Buxus microphylla, was developed as a safe and effective cardiovascular drug in China. B. microphylla has also been used to relieve various pain symptoms for centuries. In this study, we examined and uncovered strong and persistent analgesic effects of cyclovirobuxine D against several mouse models of pain, including carrageenan- and CFA-induced inflammatory pain and paclitaxel-mediated neuropathic hypersensitivity. Cyclovirobuxine D shows comparable analgesic effects by intraplantar or intraperitoneal administration. Cyclovirobuxine D potently inhibits voltage-gated Cav2.2 and Cav3.2 channels but has negligible effects on a diverse group of nociceptive ion channels distributed in primary afferent neurons, including Nav1.7, Nav1.8, TRPV1, TPRA1, TRPM8, ASIC3, P2X2 and P2X4. Moreover, inhibition of Cav3.2, rather than Cav2.2, plays a dominant role in attenuating the excitability of isolated dorsal root ganglion neurons and pain relieving effects of cyclovirobuxine D. Our work reveals that a currently in-use cardiovascular drug has strong analgesic effects mainly via blockade of Cav3.2 and provides a compelling rationale and foundation for conducting clinical studies to repurpose cyclovirobuxine D in pain management.Cyclovirobuxine D (CVB-D), the main active constituent of traditional Chinese medicine  Pain is a major health and socioeconomic burden worldwide, and there has been intensive efforts to develop novel therapeutics for pain treatment and management . Commonlω-conotoxin (Ziconotide)  , morphinonotide)  or develonotide)  and amitonotide) ). In thiBuxus microphylla var. Sinica (Chinese Boxwood) is a traditional Chinese medicine (TCM) widely used in cardiovascular disease management for centuries  calcium channels , high-voltage-activated (HVA) calcium channels (Cav2.2), voltage-gated sodium channels (Nav1.7 and Nav1.8), transient receptor potential (TRP) channels , acid-sensing ion channels (ASIC3), and purinergic channels (P2X2 and P2X4)  . Our stuelopment .l-cysteine (A600132) was obtained from Sangon Biotech. Z944 hydrochloride (SML2635), SNX-111 (C1182), (±)-Menthol (63670-100G-F) , CsCH3SO3 (C1426), Mg-ATP (A9187), CsOH (232041), and Complete Freund’s adjuvant  were purchased from Sigma-Aldrich. Other chemicals used in electrophysiology were purchased from Sangon Biotech, including NaCl (A501218), KCl (A501159), CaCl2 (A501330), MgCl2 (A100288), HEPES (A100511), glucose (A501991), TEA-Cl (A500933), TEA-OH (A501767), NaOH (A100583), KOH (A610441), CsCl (A620054), EGTA (A600077), BaCl2 (A602020), K-gluconate (A507810).Cyclovirobuxine D , Capsaicin (HY-10448) and Paclitaxel (HY-B0015) were purchased from MedChem Express. Allyl isothiocyanate  was obtained from Ai Keda Chemical Technology Co. AMG 517 (S7115) was purchased from Selleckchem. A-967079 (B7702) and ATP disodium salt (B3304) were obtained from APExBIO. Carrageenan (S51730) was purchased from Shanghai Yuanye Bio-Technology Co. Ltd. C57BL/6J mice weighing 18–22 g and aged 6–8 weeks were purchased from SKbex Biotechnology Co., Ltd. and maintained in the animal services facility of Kunming Institute of Zoology. Mice of both genders were randomly divided into 6 to 24 animals per group. We used half male and half female mice for groups with an even animal number, and groups with an odd animal number contained one more mouse with the different gender. The mice were maintained at a constant temperature (22 °C) and humidity (55%) with a 12 h light-dark cycle. Animal husbandry was performed exclusively by the experimenter for the entire duration of the animals’ stay at the housing facility. Lab coats and gloves were changed between cohorts. The housing room was used exclusively for this study and was open to only the experimenter. The mice were acclimatized to the testing facility for at least 1 week before the experiments. Each animal was housed in a single cage to prevent fighting and to freely consume food and water.All the procedures and care and handling of the animals were approved by the Animal Care and Use Committee at Kunming Institute of Zoology, Chinese Academy of Sciences (IACUC-RE-2022-07-002), and the principles of laboratory animal care  were followed. All animal experiments were performed at room temperature (approximately 23°C).Before measurement of capsaicin- or AITC-induced acute pain, mice were acclimated to a Plexiglas chamber for at least 30 min. Based on toxicological study of CVB-D , saturatCapsaicin-induced thermal hypersensitivity was conducted as described previously . Mice fiPeripheral inflammatory painmodels were generated by i. pl. injection of carrageenan or CFA into the hindpaws of mice. We used 3% carrageenan in saline to induce sub-acute inflammation as described previously . Mice fiIn CFA model, mice firstly received a single i. pl. injection of a vehicle , CVB-D  or Z944 . Thirty minutes later, 20 μL of CFA  or saline was administrated by i. pl. injection. The time of receiving such treatments was set as Day 0. Mechanical and thermal hypersensitivity were studied within a 2-week period after CFA injection. Mechanical hypersensitivity was examined on Day 0, Day 1, Day 2, Day 4, Day 7, Day 10, and Day 14, and thermal hypersensitivity was evaluated on Day 0, Day 1, Day 2, Day 4, Day 7, and Day 12.For assessment of mechanical hypersensitivity, mice were placed in a plastic cage with a wire mesh bottom and were allowed to acclimate until cage exploration and major grooming activities ceased. 50% mechanical paw withdrawal threshold (PWT) was assessed with von Frey filaments  using the up-down method . Thermall-cysteine-induced hypersensitivity model, mice were given l-cysteine based on the protocol as described previously  or saline by i. pl. injection. Behavioral assessments (von Frey test or hot plate assay) were conducted at 10 min after hypersensitivity induction using the same approaches as described in For eviously . In brieMice were given paclitaxel based on the protocol as described previously . In brieBlinding was not carried out for above-mentioned mouse models, as the tester also conducted the modeling and all animal husbandry. In addition, the effects of these models are obvious when handling the animals and performing testing.d-lysine-treated 35 mm dishes containing DMEM/F-12  supplemented with GlutaMAX  and 10% fetal bovine serum . Electrophysiological experiments were performed after 2 h of culture.Inhalation anaesthesia was firstly applied to adult mice. Then dorsal root ganglia were dissected, rinsed with Hank’s buffer , and digested in the same buffer containing 1.5 mg/ml collagenase P  for 25–45 min at 37°C. Partially digested tissues were centrifuged at 200 × g for 3 min, and the pellets were resuspended in 0.25% trypsin-EDTA  and digested for an additional 5 min at 37°C. The digested ganglia were spun down, resuspended, and triturated with plastic pipette tips to release the neurons. The cells were filtered through a 70-μm cell strainer , plated into 24-well plates, then aliquot and cultured in poly-2X2 (NM_053656.3), human P2X4 (NM_002560.3), rat ASIC3 (NM_173135.1), rat Nav1.7 (NM_133289.1), human Nav1.8 (NM_006514.3), human Cav3.1 (NM_198387.3), human Cav3.2 (NM_021098.3), human Cav3.3 (NM_021096.4) and human Cav2.2 (NM_000718.4) (with rat β3 (NM_012828.3) and rabbit α2δ (NM_001082276.1) subunits), together with pEGFPN1  plasmids using LipoD293 in vitro DNA Transfection Reagent  and used within 48 h.HEK 293T cells (American Type Culture Collection (ATCC) were grown in DMEM  plus 10% fetal bovine serum  and 1% penicillin (100 U/ml)/streptomycin . HEK 293T cells were transiently transfected with pCDNA3.1-human TRPA1 (NM_007332.3), human TRPV1 (AJ277028.1), human TRPM8 (NM_024080.5), rat PThe patch clamp amplifier Axopatch 200B  and Double IPA , which is an integrated patch clamp amplifier with data acquisition system, were used for cell electrical signal amplification. A digital-to-analog converter Digidata 1440A  was used for digital electrical signal conversion when Axopatch 200B was used. Currents were low-pass filtered at 2 kHz and sampled at 10 kHz. Recordings were included for analysis only if access resistance began below 25 MΩ and did not change by more than 30% during the recording period. pCLAMP 10 software  and SutterPatch2.1 software (Sutter Instrument) were used for data acquisition and analysis. All experiments were performed at room temperature (approximately 23°C). For patch-clamp recordings, pipettes were fabricated from borosilicate glass  using a micropipette puller , and were fire-polished to resistances of 4–6 MΩ for whole-cell recording. All the experiments where comparisons in different conditions were made were conducted in a double-blind way.v3.1-3.3) current measurements, the extracellular solution contained (in mM) 142 CsCl, 1 MgCl2, 2 CaCl2, 10 Glucose and 10 HEPES (pH 7.4 adjusted with CsOH). The intracellular solution contained (in mM) 142 CsCl, 2 MgCl2, 11 EGTA, 5 Na2-ATP, 10 HEPES (pH 7.4 adjusted with CsOH). Peak currents of Cav3.1 and Cav3.2 were elicited by 150-ms depolarizations to −40 mV at 4-s intervals from a holding potential (HP) of −100 mV. For recording peak current of Cav3.3, the stimulus time was set to 400 ms. For studying state-dependent of Cav3.2, the HP was set to −100 mV and −75 mV, respectively. For studying use-dependent of Cav3.2, the stimulus frequencies were set to 0.1 Hz and 1 Hz, respectively. I-V curves of Cav3.1 and Cav3.2 were evoked by 150-ms depolarizations from −80 mV to + 60 mV in 10-mV increments with a 4-s interval from a HP of −100 mV. For recording I-V curve of Cav3.3, the stimulus time was set to 400 ms.For T-type calcium channels  was calculated using the equation: G = I/(V − Vrev), where V is the test voltage and Vrev was calculated by linear extrapolation of peak currents with depolarization potentials from 10 to 40 mV. Normalized G was then plotted against voltage, and activation curves were obtained by Boltzmann fitting: G/Gmax = 1/{1 + exp [(V1/2 − V)/k]}, where Gmax is the maximal conductance, V1/2 is the potential of half-maximal activation, and k is the slope factor.Voltage-dependent activation of Cav3.2 was studied by comparing the current amplitude (P1) elicited by a voltage step to −40 mV (10 ms) in the absence of fast inactivation to a second step (P2) following a series of 150-ms inactivating steps ranging from −100 to −30 mV in 5-mV increments with a 4-s interval from a HP of −110 mV. P1 was followed by a 1-s recovery step to −110 mV to remove any inactivation induced by P1. The P2/P1 ratio was used as a measure of the fraction of available channels. Normalized residual current was plotted against the voltage of the conditioning pulses. Inactivation curves were fitted with Boltzmann function in the form of I/Imax = 1/{1 + exp [(V1/2 − V)/k]}.Inactivation of Cav2.2) current measurements, the extracellular solution contained (in mM) 105 CsCl, 40 TEA-Cl, 2 BaCl2, 1 MgCl2, 10 d-glucose, and 10 HEPES (pH 7.4 adjusted with CsOH). The intracellular solution contained (in mM) 130 CsCH3SO3, 10 TEA-Cl, 10 EGTA, 10 HEPES, 5 MgCl2, and 5 Na2-ATP (pH 7.4 adjusted with CsOH). Peak currents of Cav2.2 were elicited by 500-ms depolarizations to 0 mV at 4-s intervals from a HP of −80 mV. For the state-dependent studies, the HP was set to −80 and −60 mV, respectively. I-V curve of Cav2.2 was evoked by 500-ms depolarizations from −60 mV to +70 mV in 10-mV increments with a 4-s interval from a HP of −80 mV.For N-type calcium channel . The intracellular solution contained (in mM) 150 NaCl, 1 MgCl2, 1 EGTA, and 10 HEPES (pH 7.4 adjusted with NaOH). For ASIC3, P2X2 and P2X4 recordings, the extracellular solution was the same as that of TPR channels. The intracellular solution contained (in mM) 140 KCl, 5 EGTA, and 10 HEPES (pH 7.4 adjusted with NaOH). The whole-cell currents of TRPA1, TRPM8 and TRPV1 were elicited by 500-ms voltage ramps from −100 to +100 mV at a frequency of 0.5 Hz with a HP of 0 mV. The whole-cell currents of ASIC3, P2X2 and P2X4 were recorded by the gap-free mode with a HP of −60 mV. For Nav1.7 and Nav1.8 recordings, the internal solution contained (in mM) 135 K-gluconate, 5 KCl, 5 Mg-ATP, 0.5 Na2GTP, 5 HEPES, 2 MgCl2, 5 EGTA, and 0.5 CaCl2 adjusted to pH 7.4 with KOH, and the bath solution contained (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, and 10 glucose, adjusted to pH 7.4 with NaOH. Peak currents of Nav1.7 and Nav1.8 were evoked by 10-ms depolarizations to −30 mV at 5-s intervals from a HP of −110 mV.For TRP channel recordings, the extracellular solution contained (in mM) 150 NaCl, 1 MgCl2, 152 TEA-Cl, and 10 HEPES adjusted to pH 7.4 with TEA-OH. The recording electrode was filled with a solution containing (in mM) 135 TEA-Cl, 10 EGTA, 40 HEPES, and 2 MgCl2 adjusted to pH 7.2 with TEA-OH. LVA calcium channel peak currents were evoked by 250-ms depolarizations to −40 mV at 4-s intervals from a HP of −100 mV. HVA calcium channel peak currents were evoked by 250-ms depolarizations to +10 mV at 4-s intervals from a HP of −80 mV. DRG neurons with diameters ranging 10–30 μm were chosen for recording.For whole-cell recording of dorsal root ganglion (DRG) neurons, 2 hours after being plated, dissociated DRG neurons were perfused with an extracellular solution containing (in mM) 10 BaClv channels recording. DRG neurons were held at 0 pA, and the threshold current of action potential was evoked using a series of 500-ms depolarizing current injections in 10-pA steps from 0 pA. Only neurons with a resting membrane potential below −40 mV, had stable baseline recordings, and showed evoked spikes over 0 mV were used for further experiments and analysis. Series resistance was compensated to above 75% for the recorded DRG neurons.For current-clamp recording of DRG neurons, the internal and bath solutions were the same as those for NaFor all electrophysiological experiments, extracellular solutions containing CVB-D, Z944, SNX-111, AITC, menthol, capsaicin, or ATP were prepared immediately before the experiments.t-test  or one-way analysis of variance (ANOVA) followed by Tukey’s and LSD test (for multi-group comparisons). Data are presented as the mean ± standard error of the mean (SEM), and a p-value <0.05 is considered statistically significant, with *p < 0.05, **p < 0.01, and ***p < 0.001. For multi-group comparisons, asterisks, pound signs or ampersand was used respectively, ns indicates no significant difference.We used sample/animal sizes that were deemed suitable for statistics and were similar to other studies in the field. Animals were randomly selected and allocated to experimental groups. Data normality was assessed by the Shapiro-Wilk method, and the equality of variances for two or more data groups was determined by F-test or Levene’s test. Statistical significance was evaluated using two-tailed p-value of p < 0.001 and p = 0.007 respectively, but not CVB-D . CVB-D (i.pl. injection) alone did not induce hypersensitive responses (p = 0.529). Thus, CVB-D does not affect TRPA1 or TRPV1-mediated acute pain.To evaluate the analgesic effect of CVB-D, we first examined its alleviationon acute pain induced by capsaicin (Cap) and AITC, which activate TRPV1 and TRPA1, respectively. Cap or AITC (i.pl. injection) caused robust acute pain responses in mice, manifested as licking or lifting of the injected hindpaws . These rp-value of each test point is 0.221, 0.551, 0.451, 0.389, 0.495, and 0.492, respectively). Thus, results from both genders were combined for data analysis and statistics. Carrageenan-induced mechanical and thermal hypersensitivity, manifested as a decreased paw withdrawal threshold (PWT) or paw withdrawal latency (PWL), respectively, peaked at 10–30 min, and gradually decayed over 2 days (sub-acute phases). CVB-D (10.07 μg) administrated alone by i. pl. injection did not affect the PWT or PWL in control animals . By contrast, it (i.pl. injection) reduced both acute  and sub-acute phases (Day 1) of mechanical hypersensitivity . Strikingly, at this dosage CVB-D prevented the development of thermal hypersensitivity . In the dosage range of 0.4–10.0 μg by i. pl. injection, CVB-D dose-dependently prolonged the PWL measured at 30 min after carrageenan injection, with an EC50 of 2.03 ± 0.53 μg . Therefore, both intraplantar and intraperitoneal administration of CVB-D produced clear analgesic effects on carrageenan-induced inflammatory hypersensitivity.We next examined the effect of CVB-D on two typical mouse inflammatory pain models induced by i. pl. injection of carrageenan and CFA, respectively. In the carrageenan-induced model, there was no noticeble difference in the mechanical hypersensitivity between male and female mice in both control and test groups  and largely eliminated thermal hypersensitivity  during 2 weeks after injection of CFA. These long-lasting effects of CVB-D hint that its actions may not only involve the initiation but also the maintenance of CFA-induced hypersensitivity.CFA-induced hypersensitivity is another well-studied chronic inflammatory pain model, with hypersensitive responses lasting for approximately 1–2 weeks . A singlThese results indicate that in addition to its cardiovascular bioactivities, CVB-D has strong analgesic effect on inflammatory pain hypersensitivity.50 value of 16.03 ± 0.11 μM  did not affect the basal whole-cell current of mock-transfected HEK 293T cells, and did not affect agonist-induced currents in HEK 293T cells expressing TRPM8 and TRPV1, which sense noxious temperature and chemicals  Yekkira. CVB-D d 0.11 μM . Of note 0.11 μM , indicatand P2X4  , consist of Caral pain . We therg Cav3.2 . This ing Cav3.2 . The remg Cav3.2 , a high g Cav3.2 . Dose-re 0.35 μM . CVB-D aectively .v2.2 (N-type) is a well-established target for antinociceptive drug development  than from a HP of −100 mV (Cav3.2 under resting state) . CVB-D has an IC50 of 1.47 ± 0.03 μM . CVB-D (2.50 μM) notably shifted the half-inactivation potential (V1/2inact) of Cav3.2 from −60.53 ± 0.47 mV to −64.19 ± 0.89 mV, as well as potently altered the slope factor of the inactivation curve from 2.57 ± 0.16 mV to 3.54 ± 0.11 mV . CVB-D showed negligible effect on the half-activation potential (V1/2act) of Cav3.2 (−51.99 ± 0.77 mV VS. −52.91 ± 0.87 mV), but it noticeably augmented the slope factor of the activation curve from 3.48 ± 0.21 mV to 5.10 ± 0.16 mV . As a result, the window current, which is represented by the overlapping area under activation and inactivation curves, was negatively shifted by CVB-D   and was almost eliminated by 5 μM Z944 (We next examined the effect of CVB-D on native Ca0–30 μm) . Whole-cvoltages . Z944-seμM CVB-D . As withreversed . On aver μM Z944 . At this μM Z944 .v2.2-mediated HVA current in DRG neurons evoked at +10 mV . This concentration of SNX-111 did not affect Cav3.2 current evoked at−40 mV (CVB-D (10 μM) also greatly inhibited SNX-111-sensitive, Cat +10 mV . This int +10 mV . On averat−40 mV .v3.2 in AP firing in these neurons. By contrast, SNX-111 did not visibly alter AP firing , and the number of AP spikes were drastically decreased . No such effects were observed in SNX-111-treated neurons  firing of DRG neurons. Freshly dissociated DRG neurons were randomly divided into four groups: control (bath solution), CVB-D (30 μM), Z944 (5 μM) and SNX-111 (0.5 μM). In control neurons, a threshold depolarizing current evoked repetitive and sustained AP firing . StrikinP firing . Comparev3.2 and Cav2.2 channels and that inhibition of Cav3.2, but not Cav2.2, greatly decreases the excitability of DRG neurons.These results indicate that CVB-D potently inhibits native Cav3.2 in CVB-D’s analgesic effects. l-cysteine promotes cutaneous mechanical and thermal hypersensitivity via a quick and specific way through Cav3.2 channels . Z944 (3.84 μg) applied by i. pl. injection showed a comparable analgesic effect on the tactile hypersensitivity as that of CVB-D  without affecting the baseline PWT or PWL .We next studied the importance of Cachannels . Adminisp-value in p-value in p-value in p < 0.001 for carrageenan VS. CVB-D, p < 0.001 for carrageenan VS. Z944, and p = 0.247 for CVB-D VS. Z944. In p < 0.001 for carrageenan VS. CVB-D, p < 0.001 for carrageenan VS. Z944, and p = 0.247 for CVB-D (i.p.) VS. Z944). Noticeably, as with CVB-D, a single dose of Z944 by i. pl. injection persistently eliminated mechanical hypersensitivity from day 1 to day 7 in CFA-induced mouse model . Z944 (i.p. injection) alone did not affect the baseline PWT . The vehicles for CVB-D and Z944 showed negligible effects on the basal mechanical sensation and carrageenan-induced tactile hypersensitivity  + saline VS. vehicle (1% DMSO) + saline of each test point is 0.963, 0.123, 0.630, 0.031, 0.235, and 1.000, respectively. p-value of vehicle (5% ethanol) + carrageenan VS. vehicle (1% DMSO) + carrageenan of each test point is 0.604, 0.517, 0.470, 0.811, 0.155, and 0.039, respectively).In carrageenan-induced mouse models, i. pl. (3.84 μg) or i. p. (3 mg/kg) injection of Z944 alleviated both acute and sub-acute phases of thermal and mechanical hypersensitivity . However, CVB-D (10.07 μg) or Z944 (3.84 μg) administrated by i. pl. injection showed a much stronger analgesic effect than SNX-111 did . Indeed, the PWL at 15 or 30 min after Cap injection was even longer than the baseline PWL (0 min)  , suggesting that Cav2.2 also contributes to the analgesic effect of CVB-D on thermal hypersensitivity.Casal horn . It has the skin . Consist (0 min) . These rv2.2 blocker SNX-111 could alleviate inflammatory hypersensitivity as CVB-D did. SNX-111 (53 ng) administrated by i. pl. injection had negligible effect on thermal or mechanical hypersensitivity in carrageenan-induced mouse model . Furthermore, a combination of SNX-111 with CVB-D or Z944 showed ignorable synergistic analgesic effect .Next, we examined whether the specific Cav3.2 plays a major role and Cav2.2 plays a minor role in the analgesic effects of CVB-D in mouse models.Taken together, these results suggest that Cav3.2 is highly involved in PTX-induced mouse neuropathy . Z944 produced a comparable analgesic effect . Given that CVB-D was also identified as a potential anti-tumor agent (Paclitaxel (PTX) is a well-known anti-tumor drug worldwide . Howeveruropathy , we testuropathy . A singlor agent , it may l-cysteine-mediated thermal and mechanical hypersensitivity in mice. Although CVB-D is a clinically used drug and has been found to have diverse actions in humans and animals, its molecular targets remain to be identified , Aδ- and C-LTMRs (v3.2 along LTMR-fiber trajectories is consistent with its key role in the transmission of low-threshold mechanical signals and in the development of mechanical hypersensitivity (v2.2 to mechanical hypersensitivity remains incompletely defined. For instance, pharmacological blockade of Cav2.2 does not attenuate mechanical hypersensitivity in CFA-induced mice (v2.2 KO mice show a comparable mechanical sensation as WT animals do (v3.2 is the major player for CVB-D’s analgesic effects on mechanical hypersensitivity.Ca C-LTMRs . The presitivity . The conced mice . Cav2.2 imals do  but exhiimals do . Our worimals do , SNX-111imals do  and admiimals do . TherefoWhile CVB-D is a safe and effective cardiovascular drug widely used in China, several animal experiments have reported some potential toxicities when it is administered systemically at relatively high doses or for prolonged durations . In thisin vitro and in vivo antiproliferative activities (Our study shows that CVB-D effectively and long-lastingly relieves PTX-induced thermal hypersensitivity . This fitivities , also raB. microphylla, exhibits strong and persistent antinociceptive actions against several mouse models of inflammatory and neuropathic pain. Second, we demonstrate that the analgesic effects of CVB-D are mainly achieved through inhibition of Cav3.2, a key target in modern analgesic drug development. Third, we uncover a potential of CVB-D to ameliorate the intractable neuropathic hypersensitivity associated with PTX chemotherapy while concurrently exerting its anticancer effects. In conclusion, we provide a convincing rationale and foundation for conducting clinical studies to repurpose CVB-D in pain management.In this study, we first demonstrate that CVB-D, a cardiovascular drug from traditional Chinese medicine


Streptomyces sp. RKND-216. Through the use of Global Natural Product Social Molecular Networking (GNPS), additional members of the levesquamide family were identified (B-G). Levesquamide B is a glycosylated analogue, isolated and structurally elucidated via spectroscopical techniques along with the putative structures of levesquamide C and D. For masses relating to the additional three levesquamides (E-G), their complete structures remain ambiguous.Levesquamide A is an isothiazolinone-containing anti-tubercular natural product isolated from  Actinobacteria are one of nature’s most prolific medicinal chemists producing a plethora of antibiotic, anticancer, antifungal and anti-infective agents +m/z 631.2181) and E ([M+H]+m/z 659.2151) were calculated to assess relative production levels in the different media  using the optimal NH4Cl concentration. Extraction of the scale-up fermentation with EtOAc yielded 1.6 g of crude extract.Supplementation of media with NH18 stationary phase over a H2O:MeOH 95:5 to 2:98 gradient over 35 min. Levesquamide A (m/z 631.2181) eluted at 31 min and its analogue, levesquamide E (m/z 659.2151) eluted at 29 min. These fractions were subjected to analysis via UHPLC-HR-ESI-MS+. which led to the identification of another levesquamide analogue (m/z 793.2729), named levesquamide B. In order to determine how many levesquamides were present we conducted a molecular networking analysis using the Global Natural Product Social Molecular Networking (GNPS) server .Automated flash chromatography was performed on a Teledyne Combiflash Rf200 using C18 RediSep columns (24 g). HPLC purifications were carried out on a Waters auto purification system coupled with an evaporative light-scattering detector and UV detector. All amino acid standards were purchased from Sigma-Aldrich. All reagents were purchased from commercial sources and used without further purification unless otherwise stated.®, Spectrum Brands) agar plates incubated at 30 °C. Approximately 1 cm2 of the agar culture was used to inoculate 7 mL of ISP2m broth in a 25 × 150 mm culture tube containing five 4 mm glass beads and incubated at 30 °C and 200 RPM. After 72 h, 1 mL of the first-stage seed was transferred to 7 mL of fresh ISP2m broth and incubated under the same conditions for 24 h. After 24 h 0.5 mL of the second stage seed as transferred into triplicate 10 mL fermentations of RKND-216 performed in BFM23m base medium and variations containing different salts  added at five different concentrations . After incubating for 10 days at 30 °C with shaking at 200 RPM, fermentations were extracted with an equal volume of EtOAc. The extracts were dried in vacuo and dissolved in 1 mL of MeOH and subjected to UHPLC-HR-ESI-MS analysis.A two-stage seed culture process was used to generate inoculum. The strain wasmaintained on marine ISP3m  broth each. After incubating for 10 days at 30 °C with shaking at 200 RPM, the culture was extracted three times with equal volumes of EtOAc. The organic layers were combined and dried in vacuo, providing 1.6 g of crude extractA two-stage seed culture process was performed as previously described above. The second-stage seeds from multiple tubes were combined, and 30 mL of seed culture was used to inoculate each of 9 Fernbach flasks containing 1 L of fresh BFM23m supplemented with 2.4 g/L NHhttps://winscp.net/eng/download.php (accessed on 14 March 2022)). The molecular network was generated using Global Natural Products Social Molecular Networking [https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=c18a7718fb9c40b0b56d95db62eef1c9 (accessed on 12 September 2022)). The MS/MS data were deposited in the MassIVE Public GNPS data sets (https://massive.ucsd.edu (accessed on 12 September 2022)) with the accession no. MSV00009031. Notable molecular network setting parameters were as follows: precursor ion mass tolerance 2.0; fragment ion mass tolerance, 0.5; minimum pairs cos, 0.7; network TopK, 10; minimum matched peaks, 6; minimum cluster size 2. The molecular network was analyzed and visualized using Cytoscape (ver. 3.8.1) [2 fragmentation patterns compared to that of levesquamide A giving arise to a total of 7 compounds within the levesquamide family.The acquired UHPLC-MS/MS chromatograms were converted from a .RAW file to an open source MS file type .mzML using the open source GUI msConvert ver. 3.0.18232), part of the ProteoWizard tool kit . The .mztworking . The spe232, partStreptomyces sp. RKND-216 crude extract was prepared for solid load injection by adsorbing on C18 and the separation was performed with a 130 g C18 column (RediSep Rf) using a mobile phase flow rate of 32 mL/min. The mobile phase consisted of a linear gradient from H2O:MeOH (95%:5%) to 100% MeOH over 30 min followed by 100% MeOH for 5 min. Levesquamide B was further purified RP-HPLC using a semi-preparative Luna C18 100 Å column . An isocratic elution with 35% H2O containing 0.1% formic acid and 65% MeOH containing 0.1% FA was used over 30 min. The eluent was monitored by evaporative light scattering detection, and UV at 277 and 348 nm while levesquamide B was collected at 19 min. Subsequent evaporation in vacuo resulted in 0.75 mg of pure levesquamide B.The entire in vacuo. The sample was re-suspended in 0.5 mL of pyridine followed by 0.5 mL of L-cysteine methyl ester stock (5 mg/mL) before heating for 1 h at 60 °C. After 1 h, 0.5 mL of o-tolyl isothiocyanate in pyridine (5 mg/mL) was added and the reaction was again heated to 60 °C for 1 h. The reaction was concentrated under reduced pressure and resuspended in MeOH for analysis via UHPLC-HR-ESI-MS. This process was repeated for all glycoside standards.Levesquamide B (0.25 mg) was dissolved in 200 µL 2 M HCl and heated to 110 °C for 16 h before allowing to return to ambient temperature and evaporated to dryness 18 ; mobile phase flow rate, 0.8 mL·min−1; injection volume, 10 μL; linear gradient, H2O:MeOH  at 0.5 min to H2O:MeOH  at 13.0 min, which was held until 15.0 min before another linear gradient to 100% MeOH (0.1% FA) at 17.0 min, which was held until 20.0 min before returning to H2O:MeOH  at 20.1 min and equilibrating for 5.0 min. The following HRMS parameters were used: positive ionization mode; mass resolution, 30,000; mass range, m/z 190 to 2000; spray voltage, 2.0 kV; capillary temperature, 300 °C; S-lens RF voltage, 60.0%, maximum injection time, 10 ms; 1 microscan.Tanakas method was conducted using a Phenomenex Kinetex PFP 100 Å C2 fragmentation data for levesquamides E, F and G. Further work must be conducted in order to determine their structures whilst exploring the antimicrobial activity of the newly described analogues.Reinvestigation of RKND-216 has demonstrated new natural products can still be attainted through currently known microbes. The levesquamides have the potential to be an important group of compounds with levesquamide A presenting promising antitubercular properties. With technological tools continuing to advance so does our biodiscovery capabilities. Using GNPS we were able to identify six new members the levesquamide family and through the use of spectroscopical methods, we have elucidated the structure of levesquamide B. We have also presented the putative structures of levesquamide C and D, along with the MS


Objective. To study the therapeutic effects of metformin in combination with medroxyprogesterone in the early endometrial cancer patients with fertility requirements. A total of 120 patients with early endometrial cancer admitted to and treated in our hospital were enrolled and evenly assigned into two groups according to different therapeutic regimens, namely, metformin group (metformin combined with medroxyprogesterone acetate) and control group . The objective response rate (ORR) and disease control rate (DCR) were 71.7% (43/60) and 90.0% (54/60) in the metformin group and 53.3% (32/60) and 78.3% (47/60) in the control group, respectively. Adverse reactions such as gastrointestinal reaction, headache, and insomnia were mainly observed in patients. The body mass index (BMI) declined from (34.43 ± 4.34) kg/m2 to (24.77 ± 2.39) kg/m2 in the metformin group and from (33.37 ± 4.49) kg/m2 to (31.28 ± 3.55) kg/m2 in the control group after treatment. After treatment, serum levels of vascular endothelial growth factor (VEGF), angiotensin-2 (Ang-2), carbohydrate antigen 125 (CA125), and CA19-9 in the metformin group were significantly lower than those in the control group . During follow-up, the pregnancy rate was 81.7% (49/60) in the metformin group and 61.7% (37/60) in the control group, and the former was prominently higher than the latter (P = 0.025). Metformin in combination with progesterone is effective in treating early endometrial cancer patients with fertility requirements, which significantly reduced the BMI of patients and increased the pregnancy rate after treatment. Endometrial cancer, one of the most common cancers of female reproductive system, accounts for about 7% of malignant tumors in females. Its incidence rate rises obviously in females aged below 40 years old in recent years, which is about 2–14% and increasing year by year [Progesterone therapy is a therapeutic method most commonly used in clinic to preserve the fertility of patients. However, adverse reactions including progesterone resistance, thrombosis, and weight gain are observed with increasing application in clinical practice, affecting the treatment efficacy and compliance of patients , 4. MetfEndometrioid well-differentiated adenocarcinoma diagnosed by histopathological examination after operation.Patients with involved myometrium <1/2 and no extra-uterine lesions and lymph node metastasis by MRI.Those who were aged <40 years old, nulliparous, and still had the willingness to bear children.Those with estrogen-dependent and progesterone receptor (PR)-positive endometrial cancer.The clinical data of 120 patients with early endometrial cancer admitted to and treated in our hospital were collected  . The incThose who received other anti-tumor therapy were excluded.Patients with severe heart, lung, liver, and kidney dysfunction, coagulation dysfunction, immune system disorders, or infectious diseases.Patients with other malignant tumors.Patients with abnormal neurological function.The exclusion criteria were as follows:n = 60) and control group . Written signed informed consent was obtained from each patient before the study. This study was approved by the ethical committee of our hospital.The enrolled patients were divided into two groups according to different therapeutic regimens, namely, metformin group  orally at 0.4–0.8 g/d. In the metformin group, metformin  was administrated orally  in addition to the treatment in the control group. During continuous treatment, the clinical symptoms of patients in the two groups were closely observed. Administration was stopped when no residual disease was found. Patients could get pregnant only after 3 months of drug withdrawal.The following indicators were calculated: complete remission (CR): lesions disappeared, with atrophy of endometrial glands and decidual response in the stroma, partial remission (PR): there were residues of hyperplastic endometrium, without atypical cell residues, stable disease (SD): histopathological examination results were consistent with those before treatment, and progressive disease (PD): myometrial infiltration was deepened or metastasis appeared. Objective response rate (ORR) = (CR + PR)/total number of cases  ×  100%; disease control rate (DCR) = (CR + PR + SD)/total number of cases  ×  100%.The levels of tumor markers, including vascular endothelial growth factor (VEGF), angiotensin-2 (Ang-2), carbohydrate antigen (CA) 125, and CA19-9, were evaluated. Besides, the body mass index (BMI) of patients was also analyzed, and the incidence rate of adverse reactions such as gastrointestinal reactions, insomnia, and headache was compared between the two groups of patients.Besides, the successful pregnancy rate, pregnancy status, and pregnancy outcome of patients in the two groups were followed up and recorded.t-test was employed for comparing the variables before and after treatment. Percentage (%) was used to express the enumeration data, and the chi-square test was used for data analysis. A significant difference was set at P < 0.05.SPSS 19.0 software  was used for statistical analyses. All quantitative data were expressed as mean ± standard deviation. Student's P = 0.038), whereas the DCR had no statistically significant difference (P = 0.132)  and 90.0% (54/60) in the metformin group and 53.3% (32/60) and 78.3% (47/60) in the control group, respectively. The ORR was remarkably higher in the metformin group than the control group (= 0.132) .Adverse reactions such as gastrointestinal reactions, headaches, and insomnia were mainly observed in patients. There were 8 (13.3%) and 5 (8.3%) cases of gastrointestinal reactions, 6 (10.0%) and 6 (10.0%) cases of headache, 5 (8.3%) and 5 (8.3%) cases of insomnia in the metformin group and control group, respectively.2 to (24.77 ± 2.39) kg/m2 in the metformin group and from (33.37 ± 4.49) kg/m2 to (31.28 ± 3.55) kg/m2 in the control group after treatment. The serum VEGF, Ang-2, CA125, and CA19-9 levels displayed no statistically significant differences . After treatment, such levels declined in both groups (VEGF: (482.31 ± 23.67) ng/L and (477.97 ± 21.89) ng/L to (287.63 ± 25.85) ng/L and (302.51 ± 30.43) ng/L, Ang-2: (1.47 ± 0.40) μg/L and (1.51 ± 0.39) μg/L to (0.39 ± 0.09) μg/L and (0.45 ± 0.11) μg/L, CA125: (53.03 ± 6.11) U/mL and (52.08 ± 5.58) U/mL to (28.78 ± 3.03) U/mL and (30.29 ± 2.21) U/mL, and CA19-9: (22.82 ± 1.96) kU/L and (22.49 ± 2.11) kU/L to (10.47 ± 1.08) kU/L and (12.63 ± 3.05) kU/L) (The BMI declined from (34.43 ± 4.34) kg/m5) kU/L) .Results showed that the pregnancy rate was 81.7% (49/60) in the metformin group and 61.7% (37/60) in the control group. Naturally pregnant patients accounted for 67.3% (33/60) and 62.2% (23/60) of the total pregnant patients, and patients who got pregnant by assisted reproductive technology accounted for 32.7% (16/60) and 37.8% (14/60) of the total pregnant patients in metformin and control groups. Abortion, premature delivery, and term delivery were found in 8.2% and 10.8%, 16.3% and 18.9%, and 75.5% and 70.3% of pregnant patients in metformin and control groups, respectively .Generally, the attack of early endometrial cancer in young patients has associations with factors such as obesity, insulin resistance, ovulation disorders, and infertility . StudiesA phase II clinical trial conducted by Mitsuhashi et al. in 2015, in which 36 patients with atypical endometrial hyperplasia/endometrial cancer  were treated with MPA in combination with metformin for 24–36 weeks, reported that CR is found in 81% (29/36) of patients within 36 weeks, with a median follow-up of 38 months, and recurrence is observed in only 3 patients .vs. 25%, P = 0.484). In the above-mentioned study, the BMI of patients is higher in metformin treatment group than that in control group (p = 0.042), and the dose of metformin is 500–1,000 mg. The insufficient dose of metformin and the heterogeneity of BMI between treatment groups may have some influence on the results of the study, so further research should be carried out for verification [To find out whether metformin in combination with progesterone has a better therapeutic effect than progesterone alone, a study was conducted on 120 patients with early endometrial cancer. The results uncovered that the 16-week CR rate is higher in metformin + MA group than that in MA group. It was found through sub-group stratified analysis that patients with atypical dysplasia benefit more from metformin: the 16-week CR rate in metformin + MA group is almost twice that in MA group. Additionally, obese and insulin-sensitive patients also benefit from it . Howeverfication . A meta-fication . Existinn = 37) and 48.4% in MA group (n = 31) [vs. 61.7%, P = 0.025), and the metformin group also had a higher proportion of natural pregnancy than the control group, but the difference was of no statistical significance. It implies that metformin improves the pregnancy outcome of patients to some extent. The main novelty of the present study was that the patients enrolled in this research were evaluated comprehensively, including the clinical efficacy, adverse reactions, tumor marker levels, pregnancy rate, and abortion results. However, limitations also existed in this study. The limited sample size, short follow-up time, and incomprehensive follow-up content might lead to the biased conclusions. Prospective clinical research data with rigorous design, high reliability, and large sample size are needed to support the conclusions drawn in this study in the future.Usually, conservative treatment with progesterone is adopted to alleviate tumor progression, conducive to pregnancy. The meta-analysis by Koska et al. showed that the pregnancy rate is 32% (111/351) after conservative treatment of endometrial cancer, implying that the traditional progesterone therapy leads to a poor pregnancy outcome. Hence, discovering new strategies to change this situation is urgent . Conside(n = 31) . The aboMetformin in combination with progesterone has exact effects in treating early endometrial cancer patients with fertility requirements, which is capable of significantly decreasing the BMI of patients and elevating the pregnancy rate after treatment.


This study aimed to develop a prediction model for the prognosis of patients with Hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF). 122 patients were divided into survival group and death group according to 90-day prognosis after diagnosis. Risk factors affecting the prognosis were identified by the logistic regression analysis and then were used to establish an FT3-related prediction model. Age, proportion of liver cirrhosis, AST, TBil, INR, Cr, Na, WBC, and several scores  of the death group were significantly higher than that of the survival group on admission. FT3 and Na were protective factors for the prognosis of patients; Age, TBil, INR, HE grading, and Cr were risk factors. FT3 levels were (2.79 ± 0.34) (95%CI 2.73–2.87) pmol/L for the survival group and (2.20 ± 0.20) (95%CI 2.11–2.29) pmol/L for the death group. The level of FT3 in survival group was significantly higher than that of the death group in patients regardless of gender, initial liver disease, and liver failure stages (P < 0.05). The ROC curve for FT3-related prognostic model score was 0.923 (95%CI 0.809–0.947), significantly higher than that of the CTP, MELD, MELD-Na, CLIF-SOFA, CLIF-C OF, and AARC scores (P < 0.001). The FT3-related prediction model has good predictive value for 90-day prognosis.         FT3 concentration was significantly decreased in death group, indicated that FT3 may be a protective predictor of the prognosis of HBV-ACLF.FT3 was significantly higher in survival group with different gender, different initial liver disease and different stages of liver failure, suggesting FT3 has good stability as a predictor.The FT3 level may facilitate prediction of prognosis of HBV-ACLF.1Acute-on-chronic liver failure (ACLF) is recognized as a complex, clinical disease relating to the acute decline of liver function and even organ failure, which is associated with an elevated short-term mortality . ACLF caMost of the prognostic prediction models that were established have different evaluation effects, including the Child–Turcotte–Pugh (CTP) score, the model for end-stage liver disease (MELD) score, the MELD-Na score, the chronic liver failure–sequential organ failure assessment (CLIF-SOFA), the CLIF-consortium organ failure score (CLIF-C OF) and the APASL ACLF research consortium (AARC) score. CTP score is calculated using bilirubin, albumin, ascites, encephalopathy, and international normalized ratio [INR]. This score can be used to assess liver cirrhosis, but the accuracy has been questioned. MELD-Na score is calculated using lab tests such as total bilirubin (TBil), the international normalized ratio (INR), serum creatinine (Cr), serum sodium (Na), and etiology. However, it has some limitations regarding the accuracy of assessment and ease of clinical application . CLIF-SOMoreover, thyroid hormone is involved in many pathophysiological mechanisms. Liver is of great importance for thyroid hormones synthesis, metabolism, and excretion . Therefo22.1We enrolled 122 consecutive HBV-ACLF patients in our hospital between September 2018 and February 2020. Inclusion criteria: (1) the diagnosis of chronic hepatitis B in accordance with the guidelines ; (2) meeThe etiology of ACLF in this study was all caused by HBV. Patients received standard medical support treatment and routine anti-HBV treatment. Patients who met the conditions of liver transplantation were included in the waiting queue for liver transplantation. AKI patients required hemodialysis treatment when they reached the standard of renal replacement therapy. Patients were followed for at least three months. According to 90-day prognosis, patients were separated into the survival (n = 77) and the death groups (n = 45). The protocol was approved by our institutional review board and informed consent was obtained from all participants.2.2The following information was collected from patients on admission: age, gender, BMI, history of alcohol intake, anamnesis, mean arterial pressure (MAP), and other general information; laboratory parameters were measured at 6:00 a.m. the next day: FT3, alanine transaminase (ALT), aspartate transaminase (AST), TBil, albumin (ALB), Cr, platelet, hemoglobin, serum sodium, and white blood cell (WBC) count were recorded; and the prothrombin time was evaluated, and the INR and HBV viral load (DNA) were calculated.2.2.1Enzyme-linked immunosorbent assay was performed using an Abbott i2000SR fully automatic chemiluminescence analyzer to determine the patient’s serum FT3 level; a Sysmex XE-2100 fully automatic blood cell analyzer was used to determine the WBC; an Olympus AU 5400 automatic biochemical analyzer was used to test the following blood biochemical indexes; an ACL TOP 700 automatic coagulation analyzer was used to measure the prothrombin time and calculate the INR; HBV-DNA and HBeAg were detected by the kit produced by Roche company, and the detection limit of HBV DNA is 500 IU/ml.2.2.2According to the results of laboratory examination and clinical manifestations of patients, Child and the Model for End-stage Liver Disease (MELD) score were calculated. According to the results of laboratory examination and clinical manifestations of patients, Child and the Model for End-stage Liver Disease (MELD) score were calculated. Each patient’s condition was evaluated by CTP score, MELD score, MELD-Na score, CLIF-SOFA score, CLIF-OF score (Chronic Liver Failure Organ Failure score), and AARC score.2.32 test; The logistic regression analysis was used to identify the independent factors affecting the prognosis. We also assessed the discrimination of the prediction model by using receiver-operating characteristic (ROC) curves. The ROC curve was constructed to compare the difference between the prediction model and the other scores in predicting prognostic value, and the Kaplan-Meier test was used between groups. A two-tailed P-value < 0.05 was used to indicate the statistical significance.All statistical analyses were conducted with SPSS 25.0 and MedCalc 19.0 software. The continuous data were expressed as mean ± standard deviation (x ± s); Comparisons between the two groups were made using a Mann–Whitney U test; a comparison check of the count data of the two groups was performed using an Χ3For all 122 patients, survival group had higher value of FT3 and Na, and death group had higher values of AST, TBil, INR, Cr, Na, WBC, BMI, MAP, hemoglobin level, and various prognostic scores. Further work showed that FT3 was significantly higher in the survival group independent on gender, initial liver disease, and liver failure stages. With the decrease of FT3 level, the mortality of patients increased. The area under the ROC curves of FT3-related prognostic model-1 score was significantly higher than that of other risk scores.3.1Among the 122 patients, 77 cases (63.1%) in the survival group, including 65 males (84.4%) and 12 females (15.6%), aged (43.66 ± 9.94) years; 45 cases (36.9%) in the death group, including 35 males (77.8%) and 10 females (22.2%), aged (48.86 ± 8.91) years. The direct death cause of death group was liver failure. Death group showed significant higher values of AST, TBil, INR, Cr, Na, WBC, BMI, MAP, hemoglobin level, and various prognostic scores than survival group on admission . After a3.2P < 0.001); 1.055 (1.012–1.089) for age (P = 0.018); 1.100 (1.022–1.184) for INR (P = 0.018); 1.119 (1.074–1.166) for TBil (P < 0.001); 4.850 (2.082–7.296) for Cr (mg/dl) (P < 0.001); 0.883 (0.818–0.952) for sodium (P = 0.034); Grade of hepatic encephalopathy (HE) was used as categorical variable (P < 0.05). The results indicated that FT3 and Na were protective factors affecting the prognosis of patients (P < 0.05); age, TBil, INR, HE grading, and Cr were risk factors (P < 0.05). The patients’ prognosis was used as a dependent variable, and factors were used as independent variables to establish the logistic proportional hazards model (As shown in ds model . FT3-rel3.3P < 0.001. The FT3 levels of the two groups are compared in FT3 levels were (2.79 ± 0.34) (95%CI 2.73–2.87) pmol/L and (2.20 ± 0.20) (95%CI 2.11–2.29) pmol/L in survival and death group, 3.3.1P < 0.001  (95% CI 2.72–2.88) pmol/L, death group was (2.00 ± 0.20) (95% CI 2.11–2.29) pmol/L,  < 0.001 . Female: = 0.007 .3.3.2P = 0.0001  (95%CI 2.73–2.87) pmol/L, death group was (2.33 + 0.22) (95%CI 2.15–2.51) pmol/L, = 0.0001 . Cirrhos < 0.001 .3.3.3P < 0.001  (95% CI 2.77–2.98) pmol/L, death group was (2.08 ± 0.11) (95% CI 1.95–2.21) pmol/L,  < 0.001 . Middle  < 0.001 . Late st = 0.018 .The above results showed that the survival group had a significantly higher FT3 level than the death group regardless of gender, initial liver disease (chronic hepatitis B or cirrhosis) and stages of liver failure.3.4The FT3 level of all patients ranged from 1.13 to 4.17 pmol/L. We were divided into four zones from high to low according FT3 level: Q1 area (11 cases) was 3.44–4.27 pmol/L; Q2 area (38 cases) was 2.61–3.43 pmol/L; Q3 area (59 cases) was 1.78–2.60 pmol/L; Q4 area (14 cases) was 0.95–1.77 pmol/L. The mortality of patients in Q1, Q2, Q3, and Q4 were 9.1%, 26.3%, 32.2%, and 50%, respectively. It is suggested that with the decrease of FT3 level, the mortality of patients increases gradually .Figure 3.5The accuracy of FT3 level in predicting patients’ 90-day prognosis was analyzed by the ROC curve . The res3.62 = 0.615, Brier score = 0.103 (Each item value of patients was substituted into the regression equation, ROC curve of prediction probability value was drawn to get the FT3-related prognostic model-1 score , AUROC w = 0.103 . The res3.7P < 0.001 (The ROC curves of FT3-related prognostic model-1 score and other competing scores on the 90 day prognosis of patients were plotted . The res < 0.001 .Figure 4ACLF is a condition of hepatic decompensation with multiple organ failure caused by a systemic inflammation . ConsideIn our study, there was a higher level of various risk index values in the death group were compared with the survival group whereas serum FT3 concentration was significantly decreased in death group. FT3 may be a protective factor for the development of HBV-ACLF, which is similar to the previous findings in this field. The reason for the decrease of FT3 may be that HBV-ACLF patients occur the euthyroid sick syndrome ,15. In aPrevious studies have identified different predictors for the ACLF patients’ prognosis. However, few researches evaluated the prognostic value of FT3 for HBV-ACLF. We studied the level of FT3 in different gender, different initial liver disease and different stages of liver failure. The results indicated that the death group had a significantly lower FT3 value than survival group, suggesting FT3 has good stability as a predictor. Its predictive ability is not affected by population, basic liver diseases and severity of liver failure.This study integrated with predictor factors analyses, we developed FT3-related prognostic model-1 score, which could predict the prognosis of patients with HBV-ACLF. We also assessed the performance of several risk scores in prediction of HBV-ACLF patients’ prognosis. The results indicated that these prognostic scores have a better ability to predict the HBV-ACLF patients’ prognosis, which is supported by previous researches confirming the efficiency of the scores ,19. At t5This study had several limitations. As it was a retrospective observational study, there were only a relatively small number of observation cases. And the study is lacking external validation, so the findings probably reflect overly optimistic model performance. In the future, a prospective study with an expanded sample size and a multicenter approach could explore the association between FT3 and the severity of HBV-ACLF patients.6The FT3-related prognostic model-1 score has better prognostic value than the CLIF-OF, CLIF-SOFA, MELD, MELD Na, and AARC scores. The FT3 level may facilitate the risk-stratification and clinical decision-making of HBV-ACLF.


KRAS/NRAS/BRAF metastatic colorectal cancer (mCRC). An over-expression of the epidermal growth factor receptor (EGFR) has been observed in colorectal cancer and is associated with aggressive disease and poor prognosis. SCT200 is a newly developed recombinant, fully humanized, anti-EGFR monoclonal antibody. This study aimed to evaluate its safety, tolerability, pharmacokinetics (PK), and efficacy in patients with wild-type This phase I study comprising dose-escalation phase and dose-expansion phase. SCT200 was administrated intravenously to groups of three to six patients. An every 3-week dosing cycle (0.5–15.0 mg/kg) and multiple dosing schedule were evaluated. Blood samples were collected at preset intervals for PK assessment, radiological imaging was used for efficacy assessment, and continuous safety monitoring was performed in each group during the study. KRAS/NRAS/BRAF mCRC receiving ≥ 1 dose of SCT200 were evaluated. Among them, 44.6% (25/56) of the patients failed at least two prior lines of chemotherapy. No dose-limiting toxicities occurred in any group. All of the patients experienced treatment-emergent adverse events (TEAEs). 96.4% (54/56) of patients experienced treatment-related adverse events (TRAEs), and 26.8% (15/56) of patients with Grade ≥ 3 TRAEs. No serious TRAEs were observed. The most common TRAEs were dermotoxicity and hypomagnesemia. PK analysis showed non-linear PK in the range of 0.5 - 8.0 mg/kg of single dose SCT200, the clearance decreased, and the elimination half-life (T1/2) prolonged following dose increase. In the multiple-dose period, the clearance decreased, peak concentration increased, and T1/2 prolonged during prolonged drug administration, and a steady state was reached after five consecutive dose of 6.0 mg/kg quaque week (QW). The objective response rate (ORR) was 30.4% . The ORR in the dose-expansion group (6.0 mg/kg QW) was 48.0% , the median progression-free survival was 5.2 months , and the median overall survival was 20.2 months .From December 16, 2014 to December 31, 2018, fifty-six patients with wild-type KRAS/NRAS/BRAF mCRC.SCT200 showed favorable safety, PK profile, and preliminary efficacy for patients with wild-type NCT02211443).This study was registered with ClinicalTrials.gov (The online version contains supplementary material available at 10.1186/s12885-022-10147-9. KRAS/NRAS/BRAF mCRC, which extended the overall survival (OS) by 6 to 8 months over chemotherapy alone [The epidermal growth factor receptor (EGFR) pathway plays a key role in tumorigenesis, cancer cell survival, migration, angiogenesis and apoptosis. EGFR overexpression has been found in 50–80% patients of colorectal cancer (CRC) and its increased levels are associated with aggressive disease and poor prognosis , 2. Amonpy alone –7.SCT200 is a fully humanized anti-EGFR monoclonal antibody which was developed by Sinocelltech Ltd., Beijing, China, with an antigen-binding epitope, physicochemical properties, and biological activity that are different from those of currently marketed anti-EGFR monoclonal antibodies. The binding affinity to EGFR (Kd = 0.08 nM) of SCT200 is comparable to that of panitumumab (Kd = 0.05 nM), and higher than that of cetuximab (Kd = 0.147 nM) and nimotuzumab (Kd = 1 nM). The tumor-targeted monoclonal antibodies may increase the anticancer effects through antibody-dependent cellular cytotoxicity (ADCC). For this purpose, SCT200 is specifically designed to enhance ADCC and complement-dependent cytotoxicity (CDC) through the Fc domain, which exhibits ADCC mediated anticancer activity at low concentration. Preclinical studies showed that SCT200 alone significantly inhibited the growth of vulvar squamous cell carcinoma and colon cancer cells in vivo, and the anticancer activity was enhanced when combining with chemotherapeutic agents (data unpublished). Comparing with cetuximab, SCT200 demonstrated superior inhibition of tumor cell growth in vitro and in vivo. The target organs of toxicity of SCT200 were mainly the skin and gastrointestinal system, and there were no non-target-related toxic effects observed in the preclinical study (data unpublished).KRAS/NRAS/BRAF mCRC who had failed prior chemotherapies (NCT02211443).This is the phase I, dose-escalation and dose-expansion study to investigate the safety, tolerability, pharmacokinetics (PK), and efficacy of SCT200 in patients with wild-type KRAS/NRAS/BRAF, measurable/nonmeasurable tumor lesions, an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0–1 and life expectancy over 3 months, with adequate organ functions and no history of other malignancies were enrolled. The main exclusion criteria were: prior treatment with EGFR inhibitors, anticancer therapy or surgery within 4 weeks, Grade ≥ 2 toxicity due to prior anticancer therapy, and any other condition deemed inappropriate by the investigator. The mutation status  of all patients were examined in the screening period, and patients with RAS  and BRAF V600E mutation were excluded from the study. The detailed study protocol Synopsis was presented in Supplementary file Patients aged 18–70 years with pathologically confirmed CRC who had prior treatment failure with fluorouracil/oxaliplatin/irinotecan, wild-type This study was approved by the Ethics Committee of the National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College , and was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. All patients provided written informed consent.The study was composed of the dose-escalation phase and dose-expansion phase. SCT200 was administrated intravenously to groups of three to six patients. The starting dose and the maximum dose of SCT200 was set as 0.5 mg/kg and 15.0 mg/kg, respectively based on the results of preclinical studies. Patients in the six low-dose escalation groups  first received a single ascending dose (SAD), were monitored for 3 weeks, and then entered the multiple ascending dose (MAD) stage. In the MAD stage, patients in 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, and 4.0 mg/kg groups were administered SCT200 once per week for 4 weeks. In the 6.0 mg/kg group, three patients were administered one dose of SCT200 per week for 6 weeks and another three patients received one dose of SCT200 in 2 week intervals for 6 weeks. The 8.0 mg/kg group was administered with SCT200 once in 2 weeks for 6 weeks. Patients in three high-dose MAD groups  were administered one dose of SCT200 per week for 6 weeks. SAD/MAD of the next dose group were started on the premise that the dose level of SCT200 on the previous dose group was safely tolerated in a 3-week observation period. In addition, a dose-expansion phase for evaluating a target dose of SCT200 was initiated when the dose escalation phase was completed. The target dose was determined based on the PK analysis of the dose escalation phase, and a total of 25 patients were administered one dose of SCT200 at the target level per week for 6 weeks. After the completion of the pre-specified multiple dosing cycles, if the tumor was in remission or stable, patients were allowed to continue the treatment of SCT200 at the respective dose levels until disease progression or intolerable toxicity occurred  version 1.1 at baseline, the end of the multiple-dose treatment cycle, and every 8 weeks after entering the multiple-dose expansion period until disease progression (PD) or intolerable toxicity occurred. Objective response rate (ORR) was defined as the percentage of complete response (CR) or partial response (PR). The disease control rate (DCR) was defined as the proportion of patients with CR or PR or stable disease (SD). Progression-free survival (PFS) was defined as the time from the first dose of SCT200 to the date of PD or death of any reason. OS was defined as the time from the first dose of SCT200 to the date of death due to any reason or last follow-up.Single-dose PK blood samples were collected within 60 min before dosing and at 0.5, 2, 6, 24, 48, 96, 168, 264, 336, 408, and 504 h after dosing. For multiple dosing, PK blood samples were collected within 60 min before each dose and 30 min after dosing. Intensive blood collection was also performed for the first and last doses.All statistical analyses were performed using SAS . Safety and efficacy evaluations were performed on all patients who received ≥ one dose of SCT200 treatment, and all AEs were recorded according to MedDRA (version 22.1). Survival was estimated using the Kaplan–Meier method, and the time to the onset of AEs of dermotoxicity and hypomagnesemia was analyzed. 95% confidence intervals (CIs) were calculated using the Clopper–Pearson method. Blood concentrations of SCT200 were measured at various time points using an enzyme-linked immunosorbent assay, and key PK parameters were calculated using a non-compartmental model .RAS  and BRAF V600E wild-type. 12.5% (7/56) of the patients had right-sided primary colon cancer and 87.5% (49/56) had left-sided primary colon cancer. 96.4% (53/56) of the patients failed at least two prior lines of chemotherapy. Fifty-five patients received multiple doses of SCT200, including 21 patients in the low-dose groups , 9 patients in the high dose groups  and 25 patients in the dose-expansion group (6.0 mg/kg). One patient in the 4.0 mg/kg group received a single dose of SCT200 and did not receive subsequent multiple doses as the patient received other anticancer therapy. After completing the pre-specified multiple-dose treatment cycles, 38 patients continued the treatment with the same treatment regimens. Reasons for terminating SCT200 treatment included PD (n = 29), voluntary withdrawal (n = 4), and withdrawal after SCT200 treatment was delayed by 2 weeks (n = 2). Three patients continued treatment until the data-cutoff date on December 31, 2018  included acute myeloid leukemia, gastrointestinal bleeding, intestinal obstruction, and ureteral calculi (one each), all of which were considered unrelated to SCT200 (Supplementary Table 7.1% (4/56) of the patients developed Grade 1–2 infusion reactions at the time of the first dose of SCT200, and no Grade ≥ 3 infusion reactions occurred. No patients reported DLT during the single and multiple dose escalation period. SCT200 dose was not escalated beyond 15.0 mg/kg, and the maximum tolerated dose (MTD) was not reached in this study. All of the patients experienced treatment-emergent adverse events (TEAEs). 96.4% (54/56) of the patients experienced treatment-related adverse events (TRAEs). The most common TRAEs of the 56 patients were dermotoxic-related AEs, including dermotoxicity with acneiform dermatitis (89.3%), paronychia (33.9%), and dry skin (21.4%). Other TRAEs included hypomagnesemia (71.4%), elevated alanine aminotransferase (ALT) (28.6%), elevated aspartate aminotransferase (AST) (17.9%), elevated bilirubin (17.9%), elevated alkaline phosphatase (10.7%), hypertriglyceridemia (25.0%), hypophosphatemia (17.9%), conjunctivitis (16.1%), and hypercholesterolemia (14.3%). Fifteen of 56 patients (26.8%) developed Grade ≥ 3 TRAEs: acneiform dermatitis (8.9%), dry skin (3.6%), and hypomagnesemia (8.9%). Serious adverse events (SAEs) (max) appeared to be approximately dose proportional, the area under the concentration–time curve (AUC) exposure disproportionally increased, clearance decreased from 2.3 mL/h/kg to approximately 0.3 mL/h/kg, and the elimination half-life was extended from 17.6 h to 75.9 h.The SCT200 single-dose PK investigation was performed at six dose levels . The concentration–time curves of different doses of SCT200 are shown in Fig. tau) increased following the dose ascending from 0.5 mg/kg to 15.0 mg/kg. The AUCtau increased disproportionally when the dose escalated from the 0.5 mg/kg to 4.0 mg/kg, showing a non-linear PK characteristic. When the dose escalated from 6.0 mg/kg to 15.0 mg/kg, the AUCtau approximately increased proportionally, showing a linear PK trend. The accumulation index fluctuated in a range from 1.3 to 2.4 when the dose escalated from 0.5 mg/kg to 15.0 mg/kg. Following the prolonged multi-dose treatment of SCT200, all groups showed a decrease in the drug clearance, increase in the peak plasma concentration, and a prolonged T1/2. Based on the PK results of SCT200 dose escalation and comparison of similar drugs, 6.0 mg/kg was determined as the target dose of SCT200 for the dose-expansion phase, and a steady state was reached after five consecutive SCT200 administrations.The drug concentration–time curves for multiple doses of SCT200 are shown in Fig. N = 6), 39.0% (95% CI: 24.2%-55.5%) and 4.4 months (95%CI:3.4–5.5) for patients with Grade 1–2 dermotoxicity (N = 41) and 55.6% (95% CI: 21.2%-86.3%) and 5 months (95%CI: 5.2-not reached) for the patients with Grade 3–4 dermotoxicity (N = 9), respectively.Efficacy evaluation was performed for all patients who received at least one dose of SCT200. Of 56 patients, 17 achieved PR, with an ORR of 30.4% (95% CI: 18.8%–44.1%), and a DCR of 69.6% (95% CI: 55.9%–81.2%). No patients in the ≤ 4 mg/kg dose groups achieved an objective response. Table KRAS/NRAS/BRAF mCRC. In patients with wild-type KRAS mCRC who have failed standard chemotherapy, anti-EGFR monoclonal antibody therapy provided a median OS of 8.1–10.4 months, a median PFS of 2.9–4.4 months, and an ORR of 12.8–27% [CRC is the third most common cancer worldwide, with 20–25% of patients initially diagnosed with stage IV disease. The 5-year OS rate of stage IV CRC was 12% , 9. Over12.8–27% .In this study, SCT200 is well tolerated with no DLT occurring during the dose-escalation phase and no serious TRAEs were observed. The MTD was not reached when the dose of SCT200 escalated to 15.0 mg/kg. The most common TRAE was dermotoxicity. The overall incidence and incidence of Grade ≥ 3 TRAEs of dermotoxicity were 89.3% and 16.1%, respectively, which are comparable to the incidence rates of cetuximab (88% and 10%) and panitumumab (86% and 12%) . PatientThe target dose of SCT200 was determined based on the PK investigation of dose-escalation phase. SCT200 was well tolerable within the dose level of 15.0 mg/kg. The target steady-state trough concentrations of cetuximab and panitumumab were 41–85 μg/mL and 50 μg/mL, respectively . Consideversus best supportive care [RAS  mCRC in the panitumumab group was 31%, 5.2 months, and 10  months, respectively. The study result of cetuximab showed that the ORR was 12.8%, median PFS was 3.7 months, and median OS was 9.5 months in patients with wild-type KRAS exon 2 mCRC [In this study, 6.0 mg/kg QW was determined as the target dose for the dose-expansion study. 96.0% (24/25) who failed at least two prior lines of chemotherapy, were enrolled to receive 6.0 mg/kg QW for six consecutive doses and then changed to 8.0 mg/kg Q2W to improve patient compliance. 48.0%  of the patients had PR. The DCR was 92.0% , the median PFS was 5.3 months (3.6–5.5), and the median OS was 20.2 months (95% CI: 12.1–not reached). In the phase III study of panitumumab n 2 mCRC . Previoun 2 mCRC . This stn 2 mCRC . This stKRAS/NRAS/BRAF mCRC.This phase I dose-escalation and dose-expansion study demonstrated that SCT200 showed a favorable safety, tolerabolity, PK profile, and efficacy to patients with wild-type Additional file 1. Protocol synopsis.Additional file 2:Supplementary Table 1. Occurrence of adverse events associated with study drugs .Additional file 3:Supplementary Table 2. Kaplan–Meier method—time to first dermotoxicity/hypomagnesemia event.


KDGal is also found in the catabolic pathway of 3,6-anhydro-l-galactose (AHG), the main component of red-algae-derived agarose. AHG is converted to 3,6-anhydrogalactonate by AHG dehydrogenase and subsequently isomerized to KDGal by 3,6-anhydrogalactonate cycloisomerase. Therefore, we used the above-described pathway to produce KDGal from agarose. Agarose was depolymerized to AHG and to agarotriose (AgaDP3) and agaropentaose (AgaDP5), both of which have significantly higher molecular weights than AHG. When only AHG was converted to KDGal, AgaDP3 and AgaDP5 remained unreacted. Finally, KDGal was effectively purified from the enzymatic products by size-exclusion chromatography based on the differences in molecular weights. These results show that KDGal can be enzymatically produced and purified from agarose for use as a precursor to high-value products.2-keto-3-deoxy sugar acids, which have potential as precursors in medicinal compound production, have gained attention in various fields. Among these acids, 2-keto-3-deoxy- Fi. FiVibriain EJY3 . In addiSdNABH were purified. The purified Aga16B and SdNABH were identified by SDS-PAGE based on their theoretical molecular weights of 63.7 kDa and 41.5 kDa, respectively  [SdNABH originating from S. degradans 2-40T) [VejAHGD originating from Vibrio sp. EJY3) [VejACI originating from Vibrio sp. EJY3) [Escherichia coli BL21(DE3) by using the heat-shock method, and transformants were selected on agar plates containing 100 µg/mL ampicillin . E. coli BL21(DE3) harboring genes encoding each of the proteins was grown at 37 °C in Luria-Bertani  medium containing 100 µg/mL ampicillin until the mid-exponential phase of growth. Protein overexpression was induced by adding 0.5 mM IPTG  for Aga16B, SdNABH, and VejACI or 0.2% l-(+)-arabinose  for VejAHGD at 16 °C for 16 h. The cell pellet was collected by centrifugation at 6000 rpm for 30 min at 4 °C and resuspended in ice-cold Tris-HCl buffer (pH 7.4). Cell disruption was then performed using a sonicator . The supernatant was collected by centrifugation at 16,000 rpm for 30 min at 4 °C to purify the soluble recombinant protein. The overexpressed recombinant protein was further purified via affinity chromatography using a HisTrap column , followed by desalting and concentration using an Amicon ultrafiltration membrane . The protein concentration was determined using a BCA protein assay kit . The purified recombinant enzymes were identified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) based on their theoretical molar mass.To produce KDGal from agarose, four recombinant enzymes were used: endo-type β-agarase (Aga16B originating from s 2-40T) , NAOH (Ss 2-40T) , AHGD (Vp. EJY3) ,27, and p. EJY3) . To prodSdNABH. To produce NAOSs from agarose, including NeoDP4 and NeoDP6, 4 mg of Aga16B was added to 100 mL of 2% (w/v) agarose  in 20 mM Tris-HCl (pH 7.4), and the reaction mixture was incubated at 50 °C overnight. To produce AHG from Aga16B reaction products, NeoDP4 and NeoDP6, 2 mg of SdNABH was then added to the Aga16B reaction mixture, followed by incubation at 30 °C for 5 h.To produce AHG as a substrate for KDGal production, we performed enzymatic depolymerization of agarose using the recombinant enzymes Aga16B and SdNABH were analyzed using high-performance liquid chromatography  equipped with a Rezex ROA-Organic Acid H+ (8%) column  and a refractive index (RI) detector . The temperature of the column and RI detector was set to 50 °C. The column was eluted using 0.005 N H2SO4 as the mobile phase at a flow rate of 0.6 mL/min.The enzymatic reaction products of Aga16B and SdNABH were also identified using thin-layer chromatography (TLC). Each enzymatic reaction product (1 µL) was loaded onto silica gel 60 TLC plates , which were developed with a mobile phase consisting of n-butanol, ethanol, and water . The plate was visualized using 10% (v/v) H2SO4 and 0.2% (w/v) 1,3-dihydroxynaphthalene in ethanol, as previously described [The enzymatic reaction products of Aga16B and escribed .VejAHGD and VejACI, were used to convert AHG to KDGal. First, to oxidize AHG into AHGA using AHGD, VejAHGD was added to the NABH reaction products containing mainly AHG, AgaDP3, and AgaDP5. The AHGD reaction mixture containing 2 mM AHG, 3 mM NAD+ as a cofactor, and 0.05 mg/mL of VejAHGD was incubated at 30 °C and 100 rpm. After 12 h, 0.05 mg/mL of VejACI was finally added to the reaction product of VejAHGD to isomerize AHGA into KDGal. Subsequently, the reaction mixture was incubated at 30 °C and 100 rpm for 12 h.Two recombinant enzymes, VejAHGD and VejACI were analyzed using gas chromatography–mass spectrometry (GC-MS). For GC-MS analysis, the reaction products of VejAHGD and VejACI were derivatized as previously described [VejAHGD and VejACI were centrifuged at 4 °C and 16,000 rpm for 5 min. Then, 20 μL of the supernatant was dried for 1 h using a speed vacuum evaporator, and methoxyamination and trimethylsilylation were subsequently performed for derivatization. Briefly, 10 μL of 40 mg/mL methoxyamine hydrochloride in pyridine  was added to the dried sample and incubated at 30 °C. After 90 min, the sample was treated with 45 μL of N-methyl-N-(trimethylsilyl)trifluoroacetamide  for 30 min at 37 °C. The chemically derivatized samples were analyzed using an Agilent 7890A GC/5975C MSD system  equipped with a DB5-MS column . The derivatized sample, 1 µL in volume, was injected into the GC column in the splitless mode. The oven was programmed to maintain an initial temperature of 100 °C for 3.5 min and then for the temperature to be increased to 160 °C at 15 °C/min and held for 20 min, increased to 200 °C at 20 °C/min and held for 20 min, and finally to 280 °C at 20 °C/min and held for 5 min. Electron ionization was performed at 70 eV, and the temperature of the ion source was 230 °C. The mass range was 85–700 m/z. The reaction products of VejAHGD and VejACI were also analyzed using TLC, as described above.To confirm the production of KDGal, the enzymatic reaction products of escribed . Before For the purification of KDGal from the enzymatic reaction mixtures, size-exclusion chromatography using Bio-Gel P-2 Gel  was performed. Distilled water was used for elution at a flow rate of 0.3 mL/min. To obtain high-purity KDGal, the fractions containing only KDGal were collected and freeze-dried. The purified KDGal was finally analyzed using TLC.


O-acetylated ManNAz (Ac4ManNAz) remains the most popular choice so far for tracking intracellular processing of sialoglycans and cell surface sialylation in various cells. Taking advantage of CuAAC, we compared the metabolic incorporation of ManNAl, ManNAz, SiaNAl, SiaNAz and Ac4ManNAz in the human colon cell lines CCD841CoN, HT29 and HCT116, and in the two gold standard cell lines, HEK293 and HeLa. Using complementary approaches, we showed marked differences in the efficiency of labeling of sialoglycoproteins between the different chemical reporters in a given cell line, and that switching the azide and alkyne bioorthogonal tags on the analogs highly impacted their metabolic incorporation in the human colon cell lines. Our results also indicated that ManNAz was the most promiscuous metabolized reporter to study sialylation in these cells.Sialylation of cell surface glycans plays an essential role in cell–cell interaction and communication of cells with their microenvironment. Among the tools that have been developed for the study of sialylation in living cells, metabolic oligosaccharide engineering (MOE) exploits the biosynthetic pathway of sialic acid (Sia) to incorporate unnatural monosaccharides into nascent sialylatedglycoconjugates, followed by their detection by a bioorthogonal ligation of a molecular probe. Among bioorthogonal reactions, the copper-catalyzed azide-alkyne cycloaddition (CuAAC) is the only ligation where both reactive tags can be switched on the chemical reporter or on the probe, making this reaction very flexible and adaptable to various labeling strategies. Azide- and alkyne-modified ManNAc and Sia reporters have been widely used, but per- Yet, identification of proteins whose sialylation is impaired is still of interest to better understand the molecular and cellular mechanisms that are affected during tumor progression, such as migration and invasion properties, and immune escape mechanisms5.The N-Acetyl neuraminic acid  is the most abundant member of the sialic acid family. This negatively charged monosaccharide is located at the non-reductive end of glycan chains carried by glycoproteins and glycosphingolipids at the surface of human cells. Sialylation plays an important role in cell–cell recognition and cell-microenvironment interactions in physiological or pathological context. For example, changes of sialylation are associated with cancer progression and metastasis, especially in colon cancer7. Sia or ManNAc analogs can both be used as two different entry points into the metabolic route  activity of the bifunctional enzyme GNE (UDP-GlcNAc 2-epimerase). In contrast, GNE is not required for Sia analogs which go through the endo-lysosomal recycling pathway before being exported into the cytosol via SLC17A5 11. Both peracetylated alkyne and azide-modified ManNAc and Sia can be used. However, differences in the efficiency of incorporation and cell surface labelling were reported, as shown for example for Ac4ManNAz and Ac4ManNAl13, or Ac5SiaNAz and Ac5SiaNAl (referred to as Ac5SiaNPoc)15. Sia reporters, whose synthesis is demanding, seem to be more efficiently incorporated into sialylated GCs compared with their ManNAc analogs14. Indeed, Sia analogs enter cells via the salvage pathway and are directly available for CMP activation in the nucleus, while the conversion of ManNAc analogs into the modified nucleotide-sugar requires three additional enzymatic steps  using metabolic oligosaccharide engineering (MOE). Such a strategy exploits the biosynthetic pathway of sialic acid to incorporate unnatural Sia derivatives into GCs in living cellsute Fig. . Synthet22. CuAAC is the only bioorthogonal reaction in which alkynes and azides can be introduced indifferently either on the metabolic analog or on the probe for the ligation, making this reaction very flexible and adaptable to various labeling strategies. In contrast, only azides are adapted for a bioorthogonal ligation via SPAAC reaction, which is easier to apply in living systems than CuAAC due to the absence of copper associated cytotoxicity, but generally suffers from poorer kinetics and specificity especially in the intracellular environment23. Albeit numerous works using MOE to label sialylated glycans have been reported, the comparison of alkyne- or azide-functionalized ManNAc and Sia free monosaccharides has not been done so far. Therefore, in this work, we utilized the advantage of CuAAC to swap the azide and alkyne tags between the unnatural monosaccharides and the probes. We compared the metabolic incorporation efficiency of ManNAl, ManNAz, SiaNAl, SiaNAz and peracetylated Ac4ManNAz in several human cell lines  and the strain-promoted [3 + 2] alkyne-azide cycloaddition (SPAAC), have been widely used in vitro and in vivones Fig. . We chos9, therefore unprotected ManNAc derivatives have also been used to label sialoglycoconjugates. In agreement with previous studies, we used 500 µM of the free-hydroxyl analogs10. Ac4ManNAz is usually used in a range of 20 to 100 µM11. However, we found that 100 µM Ac4ManNAz reduced approximately by 40% the cellular growth of CCD841CoN, HT29 and HCT116 human colon cell lines  rather than relative MFIs . Sialylation occurs in the Golgi complex, making its organelle the first in which incorporated chemical reporters can be visualized onto GCs. Therefore, the Golgi marker TGN46 was used to colocalize the Strept signal in the Golgi  by Western blot. Cells were cultured for 24 or 48 h with the chemical reporters and click chemistry reaction was performed with the appropriate biotinylated probe onto whole cells. A large range of tagged sialoglycoproteins was detected in CCD841CoN cells whatever the analog, although the use of SiaNAz gave a fainter signal for the same time of exposure  in cells39. This is also the case for ST6Gal-I and ST3Gal-IV whose in vitro activity is differentially affected by C-9 modified neuraminic acids40.Impairment in the metabolic incorporation of the alkyne-modified precursors in HT29 cells might be due to a defect in the late steps of sialylation. It is unlikely to be due to a low activity of CMAS towards alkyne-substrates to produce CMP-Neu5Ac derivatives. Indeed, we previously showed that the activity of a recombinant bacterial CMAS was as effective for SiaNAz and SiaNAl as for Neu5Ac30. In contrast, GNE1 exists in different oligomeric states: the hexameric state is the fully functional active form of GNE in vitro, but how the oligomerization of GNE affects its functional activity into cells is not yet understood30. Interestingly, in vitro assays showed that GNE has a strongly reduced kinase activity towards ManNAl (ManNPoc) or ManNBut, compared to ManNAc41. In HT29 cells, the presence of GNE2 into the oligomeric complexes of GNE might change the promiscuous kinase activity of GNE towards ManNAl. Further investigations are needed to explore this hypothesis.Strikingly, HT29 is the only cell line used in this work to express two isoforms of GNE, GNE1 and GNE2. GNE2 has additional N-terminal 31 amino-acids compared to the ubiquitously expressed GNE1 (commonly referred to as GNE). GNE2 displays a drastically reduced epimerase activity and is found only as a dimer4ManNAz has been widely used for MOE targeting sialylation. Here we show that ManNAz is a good alternative for the labeling of cell surface sialylated GCs, especially for the colon cell lines whose proliferation is sensitive to high concentration of Ac4ManNAz (≥ 100 µM). It may come from the perturbation of intracellular pH due to acetic acid release into cells, and the cross-reactivity with cysteine residues of cytosolic proteins28. In addition, Ac4ManNAz metabolic incorporation results in a diffuse fluorescence in the cytoplasm and nucleus of HT29 and HCT116 cells. In contrast, distinct subcellular compartments such as Golgi apparatus are clearly labeled in colon cells treated with ManNAz. This may reflect the marked difference of metabolism kinetics of these two compounds. Indeed, ManNAz enters cells by an active transport mechanism that remains to be identified9 but is then directly incorporated into the sialic acid de novo metabolic pathway. On the contrary, Ac4ManNAz enters by passive diffusion, but requires non-specific cytosolic esterase activity to be fully deacetylated before being incorporated in the metabolic pathway. Diffuse cytoplasmic staining and relatively low labeling of GCs observed in colon cancer cells may come from an inefficient and/or partial deacetylation of Ac4ManNAz, which might perturb its metabolic incorporation and even induce cytotoxicity in these cells. As previously performed in cultured cells and organisms, longer incubation period could address this issue12.So far, Ac4ManNAl (Ac4ManNPoc) resulted in biosynthesis of not only CMP-SiaNAl, but also UDP-HexNAl42. The latter may come from the hijacking of unnatural ManNAc by GlcNAc-2-epimerase (RENBP), leading to the biosynthesis of chemically modified UDP-GlcNAc through the salvage pathway43 23. Interestingly, OGT is overexpressed in colon cancer cells24; this could explain in part the higher cytosolic and nuclear signal observed in the two cancer cell lines cultured with Ac4ManNAz, compared to the non-cancerous CCD841 cell line. It is worth noting that the low relative abundance of unnatural UDP-GlcNAc compared to its natural counterpart may not be sufficient to detect the incorporation of modified GlcNAc into the N- and O-linked glycans on cell surface, as evidenced by a glycomic study in prostate cancer cells cultured with Ac4ManNAz44.Another hypothesis regarding the diffuse staining of labeled GCs into cells is supported by a recent study showing that incubation of human fibroblasts with Acy43 Fig. . Cytosol44, thus facilitating the analysis of metabolically modified glycans by mass spectrometry.Numerous unnatural ManNAc and Neu5Ac analogs are available to study sialylation in cells and organisms using MOE. However, as highlighted in this work, caution should be taken to carefully choose the chemical reporter for a given cellular model. Among the five reporters that we tested in this study, ManNAz is the most appropriate analog to label sialylated GCs that are being processed into cells and exposed at the cell surface of human colon cells. Also, the azido group offers the great advantage to be converted to an amine group in presence of a reducing agent4ManNAz which was dissolved in DMSO. Acetylene-polyethylene glycol (PEG)4-Biotin  and AF594-Picolyl-Azide  were purchased from Jena Bioscience . Azide-PEG3-biotin , Azide-fluor 545 , Fluor 488-Alkyne  were from Merck-Sigma-Aldrich. Streptavidin DyLight® 488 and lectins  were from Vector Laboratories .Stock solutions (50 mM) of the chemically modified monosaccharides were prepared in PBS, except for AcN-(4-pentynoyl mannosamine) and SiaNAl (N-(4-pentynoyl) neuraminic acid) were synthesized as previously described9.ManNAl , HOBt  and DIPEA  in DMF , D-mannosamine hydrochloride  was added and the reaction was stirred at room temperature (r.t.) for 19 h under argon. The total consumption of mannosamine was assessed by silica thin layer chromatography (TLC) (CH2Cl2/MeOH 9:1 v/v) before solvent removal under reduced pressure. The reaction crude was purified by silica flash column chromatography , fractions containing the product were gathered and concentrated under reduced pressure, affording ManNAz as a white powder .2-azidoacetic acid  was dissolved along with DIC  δ = 5.10 , 5.01 , 4.46 , 4.33 , 4.10–3.98 , 3.88–3.71 , 3.56 , 3.46 , 3.38 .13C NMR  δ = 171.87 , 170.97 , 92.89 , 92.83 , 76.40 , 72.03 , 71.97 , 68.82 , 66.76 , 66.52 , 60.41 , 60.39 , 54.28 , 53.37 , 51.72 , 51.65 .N,N-dimethylaminopyridine, catalytic amount) were dissolved in pyridine (5 mL). Acetic anhydride  was added dropwise and the reaction was stirred at r.t. for 16 h under argon. The reaction crude was concentrated under reduced pressure and purified by silica flash column chromatography . Fractions containing the product were gathered and concentrated under reduced pressure, affording Ac4ManNAz as a white powder.ManNAz  and DMAP  δ = 6.64 , 6.56 , 6.05 , 5.89 , 5.35 , 5.22 , 5.17 , 5.06 , 4.73 , 4.62 , 4.30–4.00 , 3.82 , 2.24–1.93 .13C NMR  δ = 170.67–166.55 , 91.31 , 90.26 , 73.40 , 71.45 , 70.27 , 68.86 , 65.10 , 64.93 , 61.75 , 61.67 , 52.62 , 52.45 , 49.72 , 49.26 , 20.90–20.56 .3, H2O 6:1:2,5) with resorcinol as revelating agent. The reaction crude was purified by anion exchange chromatography . Fractions containing the product were identified by TLC as previously then gathered and concentrated under reduced pressure. The white powder obtained was desalted using size exclusion chromatography (P2) with ultrapure water as eluent. Fractions containing the product were gathered, concentrated under reduced pressure then freeze-dried, affording the product as a white powder .ManNAz  was dissolved in 600 µl PBS (pH 7.6) with sodium pyruvate  in a 5 ml capped tube. 5 units of Neu5Ac aldolase  were added and the reaction was stirred at 37 °C for 16 h. The formation of the product was checked by TLC  δ 4.09–3.88 , δ 3.78 , δ 3.65 , δ 3.55 , δ 3.45 , δ 2.18 , δ 1,75 .13C NMR  δ 70.8 , δ 69.82 , δ 68.43 , δ 67.01 , δ 63.10, δ 52.21, δ 51.9 , δ 39.32 .l-Glutamine . Cells were cultured at 37 °C in a 5% CO2-enriched humidified atmosphere.All the cell lines were purchased from ATCC. HT29 cells (derived from a colon adenocarcinoma), HCT116 cells (derived from a colon carcinoma), HeLa (derived from a cervix adenocarcinoma) and HEK293 human cells (derived from a human embryonic kidney) were maintained in Dulbecco’s modified Eagle’s medium  . CCD841CoN cells  were maintained in Eagle’s Minimum Essential Medium  (Lonza). All media were supplemented with 10% (v/v) fetal calf serum and 2 mM 4 cells/well). HT29 (105/well), HCT116 (8 × 104/well), HEK293 (5 × 104/well) and HeLa (3.5 × 104/well) were seeded in 12-well plates. One day later, ManNAc  or the chemical reporter was added in cell culture medium  for the indicated times. Then, cell monolayers were rinsed once with ice-cold PBS and cells were gently scrapped in 1 mL DPBS−/−  before centrifugation in microtubes at 600 g and 400 g for 5 min at 4 °C for respectively CCD841CoN and cancer cells. Cells were then gently resuspended in 20 µL PBS.CCD841CoN cells were seeded in 6-well plates , 300 µM BTTAA (stock solution 6 mM), 2.5 mM sodium ascorbate (stock solution 100 mM) (80 µl/tube). For the negative control, click reaction was performed on cells cultured with ManNAc. After the biorthogonal labelling one hour at r.t., 1 mL DPBS was added in the microtube, cells were centrifuged  and then lysed in 35 µl of 2X Laemmli buffer. Samples were boiled before SDS-PAGE. For flow cytometry analysis, CuAAC was performed 45 min at r.t. in the dark using a fluorescent probe  in the click buffer. Cells were then washed three times in 1 mL DPBS and centrifuged . Finally, cells were resuspended in 100 µL DPBS and kept in ice until flow cytometry analysis.For Western blotting, CuAAC was performed onto cells using either 50 µM azido-PEG3-biotin or alkyne-PEG4-biotin diluted in a freshly prepared click buffer containing 100 mM K45. As a negative control, cells were incubated without the protease in the same buffer. After incubation, cells were washed twice in 1 mL of DPBS and centrifuged . Cell pellets were gently resuspended in 20 µL PBS before CuAAC.To cleave plasma membrane proteins prior to the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), harvested and pelleted cells were treated with 0.1% (w/v) bromelain  for 15 min at 37 °C in 100 µL or 150 µL of DPBS-0.1% (w/v) BSA, for respectively CCD841CoN and cancer cell lines6 CCD841CoN cells, 107 HeLa cells, and 5 × 106 cells for HT29, HCT116 and HEK293 cell lines).Cells were incubated for 15 min at 37 °C with or without bromelain (0.1% in 1 mL DPBS-0.1% BSA) before CuAAC with alkyne-PEG4-biotin (50 µM in 1 mL click reaction buffer per tube). Ten percent of the samples were set aside for SDS-PAGE and Western blotting. After the last centrifugation, cell pellets were frozen in 1 mL of water and lyophilized. Then glycolipids were extracted by the sequential addition of 1 mL CHCl3/CH3OH , CHCl3/CH3OH , and CHCl3/CH3OH/H2O (1:2:0.8), followed by centrifugation 25. Combined supernatants were dried under a nitrogen stream and the resulting glycolipids were dissolved in 100 µL CH3OH. PVDF membrane  was dipped in methanol for 1–2 min and allowed to dry for a few minutes at r.t. To detect labeled sialylated glycolipids (gangliosides), samples (10 µL) were blotted onto the PVDF membrane and Western blotting was performed as described below.Cells were cultured in T25 or T75 flasks with 500 µM ManNAc (negative control) or ManNAz for 48 h, and harvested as described above  BSA. Cells were washed three times in DPBS and resuspended in 100 µL of PBS before flow cytometry analysis.N-glycans were released from cell surface glycoproteins using PNGase F , according to manufacturer’s instructions. Briefly, after CuAAC with the alkyne-PEG4-biotin, cell pellets were resuspended in water (10 µL per 104 cells). 10× denaturing buffer was added to each sample and microtubes were incubated at 95 °C for 10 min. PNGase F digestion was performed on 10 µL of denaturated proteins (~ 30 µg) by adding 2 µL of 10× reaction buffer, 2 µL of 10% NP-40 solution, 1 µL of PNGase F and 4 µL of water . After incubation at 37 °C for one hour, reaction was stopped by adding 5× Laemmli buffer and samples were heated at 95 °C for 7 min before SDS-PAGE.46: GNE , E-cadherin , GAPDH , alpha-tubulin . For detection of O-linked glycoproteins, membranes were incubated overnight at 4 °C in blocking buffer (1% (w/v) BSA in PBS-Tween 0.05% (v/v)) before incubation with Jacalin-FITC (1:100) in blocking buffer for one hour at r.t. After washes, fluorescence was read with ImageQuant™ LAS 4000 . N-linked glycoproteins were revealed using L-PHA-biotin (1:500 in PBST with 1% BSA), followed by incubation with the anti-biotin-HRP. Densitometry analysis was done using the ImageJ® software.Proteins were separated by 8% SDS-PAGE and transferred onto nitrocellulose membrane . Membranes were stained with Ponceau Red (0.1% (w/v) Red Ponceau S in 5% (v/v) acetic acid) to confirm equal loading, washed with TBST  before blocking in TBST with 5% (w/v) nonfat dry milk. Membranes were incubated overnight at 4 °C with peroxidase-conjugated monoclonal mouse anti-biotin   in blocking buffer. After four washes with TBST, chemiluminescence signal  was detected on a CCD camera  using the FUSION-Capt Solo software for acquisition and processing merge images. Western blot with the following antibodies were done as previously describedCell surface fluorescence was measured on a BD ACCURI C6 Plus flow cytometer , either in the FL1 channel for labeling with the Alkyne-AF488 probe and lectin-FITC, or in the FL2 channel for labeling with the picolyl-azide-AF594 probe. Data were analyzed with FlowJo software (vX 0.7). Cell population was gated based on SSC/FSC dot plot to remove cell debris and doublets. The means of fluorescence intensity (MFI) were obtained for each condition (FL1A or FL2A), unspecific MFI was quantified from cells incubated with ManNAc (negative control). Raw values of MFI are indicated on the respective histograms in order to be able to compare the MFI obtained with a given fluorescent probe for the different analogs performed in the same experiment. Histograms with the relative MFIs compared to the negative controls are presented in Fig. 3/well; HT29, 30 × 103/well; HCT116 and HEK293, 23 × 103/well; HeLa 20 × 103/well). One day later, chemical reporters were added in the cell culture medium for the indicated times. Then cells were gently washed in DPBS and fixed in 4% (w/v) paraformaldehyde (PAF) for 30 min at r.t. Coverslips were then washed three times in PBS (5 min) before quenching with 100 mM glycine for 20 min at r.t. After three washes with PBS, cells were permeabilized with 0.5% (v/v) Triton X100 in PBS for 10 min. After three washes in PBS, coverslips were incubated for 30 min at r.t. in the dark and under gentle agitation with 100 µl of the freshly prepared click reaction mix containing the appropriate biotinylated probe (50 µM). After three washes with PBS, immunofluorescence was performed as previously described with the rabbit polyclonal anti-TGN46  and the goat anti-Rabbit IgG Alexa Fluor 568® (1:600)46. Streptavidin-DyLight®488 (1:600) was added at the same time to reveal click chemistry reaction. Nuclei were stained with DAPI (50 µg/mL) and coverslips were mounted on microscope slides using a drop of mounting medium . Images were acquired on a Nikon A1R confocal microscope  using NIS-Elements software, and further analyzed with ImageJ® software.Cells were seeded in 24-wells plate on a 10 mm glass coverslip  of these regions. Results are presented according to the mean of the integrated density (IntDen), representing the concentration of the signal of streptavidin in the ROI. Standard deviation was obtained from at least two images of two independent experiments.We developed a home-made macro on Image J® to quantify the relative fluorescence signal of streptavidin in whole cell vs. Golgi apparatus (10.5281/zenodo.6778042). Briefly, a threshold value was first applied to confocal images based on the streptavidin channel intensity of each condition’s control to get rid of the background signal emanating from the binding of the streptavidin into cells . Regions of interest (ROI) of the whole cells were generated on the thresholded images. Golgi ROIs were created using the TGN46 detection channel. These ROIs allowed us to discriminate regions t-test was used. p-values were calculated and reported accordingly .Unpaired/two-tailed Supplementary Legends.Supplementary Figure 1.Supplementary Figure 2.Supplementary Figure 3.Supplementary Figure 4.Supplementary Figure 5.Supplementary Figure 6.Supplementary Figure 7.Supplementary Figure 8.Supplementary Information 10.