The levels of secreted enzyme activity under the control of glyceraldehyde-3-phosphate dehydrogenase (GAPP) and alcohol oxidase 1 (AOX1P) promoters were compared. The Pc Xyn11A was produced as a soluble protein and the total xylanase activity under the control of GAPP and AOX1P was 34- and 193-fold, respectively, higher than that produced by the native strain of P. citrinum. The Pc Xyn11A produced under the control of the AOX1P reached a maximum activity of 676 U/mL when induced with 1% (v/v) methanol every 24 h for 5 days. The xylanase was purified by ion exchange chromatography and then characterized. The enzyme was optimally active at 55 °C and pH 5.0 but stable over a broad pH range (3.0–9.0), retaining more than 80% of the original activity after 24 h or after pre-incubation at 40 °C for 1 h. With birchwood xylan as a substrate, Pc Xyn11A showed a Km(app) of 2.8 mg/mL, and a kcat of 243 s−1. The high level of secretion of Pc Xyn11A and its stability over a wide range of pH and moderate temperatures could make it useful for a variety of biotechnological applications.Most common industrial xylanases are produced from filamentous fungi. In this study, the codon-optimized  Plant cell walls are the main polysaccharide-containing renewable resource on earth, and are generated by photosynthesis . HemicelXylanases are widely used in various biotechnological applications. Examples include serving as bio-bleaching agents in the pulp and paper industry , improviTrichoderma spp. and Aspergillus spp [Penicillium citrinum appears to be an interesting xylanase-producing fungus since its xylanases are stable over a wide pH range [P. citrinum [Xylanases are widespread in nature and have been reported in many microorganisms ,13,14,15llus spp . Howeverllus spp ,18,19,20pH range , but thecitrinum .Pichia pastoris is a useful alternative for producing genetically engineered enzymes for research as well as industrial purposes [P. pastoris, as an excellent host for the production of recombinant proteins, provides additional benefits, such as a rapid growth rate to high cell density in inexpensive and non-complex culture medium, and ease of purification of heterologously-expressed recombinant proteins [Heterologous gene expression by the methylotrophic yeast purposes . It is cpurposes . Moreoveproteins ,26.Codon optimization technology is a commercialized algorithm for heterologous gene expression. The DNA sequence used to encode a protein varies between different organisms. To obtain a high expression level in the new host, changing the foreign DNA sequence into that which matches the host requirements for expression is frequently required ,28.xynA gene encoding a GH family-11 xylanase A from the fungus P. citrinum (Pc Xyn11A) strain FERM P-15944 was synthesized by optimization of codon usage and then cloning in the heterologous yeast host P. pastoris. A comparison of the expression level from a constitutive and inducible expression system, under the control of glyceraldehyde-3-phosphate dehydrogenase (GAPP) and alcohol oxidase 1 (AOX1P) promoters, respectively, was performed in order to improve the production and to characterize the Pc Xyn11A enzyme. This is the first report on the expression of an optimized codon xynA gene from P. citrinum in P. pastoris.In this study, the xynA gene from P. citrinum FERM P-15944 was codon optimized to enhance the translation efficiency, and was then expressed in P. pastoris X-33 to improve the expression level and study xylanase A. The mature protein-coding xynA gene consists of 573 nucleotides and encodes a protein of 190 amino acid residues. The optimized DNA sequence, aligned with the native gene, and the protein alignment, are shown in P. pastoris, 138 codons were changed. However, the alignment clearly showed that both sequences encoded a protein with the same amino acid sequences. The codon usage bias was changed by upgrading the codon adaptation index (CAI) from 0.69 to 0.83, giving a smoother GC content curve and eliminating the negative cis-acting elements. The potential N- and O-glycosylation sites of Pc Xyn11A were predicted using the NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) and the NetOGlyc 4.0 Server (http://www.cbs.dtu.dk/services/NetOGlyc-4.0/), respectively. The results showed that there was no N-glycosylation, whilst there were four possible O-glycosylation sites.The P. pastoris in this study. Both the pGAPZα A and pPICZα A plasmids contain the native Saccharomyces cerevisiae α-factor secretion signal for extracellular production of protein and the Zeocin resistance gene for positive selection in E. coli and P. pastoris. The gene encoding the mature Pc Xyn11A was cloned in frame with the α-factor secretion signal under the control of GAPP or AOX1P to produce the recombinant expression plasmids pGAPZαA-xynA and pPICZαA-xynA, respectively. After transformation into P. pastoris X-33, the transformants were selected on a YPDS agar plate containing 100–2000 µg/mL Zeocin.Several studies have reported that heterologous protein production by GAP is better than AOX1system ,30,31,32GAPP is a constitutively expressed promoter, the transformants were preliminary selected on YP with 0.2% Azo-xylan agar plates, where all transformants produced xylanase activity, as determined by the clear halo indicating that the substrate was hydrolysed and released soluble dye-labelled fragments around the colony. The negative control, untransformed P. pastoris X-33, did not show any clear zone. For pPICZαA-xynA, the transformants grew on all YPDS agar plates containing 100–2000 µg/mL Zeocin, although the number of transformants decreased when the concentration of Zeocin was increased.For pGAPZαA-xynA, YPDS agar plates containing 100 µg/mL Zeocin showed the highest number of transformants, and this number decreased as the concentration of Zeocin increased, with no colonies growing at 2000 µg/mL Zeocin. As P. pastoris X-33 had no detectable extracellular xylanase activity. For the constitutive expression (GAPP), the growth of all transformants reached a steady state after 48 h, whereas for the methanol-inducible expression (AOX1P), all transformants were still in the growth phase after 5 days with induction with methanol every 24 h. The maximum xylanase activity of the pGAPZαA-xynA transformants (Pp-gap-xynA) was from a clone picked on a YPDS agar plate containing 100 µg/mL Zeocin. After 48 h of culture, the xylanase activity reached 119.5 µmol/mL/min, which was 34-fold higher than that of the native strain P. citrinum at 72 h [All transformants were cultured and monitored every 24 h for growth and xylanase activity in the supernatant. The wild type  at 72 h .v/v) methanol in BMMY and induced with 1% (v/v) methanol every 24 h for 5 days, an activity that was 193-fold higher than that of the native fungus P. citrinum [For pPICZαA-xynA-containing clones, in order to maximize the Pc Xyn11A production level, the methanol concentration was varied in BMMY medium during induction. The maximum xylanase activity (676 µmol/mL/min) of the pPICZαA-xynA transformants was obtained from a clone selected on a YPDS agar plate containing 500 µg/mL Zeocin and cultured with 1%  by fusion with the signal peptide of Aureobasidium pullulans XynI in place of the native one of xynA from P. citrinum. However, the obtained xylanase activity from this heterologous construct in E. coli was 7.1- and 40.2-fold lower than that of Pc Xyn11A under the GAPP and AOX1P, respectively, in this study. The xynA from P. citrinum has also been investigated in Yarrowia lipolytica to enhance the xynA protein production by fusion of the preproLIP2 secretion signals compared to the native one of xynA from P. citrinum [AOX1P reported in this study was 17.7- and 3.7-fold higher than that of the recombinant XynA using native and preproLIP2 secretion signals, respectively  at 55 °C, designated as the optimum temperature for the enzyme activity, and then decreased to about 70% activity at 60 °C . The enzP. citrinum [Aspergillus awamori VTCC-F312 [P. citrinum produced in Y. lipolytica [Y. lipolytica at pH 10.0 (30% after 24 h). This could be due to the post-translational modifications. Glycosylation is one of the most important post-translational modifications in eukaryotic cells, with significant effects on protein folding, conformation, activity and stability. The modification of proteins through enzymatic glycosylation is controlled by factors that differ greatly among cell types and species [A. awamori VTCC-F312 (pH 4.0–8.0) [Achaetomium sp. Xz-8 (pH 5.0–10.0) [Aspergillus fumigatus MKU1 (pH 4.0–8.0) [Bacillus pumilus ARA (pH 5.8–8.2) [Bacillus sp. GA1(6) (pH 4.0–7.0) [Penicillium oxalicum (pH 3.0–5.0) [Aspergillus niger was used to improve dough characteristics and bread quality [Endoxylanases previously reported from fungal sources usually show an optimum pH in the range of 4.0–7.0 and an optimum temperature between 40 °C and 60 °C . The Pc citrinum , AspergiTCC-F312  and P. cpolytica . However species ,44. More4.0–8.0) , Achaeto.0–10.0) , Aspergi4.0–8.0) , Bacillu5.8–8.2) , Bacillu4.0–7.0)  and Peni3.0–5.0) . These r quality . It coulKm(app), Vmax and kcat were calculated to be 2.8 mg/mL, 310.7 µmol/min and 243 s−1, respectively. The Pc Xyn11A kcat was higher than that previously reported for some other xylanases, such as those from Malbranchea cinnamomea and Bacillus cellulosilyticus with kcat values of 4.26 and 165.8 s−1, respectively [Km(app) of the Pc Xyn11A of this study was lower than that of the native Aspergillus niger xylanase (Native XylA), recombinant A. niger xylanase produced in Pichia pastoris (reXylA), Nonomuraea flexuosa xylanase produced in Trichoderma reesei (Nf Xyn11A) and P. citrinum xylanase produced in Y. lipolytica (re-xynA) [Km(app) of Pc Xyn11A expressed in P. pastoris and Y. lipolytica could be due to the difference in glycosylation, as glycans can alter the substrate recognition, specificity and binding affinity, and turnover rates [Km(app) of Pc Xyn11A in this study indicated that it had a higher affinity for birchwood xylan. The Pc Xyn11A also had a higher turnover number (kcat) and catalytic efficiency (kcat/Km(app)), indicating that it has higher catalytic activity.The Pc Xyn11A kinetics were evaluated at 40 °C and a pH of 5.0 using nine different birchwood xylan (substrate) concentrations. The ectively ,51. In cre-xynA) ,52,53, aer rates . The lowxynA gene from P. citrinum FERM P-15944 was synthesized with codon optimization for expression in P. pastoris based on the nucleotide database (GenBank: accession no. AB198065.1) [P. pastoris expression kit, including the P. pastoris strain X-33, constitutive expression vector (pGAPZα A), methanol-inducible expression vector (pPICZα A) and Zeocin were all from Invitrogen . All restriction enzymes and Phusion High-Fidelity DNA polymerase were from New England BioLabs . Birchwood xylan was from Sigma-Aldrich , Azo-xylan was from Megazyme .The 98065.1)  and intrPichia pastoris was cultured in YPD medium 1% (w/v) yeast extract, 2% (w/v) bacteriological peptone and 2% (w/v) dextrose at 30 °C with shaking at 200 rpm. The transformants were selected on YPDS plates (YPD with 1 M sorbitol) containing 100–2000 µg/mL Zeocin. The pGAPZα A transformants were chosen for preliminary selection on YP (YPD without dextrose) with 0.2% (w/v) Azo-xylan.Escherichia coli DH5α (Gibco) was used for vector propagation and was grown in a Luria-Bertani (LB) medium at 37 °C with either kanamycin (50 µg/mL) to select for pUC57-Kan or Zeocin (25 µg/mL) to select for pGAPZα A and pPICZα A transformants.xynA gene was amplified without a signal peptide or stop codon from pUC57-Kan containing the optimized codon xynA gene by PCR using the following specific primers: xynA-F (5′-GCGGTACCGAGTCATATACTTCTTCCTCAACC-3′), and xynA-R (5′-GAGCGGCCGCGGAAACGGTAATGTCAGCG-3′). KpnI-HF and NotI-HF restriction sites (underlined) were added to the forward and reverse primers, respectively. The PCR was thermal cycled at 98 °C for 30 s followed by 30 cycles of 98 °C for 10 s, 54.1 °C for 30 s and 72 °C for 20 s, and then a final 72 °C for 10 min. The amplified fragments were purified and then digested with KpnI-HF/NotI-HF before cloning into the pGAPZα A or pPICZα A expression vectors predigested with the same restriction enzymes to yield the pGAPZαA-xynA and pPICZαA-xynA plasmids, respectively. The plasmids were transformed into E. coli DH5α competent cells and selected by plating on low-salt LB agar plates (1% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.5% (w/v) NaCl, 1.5% (w/v) agar) containing 25 µg/mL Zeocin to select for the presence of the recombinant plasmid and isolate single colonies. The insertion was verified by double restriction enzyme digestion and commercial DNA sequencing .The synthetic AvrII and PmeI, respectively, for integration into P. pastoris genome. Each linearized DNA was transformed into P. pastoris strain X-33 using the lithium acetate and dithiothreitol method by electroporation [P. pastoris X-33 cells using a Gene Pulser Xcell (Bio-Rad) and immediately recovered in 1 mL of 1 M sorbitol for 2 h at 30 °C. Transformants were selected on YPDS agar plates containing 100–2000 µg/mL Zeocin. Transformants under the control of GAPP were chosen for preliminary selection on YP agar plates containing 0.2% (w/v) Azo-xylan.The pGAPZαA-xynA and pPICZαA-xynA plasmids were linearized with poration . BrieflyGAPP), P. pastoris transformants were cultured in YPD medium for 4 days, whereas for methanol-inducible expression (AOX1P), P. pastoris transformants were grown using buffered glycerol-complex medium (BMGY: 1% (w/v) yeast extract, 2% (w/v) peptone, 100 mM KH2PO4/KOH buffer (pH 6.0), 1.34% (w/v) Yeast Nitrogen Base, 4 × 10−5% (w/v) biotin, and 1% (v/v) glycerol) and buffered methanol-complex medium (BMMY: 1% (w/v) yeast extract, 2% (w/v) peptone, 100 mM KH2PO4/KOH buffer (pH 6.0), 1.34% (w/v) Yeast Nitrogen Base, 4 × 10−5% (w/v) biotin, and 0.5% (v/v) methanol). Absolute methanol was added every 24 h to maintain induction for 5 days, at an initial final concentration of 0.5–1% (v/v) in BMMY medium, and then supplemented at 0.5–3% (v/v) for the daily induction to optimize recombinant xylanase (Pc Xyn11A) production. All enzyme productions were performed in a baffled shaker flask at 30 °C with constant shaking at 200 rpm. The culture supernatant was collected at periodic intervals to analyze the expression level to determine the optimal time for post-induction harvest. When the maximum xylanase activity was observed, the culture supernatant was collected to analyze the production level and to allow purification and characterization of the produced Pc Xyn11A.For constitutive expression (w/v) birchwood xylan in deionized water, 0.4 mL of 0.15 M acetate buffer (pH 5.0), and 0.1 mL of suitably diluted enzyme. Reactions were incubated at 50 °C for 5 min and aliquots (75 µL) were taken every minute. The reaction was stopped by transferring it into an equal amount of DNS  before boiling for 5 min [The xylanase activity was assayed by following the release of reducing sugars from xylan. The reaction mixture consisted of 0.5 mL of 4% (or 5 min . After cg for 10 min. The supernatant was concentrated and exchanged against 20 mM acetate buffer (pH 5.5) by ultrafiltration through regenerated cellulose membrane ultra-centrifugal filters with a 10 kDa molecular weight cut-off  and loaded onto a HiLoadTM 16/10 Q Sepharose High Performance column  previously equilibrated with 20 mM acetate buffer (pH 5.5). The adsorbed proteins were eluted with a linear gradient of 0–0.6 M NaCl in the same buffer at a flow rate of 2 mL/min.All purification steps were performed at 4 °C. Yeast cells from liquid cultures were sedimented by centrifugation at 4500× ® TGX Stain-FreeTM gels to determine the molecular weight and confirm the apparent purity (size homogeneity) of the enzyme. The zymographic analysis for xylanase activity was performed on 10% polyacrylamide gel containing 1% (w/v) Azo-xylan as substrate. The electrophoresed gel was soaked in 25% (v/v) isopropanol to remove the SDS and renature the protein and then washed four times with 60 mM acetate buffer pH 5.0 for 30 min each, before being incubated in the same buffer at room temperature overnight. The activity bands were observed as clear and colorless areas.The MWCO concentrated culture supernatant (crude Pc Xyn11A preparation) and purified Pc Xyn11A were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using Mini-ProteanThe effect of pH on the xylanase activity of the purified Pc Xyn11A was determined within the pH range of 3.0–7.0 using xylan as a substrate in citric acid–sodium citrate buffer (pH 3.0–6.0) and sodium phosphate buffer (pH 6.0–7.0) at 50 °C, whilst the optimum temperature of the purified Pc Xyn11A was evaluated at 25 °C (room temperature) to 60 °C using xylan as the substrate at the determined pH optimum.The effect of pH on the purified Pc Xyn11A xylanase stability was evaluated by measurement of the residual activity after 24 h pre-incubation at 25 °C in 60 mM of the appropriate buffers: citric acid–sodium citrate buffer (pH 3.0–6.0), sodium phosphate buffer (pH 6.0–8.0) and sodium carbonate (pH 9.0–10.0), whilst the thermostability was determined after pre-incubation in citric acid–sodium citrate buffer (pH 5.0) at 40–60 °C. Each data point represents the mean of three independent replicates and the error bars indicate the standard deviation.Km, kcat and Vmax) of the purified Pc Xyn11A enzyme were determined using birchwood xylan at 0.1–2% (w/v) as the substrate in 60 mM citric acid-sodium citrate buffer at pH 5.0, 40 °C for 10 min. The kinetic analysis was then performed using Michaelis–Menten and Lineweaver–Berk plots. Each data represents the mean of three independent experiments.The kinetic parameters  or methanol-inducible (AOX1P) promoters. Both promoters demonstrated efficient expression of the codon optimized xynA gene. The total xylanase activity produced with the AOX1P was 193-fold higher than with the native P. citrinum strain. The high level of secretion and the stability over a wide range of pH and at moderate temperatures of the Pc Xyn11A make it potentially useful for a variety of biotechnological applications.This is the first report on the expression of the codon-optimized


Finally, we established the measurement of PRR activation in tissue sections via PLA upon IVT mRNA intramuscular (i.m.) injection in mice. Overall, our results indicate that PLA is a valuable, versatile, and sensitive tool to monitor PRR activation for vaccine, adjuvant, and therapeutic screening.The characterization of innate immune activation is crucial for vaccine and therapeutic development, including RNA-based vaccines, a promising approach. Current measurement methods quantify type I interferon and inflammatory cytokine production, but they do not allow for the isolation of individual pathways, do not provide kinetic activation or spatial information within tissues, and cannot be translated into clinical studies. Here we demonstrated the use of proximity ligation assays (PLAs) to detect pattern recognition receptor (PRR) activation in cells and in tissue samples. First, we validated PLA’s sensitivity and specificity using well-characterized soluble agonists. Next, we characterized PRR activation from   The innate immune system is the cell’s frontline mechanism of protection against foreign pathogens.The primary controllers of the innate immune system are pattern recognition receptors (PRRs). Among these, Toll-like receptors (TLRs) are transmembrane receptors on the cell surface, such as TLR4, which recognizes bacterial wall lipids, or in endosomes, such as TLR7, which detects single-stranded RNA (ssRNA). Cytoplasmic PRRs include retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated protein 5 (MDA5), which recognize short double-stranded RNA (dsRNA) with 5′ triphosphorylated (5′ppp) ends or longer dsRNA, respectively. Once activated, PRRs form complexes with adaptor molecules to begin signaling cascades that ultimately lead to type I interferon (IFN) and cytokine induction A.1, 13, 18ELISAs or intracellular immunofluorescence is traditionally used to assess PRR activation by measuring downstream type I IFN and inflammatory cytokines synthesis.in situ with fluorescent oligonucleotides. PLA is sensitive, due to the need of close proximity, and quantitative, as the PLA puncta are measurable. PLA can be used in tissue sections, which is critical for clinical studies.in vivo context.To overcome these limits, we used a proximity ligation assay (PLA) to detect PRR activation in a sensitive, kinetic, and quantitative manner by measuring the formation of specific PRR complexes. While some proteins are ubiquitous across pathways, the complexes they form are unique to each pathway. The formation of such complexes is typically measured by immunoprecipitation, which lacks cell-to-cell variability or spatial information.in vitro-transcribed (IVT) mRNA vaccine model, in both a cellular model and in vivo mouse studies. IVT mRNA encodes for the antigenic protein(s) and is concurrently detected by anti-viral PRRs, which activate innate immune responses, facilitating the development of protective immune responses.in vitro and in vivo during development and screening.Here we applied PLA to the study of immune responses elicited by an in vitro by quantifying TLR7, RIG-I, MDA5, or TLR4 complex formation. Upon activation, TLR7 complexes with myeloid differentiation response gene 88 (MyD88). MyD88 then interacts with the interleukin-1 receptor-associated kinase proteins , forming the myddosome and beginning a pathway that leads to type I IFN and inflammatory cytokine induction. TLR4 can associate with MyD88 and TIR-domain-containing adaptor protein (TIRAP) and follow similar pathways to lead to inflammatory cytokine production. TLR4 also induces type I IFN through a MyD88-independent pathway by associating with TIR-domain-containing-adaptor protein-inducing IFN-β (TRIF) and TRIF-related adaptor molecule (TRAM).PLA’s efficiency and specificity in detecting PRR activation pathways were first assessed Activation of TLR7, RIG-I, and MDA5 was induced via IVT mRNA transfection. As TLR4 is not stimulated by nucleic acids, lipopolysaccharide (LPS) was used as an agonist. PRR activation was quantified in wild-type (WT) cells and cells expressing modulated PRR levels. TLR7 PLA was evaluated in NIH/3T3 cells, which naturally express low levels of TLR7, and in cells transfected with a TLR7-encoding plasmid. RIG-I and MDA5 activation was assayed in RAW-Lucia ISG macrophages, using either the WT cell line or the commercially available RIG-I- or MDA5-KO cell lines. As TLR4-KO cells were not readily available, small interfering RNA (siRNA) was used to knock down TLR4 in RAW 264.7 macrophages for comparison with WT cells. Lipofectamine-only controls were included in each condition. TLR7-IRAK4 interactions were significantly higher in cells that were both stimulated by IVT mRNA and that expressed high levels of TLR7, in comparison to cells with basal levels of TLR7 or unstimulated cells A. SimilaWe subsequently evaluated PLA’s ability to sensitively measure PRR activation kinetics. Well-characterized soluble PRR agonists were delivered to RAW 264.7 macrophages, and PRR activation-induced complexes were measured over several time points. 8 μg/mL imiquimod was used to activate TLR7, 10 μg/mL 5′ppp dsRNA was used to activate RIG-I, while 0.1 μg/mL polyinosinic:polycytidylic acid [poly(I:C)] was utilized as an MDA5 agonist. 1 μg/mL LPS was used to stimulate the TLR4-TIRAP pathway, while 10 μg/mL LPS was used to stimulate the TLR4-TRAM pathway. To assess kinetics, PRR activation was measured at 30 min and 6 hr after delivery of the agonists. Additionally, at 6 hr, the agonists were removed, and the cells were incubated with fresh media for 3 hr to allow for the recovery of base levels of PRR activation. PRR activation was detected for TLR7, RIG-I, MDA5, and both TLR4 pathways as early as 30 min after agonist delivery A and S1.By fluorescently labeling organelles prior to PLA, we demonstrated the colocalization of PLA puncta with endosomes and mitochondria B. TLR7-INext, we compared PRR activation by PLA to tumor necrosis factor alpha (TNF-α) production by ELISA. RAW 264.7 macrophages were stimulated with 8 μg/mL imiquimod, 10 μg/mL 5′ppp dsRNA, 0.1 μg/mL poly(I:C), and 1 or 10 μg/mL LPS. Supernatants were collected and cells were fixed at 30 min or 6 or 24 hr post-stimulation. An ELISA was performed for each time point C. TNF-α Critically, only the expected agonists triggered specific pathways when measured by PLA, with the exception of TLR7-IRAK4, whose activation was also observed upon the delivery of poly(I:C), likely due to the transfection agent needed for its cellular entry. These data demonstrate that PLA is superior to conventional methods, such as ELISAs, due to its ability to isolate specific pathways, as well as to detect early activation kinetics.We next explored PLA’s sensitivity in quantifying innate immune responses to delivered IVT mRNA. It has been shown that incorporating modified nucleosides into IVT mRNA reduces PRR activation.We also examined PLA’s sensitivity in quantifying PRR activation due to different IVT mRNA sequences. GFP- or luciferase-encoding IVT mRNA, both containing modified nucleosides but with different 3′ UTRs and coding regions, was delivered to RAW 264.7 macrophages with Lipofectamine 2000 and fixed after 30 min and 6 hr. Differences in IFN-α production between GFP and luciferase mRNAs have previously been discussed in the literature with both unmodified and modified mRNAs.Next, we aimed to quantify PRR activation via PLA in muscle tissue after intramuscular delivery of mRNA in mice. To visualize mRNA distribution in the muscle, IVT mRNA encoding for firefly luciferase was pre-labeled with MTRIPs targeting three regions within the 3′ UTR A and S3B+ cells in the muscle  either naked or formulated with the nanoparticle CholK, for improved delivery, especially to lymph nodes over naked delivery.e muscle B. Mice iBased on the upregulated expression of PRRs as evidenced by immunofluorescence, as well as by the uptake of mRNA by infiltrating immune cells, we next examined the PRR response via PLA at 1.5, 6, and 16 hr following i.m. injection of fluorescently labeled IVT mRNA, either naked or complexed with CholK, as well as after a sham injection. We noticed that, in tissue sections, PRR activation did not result in the formation of single PLA puncta but rather in large aggregates. Therefore, PRR activation was quantified by measuring the total PLA signal volume.Naked mRNA induced significant TLR7 activation at 6 hr and RIG-I activation at 16 hr post-delivery, respectively . mRNA fo+, CD11b+, and CD19+CD3− (B cell) lymph node cells. Next, PRR activation was assessed in the lumbar aortic lymph nodes excised 16 hr post-injection  was used on euthanized mice to determine IVT mRNA trafficking to the draining lymph nodes  and, spenjection . Both nain vitro, as well as over time in response to known agonists. This validated the specificity of PLA to detect individual PRR pathways, as interactions unique to each pathway were identified and quantified, indicating both the antibodies’ specificity for the target complex molecules and the assay’s specificity for complex formation. Furthermore, this demonstrated the versatility of PLA, as it was successfully optimized for several PRR pathways whose activation occurred in distinct cellular compartments: TLR4 on the cell surface membrane, TLR7 in the endosome, and RIG-I and MDA5 in the cytoplasm. Immunofluorescence colocalization of PLA puncta with cellular organelles further verified the ability of PLA to distinguish between pathways in different cellular compartments. This provides support to the translatability of PLA to detect other PRR pathways not detailed in this work.Here we demonstrated the use of PLA as a sensitive, quantitative, and specific tool to measure PRR activation in cells and in tissue upon IVT mRNA delivery. First, we compared PRR activation with modulated protein levels in vitro. This sensitivity is critical because the formation of these complexes is transient and unique to each pathway. PLA was able to specifically identify the individual pathway, as interactions for each pathway were not detected if non-expected agonists were used. Furthermore, the results support the sensitivity of PLA to distinguish levels of PRR activation over time at earlier time points than ELISAs. Comparing the strength of responses induced by two candidate agonists, for example, would provide critical information during vaccine and therapeutic development.PLA was also shown to be able to distinguish TLR4 sub-pathways, confirming its ability to isolate unique activation routes. Moreover, PLA successfully detected activation kinetics and quantitatively measured the turning on and turning off of individual PRR pathways Subsequently, we examined PLA’s suitability in measuring the PRR response of synthetic IVT mRNAs. We compared unmodified mRNA to mRNA with modified nucleosides previously seen to be immunomodulatory.We also demonstrated that PLA can measure differences in PRR activation induced by two model reporter mRNAs. We confirmed that mRNA sequence is a determinant in PRR activation, supporting differences in IFN-α production previously observed in the literature.Both charge ratio and size of lipoplexes have been observed to affect transfection time and efficiency.in vitro is that they could be performed in a 96-well glass-bottom plate using the manufacturer’s protocol. Several interacting protein pairs can be assessed in the same plate and imaged directly in the plate. The Volocity acquisition software could be set to automatically acquire images in each well, allowing for an automated imaging process. As a result, PLA allows the measurement of PRR activation in a high-throughput screening fashion, establishing it as an ideal approach to quickly characterize agonist-induced PRR activation in cells prior to their use in in vivo experiments.Another advantage of PLA measurements in vivo in tissue. First, we verified the successful delivery of the IVT mRNA into muscle via i.m. injection, and we assessed the mRNA-induced recruitment of immune cells. CholK-mediated delivery enhanced immune cell infiltration over naked delivery, as supported by flow cytometry and immunofluorescence. Furthermore, mRNA complexed with CholK was retained in the muscle longer than free mRNA, inducing a stronger RIG-I response at 6 hr and a stronger MDA5 activation at 16 hr. We hypothesize that CholK enhanced cytoplasmic delivery of the mRNAs in tissue, causing the stronger cytoplasmic PRR responses of complexed mRNA over naked mRNA, as supported by our prior work.We next demonstrated the use of PLA as a PRR quantification tool Type I IFN is also known to promote migration of antigen-presenting cells to lymph nodes.in vitro in cells and in tissue sections. This valuable tool allows for sensitive, specific, and quantitative measurements of the kinetics of PRR activation. PLA can be used in a high-throughput screening manner, to compare the effect of multiple agonists, adjuvants, or therapies easily and rapidly in vitro. Finally, it allows insights into the spatial context where the activation occurs, both in single cells and across whole tissues. We believe PLA has the potential to be a crucial tool in therapeutic and vaccine development.In conclusion, we demonstrated PLA’s use as a method of detection of innate immune responses both Primary antibodies used were as follows: mouse anti-RIG-I , goat anti-MDA5 , rabbit anti-MAVS , rabbit anti-TLR7 , mouse anti-IRAK4 , rabbit anti-TLR4 , goat anti-TIRAP , and goat anti-TRAM .− control. After 48 hr, cells were transfected with 2.5 μg luciferase IVT mRNA/well using Lipofectamine 2000; 24 hr later, cells were fixed with 1% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and then assayed for TLR7 and IRAK4 proximity.NIH/3T3 mouse fibroblast cells  were maintained in High Glucose DMEM  with 20% bovine calf serum . NIH/3T3 cells do not express detectable levels of TLR7RAW-Lucia ISG mouse macrophage cells, which express both RIG-I and MDA5, were used as WT. RAW-Lucia ISG-KO-RIG-I cells and RAW-Lucia ISG-KO-MDA5 cells were used as KO controls. All RAW-Lucia ISG cell lines were purchased from InvivoGen and maintained in DMEM with 10% fetal bovine serum (FBS)  and 100 μg/mL Normocin . In all cases, cells were seeded onto glass coverslips in a 24-well plate and transfected with 2.5 μg luciferase IVT mRNA per well using Lipofectamine 2000. Cells were fixed with ice-cold methanol at 24 hr following IVT mRNA transfection for 15 min at −20°C, permeabilized with 0.2% Tween-20 for 5 min, and then assayed for RIG-I and MDA5 proximity with MAVS. We found that methanol fixation, rather than paraformaldehyde fixation, was essential for performing the MDA5 and RIG-I PLAs (data not shown).RAW 264.7 macrophages , a commonly used cell line for studying the immune response, were used. They were maintained in DMEM with 10% FBS and 1% penicillin streptomycin (PS) . Cells were transfected with and without 100 nM TLR4 siRNA  delivered with Lipofectamine 2000. Cells were plated in a 96-well glass-bottom plate. 24 hr after siRNA delivery, 1 μg/mL LPS was added to TLR4-TIRAP conditions and 10 μg/mL was added to TLR4-TRAM conditions. After 30 min, cells were fixed with 1% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and then assayed for TLR4 proximity with TIRAP or TRAM.After permeabilization, cells were blocked against nonspecific binding with 1% BSA, 2% donkey serum, and 0.2% gelatin in 1× PBS for 30 min at 37°C. Primary antibodies were then incubated with cells in 1% BSA, 0.2% donkey serum, and 0.2% gelatin in 1× PBS at the indicated concentrations for 30 min at 37°C. Cells were washed in wash buffer A  for 10 min at room temperature (RT) and incubated with secondary antibodies  at manufacturer-recommended concentrations in 0.2% Tween-20 in 1× PBS for 30 min at 37°C. Cells were again washed in wash buffer A for 10 min at RT and then incubated with the ligase and ligase buffer, as specified by the manufacturer  for 30 min at 37°C. Next, cells were washed in wash buffer A for 10 min at RT and incubated with the polymerase and rolling circle amplification buffer, as specified by the manufacturer  for 100 min at 37°C. Finally, cells were washed in wash buffer B  for 20 min at RT and then 0.01× buffer B for 4 min at RT. Mounting medium with DAPI  was added and cells were imaged.In vitro images were taken using a Hamamatsu Flash 4.0 version (v.)2 sCMOS camera on a PerkinElmer UltraView spinning-disk confocal microscope on a Zeiss Axiovert 200 M body. A 63×, numerical aperture (NA) 1.4 Zeiss Plan-Apochromat oil objective was used for all images. Imaging was controlled by Volocity acquisition software . Image stacks were recorded at 200-nm intervals.After images were acquired, Volocity acquisition software was used to draw regions of interest (ROIs) around each cell. Volocity software then was used to quantify the number of fluorescent puncta in each ROI above a specified signal threshold. Thirty cells were counted for each experimental condition. Two different statistics methods were used, depending on the number of time points in the experiment. For single-time point experiments, a one-way Kruskal-Wallis ANOVA test was used with a Dunn’s multiple comparisons test. For multiple-time point experiments, a two-way ANOVA was used with a Tukey’s multiple comparisons test. Statistics were performed in GraphPad Prism 7.0.RAW 264.7 macrophages were plated in a 96-well glass-bottom plate. Soluble agonists included 8 μg/mL imiquimod  for TLR7, 10 μg/mL 5′ppp dsRNA and LyoVec  for RIG-I, 0.1 μg/mL poly(I:C) and LyoVec  for MDA5 agonist, 1 μg/mL LPS  for the TLR4-TIRAP pathway, and 10 μg/mL LPS  for the TLR4-TRAM pathway. Cells were fixed at 30 min and 6 hr after delivery with 1% paraformaldehyde for 10 min for TLR7 and TLR4 or ice-cold methanol at −20°C for 20 min for RIG-I and MDA5. Also at 6 hr, agonists were removed and fresh media were added to cells for 3 hr before fixing with paraformaldehyde or methanol. Cells were then permeablized as before and assayed for their respective interactions.A549 cells  were incubated with CellLight Early Endosome-GFP, BacMAM 2.0  at 50 particles per cell for 16 hr prior to stimulation. Cells were then stimulated as indicated above for TLR7 and TLR4 and fixed 30 min post-delivery with 1% paraformaldehyde for 10 min. Cells were then permeablized as before and assayed for their respective interactions. RAW 264.7 macrophages were incubated with 200 nM MitoTracker Deep Red FM  for 15 min and then stimulated with 0.1 μg/mL poly(I:C) and LyoVec for 30 min before fixation with ice-cold methanol at −20°C for 20 min. Cells were then permeablized as before and assayed for their RIG-I or MDA5 interactions.RAW 264.7 macrophages were plated in a 96-well glass-bottom plate. Soluble agonists included the following: 8 μg/mL imiquimod , 10 μg/mL 5′ppp dsRNA and LyoVec , 0.1 μg/mL poly(I:C) and LyoVec , and 1 and 10 μg/mL LPS . Supernatants were collected at 30 min and 6 and 24 hr post-stimulation before fixation, as previously described. A TNF-α ELISA  on the supernatants was then performed according to the manufacturer’s instructions.IVT mRNA encoding firefly luciferase was provided by CureVac AG . It included a 5′ cap, the 3′ UTR from human albumin, as well as a poly(A) tail. All GFP mRNA contained identical sequences, a 5′ cap, the 3′ UTR sequence from mouse alpha globin, and a poly(A) tail. If modified, IVT mRNAs were made with 5-methylcytosine and pseudouridine.RAW 264.7 macrophages were plated in a 96-well glass-bottom plate. 200 ng per well of modified IVT luciferase mRNA, modified IVT GFP mRNA, or unmodified IVT GFP mRNA was delivered with Lipofectamine 2000. Cells were fixed, permeablized, and assayed for interactions at 30 min or 6 or 24 hr after delivery, as previously detailed.in vivo experiments was labeled with MTRIPs for the detection by histology. A cartoon showing MTRIP assembly is shown in Luciferase IVT mRNA for CholK was prepared in five steps starting from Kanamycin A, according to the schematic described in 3N  and a solution of N-benzyloxycarbonyloxy-5-norbornene-2, 3-dicarboximide  in dimethylformamide (DMF) (15 mL) were successively added, and the mixture was stirred at RT for 24 hr. A solution of 2-(trimethylsilyl)ethyl-4-nitrophenylcarbonate  in DMF (20 mL) and more Et3N  were then added, and the yellow solution was stirred at 55°C for 48 hr. The mixture was then concentrated and the residue taken up with EtOAc, before washing with NaOH 1M (3 times), water, and brine. The organic fraction was dried over MgSO4 and concentrated in vacuo. The residue was purified by flash chromatography (dethylene dichloride [DCM]/methanol [MeOH]) to afford the expected product . 1H nuclear magnetic resonance (NMR)  results were as follows: δ 0.02–0.05 , 0.98–1.02 , 1.50–1.53 , 2.04–2.08 , 3.19 , 3.41–3.75 , 4.11–4.16 , 5.03–5.10 , 7.29–7.38 ; mass spectrometry (MS) (ESI+) 1,074.5 [M+Na]+.Kanamycin A  was dissolved in water (10 mL). EtKanamycin-Cbz-Teoc3  in degassed wet MeOH  was added 10% Pd/C . 3 cycles of vacuum-N2 were applied, followed by 3 cycles of vacuum-H2. The mixture was stirred overnight, then filtered over a pad of cellite and washed with EtOH. The filtrate was concentrated in vacuo and the residue purified by flash chromatography (DCM-MeOH) to afford the expected free amine . 1H NMR  results were as follows: δ 0.00–0.02 , 0.93–0.95 , 1.46–1.52 , 1.98–2.07 , 2.59–2.64 , 2.92–2.96 , 3.04–3.09 , 3.32–3.57 , 3.60–3.69 , 4.03–4.13 , 5.01 , 5.12 ; MS (ESI+) 939.6 [M+Na]+.To a solution of 3N  and cholesteryl chloroformate  were added to a solution of Kanamycin-Teoc3  in tetrahydrofuran  and DMF (15 mL). The mixture was stirred at RT overnight, then concentrated in vacuo. The residue was purified by flash chromatography (DCM-MeOH) to afford the expected product . 1H NMR  results were as follows: δ 0.00–0.02 , 0.67 , 0.82–1.59 , 1.84–2.02 , 2.27–2.29 , 3.07–3.09 , 3.33–3.39 , 3.53–3.69 , 3.86–3.89 , 4.02–4.11 , 4.55 , 5.01–5.01 , 5.34 ; MS (ESI+) 1,352.0 [M+Na]+.Et(Cholesteryloxy-3-carbonyl)]Kanamycin-Teoc3  in DCM (6 mL) was slowly added trifluoroacetic acid  at 0°C. The solution was stirred at RT for 2 hr and then concentrated. After 3 consecutive additions of MeOH and subsequent evaporations, the residue was purified by flash chromatography  and lyophilized to give CholK . 1H NMR  results were as follows: δ 0.72 , 0.87–1.63 , 1.85–2.07 , 2.32–2.34 , 2.45–2.55 , 3.17–3.19 , 3.35–3.88 , 4.30–4.50 , 5.10 , 5.26 , 5.38 ; MS (ESI+) 919.4 [M+Na]+.To a suspension of CholK was prepared by In-Cell-Art . To prepare RNA-CholK nanoparticles, IVT mRNA and CholK were individually diluted in RiLa to 20 μL and mixed to generate a low charge ratio (+/−) particle (10 μg mRNA and a 1:10 charge ratio [+/−] of CholK:mRNA). The CholK solution was then added to IVT mRNA, mixed well, and allowed to incubate for 15 min prior to injection. Size and zeta potential of particles were measured with the Malvern Instruments Zetasizer Nano ZS. Particles were diluted in PBS to measure size or deionized water to measure zeta potential.Female BALB/c mice  were anesthetized with 2.5% isoflurane. The region around their anterior tibialis was shaved and then swabbed with isopropanol. 10 μg (40 μL) IVT mRNA diluted in RiLa was then injected into the anterior tibialis using a 29G needle. The alternate leg served as a sham injection control. Mice were housed and manipulated under specific-pathogen-free conditions in the animal care facilities of Georgia Institute of Technology. All experiments were in accordance with the Institutional Animal Care and Use Committee.For histological PLAs, samples were initially deparaffinized. Antigen retrieval and permeabilization were performed prior to PLA as described above. To perform PLA, samples were blocked for 1 hr with a solution containing 0.5% Tween-20, 0.1% Triton X-100, 0.1% gelatin , 2% donkey serum , and 1% BSA  in PBS. Samples were then stained with the indicated antibodies  overnight at 4°C, diluted 1:500 in a solution of 0.25% gelatin, 0.5% donkey serum, and 1% BSA in PBS. All remaining steps were in accordance with the manufacturer’s instructions .Images were acquired with an UltraVIEW spinning disk confocal microscope utilizing a Hamamatsu Flash 4.0 v.2 sCMOS camera. A 40×, NA 1.3 Zeiss EC Plan-Neofluar oil objective was used for all tiled images, and a 63×, NA 1.4 Zeiss Plan-Apochromat oil objective was used for all other images. Imaging was controlled by Volocity acquisition software . Image stacks were recorded at 350-nm intervals. Tiled images were stitched using Volocity. Six images were acquired of each tissue section, and a total of four animals were used per group per time point. The PLA signal-imaging frame was quantified using Volocity software. The PLA puncta were identified as objects with a signal above a minimum threshold. The sum of PLA signal volume was measured for each frame. Two different statistical methods were used, depending on the number of time points in the experiment. For single-time point experiments, a one-way Kruskal-Wallis ANOVA test was used with a Dunn’s multiple comparisons test. For multiple-time point experiments, a two-way ANOVA was used with a Tukey’s multiple comparisons test. Statistics were performed in GraphPad Prism 7.0.RAW 264.7 macrophage cells were seeded onto 96-well plates. Cells were transfected with 250 ng/well modified or unmodified GFP IVT mRNA with and without MTRIP labels using Lipofectamine 2000. Cells were processed for PLAs 2 hr later, as described above.Extracted tissue was fixed overnight using 4% paraformaldehyde in PBS. Tissue was embedded in paraffin and sectioned to 5 μm thick. To stain tissue, sections were deparaffinized, antigen retrieval was performed in citrate buffer  for 20 min under steam, and then tissue was permeabilized in PBS with 0.1% Tween-20  (PBST) for 10 min. Tissue was blocked with 5% donkey serum in PBST, incubated with primary antibodies (at a 1:200 dilution) overnight at 4°C in PBST, washed three times in PBST, incubated with the secondary antibody for 1 hr in PBST, stained with DAPI, and then mounted with prolong gold .Lymph nodes and muscle were removed with the assistance of the Fluobeam NIR imaging system , at the indicated time points. Muscle tissue was dissociated with Type IV collagenase  for 1.5 hr at 37°C with gentle agitation. Lymph node and muscle samples were then dissociated by straining through a 40-μm cell strainer with gentle agitation from a syringe plunger. Cells were washed in PBS + 2% FBS, blocked with Fc block  according to the manufacturer’s directions, and then stained with a panel of antibodies diluted 1:100 in PBS with Fc block for 30 min on ice. Cells were washed with PBS + 2% FBS, fixed with 4% paraformaldehyde in PBS for 10 min, washed with PBS + 2% FBS, and stored at 4°C until analysis. Flow cytometry was performed with a BD LSRII and analyzed with FlowJo software. Samples were stained using two different antibody panels, either anti-F4/80 BV421, anti-LYVE1 Alexa 488 , I-Ad PE, anti-CD11c APC-Cy7, and anti-CD11b Alexa 647 or anti-CD3 BV421, anti-CD45R Alexa 488 , anti-CD335 PE, anti-CD11b Alexa 647, and anti-CD19 APC-Cy7. IVT mRNA was labeled with DyLight 680. All antibodies were purchased from BD Biosciences unless otherwise noted. ANOVA followed by Hsu’s multiple comparison with best post analysis was performed to determine significance using JMP Pro software .E.L.B., K.H.L., and P.J.S. planned the experiments and interpreted the data. E.L.B., K.H.L., S.M.B., and D.V. performed experiments. P.B. provided RNA and B.P. provided delivery reagents. E.L.B., K.H.L., C.Z., and P.J.S wrote and edited the paper.P.B. is an employee of CureVac, which commercializes RNA-based vaccines. B.P. is an employee of In-Cell-Art, which commercializes lipidic aminoglycoside derivatives.


Average urine concentrations were 47.7 ± 39.3 and 417 ± 296 μg/L for cyclopropylfentanyl and cyclopropyl norfentanyl, respectively (n = 11), 995 ± 908 μg/L for methoxyacetylfentanyl, (n = 3), 1,874 ± 1,710 and 6,582 ± 3,252 μg/L for acetylfentanyl and acetyl norfentanyl, respectively (n = 5), 146 ± 318 and 300 ± 710 μg/L for fentanyl (n = 5) and norfentanyl (n = 6), respectively, 84.0 and 23.0 μg/L for furanylfentanyl and furanyl norfentanyl, respectively (n = 1), and 50.5 ± 50.9 μg/L for 4-ANPP (n = 10). Average hair concentrations were 2,670 ± 184 and 82.1 ± 94.7 ng/g for fentanyl and norfentanyl, respectively (n = 2), and 10.8 ± 0.57 ng/g for 4-ANPP (n = 2).Recently, synthetic opioid-related overdose fatalities, led by illicitly manufactured fentanyl and analogs, increased at an alarming rate, posing a global public health threat. New synthetic fentanyl analogs have been constantly emerging onto the drug marked for the last few years, to circumvent the laws and avoid analytical detection. Analytical methods need to be regularly updated to keep up with the new trends. In this study, we aimed to develop a new method for detecting the newest fentanyl analogs with a high sensitivity, in whole blood, urine, and hair. The method is intended to provide to clinical and forensic toxicologists a tool for documenting consumption. We developed a comprehensive ultra-high-performance liquid chromatography-tandem mass spectrometry method for quantifying fentanyl and 22 analogs and metabolites. Urine samples were simply diluted before injection; a liquid-liquid extraction was performed for blood testing; and a solid phase extraction was performed in hair. The chromatographic separation was short (8 min). The method was validated with a high sensitivity; limits of quantifications ranged from 2 to 6 ng/L in blood and urine, and from 11 to 21 pg/g in hair. The suitability of the method was tested with 42 postmortem blood, urine, or hair specimens from 27 fatalities in which fentanyl analogs were involved. Average blood concentrations (±SD) were 7.84 ± 7.21 and 30.0 ± 18.0 μg/L for cyclopropylfentanyl and cyclopropyl norfentanyl, respectively ( Fentanyl is a μ-opioid receptor agonist with strong anesthetic and analgesic properties, with a 50- to 100-fold higher potency than that of morphine. It has been used as a medication for pain management since the 1960s and has been among the most prescribed opioids for the last 3 decades  they are likely taken in combination with another opioid and may be unnoticed  to quantify fentanyl and 22 analogs and metabolites in whole blood, urine, and hair. We aimed to provide a fast, simple, and sensitive analytical tool for clinical and forensic toxicologists to document the consumption of the most recent fentanyl analogs described in the scientific literature: acetylfentanyl, acetyl norfentanyl, alfentanil, butyrylfentanyl, butyrylfentanyl carboxy metabolite, butyryl norfentanyl, carfentanil, cyclopropylfentanyl, cyclopropyl norfentanyl, despropionylfentanyl (4-ANPP), despropionyl para-fluorofentanyl, fentanyl, furanylfentanyl, furanyl norfentanyl, furanylethyl fentanyl, β-hydroxyfentanyl, β-hydroxythiofentanyl, methoxyacetylfentanyl, methoxyacetyl norfentanyl, norfentanyl, phenylacetyl fentanyl, sufentanil, and valerylfentanyl carboxy metabolite) and deuterated internal standards  were purchased from Cayman Chemical  and stored at −20°C until use. LC-MS grade water, acetonitrile, methanol, and formic acid and LC grade acetone and dichloromethane were obtained from Sigma-Aldrich® . Ammonium acetate buffer was prepared with ≥97% purity ammonium acetate salt (Sigma-Aldrich®) dissolved in LC-MS water. VMA-T M3®, Washing Solution, and Multimatrix Eluent were acquired from Comedical® s.r.l. ; their composition is not disclosed.Working standards . Low, medium, and QC working solutions were daily prepared from the standard stock solution in methanol. IS working solution was daily prepared from the IS stock solution in methanol to reach a concentration of 5 μg/L in urine and blood samples and 5 ng/g in hair samples.Blank human blood, urine, and hair were obtained from the laboratory storehouse of blank biological samples. Pools of blank samples were prepared using 20 different post mortem blood, urine, or hair samples from the Section of Legal Medicine , pre-screened for the presence of any drug of abuse and pharmaceutical. Postmortem blood, urine, and hair specimens from authentic cases of consumption were provided as discarded material by the Institute of Forensic Medicine of Strasbourg (France), and the Department of Medical and Health Sciences, Division of Drug Research of Linköping University (Sweden). Demographics, detection of other drugs, and cause of death were not specified.v/v) in polypropylene microcentrifuge tubes. Tubes were capped, vortexed for 10 s, and centrifuged at 15,000 g for 5 min. Supernatants were transferred onto conical glass tubes and evaporated to dryness under nitrogen at 45°C. Samples were reconstituted with 1 mL mobile phase A:B 95:5 (v/v) and centrifuged at 15,000 g for 5 min. Supernatants were transferred into autosampler glass vials, prior to injection onto the chromatographic system.Blood samples (100 μL) were fortified with 5 μL IS working solution, 70 μL M3® reagent , tubes were capped, vortexed for 10 s, and centrifuged at 15,000 g for 5 min. Supernatant was transferred into autosampler glass vials, prior to injection onto the chromatographic system.Urine samples (100 μL) were fortified with 5 μL IS working solution in conical glass tubes and vortexed. After adding 3 mL mobile phase A:B 95:5 (Hair samples were washed twice with dichloromethane and dried under nitrogen at 45°C. An amount of 25 mg was cut into pieces (<5 mm) in glass tubes and fortified with 5 μL IS working solution. After addition of 500 μL M3® reagent, tubes were capped and incubated at 100°C for 1 h, for complete hair digestion. Tubes were cooled down at room temperature and samples underwent solid phase extraction on 30 mg/1 mL Oasis® PRIME HLB cartridges (Waters®): samples were diluted with 0.5 mL M3® reagent and loaded onto the cartridges; cartridges were then washed with 0.5 mL Comedical® Washing Solution and dried under nitrogen; elution was performed with 0.5 mL Multimatrix Eluent. Eluates were diluted with 9.5 mL water and 1 mL was transferred into autosampler glass vials, prior to injection onto the chromatographic system.UHPLC-MS/MS analysis was performed on a Waters® Xevo® TQ-S micro mass spectrometer (triple quadrupole) equipped with an electrospray ionization source in positive ion mode (ESI+) and interfaced with an ACQUITY UPLC® I-Class . Data were acquired with MassLynx® software version 4.1 (Waters®).18 column from Waters® . Run time was 8 min with a gradient mobile phase composed of 0.1% formic acid in 5 mM ammonium acetate buffer (A) and 0.05% formic acid in acetonitrile (B) at a flow rate of 0.35 mL/min. Initial conditions were 5% B, held for 1 min, increased to 30% B within 3.5 min, increased to 95% B within 0.5 min, held for 0.5 min, returned 5% B within 0.1 min, and then held for 2.4 min. LC flow was directed to waste the first 1.5 min of the separation and after 6 min. Autosampler and column oven temperatures were 10 and 50°C, respectively. The injection volume was 10 μL for blood and urine samples, 1 μL for hair samples.Separation was performed on an ACQUITY UPLC® BEH CThe mass spectrometer operated in scheduled multiple reaction monitoring (MRM) mode, with two transitions for each analyte and one transition for each IS . MS parav/v) before sample treatment. Calibration points and QC samples were prepared by two different staff members.The method was validated in whole blood, urine, and hair following the most recent criteria for method development and validation in analytical toxicology ; average urine concentration was 50.5 ± 50.9 μg/L (n = 10); average hair concentration was 10.8 ± 0.57 ng/g (n = 2). Samples from 12 cases tested positive for cyclopropylfentanyl and cyclopropyl norfentanyl; average blood concentrations were 7.84 ± 7.21 and 30.0 ± 18.0 μg/L, respectively (n = 8); average urine concentrations were 47.7 ± 39.3 and 417 ± 296 μg/L, respectively (n = 11). Samples from 8 cases tested positive for fentanyl and norfentanyl and one case for norfentanyl only; blood concentrations were 33.7 and 7.17 μg/L, respectively (n = 1); average urine concentrations were 146 ± 318 and 300 ± 710 μg/L, respectively ; average hair concentrations were 2,670 ± 184 and 82.1 ± 94.7 ng/g, respectively (n = 2). Samples from 5 cases tested positive for acetylfentanyl and acetyl norfentanyl; average blood concentrations were 40.2 ± 38.6 and 44.5 ± 21.1 μg/L, respectively (n = 3); average urine concentrations were 1,874 ± 1,710 and 6,582 ± 3,252 μg/L, respectively (n = 5). Samples from 4 cases tested positive for methoxyacetylfentanyl; average blood concentration was 4.08 ± 2.30 μg/L (n = 4); average urine concentration was 995 ± 908 μg/L (n = 3). One sample tested positive for furanylfentanyl and furanyl norfentanyl; blood concentrations were 3.60 and 0.90 μg/L, respectively; urine concentrations were 84.0 and 23.0 μg/L, respectively. One sample tested positive for sufentanil; blood concentration was 0.67 μg/L.Fentanyl analogs' concentrations measured in authentic human specimens are reported in 18 column, following an automated solid phase extraction (0.5 mL urine) with C18 cartridges. The method was successfully validated for 8 analogs with acceptable accuracy and precision; LODs ranged from 00.003 to 0.07 μg/L and LOQs were 0.01 μg/L; recoveries ranged from 64 to 114% and ME ranged from 11 to 96%  , and the extracts were analyzed by LC-MS/MS with an 11 min separation on a C18 column and a data-dependent acquisition. The method was validated for 10 analogs with insufficient sensitivity for several analytes (LODs ranged from 0.1 to 0.5 μg/L in blood)   , but higher voltages did not provide a better signal so we opted for a low voltage to limit unwanted side reactions and preserve the source. The same capillary voltage was used in other published methods for quantifying fentanyl and analogs in blood and urine, using Waters® technology , although the drug has been on the drug market for only 2 years , acetylfentanyl (n = 5), methoxyacetylfentanyl (n = 4), furanylfentanyl (n = 1), and sufentanil (n = 1); the other detected substances were metabolites. Drugs were mostly taken alone, although co-consumption of fentanyl and acetylfentanyl was frequent (n = 4). Demographics, co-administration of drugs, and cause of death were not specified. Moreover, fentanyl is subject to extensive postmortem redistribution . This is not surprising, considering that 4-ANPP is a metabolite of several fentanyl analogs and a well-known chemical intermediary of fentanyl synthesis by Siegfried method . Cyclopropylfentanyl was the most prevalent analog; acetylfentanyl, fentanyl, furanylfentanyl, methoxyacetylfentanyl, sufentanil, and metabolites were also found. Drugs were mostly taken alone, although co-consumption of fentanyl and acetylfentanyl was frequent. We report the first concentrations of cyclopropyl norfentanyl in blood and urine, and the first concentration of furanyl norfentanyl in blood. Cyclopropyl norfentanyl proved to be a good marker of cyclopropylfentanyl intake. On the contrary, methoxyacetyl norfentanyl was not a suitable marker of methoxyacetylfentanyl consumption. Similarly, furanyl norfentanyl was a poor biomarker of furanylfentanyl intake, and 4-ANPP may be more suitable to document furanylfentanyl consumption, although it is also the metabolite of several other fentanyl analogs.FB, MG, and SP designed the study; JC, RG, AT, and RP approved the design. FB, MG, and SP developed the analytical method and RG, AT, and RP revised the method and performed the analyses. JC drafted the manuscript and all the other authors contributed to manuscript intellectual content and revision.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Conus snails produce venoms containing numerous peptides such as the α-conotoxins (α-CTXs), which are well-known nicotinic acetylcholine receptor (nAChR) antagonists. Thirty-eight chromatographic fractions from Conus princeps venom extract were isolated by RP-HPLC. The biological activities of 37 fractions (0.07 µg/µL) were assayed by two-electrode voltage clamp on human α7 nAChRs expressed in Xenopus laevis oocytes. Fractions F7 and F16 notably inhibited the response elicited by acetylcholine by 52.7 ± 15.2% and 59.6 ± 2.5%, respectively. Fraction F7 was purified, and an active peptide (F7-3) was isolated. Using a combination of Edman degradation, mass spectrometry, and RNASeq, we determined the sequence of peptide F7-3: AVKKTCIRSTOGSNWGRCCLTKMCHTLCCARSDCTCVYRSGKGHGCSCTS, with one hydroxyproline (O) and a free C-terminus. The average mass of this peptide, 10,735.54 Da, indicates that it is a homodimer of identical subunits, with 10 disulfide bonds in total. This peptide is clearly similar to αD-CTXs from species of the Indo-Pacific. Therefore, we called it αD-PiXXA. This toxin slowly and reversibly inhibited the ACh-induced response of the hα7 nAChR subtype, with an IC50 of 6.2 μM, and it does not affect the hα3β2 subtype at 6.5 μM. The nicotinic acetylcholine receptors (nAChRs) play important roles in neuronal signaling. They modulate the release of neurotransmitters, such as dopamine, norepinephrine, γ-amino butyric acid, and acetylcholine (ACh). Therefore, they are implicated in a variety of pathophysiologies . ACh is Conus are venomous species distributed throughout tropical and subtropical waters. To date, more than 800 species of cone snails have been described, with more species likely remaining to be discovered ” the distinct annotations of these hits identify them as αD-conotoxins (data not shown). These toxins are covalent dimers of identical or similar polypeptide chains of ~50 residues containing 10 disulfide bonds in total [The BLASTP similarity search revealed 25 significant hits  [Thus, we decided to compare the partial sequence of toxin F7-3, now renamed to αD-PiXXA, to the predicted mature α-D-conotoxin identified in the 549_4_2)  , a free C-termini  , and assConus princeps, a worm-hunter of the Eastern Pacific, from which one toxin, γ-CTX PiVIIA, has been recently purified and characterized at the chemical and electrophysiological levels [C. princeps are predicted to target α7 nAChRs  [This work continues the study of the venom components of l levels ,20. Othel levels ,18,21. Cubtypes) , and theubtypes) , in thisConus species, for example, α-PnIA and α-PeIA of C. pennaceus and C. pergrandis, respectively [There are reports on short (containing four Cys residues) αA-CTXs inhibiting human α7 nAChRs from molluscivorous and piscivorous ectively ,24,25. Iectively ,27,28, bThe use of human α7 nAChRs in biological screening assays is relevant due to the potential application of α-CTXs as potential drugs, or leads for them, for human disease treatments . In thisC. princeps at the protein level and they refer to the same peptide with activity over two different molecular targets [50 = 6.2 μM, whereas it did not significantly affect the hα3β2 at 6.5 μM, which suggests that, regarding these two subtypes, it can be considered to be selective for hα7 nAChRs. However , it might also affect the hα9/α10.As mentioned above, there are only two reports on α-CTXs from  targets ,20. TherThe amino acid sequence of this toxin , unexpectedly, but undoubtedly, revealed that it is an αD-CTX, and, indeed, the first αD-CTX isolated from a species of the Eastern Pacific, αD-PiXXA. The short and long α-CTXs previously mentioned  inhibit the hα7 subtype with higher affinity than αD-PiXXA ,24,27,28Conus vexillum [Conus mustelinus, and αD-Cp from Conus capitaneus [50), but also inhibit α3β2 and other nAChRs subtypes with clearly lower affinities. Nevertheless, it should be pointed out that the effects of all these toxins have been assessed only over rat subtypes. Interestingly, αD-GeXXA from C. generalis has been shown to inhibit the human α9/α10 subtype with higher affinity than the human α7 subtype [A few αD-CTXs from species of the Indo-Pacific  have been characterized at the electrophysiological level: αD-VxXIIB (currently named αD-VxXXB) from vexillum , αD-Ms fpitaneus , inhibit subtype , and αD- subtype .Conus subgenera: Rhizoconus  [Strategoconus  [Elisaconus (litteratus), Stephanoconus , Splinoconus (tribblei) [Ductoconus . Interestingly, αD-conotoxins were not detected in any of the four piscivorous and two molluscivorous species studied by RT-PCR [Strategoconus generalis [Stephanoconus imperialis, Elisaconus litteratus, and Rhizoconus miles [Stephanoconus and Strategoconus), intermediate-  and late-diverging (Elisaconus) among the Conus genus [Rhizoconus species [Conus princeps αD-PiXXA is a minor component of venom duct extract  ,17,34,35anorbis) ,16,35, Eeneralis , Stephanus genus , which ius genus ,13,17 an extract , which sRhizoconus subgenus  are more related to each other than to those from both Strategoconus  and Ductoconus (princeps). This is not unexpected and has also been observed by Prashanth et al. in a study including species belonging to the Stephanoconus, Strategoconus, Splinoconus, and Elisaconus subgenera in a phylogenetic tree including toxins from six Rhizoconus species [In order to gain insight into the structure-activity relationships of these infrequent, dimeric, vermivorous toxins, we compared the sequences of the αD-CTXs that have been characterized at the electrophysiological level. The multiple alignments of PiXXA with VxXXB, Ms, Cp, and GeXXA  clearly  species .Rhizoconus species (even as conserved substitutions), such as Val-2, His-3, Asp-33, and Thr-40 , we cannot point to any of these residues as responsible for the highest affinity of αD-GeXXA for the α9α10 subtype. As mentioned above, the amount of natural toxin prevented testing αD-PiXXA over this subtype.αD-GeXXA is the one toxin that has been tested over α9α10 nAChRs (rat and human) and shown to be more potent over this subtype than over α7 (human) . DespiteC. princeps from Mexican coasts. Like the αD-CTXs from species of the Indo-Pacific , αD-PiXXA has a clear antagonistic effect over α7 nAChRs (human). Due to their remarkably different three-dimensional structure with respect to other nAChR-affecting toxins and their allosteric mechanism of action, αD-conotoxins are expected to contribute to our growing knowledge of ligand interactions with nAChRs [C. princeps could provide novel molecular tools and therapeutic agents, or leads for rationally designing them, for the treatment of a range of neurological disorders that involve these receptors [In summary, this work is the first report of αD-CTXs from species of the Eastern Pacific, specifically from the vermivore h nAChRs . The isoeceptors .Genetic engineering products: Wizard Plus SV Minipreps DNA Purification System was purchased from Promega Corp. ; Not I restriction enzyme from Thermo Fisher Scientific , mMESSAGE mMACHINE kit for high yield capped RNA transcription from Ambion Inc. ; QIAquick PCR Purification and RNeasy Mini kits from Qiagen . Gentamycin was obtained from Life Technologies Co. . High Performance Liquid Chromatography (HPLC) reagents: Trifluoroacetic acid (TFA) was acquired from Thermo Scientific ; HPLC-grade acetonitrile (ACN) from Sigma Chemical Co. . Bradford protein assay kit from Thermo Scientific : oxidized insulin B chain, bovine serum albumin (BSA), type I collagenase, and acetylcholine (ACh) from Sigma Chemical Co. . All other reagents were analytical grade.C. princeps specimens were captured off the coasts of the Mexican Eastern Pacific Ocean  and conserved at −70 °C. The venom ducts of 35 specimens were isolated and homogenized in 4.0 mL of 40% (v/v) ACN in water containing 2.0% (v/v) TFA. The homogenate was centrifuged at 10,000 g for 15 min, and the crude venom extract (supernatant) used for fractionation by HPLC as described below. Protein content was analyzed by the Bradford method using BSA as standard [standard .v/v) TFA) before of the fractionation by Reversed Phase-High Performance Liquid Chromatography (RP-HPLC) in a Waters 600 HPLC System  using a Vydac Peptide & Protein C18 column . The column was equilibrated at room temperature with Solution A. Fractions of the crude venom extract were eluted with a linear gradient from 0% to 100% (v/v) of Solution B (90% ACN in water containing 0.085% (v/v) TFA), over 200 min, after an isocratic step of 0% Solution B for 10 min, at a flow rate of 1 mL/min. The elution profile was monitored at 220 nm. Fractions were quantified by comparison of areas yielded by the HPLC system in comparison with a standard curve of oxidized bovine insulin B chain.Aliquots (1.5 mg of protein/534 μL) of the obtained crude venom extract were centrifuged at 10,000 g for 15 min and the supernatant was taken to 5 mL with Solution A (aqueous solution with 0.1% (v/v) of Solution B, over 200 min, after an isocratic step at 10% Solution B for 10 min at a flow rate of 1.0 mL/min. A second step of purification was done using a linear gradient from 0% to 20% (v/v) of Solution B, over 200 min, after an isocratic step at 0% Solution B for 10 min at a flow rate of 0.8 mL/min in the same HPLC system. The elution profile was monitored at 220 nm. Fractions were quantified as described above for the crude venom extract. The first step of purification was performed by RP-HPLC using a linear gradient from 10% to 30% ; then they were defolliculated with 1.25 mg/mL type 1 collagenase in OR-2 solution for 40 min at room temperature and slowly stired. The Xenopus oocytes were then washed five times with 5 mL of OR-2 solution. Stage VI oocytes were selected and incubated in ND-96 /gentamycin (8 µg/mL) solution, at 15 °C. Oocytes were injected 1 day after harvesting.Ovarian lobes from cDNA clones encoding neuronal human α3 and α7 and β2 nAChR subunits were kindly provided by Dr. J. Michael McIntosh . After amplification, purification, linearization, and purification of linearized cDNAs, cRNA was obtained using the in vitro RNA transcription kit, according to the protocol of the manufacturer. cRNAs were purified and confirmed on 1.5% agarose/buffer MOPS  gels, and stored at −80 °C. For expression of neuronal human α7 and α3β2 at 55.8 ng and 13.3 ng of the corresponding cRNAs, respectively, were injected into each oocyte with a Drummond Nanoject II Auto-Nanoliter injector . Each injected oocyte was incubated in ND-96/gentamycin solution at 15 °C . Oocyte C. princeps chromatographic fractions on neuronal human α7 nAChRs expressed by Xenopus laevis oocytes and the purified peptide on hα7 and hα3β2 nAChRs [n ≥ 3).A two-electrode voltage clamp amplifier was operated  to measure the effects of 2 nAChRs . Resistan = 2−4 oocytes for each data point).The concentration-response curve for the purified toxin was determined with 0.1, 0.2, 0.3, 1.0, 3.0, 6.0, and 10.0 μg . All electrophysiological data were pooled  was determined by a LC-MS system composed by a nano-flow liquid chromatographer Dionex 3000 and a hybrid Orbitrap Velos mass spectrometer with nano-electrospray ion source, both from Thermo-Fisher . Calibration of the mass spectrometer was carried out using Calmix solution consisting of 10 different calibrants allowing measurements with accuracies better than 5 parts per million. The nano-flow chromatographic system was operated at 350 nL/min, using an isocratic gradient of 50% Solvent B  for 30 min. A micro-needle  was used in the nano-electrospray ionization source operating with 2.0 kV of spray voltage.v/v) aqueous ACN containing 1.0% acetic acid. An aliquot of 4 µL was taken and diluted with 10 µL of 37% aqueous ACN. The mixture was then loaded onto a TFA-treated glass fiber disk  previously treated with polybrene  according to manufacturer’s protocol. The Glass Fiber Disk protocol was used for 32 cycles.The amino acid sequence of the purified peptide was determined by automated Edman degradation. A PPSQ-31A Protein Sequencer  was used. The purified peptide  was sequenced without reduction and alkylation; the sample was dissolved in 5 µL of 60%  value was determined using Prism 2.0 , by fitting the data obtained from the concentration-response relationship to the equation (1):R is the response (percent inhibition) at a given concentration (C) of αD-PiXXA, Rmin is the minimum response, Rmax is the maximum response, and IC50 is the concentration that produces 50% of Rmax. Standard errors of the mean were used for this calculation.All analyses were carried out at least in triplicate, mean values ± standard deviations were reported. Differences were statistically accepted at P < 0.05. The half-maximal inhibitory concentration (IC


Purpose: Mitochondrial dysfunction in adipose tissue has emerged as key to the development of obesity and diabetes. Salvianolic acid B  is a water-soluble ingredient derived from Salvia miltiorrhiza that has been shown to possess potential anti-obese and anti-diabetic activities. However, the cellular mechanism of SalB on mitochondrial function with respect to these metabolic disorders has not been elucidated. Therefore, we aim to investigate the effects of SalB on mitochondrial function in 3T3-L1 adipocytes and analyze the underlying molecular mechanism.Methods: The effects of SalB on adipocyte differentiation, glucose uptake, and glycerol release were evaluated in 3T3-L1 adipocytes. Differentiated adipocytes were treated with SalB (50 μM) with or without PPARγ antagonist  for 48 h, and mitochondrial oxygen consumption rate (OCR) as well as extracellular acidification rate (ECAR) were assessed using an XF Extracellular Flux Analyzer. The mitochondrial distribution of adipocytes was assessed using Mito Tracker Green (MTG) and observed under a fluorescent microscope. In addition, the mRNA expression levels of peroxisome proliferators-activated receptor γ/α (PPARγ/α), CCAAT/enhancer binding proteinα (C/EBPα), Nuclear respiratory factor 1/2 (NRF1/2), Uncoupling protein 2 (UCP2), and phosphofructokinase 2/fructose-2, 6-bisphosphatase 2 (PFKFB2) were detected by RT-PCR. Finally, changes in the protein levels of peroxisome proliferators-activated receptor γ coactivator-1α (PGC-1α) were determined by western blotting and immunofluorescence analysis.Results: Treatment with SalB increased glucose uptake and mitochondrial respiration, reduced glycerol release and promoted adipocyte differentiation by increasing mRNA expression of adipogenic transcription factors including PPARγ, C/EBPα, and PPARα. Furthermore, SalB enhanced adipocytes mitochondrial content, mitochondrial respiration and glycolysis capacity, which had been attenuated by GW9662 treatment through the increased expression of PGC-1α.Conclusion: Our results provide novel insights into the role of PGC-1α and mitochondria as probable mediators of SalB activity in the regulation of adipocyte differentiation in 3T3-L1 adipocytes. Obesity is becoming a global public health problem due to the current lifestyle of energy-rich food consumption and physical inactivity, it is predicted that up to 58% of the world’s adult population will be overweight or obese by 2030 . ObesityMitochondria are essential organelles that provide energy for cellular metabolic activity in form of adenosine triphosphate (ATP) and are considered the “power house” of the cell . In adipFigure 1) the most abundant and bioactive member of polyphenolic compounds derived from the root of Salvia miltiorrhiza (Danshen) and has been used widely and successfully in Traditional Chinese Medicine clinical practices for cardio-cerebral vascular diseases . Dulbecco’s modified Eagle’s medium (DMEM), Penicillin-streptomycin and 0.25%Trypsin-EDTA were purchased from Gibco . Fetal bovine serum (FBS) was purchased from Tianhang Biotechnology Co., Ltd. . Dexamethasone (DEX), insulin, and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma . Oil Red O was purchased from Solarbio . Glucose Oxidase Method kit was obtained from Applygen Technologies Inc. . Enzychrom Glycerol Assay Kit was purchased from BioAssay Systems . GW9662 was bought from Selleck . Mito Tracker Green (MTG) was obtained from KeyGEN BioTECH . The primers were designed and synthesized by Sangon Biotech . Antibody to PGC-1α was purchased from Proteintech . Fluorescein-Conjugated Goat anti-Rabbit lgG (H+L) was purchased from ZSGB-BIO .3T3-L1 preadipocytes were grown in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified atmosphere with 5% CO2. After reaching 80% confluence, the cells were induced to differentiate in basic medium containing 10 μg/ml insulin, 0.5 mM IBMX and 0.4 μg/ml DEX for 2 days. The cell culture medium was subsequently exchanged with DMEM containing 10 μg/ml insulin for additional 2 days. Thereafter, the medium was replaced every 2 days until the cells exhibited an adipocyte phenotype. Various concentrations of SalB were added to the culture medium in differentiated adipocytes. The cell viability of SalB was determined using a Cell Counting Kit-8 (CCK-8) assay.In order to investigate the intracellular lipid accumulation, Oil Red O staining was performed. Briefly, differentiated 3T3-L1 adipocytes were treated with SalB . After 48 h, the cells were washed twice with PBS and fixed in 4% formaldehyde at room temperature for 30 min, then incubated for 1 h in filtered Oil Red O solution (isopropanol: water = 3:2). After staining, fat droplets were visualized as a “ring” and the images were obtained by light microscopy . Oil Red O dye was eluted using isopropanol and the absorbance was determined by Microplate Reader.Differentiated 3T3-L1 adipocytes were incubated in DMEM without or with SalB  for 48 h. Samples of the medium were collected and measured for glucose concentration and glycerol release using the Glucose Oxidase Method kit and Enzychrom Glycerol Assay Kit at 24 and 48 h according to the manufacturer’s instructions.4/well in XFe24 microplate coated with 0.2% gelation, the cells were differentiated as described above and treated with drugs for 48 h. The cells were washed twice and media was replaced with XF Assay medium containing 4.5 g/L glucose, 4.0 mM Glutamine and 1.0 mM Sodium pyruvate (adjust the pH to 7.35 ± 0.05 using 1 mol/L NaOH). The plates were placed in a 37°C incubator without CO2 for one hour prior to the assay. OCR measurements were performed using Seahorse Biosciences XFe Analyzer. All experiments were performed at 37°C. After measurement of basal respiration, oligomycin (2.0 μM), FCCP (1.0 μM), rotenone/antimycin A (1.0 μM/1.0 μM) were added sequentially to measure ATP production, maximal respiratory, and non-mitochondrial respiration (NMR), respectively. These respiratory parameters of mitochondrial function were calculated as described previously , oligomycin (5 mM), and 2-DG (100 mM) were added sequentially to determine glycolysis, glycolytic capacity, and glycolytic reserve. The parameters of glycolytic rates were analyzed as described previously .Following exposure of 3T3-L1 cells to SalB (50 μM) and/or GW9662 (20 μM) for 48 h, the cells were washed with PBS, fixed in 4% paraformaldehyde, and permeabilized with 0.2% Triton-X 100 at room temperature. They were then blocked with 10% normal goat serum for 1 h and incubated overnight at 4°C with PGC-1α antibody (1:100 dilution). The secondary antibody, goat anti-rabbit FITC-conjugated (1:50 dilution) was added to the cells followed by incubation for 60 min. Nuclei were stained with DAPI for 5 min and viewed under confocal laser scanning microscope (Zeiss LSM 780). The fluorescence intensity of PGC-1α was calculated using Image Pro Plus software.3T3-L1 adipocytes were lysed in RIPA buffer with protease inhibitor cocktail and quantified using the BCA Protein Assay Kit. Twenty micrograms of total protein per well was separated by SDS-PAGE. The protein was then transferred to PVDF membranes, and then incubated overnight at 4°C with antibodies of PGC-1α (1:500 dilution). Secondary goat anti-rabbit immunoglobulin G antibody was incubated at a dilution of 1:1000 for 1 h. Protein bands were visualized using ImageJ software. β-actin (1:1000 dilution) was used as the internal control.3T3-L1 adipocytes  were treated with SalB (50 μM) and/or GW9662 (20 μM) for 48 h. The cells were then incubated with 200 nM MTG in low-glucose DMEM medium for 30 min at incubator. Mitochondria were viewed under confocal laser scanning microscope. The intensity of MTG fluorescence was analyzed using Image Pro Plus 6.0 software.Table 1. All PCR results were normalized to ARBP gene expression.Total RNA was isolated from 3T3-L1 adipocytes using TriPure reagent  according to the manufacturer’s protocols. Following isolation, 2 μg total isolated RNA was reverse-transcribed to synthesized cDNA using commercial kit. Subsequently, the diluted cDNA was used as the template to amplify the target genes. The samples were analyzed on an ABI 7500 Sequence Detection System. The primers sequences used in the experiments were summarized in P < 0.05.All data were plotted as mean ± SEM using Graph Pad Prism Software (Version 6.0). One-way ANOVA test was performed between multiple group comparisons, followed by Dunnett’s test for the comparison between two groups. Differences were considered statistically significant at Figure 2A, there was no significant difference in adipocyte viability at concentrations of 25, 50, and 100 μM of SalB compared with the control group (P > 0.05). In contrast, cell viability decreased by 17.8% when the cells were incubated in medium with 125 μM SalB (P < 0.01). 3T3-L1 preadipocytes were incubated with the cocktail method without or with SalB  for 8 days, and the accumulated oil droplets were visualized by staining with Oil Red O to identify differentiated cells. Based on microscopic observation, treatment with different concentrations of SalB promoted adipocyte differentiation and contributed to the accumulation of intracellular lipid droplets . In this study, various doses of SalB also increased the mRNA expression of adipogenic transcription factors . In detail, the expression of C/EBPα increased by 170.8% with the addition of 100 μM SalB (P < 0.01), while 25 and 50 μM SalB increased C/EBPα expression of 41.0 and 29.4%, respectively, compared to control group (P > 0.05). Furthermore, the expressions of PPARγ increased by 128.5, 143.9, and 121.4% (P < 0.01) and the expressions of PPARα increased by 80.8, 112.8, and 184.5% (P < 0.01), at dose of 25, 50, and 100 μM, respectively .The cell viability assay was simply and accurately determined using the CCK-8 reagent. As shown in P > 0.05). After 48 h treatment, the glucose uptake increased by 75.8, 97.4, and 66.6% at dose of 25, 50, and 100 μM . Of note, the optimal dose of SalB that increased glucose uptake was at 50 μM when the cells were incubated in medium for 24 h and 48 h. In addition, SalB treatment significantly reduced glycerol release by 33.2, 42.8, and 57.4% when the cells were incubated in medium with 25, 50, and 100 μM SalB for 24 h. Moreover, glycerol release was reduced by 30.4, 53.8, and 60.8% when the cells were incubated in medium with 25, 50, and 100 μM SalB for 48 h . These results suggest that SalB could regulate obesity induced glycolipid metabolic disorders.Glucose uptake increased by 22.7 and 28%, respectively, when the cells were incubated in medium with 25 and 50 μM SalB for 24 h. However, the higher dose SalB (100 μM) did not significantly increase (10.4%) glucose uptake compared to the control group . In detail, as shown in Figure 4B, basal mitochondrial respiration of SalB treated adipocytes were increased by 9.68% (P < 0.05) and 26.9% (P < 0.01), respectively, when the cells were incubated in medium with 25 and 50 μM SalB, while 100 μM SalB treated cells reduced it by 15.6% (P < 0.01) compared to control group. ATP production of SalB concentration 25, 50, and 100 μM increased by 37.5% (P < 0.01), 42.7% (P < 0.01), and 11.2% (P > 0.05), respectively. The H+ leak of SalB treated cells at concentrations of 25 and 50 μM was increased by 16.1% (P > 0.05) and 54.4% (P < 0.01), respectively, and 100 μM SalB reduced it by 56.4% (P < 0.01). The maximal mitochondrial respiration of SalB treated 3T3-L1 adipocytes was increased by 10.8% (P < 0.01) and 22.4% (P < 0.01), respectively, at dose of 25 and 50 μM, while 100 μM SalB decreased it by 16.7% (P < 0.01). The spare respiration capacity at a SalB concentration of 25 μM was increased by 24.6% (P > 0.05), but treatment with 50 and 100 μM SalB reduced it by 59.6% (P < 0.01) and 44.8% (P > 0.05), respectively. Treatment with 25 and 50 μM SalB increased NMR by 15.7% (P < 0.01) and 22.9%(P < 0.01), respectively. However, 100 μM SalB reduced it by 10.7% (P < 0.01).To explore the effect of SalB on mitochondrial respiration, OCR was measured using a Seahorse XF analyzer in 3T3-L1 adipocytes. The OCR of cell treated with SalB (25 and 50 μM) treatment remained higher than control group, but high dose SalB (100 μM) decreased the OCR in 3T3-L1 cells . The positive effect of SalB on mitochondrial respiration was attenuated by treatment with the PPARγ antagonist.To further explore the effects of SalB on mitochondrial function through the regulation of PPARγ activity in 3T3-L1 adipocytes. PPARγ antagonist (GW9692) was added with or without SalB. As shown in Figure 6A and Supplementary Table S3). Treatment with 50 μM SalB for 48 h markedly enhanced the basal rates of glycolysis and glycolytic capacity by 18.3 and 12.1%, respectively (P < 0.05). In addition, treatment with 20 μM GW9692 resulted in a significant reduction in glycolysis and glycolytic capacity by 97.2% (p < 0.01) and 28.3% (p < 0.01), respectively, following oligomycin injection. Interestingly, these two parameters were increased after supplementation with SalB compare to the GW9662 group . However, the glycolytic reserve of ECAR exhibited no significant differences (P > 0.05). We further investigated the genes related to mitochondrial OxPhos and glycolysis. As shown in Figure 7, we found that SalB increased mRNA expression of NRF1, NRF2, UCP2, and PFKFB2 by 96.0, 104.8, 80.9, and 39.2%, respectively, compared with the control group. GW9662 treatment decreased the expression of these genes by 44.1, 19.5, 17.9, and 27.6%, respectively. Furthermore, GW9662 combined with SalB increased the expression of these genes by 122.7, 45.7, 75.1, and 32.5%, respectively, compared with the GW9662 group. These results suggest that SalB can improve mitochondrial glycolytic function through upregulating the related genes expression after inhibiting the PPARγ activation.Extracellular acidification rate was used to determine the changes in the glycolytic rate in 3T3-L1 adipocytes  and GW9662 treatment reduced it by 20.0% (P > 0.05). GW9662 combined with SalB significantly increased fluorescence density by 86.1% (P < 0.01) compared with GW9662 group. In addition, SalB treatment significantly increased the protein expression of PGC-1α by 79.0% (P < 0.01), however, GW9662 treatment reduced its expression level by 35.0% (P < 0.05) compared with the control group. Furthermore, PGC-1α expression was increased approximately 1.63-fold after supplemented with SalB compared to GW9662 group (P < 0.01). These results indicate that SalB increase the expression of PGC-1α and ameliorate damage induced by PPARγ antagonist.As shown in Figure 9, treatment with SalB in 3T3-L1 cells increased the fluorescence intensity by 41.9% (P < 0.01), however, GW9662 significantly reduced it 34.6% (P < 0.01) compared with the control group. When GW9662 combined with SalB, the adipocyte mitochondrial fluorescence intensity increased by 90.4% compared to GW9662 group (P < 0.01).To further investigate the effects on SalB on mitochondrial distribution, we stained adipocytes mitochondria using MTG reagent. As shown in Recently, more research has revealed that many diseases such as obesity, diabetes, neurodegenerative diseases, aging, and cardiovascular diseases have a close relationship with mitochondrial function . MitochoHere, we aimed to investigate whether SalB treatment combined with PPARγ antagonists could further alter mitochondrial morphology, content, respiratory, and glycolytic capacity. Interestingly, our results indicated that SalB significantly enhanced basal respiration, ATP production, uncoupling capacity and glycolysis capacity as well as promoted the 3T3-L1 cell differentiation. Furthermore, SalB also strikingly increased the morphology and quantity of mitochondria especially after the addition of GW9662 (PPARγ antagonist) in 3T3-L1 adipocytes.Adipose tissue is considered to be a major endocrine organ affecting the whole body metabolism, in which oxygen consumption (oxidative phosphorylation) and glycolysis are involved. Fortunately, the assessment of real-time changes in cellular metabolism has been greatly facilitated with the introduction of the XF Seahorse extracellular flux analyzer, which measures OCR and ECAR . MitochoEmerging evidence has demonstrated that PPARγ is a ligand-activated transcription factor that mainly predisposes the adipogenic differentiation molecular network . PPARγ aTaken together, our results suggest that treatment with SalB exerts some protection on mitochondrial dysfunction and adipogenesis through a pathway involving PGC-1α. Furthermore, these also raise the possibility that SalB could be useful as an alternative medicine against obesity and its associated diseases.YP and WZ conceived the study and drafted the manuscript. NY and YP carried out the experiments and performed the statistical analysis. TA, JM, YG, and BL contributed reagents, materials, and analysis tools. FM, JL, CW, and DZ analyzed the data. SG and GJ designed the experiments and reviewed drafts of the paper. All authors gave their final approval for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.