MFN, MFN2, and OPA1. In addition, elevation of the fission genes DRP1 and FIS1 during STZ exposure was prevented. Acetate showed more efficiency in enhancing metabolism and inhibiting ROS, while butyrate had less effect but was stronger in inhibiting the SCFA receptor GPR41 and NO generation. Our data suggest that SCFAs play an essential role in supporting β-cell metabolism and promoting survival under stressful conditions. It therewith provides a novel mechanism by which enhanced dietary fiber intake contributes to the reduction of Western diseases such as diabetes.Islet dysfunction mediated by oxidative and mitochondrial stress contributes to the development of type 1 and 2 diabetes. Acetate and butyrate, produced by gut microbiota via fermentation, have been shown to protect against oxidative and mitochondrial stress in many cell types, but their effect on pancreatic β-cell metabolism has not been studied. Here, human islets and the mouse insulinoma cell line MIN6 were pre-incubated with 1, 2, and 4 mM of acetate or butyrate with and without exposure to the apoptosis inducer and metabolic stressor streptozotocin (STZ). Both short-chain fatty acids (SCFAs) enhanced the viability of islets and β-cells, but the beneficial effects were more pronounced in the presence of STZ. Both SCFAs prevented STZ-induced cell apoptosis, viability reduction, mitochondrial dysfunction, and the overproduction of reactive oxygen species (ROS) and nitric oxide (NO) at a concentration of 1 mM but not at higher concentrations. These rescue effects of SCFAs were accompanied by preventing reduction of the mitochondrial fusion genes  Acetate and butyrate are short-chain fatty acids (SCFAs) and the main large intestinal microbiota fermentation products of soluble fibers. SCFAs have been reported to attenuate various inflammatory responses and metabolism disorders ,2. For eOPA1) and mitofusin (MFN) 1 and 2, and fission proteins, e.g., dynamin-related protein 1 (DRP1) and fission protein 1 (FIS1) . W. W22]. W < 0.05) C, ATP-li < 0.05) D, and sp < 0.01) E. Butyrap < 0.001), reduced ATP production by 63.0 ± 7.8% (p < 0.001), and spare respiration by 58.3 ± 4.7% (p < 0.001) (p < 0.01) higher basal respiration, 53.8 ± 5.6% (p < 0.01) higher ATP-linked respiration, and 46.5 ± 3.5% (p < 0.001) higher spare respiration than cells exposed to STZ without butyrate.To investigate whether the tested SCFAs also protect β-cell respiration under STZ-induced stress, MIN6 cell OCR was quantified before and after exposure to STZ with and without exposure to SCFA. STZ has a strong negative impact on OCR B, and it< 0.001) B–E. AcetIn order to gain insight into the possible mechanisms involved in the rescuing effects of acetate and butyrate on mitochondrial respiratory functions after the STZ exposure of β-cells, we studied the effect of acetate and butyrate on mitochondrial fission and fusion, as these are important processes for functional mitochondria after cell damage . Islets MFN1, MFN2, and OPA1 were found to be statistically significantly downregulated in human islets by respectively 65.3 ± 14.0% (p < 0.01), 75.5 ± 12.5% (p < 0.001), and 57.1 ± 11.8% (p < 0.01)  and 66.0 ± 12.4% (p < 0.001). This indicates that STZ induces a reduction in fusion and a stimulation of fission in human islets. Under STZ-induced mitochondrial stress, acetate prevented against the STZ-induced reduction of the fusion genes MFN1 (p < 0.001) and MFN2 (p < 0.001), which were comparable with the untreated control groups. The reduction of OPA1 was prevented by 45.3 ± 12.9% (p < 0.05). Additionally, acetate prevented the increase in the fission genes DRP1 with 65.3 ± 5.9% (p < 0.01) and FIS1 with 55.5 ± 7.1% (p < 0.05). In addition, butyrate showed similar pro-fusion and anti-fission effects. Butyrate almost completely prevented the STZ-induced decrease in the fusion genes MFN1 (p < 0.01) and MFN2 (p < 0.001) compared to islets exposed to only STZ. The decrease of another mitochondrial fusion gene OPA1 was prevented by 47.1 ± 17.6% (p < 0.05) compared to islets with only STZ-exposure. In addition, butyrate moderated the upregulation of fission gene DRP1 (p < 0.01) to control level and FIS1 by 47.1 ± 17.6% (p < 0.01).However, this was different following STZ exposure. After incubation with STZ, the fusion genes  < 0.01) C. FurtheMFN1, MFN2, and OPA1 were found to be statistical significantly downregulated in β-cells by respectively 23.4 ± 2.7% (p < 0.01), 36.2 ± 5.4% (p < 0.001), and 56.9 ± 2.3% (p < 0.001) (DRP1 and FIS1 were upregulated by 48.1 ± 4.3% (p < 0.001) and 43.9 ± 4.0% (p < 0.001). Both acetate and butyrate showed pro-fusion and anti-fission effects in STZ-treated MIN6 cells, which prevented the decrease in the fusion genes MFN1 (p < 0.001), MFN2 (p < 0.001), and OPA1 (p < 0.001); it also prevented the increase in the fission genes DRP1 (p < 0.001) and FIS1 (p < 0.001) compared to cells with only STZ-exposure.We repeated the experiments with MIN6 β-cells. In addition, here, the presence of STZ showed a pro-fission and anti-fusion effect. The fusion genes < 0.001) D. The exp < 0.001) (p < 0.001) as well as by butyrate (p < 0.001). Notably, acetate showed a significantly more effective anti-ROS (reactive oxygen species) effect in human islets (p < 0.05). STZ had a strong impact on NO synthesis in human islets, which was enhanced by 68.6 ± 4.2% (p < 0.001) (p < 0.001). Butyrate more effectively inhibited the STZ-induced NO production (p < 0.001), whose efficiency was 39.0 ± 5.1% (p < 0.001) higher than that of acetate  A. This i< 0.001) B. Acetat acetate B. p < 0.001) in the STZ-treated MIN6 cells. Acetate inhibited this STZ-induced increase (p < 0.001), as well as butyrate (p < 0.001). STZ enhanced NO in MIN6 cells with 36.8 ± 3.5% (p < 0.001) (p < 0.001) as well as butyrate (p < 0.001). In the MIN6 cell line, there was no significant difference in the ability of acetate and butyrate to reduce free radical generation. Our findings suggest that the tested SCFAs inhibit STZ-induced oxidative and nitrosative stress in islet β-cells by reducing ROS and NO levels, but the efficiency in human islets is SCFA-dependent.We also studied ROS and NO generation induced by STZ in a MIN6 cell line. As shown in < 0.001) D. AcetatPancreatic β-cells are very sensitive to oxidative stress-induced mitochondrial dysfunction due to a relatively weak enzymatic anti-oxidative defense mechanism along with an inefficient repair system for oxidative DNA damage ,26. ThisMFN1, MFN2, and OPA1) and a downregulation of the mitochondrial fission genes (DRP1 and FIS1) [Both human islets and a β-cell line were studied. Despite amble experience with isolating β-cells from human islets , we werend FIS1) ,35. The nd FIS1) ,37.The SCFAs effects were dose-dependent; acetate and butyrate at a concentration of 1 mM enhanced β-cell viability and showed slightly improved metabolism at 2 mM, but no protective effects were observed at 4 mM. The physiological concentrations of acetate and butyrate in the portal and peripheral circulation were also found to be approximately 1 mM ,39. The Acetate had a different effect than butyrate. Acetate showed more efficacy in supporting energy metabolism and anti-oxidative effects than butyrate. This might be caused by adaptations of β-cells to lower concentrations of butyrate in the pancreatic circulation. Butyrate is present at relatively lower concentrations compared to acetate because of the preferential use of butyrate by colonic epithelial cells for energy production . AlthougRecently, acetate and propionate have been reported to improve insulin secretion and to protect against apoptosis in β-cells damaged by the cytokines interferon gamma, tumor necrosis factor alpha, and interleukin 1 beta and the fatty acid sodium palmitate ,29. PingIn conclusion, this study provides new insights into how SCFAs can contribute to the maintenance of health. It shows that acetate and butyrate support β-cell metabolism, modulate mitochondrial efficiency and dynamics, and by that provide islet cells with enhanced anti-oxidative responses by which less ROS and NO is generated when exposed to the β-cell specific toxin STZ. Therefore, they support pancreatic β-cells function and prevent STZ-induced cell apoptosis.l-glutamine, 50 U/mL penicillin, and 50 µg/mL streptomycin . Cells were cultured at 37 °C in a humidified atmosphere containing 95% air and 5% CO2.The MIN6 cell line was purchased from American Type Culture Collection . MIN6 cells (passages 30–45) were cultured in (Dulbecco’s Modified Eagle Medium) DMEM High-glucose medium , containing 15% fetal bovine serum , 50 µmol/L β-mercaptoethanol, 2 mmol/L Human islets of Langerhans were isolated from cadaveric pancreata at the Leiden University Medical Center, as previously described  and provThe effect of SCFAs on β-cell viability was determined by the cell proliferation reagent WST-1 . Briefly, human islets (15 islets/well) and MIN6 cells (1 × 104 cells/well) were seeded in 96-well plates. Islets and cells were cultured overnight and the following day incubated with or without the acetate (Sigma-Aldrich) or butyrate (Sigma-Aldrich) at 1, 2, and 4 mM for 24 h followed by WST-1 assay. In order to investigate the protective effect of tested SCFAs, a one-hour preincubation of SCFA was followed by a 24-h co-incubation of the SCFAs and 5 mM STZ (Sigma-Aldrich), since STZ is widely reported as causing β-cell destruction and reduced insulin secretion . Cells w® 488 Annexin V and 1 µg/mL propidium iodide. The staining of human islets was analyzed using a Leica DM4000 B microscope . The percentage of Annexin V positive area and islet size were measured using ImageJ software . MIN6 cells were analyzed by flow cytometry . The flow cytometric results were analyzed using FlowJo® 10.4.2 software , and the number of apoptotic cells was expressed as the percentage of the total number of cells. The effect of acetate and butyrate on β-cell apoptosis was analyzed using an Annexin V  and PI  staining. Briefly, human islets were seeded on gelatin-coated coverslips with a diameter of 10.2 mm (15 islets/slide). MIN6 cells (1 × 106 cells/well) were cultured in 6-well plates. Islets and MIN6 cells were incubated with or without acetate or butyrate at 1, 2, and 4 mM for 24 h followed by Annexin V/propidium iodide (PI) staining. In order to investigate the rescue effects of SCFAs, one-hour pre-incubation of tested SCFA was followed by a 24-h co-incubation of the SCFAs and 5 mM STZ. Cells without tested SCFAs and STZ incubation were used as negative control. Cells with only STZ incubation were used as positive control. After incubation, islets and MIN6 cells were incubated for 15 min with 4.5 µg/mL Alexa Fluor2 incubator. Plates were transferred to a Seahorse Bioscience XFe24 extracellular flux analyzer (Seahorse Bioscience) and subjected to an equilibration period. After measuring the basal oxygen consumption rate (OCR) for three cycles, oligomycin  was added to determine the proportion of respiration used to generate ATP. Then, carbonyl cyanide-4-(trifluoro methoxy) phenyl hydrazone  was added to determine the maximal respiration by mitochondria. After that, rotenone  and antimycin A  were added to measure the non-mitochondrial respiratory rate.The effect of acetate and butyrate on mitochondrial function was measured by seeding MIN6 cells in XFe24 cell culture microplates  at 4 × 104 cells/well. Cells were cultured as described above. Cells with STZ incubation were used as positive controls, and cells without SCFAs and STZ incubation were used as negative controls. The cells were washed with extracellular flux (XF) basic medium (Seahorse Bioscience) supplemented with 3 mM glucose, 2 mM glutamine, and 2 mM sodium pyruvate followed by an incubation in this medium for 60 min at 37 °C in a non-COIntracellular ROS was detected using a DCFDA Cellular ROS Detection Assay Kit  according to the instructions of the kit manufacturer. Briefly, MIN6 cells (1 × 104/well) and human islets (10 islets/well) were seeded in black 96-well immunoplates (Thermo Scientific). Both were cultured and incubated with the tested SCFAs and STZ as described above. They were incubated with 20 µM 2′,7′-dichlorofluorescin diacetate (DCFDA) at 37 °C for 30 min, and fluorescence was measured  using a fluorescence plate reader .The NO concentration in the supernatant of the MIN6 cells and the human islets was measured with a Nitric Oxide Assay Kit  according to the manufacturer’s instructions. MIN6 cells and human islets were cultured and incubated with the tested SCFAs and STZ as described above. Briefly, nitrate in the supernatants was converted to nitrite using nitrate reductase. Subsequently, using Griess reagents, the nitrite was converted to a deep purple azo compound. Then, a Bio-Rad Benchmark Plus microplate spectrophotometer reader at 540 nm was used to measure the level of azo compounds, which reflected the NO amount in the sample.® 488  or goat-anti-guinea pig Alexa Fluor® 488  followed by a 1-min incubation with  DAPI . The staining of GPR41 and 43 was analyzed using a Leica DM4000 B microscope through the hanging drop method [Immunofluorescent staining was performed to stain for the SCFA receptors GPR41 and GRP43 and insulin. Human islets were cultured and incubated with the tested SCFAs and STZ as described above. After that, islets were fixed for 15 min with 4% paraformaldehyde , and non-specific binding was blocked by incubating with 1% bovine serum albumin (Sigma-Aldrich) for 1 h. The cells and islets were incubated for 1 h at room temperature with the primary antibodies rabbit anti-GPR41 , rabbit anti-GRP43 , and guinea pig anti-insulin . After washing, the cells and islets were incubated for 1 h at room temperature with donkey-anti-rabbit Alexa Fluorp method . To this®-FL polyvinylidene fluoride membranes (Merck) and blocked with Odyssey® blocking buffer  for 1 h at room temperature. Then, membranes were incubated with the primary antibody rabbit anti-GPR41  and rabbit anti-GPR43  in 1× PBS with 0.1% Tween. The mouse anti-β actin antibody  was used as a housekeeping protein. After 4 °C incubation overnight, membranes were washed in Tris-buffered saline (pH 7.4) and incubated for 1 h at room temperature with secondary antibody IRDye 800CW or 680 CW  in the dark. Membranes were analyzed using a LI-COR Odyssey Scanner (LI-COR).Western blot was performed to analyze the protein expression of the SCFA receptors GPR41 and GPR43. MIN6 cells were cultured as described above and were lysed afterwards using radioimmunoprecipitation assay buffer  supplemented with protease inhibitor (Roche). The protein concentration was determined using the BCA™ Protein Assay Kit (Thermo Scientific). Proteins were separated by loading a 10% polyacrylamide gel with 20 µg protein per well. The electrophoresis-separated proteins were subsequently transferred to ImmobilonMFN1, MFN2, DRP1, OPA1, and FIS1 . The total RNA was isolated following the manufacturer’s instructions, followed by obtaining reverse-transcribed cDNA using SuperScript II Reverse Transcriptase (Invitrogen). qPCR was performed with a FastStart Universal SYBR-Green Master kit (Roche) for the genes p-values < 0.05 were considered to be statistically significant ; ns, not significant. The data were analyzed using GraphPad Prism .Parametric distribution of data was tested using the Kolmogorov–Smirnov test. Data are expressed as mean ± standard error of mean (SEM). Statistical differences of parametric data were analyzed using one-way ANOVA, while nonparametric data were analyzed with a Kruskal–Wallis test.


Haplopelma schmidti, and conjugated to Cy7.5 dye. Hs1a-FL was injected intravenously in mice, and harvested nerves were imaged microscopically and with epifluorescence.Accidental peripheral nerve injury during surgical intervention results in a broad spectrum of potentially debilitating side effects. Tissue distortion and poor visibility can significantly increase the risk of nerve injury with long-lasting consequences for the patient. We developed and characterized Hs1a-FL, a fluorescent near-infrared molecule for nerve visualization in the operating theater with the aim of helping physicians to visualize nerves during surgery. Hs1a was derived from the venom of the Chinese bird spider, V1.7, present on the surface of human and mouse nerves. Hs1a-FL allowed epifluorescence visualization of sciatic mouse nerves with favorable nerve-to-muscle contrast.Hs1a-FL showed specific and stable binding to the sodium channel NaV1.7-targeted tracers have the potential to be adopted clinically for the intraoperative visualization of peripheral nerves during surgery, providing guidance for the surgeon and potentially improving the standard of care.Fluorescent Na Unintentional resection or injury of nerves during medical interventions is a significant concern during surgery , 2. DuriFor example, in head and neck surgery, iatrogenic nerve injuries can result in facial paralysis, hoarseness or weakening of voice, respiratory distress, and other neurological complications with harsh lifestyle implications for the patient –8. As a Haplopelma schmidti. Hs1a proved to be a potent and subtype-selective inhibitor of sodium channel NaV1.7, a key signal-transmitter located on nerve surfaces  3850.74, [C164H251N49O47S6 + 3H]3+ 1284.58, found [M + 3H]3+ 1285.00, [C164H251N49O47S6 + 4H]4+ 963.69, found [M + 4H]4+ 964.20, [C164H251N49O47S6 + 5H]5+ 771.15, found [M + 5H]5+ 772.60, [C164H251N49O47S6 + 6H]6+ 642.79, found 643.25.Recombinant Hs1a was produced via expression in the periplasm of peptides . The recV1.7, as previously described  4482.12, [C209H298N51O48S6 + 3H]3+ 1495.04, found [M + 3H]3+ 1495.45, [C209H298N51O48S6 + 4H]4+ 1121.53, found [M + 4H]4+ 1121.75, [C209H298N51O48S6 + 5H]5+ 897.42, found [M + 5H]5+ 897.75, [C209H298N51O48S6 + 6H]6+ 748.02, found [M + 6H]6+ 748.25Hs1a was discovered in a high-throughput fluorescent-based assay to screen spider venoms against hNaescribed . RecombiV channel β1 subunit (hNaVβ1) in combination with the α subunit hNaV1.1, hNaV1.2, hNaV1.3, hNaV1.4, hNaV1.5, hNaV1.6, or hNaV1.7  were cultured in DMEM/F-12 media (1:1), supplemented with 10% fetal bovine serum, 400 mg/mL geneticin, and 100 mM non-essential amino acids  at 37 °C and in 5% CO2.HEK293 cells stably expressing the human Na2, 1 mM MgCl2, 10 mM HEPES, 4 mM KCl, and 145 mM NaCl at pH 7.4, and the intracellular solution was 140 mM CsF, 1 mM/5 mM EGTA/CsOH, 10 mM HEPES, and 10 mM NaCl at pH 7.3. Hs1a-FL were dissolved in extracellular solution with 0.1% bovine serum albumin (BSA). Concentration-response data were obtained using five concentrations of peptide (2 nM to 10 μM). HEK293-hNaV cells were clamped at a holding potential of − 60 mV for NaV1.1, − 65 mV for NaV1.2, − 60 mV for NaV1.3, − 75 mV for NaV1.4, − 105 mV for NaV1.5, − 60 mV for NaV1.6, and − 75 mV for NaV1.7. For each concentration, 10 μL of peptide was added for 6 s before applying the following voltage protocol: − 80 mV for 10 ms, − 120 mV for 200 ms, 0 mV for 20 ms, then return to − 80 mV potential. This was repeated once every 60 s during liquid applications. Cells were otherwise held at the holding potential when the above voltage protocol was not executed. Upon establishment of the whole cell recording configuration, a total of five applications of the extracellular solution ), all containing 0.1% BSA (except for the TTX solution) were made on each cell. The voltage protocol was executed 10 times after each application. Currents were sampled at 25 kHz and filtered at 5 kHz with an 8-pole Bessel filter. The series resistance compensation level was set at 80%. All experiments were performed at room temperature (~ 22 °C). IC50 values were determined from non-linear regression of concentration-response data using GraphPad Prism.Whole-cell patch-clamp experiments were performed at room temperature using a QPatch 16x automated electrophysiology platform  using 16-channel planar patch-chip plates (QPlates) with a patch-hole diameter of 1 μm and resistance of 2 MΩ. Whole-cell currents were filtered at 5 kHz (8-pole Bessel) and digitized at 25 kHz. A P4 online leak-subtraction protocol was used with non-leak-subtracted currents acquired in parallel. The extracellular solution was 2 mM CaClFemale athymic nude mice   were allowed to acclimatize at the MSKCC vivarium for 1 week with ad libitum food and water prior to the experimental procedure. For imaging experiments, animals were sacrificed 30 min post-tail vein injection of Hs1a-FL, Hs1a/Hs1a-FL, or PBS. All animal experiments were performed in accordance with institutional guidelines and approved by the MSKCC Institutional Animal Care and Use Committee, following the NIH guidelines for animal welfare.Post-euthanasia, a representative mouse was fast frozen in hexanes with dry ice. Coronal cryosectioning and white-light imaging were performed by EMIT using a Xerra imager; following each sequential removal of 50-μm-thick slices, the tissue-embedded block was imaged at 30 μm in-plane resolution. A 3D image volume of the mouse was generated through multiplanar reformation using 3D Slicer software for anatomic visualization Fig. a.V1.7 in mouse sciatic nerve tissue. Anti-NaV1.7 antibody [N68/6] (Abcam ab85015) was found to specifically bind to mouse NaV1.7 (0.5 μg/mL). Paraffin-embedded formalin-fixed 5 μm sections were deparaffinized with EZPrep buffer. For IHC detection, a 3,3′-diaminobenzidine (DAB) detection kit  was used according to the manufacturer’s instructions. These experiments were performed at the MSKCC Molecular Cytology Core Facility using the Discovery XT processor . Adjacent sections were stained against IgG, to control for non-specific binding to NaV1.7. Sections were counterstained with hematoxylin and eosin (H&E) and coverslipped with Permount  for morphological evaluation of tissue characteristics.Immunohistochemical (IHC) staining experiments were used to detect the expression and abundance of sodium channel NaFor the confocal microscopy experiments, 5 μm cryosections of sciatic nerve tissues embedded in optimal cutting temperature compound (OCT) were used to determine the distribution and localization of Hs1a-FL from mice previously injected with the fluorescent agent Hs1a-FL , the blocking solution Hs1a/Hs1a-FL , or 100 μL of PBS. These resected nerves were incubated with Hoechst 33342  to counterstain nuclei, which were subsequently embedded in Mowiol mounting medium. Fresh tissues were counterstained with Hoechst 33342  and samples placed directly on a microscope slide for detection of the fluorescence signal of the fluorescent peptide.n = 3). A second group of animals was injected with Hs1a and Hs1a-FL  or PBS (n = 3). Animals were sacrificed 30 min post-injection and epifluorescence images obtained. Epifluorescence images of the right sciatic nerve (RSN) and left sciatic nerve (LSN) were obtained in situ from all the mice in the study. Epifluorescence images of the biodistribution included RSN, LSN, muscle, heart, kidney, liver, and brain and were acquired with an IVIS Spectrum imaging system (PerkinElmer) using a predetermined filter set . Autofluorescence was removed through spectral unmixing. Semiquantitative analysis of the Hs1a-FL signal was conducted by measuring the average radiant efficiency (in units of [p/s/cm2/sr]/[μW/cm2]) in regions of interest (ROIs) that were placed on all resected nerves and as well in all organs from the biodistribution under white light guidance.One group of animals was intravenously injected with Hs1a-FL , but it did not inhibit NaV1.4 and NaV1.5, which are found primarily in the muscle and heart, respectively at concentrations up to 3 μM  shifted from 12 min for the unmodified Hs1a to 16 min for Hs1a-FL , which was also present in the starting material . LC/MS spectra for both Hs1a and Hs1a-FL showed clean peak families confirming the peptides’ calculated masses of 3850.74 Da and 4482.12 Da for Hs1a and Hs1a-FL, respectively  or in combination with an excess of unmodified peptide  and sacrificed 30 min after injection. Nerves were surgically harvested and flash-frozen in OCT blocks. Blocks were then sliced on a cryotome at a 10-μm thickness and imaged. Nerves were imaged to detect fluorescent signal and H&E stained to enable the visualization of Schwann cells within the nerve structure. Anti-Naity Fig. b.Fig. 35 and 0.09 ± 0.03 × 105 for Hs1a-FL and co-injection (blocking solution), respectively; unpaired t test, p value < 0.001, Fig. 7 and 0.002 ± 0.001 × 107, 1.4 ± 1.1 × 107 and 0.005 ± 0.004 × 107, 0.2 ± 0.1 × 107 and 0.001 ± 0.0005 × 107, and 0.8 ± 0.5 × 107 and 0.004 ± 0.0003 × 107 for organs injected with fluorescent agent and with PBS, respectively; Fig. Mice were injected intravenously with Hs1a-FL alone  or in combination with an excess of unmodified peptide  and sacrificed 30 min after injection. The RSN and LSN were resected and epifluorescence imaging performed using an IVIS Spectrum imaging system . In mice receiving just the imaging agent, we observed accumulation of Hs1a-FL in the resected sciatic nerves, which were clearly visible Fig. c, whereaV1.7-expressing nerve and unspecific liver signal. However, this limitation could be overcome in humans thanks to more favorable anatomy and nerves with larger diameters. Due to the preclinical model chosen, the sciatic nerves represent the largest peripheral nervous system target for our molecule in vivo. Hs1a is a peptide derived from the venom of a Chinese tarantula, with a low nanomolar affinity for neuronal sodium channels. Here, we showed that conjugation of Hs1a and Cy7.5 dye yields a fluorescent tracer for nerve imaging. The synthesis of Hs1a-FL was straightforward and efficient. We evaluated Hs1a-FL for ex vivo imaging of resected sciatic nerves via microscopy as well as imaging of exposed nerves via epifluorescence. The chosen mouse model for the in vivo investigation was athymic nude mice for their versatility and ease of use. Furthermore, nude mice do not have a furred skin and therefore allow an easier surgical exposure of the nerves without the need of hair removal. The mouse model should not affect the efficacy of Hs1a-FL in imaging nerves. Hs1a-FL was shown to specifically bind to NaV1.7 channels in vivo post-intravenous injection, and its binding could be blocked by competition with an excess dose of the unlabeled Hs1a. A more specific nerve agent could improve imaging potential and significantly limit background noise and possible toxicity. It will be interesting to investigate in the future whether blood pools or coagulated blood could represent a limitation in the signal-to-background ratio in the operation room and to identify potential changes in the binding affinity against different NaV channels after different dyes conjugation. The near-infrared emission spectrum of Cy7.5 may enable the use of this agent for visualization of nerves that are beneath the tissue surface during surgical interventions. The nerve-to-muscle ratio was calculated and showed a blockable signal of ~ 4 for Hs1a-FL-injected mice. A broader investigation is needed to identify the most ideal nerve imaging agent for intraoperative applications. This study represents the scientific basis for the development of a set of nerve-targeting agents derived from naturally available peptides.In the present study, we developed and characterized Hs1a-FL, a near-infrared imaging agent to target human nerves. A limitation of the present study is that, in a small rodent model, the high liver accumulation makes it difficult to discern the signal coming from NaHs1a-FL proved to be a useful tool for nerve visualization. This nerve-targeting agent should be explored further with a view to developing tools that can assist surgeons to identify peripheral nerves and avoid surgical morbidity due to nerve injury.Additional file 1. Fluorescence labeling of a NaV1.7-targeted peptide for near-infrared nerve visualization.


SUMOylation is a post-translational modification (PTM) whereby members of the Small Ubiquitin-like MOdifier (SUMO) family of proteins are conjugated to lysine residues in target proteins. SUMOylation has been implicated in a wide range of physiological and pathological processes, and much attention has been given to its role in neurodegenerative conditions. Due to its reported role in neuroprotection, pharmacological modulation of SUMOylation represents an attractive potential therapeutic strategy in a number of different brain disorders. However, very few compounds that target the SUMOylation pathway have been identified. Guanosine is an endogenous nucleoside with important neuromodulatory and neuroprotective effects. Experimental evidence has shown that guanosine can modulate different intracellular pathways, including PTMs. In the present study we examined whether guanosine alters global protein SUMOylation. Primary cortical neurons and astrocytes were treated with guanosine at 1, 10, 100, 300, or 500 μM at four time points, 1, 6, 24, or 48 h. We show that guanosine increases global SUMO2/3-ylation in neurons and astrocytes at 1 h at concentrations above 10 μM. The molecular mechanisms involved in this effect were evaluated in neurons. The guanosine-induced increase in global SUMO2/3-ylation was still observed in the presence of dipyridamole, which prevents guanosine internalization, demonstrating an extracellular guanosine-induced effect. Furthermore, the A1 adenosine receptor antagonist DPCPX abolished the guanosine-induced increase in SUMO2/3-ylation. The A2A adenosine receptor antagonist ZM241385 increased SUMOylation per se, but did not alter guanosine-induced SUMOylation, suggesting that guanosine may modulate SUMO2/3-ylation through an A1-A2A receptor interaction. Taken together, this is the first report to show guanosine as a SUMO2/3-ylation enhancer in astrocytes and neurons.The online version of this article (10.1007/s11302-020-09723-0) contains supplementary material, which is available to authorized users. SUMOylation is a post-translational modification (PTM) whereby the Small Ubiquitin-like MOdifier (SUMO) peptide is conjugated to target proteins at lysine residues . SUMO coAccumulating evidence has demonstrated that guanosine, an endogenous nucleoside, may be a therapeutically useful compound in a number of disorders –24. Due 2+-activated K+ channel (BK) [Although a specific receptor for guanosine has not been identified , a numbenel (BK) .Here, we sought to evaluate whether guanosine may modulate global protein SUMOylation in neurons and astrocytes, and further determine whether adenosine receptors mediate these effects.4 cells/well) were plated on 6-well plates previously treated with poly-L-lysine . Plating medium consisted of Neurobasal Medium (Gibco) containing 10% horse serum (Gibco), B27 , penicillin-streptomycin , and 5 mM Glutamax (Gibco). After 24 h, plating medium was replaced with 3 mL of feeding medium  with no further medium changes. Neurons were used for experiments at 14 days in vitro. Cortical astrocytes were prepared as described previously [4 cells per well) were plated on 6-well plates previously treated with poly-L-lysine . Plating medium consisted of Dulbecco’s modified Eagle’s medium (DMEM) nutrient mixture F-12 (Gibco), supplemented with 10% fetal bovine serum (Gibco). Cell culture medium was changed 24 h after plating and changed subsequently three times a week. Astrocytes were used for experiments at 14 days in vitro. Ethics committees previously approved all procedures used in this study (CEUA 955 – UFSC and UB/18/004 – University of Bristol).Cortical neurons were prepared as described previously . Brieflyeviously . BrieflyUnless otherwise specified, the drugs used in the experiments were obtained from Sigma: guanosine (G6752), adenosine (A9251), dipyridamole , DPCPX  and ZM 241385 . The concentration curves  for guanosine, adenosine and guanine were based on previous studies , 41, 42.For immunoblotting, neurons and astrocytes were lysed in 250 μL sample buffer solution (1×) containing 2% SDS (w/v), 5% glycerol (v/v), 62.5 mM Tris-HCl pH 6.8, and 5% (v/v) β-mercaptoethanol. Lysates were collected and heated to 95 °C for 10 min prior to gel electrophoresis. Proteins were separated by SDS-PAGE (10–15% gels). PDVF membranes were blocked in 5% (w/v) non-fat milk powder or bovine serum albumin  in PBS-T. The following primary antibodies were incubated overnight at 4 °C: SUMO1 , SUMO2/3 , SENP3 , and GAPDH . Ponceau S  was also used for protein staining . After t5 cells/well) previously treated with nitric acid and poly-L-lysine (0.1 mg/mL). Neurons were washed with PBS once and fixed with 4% paraformaldehyde (PFA) for 20 min. After three washes with PBS, PFA was quenched by incubation with PBS containing 20 mM glycine for 10 min, before cells were permeabilized with PBS containing 0.05% Triton for 5 min. Next, blocking solution consisting of 5% BSA diluted in PBS was added to the coverslips for 1 h at room temperature. Neurons were then incubated with anti-β-tubulin III  and astrocytes with anti-GFAP  for 1 h. Cells were then washed twice with PBS containing 0.1% tween 20 for 5 min and incubated with Alexa Fluor 594 fluorescent antibodies  for 1 h at room temperature. For nuclear staining, Hoechst 33342 was present in the mounting media. Images were acquired on a confocal microscope . To analyze morphology, three independent experiments were performed [For immunofluorescence, neurons and astrocytes were plated on glass coverslips -2,5-diphenyltetrazolium bromide) assay . After thttps://www.graphpad.com/quickcalcs/Grubbs1.cfm). After confirming data normality (Kolmogorov-Smirnov test), one-way analysis of variance (ANOVA) was performed and Newman-Keuls was used as a post hoc test to determine significant differences among groups. Data are presented as mean + standard error of the mean (S.E.M.) and statistical significance expressed by *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. GraphPad Prism 5.0 was used for graphs and statistics [All results were included in the statistical analysis except for those significantly detected as outliers . However, increased global SUMO2/3 conjugation was not observed at longer time points .Although SUMOylation is a PTM known to be involved in a wide range of cellular functions , 3, 48, nts Fig. , suggestF = 9.025; p = 0.0002), but this effect was transient and not observed at later time points  and led to a similar trend towards decreased conjugation in astrocytes; however, this trend was not statistically significant . Furthermore, 48 h treatment with 100 μM guanosine did not obviously affect astrocyte or neuronal morphology, as determined by GFAP or β-tubulin III staining, respectively . These results suggest that the guanosine-induced increase in SUMO2/3 conjugation does not require guanosine internalization and indicates that guanosine is acting through a membrane receptor interaction.Guanosine uptake is mediated through nucleoside transporters present in the cell membrane , 54. In F = 3.086; p = 0.0571), suggesting guanosine is enhancing SUMO2/3 conjugation through A1 receptors. In the same way, neurons were treated with guanosine in the presence or absence of the A2A antagonist ZM241385 (50 nM) for 1 h. ZM241385 did not affect the guanosine-induced increase in SUMO2/3 conjugation . However, ZM241385 per se increased global SUMO2/3 conjugation  or SUMO2/3 .Next, we sought to investigate whether the guanosine-induced increase in global SUMO2/3 conjugation was mediated by A1 and/or A2A adenosine receptors. Neurons were treated with guanosine (500 μM) in the presence or absence of the A1 receptor antagonist DPCPX (100 nM) for 1 h. DPCPX abolished the effect of guanosine on SUMO2/3 conjugation  for 1 h did not modulate global SUMO2/3 or SUMO1 conjugation. However, it remains possible that adenosine could modulate SUMOylation at shorter time points, especially considering that G protein-coupled receptors, such as adenosine receptors, may suffer from desensitization and internalization in response to continuous exposure to agonist, preventing the observation of downstream effects –75. AnotTo the best of our knowledge, there is, to date, only one experimental demonstration that SUMOylation can be modulated through adenosinergic signaling. The protein IκBα , an important modulator of inflammatory responses, can be SUMO1-ylated in response to adenosine signaling. Following hypoxia and reoxygenation, there was an increase in SUMO1 conjugation to IκBα in HeLa cells, and treatment with NECA, a non-specific adenosine receptor agonist, increased SUMO1 conjugation to IκBα in a concentration-dependent manner. In contrast, a nonselective adenosine receptor antagonist, 8-phenyltheophylline, abolished NECA-induced IκBα SUMO1-ylation . HoweverThe importance of the SUMOylation pathway for neuronal function and dysfunction is well demonstrated in the literature , 77, 78.ESM 1a. 1 h, b. 6 h, c. 24 h and d. 48 h. SUMO1 conjugation optical density quantification for e. 1 h, f. 6 h, g. 24 h and h. 48 h. (PNG 1130 kb)Evaluation of guanosine effects on global SUMO1 conjugation in astrocytes. Representative SUMO1 Western blots from astrocytes treated with guanosine (1 – 500 μM) for High Resolution Image (TIF 282 kb)ESM 2i. 1 h, j. 6 h, k. 24 h and l. 48 h. Representative Ponceau staining used to normalize the Western blots for global SUMO2/3-conjugated proteins in astrocytes at m. 1 h, n. 6 h, o. 24 h and p. 48 h. Results expressed as mean + standard error of the mean (n = 3-5 independent experiments). One-way ANOVA did not identify any significant differences between groups. C: Control. kDa: Kilodaltons. (PNG 1493 kb)Ponceau staining was used as a loading control. Representative Ponceau staining used to normalize the Western blots for global SUMO1-conjugated proteins in astrocytes at High Resolution Image (TIF 409 kb)ESM 3a. SUMO1 and b. SUMO2/3 conjugation and optical density quantifications for c. SUMO1 and d. SUMO2/3 from neurons treated with adenosine (1 – 500 μM) for 1 h. GAPDH was used as a loading control. Results expressed as mean + standard error of the mean (n= 3-5 independent experiments). One-way ANOVA did not identify any significant differences between groups. C: Control. kDa: Kilodaltons. (PNG 574 kb)Adenosine does not change global SUMO1 nor SUMO2/3 conjugation in neurons at 1 h. Representative Western blots of High Resolution Image (TIF 122 kb)


Biology utilizes multiple strategies, including sequestration in lipid vesicles, to raise the rate and specificity of chemical reactions through increases in effective molarity of reactants. We show that micelle-assisted reaction can facilitate native chemical ligations (NCLs) between a peptide-thioester – in which the thioester leaving group contains a lipid-like alkyl chain – and a Cys-peptide modified by a lipid-like moiety. Hydrophobic lipid modification of each peptide segment promotes the formation of mixed micelles, bringing the reacting peptides into close proximity and increasing the reaction rate. The approach enables the rapid synthesis of polypeptides using low concentrations of reactants without the need for thiol catalysts. After NCL, the lipid moiety is removed to yield an unmodified ligation product. This micelle-based methodology facilitates the generation of natural peptides, like Magainin 2, and the derivatization of the protein Ubiquitin. Formation of mixed micelles from lipid-modified reactants shows promise for accelerating chemical reactions in a traceless manner. Sequestration of reactants in lipid vesicles is a strategy prevalent in biological systems to raise the rate and specificity of chemical reactions. Here, the authors show that micelle-assisted reactions facilitate native chemical ligation between a peptide-thioester and a Cys-peptide modified by a lipid-like moiety. Many reactions that would otherwise not occur in practical time periods are facilitated by templating. Nucleic acid8 and protein templates11 are widely appreciated in biology and biomimetic chemistry, and a number of seminal studies have exploited such templates for programming the synthesis of complex molecules such as oligopeptides14. All living cells possess lipid membranes, which in addition to acting as compartments and barriers, also act as scaffolding structures for guiding biological reactions. By restricting substrates in two-dimensions, local concentration is increased. Signaling from clusters of membrane bound proteins benefits from this phenomenon, as does the biosynthesis of membrane components such as the bacterial cell wall. Given that templated protein synthesis is a principal tenet of the central dogma of molecular biology, it would be of particular interest to understand if lipid moiety mediated self-assembly of mixed micelles15 can drive the selective formation of discreet polypeptides. Here, the mixed micelles would be composed of two different lipid detergents, bearing polar head groups that would be reactive with one another. If successful, mixed micelle assistance could provide a straightforward mechanism to accelerate sluggish chemical reactions and could be potentially be a useful synthetic method if mechanisms existed to make the process traceless through removal of lipid auxiliary groups.Templated chemistry is a hallmark of biological processes, bringing reactants into close proximity with one another, thus greatly increasing effective molarity and dramatically raising the rate of reaction16. NCL is a chemoselective reaction that occurs at neutral pH between two unprotected peptides, one bearing an N-terminal cysteine residue and the other containing a C-terminal thioester. NCL is a robust method19 for the synthesis and derivatization of large peptides, proteins24, and even nucleic acids26 and phospholipids29. Despite its widespread use in chemical protein synthesis, NCL has several limitations. First, C-terminal peptide thioesters are commonly prepared as alkyl thioesters, which simplifies synthesis, purification and storage, but results in precursors with low reactivity30. Second, although some thiol-free NCL strategies have been developed recently33, addition of thiol catalysts is often required in conventional NCL method to promote the in situ formation of more reactive thioesters34. Thiol catalysts, such as MPAA30 introduced by Kent and coworkers, have been widely applied in synthetic protein chemistry36. Despite their proven robustness, thiol catalysts may have the potential for unwanted side reactions33. Furthermore, thiol additives can interfere with the NCL-desulfurization synthetic strategy38 by acting as radical scavengers39, which may necessitate a removal step prior to desulfurization40. Third, the modest reaction rates of NCL make it necessary to use millimolar concentrations of the substrate peptide fragments for practical application42.To address these issues, we focused on applying lipid modification to accelerate oligopeptide coupling, specifically through native chemical ligation (NCL)43 and in the absence of thiol additive catalysts, would be of considerable value. Herein, we describe a strategy for lipid-facilitated acceleration of NCL via micelle mixing , which is capable of forming micelles in aqueous solution. Addition of an Cys-peptide containing a photolabile lipid45 moiety near the N-terminus  leads to rapid NCL. We hypothesize that the close proximity50 of the alkyl thioester and cysteine within formed micelles increases the rate of transthioesterification substantially, even at low concentrations of reagents (1 mM as standard concentration) and in the absence of thiol catalysts. Although there are several reported cases53 of facilitated NCL in hydrophobic environments or in lipid bilayers, in our work, the surfactants are the reacting peptide segments, to which lipid moieties are covalently attached. Micelles are formed by the lipid-modified reactants themselves, which upon addition to the same solution, fuse to form mixed micelles. During NCL, the lipid moiety attached to the thioester peptide is eliminated in the initial thiol-thioester exchange. After NCL, the lipid moiety maintained on the product can be removed by a photochemical process. Thus, a traceless synthetic system can be established. We demonstrate the utility of this strategy through the successful one-pot synthesis of the natural antimicrobial peptide Magainin 2 and derivatization of the natural protein Ubiquitin (Ubi). Our studies suggest that micelle-mixing assembly may be a broadly applicable method for the acceleration of chemical reactions.Development of a lipid-facilitated NCL methodology that allows the rapid production of ligated polypeptides, using low concentrations of reactantsing Fig. . Our app1a), C16 (1b), and C18 (1c). We note that LYRMG peptide thioesters with Cn alkyl chain will henceforth be denoted as LYRMG-αCOSCn; e.g. LYRMG-αCOSC8 (1a). As a control, we prepared LYRMG-αCOSC2 (1d), a short-chain peptide thioester containing an ethyl moiety on its C-terminus, which lacks the necessary hydrophobicity for micelle formation. This behavior was confirmed by dynamic light scattering (DLS) measurements, showing that at 1 mM concentration, no micelle formation was observed for 1d. Additionally, we synthesized Cys-peptides  using a standard Fmoc-SPPS protocol. These peptide segments were adapted from the peptide model sequence CRANK-OH18. In our case, we substituted the arginine for the unnatural amino acid Dap(PhCn), which bears a photolabile lipid group45 at the Nβ position of 2,3-diaminopropionic acid (Dap). The photolabile group possesses a linear aliphatic alkyl chain of either C8 (2a) or C16 (2b). Peptide 2c, where the photolabile group is absent, was also prepared as a control.To explore the potential of lipid-facilitated NCL, solid-phase peptide synthesis (SPPS) was used to construct the peptide LYRMG-OH, which was subsequently modified with linear aliphatic alkyl chains of different lengths to form the corresponding thioesters Fig. –C8 (1a),1a–d) and the cysteine-functionalized peptide substrates (2a–c) in a thiol-additive-free aqueous phosphate buffered system at pH 7.0, containing tris(2-carboxyethyl) phosphine hydrochloride (TCEP·HCl) to maintain a reducing environment.We tested the NCL reactions between the oligopeptide thioesters (2b) was treated with various oligopeptide LYRMG-αCOSZ thioesters (1a–d) , but only 60% ligation yield in 180 min. The reaction of 1c and 2b was slow; less than 50% ligation yield could be achieved after 960 min. Use of thioester 1d, which lacks a long aliphatic chain, presented less than 20% ligation yield with 2b after 960 min, indicating the importance of mixed micelle self-assembly in accelerating the reaction.Cysteine-based oligopeptide CDap(PhC16)ANK (–d) Fig. . The lig1a (C8) promoted the greatest acceleration of the NCL. Therefore, we studied its reactivity against various cysteine-based peptide analogs (2a–c)  Fig. . The lig1c) < LYRMG-αCOSC16 (1b) < LYRMG-αCOSC8 (1a)]. Micelles composed of shorter alkyl thioester chains are predicted to have a higher rate of dissociation55, resulting in facilitated micelle mixing, potentially explaining this result. However, when 1a was reacted with various Cys-peptides differing in lipid chain length  . When the concentrations of the reactants  are below the CMCs of both reactants [conditions in which both 1a and 2b do not form micelles], ligations once again become slow . The significant difference in NCL kinetics at various reactant concentrations provides further evidence for the role of mixed micelle self-assembly in facilitating the reaction. In comparison, MPAA-catalyzed NCL reactions between the unlipidated precursors LYRMG-αCOSC2 (1d) and CDapANK (2c) were also performed  and LYRML-αCOSC8 (6a) and treated them at room temperature with the N-terminal cysteine peptide CDap(PhC16)ANK (2b). The desired ligated polypeptides  were afforded in good yields (>80%) after 900 min. Control reactions of LYRMN-αCOSC2 (5b) and LYRML-αCOSC2 (6b) with 2b did not generate the corresponding ligation products, supporting the role of lipid modification in acceleration of these ligations.To test the robustness and widen the applicability of our methodology, we generated more complex peptide ligation sites such as Asn-Cys and Leu-Cys Fig. . We syntS10) NK (30. NCL reaction between CK(PhC16)ANC (S12)  demonstrated that our lipid-facilitated methodology is also compatible with the presence of non N-terminal cysteines  are removed during the reaction process.Without purification, the nitrobenzyl photocaging group was removed using a standard photocleavage protocoleld Fig.  and 51. Nε position of the lysine, we successfully synthesized the decapeptide LYRMGCKANK (S6) , which bears the photolabile lipid group at the 61. This natural antimicrobial peptide has been reported to be a difficult sequence to synthesize by Fmoc chemistry SPPS63. We devised a straightforward one-pot synthesis of Magainin 2 based on our lipid-facilitated NCL methodology    .Encouraged by these results, we next applied our methodology to the total synthesis of a natural peptide product. Magainin 2 is a 23aa cationic and amphipathic polypeptide that acts as a potent antibiotic in a variety of organisms Fig. 60,61. Thogy Fig. . The pep67 has been extensively used for the site-specific introduction of probes, unnatural amino acids, and complex post-translational modifications. The recombinant protein-derived thioesters are usually prepared from a modified Cys-intein fusion protein, which can be cleaved with thiol to provide the corresponding thioester moiety. Thus, we obtained the Ubiquitin-derived thioester Ubi-αCOSC8  was subsequently treated with the Cys-peptide CK(PhC16)ANK  to afford the ligation product, lipidated protein Ubi-CK(PhC16)ANK , within 5 h at 37 °C without thiol additives  and peptide CKANK  were enough to efficiently facilitate the reactions with all the peptides studied. We believe that the hydrophobic character of the peptide fragments may also play a key role in the acceleration of the NCL. Therefore, incorporation of hydrophobic residues  could considerably increase the rate of the peptide ligationsS3a was synthesized according to Supplementary Fig. Here, we describe the representative procedure for the preparation of Cn-photocaged amino acids. Amino acid S1a): a solution of 4-methyl-3-nitrophenol  in CH3CN (200 mL) was treated with K2CO3  and 1-bromohexadecane . Then, the reaction mixture was stirred for 24 h at reflux . Afterwards, the resulting suspension was filtered to remove the K2CO3. The solvent was removed in vacuo and the crude was purified by flash chromatography (0%–10% EtOAc in hexanes) to afford S1a as a yellowish solid . 1H NMR : 7.44 , 7.16 , 7.00 , 3.92 , 2.47 , 1.83-1.68 , 1.43–1.36 , 1.26-1.19 , 0.83  : 157.61, 149.31, 133.37, 125.29, 120.44, 109.59, 68.63, 31.95, 29.74, 29.73, 29.72, 29.70, 29.69, 29.68, 29.63, 29.61, 29.58, 29.41, 29.40, 29.36, 29.07, 29.05, 25.96, 22.72, 19.80, 14.16 -1-methyl-2-nitrobenzene (S2a): a solution of S1a  in CCl4 (15 mL) placed on a flame-dried two-neck round bottom flask with a condenser was successively treated with recrystallized N-bromosuccinimide  and azobisisobutyronitrile . The resulting suspension was irradiated with white light (250 W Philips tungsten bulb) for 10 h. After this period of time, the light was removed and the reaction was cooled to rt. The obtained succinimide was filtered off and washed with CH2Cl2 (15 mL). The filtrate was then added to a separatory funnel and washed with NaHCO3 (sat.) (10 mL), H2O (10 mL) and NaCl (sat.) (10 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated in vacuo. The resulting crude material was then purified by flash chromatography (0-10% EtOAc in hexanes) to afford S2a as an off-white solid . 1H NMR : 7.54 , 7.43 , 7.11 , 4.80 , 4.01 , 1.86-1.76 , 1.51–1.42 , 1.39–1.19 , 0.88  : 157.60, 149.31, 133.37, 125.29, 120.43, 109.60, 68.63, 31.80, 31.76, 29.31, 29.23, 29.04, 25.96, 22.67, 19.79, 14.12 -4-(hexadecyloxy)-2-nitrobenzene (S3a): to a solution of S2a  in MeOH:CH2Cl2  was added Nα-Fmoc-l-2,3-diaminopropionic acid , and the mixture was stirred at rt for 5 min. The resulting suspension was then treated with N,N-diisopropylethylamine , which caused the suspension to gradually clear. The reaction mixture was stirred at rt for 20 h shielded from light. Afterwards, the solvent was removed in vacuo, while maintaining the temperature of the water bath around 20 °C to avoid potential decomposition and/or side reactions. Then, the obtained crude was purified by flash chromatography (0%–5% MeOH in CH2Cl2), affording S3a as a yellowish foam . 1H NMR : 7.63 , 7.49 , 7.27 , 7.19 , 6.94 , 6.56 , 4.31 , 4.20 , 4.16-4.06 , 4.06 , 3.73 , 3.50-3.34 , 1.58 , 1.19 , 0.80  : 172.87, 160.55, 156.51, 149.39, 143.81, 141.15, 135.39, 127.65, 127.11, 125.31, 120.46, 119.84, 117.51, 111.47, 68.91, 67.39, 52.06, 49.61, 48.84, 46.95, 31.95, 29.75, 29.60, 29.40, 28.91, 25.87, 22.75, 14.19  700.3967, found 700.3963.(R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((4-(hexadecyloxy)-2-nitro-benzyl)amino)propanoic acid  protocols . The resin was dried under high vacuum and the loading was determined by quantification of the Fmoc group. For this, a small portion of the resin (~2 mg) was treated with a solution of 20% piperidine in DMF (1 mL) for 30 min. To an aliquot of this solution (100 μL), 900 μL of DMF was added and the absorbance was read at 301 nm. The concentration of the dibenzofulvene-piperidine adduct was obtained by using the extinction coefficients (ε) tabulated in the literature. Thus, the resin loading was estimated to be 0.66 mmol/g.Here, we describe the representative procedure for the preparation of CDap(PhCn)ANK derivatives . Peptide N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate , 1-hydroxybenzotriazole  and DIEA  in DMF (3 mL). The resin was shaken for 45 min and then washed with DMF (3 mL). The procedure was repeated with each corresponding amino acid . Note: When coupling with Fmoc-Dap(PhC16)-OH or Boc-Cys(Trt)-OH, the reaction time was prolonged to 6 h.A portion of resin (0.1 mmol loaded Fmoc-Lys(Boc)-OH) was used for the synthesis of the desired peptide. The Fmoc group was removed by treatment with 20% piperidine in DMF (3 mL) for 30 min. The resin was washed with DMF (6 mL) and then treated with a solution of Fmoc-protected amino acid (0.4 mmol), 2O:1,2-ethanedithiol(EDT); 82.5: 5: 5: 5: 2.5; 3 mL per 100 mg of resin] for 2 h and then filtered. The resin was washed with TFA (0.5 mL) and the combined fractions were evaporated to 1–2 mL by bubbling argon. The concentrated solution was added dropwise to cold Et2O (10 mL of Et2O per ml of TFA). The resulting precipitate was centrifuged for 10 min at 1008 × g. The supernatant was discarded, then fresh Et2O was added, and the suspension was sonicated and centrifuged. The resulting solid was dried under vacuum. The sample was dissolved in MeOH and purified by semipreparative HPLC using a C18 column [gradient of H2O with 0.1% formic acid and MeOH with 0.1% formic acid 95:5 (0 min) to 5:95 (20 min)] to give CDap(PhC16)ANK (2b) as a white solid . Analytical HPLC: tR = 2.85 min  :phenol:thioanisole:H1a was prepared manually by standard Fmoc chemistry solid phase peptide synthesis (SPPS) protocols . The resin was dried under high vacuum and the loading was determined by quantification of the Fmoc group. For this, a small portion of the resin (~ 2 mg) was treated with a solution of 20% piperidine in DMF (1 mL) for 30 min. To an aliquot of this solution (100 μL) was added 900 μL of DMF and the absorbance was read at 301 nm. The concentration of the dibenzofulvene-piperidine adduct was obtained by using the extinction coefficients (ε) tabulated in the literature. Thus, the resin loading was estimated to be 0.70 mmol/g.Here, we describe the representative procedure for the preparation of LYRMX-αCOSZ derivatives . Peptide tBu)-OH and Boc-Leu-OH].A portion of resin (0.1 mmol loaded Fmoc-Gly-OH) was used for the synthesis of the desired peptide. Fmoc group was removed by treatment with 20% piperidine in DMF (3 mL) for 30 min. The resin was washed with DMF (6 mL) and then treated with a solution of Fmoc-protected amino acid (0.4 mmol), HBTU , HOBt  and DIEA  in DMF (3 mL). The resin was shaken for 45 min and then washed with DMF (3 mL). The procedure was repeated with each corresponding amino acid  to give LYRMG-αCOSC8 (1a) as a white solid . Analytical HPLC: tR = 4.35 min  -Arg(Pbf)-Met-Gly-αCOSC8  was deprotected by treatment with freshly prepared cocktail K  and 2b  were dissolved in separate vials using freshly prepared ligation buffer . After sonication of both mixtures for 10 minutes, the solutions from both vials were combined . The resulting solution was gently shaken at rt. Aliquots were taken at various time intervals and analyzed by analytical HPLC-MS. The yield was calculated based on the peak areas of [2b + 3b] versus the desired ligation product 3b at 210 nm. Analytical HPLC: tR = 13.43 min   to 5:95 (10 min to 15 min)], giving 3b  as a white solid.Here, we describe the representative procedure for the preparation of LYRMGCDap(PhC16)ANK derivatives by NCL reaction . Peptide 4 was synthesized according to Supplementary Fig. 3b) was directly transferred to a quartz tube. Then, the solution was irradiated with UV light (365 nm) for 5 min45. Afterwards, an aliquot was taken and analyzed by analytical HPLC-MS and the formation of the peptide 4 was observed. Analytical HPLC: tR = 5.0 min  . Peptide 1a, 1b, 2a, 2b) subjected to CMC determination was initially lyophilized, and then dissolved in separate vials by addition of freshly prepared ligation buffer. The resulting aqueous solutions (100 µL) of LYRMG-αCOSC8  and CDap(PhC16)ANK  were analyzed by DLS in order to determine the CMCs68. This technique allowed us to measure the CMCs at 25 °C, since the scattered light intensity (measured at 90°) is dependent on the molecular weight and size of the particle, it increases when monomers start to aggregate in solution. Therefore, in our particular case, light scattering intensity values were plotted versus the concentration of the lipid-modified peptide acting as a surfactant  and CK(PhC16)KFGKAFVGEIMNS (12).Magainin 2 was synthesized on a one-pot-3-step reaction (NCL → photocleavage→desulfurization) from peptides GIGKFLHS-αCOSC8 (12): Peptide 12 was synthesized according to Supplementary Fig. tBu)-2-Chlorotrityl chloride resin (S7a) was obtained (loading: 0.46 mmol/g). Then, the Alloc group on the peptidyl-resin  was removed with tetrakis(triphenylphosphine)palladium(0)  in the presence of phenylsilane . Once the deprotection was completed , the resin was washed with sodium diethyldithiocarbamate trihydrate in DMF  to remove palladium residues. Afterwards, the resin S7b was suspended in DMF, and 2 equiv of DIEA and 1 equiv of 1-(bromomethyl)-4-(hexadecyloxy)-2-nitrobenzene (S2a) were successively added. After shaking at rt for 8 h, the resin was successively washed with DMF and DCM, and finally dried under vacuum for 12 h.Preparation of CK(PhC16)KFGKAFVGEIMNS (S7c) by treatment with freshly prepared cocktail K  for 2 h, and then filtered. The resin was washed with TFA (0.5 mL) and the combined fractions were evaporated to 1–2 mL by bubbling argon. The concentrated solution was added dropwise to cold Et2O (10 mL of Et2O per ml of TFA). The resulting precipitate was centrifuged for 10 min at 1008 x g. The supernatant was discarded, then fresh Et2O was added, and the suspension was sonicated and centrifuged. The resulting solid was dried under vacuum. The sample was dissolved in MeOH and purified by HPLC using a Zorbax SB-C18 semipreparative column , obtaining CK(PhC16)KFGKAFVGEIMNS (12) as a white solid  . Analytical HPLC: tR = 2.58 min  : using the general procedure II, 1.31 mg of the 8-mer thioester peptide GIGKFLHS-αCOSC8 (11) were obtained . Analytical HPLC: tR = 2.31 min   and CK(PhC16)KFGKAFVGEIMNS  were dissolved in separate vials  using freshly prepared and degassed ligation buffer . After sonication of both mixtures for 10 min, the solutions were combined . The resulting solution was gently shaken at 30 °C for 24 h under argon. An aliquot was taken and analyzed by HPLC-MS  was verified to be the peak with tR = 14.94 min . MS (ESI) [C137H217N31O32S2] calculated: 958.5 [M + 3 H]3+, 965.9 [M + 2 H + Na]3+, 719.2 [M + 4 H]4+; found 958.2 [M + 3 H]3+, 966.0 [M + 2 H + Na]3+, 719.3 [M + 4 H]4+ : Native chemical ligation (NCL): The lyophilized peptides GIGKFLHS-αCOSC8 11, 0. µmol and14) was verified to be the peak with tR = 9.66 min . MS (ESI) [C114H180N30O29S2] calculated: 1249.7 [M + 2 H]2+, 833.4 [M + 3 H]3+, 625.3 [M + 4 H]4+; found 1249.6 [M + 2 H]2+, 833.6 [M + 3 H]3+, 625.2 [M + 4 H]4+ , 40 µL of 6 M guanidinium chloride (GuHCl) aqueous solution, 8 μL of 2-methyl-2-propanethiol and 8 μL of radical initiator 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride . Then, the reaction mixture was stirred at 37 °C. After 120 min, an aliquot was taken and analyzed by HPLC-MS . Analytical HPLC: tR = 9.58 min . MS (ESI) [C114H180N30O29S] calculated: 822.8 [M + 3 H]3+, 617.3 [M + 4 H]4+; found 822.9 [M + 3 H]3+, 617.3 [M + 4 H]4+  to 5:95 (15 min to 18 min)], giving 15  as white solid. Note: In conventional conditions70, SDS  is difficult to be removed by HPLC. In our case, the concentration of SDS is 2 mM, which is far below the SDS concentration used in conventional methods. Exhaustive analysis of the obtained HPLC-MS traces showed that the retention time of SDS is 15.5 min, and the final product was SDS free.Desulfurization: After photocleavage, to the vial containing 400 µL of the reaction mixture was added 40 µL of 0.5 M Bond-breaker15) was also corroborated by high resolution mass spectrometry (ESI-TOF) analysis  (lipid-facilitated NCL → photocleavage→desulfurization→purification) are shown in Supplementary Fig. The full spectra of the synthesis of Magainin 2 (17) was synthesized by NCL between the protein thioester Ubi-αCOSC8 (16) and CK(PhC16)ANK (S4).Ubi-CK(phC16)ANK (16): BL21 (DE3) cells were transformed with the Ubi-AvaE-6xHis plasmid by 30 s heat shock at 42 °C and plated overnight on an ampicillin LB-agar plate (100 µg/mL) at 37 °C. Overnight LB cultures (100 µg/mL ampicillin) were inoculated with a single colony from the plate and grown overnight at 37 °C with shaking at 7 × g. Growth TB cultures (100 µg/mL ampicillin) were inoculated with a small amount of overnight culture (1 mL LB/100 mL TB) and incubated at 37 °C with shaking at 7 × g until mid-log phase was reached (OD600 = 0.6–0.8) prior to induction by the addition of 0.5 mM IPTG. Cells were incubated at 25 °C with 7 × g shaking overnight. Bacteria was then harvested by centrifugation at 6000 × g, 4 °C for 15 min. Pellets were resuspended in lysis buffer  and probe-sonicated . The soluble fraction was separated by centrifugation at 7000 × g for 1 h at 4 °C and incubated with pre-equilibrated Co-NTA affinity resin for 1 h at 4 °C with rocking. Beads were rinsed thoroughly with wash buffer  before elution of target protein with elution buffer . The eluate was concentrated, and buffer exchanged into 1X DPBS via Amicon Ultra 15 mL centrifugal filters (3 K MWCO) and subjected to sodium 2-mercaptoethanesulfonate (MesNa) thiolysis  for 48 h at 25 °C. The ubiquitin-thioester was then separated from the intein by C4 reverse-phase purification using a Biotage Isolera Spektra FLASH system. Characterization of the ubiquitin-thioester was performed using C4 analytical RP-HPLC and ESI-MS. Lyophilized Ubi-MesNa was dissolved to 5 mM in DMSO containing 50 molar equivalents of 1-octanethiol and 2 mM TCEP·HCl. The pH was adjusted to 7.0 and incubated for 1 h at 25 °C with monitoring by C4 analytical RP-HPLC. A separate product peak appeared with a later retention time in an ~75% yield. Unreacted Ubi-MesNa could be purified and recycled in subsequent thiol exchange reactions to improve yield. The Ubi-αCOSC8 was separated from the MesNa thioester by C4 reverse-phase purification using a Biotage Isolera Spektra FLASH system. Prior to lyophilization, the Ubi-αCOSC8 was purified by semipreparative HPLC using a C18 column  to give Ubi-αCOSC8 (16). Analytical HPLC: tR = 12.30 min . The expected mass of Ubi-αCOSC8 (16) was corroborated by mass spectrometry (ESI-TOF) : The lyophilized peptides Ubi-αCOSC8  and CK(PhC16)ANK  were dissolved in separate vials  using freshly prepared and degassed ligation buffer . The solutions were combined  for 1 min, then 5%–70% MeCN (0.1% TFA) over 19 min; Eclipse Plus C8 analytical column] was collected, and then subjected to analysis by mass spectrometry (ESI-TOF)  was corroborated ANK  Fig. . The expted Fig. .Supplementary InformationPeer Review File


Formosa algae KMM 3553T. They were classified to PL7 and PL6 polysaccharide lyases families and expressed in E. coli. The recombinant ALFA3 appeared to be active both on mannuronate- and guluronate-enriched alginates, as well as pure sodium mannuronate. For all substrates, optimum conditions were pH 6.0 and 35 °C; Km was 0.12 ± 0.01 mg/mL, and half-inactivation time was 30 min at 42 °C. Recombinant ALFA4 was active predominately on pure sodium mannuronate, with optimum pH 8.0 and temperature 30 °C, Km was 3.01 ± 0.05 mg/mL. It was stable up to 30 °C; half-inactivation time was 1 h 40 min at 37 °C. 1H NMR analysis showed that ALFA3 degraded mannuronate and mannuronate-guluronate blocks, while ALFA4 degraded only mannuronate blocks, producing mainly disaccharides. Products of digestion of pure sodium mannuronate by ALFA3 at 200 µg/mL inhibited anchorage-independent colony formation of human melanoma cells SK-MEL-5, SK-MEL-28, and RPMI-7951 up to 17% stronger compared to native polymannuronate. This fact supports previous data and suggests that mannuronate oligosaccharides may be useful for synergic tumor therapy.A bifunctional alginate lyase (ALFA3) and mannuronate-specific alginate lyase (ALFA4) genes were found in the genome of polysaccharide-degrading marine bacterium  They are produced by organisms, which use alginic acids as a source of energy, and by alginate-producing bacteria to form exo-polysaccharide (EPS) as the main part of biofilms . Their mnate (Δ) . (Pseudomonas aeruginosa strain PDO486 (DP 263) [According to classical nomenclature, alginate lyases are divided into M-specific EC 4.2.2.3) and G-specific (EC 4.2.2.11), however, there are also lyases with wide specificity that cleave bonds between М- and G- residues. They also have different types of action (exo- or endo-), and alginate oligosaccharide lyases (4.2.2.26) have the exo- type of action. Not all alginate lyases digest acetylated substrate [.2.3 and http://www.cazy.com): PL5, PL6, PL7, PL14, PL15, PL17 and PL18 [n toroid folds [By the amino acid sequence homology, alginate lyases can be divided into several structural families of polysaccharide lyases (PL)  stimulate the immune system, increasing the production of cytokines  in mural macrophages. The most active are unsaturated guluronate G8 and unsaturated mannuronate M7 oligomers [Agarivorans sp. L11 (only DP 5) inhibit the growth of human bone cancer cells MG-63 by 60–70% [Sphingobacterium sp. (DP 2–4) show antioxidative properties in vitro [Products of action of alginate lyases reveal multiple biological activities, presumably, due to their evenly distributed negative charge and ability to strongly bind with a protein molecule, modifying its conformation. For example, those obtained by sp. DP 3– stimulatligomers . Productin vitro . Alginatin vitro ,14 and ein vitro . Computer analysis of the distribution of alginate lyases and alginate lyase-encoding genes among different groups of living organisms reveals that there is still a great potential in experimental characterization of amino acid sequences with potential alginate degrading activity. There are more than 50 thousand known amino acid sequences, identified as alginate lyases, and 47 thousand of them belong to bacterial genomes. Just 260 enzymes have been functionally characterized: 181 from EC 4.2.2.3, 70 from EC 4.2.2.11 and 9 from EC 4.2.2.26. According to the CAZY database [Formosa algae KMM 3553T  is one of the potential sources of genetic material for the expression of alginate lyases; these bacteria inhabit thalli of brown algae Fucus evanescens in the cold waters of Sea of Okhotsk [Thus, the class of alginate lyases is still insufficiently studied, while its practical application is of high importance. Polysaccharide-degrading marine bacterium  Okhotsk . F. algae can be described as a psychrotolerant organism, environmentally adapted and evolutionally specialized for inhabiting and utilizing brown algae, and containing enzymes, that are active at low temperatures. On the other hand, its optimum temperature of growth is 23 °C, so there can be other enzymes, stable at room temperature, that can be used in biotechnology.The strain has been characterized previously as the producer of various carbohydrate-degrading enzymes: fucoidanases FFA1 , FFA2 1, and endF. algae’s enzymatic machinery for biotechnology applications and the discovery of new alginate lyases ALFA3 and ALFA4.In this study, we report on the progress of research on The yield of the target enzyme was 72 mg/L of cultural media for ALFA3 and 12 mg/L for ALFA4. Molecular weights, structural families of amino acid sequences, and substrate specificity are presented in ALFA3 digested three types of sodium alginates  with equal effectiveness: 21.5 U/mg for M- and MG- and 18.7 U/mg for G-. The detected difference indicated that this enzyme belonged to EC 4.2.2.3 . Optimum pH was 6.0 for all types of substrates .+ and Ca2+ in concentrations up to 0.5 M did not influence the activity. Km was 0.12 ± 0.01 mg/mL Kinetic analysis of substrate digestion for ALFA3revealed that the depth of substrate cleavage strongly depended on M/G ratio: while sodium polymannuronate was completely digested to oligosaccharides in 24h (hydrolysis depth 88.3%), guluronate-enriched substrate contained a considerable high-molecular fraction (hydrolysis depth 67.9%), while this fraction was much smaller for the mannuronate-enriched substrate (hydrolysis depth 87.3%) . These fPrecipitation of alginate poly- and oligosaccharides with ethanol provides from incubation mixture is a controllable and simple method for isolating low-molecular fraction with a defined range of alginate oligosaccharides (AOS) . In comp1H NMR analysis of digestion products for guluronate-enriched substrate (M:G~1:2) showed that it was successfully hydrolyzed by ALFA3 . For ALFA3 predominantly H4 ΔM signals (5.87 and 5.95 ppm) above H4 ΔG (5.76 and 5.80 ppm) are observed A, while Classification of alginate lyases into M-specific (EC 4.2.2.3), G-specific (EC 4.2.2.11), and alginate oligosaccharide lyases (4.2.2.26) prompted testing of the ALFA3 and ALFA4 substrate specificity on three different types of substrate . ConsideIn the present study, the alginates and their products did not possess cytotoxic effect against melanoma SK-MEL-5, SK-MEL-28, and RPMI-7951 cell lines at concentrations up to 800 µg/mL.Colony formation and growth of SK-MEL-5, SK-MEL-28, and RPMI-7951 cells was tested in the presence of non-toxic concentrations (200 µg/mL) of alginates and their derivatives using soft agar. The results showed that alginates and their products inhibited colony formation of these cells by less than 15%, although RPMI-7951 cells were more sensitive to the investigated compounds. Polymannuronate and products of its digestion by ALFA3  demonstrated higher activity and decreased the number of melanoma RPMI-7951 cells colonies by 20% and 37%, respectively .Strongylocentrotus intermedius  did not affect the development rates and survivability of fertilized eggs of the sea urchin ermedius . Both trF. algae KMM 3553T. Our results with different digestible substrates determined that ALFA3 is a bifunctional endolytic enzyme, while ALFA4 is an M-specific endolytic enzyme. Slight differences in substrate specificity allowed us to classify ALFA3 and ALFA4 as EC 4.2.2.3 . These bifunctional alginate lyases form a sizable group in the pool of alginolytic enzymes. While there is no specific EC number assigned to this group, these enzymes can be classified as either mannuronate-specific (EC 4.2.2.3) or guluronate-specific (EC 4.2.2.11). Previously four alginate lyases from marine bacterium Vibrios plendidus 12B01 were isolated, and two of them could cleave the bond between M and G subunits, while the other two were guluronate-specific [Flammeovirga sp. strain MY04 [Two recombinant alginate lyases (ALFA3 and ALFA4) were cloned from the genome of marine bacterium specific . Recentlain MY04  suggestiThe relatively low molecular weight of these enzymes makes its recombinant production reliable and efficient due to fewer resources required for their synthesis. Sargassum filipendula to 2.28 in Saccharina japonica [Alginic acids present up to 40% of brown algae dry weight with M/G ratio ranging from 0.21 in japonica , so M-spSignificantly, the optimum pH of ALFA3 was rather sharp for pure M- and G-enriched substrate, but it was much wider (6 to 9) for MG-substrate. This phenomenon can be explained by the differences in optimum conditions for digestion of MM and MG bonds, but this hypothesis requires further investigations.Analyzing final digestion products of different substrates by ALFA4, we realized that the enzyme produced alginate oligosaccharides predominately from mannuronate-enriched and guluronate-enriched substrates but not from pure polymannuronate. Considering that ALFA4 has the highest activity with mannuronate-enriched substrate, a lower one with pure polymannuronate and relatively insignificant with guluronate-enriched one, it is possible to hypothesize that ALFA4 cleaves MM-bonds but needs some G-links for more effective substrate binding.The initial substrates and products of their enzymatic digestion showed only moderate toxicity on human melanoma cells and almost no toxicity on fertilized eggs of sea urchin.There was no difference if the substances were applied on fresh-fertilized eggs or blastulas (12 h after fertilization). In some cases, the cells or blastulas survived for 6–7 days longer than controls after treatment with products of alginate enzymatic digestion (both HMW and LMW in concentrations 0.5-1.0 mg/mL); this effect may be explained by the use of the alginates as a source of energy. If this is confirmed, the products of enzymatic digestion may be characterized as potentially non-toxic for healthy cells, opening the possibility for further long-term bioassays with healthy human cells.F. algae genome, all alginate lyases are in different gene clusters. This distribution suggests that algal alginic acids play a facultative role in the nutrition of this bacterium. In F. algae, there is a wide range of alginate oligosaccharides with DP from 1 to 20. This observation can be used for screening of products’ biological activity.In conclusion, in all cases of alginate substrate digestion with recombinant alginate lyases ALFA3 and ALFA4 of marine bacterium F. algae KMM3553T strain was grown as described by Ivanova et al. [a et al. . Its gena et al. . The stra et al.  based onALFA3_dir 5’-CCGAGAACCTTTACTTCCAGGGGGAAGTTACAGATGATAATGCCGAC-3’ALFA3_rev 5’-TCTTAGATTCTGTGCTTTTAAGCAGAGATTACCTATTATAAACTATGTTCGGTTTTTACCTTAT-3’ALFA4_dir 5’- CCGAGAACCTTTACTTCCAGGGGGAAGATCCAGACGACATAGAAGAC-3’ALFA4_rev 5’-TCTTAGATTCTGTGCTTTTAAGCAGAGATTACCTATTACTTTAAGTCTGCCGCCAA-3’http://www.ebi.ac.uk/interpro/) [E. coli Transformation Kit & Buffer Set . Plasmids were cloned in E. coli XL10 Gold cells  and extracted with QuantumPrep® Plasmid Miniprep Kit . The PCR was done using Phusion HF polymerase . Thirty-five cycles of amplification were done to obtain the necessary amount of insert and vector. PCR reaction was controlled by electrophoresis in agarose gel. Sequence analysis was carried out using the NCBI BLAST . Domain terpro/) . Multiplterpro/) . CompeteE. coli Arctic Express (DE3)  cells with plasmids pColdII-ALFA3 and pColdII-ALFA4 were grown overnight at 37 °C on LB plates containing 100 µg/mL ampicillin. A single colony was picked and grown at 200 rpm in LB with 100 µg/mL ampicillin at 37 °C for 12 h, then transferred to fresh LB with 100 µg/mL ampicillin. Cells were grown up to an optical density of approximately 0.6 OD600; expression was induced by IPTG (0.2 mM) and carried out for 16 h at 16 °C. Recombinant cells were collected by centrifugation at 6000 relative centrifugal forces (rcf) for 20 min at 4 °C, resuspended in 20 mM phosphate buffer (pH 7.4), disrupted by sonication, and centrifuged at 6000 rcf during 20 min at 4 °C. The resulting supernatant was applied to a 2 mL column of Ni-NTA agarose  pre-equilibrated with 10 mM sodium phosphate buffer (pH 7.4) with 0.5 M NaCl and 20 mM imidazole. Chromatography was performed with a linear gradient of 20–500 mM imidazole , the flow rate of 0.5 mL/min. His-tag was removed by TEV-protease . The enzyme was re-equilibrated in succinate buffer pH 6.0. This method produced pure alginate lyases ALFA3 and ALFA4  as a standard . The hom2O with a volume ratio of 2:1:1. The gel images were visualized using a calibrated densitometer DS-800 (Bio-Rad).The substrates and products of their hydrolysis  were mixed with 5 μL of loading buffer containing a 20% solution of glycerol in water and 0.02% of phenol red. The samples (10 μL) were separated by electrophoresis through a 5% (w/v) stacking gel with 50 mM Tris–HCl buffer pH 6.8 and 27% (w/v) resolving polyacrylamide gel with 150 mM Tris–HCl buffer pH 8.8. The gel was 1 mm thick. The gel was stained with 0.02% O-toluidine blue  and 0.3% alcian blue in EtOH, AcOH, and HSaccharina cichorioides in the Laboratory of Enzyme Chemistry, G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Russia PIBOC FEB RAS. Guluronate-enriched sodium alginate , and mannuronate-enriched sodium alginate  were also used.Sodium polymannuronate was isolated from brown alga The activity of alginate lyase was deduced from the increase in reducing sugars . The incThe optimum pH values for ALFA3 and ALFA4 were determined by the standard activity assay for 1 h at 37 °C using 0.2 M citrate-phosphate buffer in the range of pH 2.0–10.0. The reaction mixture contained 100 µL of the enzyme solution, 200 µL of the substrate solution (4 mg/mL), and 300 µL of a buffer with various pH.The optimum temperature was determined by the standard activity assay for 1 h at temperatures between 25 and 55 °C with 5 °C intervals and the concentration of reducing sugars was measured.Thermal stability was estimated by measuring the residual activity by standard assay after pre-incubation of the enzyme at temperatures between 10 and 70 °C for 1 h. Km was calculated from the Lineweaver-Burk plot using data collected by measuring the rate of mannuronate-enriched sodium alginate hydrolysis under the standard assay conditions for 1 h at 37 °C from 0.025 to 2.0 mg/mL.Classification of the enzymes by amino acid sequences was done using InterPro . ClassifFor exhaustive hydrolysis, 100 µL of enzyme solution (1 mg/mL) was added to a solution of different sodium alginate substrates , and the mixture was incubated at 37 °C. Aliquots (50 μL) were taken at 0, 0.5, 1.5, 3.5, 6, 24, 48, 72, and 96 h, and the reaction was terminated by heating in a solid-state thermostat  for 1 min at 99 °C. Products were analyzed by C-PAGE. After 96 h, the reaction mixture was concentrated by rotary evaporator  at 50 °C and lyophilized.Fractionation of alginate oligosaccharides was performed with ethanol precipitation. After the end of reaction, the enzyme was inactivated by heating the reaction mixture at 90 °C during 1 h and then cooling it to the room temperature. Ninety-five percent ethanol was added to aliquots of inactivated reaction mixture in ration (ethanol:mixture) from 0.1:1 to 0.9:1, so the final concentrations of ethanol were from 8.6 to 45%. The aliquots were stirred well and then incubated at 4 °C for 18 h. After incubation, the aliquots were centrifuged at 9000 rcf during 30 min, and the supernatants were collected, concentrated by rotary evaporator  at 50 °C, lyophilized, resuspended in water (up to 4 mg/mL), and analyzed by C-PAGE.1H NMR spectra were recorded using a Bruker DRX 500 spectrometer 500 MHz . There were taken the spectra of the native substrates and the reaction mixtures (96h of reaction) . The reactions of hydrolysis  were run in 25 mM succinic buffer pH 6.0 at 37 °C, so after resuspension, pH was close to 6.0. The products of the exhaustive hydrolysis were freeze-dried, resuspended in D2O, and the spectra were taken at 35 °C. The volume of the sample was 0.5 mL.l-glutamine, penicillin-streptomycin solution, trypsin, fetal bovine serum (FBS), sodium hydrocarbonate (NaHCO3), and agar were purchased from Biolot .Dulbecco’s Modified Eagle Medium (DMEM), phosphate-buffered saline (PBS), Human melanoma SK-MEL-5 (ATCC® no. HTB-70™), SK-MEL-28 (ATCC® no.HTB-72™), RPMI-7951 (ATCC® no. HTB-66™) cell lines were purchased from the American Type Culture Collection .l-glutamine, and penicillin-streptomycin solution. The cell cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2.Human melanoma SK-MEL-5, SK-MEL-28, and RPMI-7951 cell lines were cultured in DMEM medium, supplemented with 10% fetal bovine serum (FBS), 200 mM 4 cells per well in 200 µL of the complete medium in 96-well plates. After 24 h incubation, the attached cells were treated with various concentrations of alginates and its products , while the control was mock-treated with the DMEM medium only. Cells were cultured for an additional 24 h at 37 °C in 5% CO2 incubator. After incubation MTS reagent (20 µL) was added to each well, and cells were incubated for 3 h at 37 °C in 5% CO2. Absorbance was measured at 490/630 nm by a microplate reader Power Wave XS . All tests were carried out in triplicate.3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was used as the indicator of cell viability determined by mitochondrial-dependent reduction of formazan. In brief, cells were seeded at a density of 1.0 × 104/mL) were seeded in six-well plate with or without alginates and its products (200 µg/mL) in 1 mL of 0.3% Basal Medium Eagle (BME) agar containing 10% FBS, 2 mM l-glutamine, and 25 µg/mL gentamicin. The cultures were maintained at 37 °C in a 5% CO2 incubator for 14 days, and the cell’s colonies were scored using a microscope Motic AE 20  and the Motic Image Plus computer program.SK-MEL-5 (A), SK-MEL-28 (B), and RPMI-7951 cells . A Student’s t-test was used to evaluate the data with the following significance levels: * p < 0.05, ** p < 0.01, *** p < 0.001.


Gekko gecko) is the source of a valuable traditional Chinese medicine, it is therefore listed as a Class II protected animal species in China. Due to increasing market demand and a declining supply of the species, a considerable number of adulterants have emerged in the market. Thus, it is necessary to establish an accurate and rapid method of identification for distinguishing G. gecko from its adulterants and for separating it from highly processed products.The dry body of the Tokay Gecko (COI sequences were analyzed by using MEGA 5.0 software. Several specific primers were designed to amplify mini-barcode regions and identify G. gecko from its counterfeits and products.A total of 274 COI sequences of G. gecko and 15 adulterants species were analyzed. G. gecko could be distinguished from its adulterants through BLAST analysis, intra- and inter-specific distance analyses, and an NJ tree based on COI sequences. Two pairs of specific primers designed for this study, COISF2/COISR2 and COISF3/COISR3, amplified 200- and 133-bp fragments of the COI region, respectively, both of which were suitable for the identification of G. gecko and its adulterants. Furthermore, COISF3/COISR3 detected G. gecko in 15 batches of products.274 G. gecko and counterfeits, and may also be used to distinguish G. gecko from its highly processed by-products.Therefore, the specific DNA mini-barcoding method developed here may be a powerful tool for the identification of  However, due to the destruction of its natural habitats and indiscriminate hunting, the wild G. gecko has been classified as a Class II protected animal species in China since 1989 [G. gecko [G. gecko increases its value; consequently, a considerable number of adulterants have emerged in the market, including Gekko swinhonis, Hemidactylus frenatus, Laudakia himalayana, Eumeces chinensis, Batrachuperus pinchonii, and other common adulterants [G. gecko from counterfeits. For example, G. swinhonis is commonly known as little Gecko, and L. himalayana is known as the Tibet Gecko [As one of the rarest Chinese medicinal materials, the dry body of Tokay Gecko ficiency , 2. Prevg tumors –5. Todayg tumors , 7. For lterants –12. Besiet Gecko . The indG. gecko involves observing its morphological characteristics and conducting microscopic analyses [G. gecko is used in related functional foods and Chinese patent medicines [COI region has the ability to distinguish G. gecko from related species and to identify other animal species successfully [COI region (> 600 bp) of highly processed materials [At present, the most frequently used method of identifying analyses –16; it ianalyses –19. No redicines , 21. Altessfully , 22, 23,aterials –27.COI region combined with a specific PCR technique was invented and used in this study to distinguish G. gecko from its adulterants and related products.Therefore, a new method using DNA mini-barcoding based on the G. gecko and its adulterants were collected from localities, such as Anguo herb market and Bozhou herb market , 19 specimens , and 203 medicinal materials belonging to 16 species of In addition, 15 batches of five kinds of related products were purchased from online stores and drugstores.COI sequences were amplified with universal primers LCO1490/HCO2198 according to Standard DNA Barcodes of Chinese Materia Medica in Chinese Pharmacopoeia [In 75% ethanol 10–30 mg of underlying muscles of specimens were rinsed then pulverized using a DNA Extraction Grinder . Genomic DNA was extracted using an Animal Genomic DNA Kit . acopoeia , 28, 29.COI sequences were verified in the NCBI database and DNA Barcoding System for Identifying Herbal Medicine (http://www.tcmbarcode.cn/en/). A total of 274 COI sequences were analyzed using MEGA 5.0 software, including calculating intra- and inter-specific distances and constructing a neighbor-joining (NJ) tree based on the Kimura 2-Parameter model. Sequentially, two pairs of specific primers of G. gecko were designed based on COI regions of G. gecko (658 bp) using Primer 5.0 software: COISF2: 5′-GCCTCCGCGAGTGTG-3′, COISR2: 5′-CGTGTATTGGGTTATGCTTGG-3′; and COISF3: 5′-AAAATGAAAACCCCAAG-3′, COISR3: 5′-TACGAACGGTCAACAA-3′. A reaction mixture comprising of 12.5 μL 2 × Taq PCR Master Mix , 1 μL of each primer (2.5 μM), 1 μL genomic DNA, and 9.5 μL ddH2O was prepared. The PCR amplification conditions of COISF2/COISR2 were initially set up as 94 °C for 5 min, followed by 40 cycles of 94 °C for 30 s, 61 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 10 min. The amplification of COISF3/COISR3 started with 94 °C for 5 min, followed by 40 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s, and a final extension temperature at 72 °C for 5 min.The attained trace files were assembled and trimmed using CodonCode Aligner V 3.0 . Then the 269 G. gecko was diluted to a series of concentrations  and then amplified with the specific primers to determine the minimum amplifiable concentration.The genomic DNA extracted from G. gecko were collected from online stores and drugstores. DNA was extracted from all samples in triplicates. Using nuclear separation liquid (100 mM Tris–HCl (pH 8.0), 20 mM EDTA (pH 8.0), 700 mM NaCl, 2% PVP-40, and 0.4% β-mercaptoethanol), 0.5–1.0 g of powdered samples, 5–6 g of pills, and 10 g of syrup were rinsed 1–5 times until the supernatant liquid became lightly colored or colorless [COI region and specific primers to examine the existence of G. gecko. A total of 15 DNA samples were detected with PCR reaction, three samples of each product.Fifteen batches of functional foods and Chinese patent medicines, including three batches of functional foods (Renshen Gejie Powder), twelve batches of Chinese patent medicines , allegedly containing olorless . GenomicCOI sequences belonging to 16 species were analyzed after being verified in the NCBI database and DNA Barcoding System for Identifying Herbal Medicine . The guanine-cytosine (GC) content for all species was lower than 50% except for H. frenatus (50.6%); G. gecko had a GC content of 48.2% and B. pinchonii had the lowest GC content (39.5%). Furthermore, the maximum intra-specific K2P distances between G. gecko and 15 adulterants were less than their minimum inter-specific distances, which indicated that every species in this study could be distinguished from other species  and COISF3/COISR3  amplified 200- and 133-bp fragments of the G. gecko than the universal primer and the required minimal template concentration was 1–10 pg/μL.To examine the sensitivity of the two specific primers, template DNA was diluted to a series of concentrations varying from 100 ng/μL to 1 pg/μL. Then, these samples were amplified with three replicates each using universal primers, LCO1490/HCO2198 and the two specific primers, COISF2/COISR2 and COISF3/COISR3 , can detect adulterants in admixtures based on mini-barcoding with universal primers efficiently –59. HoweG. gecko and its 15 adulterants were distinguished from each other through DNA barcoding based on COI sequences. Two pairs of specific primers COISF2/COISR2 and COISF3/COISR3 of G. gecko were designed to amplify 200-bp and 133-bp fragments of COI region, both of which were suitable for the identification of G. gecko and its adulterants. Importantly, specific primers COISF3/COISR3 could distinguish G. gecko from its products. Consequently, the specific DNA mini-barcoding method established from this study is a powerful tool for the identification of G. gecko and its adulterants. Therefore, this method could complement the traditional identification methods and lead to the selective identification of G. gecko from highly processed products. This study also provides insight into the identification of Chinese patent medicines.In this study, Additional file 1. Information of Gekko gecko and its adulterants, COI sequences analysis, figures of amplification of Gekko gecko from related products with COI universal primers and specific primers. Table S1. The information of Gekko gecko and its adulterants in this study. Table S2.COI sequences characteristics and K2P distances of Gekko gecko and its adulterants. Figure S1. The Neighbor-joining (NJ) tree based on haplotypes of Gekko gecko and its adulterants’ COI sequences. The bootstrap values (1000 replicates) were showed (≥50%) for each branch. The number of samples producing each haplotype was noted in “”. Figure S2. Alignment of specific primers COISF2/COISR2 binding regions of G. gecko and adulterants. The number of samples producing each haplotype was noted in “”. Figure S3. Alignment of specific primers COISF3/COISR3 binding regions of G. gecko and adulterants. The number of samples producing each haplotype was noted in “”. Figure S4. Amplification of Gekko gecko from related products. a Amplification with COI universal primer pair LCO1490/HCO2198. b Amplification with COI specific primer pair COISF2/COISR2. c Amplification with COI specific primer pair COISF3/COISR3. 1-3: Renshen Gejie Powder, 4-6: Shenge Pingchuan Capsule, 7-9: Gejie Dangshen Syrup, 10-12: Gejie Dingchuan Pill, 13-15: Gejie Dingchuan Capsule, CK: negative control.