Major spheroid characteristics could be preserved with this new conservation method, allowing their use in high throughput screening tests.Cancer spheroids are reproducible and relevant multicellular in vitro preclinical models. Thus, their use is required more and more for drug development processes in oncology in order to improve the prediction of anticancer drugs responses. Moreover, spheroid models allow for the reduction in animal experimentation, in accordance with the rule of Reduce, Refine, Replace (3Rs). In order to optimize and extend the use of these spheroid models, this works was focused on the development of an original methodology to keep these cancer spheroids in the long term. This innovative concept is based on a cold storage for up to 7 days of Triple-Negative Breast Cancer (TNBC) spheroids cultured in the synthetic serum-free OptiPASS® serum-free culture medium to preserve the spheroid characteristics during 3-, 5- or 7-day-long storage. Indeed, after storage they were returned to normal culture conditions, with recovered spheroids presenting similar growth rates (recovery = 96.2%), viability (Live/Dead® profiles) and metabolic activities (recovery = 90.4%) compared to nonstored control spheroids. Likewise, both recovered spheroids (after storage) and nonstored controls presented the same response profiles as two conventional drugs, i.e., epirubicin and cisplatin, and two anti-PARP1 targeted drugs—i.e., olaparib and veliparib. This new original storage concept seems to induce a temporary stop in spheroid growth while maintaining their principal characteristics for further use. In this way, this innovative and simple storage concept may instigate future biological sample preservation strategies.Cancer spheroids are very effective preclinical models to improve anticancer drug screening. In order to optimize and extend the use of spheroid models, these works were focused on the development of a new storage concept to maintain these models in the longer term using the Triple-Negative Breast Cancer MDA-MB-231 spheroid models. The results highlight that the combination of a temperature of 4 °C and oxygen-free conditions allowed the spheroid characteristics of OptiPASS Cancer remains a global health care issue, with 18.1 million new diagnosed cases and 9.6 million deaths worldwide in 2018 . RecentlIn this context, three-dimensional (3D) cell cultures models appeared as a pertinent in vitro option. More precisely, multicellular spheroids are compact round cell aggregates generated from cancer cell lines ,11. Thei® medium [® cultured models compared to others cultured in the serum-supplemented medium. Moreover, OptiPASS® medium improved the spheroids’ drug sensibility threshold and also allowed a prolonged culture of spheroids. Alongside this, other studies recently highlighted an innovative approach to store colorectal multicellular tumor spheroids under cold (4 °C) and anoxic conditions [The predictability and reproducibility of the 3D culture models’ drug responses depend on various parameters, such as the composition of the culture medium ,20. The ® medium . These wnditions . This me® medium in combination with anoxia and cold storage conditions, for the preservation of TNBC MDA-MB-231 spheroids, over time.In the context of the development and optimization of predictive preclinical models in oncology, this work aimed to evaluate the potential of using the synthetic OptiPASS®, HTB26™) was stored in the Biological Resource Center of Jean Perrin Comprehensive Cancer Center under the No. BB-0033-00075 . After absence of mycoplasma contamination check , MDA-MB-231 cells were cultured in RPMI 1640 medium  supplemented with 10% decomplemented fetal calf serum  and 20 µg/mL Gentamycin  at 37 °C under 5% CO2 in humid incubator.The triple-negative breast cancer MDA-MB-231 cell line  were seeded in ultralow attachment round-bottom microplates  at a concentration of 1000 cells/well. After 24 h at 37 °C in humid incubator, Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix  diluted in cold RPMI1640 or cold OptiPASS® culture medium was dispensed in each well for a final concentration of 2%. Microplates were then shacked for 20 min at 200rpm to promote the formation of a unique cell aggregate per well. The day after, compact spheroids were observable in bright field microscopy.MDA-MB-231 spheroids were formed as previously described . Briefly® fluorescence microscopy test. Metabolism activity of spheroids was also quantified at D3, D7 and D14 with resazurin metabolic activity test.For both conditions, spheroid size and integrity were measured by bright field microscopy at days D1, D3, D4, D7, D10 and D14. Spheroid viability was assessed at D3, D4, D7 and D14 using Live/Dead2) was checked with monogaz O2 detector   for 11 other days, giving a total culture time of 14 days . For the oxygen-free storage condition, microplates were packed in a hermetically closed plastic bag  with anaerobic gas generating AnaeroGen™ 2.5L  before being kept at 4 °C for storage period of 1, 3, 5, 7, 14 or 30 days . The conNC, USA)  and Resa 14 days . For con®-M = 4X) coupled with Gen5 3.08 software . The “Cellular analysis” tool was applied to a stitched picture to calculate the object size of each well . Spheroid size was recorded for each condition on the 1st, 3rd, 4th, 7th, 10th, and 14th days of culture  allowing growth kinetic curves to be set. Growth curve slopes were calculated for each spheroid condition between the 4th and the 14th culture days (corresponding to the poststorage period) [Each spheroid of the microplates was automatically imaged using bright field microscopy module of the Cytation™3MV cell analyzer (Biotek period) .® fluorescent microscopy kit . Spheroids were incubated for 30 min at room temperature in a working solution containing 1 µM calcein-AM and 2 µM ethd-D1 in D-PBS . They were then transferred to a µ-Slide 8 Well  to be imaged using the fluorescent microscopy module of the Cytation™3MV cell analyzer (Biotek®-M = 4X-fluorescence filters = green fluorescent protein and propidium iodide).The spheroid cell viability was monitored after 3, 4, 7 and 14 days of culture  using Live/DeadAdditionally, global spheroid metabolic activity was measured at days 3, 7 and 14  by transferring each spheroid to 100 µL of a sterile 60 µM resazurin solution in D-PBS . This test was based on the transformation of resazurin to resorufin by the mitochondrial respiration chain in living cells. The resorufin concentration, which can be measurable by fluorimetry, was directly proportional to the metabolic activity in spheroids . After 1® medium without storage (control spheroids) or stored for 3, 5 or 7 days at 4 °C in oxygen-free conditions were harvested at days 4, 7 and 14.Spheroids cultured in OptiPASSFor ki67 immunostaining, after a 12 h fixation in paraformaldehyde 4% , spheroids were successively incubated in 15 and 30% sucrose D-PBS solutions to be dehydrated. Spheroids were then placed in cryostat embedding medium to be quickly frozen in liquid nitrogen and cut into 10 µm sections with a cryostat device . Obtained slides were then incubated in saturation/permeabilization solution  for 1 h. They were then incubated with PBS 0.1% BSA diluted primary antibodies anti-ki67 , or Mouse IgG1 isotypic control for 1 h. After three washes in PBS, slides were incubated with PBS 0.1% BSA diluted secondary fluorescent antibodies antirabbit  for 1 h, followed by a nuclear counterstaining with Hoechst33258  for 5 min. Negative controls with no primary antibodies were also performed. Slides were mounted with Prolong Diamond Antifade  and imaged with Cytation™3MV fluorescent microscopy module (M = 10X-fluorescence filters = Texas Red and DAPI). Thanks to the “cellular analysis” algorithm of Gen5 3.08 software, nuclei were detected and localized on DAPI channel . For ki67 staining, some nuclei presented intense and specific staining compared to other ones, agreeing with an on/off activation of the transcription factor expression depending on cell cycle. Thereby, adapted algorithm was used to select nuclei subpopulation presenting activated ki67 expression (Texas Red fluorescent intensity > 3000). For the quantification of ki67 expression in each spheroid, the following formula was applied:® orange reagent  was used. This probe was attached to a nitro group that was specifically reduced by nitro reductase activity in hypoxic cells, releasing an orange fluorescence. Spheroids were exposed with Hypoxia Red reagent at a concentration of 250 µM diluted in OptiPASS® medium for 4 h in humid incubator at 37 °C and 5% CO2 . Mean fluorescence intensity of spheroids was calculated with “cellular analysis” algorithm of Gen5 3.08 software .For spheroid global hypoxia level evaluation, ROS-IDd 5% CO2 . They we® medium for final treatment concentrations of 0.1, 1 or 10 µM. Olaparib  and veliparib  were solubilized in DMSO to prepare 50 mM stock solution. Adapted dilutions were prepared in OptiPASS® medium for final treatment concentrations of 0.5, 5 or 50 µM. With the exception of epirubicin treated samples, the final DMSO concentration remained constant at 0.1% in all treated spheroid conditions.Epirubicin  and cisplatin  were solubilized in distilled water and DMSO, respectively, to prepare 10 mM stock solution. Adapted dilutions were prepared in OptiPASS® medium without the storage step or stored for 3, 5 or 7 days at 4 °C in oxygen-free conditions were treated for 5 days (between the 5th and the 10th culture days), with increased concentrations of selected anticancer drugs. The size of the untreated control and treated spheroids was monitored daily using Cytation™5MV  coupled with BioSpa™ automated cell incubator  and Gen5 3.08 software . Growth curve slopes were calculated for each spheroid between the 5th and the 10th culture days. At the end of the treatment, spheroid viability was assessed by the Live/Dead® and resazurin tests as described previously. Normalized viability was determined by the percentage of treated spheroids resorufin values on untreated control values.MDA-MB-231 spheroids cultured in OptiPASSt-test or two-way ANOVA  was used to evaluate statistical significance. Results were considered statistically different when p < 0.05 (*). Stronger differences were noted as follows: p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****) and p < 0.00001 (*****). Nonsignificant results were noted as “ns”.Results were presented as mean ± standard deviation. All experiments were performed independently on at least three separate occasions. An unpaired 2-sided Student’s ® medium were firstly stored in normoxic conditions at 4 °C, for one to three days. Then, 3D cell cultures were then exposed to standard cell culture conditions—i.e., at 37 °C under 5% CO2 [® medium, respectively.MDA-MB-231 spheroid models cultured for three days in RPMI1640 or OptiPASSp = 10−113 and 10−86, 2-sided t-test). Then, spheroid cell proliferation was analyzed between the 4th and the 14th days of culture by a growth curve slope analysis. Slopes of nonstored control spheroids were 47.3 ± 10.9 and clearly superior to one- or three-day storage conditions, at 3.0 ± 7.2  and 14.7 ± 8.6 , respectively  at D3 to 21.4 ± 2.8 × 103 UF at D7 and to 52.0 ± 5.5 × 103 UF at D14 (3 UF) and D14 (0.2 ± 0.1 × 103 UF)  compared to 16.1 ± 4.5 × 103 UF at D3 (before storage)  d.® culture medium, after one day of cold storage, the spheroid compaction aspect was similar to controls at day 4 and day 14    than controls (43.1 ± 11.6)     e. In con t-test) e. For gr t-test) f. For sp t-test) f. The spd day 14 g. In cond day 14 g. Finallnd 10−6) h. Similaectively h. In conage step h.® medium culture, these parameters seemed to be partially preserved for only one day of cold and normoxic storage of MDA-MB-231 spheroids.All these results suggest a clear loss of spheroid proliferation capacity, cell viability and cell metabolic activity, after one- or three-day cold and normoxic storage in RPMI 1640 medium. In contrast, in OptiPASS® media and were then hermetically packed using the Anaerogen™ oxygen-consumer. Spheroids were then stored for three days at 4 °C. Then, microplates were incubated in 37 °C and 5% CO2 and spheroid recovery was analyzed in the same conditions as previously presented.In order to achieve cold and oxygen-free conditions, spheroids in microplates were first cultured for 3 days under normal conditions in both RPMI1640 and OptiPASSp = 10−10, 2-sided t-test). Viability/mortality spheroid cell analysis by live/dead tests showed, for oxygen-free cold-stored spheroids, an absence of viable green cells at D4 and a low proportion of viable green cells at D14  .In RPMI 1640 medium, oxygen-free spheroids that were cold-stored for 3 days presented less cohesive structures than nonstored spheroids at D4 a. These s at D14 a. In cons at D14 a. In par t-test) b. FinallF at D14 c. These ® culture medium, after an oxygen-free 3-day long cold storage step, compact and cohesive spheroids, comparable to nonstored controls, were detected with bright field microscopy at D4 and D14 . Live/Dead® tests showed a vast majority of green/viable cells giving similar viability profiles to nonstored controls , showing similar proliferation rates between the two conditions .In OptiPASS and D14 d. The mecontrols d. Growthnditions e. Simila® medium, the combination of oxygen-free and cold storage over 3 days made it possible to preserve the properties of the MDA-MB-231 spheroids in comparison to nonstored controls. This last condition for the storage of MDA-MB-231 spheroids had no impact on these culture parameters, and longer storages in OptiPASS® medium were then studied.All these results showed that in OptiPASS® medium cultured MDA-MB-231 spheroids were maintained for 3 days under normal conditions and then exposed to oxygen-free conditions at 4 °C for 5, 7, 14 or 30 days. After each storage, microplates were exposed to 37 °C and 5% CO2. This is the same as previous parameters—i.e., spheroid integrity, growth, viability/mortality profile, metabolic activity and also ki67 proliferation marker expression—and spheroid hypoxia levels were analyzed at different times in comparison to nonstored spheroid controls.OptiPASSp = 10−9, 10−18, 10−44 and 10−7, 2-sided t-test). Otherwise, at D14, the spheroids stored for 5, 7 and 14 days showed regularly round and cohesive shapes with a mean size in the same range as nonstored controls—831.3 ± 51.3 µm, 845.0 ± 70.8 µm and 825.5 ± 140.2 µm, respectively, compared to 908.1 ± 52.1 µm for controls . For 30-day storage, spheroids presented a compaction loss aspect .First, concerning spheroid topology in terms of cell mass cohesion and cellular compaction, no difference was detected at D4 between the spheroids stored for 5 and 7 days and the nonstored controls with compact and ovoid spheroids a. In cons aspect a with a p = 0.09 and 0.07, 2-sided t-test)  and of spheroids stored for 30 days   b. Howeve t-test) b. In par storage c. For ex storage c.3 at D7 and 34.4 ± 3.0 × 103 at D14 for spheroids stored for 5 days compared to 22.4 ± 3.4 × 103 and 38.9 ± 4.5 × 103 for nonstored spheroid controls . However, the spheroids stored for 14 days showed significantly decreased metabolic activity, with 11.1 ± 2.4 × 103 at D7 and 17.6 ± 3.0 × 103 at D14 . Finally, the results obtained for the spheroids stored for 30 days were dramatically lower than those of nonstored control and close to blank background , presenting an almost total loss of spheroid viability after the storage step.These viability profiles were then confirmed with the resazurin metabolic activity test performed on D3, D7 and D14. The spheroids stored for 5 and 7 days presented metabolic activity evolutions similar to nonstored controls d, with f® cultured MDA-MB-231 spheroids in terms of proliferation, viability and metabolic activity, phenotypic characterization was then studied for these three storage conditions in comparison to nonstored control conditions.Regarding the absence of an impact of 3-, 5- and 7-day-long cold and oxygen-free storage on OptiPASS2 UF at D4, to 9.2 ± 5.7 × 102 UF at D7 and to 0.2 ± 0.2 × 102 UF at D14. This evolution was similar to those seen for spheroids stored for 3 days, with values of 22.2 ± 6.0 × 102 UF at D4 , 9.4 ± 1.1 × 102 UF at D7  and 6.2 ± 4.3 × 102 UF at D14 . These results were also similar for spheroids stored for 5 and 7 days spheroids    h. In the t-test) h.® synthetic medium in oxygen-free and cold conditions had the ability to preserve MDA-MB-231 spheroids for a maximum time of 7 days. For the studied parameters, under these storage conditions, spheroids would have the same characteristics as nonstored control spheroids in terms of proliferation, viability metabolic activity, ki67 expression rate and hypoxia level.These results suggest that OptiPASS® medium, these spheroids were treated with two conventional chemotherapeutics—i.e., epirubicin and cisplatin at 0.1, 1 and 10 µM or two anti-PARP targeted chemotherapeutics—i.e., olaparib and veliparib at 0.5, 5 and 50 µM. Drug cytotoxicity action on spheroids was assessed by the analysis of growth curve slopes, spheroid viability/mortality profile and cell metabolic activity in comparison with nonstored control spheroids.In order to evaluate the drug sensitivity of MDA-MB-231 spheroids after oxygen-free 4 °C storage for 3, 5 or 7 days in OptiPASSp ≥ 0.9, >0.9, 0.9 and >0.9, ANOVA) . Similarly, the epirubicin IC50 values were 2.19 µM for nonstored spheroid controls and 2.29, 2.20 and 2.29 µM for 3-, 5- and 7-day-long storage conditions, respectively.When the epirubicin concentration increased, dose-dependent decreases in growth curve slopes was detected for nonstored spheroids with 54.8 ± 4.7 for control, 47.5 ± 3.3 for 0.1 µM, 36.9 ± 3.7 for 1 µM and 23.3 ± 3.3 for 10 µM of epirubicin, respectively a. The sa, ANOVA) a. In par, ANOVA) b. The sa, ANOVA) c. Pooledp = 0.68, 0.74 and 0.56, ANOVA   . Compa, ANOVA) d. Likewidetected e. For me, ANOVA) f.p = 0.36 and 0.93, ANOVA). In contrast, with 50 µM anti-PARP1 growth curve slopes decreased significantly to 39.4 ± 3.7  compared to DMSO control  . Otherwise, for 50 µM olaparib metabolic activity decreased significantly to 72.3 ± 9.7%    g. Live/Dnditions h. After , ANOVA) i. Compar, ANOVA) i. These p = 0.46, ANOVA) and to 51.4 ± 3.5 with 5 µM . For 50 µm of this anti-PARP1, growth curve slopes decreased significantly to 39.4 ± 3.1    j. Then, nditions k. The no, ANOVA) l. Close ®. This suggests that selected spheroids storage conditions in OptiPASS® medium do not impact MDA-MB-231 spheroids’ anticancer drug sensitivity.These results indicated different MDA-MB-231 spheroid drug responses according to the selected anticancer agent. Indeed, a strong cytotoxic effect was detected after epirubicin treatment, while no effect was detected after cisplatin treatment. For both anti-PARP1 targeted therapy drugs, only the highest-tested concentration had an impact on spheroid growth and metabolism activity. Otherwise, and very interestingly, spheroid drug responses were similar between nonstored spheroids and 3-, 5- or 7-day-long oxygen-free cold-stored spheroids cultured in OptiPASS® [® medium. These results also highlight the key role of the culture medium in the optimization of the culture model’s predictive response [Spheroid in vitro models remain particularly relevant for preclinical evaluation of anticancer treatments, combining production reproducibility and predictivity of response at the same time . Their u® . Our resresponse . This apTo optimize the use of a high number of 3D culture models in high throughput screening assays, a concept of spheroid storage should be considered. In this field, cryopreservation methods represented the gold standard to maintain the viability/integrity of biological samples for several years or decades. However, in the case of 3D culture models, these techniques are time consuming, difficult to apply and often require specific equipment. Moreover, the compulsory use of cytotoxic cryoprotectants causes a partial cell viability recovery after thawing ,32. Ther® medium, in comparison with RPMI FBS-supplemented medium preconized for this cell line.In this context, with the objective of developing a new storage concept of 3D cell culture models, our works were focused on the development of a spheroid storage methodology using the TNBC MDA-MB-231 cell line as a model in the new synthetic OptiPASS2 conditions at 37 °C. Spheroid recovery was evaluated with several parameters such as (i) spheroid growth via spheroid growth curve slopes between day 4 and day 14, (ii) cell viability via live/dead test and (iii) metabolic activity via resazurin test.For this, several culture conditions were studied. Indeed, the spheroids were cultured in both tested medium cultures in microplates under conventional conditions at 37 °C in each culture medium for 3 days. Then, they were stored at 4 °C with or without anoxia for 1, 3, 5, 7, 14 or 30 days. After each conservation period, spheroids were placed back under 5% CO® culture medium led to an incomplete preservation of spheroid growth curve slopes (decreases of 93.7 and 101.5%) and metabolic activity , respectively. Indeed, the cold-only storage condition is far from sufficient to obtain usable spheroids for further experiments.Firstly, the 3-day storage at 4 °C in normoxic conditions in both RPMI1640 and OptiPASS® medium preserved the properties of the MDA-MB-231 spheroids. Indeed, in this condition, between day 4 and day 14, no impacts on spheroid integrity in growth curve slopes (recovery of 96.8%) and cell metabolic activity (preservation of 91.5%) were detected. All these results show that the properties of the MDA-MB-231 spheroids can be preserved after their storage for 3 days in OptiPASS® medium at 4 ° C and under oxygen-free conditions for further recovery.Secondly, in order to optimize the spheroid storage step in both media, spheroids were placed in an oxygen-free environment at 4 °C for 3 days, according to previous works of Gomes et al. . The res® medium at 4 °C and in oxygen-free conditions, longer storage times of 5, 7, 14 and 30 days were analyzed in this medium. Interestingly, after 5- and 7-day-long cold and oxygen-free storage in OptiPASS®, the spheroid growth curve slopes  and the metabolic activity  were preserved compared to nonstored controls, confirming an absence of impact on this spheroid growth parameter. In contrast, the longer storage times, i.e., 14 and 30 days, did not allow spheroid growth curve slopes  and metabolic activity  to be maintained compared to nonstored controls. Therefore, in MDA-MB-231 spheroid models, cell proliferation, cell viability and cell metabolic activity parameters may be preserved after storage times of 3, 5 or 7 days in OptiPASS® medium at 4 °C and in oxygen-free conditions. This conservation methodology was then selected for the following experiments.Then, in order to optimize the conservation in OptiPASSMDA-MB-231 spheroid recovery performances were then assessed with ki67 expression rate and spheroid global hypoxia level analyses, reflecting the cell proliferation capacity and global hypoxia status ,31. Afte® medium at 4 °C and in oxygen-free conditions, were treated with increasing concentrations of two classic chemotherapies, i.e., epirubicin or cisplatin, and two PARP1 inhibitors—i.e., olaparib or veliparib. In presence of various drugs, whatever the storage conditions, recovered spheroids presented similar cell proliferation decreases  and cell metabolic activity decreases  compared to nonstored controls. All these results indicate that this new methodology combining a spheroid storage at 4 °C and oxygen-free atmosphere in OptiPASS® medium does not influence spheroid responses to various drugs in terms of growth, viability, and metabolic activity. In terms of the unchanged model’s behavior and drug sensitivity profiles, stored spheroids can be used in drug screening tests the same way as nonstored spheroids.Thereafter, to ensure that the storage step did not affect spheroids sensibility threshold to anticancer drugs, the spheroid drug response was analyzed. For this, MDA-MB-231 spheroids, after 3, 5 and 7 days of storage in OptiPASS® serum-free medium, in comparison to serum-supplemented media. This culture medium with an original confidential formulation (developed by BIOPASS company) probably plays a major role in the spheroid recovery process. Indeed, serum-free media enriched in antioxidants substrates have already been described as improving the preservation of stromal stem cell monolayer cultures at 4 °C [In our experimental conditions, the comparison of both media culture performances highlighted that our innovative spheroid storage concept is principally permitted thanks to the new OptiPASS at 4 °C . Otherwi at 4 °C . For bas at 4 °C . This gl at 4 °C ,34,37.® medium. Indeed, the spheroid size ratio measured between the day when spheroids were placed at 4 °C and the day after their return in culture incubator was close to 1 , while it was of 1.22 ± 0.18 for nonstored controls (p < 0.0001). Thereby, this methodology allowed the cessation of spheroid growth while maintaining their ability to restart after their return to normal cell culture conditions. It should be considered as a break in spheroid culture time, allowing the MDA-MB-231 model to be preserved for up to 7 days. This mechanism can be related to the tumoral cellular quiescence phenomenon. Indeed, the stress caused by cold and oxygen-free conditions may have induced spheroid cell entry in dormancy, which is at the origin of a reversible arrest of spheroid growth. Moreover, cellular quiescence generally occurs in response to stress or to a hostile environment and drives cancer cells to enter in a dormant state, corresponding to a reversible cell cycle arrest in order to survive and being reactivated in favorable conditions [Furthermore, this new methodology of storage spheroids was relatively simple to apply and did not require expensive specific equipment. Especially, there is no need to transfer spheroid models to different supports or to change the cell culture medium. This point may be very important, especially for high added-value models, allowing the reduction in spheroid manipulations and thus limiting spheroid disturbances or losses. Moreover, the oxygen-free atmosphere generating device allows the induction of anoxic conditions very quickly and reproducibly. Indeed, once the system is closed, oxygen pressure decreases to 0% after less than 20 min . This conditions . This menditions ,43. Thisnditions . This st® medium had already demonstrated the improvement in growth of TNBC spheroids while maintaining their drug sensitivity in normal culture conditions [Otherwise, in our previous studies, the serum-free OptiPASSnditions . MoreoveTherefore, this concept opens the way (i) to the development of new tumor dormancy models, (ii) to the preservation of precious biological materials in the laboratory, and also (iii) to easy handling during interlaboratory transport for several days. These works are currently underway.


The present study emphasizes the effect of withering time set at 4 ± 0.5 h (WT4), 6 ± 0.5 h (WT6), 8 ± 0.5 h (WT8), 10 ± 0.5 h (WT10), and 12 ± 0.5 h (WT12) on the sensory qualities, chemical components, and nutritional characteristics of black tea. The sensory evaluation revealed high total quality scores at WT8 and WT10. Polysaccharides, amino acids, and soluble sugars significantly increased with an increase in withering time, and an apparent peak value was obtained at WT10. However, polyphenols, flavonoids, glycosides, organic acids, catechins, alkanoids, and theaflavins decreased with an increase in withering time. With an increase in withering time, the content of aromatic substances showed a trend of increasing first and then decreasing. The peaks of alcohols, aldehydes, and acids appeared at 10 ± 0.5 h, 10 ± 0.5 h, and 8 ± 0.5 h, respectively. The content of esters, ketones, and hydrocarbons showed a downward trend with an increase in withering time. Aroma analysis revealed that withering time could not exceed 10 ± 0.5 h. Black tea withered up to WT10 showed enhanced inhibition of α-glucosidase and α-amylase activity with good sensorial attributes. Glucose uptake inhibition capacity increased up 6 ± 0.5 h and then decreased, while antioxidant capacity decreased with an increase in withering time. The overall results show that the 8 ± 0.5 h to 10 ± 0.5 h withering time could improve black tea quality and nutritional characteristics. Black tea is the most popular type of tea all over the world, owing to its unique aroma and taste, attributed to secondary metabolites such as polyphenols, flavonoids, alkanoids, glycosides, terpenoids, and their precursors ,2. TheseThere are four main stages in black tea processing, i.e., withering, maceration/rolling, fermentation and drying/firing. As a key step, withering decreases the moisture content of fresh leaves about 75%) to the average amount in withered leaves 60–65%) [0–65% [6]5% to theIn fact, previous studies have shown that the hydrolysis of biological macromolecules is the vital chemical reaction during the withering process . PolysacType 2 diabetes mellitus (DM) is a prevalent chronic disease worldwide . The decTheaflavin standards and the catechin standard were obtained at Yuanye Biotechnology Co., Ltd. . The amino acid standards were obtained at Chemfaces . The phenolic acid standards, alkanoid standards, and flavonoid standards were all obtained at J&K Scientific Ltd. ; glycoside standards were obtained at National Glycoengineering Research Center . The internal standard (D-acetaminophen) was purchased from Aladdin . 2,2-Diphenyl-1-picrylhydrazyl , α-amylase , and α-glucosidase  were bought at Sigma-Aldrich Chemical Co., Ltd. Caco-2 cells were purchased from the Chinese Academy of Sciences and Dulbecco’s modified Eagle medium (DMEM) was obtained from HyClone Laboratories Inc. . The rest of the reagents were of analytical grade.(Camellia sinensis L.) were picked in September 2019 from the experimental tea garden of Huazhong Agricultural University in Hubei, China, with two leaves and one bud. A bunch of fresh leaves was distributed equally in five portions and treated in a 6CWD-200 withering trough , with the withering time set at 4 h ± 0.5 h (WT4), 6 h ± 0.5 h (WT6), 8 h ± 0.5 h (WT8), 10 h ± 0.5 h (WT10), and 12 h ± 0.5 h (WT12). Air with a temperature of 32 °C was blown through the leaves and turned off for an hour once every two hours. During the pause, leaves were shuffled to redistribute the moisture content uniformly. In all five treatments, moisture content decreased from 75 ± 0.67% to 64.53 ± 0.15%, with the final moisture loss rate at 2.71 ± 0.34%/h (WT4), 1.90 ± 0.15%/h (WT6), 1.25± 0.08%/h (WT8), 1.04 ± 0.05%/h (WT10), and 0.94 ± 0.04%/h (WT12), respectively. The rate of water loss in withering was monitored by adjusting the leaf layer thickness, time of airflow at the equal temperature, and wind speed, ensuring that all treatments reached the time level at an equal moisture content (64.53 ± 0.15%). This treatment aimed to monitor the water loss rate and prevent any impact of different withering moisture content on the outcomes. Withered leaves were pulverized into small pieces by a 600 T Pulverizer , and then the grinded withered leaves were sieved through 2 mm meshes and placed in a 6CFJ-100 fermenter  for two hours at a temperature of 32 °C and relative humidity of >92%. Fermented tea was dried with a 6CZG-60 drying cabinet at 110 °C for 10 min and then cooled outside of the cabinet to room temperature, and dried again at 90 °C for one hour to obtain black tea with a moisture content around 5%. The experiments were conducted in triplicate. The produced tea was finally kept in a freezer (−20 °C) for subsequent analysis.Fresh leaves of Echa NO. 10 cultivar The professional evaluation was conducted in accordance with the Chinese National Standard (GB/T23776-2018) . BrieflyCustomer sensory evaluation was conducted by 58 people who were familiar with black tea characteristics. These evaluators were of both genders  and ranged from 18 to 53 years old. Before starting the evaluation, all evaluators were given evaluation guidelines. Samples were blindly coded in the preparation room, and the coded samples were served to the evaluators in their respective booths through the small windows. The tea aroma, taste, color, and overall acceptance were scored using 9 hedonic points . Moisture content was determined using the existing method (GB/T 8304-2013) . The tot2 spectra, accurate mass, and respective standards. Untargeted nonvolatile constituents in black tea were determined . In shor2 spectra, and accurate mass were used to identify volatile compounds.Volatile compounds were determined by gas chromatography–mass spectrometry (GC–MS) , using a modified method . Briefly4 cells/cm2 using 24-well transwell inserts, then incubated in 95% of air and 5% of CO2 for 21 days. In glucose uptake testing, tea extract was dissolved in DMEM with the concentration of 8 mg/mL and 0.6 mL of the solution, and 1.2 mL of PBS was placed into the apical and basal sides of the monolayer, respectively, then incubated for 2 h at 37 °C  value > 500 Ω. Cm2 were used). Then, solution (10 μL) from the basolateral side was taken to determine the content of glucose using a glucose assay kit.The DPPH free radical scavenging assay, α-amylase activity inhibition and α-glucosidase activity inhibition assays were determined according to the method used previously . Brieflyp-value (p ≤ 0.05) were used to find statistical significance.Statistical analysis software  was used. All data were expressed as the mean ± standard deviation of autonomous triplicate experiments. One-way analysis of variance (ANOVA) was used to analyze the results and the figures were drawn using OriginPro 9.1 and GraphPad prism 8.0.2 (263). Duncan’s test and the p < 0.05) difference between the two treatments. Infused leaves were slightly uneven and red-blown at WT4 and WT6, with a reddish and dull appearance observed at WT12. The cumulative scores pattern showed the highest score at WT10 (86.88) followed by WT8 (86.74). The withering time exhibited a significant effect on the sensory quality, and this finding suggested that WT8 and WT10 could develop the sensory attributes of black tea.The sensory evaluation was carried out using two groups: professionals and consumers. p > 0.05) among all sensory indices evaluated (2 = 0.007) at WT10, followed by aroma (Pr > X2 = 0.011) at WT12  decrease in the following order: WT4 (13.47 mg/g) > WT6 (12.98 mg/g) > WT8 (12.73 mg/g) > WT10 (12.51 mg/g) > WT12 (11.69 mg/g) (p < 0.05) increased with withering time (p ˂ 0.05) increase, including L-tryptophan, L-valine, L-leucine, L-proline, L-phenylalanine, L-tyrosine, L-isoleucine, L-glutamic acid, and GABA, while three of them  decreased significantly (p ˂ 0.05) as the withering time increased. All detected amino acids were proteinogenic amino acids, except L-theanine and GABA.The catechins content declining with the increase in withering time and overall content demonstrated a significant (69 mg/g) . During ing time e. Figurep ˂ 0.05) reduced with withering time: WT4 (4.27 mg/g) > WT6 (4.18 mg/g) > WT8 (4.10 mg/g) > WT10 (4.03 mg/g) > WT12 (3.89 mg/g)  were the main contributors to the total relative flavonoid glycosides content detected. The relative content of total flavonoid glycosides significantly (89 mg/g) . p ˂ 0.05) up to WT6 and then kept reducing insignificantly (p > 0.05), knowing that organic acid imparts the sour taste in the balance of the complex characteristics of black tea.After withering, the seven organic acids detected included p-coumaric acid, salicylic acid, gallic acid, D- (-)-quinic acid, and chlorogenic acid, all of which were increased, while caffeic acid and shikimic acid decreased with an increase in withering time . The cumThe relative total flavonoid content in black tea decreased from WT4 (0.67%) to WT12 (0.39%) and the highest decrease was observed between the first and the second treatments, by comparing two successive treatments b. With ap < 0.05) increase in theabrownins - and 2-isopropenyl-5-methyl-4-hexenyl acetate were not detected in WT4, while bicyclo [3.1.1]hept-2-ene-2-ethanol, 6,6-dimethyl- and 1-dodecanol, 3,7,11-trimethyl- were not detected in WT4 and WT6 treatments. However, all these compounds were found in the remaining treatments. This absence of some compounds in WT4 and WT6 might be caused by insufficient withering time for their synthesis or formation. The major contributors of tea aroma considered in this study were 2,4-heptadienal, -, benzyl alcohol, 2-octenal, (E)-, benzeneacetaldehyde, cis-4,5-epoxy-(E)-2-decenal, phenylethyl alcohol, methyl salicylate, geraniol, geranyl acetone, geranyl formate, and linalool/linalool oxides. They increased with the increase in withering time, up to WT10 . A signip ˂ 0.05) in the free radical scavenging activity of black tea among five withering treatments  was used to analyze the antioxidant activity and the results show a significant difference . The higher the IC50 value, the lower the scavenging or inhibition potential and vice versa. The results show that black tea has a higher potential of α-glucosidase inhibition than α-amylase inhibition , and oxidation products of catechins  . The decThe increasing tendency of amino acids was due to the degradation of protein molecules by peptidase into simple amino acids; during the withering of black tea, protein reduced by about 1.2% of its total content . The metA reduction in L-theanine during withering was previously reported  and in tp < 0.05) increased as withering time increased  oxidizencreased f. This rThe most abundant subclass of flavonoid detected was flavonols , which are spatially distributed in mesophyll cells and present in glycoside form, especially kaempferol O-glycosides, myricetin O-glycosides, and quercetin O-glycosides ,24. HoweThe gallic acid increase was caused by the decomposition of gallated catechins. Gallic acid is a vital phenolic acid in black tea that contributes to its antioxidant property . Furtherp < 0.05) with an increase in withering time up to WT10. The accumulation of these terpenoid volatiles compounds correlated with the upregulation in genes responsible for the biosynthesis of terpenoid volatile constituents in tea during withering, through the plastidial methyl-erythriol phosphate (MEP) and cytosolic mevalonic acid (MVA) pathways [Terpenoid volatile derivatives such as geraniol, linalool, linalool oxides, volatile phenylpropanoids/benzenoids viz. benzyl alcohol, and phenylethyl alcohol  increasepathways . Alcoholpathways ,40. Phenpathways . Usuallypathways .DPPH was used to analyze the antioxidant activity due to its potential to attract the proton from the antioxidant . The decα-amylase and α-glucosidase play an essential role in the degradation of starch into glucose . α-amylaThe reduction in inhibitory activity is due to the decrement in gallated catechins, theaflavins, and flavonoids, which exhibit potent inhibitory effects against α-amylase and α-glucosidase activities ,45. The Withering time significantly affects the sensory qualities, chemical components and nutritional characteristics of black tea. It was observed that withering is practiced to condition the fresh tea leaves physically and chemically for subsequent stages to obtain high-quality tea. Based on tea sensory qualities, internal quality analysis and the biological activity of tea, the withering time of black tea should not exceed 10 ± 0.5 h and not be less than 6 ± 0.5 h. The recommended range is 8 ± 0.5 h–10 ± 0.5 h, of which 8 ± 0.5 h is the appropriate withering time. It is possible to make functional black tea with potential hyperglycemic activity by controlling withering time, and this will provide more choices for different people.


A. niger CGMCC 10142. HGT1-overexpressing strains using the PglaA and Paox1 promoters were constructed to verify the glucose transporter functions.Glucose transporters play an important role in the fermentation of citric acid. In this study, a high-affinity glucose transporter (HGT1) was identified and overexpressed in the industrial strain A. niger 20-15 exhibited a reduction of the total sugar content and residual reducing sugars by 16.5 and 44.7%, while the final citric acid production was significantly increased to 174.1 g/L, representing a 7.3% increase compared to A. niger CGMCC 10142. Measurement of the mRNA expression levels of relevant genes at different time-points during the fermentation indicated that in addition to HGT1, citrate synthase and glucokinase were also expressed at higher levels in the overexpression strains.As hypothesized, the HGT1-overexpressing strains showed higher citric acid production and lower residual sugar contents. The best-performing strain Aspergillus niger CGMCC 10142.The results indicate that HGT1 overexpression resolved the metabolic bottleneck caused by insufficient sugar transport and thereby improved the sugar utilization rate. This study demonstrates the usefulness of the high-affinity glucose transporter HGT1 for improving the citric acid fermentation process of The online version contains supplementary material available at 10.1186/s12934-021-01659-3. Aspergillus niger fermentation on the industrial scale. As a saprophytic fungus, A. niger secretes abundant hydrolytic enzymes which are beneficial for CA production on complex substrates [Citric acid (CA) is a naturally occurring tricarboxylic acid that is widely used in food, chemistry, medicine, and other fields, whose high demand places it among the most commonly used organic acids in the world , 2. Morebstrates , 4.A. niger is one of the most important industrial microorganisms, with tremendous commercial value. With the development of sequencing and genomic technologies, rational molecular evolution and modification instead of random mutagenesis are increasingly being applied to increase the CA fermentation efficiency of A. niger [A. niger [A. niger includes a complex network of replenishment pathways, which can overcome the effects of individual gene overexpression, leading to limited improvement of citric acid production. For example, the overexpression or deletion of citrate synthase did not improve citric acid production [A. niger . To dateA. niger –8, mostlA. niger –11. ReceA. niger , 13. Theoduction , 15. ThuAccordingly, Torres et al. calculated that the key limiting points of citric acid production are the intake and phosphorylation of hexoses . If a hiA. niger, a low-affinity glucose transporter with Km of 3.67 mmol/L, and a high-affinity glucose transporter with Km of 260 µmol/L. Torres et al. found that a high glucose concentration (> 50 g/L) is needed for the low-affinity glucose transporter to become active, which is capable of providing the high flux of glucose required for citrate production [A. niger H915-1 during the fermentation process revealed that a low-affinity glucose transporter maintained high transcript levels, while 5 high-affinity glucose transporters did not [A. niger with a low-affinity glucose transporter with high Km and high-affinity glucose transporters with extremely low-expressed levels, can explain the limited citrate yields and large amounts of residual glucose in the fermentation broth [A. niger is crucial for increasing CA production and may be one of the limiting factors for obtaining higher CA yields [There are two types of glucose transporters in oduction . Transcr did not . This naon broth , 22. ConA yields , 22.A. nidulans, A. niger, and T. reesei can also transport glucose [A. niger. Based on a conserved protein domain search, 86 putative sugar transporter genes were identified and annotated in the genome of A. niger [A. niger by membrane‑associated proteome analysis and biochemically characterized [A. niger, and its disruption resulted in a two- to fivefold reduction of cellular glucose uptake [A. niger CBS 513.88 as well as the original industrial acid-producing strain A. niger ATCC 1015 [A. niger CGMCC 10142. Additionally, two glucose transporters were identified in A. niger H915-1, a low-affinity glucose transporter with high expression at high glucose concentration, and a high-affinity glucose transporter with extremely low-expression level [Kluyveromyces lactis, was also found in the A. niger CBS 513.88 and A. niger CGMCC 10142 [Many putative sugar transporters have been identified in fungi. Interestingly, several newly discovered xylose transporters from  glucose , 24. Add glucose . HoweverA. niger . Two newcterized . Additioe uptake . This prTCC 1015 , but it on level . Liu et on level . NotablyCC 10142 , 32.A. niger CA industry is corn steep liquor with high amounts of total sugars, including glucose, disaccharides, and polysaccharides. A. niger secretes abundant glucoamylases that can hydrolyze polysaccharides into readily bioavailable glucose. Thus, the main carbon source at the beginning of the fermentation is glucose, and an adequate supply of carbon sources can greatly increase the CA production. Undoubtedly, the transport rate of glucose understandably becomes a limiting factor preventing the further increase of CA yields. Therefore, it is desirable to accelerate the transport of glucose during the fermentation of A. niger and thereby improve the citric acid production performance.The primary substrate used in the A. niger CGMCC 10142 was used as the starting strain for metabolic engineering. The gene encoding the high-affinity glucose transporter HGT1 was identified and cloned from the parental strain. After sequence alignment, the strong endogenous PglaA and Paox1 promoters were selected to overexpress HGT1. Subsequently, HGT1-overexpressing strains were successfully constructed and characterized. The transcription levels of key enzyme genes involved in CA production were analyzed during the fermentation by real-time qPCR. Furthermore, the biomass and residual sugars were quantified and compared between the engineered strains and the parental strain.Here, the industrial CA-production strain The low-affinity glucose transporter is the limiting factor for glucose import into the cell in fermentation at relatively higher glucose concentrations (> 50 g/L) . By contA. niger CGMCC 10142 was re-sequenced by our team and further analyzed to screen essential high-affinity glucose transporters. The high-affinity glucose transporter HGT1 was identified at the locus contig1  and a BLASTn showed that it was 100% identical to its ortholog from A. niger CBS 513.88 (GenBank: AM269996.1). It is remarkable that the high-affinity glucose transporters show practically no-transcription at 12 h of fermentation. To improve the glucose utilization rate, the two plasmids p20 and p21 for the overexpression of HGT1 using the promoters PglaA and Paox1 were successfully constructed . Both plasmids were verified by PCR and sequenced as shown in Figures S2a and S2b.The genome of the industrial CA production strain A. niger HGT1 (XP_001391024.1) was aligned with those of MstA (AAL89822.1) [A. nidulans MstE (XP_663464.1) [A. niger and its homolog (AAC49461.1) from Kluyveromyces lactis reached 40.87% [A. niger was predicted through homology modeling in Swiss-model using the sugar transport protein STP10 (PDB:6H7D_A) [The amino acid sequence of 89822.1) , LGT1 (X89822.1) , and A. 63464.1) . The resd 40.87% . In this:6H7D_A)  and D-xyA. niger and A. nidulans. The HGT1 of A. niger CGMCC 10142 corresponds to CBS 513.88 HGT1 (XP_001399197.1) of A.niger. Several other glucose transporters that are close to HGT1 included XP_001394117.2 and XP_001390064.1. What’s more, the MstA in A. niger and most of the sugar transporters identified in A. nidulans were located in the same branch of the phylogenetic tree. Additionally, several different sugar transporters were identified in A. nidulans and A. niger, which was consistent with previous studies. Several different high-affinity sugar transporters are encoded in the genome of A. niger, and they may not be closely related according to the phylogenetic analysis (collapsing branches at an average branch length distance < 0.6). It is possible that sugar assimilation constraints under different environmental conditions led to the evolution of a diverse array of glucose transporters, whereby their transcriptional regulation and ecological role remain mostly unclear.A phylogenetic tree of sugar (glucose) transporters from different fungi is shown in Fig. glaA-HGT1, Paox1-HGT1, and HGT1-hyg fragments of the transformants were amplified to verify successful cloning of the cassette into A. tumefaciens . Then, the integration location was determined using primers spanning the total length of ku70. If the plasmid was homologously recombined to the ku70 gene, a 5710 bp fragment including the homologous bodies of ku70 and the HGT1-overexpression cassette could only be amplified by PCR, while the HGT1-overexpression cassette was randomly integrated into the genome, only an 1800 bp fragment of ku70 gene itself can be amplified. As shown in Additional file ku70 (1800 bp), and not the target fragment (5710 bp) was amplified from the genomic DNA of A. niger. Thus, the results confirmed that the whole PglaA/Paox1-HGT1-hyg segment was randomly inserted into the genome of the transformants. Furthermore, the 8 genetically modified strains were passaged for 15 generations and transformants’ genetic stability was confirmed by validating the hyg gene sequence as shown in Additional file Firstly, The PA. niger 20-15, 20-16, 20-25, 20-27, 20-29, 21-8, 21-28, and 21-32) were selected for shake flask fermentation to screen higher yielding transformants , 144.6 g/L (+ 11.1%), 146.7 g/L (+ 12.8%) and 145.3 g/L (+ 11.7%), respectively  and 4.4% (P < 0.05) improvements compared to the 162.3 g/L CA titer of strain 10142. The difference between A. niger 20-15 and 21-8 was not statistically significant (P > 0.05). The productivity at each fermentation stage was calculated, as shown in Figs. A. niger 20-15 reached 4.4 g/L/h, which was higher than both A. niger 21-8 and 10142, which respectively reached 4.1 g/L/h at 24 h of fermentation. The highest productivity of A. niger 21-8 reached 4.2 g/L/h, which was approximately equal to A. niger 20-15 but higher than A. niger CGMCC 10142 with 4.0 g/L/h at 32 h, and remained at a high level at 40 h. This indicated that CA production at 24 h was higher in A. niger 20-15 than in A. niger 21-8 or A. niger CGMCC 10142, probably due to the fact that 21-8 did not have such a strong HGT1 overexpression promoter. The biggest differences in productivity were observed between 56 to 64 h at the end of fermentation. During this time, the CA productivity of A. niger 20-15 and A. niger 21-8 was 1.6-0.6 g/L/h and 1.0-0.7 g/L/h, respectively, which was 2.1-1.5 and 1.3-1.8 times higher than that of A. niger CGMCC 10142, which reached 0.7-0.4 g/L/h. There was a significant difference among the three strains (P < 0.01) at 56 h, and there was also a significant difference between A. niger 21-8 and A. niger CGMCC 10142 (P < 0.01) or A. niger 20-15 (P < 0.05). However, there was no significant difference between A. niger 20-15 and A. niger CGMCC 10142 (P > 0.05) at 64 h. These results further confirmed that the overexpression of HGT1 promoted CA production at the later stage of fermentation when the carbon source was nearing exhaustion.The CA production in the bioreactor-scale fermentation was also measured every 8 h, as shown in Figs. A. niger 20-15, which were higher than the corresponding values for A. niger 21-8, at 33.8, 18.9, and 8.8 g/L, but lower than those of A. niger CGMCC 10142, at 37.4, 22.9, and 12.7 g/L, respectively , citrate synthase (CS) gene (XM_001393946), and glucokinase gene (XM_001395875.2) (The GenBank access numbers are derived from A. niger CBS 513.88) during the fermentation process were measured to explore whether the overexpression of HGT1 influenced the metabolism and accumulation of CA in A. niger. Mycelial pellet samples of A. niger 20-15 (PglaA) and 21-8 (Paox1) were taken at 12 and 48 h, separated from the culture supernatant through filtration, and the total RNA was extracted for real-time quantitative PCR.The relative transcription levels of the HGT1 in the overexpression strains was much higher than in the control at 12 and 48 h. The transcription level of HGT1 in A. niger 20-15 was about 219 times and 208 times higher than in the original strain at 12 and 48 h, respectively, which indicated that the glucose utilization efficiency of the HGT1 overexpression strain was also higher than that of A. niger CGMCC 10142. Moreover, the HGT1 overexpression strain A. niger 20-15 with the PglaA promoter showed 2.5 times higher transcription than the strain A. niger 21-8 with the Paox1 promoter at 12 h, as well as 5 times higher at 48 h. PglaA is a widely used strong fungal promoter that can be induced by starch or dextrose [aox1 is the native promoter of alternative oxidase in A. niger [glaA) with higher HGT1 transcription level didn’t present a significantly more CA yield than 21-8 (Paox1)  Fig. A, probabA. niger 20-15 was 8.4 times higher than in the parental strain, while that of A. niger 21-8 was 1.4 times higher at 48 h, which indicated that the overexpression strains had a higher glucose utilization rate and improved CA production ability. These results indicated that the overexpression of HGT1 increased the glucose uptake efficiency, which provided adequate carbon flux for increased CA production in the transformants. Glucokinase plays a key role in the metabolic activation of glucose to glucose 6-phosphate, and its activity is controlled via feedback inhibition by glucose 6-phosphate and ADP. The increased glucokinase activity could therefore directly improve the CA production at the end of the fermentation process.Notably, the relative expression levels of glucokinase in the HGT1 overexpression strains were higher than in the parental strain, especially at 48 h Fig. B. The glA. niger strain 20-15 was 6.4 times higher than that of the parental strain at 48 h. CS is necessary for the synthesis of CA as indicated by its name and therefore plays a key role in CA metabolism. When the cs gene is deleted in A. niger, CA production from 98.7 falls to 64.3 g/L [As shown in Fig. 64.3 g/L . High exaox1 and glaA have strong promoters available for A. niger engineering, these genes may be also expressed in A. niger, which may affect citric acid production [aox1 may not only drive the expression of the HGT1 gene but also activate its own aox1 gene to some extent, and thereby indirectly increase the fermentation performance. As was observed in A. niger 21-8, higher transcription levels of glucokinase and CS than A. niger 20-15 at 12 h when the mycelium grows vigorously and dissolved oxygen becomes limited. In view of this, it may be useful to verify the relative expression of aox1 and related enzymes in future studies. What’s more, the exact insertion locus or copy numbers of the overexpression locus may also influence the expression levels. The enzymes that are important for CA synthesis, such as CS and glucokinase were expressed at higher levels in the HGT1 overexpression strains than in A. niger CGMCC 10142. It is possible that sufficient glucose increased the expression of these enzymes and thereby improved the CA production performance of A. niger [Though some genes such as oduction , 39. We A. niger 20-15 and 21-8 both increased to 102.4% and 100.4%, which was close to the theoretical glucose-CA conversion rate of 106.6%  + 1.5O2 → C6H8O7 (MW: 192.12) + 2H2O) when all the resources are used to produce CA and none for growth [A. niger 20-15 and 4.4% for A. niger 21-8. Therefore, sugar intake may still be a bottleneck in the fermentation process.Glucose transport is the first step from sugar to CA, but it does not influence the CA fermentation directly. In this study, we found that the overexpression of HGT1 improved the CA fermentation performance in terms of the total consumption of reducing sugar and total residual sugar remaining in the fermentation broth. Furthermore, the final glucose-CA conversion rates of r growth . FinallyIn this study, we investigated the effects of overexpressing the glucose transporter HGT1 on industrial CA fermentation and found that it is beneficial for CA production in both shake flasks and a 30 L laboratory-scale bioreactor. The increase in CA production can be explained by an increased sugar utilization rate and indirect positive effects on the expression of key metabolic genes. Overexpression of glucose transporter genes can be used to reduce the cost of industrial-scale production and increase the yield of citric acid.A. niger CGMCC 10142 was cultivated on potato dextrose agar (PDA) at 35 ℃. Escherichia coli DH5α was cultured in LB medium at 37 ℃ for plasmid propagation. Agrobacterium tumefaciens AGL1 was cultured in LB medium at 28 ℃ for plasmid transformation. All the reagents were analytically pure. Hygromycin was purchased from Solarbio. Co., Ltd (China). The reagents for plasmid construction and real-time qPCR were bought from Takara Co., Ltd (China).All the strains and plasmids used in this work were obtained from the Tianjin Key Laboratory of Industrial Fermentation Microbiology, Tianjin, China, or constructed in this work, as shown in Table A. niger CGMCC 10142 or its transformants were obtained from cultures on PDA plates which were grown at 35 °C for 4 days, and cultured in corn steep liquor  with a concentration up to 1 × 105 spores/mL. A. niger was cultured at 35 ℃ for 72 h under constant shaking at 330 r/min in 500 mL shake flasks containing 50 mL of medium. The amount of 1 × 105 spores/mL was inoculated in 30 L fermenters containing 20 L corn steep liquor, and cultivated at 0.1 MPa, 330 L/h aeration rate, 350 r/min, and 35 °C for 64 h, according to the previous report [Spores of s report .HGT1 gene, glaA promoter (PglaA), and aox1 promoter (Paox1) were cloned from the genome of A. niger CGMCC 10142. The genomic DNA was extracted from young hyphae grown on PDA medium. The A. niger CBS 513.88 genome was used as a reference to design primers. After sequencing at BGI Genomics Co., Ltd (China), we conducted sequence alignment based on NCBI to investigated the coherence of the HGT1 sequence. The plasmid p80-HSVtk which contains HSVtk, ku70 upstream sequence, hygromycin resistance marker (hyg), and ku70 downstream sequence was used as the parental plasmid. The ku70 deletion leads to improved efficiency of transformation and the HSVtk insertion leads to an increased rate of homologous recombination [Kpn I after which promoter and HGT1 coding sequence were ligated using the Multis kit  to form the plasmids p20 and p21  was measured to determine the reducing sugar. Total sugar in supernatants needs adding equal volumes 6 mol/L HCl and heating for 30 min in a boiling-water bath, adjust the pH to neutral, then were also determined by DNS method. Standard measurements were performed using the same procedure.The cell pellets were checked under a microscope every 8 h in the 30 L fermentation process. Total sugar, reducing sugar and CA concentrations were also measured every 8 h. The contents of residual total sugar and residual reducing sugar were determined using the DNS  method . Briefly340. After that, 20 μl solution 2 (containing 0.8 U citrate lyase) was added for a further 5 min. The change in A 340 was monitored after the reaction with citrate lyase. Standard measurements were performed using the same procedure.Citric acid was measured using a commercially available test kit . Briefly, to 0.2 mL sample, 1 mL solution 1 (containing glycylglycine buffer (pH = 7.8), 11.33 U L-malate dehydrogenase, 23.3 U L-lactate dehydrogenase, 0.42 mg NADH) and 1.8 mL double-distilled water were added. After incubation at 20–25℃ for 5 min, read the A A. niger, real-time quantitative PCR was conducted during the fermentation. The relative expression of HGT1 and the genes encoding key rate-limiting enzymes related to the citric acid cycle, citrate synthase and glucokinase, were measured. The primers used for the qPCR are listed in Table To further investigate how the glucose transporter affects the metabolism and accumulation of CA in 2) of the slope of the standard curve was used to calculate each gene’s PCR amplification efficiency as described before [The relative gene transcription levels were calculated using the All the results were based on three independent experiments, and samples with P < 0.05 were considered significant.Additional file 1: Figure S1. The recombinant plasmid map of p20 and p21. (a) The recombinant plasmid (p20) of overexpression HGT1 transcripted by glaA promoter (PglaA); (b) The recombinant plasmid (p21) of overexpression HGT1 transcripted by aox1 promoter (Paox1). Figure S2. Verification of HGT1 gene overexpression plasmids and strains. (a) Verification of the plasmid p20, M: DNA marker III, 1: hyg (1389 bp), 2: PglaA -HGT1 (3312 bp), 3: HGT1-hyg (3217 bp), 4: PglaA–hyg (4710 bp); (b) Verification of the plasmid p21, M: DNA marker III, 1: Paox1-hyg (4612 bp), 2: HGT1-hyg (3217 bp), 3: hyg (1389 bp), 4: Paox1-HGT1 (3223 bp); (c) Verification of A. tumefaciens p20 and p21, M: DNA marker III, 1: p20 plasmid control, 2-3: A. tumefaciens p20 verifying of PglaA-HGT1 (3312 bp), 4: p21 plasmid control, 5-6: A. tumefaciens p21 verifying of Paox1-HGT1 (3217 bp); (d) Verification of A. niger p20 transformants, M: DNA marker III, 1: p20 plasmid control, 2: negative control, 3-10: p20 transformants genome verifying of PglaA-HGT1 (3312 bp); (e) Verification of A. niger p21 transformants M: DNA marker III, 1: p21 plasmid control, 2: negative control, 3-10: p21 transformants genome verifying of Paox1-HGT1(3217 bp); (f) HTG1-hyg verifation M: DNA marker III 1: plasmid control, 2: negative control, 2-10: PCR product of HTG1-hyg (3217 bp) from transformants genome; (g) Verifying from ku70 upstream to downstream of the transformants M: DNA marker III 1: plasmid control (5710 bp), 2 negative control (1800 bp), 3-10: transformants genome ku70 amplification. Figure S3. Multiple sequence alignment of glucose transporters. Figure S4. Validating hyg gene in the HGT1 overexpression mutants at the 15th generation. M: DNA marker III, 1: positive control (1389 bp), 2: negative control, 3-10: amplified hyg gene using the genomic DNA of overexpressed mutants. Genomes of all strains were gained from a 15-generation subculture. Figure S5. Measurement of diameter size of mycelial balls of citric acid in the 30 L bioreactor fermentation.Additional file 2: Table S1. The initial screening of high-yield citric acid transformants.


Due to the scarcity of therapeutic approaches for COVID-19, we investigated the antiviral and anti-inflammatory properties of curcumin against SARS-CoV-2 using in vitro models. The cytotoxicity of curcumin was evaluated using MTT assay in Vero E6 cells. The antiviral activity of this compound against SARS-CoV-2 was evaluated using four treatment strategies . The D614G strain and Delta variant of SARS-CoV-2 were used, and the viral titer was quantified by plaque assay. The anti-inflammatory effect was evaluated in peripheral blood mononuclear cells (PBMCs) using qPCR and ELISA. By pre–post infection treatment, Curcumin (10 µg/mL) exhibited antiviral effect of 99% and 99.8% against DG614 strain and Delta variant, respectively. Curcumin also inhibited D614G strain by pre-infection and post-infection treatment. In addition, curcumin showed a virucidal effect against D614G strain and Delta variant. Finally, the pro-inflammatory cytokines  released by PBMCs triggered by SARS-CoV-2 were decreased after treatment with curcumin. Our results suggest that curcumin affects the SARS-CoV-2 replicative cycle and exhibits virucidal effect with a variant/strain independent antiviral effect and immune-modulatory properties. This is the first study that showed a combined  effect of curcumin during SARS-CoV-2 infection. However, additional studies are required to define its use as a treatment for the COVID-19. Betacoronavirus genus [Since March 2020, the World Health Organization (WHO) has declared COVID-19 as a pandemic [us genus ,4,5, whius genus . This vius genus , which pus genus ,8,9. Spius genus .After the virus enters the target cell, multiple interactions between viral components and host proteins triggers recognition by the immune system. Indeed, in severe COVID-19 cases, the uncontrolled viral replication induces aberrant pro-inflammatory responses, thus contributing to tissue damage ,12. AccoDespite the unprecedented advances in the development of vaccines to prevent SARS-CoV-2 infection, their availability is still limited in some countries worldwide, making it necessary to search for alternative therapeutic strategies to control and reduce morbidity and mortality in COVID-19 patients. Besides, there are not enough data available about the effectiveness of therapeutic strategies with the current and upcoming variants. Thus, it is necessary to search for new therapeutic approaches.Currently, several authors have suggested the potential of phytochemical compounds in the treatment against SARS-CoV-2 infection, which may prevent the onset of COVID-19 or its severity ,16. AmonAdditionally, curcumin exhibits its inhibitory activity against the replication of diverse viruses, such as dengue virus, hepatitis B virus, zika virus, influenza A virus, and chikungunya virus ,24. CurcAlthough curcumin has shown broad antiviral activity , and itsCurcumin was cytotoxic at 20 and 40 µg/mL  after 48 h of treatment on Vero E6. Contrarily, the viability of Vero E6 was higher or equal to 80% at curcumin concentrations of 10 µg/mL or lower . The CC5p = 0.0095), 51.3% (p = 0.0095), 22.2% (p = 0.0095), and 27.8% (p = 0.0095) against SARS-CoV-2 D614G strain at concentrations of 10, 5, 2.5, and 1.25 µg/mL, respectively (p = 0.0095) was observed for chloroquine treatment   were evaluated through four treatment strategies and using a MOI of 0.01. By pre–post infection treatment  curcumin exerted antiviral activity of 99.0%  .p = 0.0095 and at 5 µg/mL of 39.3%, p = 0.0095  was obtained at 25 µg/mL of heparin (p = 0.0095) was obtained by post-infection treatment with chloroquine , 5 , and 2.5 µg/mL  of curcumin (p = 0.004) was observed by co-treatment with chloroquine  (As can be seen from curcumin . An inhiibition) .p = 0.0007), 5 , 2.5 , and 1.25 µg/mL , by pre–post infection treatment . In addition, curcumin showed antiviral activity of 99.9% (p = 0.0012), 99.1% (p = 0.0012), 31.9% (p = 0.0233), and 56.5% (p = 0.0017) against Delta variant, at concentrations of 10, 5, 2.5, and 1.25 µg/mL, respectively, using a co-treatment strategy. The EC50 value calculated for curcumin was 1.66 µg/mL, with an SI of 9.94, by the co-treatment (p = 0.0007) and co-treatment   .p = 0.0022, p < 0.001, p = 0.0022, p = 0.0050) were found in PBMCs pretreated with 10 µg/mL of curcumin compared with cells stimulated only with the virus. No significant changes in the mRNA expression of TNF-α was found  quantification. Significant decreases in IL-1β mRNA  supplemented with 2% heat-inactivated fetal bovine serum (FBS) (GIBCO), 1% penicillin–streptomycin (GIBCO), and 2 mM L-glutamine (Sigma-Aldrich) at 37 °C with 5% CO2. With three passages per week. Vero E6 cells were donated by Instituto Nacional de Salud, Bogotá-Colombia . Infections were carried out with viral stocks produced from two Colombian SARS-CoV-2 isolates: D614G strain (EPI_ISL_536399) [_536399)  and DeltCurcumin was purchased from MERCK . Curcumin powder was solubilized in dimethyl sulfoxide  at 10 mg/mL. The stock was maintained at 4 °C protected from light until use. The working solution was prepared, diluting the stock at 40 µg/mL in DMEM supplemented with 2% FBS.4 cells/well in DMEM supplemented with 2% FBS. The plates were incubated for 24 h at 37 °C with 5% CO2. After incubation,100 µL/well of serial double dilutions of curcumin ranging from 1.25 to 40 µg/mL was added to cell monolayers and incubated for 48 h. Subsequently, the supernatant was removed, the cells were washed twice with phosphate buffered saline (PBS) , and 30 µL/well of MTT (2 mg/mL) was added. After the addition of MTT, the plates were incubated for 2 h at 37 °C, with 5% CO2, protected from light. Finally, 100 µL/well of DMSO was added. Plates were read at 570 nm using a MultiskanTM GO Microplate Spectrophotometer . The average absorbance of untreated cells was used as viability control. The cell viability of each treated well was calculated based on the viability control. Concentrations with cell viability after treatment of 80% or more were considered non-cytotoxic. For the MTT assay, two independent experiments with four replicates each were conducted (n = 8).The cytotoxicity of curcumin on Vero E6 cells was evaluated using an MTT assay . Briefly4 cells/well) and incubated for 24  h, at 37 °C with 5% CO2. Then, serial double dilutions of curcumin (from 1.25 to 10 µg/mL) were prepared and added to cell monolayers (50 µL/well) for 1 h. After pretreatment, the curcumin-containing medium was aspirated from cell monolayers, and the virus was added at 0.01 MOI (multiplicity of infection) in 50 µL of DMEM supplemented with 2% FBS. After 1 h of viral adsorption at 37 °C, the inoculum was removed and replaced by 150 µL/well of the same pre-treatment dilutions. Finally, the plates were incubated for 48 h at 37 °C under a 5% CO2 atmosphere.The antiviral activity of curcumin was initially evaluated using a pre–post infection treatment strategy . Briefly2. (ii) Pre-infection treatment: 150 µL/well of serial double dilutions of curcumin was added to cell monolayers for 24 h before viral infection. (iii) Post-infection treatment: 150 µL/well of serial double dilutions of curcumin was added to cell monolayers for 48 h after infection. In all cases, two independent experiments with four replicas each were performed (n = 8). Chloroquine (100 µM) [To determinate which steps of the viral cycle are being affected by curcumin treatment, the antiviral activity was evaluated using three extra strategies: (i) Co-treatment: Serial double dilutions of curcumin were mixed with SARS-CoV-2 (MOI 0.01) and incubated for 1 h at 37 °C (1:1 ratio). After incubation, 50 µL/well of the virus-treatment mixture was added to cell monolayers and incubated at 37 °C for 1 h. After 1 h of viral adsorption, the mixture (curcumin–virus) was removed and replaced by 150 µL/well of fresh medium. The plates were incubated for 48 h at 37 °C with 5% CO(100 µM)  or hepar(100 µM)  were use5 Vero E6 cells/well were seeded in 24-well plates for 24 h, at 37 °C, with 5% CO2. Subsequently, 10-fold serial dilutions of the supernatant obtained from all antiviral assays (200 µL/well) were added to cell monolayers and incubated for 1 h at 37 °C with 5% CO2. Then, the viral inoculum was removed and replaced with 1 mL of semi-solid medium (1.5% carboxymethyl-cellulose in DMEM 1× with 2% FBS and 1% Penicillin–Streptomycin). The cells were incubated for 3 d at 37 °C and then washed twice with PBS and fixed-stained with 4% Formaldehyde/1% crystal violet solution; then, the viral plaques were counted. The difference between viral titer after curcumin treatment and untreated control was expressed as an inhibition percentage. Two independent experiments with two replicates each were conducted (n = 4).The viral titer of antiviral assay supernatants was determined by plaque assay. Briefly, 1.2 × 10Approximately 20 mL of total peripheral blood from each healthy adult donor (n = 3) was obtained by venipuncture in vacutainer tubes. PBMCs were isolated using the Ficoll-Histopaque density gradient method  as previously described .6 cells/well) in RPMI1640 medium (Sigma-Aldrich) supplemented with 5% FBS. The cells were stimulated with curcumin (5 and 10 µg/mL) for 1 h. After pre-treatment, the SARS-CoV-2 was added at a MOI of 0.1 and incubated for 24 h, at 37 °C with 5% CO2. Three independent experiments with two replicates were conducted (n = 6). The cells without any treatment were used as negative controls.To evaluate the anti-inflammatory effect of curcumin, the PBMCs were seeded in 24-well plates  as previously described . BrieflyReal-time PCR was performed using Maxima SYBR Green qPCR master mix kit . The mRNA PGK (phosphoglycerate kinase) was used as the housekeeping gene to normalize the RNA content . The ampConcentrations of IL-1β, IL-6, and IL-8 were quantified in PBMCs culture supernatants using ELISA kits  and according to the manufacturer’s instructions.p value ≤ 0.05 was considered significant. The EC50 (50% effective concentration) and CC50 (50% cytotoxic concentration) values were determined by non-linear regression analysis using a sigmoidal model. The selectivity index (SI) was calculated from the CC50/EC50 ratio.All data were analyzed with GraphPad Prism . Data were presented as median ± IQR (interquartile range) or mean ± SEM (standard error of the mean), as indicated in each figure. Statistical differences were evaluated by Student′s t-test or Mann–Whitney U test based on Shapiro–Wilk normality test. A This study was approved by the ethics committee of the Universidad Cooperativa de Colombia (Acta BIO106). It was carried out keeping good records, practicing good data collection and management, transparency of data-sharing, and realistic representation of study results. The data were analyzed anonymously. All donors were adults, read, and signed an informed consent. All research protocols were made according to the principles of the Declaration of Helsinki.


Background and Objective: HEC30654 is a selective 5-HT6 receptor antagonist that was safe and well-tolerated in preclinical models of Alzheimer’s disease. The objective of this double-blind, randomized, placebo-controlled clinical trial was to evaluate the safety, tolerability, and pharmacokinetic profile of HEC30654 after single ascending doses in healthy Chinese subjects.Methods: Healthy volunteers received a single oral dose of HEC30654 . Safety and tolerability assessments included adverse events, vital signs, and findings on electrocardiograms, electroencephalograms, physical examination, and clinical laboratory tests. Pharmacokinetic analysis of HEC30654 and its major metabolite HEC93263 were conducted in blood, urine, and fecal samples.Results: Single doses of HEC30654 up to 30 mg were generally safe and well tolerated, but dose escalation was terminated early as the 60 mg HEC30654 treatment group met the pre-defined stopping rules specified in the protocol. Median tmax of HEC30654 was 6 h , t1/2 of 10–60 mg HEC30654 ranged from 52.1 to 63.8 h. Exposure to HEC30654 across the dose range explored in this study increased more than in proportion to dose. Metabolism of HEC30654 to HEC93263 was slow (<10%), and HEC30654 was mainly eliminated unchanged through feces.Conclusion: Single doses of HEC30654 up to 30 mg were generally safe and well tolerated. Based on preclinical efficacy in various models of cognition, HEC30654 may represent a therapeutic option for symptomatic treatment of cognitive disorders. Alzheimer’s disease (AD) is a chronic neurodegenerative disease that manifests as a progressive impairment of memory and cognitive function, behavioral changes, and psychiatric symptoms, including depression and psychosis. The pathogenesis of AD remains to be elucidated, but involves amyloid plaques, neurofibrillary tangles, and profound neuronal loss . Current6 receptor antagonists have emerged as potential treatment strategies in AD, with some 5-HT6 receptor antagonists, such as Lu-AE58054  and masupirdine (SUVN-502), being investigated in Phase II or Phase III clinical trials  in patients with AD  and antagonistic activity (IC50 = 15 nM) than LuAE58054 . The metabolite of HEC30654, HEC93263, is a weak 5-HT6 receptor antagonist, with an IC50 of 1.7 μM HEC30654 has weak affinity for histamine  receptors, cholinergic  receptors, and 5-HT  receptors, and a strong affinity for 5-HT2A and 5-HT2B receptors; however, HEC30654 has weak antagonistic activity against 5-HT2A and 5-HT2B receptors, with an IC50 of 0.12 and 0.99 μM, respectively. In pre-clinical evaluations, rodent behavioral testing showed HEC30654 significantly improved scopolamine-induced cognitive dysfunction with greater efficacy than LuAE58054.HEC30654 is a 5-HT1/2) of 4–8 h after intravenous administration, and bioavailability of HEC30654 after oral administration was 12.2% or 51%, respectively. In animals and humans, HEC30654 had a plasma protein binding rate of >93%. Previous studies revealed that phase 1 metabolism of HEC30654 may involve CYP3A4, CYP2D6, and CYP2B6. In rats, mean total recovery of radioactivity in urine and fecal samples after a single oral administration of 89.7 μCi/6.04 mg/kg [14C] HEC30654 was 95.0%, with urine and feces accounting for 2.46 and 91.0%, respectively.In rats and dogs (beagles), HEC30654 had a terminal half-life ; and 3) physical examination, medical history, and clinical laboratory test results showed no clinically significant abnormal findings at screening. Key exclusion criteria were: 1) long-term history of smoking or alcohol and/or drug abuse; 2) use of any medication during the 14 days prior to the initial dose of study drug or while participating in the study; 3) blood donation during the 3 months prior to the study; 4) exposure to any CYP3A4, P-gp or Bcrp inducers or inhibitors during the 3 months prior to the initial dose of study drug; or 5) participation in a clinical trial of another investigational drug during the 3 months prior to this study.Healthy Chinese volunteers were eligible to participate in this study. Key inclusion criteria were: 1) males and females (not pregnant or lactating) aged 18–45 years; 2) body mass index (BMI) 18–28 kg/mhttp://www.chinadrugtrials.org.cn/ and was conducted in accordance with the World Medical Congress Declaration of Helsinki and Good Clinical Practice guidelines. All study subjects provided written informed consent.This single-center clinical trial was conducted at the Jilin University First Affiliated Hospital-Phase I Clinical Research Center, Changchun City, China between Nov 21, 2018 and Jul 17, 2019. The clinical study protocol was reviewed and approved by the Ethics Committee at the Jilin University First Affiliated Hospital-Clinical Research Institute. The clinical trial was registered at This single ascending dose, double-blind, randomized, placebo-controlled clinical trial investigated the safety, tolerability, and PK of HEC30654 in healthy subjects. The study planned to randomize eligible healthy subjects to one of seven treatment groups. In the first treatment group, three sentinel subjects were to receive a single oral dose of 5 mg HEC30654. In the six remaining treatment groups, ten subjects were to receive a single oral dose of 10, 15, 30, 60, 90 or 120 mg HEC30654 (n = 8 per group) or matching placebo (n = 2 per group) after an overnight fast.Dose escalation was dependant on the safety and tolerability of the previous doses defined according to the National Cancer Institute Common Terminology Criteria for the Classification of Adverse Events (NCI CTCAE) v.4.03. Dose escalation was stopped based on the occurrence of the following drug-related adverse events (AEs): >50% of subjects in the previous treatment group experienced Grade ≥2 drug-related toxicities, or >25% of subjects in the previous treatment group experienced Grade 3–4 drug-related toxicities, or one drug-related serious adverse event (SAE).The trial was terminated early with subjects in five treatment groups receiving a single dose of 5, 10, 15, 30, or 60 mg HEC30654.Safety and tolerability, including AEs, vital signs , and findings on electrocardiograms, electroencephalograms, physical examination, and clinical laboratory tests  were evaluated according to NCI-CTCAE, 4.03. Incidence, severity, and relationship of AEs to study drug were recorded. Tolerability was evaluated on Day 2 and Day 6 for subjects administered 5, 10, 15, or 30 mg HEC30654, and Day 2, Day 6, and Day 10 for subjects administered 60 mg HEC30654.via an indwelling intravenous angiocatheter into K2EDTA containing tubes. For subjects administered 5, 10, 15, or 30 mg HEC30654, blood samples were collected at 0 h (pre-dose) and 0.5, 1, 1.5, 2, 4, 6, 8, 10, 12, 24, 48, 72, 96, and 120 h after dosing. For subjects administered 60 mg HEC30654, according to the t1/2 and Tmax of previous dosages, the time points were adjusted to 0 h (pre-dose) and 1, 2, 4, 6, 8, 10, 12, 24, 48, 72, 96, 120, 144, 168, 192, and 216 h after dosing. After discarding the first 0.5–1 ml of blood, samples were centrifuged at 1,800 g for 10 min at 4°C, and plasma was stored as two equal aliquots in polypropylene tubes at −80°C until analysis. For subjects administered 60 mg HEC30654, urine and fecal samples were collected during the following time intervals: 0, 0–4, 4–8, 8–12, 12–24, 24–48, 48–72, 72–96, 96–120, 120–144, 144–168, 168–192, 192–216 h (urine) and 0–216 h (feces).Blood samples (4 ml each) were collected The concentrations of HEC30654 and its metabolite HEC93263 in plasma and feces were determined using an ExionLC HPLC system  equipped with an AB 4000 QTrap MS detector . The concentrations of HEC30654 and its metabolite HEC93263 in urine were determined with a Nexera X2 HPLC system  equipped with an API 4000 MS detector . The calibration ranges of the assays for HEC30654 and HEC93263 were 0.500–500 ng/ml in plasma, 2.00–2,000 ng/ml in urine, and 5.00–5,000 ng/ml (HEC30654) and 1.00–1,000 ng/ml (HEC93263) in feces. For HEC30654 and HEC93263 in plasma, urine and feces, accuracies were 3.33–12.8%, –11.93–6.67% and –6.00–8.88%, and precision was within 6.02, 10.5 and 9.10% CV, respectively.max), time to peak plasma concentration (Tmax), area under the plasma concentration–time curve from time 0 to the last timepoint with a quantifiable concentration (AUC0-t), AUC from time 0 to infinity (AUC0-∞), t1/2, clearance (CL/F), and apparent volume of distribution (Vz/F).Standard non-compartmental methods using WinNonlin version 6.4  were used to analyze plasma PK data, including peak plasma concentration . Continuous variables were reported as number, mean with standard deviation, median, maximum, and minimum. Categorical variables, were reported as frequency and percentage. Dose proportionality was explored using a regression power model, relating log-transformed CThis study included a total of 43 healthy subjects enrolled into five treatment groups. Baseline demographic characteristics were well balanced across the treatment groups . All subTreatment-emergent AEs (TEAEs) were reported in 33.3, 50.0, 25.0, 12.5, 100, and 37.5% of subjects, and drug-related TEAEs were reported in 33.3, 37.5, 12.5, 12.5, 100, and 25% of subjects administered 5, 10, 15, 30, or 60 mg HEC30654 or placebo, respectively. No dose-dependent TEAEs were observed .Single doses of HEC30654 up to 30 mg were generally safe and well tolerated. 100% of subjects administered 60 mg HEC30654 reported TEAEs, all of which were assessed as related to study drug. TEAEs most frequently reported by subjects administered 60 mg HEC30654 included lymphocytopenia (100%), leukopenia (87.5%), elevated C-reactive protein (75.0%), neutropenia (62.5%), fever (100%), headache (75.0%), and myalgia (75.0%). 100% of subjects in the matching placebo group reported TEAEs (100%), all of which were assessed as related to study drug. TEAEs most frequently reported by subjects administered matching placebo included lymphocytopenia (100%), leukopenia (50.0%), elevated C-reactive protein (100%), neutropenia (50%), fever (100%), headache (100%), and myalgia (50%). Most of the TEAEs reported by subjects administered 60 mg HEC30654 or matching placebo were not reported by subjects in the other treatment groups. There was no clinically significant changes for vital signs, electrocardiograms and electroencephalograms throughout the study for each group.All the subjects in 60 mg group occurred grade ≥2 drug related AEs, and three of them occurred grade ≥3 drug related AEs . As the max of HEC30654 was 6 h , t1/2 of 10–60 mg HEC30654 ranged from 52.1 to 63.8 h, and there was no dose proportional increase . The CL/F of HEC30654 was 31.8–63.1 L/h with a large Vz/F 3840–6420 L, suggesting a wide distribution in the body. The AUC0-∞ and Cmax of HEC30654 increased in a dose-dependent manner, but the increases were slightly greater than dose proportional in 60 mg. Dose proportionality over the 5–60 mg HEC30654 dose range was evaluated with a power model. The slope (90%CI) for lnAUC0-∞ was 1.17 (1.07–1.28) and lnCmax was 0.92 (0.76–1.07). The 90% CI were outside the respective acceptance intervals of 0.86–1.14 and 0.91–1.09; however, as the 90%CI of the slope for lnCmax contained the value of 1, dose proportionality may be claimed for that parameter.The mean plasma concentration-time profiles of HEC30654 after administration of single escalating doses to healthy subjects under fasted conditions are shown in max of HEC93263 was 6 h and t1/2 was 30.3 h. The rate of HEC30654 metabolism to HEC93263 was low, as suggested by Cmax (8.9%) and AUC-0–∞ (3%) values.HEC93263 exposure was low. Metabolite PK parameters could only be calculated for 60 mg HEC30654 . Median HEC30654 and HEC93263 were detected in urine and fecal samples. The percentage cumulative recovery of HEC30654 in the urine and feces from 0 to 216 h was 0.998 and 21.4%, respectively. The percentage cumulative recovery of HEC93263 in the urine and feces from 0 to 216 h was low (<0.1%) .This Phase I clinical trial was designed to characterize the safety, tolerability, and PK of HEC30654 in healthy Chinese volunteers.HEC30654 was safe and well-tolerated in preclinical animal studies. The non observed adverse effect level (NOAEL) in rat and dog were 34.5 mg/kg and 2.3 mg/kg, corresponding to maximum recommended starting doses (MRSD) of 29.2 and 6.3 mg in humans, considering a safety factor of 10. The initial effective dose in rats was 1.5 mg/kg/day, and according to physiologically based PK modeling, the initial effective dose in humans was 5 mg/day. The maximal tolerated dose (MTD) in rat and dog were 690 mg/kg and 1,150 mg/kg, corresponding to MTDs of 6,624 mg and 37,260 mg in humans. Based on these findings, this study planned to randomize healthy subjects to receive a single oral dose of 5 10, 15, 30, 60, 90, or 120 mg HEC30654 or matching placebo. However, the 60 mg HEC30654 treatment group met the pre-defined stopping rules specified in the protocol; therefore, the study was terminated early.In this study, single doses of HEC30654 up to 30 mg were generally safe and well tolerated, but dose escalation was stopped at 60 mg based on the occurrence of drug-related AEs. All the subjects in 60 mg group occurred grade ≥2 drug related AEs, and three of them occurred grade ≥3 drug related AEs. TEAEs most frequently reported by subjects administered 60 mg HEC30654 included lymphocytopenia, leukopenia, elevated C-reactive protein, neutropenia, fever, headache, and myalgia. The incidence of these TEAEs in the other treatment groups was rare.The TEAEs reported by the patients administered 60 mg HEC30654 were inconsistent with the AEs observed in patients receiving other 5-HT6 receptor antagonists , findinThe TEAEs reported by patients administered 60 mg HEC30654 or matching placebo manifested as concentrated outbreaks, with similar symptoms, and self-limiting processes. Various viral antibodies were detected, but their titer suggested no clinical significance. Other subjects and staff in the same setting showed no symptoms, so the relevance of these findings is unclear.in vitro findings.After single administration, the half-life of HEC30654 ranged from 52.1 to 63.8 h making HEC30654 compatible for once-daily dosing. In comparison, in a Phase II trial, the 5-HT6 receptor antagonist LuAE58054 required thrice daily dosing . ExposurHEC30654 exhibited an acceptable safety, tolerability and pharmacokinetic profile in healthy subjects in the dose range 5–30 mg. Based on preclinical efficacy in various models of cognition, HEC30654 may represent a therapeutic option for symptomatic treatment of cognitive disorders.


Ligustrazine, an alkaloid monomer extracted from Chuanxiong Rhizoma, has the function of protecting nerve cells. However, the effect and mechanism of ligustrazine on retinal ischemia/reperfusion (I/R) injury still need to be clarified. In our study, retinal ganglion cells (RGC-5) were used to establish a retinal I/R injury model by anaerobic cultivation. Cell viability, autophagy, and apoptosis were evaluated by cell counting kit 8 assay, transmission electron microscopy, and TUNEL staining after treatment with ligustrazine, PI3K inhibitor Ly294002, and/or mTOR inhibitor rapamycin, respectively. Besides, the levels of PI3K/Akt/mTOR pathway and autophagy-related proteins were determined by western blot. Moreover, one-way ANOVA was adopted for inter-group comparisons of measurement data. Our results demonstrated that low-concentration ligustrazine significantly enhanced cell viability and suppressed cell autophagy and apoptosis of RGC-5 cells after I/R injury, suggesting the protective effect of low-concentration ligustrazine on retinal I/R injury. Moreover, the alleviating effect of ligustrazine on RGC-5 with retinal I/R injury was mechanistically associated with the activation of the PI3K/Akt/mTOR pathway. In conclusion, low-concentration ligustrazine has a significant protective effect on RGC-5 cells with retinal I/R injury by activating the PI3K/Akt/mTOR pathway.  At presLigusticum chuanqiong Hort, as a type of traditional Chinese medicine, has effects such as activating blood circulation, expelling congestion, and relieving pain , ligustrazine group , and ligustrazine+mTOR inhibitor group [10 μg/mL ligustrazine and 10 μM rapamycin ]. In the normal (control) group, only an equivalent volume of DMSO was added to RGC-5 cells without hypoxia treatment. In addition to the RGC-5 cells in the normal group, the complete medium was replaced with low-glucose DMEM medium. RGC-5 cells were treated with ligustrazine and DMSO for 30 min, and the experiments were repeated five times.2.4.2 humidified incubator. After these experimental treatments for 24 h, 10 μL of CCK-8 reagent  was added to each well, and the cells were cultured for 2 h in the incubator. Absorbance  was measured at 450 nm using a microplate reader , following which cell viability was calculated.RGC-5 cells in the logarithmic phase of growth  were seeded in a 96-well plate and incubated for 12 h at 37°C in a 5% CO2.5.RGC-5 cells in each group were collected and fixed with 2.5% glutaraldehyde at 4°C for 2 h. After rinsing with phosphate-buffered saline (PBS) three times, the cells were fixed with 1% osmic acid at 4°C for 2 h. The cells were then dehydrated with a continuous gradient of ethanol and embedded in Embed-812 medium for 48 h. Ultrathin sections (60–80 nm) were prepared and stained with 2% uranium acetate and lead citrate for 15 min. Sections were then dried overnight at room temperature. Autophagosomes were also imaged using TEM.2.6.Cells in each group were fixed with 3% paraformaldehyde  at 4°C for 40 min according to the manufacturer’s instructions. After washing with PBS three times, 0.1% TritonX-100 and 0.1% sodium citrate solutions were added to penetrate at 4°C for 5 min. Cells were then washed again and treated with fluorescein-labeled nucleotide (dUTP) and TdT at 37°C for 60 min. Green fluorescent TUNEL-positive cells were analyzed using a fluorescence microscope.2.7.g for 15 min at 4°C. After the protein concentration was determined by the Pierce™ BCA Protein Assay Kit , total protein (30 μg) was separated by SDS-PAGE and transferred to a PVDF membrane (Millipore). The membranes were blocked with 5% skim milk powder at room temperature for 2 h and incubated with primary antibodies at 4°C overnight and secondary antibody  at room temperature for 2 h. Enhanced chemiluminescent reagent was used to develop images. The following primary antibodies were used: p-AKT , AKT , p-PI3K , PI3K , p-mTOR , mTOR , LC3 , Beclin-1 , SQSTM1/p62 , and GAPDH .After RGC-5 cells in each group were collected, they were rinsed with pre-cooled PBS three times and lysed with RIPA buffer  containing a protease inhibitor on ice for 1 h. The supernatant was centrifuged at 12,000 × 2.8.P-values of < 0.05 were considered to be statistically significant.Statistical analysis was performed using SPSS 18.0. All data were expressed as mean ± standard deviation. One-way ANOVA was performed for inter-group comparisons of measurement data. 3.3.1.p< 0.01, The retinal I/R injury is a frequent cause of blindness, and it mainly occurs in ischemic ophthalmopathy, particularly in glaucoma with intraocular hypertension . Because3.2.p< 0.01, The PI3K/Akt pathway is a signal transduction pathway involved in the regulation of cell growth, proliferation, and differentiation ,29. Akt 3.3.p< 0.05). Therefore, we verified that the retinal I/R injury prominently accelerates RGC autophagy, which is reversed by the low concentrations of ligustrazine. Hence, the low concentration of ligustrazine protects RGCs from retinal I/R injury by blocking autophagy.Previous studies have suggested that autophagy is closely related to the pathophysiology of glaucoma ,23,40. FApoptosis is one of the major mechanisms underlying the death of RGCs with retinal I/R injury and a common pathway of RGC degeneration . ApoptosIn short, ligustrazine positively regulates the PI3K/Akt/mTOR pathway in RGCs with retinal I/R injury. Ligustrazine reduces autophagy and apoptosis in RGCs. Vice versa, the effect of ligustrazine on PI3K/Akt/mTOR is inhibited when RGCs are treated with Ly294002 and rapamycin.3.4.p< 0.05, p< 0.01, p< 0.05, p< 0.01, Next, we confirmed whether the PI3K/Akt/mTOR pathway plays a role in the protective effects of ligustrazine on I/R-injured RGCs. Our results certified that the expression levels of p-PI3K, p-Akt, and p-mTOR were dramatically reduced in RGCs after retinal I/R injury compared with that in normal RGCs, while the increase of p-PI3K, p-Akt, and p-mTOR expressions could be attenuated by low-concentration ligustrazine in RGCs after I/R injury; moreover, we uncovered that the upregulation of p-PI3K, p-Akt, and p-mTOR expressions that is mediated by ligustrazine could be prominently reversed by Ly294002 or rapamycin  or mTOR inhibitor (rapamycin) could reverse the enhancement of viability and suppression of autophagy and apoptosis of RGCs after retinal I/R injury, which were mediated by low-concentration ligustrazine, thereby uncovering a novel mechanism by which low-concentration ligustrazine protects the retina against I/R injury by activating the PI3K/Akt/mTOR pathway. Therefore, we suggested that appropriate ligustrazine might serve as a crucial protective agent for retinal I/R injury.Click here for additional data file.