (18.5%).3 increased from 52 to 62% compared to the original patent.This article describes our efforts to develop an efficient and practical microwave-promoted method to synthesize rilpivirine using less toxic organic reagents and low boiling solvents. The last step's reaction time decreased from 69 h to 90 min through this optimized synthetic procedure, and the overall yield improved from 18.5 to 21%. In addition, the yield of intermediate Overall, through a series of process optimization, we have developed a practical synthesis method of rilpivirine, which is easy to scale with higher yield and shorter reaction time. E)-4-((4-((4-(2-cyanovinyl)-2,6-dimethylphenyl)amino)pyrimidin-2-yl)amino)benzonitrile), is a successful second-generation NNRTI of diarylpyrimidine family , etc.) and toxic materials ] which lead to a high price of rilpivirine. Therefore, a new synthetic route is needed urgently for a more economical, less toxic and more productive rilpivirine synthesis.HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are an integral part of highly active antiretroviral therapy (HAART) due to its high tolerability and low toxicity. However, the rapid emergence of drug-resistant HIV-1 strains limited their clinical use . RilpiviE)-3-acrylonitrile hydrochloride (intermediate 2); (b) synthesis of 4-((4-chloropyrimidin-2-yl)amino)benzonitrile (intermediate 3); (c) synthesis of rilpivirine (1).The current procedure for the synthesis of rilpivirine is mainly divided into three steps: (a) synthesis of  and acrylamide (5) under inert conditions to achieve (2E)-3-prop-2-enamide (6). Dehydration of compound 6 by treating with POCl3 yielded (2E)-3-prop-2-enenitrile (7). Finally, the desired intermediate 2 ((2E)-3-prop-2-enenitrile hydrochloride) with 77% yield was achieved by treating compound 7 with HCl in diisopropyl ether under inert condition. The application of palladium acetate  and tri-O-methylphenylphosphine  ligand has greatly improved the yield of trans-isomer. However, these two materials are relatively expensive to use in the industry and environmentally polluting benzonitrile). The starting materials 2-thioxo-2,3-dihydropyrimidin-4(1H)-one (10) and iodomethane were blended in the presence of NaOH base to attain compound 11. Further, the nucleophilic substitution of 11 with p-aminobenzonitrile (12) in DME afforded compound 13(iia). Finally, the target intermediate 3 was obtained by chlorinating the hydroxy group of 13 with POCl3 under reflux conditions. This route's advantage is that the reaction step is simple and short, and the total yield is up to 47%, and the disadvantage is that the reaction produces methanethiol, which is highly toxic and smelly. Complete elimination of this odour is highly challenging, which may increase the cost of large-scale industrial production. In addition, the boiling point of DME used in the reaction process is too high benzonitrile (20). The target intermediate 3 was achieved by reacting 20 with pyridine, followed by POCl3 under reflux condition. The reaction is relatively simple, but the process is too long and reducing the yields. Also, the highly toxic phosphorus oxychloride reagent is used repeatedly, which is harmful to the environment and public health. The entire route has a long reaction time and consumes a lot of energy, which is not suitable for industrial production [In Scheme oduction .Scheme 51) was achieved by allowing the reaction between (2E)-3-prop-2-enenitrile hydrochloride (2) and 4-[(4-chloropyrimidin-2-yl)amino]benzonitrile (3) in acetonitrile under reflux condition for 69 h  the starting material (14) used for the preparation of intermediate 3 is relatively expensive and the reaction temperature is too high; (c) when uracil is used as a starting material, the synthetic route in Scheme There are many shortcomings in the synthetic strategy of rilpivirine. Notably, (a) intermediate 3 and improve the reaction conditions for the nucleophilic substitution reaction in the last step. To achieve the desired intermediate 3, initially, a fusion reaction was carried out between 2-(methylthio)-4(3H)-pyrimidinone (11) and p-aminobenzonitrile (12) under an inert atmosphere, resulted in compound 13. After that, compound 13 reacted with POCl3 under reflux conditions to yield the target intermediate 3. As well as intermediate 2 was obtained as illustrated in Scheme 2 and 3 through microwave-irradiation leads to the rilpivirine formation with improved yield (21%) and shorter reaction time (90 min).A novel synthetic procedure for the rilpivirine is successfully demonstrated in six steps with improved yield (21%). The method Scheme  is devel13 was carried out through melting/fusion method with the temperature ranging from 160 °C to 200 °C. Herein, we investigated the effect of reaction temperature and reaction time on the yield of compound 13 . When we led our first attempt, dioxane, acetonitrile, and NMP solvents were used. It was observed that the reaction with acetonitrile solvent provided good yields, NMP with moderate to good and with dioxane the reaction is not formed. However, when we tried to use these three kinds of solvents for microwave reaction, the reaction results were not satisfactory. Table Considering that different solvents are used as the reaction, none of the reactions was ideal. We speculated that the reaction temperature and time have a significant influence on the reaction yield. Since the acetonitrile yield as solvent was similar to NMP and considering industrial production cost, we choose low-boiling acetonitrile as the reaction solvent for further exploration. Therefore, a single variable method was applied to investigate the effect of reaction temperature and time on yield. The results are shown in Table We can conclude that the yield was improved with the increased reaction time and temperature. But there is a decreasing tendency of the yield when the temperature is 150 °C and reaction time is more than 120 min. Accordingly, the amination reaction's optimized conditions were determined as follows: microwave-irradiation reaction with acetonitrile solvent at 140 °C for 90 min produced the amination product with 71% yield, which was higher than that of the traditional method (69%).13) the reaction was carried out at 160 °C for 2 h, and then the temperature was raised to 180 °C for 4 h. After the treatment, a small amount of N,N-dimethylformamide was added to dissolve the residues, subsequently, the mixture was filtered and washed with acetonitrile to give the product with 70% yield; (2) the total yield of the intermediate 3 increased from 52 to 62% compared to the original patent. The total synthesis yield of the intermediate 2 and rilpivirine is equivalent to original patents. (3) For the rilpivirine synthetic step, a microwave-irradiation method is selected instead of the traditional method in the original patent, and the yield of amination reaction is increased to 71%. Therefore, the total synthetic yield of rilpivirine risen from 18.5 to 21%. The method proceeded in six linear steps with a gram scale and did not use high toxicity starting materials and high boiling-point solvent. This efficient and environmental-friendly process and the optimum conditions for rilpivirine preparation may form the basis for future manufacturing route.In conclusion, we provide a synthetic method for rilpivirine, an essential drug in the treatment of acquired immunodeficiency syndrome (AIDS). The experiment mainly optimized the route of rilpivirine and the key process parameters of each step by controlling the single variable method to determine the optimal experimental conditions. The total yield was improved from 18.5 to 21%, and the reaction time of the last step decreased from 69 h to 90 min. The details are as follows: (1) synthesis of 4-((4-hydroxypyrimidin-2-yl)amino)benzonitrile  with tetramethylsilane as the internal reference, and J values were reported in hertz (Hz). Melting points (m.p.) were determined on a micro melting point apparatus . Mass spectra were recorded on an LC Autosampler device: Standard G1313A instrument. The microwave reaction was conducted on a CEM Discover  instrument and the conventional high-pressure reaction was performed on Parr 4590 device. Rotary evaporators were served in the concentration of the reaction solutions under reduced pressure.Thin-layer chromatography (TLC) was performed on Silica Gel GF254 for TLC (Merck), and spots were visualized by irradiation with UV light (λ = 254 nm). 4 , acrylamide 5 , sodium acetate , palladium acetate  and tris(2-methylphenyl)phosphine  was placed in a round bottom flask, and N,N-dimethylacetamide was slowly added to the round bottom flask under nitrogen, then the mixture was heated to 130 °C for 24 h. After the completion of the reaction, the mixture was cooled to room temperature, filtered over celite, evaporated. The residue was recrystallized in ethyl acetate and petroleum ether to give the product (E)-3-acrylamide (6), 4.8 g, yield: 51%.4-Bromo-2,6-dimethylaniline 6 slowly at 0 °C and then refluxed for 8 h at 40 °C. After completion of the reaction was monitored by TLC, and the reaction mixture was slowly poured into the ice-water mixture and stirred for 1 h and then filtered to obtain the crude (E)-3-acrylonitrile (7) as white crystal solid.To a round-bottomed flask consisting of 10 mL of phosphorus oxychloride was added compound 7 was dissolved in anhydrous ethanol, and then added ethanolic hydrochloric acid solution, and stirred for 30 min, then filtered to obtain (E)-3-acrylonitrile hydrochloride (2), 5.0 g. Yield: 95%. mp: 78–80 °C. 1H NMR : δ 7.22 , 7.05 , 5.59 , 3.95 , 2.18 . 13C NMR : δ 150.94, 146.21, 129.86, 128.02, 123.28, 121.50, 119.60, 90.28, 77.38, 77.06, 76.74, 17.50. ESI-MS: m/z 173.54 [M+H]+, C11H12N2 (172.10).The obtained crude product The solvent of anhydrous ethanol was stirred under ice bath, and then 41 mL of acetyl chloride reagent added slowly dropwise and stirred for 30 min to obtain an ethanolic hydrochloric acid solution.11  and p-aminobenzonitrile 12  were taken together, stirred under the inert condition at 160 °C for 2 h and then increased the temperature to 180 °C for 4 h. Then the reaction was allowed to room temperature and added an appropriate amount of N, N-dimethylformamide. The resulted mixture was kept in the ultrasonic vibrator for 30 min. Then the mixture was filtered and washed with acetonitrile and dichloromethane, dried in vacuo to give 4-((4-hydroxypyrimidin-2-yl)amino)benzonitrile (13) as a white solid .Starting materials 2-methylthio-4-pyrimidinone 13 was dissolved in 20 mL of phosphorus oxychloride at 0 °C and then refluxed for 1h. After completion, the mixture was cooled to room temperature, slowly poured into 100 g of crushed ice and stirred for half an hour. After filtration, the obtained filter cake was dissolved in 50 mL of water, and the pH was adjusted to 7–8 with potassium carbonate and filtered. The filtrate was washed with acetonitrile and dried to obtain 4-((4-chloropyrimidin-2-yl)amino)benzonitrile (3) as a white solid (9.9 g). Yield: 89%. mp: 209-210 °C. 1H NMR : δ 10.58 , 8.55 , 7.87 , 7.13 ; ESI-MS: m/z 231. 2 [M+H]+, C11H7ClN4 (230.04).Compound 2  and 3  along with 10 mL acetonitrile was transferred into 20 mL microwave vial and set the reaction temperature at 140 °C, 2 h. After completion, the reaction mixture was cooled to room temperature and then 10% potassium carbonate solution was added dropwise to the vial to adjust the pH to 8–9. The obtained filtrate was washed with acetonitrile to obtain the target molecule rilpivirine as a white solid (1.28 g). Yield 71%. mp: 138–140 °C. 1H NMR : δ 9.62 , 8.95 , 7.98 , 7.69 , 7.49 , 6.46 , 2.18 . ESI-MS: m/z 367.24 [M+H]+, C22H18N6 (366.16).A mixture of compound


To confirm that this ratio is robust in the setting of treatment and hormonal control, we used ex vivo perfused mouse livers from BLKS mice (glucagon treated and metformin treated), and db/db mice. We confirm that the ratio of signal intensities of 6C to 3C in 13C nuclear magnetic resonance spectra post HP DHA administration is sensitive to hepatic gluconeogenic state. This method is directly applicable in vivo and can be implemented with existing technologies without the need for substantial modifications.Type II diabetes and pre-diabetes are widely prevalent among adults. Elevated serum glucose levels are commonly treated by targeting hepatic gluconeogenesis for downregulation. However, direct measurement of hepatic gluconeogenic capacity is accomplished only via tracer metabolism approaches that rely on multiple assumptions, and are clinically intractable due to expense and time needed for the studies. We previously introduced hyperpolarized (HP) [2- Diabetes is a widely prevalent ailment affecting more than 10% of adults in the United States, with about 20% of those undiagnosed. A further 34% of the adult population are pre-diabetic based on fasting glucose or A1C levels dihydroxyacetone provides unique insights into hepatic physiology propionate anaplerosis was more limited, as evidenced by low amounts of larger mass  isotopomers  from non-gluconeogenic livers using HP DHA as a substrate. The ratio of integrals of hexose resonances to three- and four-carbon metabolites derived from HP DHA (identified in db/db (0.56 ± 0.24) and glucagon treated livers (0.53 ± 0.22) compared to metformin-treated livers (0.15 ± 0.14), as shown in Based on the db/db mouse model. Livers from db/db are almost twice as large as livers from the control BLKS mice. Since db/db mice livers have been shown to contain higher amounts of lipid and glycogen but similar amounts of total protein DHA (and no deuterium incorporation) will also produce M+1 mass isotopomer. Considering that the HP spectra from db/db livers show, in real time, glucose production, the observed M+1 enrichment points to DHA being a major contributor to glucose enrichment following the injection.For complete analysis of the data, the two distinct segments of the study should be considered—(i) liver perfusion prior to HP DHA administration and (ii) post HP DHA administration. Analysis of freeze clamped liver tissue provides the most accurate picture of DHA metabolism while analysis of the perfusate primarily yields information about liver metabolism prior to DHA administration, as it essentially “integrates” for metabolism over the full perfusion length. The observations from the HP experiments are validated by GC-MS measurements of glucose enrichment in the liver. GC-MS measured glucose enrichment in the liver yields insight on glucose production during the period of DHA administration and for the remainder of the perfusion. All three groups show higher enrichment of M+1 species, indicating glucose production primarily from hyperpolarized DHA . Althougdb/db livers was significantly higher in the perfusate, as measured from C1–3 and C4–6 fragments  in G3P and succinate. In db/db livers, the higher M+1 mass isotopomer of G3P  between diabetic livers and non-diabetic livers is unsurprising, as this matches previous observations ,34. The ormation . Multiplormation ,28,36.2O administration to reach ~0.5% body water enrichment). Blood glucose is then analyzed using NMR spectroscopy or mass spectrometry. In contrast, the method described in this work is straightforward since very little sample processing or extensive data analysis is required. Hyperpolarized techniques have been shown to accurately reflect the metabolic state of various tissues under in vivo and ex vivo modalities. In this study, we have demonstrated that hyperpolarized DHA is sensitive to modulations of hepatic glucose output and propose a simple metric to distinguish gluconeogenic and non-gluconeogenic livers.Regulating hepatic gluconeogenesis is an important target for treating Type II diabetes and is an important co-variate in the emerging pathology of nonalcoholic steatohepatitis. Numerous methods are available to measure hepatic glucose output and have been evaluated in detail elsewhere . The mosm+/+LeprdbJ (db/db). C57BLKS/J mice are the appropriate background control for the db/db strain. All male mice were 10–13 weeks old. Mice were anesthetized using isoflurane followed by an intraperitoneal injection of heparin. About 10 min after heparin injection, a celiotomy was performed under anesthesia (lidocaine was administered subcutaneously prior to making the first incision) exposing the liver and the portal vein. The portal vein was then cannulated and perfusion was started. The liver is then excised from the body and connected to a glass perfusion column. This glass perfusion apparatus is then moved into the bore of an NMR magnet.Experiments involving mice were handled in compliance with the University of Florida Institutional Animal Care and Use Committee (protocol number #201909320). Strains used were C57BLKS/J (Stock No. 000662) and BKS.Cg-Dock73, 112 mM NaCl, 4.7 mM KCl, 1.2 mM each of MgSO4 and KH2PO4 and 0.5 mM sodium-EDTA, 1.25 mM CaCl2), 6 mM sodium lactate, 0.6 mM sodium pyruvate, 0.2 mM [U–13C] sodium propionate, 10% (v/v) D2O, and 0.63 mM mixed fatty acids , palmitoleic acid (5.2%), stearic acid (2.7%), oleic acid (27%), linoleic acid (37.7%), γ-linolenic acid (2.4%), and decosahexanoic acid (2.8%)) along with 2% (w/v) bovine serum albumin. For perfusions with livers from BLKS mice, either 10 mM metformin hydrochloride or 50 nM glucagon was also added. Perfusate was oxygenated using 95% O2/5% CO2 mixed gas for the duration of perfusion. Oxygen consumed by the liver during perfusion was measured every 15 min using an Oxygraph+ setup . At the end of the perfusion, the livers were freeze-clamped using liquid nitrogen and stored at −80 °C until needed. The number of mice used in each group was db/db (n = 6), glucagon (n = 5), and metformin (n = 6).Livers were perfused with perfusate containing Krebs-Henseleit electrolytes  was dissolved to a final concentration of 4 M in a 2:1 mixture of dimethyl sulphoxide and water doped with 15 mM trtiyl radical ]-benzo--bis--dithiole-4-yl]-methyl sodium salt; Oxford Instruments, Abingdon, UK) and 1 mM ProHance. This sample was inserted into HyperSense polarizer . The frozen sample was hyperpolarized at 1.4 K by applying microwave irradiation (94.116 GHz at 100 mW) until steady state was achieved (~1.5–2 h). The hyperpolarized sample was rapidly dissolved using 4 mL of hot 20 mM PBS (pH 7.4) and transferred to a 14 T NMR magnet. 3 mL of the dissolved sample was mixed with 20 mL of oxygenated Krebs-Henseleit electrolytes and injected directly into the perfused liver . NMR signal acquisition was started prior to injection.[2–23Na signal. Typically, linewidths of around 18 Hz in 23Na spectrum were achieved (translating to similar linewidths in 13C spectra). 13C spectra  were recorded using a 45° {x, −x} binomial excitation scheme centered on DHA resonance  with 1H decoupling  during acquisition and a repetition time of 3 s. Spectra were processed with 15–20 Hz exponential line broadening and baseline corrected using a polynomial function. Peak areas of metabolites were obtained by fitting mixed Lorentzian-Gaussian line shapes to 13C sum spectra . We do not include 2H NMR spectra of the glucose in our analysis, as we achieved unexpectedly high enrichments of both 13C and 2H. Excessive 13C enrichment rendered the 2H spectra unusable due to overlap between the peaks and adjacent 13C j-coupled satellites.Spectra in hyperpolarized DHA experiments were collected in a custom-built 20 mm broadband probe  installed in a 14 T magnet equipped with an Avance III NMR console . Prior to injection of HP DHA, shimming was carried out using the v/v) perchloric acid. Samples were centrifuged. 1 mL of supernatant was collected and pH adjusted to 7 using 5 M KOH. The samples were centrifuged to pellet out potassium perchlorate salt before collecting 1 mL of supernatant. The supernatant was dried in a lyophilizer . The dried solution was reconstituted in 100 μL of 50:50 Acetonitrile water and then allowed to sit in a −20 °C freezer for at least 2 h before centrifugation and collection of 80 μL of the resulting supernatant. All centrifugation steps were carried out at 4 °C and 10,000× g for 30 min.Protein in 1 mL of perfusate was precipitated by adding 100 μL of 70% (v/v/v) solvent mixture. Samples were homogenized with a bead homogenizer  for 3 × 20 s cycles, and were cooled on ice for 5 min between each cycle. The samples were then centrifuged at 10,000× g at 4 °C for 30 min. 900 μL of supernatant was recovered and lyophilized . The dried precipitate was reconstituted in 150 μL of acetonitrile:water (1:1 v/v) mixture, followed by incubation at −20 °C for at least 2 h. Reagents, unless otherwise specified, were purchased from Fisher Scientific, Waltham, MA, USA.Freeze clamped perfused liver samples were stored at −80 °C prior to analysis. 110 +/− 10 mg of perfused livers were transferred into conical bottom microcentrifuge tubes with screw caps. 1.0 mm zirconium oxide homogenization beads were added to each microcentrifuge tube along with 1 mL of cold degassed acetonitrile:isopropanol:water (3:3:2 db/db perfused livers and 25 μL of extracted perfusate for BLKS livers (perfused with metformin or glucagon) were loaded into 0.5 mL V-vials  along with 400 ng of 2-hydroxyisovalerate and 400 ng of norleucine (added from stock solutions). These samples were dried under a gentle stream of air. The dried samples were then reconstituted in 50 μL of methoxyamine HCL in pyridine  and agitated using a microstirbar  for 1.5 h at 30 °C. 50 μL of N-Methyl-N-trimethylsilyl-trifluoroacetamide  was added to the reaction mixture and the reaction mixture was allowed to react for 30 min at 37 °C.1 μL of extracted perfusate for For analysis of amino acids and TCA cycle intermediates, ~8 mg of liver extract was dried down by airstream and was reconstituted in 50 uL of methoxyamine HCL in pyridine  for 1.5 h at 30 °C, while stirred by microstirbar. Then 50 μL of dimethyl tert butyl silyl trifluoroacetamide  was added and allowed to react at 70 °C for 30 min while stirring. After derivatization, 80 μL of the sample was transferred to a GC vial for analysis by GC-MS.Using the same amount of extract as the methoxyimino penta-trimethyl silyl Derivatization for each sample group, the samples were dried in 0.5 mL V-vials and reconstituted in 50 μL of 20 mg/mL hydroxylamine hydrochloride  in pyridine  and agitated with a microstirbar at 90 °C for 1.5 h. 100 μL of propionic anhydride  was then added and samples were agitated with a microstirbar at 70 °C for 30 min. The samples were dried down by a gentle stream of air and then reconstituted in 100 μL of ethyl acetate for GC-MS analysis.After the appropriate derivatization reaction  was complete, 5 μL of each sample was pooled together to make a perfusate “pooled sample” and 80 μL of each individual sample was loaded into a 150 μL glass insert  inside of a GC vial with a 9 mm PTFE/red rubber septum . The samples were loaded onto the AI/AS 1300 autosampler to await auto injection into the Trace1310 Gas chromatograph system . Samples of 1 μL volume were injected into the injection port with a splitless single taper liner at 250 °C and a splitless time of 60 s and column flow at 1 mL/min with a 30 m RTX-5MS integra Guard column  and 10 m guard column.For MSTFA derivatized samples, the oven was held at 60 °C for 1 min, followed by a 10 °C/min ramp up to 325 °C followed by a 5 min bake out. For MTBSTFS derivatized samples, the following parameters were used: 60 °C hold time = 1 min, ramp of 15 °C/min up to 320 °C followed by a 5 min bakeout at 320 °C. Aldonitrile pentapropionate derivatized samples were run on the same GC-MS instrument with these parameters: start temperature of 80 °C with a hold time = 1 min, 20 °C per minute ramp up to 280 °C, followed by a 5 min bakeout at 280 °C.m/z filter of 40–600 mass-to-charge ratio was used and individual metabolites were identified and quantified by area using their known quantification ions as in For all samples, the transfer line was maintained at 280 °C and the ion source was maintained at 230 °C. The solvent delay on the mass spectrometer was set to 10 min. A Peak areas were integrated using Xcalibur (version 4.1) batch processing with genesis peak fitting, a 5 s time interval, height cutoff at 5.5% of the peak with valley detection enabled, and a S/N cutoff threshold of 3.im) to the sum of all mass isotopomers . Statistical analysis was carried out using R (v3.6.0). Figures were prepared using in house python (v3.8) scripts (libraries utilized were numpy (v1.19.2) , pandas