Background: Thiazole is a good pharmacophore nucleus due to its various pharmaceutical applications. Its derivatives have a wide range of biological activities such as antioxidant, analgesic, and antimicrobial including antibacterial, antifungal, antimalarial, anticancer, antiallergic, antihypertensive, anti-inflammatory, and antipsychotic. Indeed, the thiazole scaffold is contained in more than 18 FDA-approved drugs as well as in numerous experimental drugs. Objective: To summarize recent literature on the biological activities of thiazole ring-containing compounds Methods: A literature survey regarding the topics from the year 2015 up to now was carried out. Older publications were not included, since they were previously analyzed in available peer reviews. Results: Nearly 124 research articles were found, critically analyzed, and arranged regarding the synthesis and biological activities of thiazoles derivatives in the last 5 years. One of the main objectives of organic and medicinal chemistry is the design, synthesis, and development of molecules having therapeutic potential as human drugs. During the past decade, combinatorial chemistry has provided access to chemical libraries based on privileged structures , with he® (Pseudomonas aeruginosa (P.aeruginosa) and used to treat complicated urinary tract infections in case when no other treatment is available  acetamide derivatives (1a–1p)  . Compoun1a, 1b, 1e, and 1f were found to be equipotent with ketoconazole (MIC 50 μg/mL) against Candida albicans , while compound 1d was found to be equipotent against Candida glabrata (C. glabrata). Furthermore, compounds 1a and 1b were also equipotent with the reference drug against Candida krusei (C. krusei) and Candida parapsilosis (C. parapsilosis), respectively. The antifungal activity of the compounds was more pronounced than their antimicrobial activity. Compounds 2a–d, 3, 4, 5a–5c) , one Gram-negative (E. coli) bacteria and two fungal species  by the tube dilution method -4H-chromen-(9), 2-bipyrazolyl-5-yl]-phenol (10), 2-[3--1-(4-fluorophenyl)-1H-pyrazol-4-ylmethylene]-benzofuran-3-one (11)  12-18 mm compared to the reference drug (IZ of 35–46 mm). Compounds 10a–b and 11 showed the highest activity among compounds tested against Bacilus subtilis (B.subtilis), . Compound 11 was also active against Staphylococcus aureus (S. aureus) (17 mm). Karale et al.  reportedone (11) , and eva12a–j) (12a–j) were tested for their in vitro antibacterial activity against both Gram-positive and Gram-negative bacteria such as Streptococcus pyogenes (S. pyogenes), S. aureus, P. aeruginosa, and Klebsiella pneumonia (K. pneumoniae) by the agar well diffusion method thiazol-3-yl]acetohydrazides  and IC50 1.05 μg/mL.Guzeldemirci et al.  synthesis 55a–I,  with the56a–j, M. tuberculosis H37Rv (ATCC 27294). All compounds were evaluated in BACTEC 12B medium using a broth microdilution assay -pyrrole-2,5-dione (62a) showed a substantial activity with MIC values of 6.25 μg/mL, being twofold less active than pyrazinamide but much less potent than the other reference drugs. Lower activity was found for N-[4-(2-amino-thiazol-4-yl)-phenyl]-benzamide and 1-[4-(2-amino-thiazol-4-yl)-phenyl]-pyrrole-2,5-dione derivatives with 4-fluoro substitutions on the benzene ring  with MIC at 12.5 μg/mL. The rest of the compounds showed only a low activity.Cordeiro et al.  reportedide 62c, , N- with an IC50 value of 43 μM. None of compounds was found to be significantly cytotoxic against Jurkat cells -1,3-thiazole (76b) with the aim to investigate in vivo their antitumor activity  in the negative control group, which decreased in the positive control group by 70.57%, while the mean values of caspase-3 activities for both therapeutic and preventive groups treatments with compounds 77a, 77b, and 78 , which was used as the reference drug. Compound 77b was further subjected to cell cycle analysis using DNA flow cytometric assay thiopyranothiazole-5-carboxylic acids. Thus, compounds 93 and 94  with EC50 values of 0.6 ÷ 2.5 μg/mL and SI = 40.0 ÷ >170.0, and against influenza virus type A  with EC50 of 0.31 ÷ 0.32 μg/mL and SI = >310.0 ÷ >320.0 respectively. In general, most of the compounds from this group showed activity against influenza virus.Lesyk et al.  synthesi3 and 94  were the95a–j, 95j was found to be active against Feline coronavirus with EC50 of 7.5 μΜ, while 95c was active against HSV-1(KOS) virus with EC50 of 9 μΜ compared to ribavirin (HCV EC50 of 2 μΜ).Güzeldemirci et al.  presenteb] thiazole-based compounds (96a–k) as potential inhibitors of influenza virus in vitro (96i–k), exhibited activity against influenza virus with an EC50 in the range of 13 ± 3 to 49 ± 6 μΜ, which is better than rimantadine (EC50 of 72 ± 8 μΜ). The highest activity was achieved for compound 97j with EC50 of 13 ± 3 μΜ and SI 77. Despite compounds 96i–k having a favorable toxicity profile (CC50 > 200 μΜ) compared to another reference drug, oseltamivir carboxylate (EC50 of 0.2 ± 0.0 and SI > 1000), they were much less potent.Galochkina et al.  describein vitro . Compoun96i–k were tested also for their ability to inhibit influenza virus neuraminidase in luminescent MUNANA-based assay  thiazole and evaluated their antiviral activity . Nevertheless, it was twice less active than UDA (EC50 2.4 μM), the second reference drug. On the other hand, two derivatives with acyl-hydrazone moiety 97c and 97d displayed weak activity against DNA viruses  in HeLa cells with an EC value of 45 μM. From these results, it is obvious that the presence of spirothiazolidinone moiety with -4hydroxyphenyl substituent at the 8-position is beneficial for the antiviral activity against Feline coronavirus, while the presence of acyl-hydrazone moiety showed a positive effect against DNA viruses.Gürsoy et al.  synthesiactivity . The stu98d is another hit compound, exhibiting activity against Coxsackie B4 virus with an EC50 value of 10 μM, being much more active than both reference drugs DS-5000 (EC50 of 84 μM) and Ribavirin (EC50 > 250 μM).Compound 99 and 100 are generally able to inhibit both RDDP and RNase H functions at µM concentrations and that these molecules are a good starting point for the design of new dual HIV-1 RT inhibitors.Meleddu et al.  synthesi101a and 101b , while three of the compounds (102a ≥ 102b > 102c) exhibited IC50 values lower than 5 nM. Two compounds 102a and 102b exhibited very good inhibitory activity with an IC50 1 nM). Moreover, the activity of compounds seemed to depend not only on the nature of substituent and its position in benzothiazole ring but also on the nature and position of substituents in the benzene ring.Petrou et al.  took int102a–g  had lowe103  is an important target for the therapy of type-II diabetes. It modulates the signal pathway of insulin and leptin in a negative way, increasing the phosphorylation of insulin receptor and its substrate, glucose uptake in insulin-responsive cells and endorsing insulin action in receptive tissues. Based on the variety in the mechanisms of PTP1B inhibitors, an enormous number of novel molecules has been designed and synthesized in the past 6 years, highlighting the importance of PTP1B in the treatment of type-II diabetes.104, 105, 104c, 104e, and 105f with IC50 values of 1.6, 1.5, and 1.9 μΜ, respectively. The SAR studies revealed that the presence of ethoxy chain between the two phenyl rings was beneficial, leading to excellent inhibitors independent of the position of this group. In the most active compound among 2-(4-fluorophenylimino)-4-thiazolidinon 104e, 2-phenylethoxy substituent is in position 4 of the 5-benzylidene ring, whereas in the most effective inhibitor of series 2-thioxo-4-thiazolidinones (compound 105f)), this group is in position 3. Kinetic studies revealed that the behavior of compounds 104 is mixed non-competitive, while compound 105 showed mixed type inhibition. According to docking studies in the active side, the [4-(2-phenylethoxy)phenyl]methylidene substituted compounds  interact with the catalytic cavity in a way similar to many published PTP1B inhibitors  thiazole derivatives for their inhibitory effects on AR. The results showed that all compounds were promising AR inhibitors with the Ki values in the range of 0.018 ± 0.005 to 3.746 ± 1.321 μM compared to the reference drug quercetin (Ki = 7.025 ± 1.780 μM). 4-(4-Cyanophenyl)-2[2- methylene) hydrazinyl] thiazole thio]-N-(thiazol/benzothiazol-2-yl)acetamides and evaluated them for their inhibitory activities on AR. Compounds 119a and 119b . These two compounds displayed competitive AR inhibition and were found to be nontoxic against healthy cells. In addition, molecular docking studies were in agreement with the biological data.In continuation of their work, Sever et al.  synthesiand 119b . were idNeglected tropical diseases (NTD) are the most widespread infections of the poor people in developing regions of sub-Saharan Africa, Asia, and tropical regions of America. Among these diseases is malaria, which is a female mosquito-borne infectious disease that affects humans and animals, being the most lethal human parasitic infection .Leishmania, which is transmitted to the mammalian hosts by the bite of the phlebotomies and fly. Leishmaniasis is a significant health problem and causes morbidity and mortality in many regions of the world thiazole derivatives, the presence of the 3-cyano group (149) was more favorable than the presence of the COOEt moiety (149). For the thiazolothienopyrimidine derivatives, a positive effect was observed with the presence of the hydroxyl group (150), while for arylhydrazone derivatives, the unsubstituted derivatives showed the best activity. Compounds 148, 150, 154b, and 154c demonstrated also high antiulcer activity.Mohareb et al.  reportedand 154c  were fou155a–n, 155f and 155g showed 81.14% and 78.8% inhibition, respectively, which was higher than indomethacin and celecoxib with 76.1% and 77.67%, respectively, after 5 h. On the other hand, compounds 155b (72.22%), 155d (68.12%), 155e (68.02%), and 155a (64.00%) exhibited activity comparable to indomethacin. The best activity against COX-2 was achieved for compound 155f with 68.3% inhibition, which was higher than indomethacin (66.23%) but less than celecoxib (72.96%). Compounds 155b, 155d, and 155f with selectivity indexes of 14.06, 16.32, and 29.00 can be considered as selective COX-2 inhibitors toward indomethacin. Among all compounds tested ex vivo against TNF-α, after docking studies on this target, the best suppression was shown by compounds 155f (70.20%) and 155g (68.43%), which was higher than that of indomethacin, which suppresses TNF-α by 66.45%. Compounds 155d, 155b, and 155c showed remarkable decreases in TNF-α concentration of 61.92–59.60%. Additionally, compounds 155f, 155g, and 155b showed the best anti-inflammatory activity and did not show any gastric ulceration, which was probably due to their COX-2 selectivity.Alam et al.  synthesi156 . These compounds were found to be more active and selective toward COX-1 than COX-2 (156a and 156b bearing the free carboxylic group to be the most potent with the number of writhes between 10.67 and 14.40 compared to diclophenac (number of writhes 24).Abdelazeem et al. , based o156  to studyan COX-2 . The ana156a and 156b showed moderate activity with edema inhibition of 53.99% and 50.30%, respectively. As far as anti-ulcer activity is concerned, the best activity was achieved for compounds 156a and 156b with ulcer indexes of 9.39 and 8.5, respectively. The molecular docking studies revealed similarity in docked poses of compound 156b and mofezolac, explaining the significant activity and selectivity of compound 156b.The anti-inflammatory activity of synthesized compounds was tested using carrageenan-induced mouse paw edema assay with diclophenac as the reference drug ,227. In 157a–m (Taking into account that it is approved by the US Food and Drug Administration (FDA), celecoxib still keeps some degree of COX-1 inhibition, being least specific among other coxibs showing a higher percentage of COX-1 inhibition than other coxibs . Oniga e157a–m  and eval50 value of 26.88 μΜ was achieved for 157c with a selectivity index (SI) of 9.24 near to that of meloxicam (SI 11.03) used as a reference, while 157a, 157e, 157f, and 157d also expressed good COX-1 inhibition. On the other hand, compound 157a with an IC50 of 23.26 μΜ was the best COX-2 inhibitor was followed by compounds 157b–d with an IC50 < 30 μΜ. The structure–activity relationships study indicated the importance of the substituents at position 4 of phenyl ring for selectivity for COX-2 without changing the affinity.The in vitro experiment revealed that the inhibitory activity of tested compounds varied. The best IC157b fitted well to the binding site of COX-2 enzyme, occupying the similar region with meloxicam. Thus, the NO2 group has a hydrogen bonding interaction with Met522 and Trp387 in the active site.The molecular docking studies showed that compound 158j, 158e, 158h, and 158c (158j (86%). Compounds 158f, 158g, and 158b displayed good anti-inflammatory activity, while the rest showed moderate inhibition.Sirsat et al.  synthesiand 158c  exhibiteKerdawi et al.  reported159a and 159b  and 159b (IC50 7.50 nΜ and 16.02 nM) were the most active COX-1/COX-2 inhibitors compared to celecoxib (IC50 15.00 μΜ and 40.00 nM) and naproxen (IC50 8.70 μΜ and 520.0 nΜ) with compound 159a being more than 10 times more active than celecoxib and 115 times more potent than naproxen.The evaluation of anti-inflammatory activity was performed by carrageenan-induced mouse paw edema ,236 withand 159b  showed t160g exhibited the best anti-inflammatory activity with 87.97% inhibition and IC50 46.29 μg/mL, as compared to reference drug  or 4-hydroxy (160f) were less potent with IC50 values of 76.12 and 62.74 μg/mL, respectively. Compounds with heterocyclic substituents  showed higher anti-inflammatory activity than compounds with aromatic substituents phenyl)imidazothiazol-5-yl)methanamine (161a) with IC50 0.08 μΜ. It was observed that large alkylamino substituents such as diethylamino (161b) and dipropylamino (161c) were not favorable for the activity, decreasing it as well as selectivity. Among derivatives with cyclic amines (161d–f), which in general showed moderate activity, the more potent and selective appeared to be morpholino derivative (161f) with IC50 0.10 μΜ and SI 551.The most potent inhibitor among all compounds tested proved to be 50 in the range of 0.07–0.99 μΜ. Among them, the best activity was achieved for compound 162b (50 of 0.07 μΜ and selectivity index (SI) of 115.4, which was higher than that of etoricoxib, 91.28, which is used as a reference drug. The next compound with good activity (IC50 0.08 μΜ) was 163a with SI 90. The good activity of compounds 163b and 163a . The rest of the compounds exhibited moderate activity.Jakob et al. , taking und 162b  with an and 163a  probably163b and 163c with IC50 values of 0.15 and 0.16 μΜ, respectively being equipotent to zileuton, which was used as the reference drug. Good activity was found also for compound 162b (IC50 0.29 μΜ). The rest of the compounds showed moderate LOX inhibition. Finally, compound 162b was tested in vivo for anti-inflammatory activity by carrageenan-induced mouse paw edema thiazoles. The results of the DPPH assay revealed that most of the compounds have moderate to good antioxidant activity compared to L-ascorbic acid, which was used as a reference drug. Compounds 168c, 168e–h, 168k, 168l, and 168p methylene)-hydrazinyl)-thiazole derivatives (169a > 169c > 169b > 169e > 169d > 1169f. Compounds 169a (50 of 5.377 μg/mL) and 178c (IC50 of 9.131 μg/mL) displayed higher antioxidant activity than Trolox standard (IC50 = 9.74 μg/mL) but worse than ascorbic acid (IC50 of 2.46 μg/mL). The antioxidant activity of the compounds 169a–f was evaluated by EPR spectroscopy, too. In the mixture with the tested compounds, the integral density of DPPH radicals is reduced compared to free DPPH solution (I = 252) and displays the oxido-reduction rate. The order of reactivity is identical to the one found by the DPPH spectrophotometric method. The reducing capacity of compounds 169a–f has been measured as ferric-reducing antioxidant power (FRAP). The reduction of Fe3+ ion from tripyridyltriazine Fe (TPTZ)3+ complex to Fe2+ ion in the blue-colored Fe (TPTZ)2+ complex is affected by the electron-donating capacity of the tested compounds 169a–f and was quantified by the spectrophotometric method. The reducing ability of the compounds can be presented as follows: 169a > 169c > 169e > 169d > 169f > 169b. Taking into account the results of all the three methods for the antioxidant activity, compound 169a is the most active of the series.In 2017, Grozav et al. , in contivatives  and eval170a and 171  exhibited very good antioxidant activity, which was almost identical with vitamin C (62.32%), while compound 170b demonstrated the lowest activity (23.51%).As a continuation of their previous work, Djukic et al.  evaluate and 171  were fou175 was found to be the best scavenger. In addition, compounds 173a, 173b, and 174a–b  are important enzymes  that catMany different compounds such as nitro , uracil 180a (180a (Ki = 0.07 nM) compared to acetazolamide (AAZ), which was used as a reference drug (Ki = 12 nM). Compounds 180e and 180c exhibited good activity against CA IX isoform  better than AAZ (Ki = 25 nM), but none of them were selective toward the enzyme. All tested compounds showed inhibitory activity in the nanomolar range against CA XII isoform with KI values ranging from 0.46 to 29.5 nM. Three compounds, 180b, 180d, and 180a, were found to be more potent  than AAZ (Ki = 5.7 nM). The binding mode in a way proves the importance of substitution at position e and 4 of the thiazolidinone moiety because it allows better accommodation in the binding pocket. The nature of the substituent at position 4 of the dihydrothiazole ring was found to play an important role for determining the activity and selectivity of these compounds.Keeping all these in mind and based on the results of Sechi , Maccion180a  showed g50 range of 26.0–31.5 nM and Ki ranging from 27.07 to 37.80 nM. The best activity was expressed by compound 181a  than that of compound 181a. Nevertheless, both compounds were more potent than the reference drug acetazolamine (AZA) with IC50 and Ki values of 40.46 and 34.5 ± 0.02 nM, respectively. Synthesized compounds were also found to be inhibitors of hCA II isoform with IC50 in the range of 21.0–24.75 nM and Ki of 11.80–25.81 nM. It is obvious that the inhibitory activity of compounds against the hCA II isoform is better than that against hCA I. Compound 181b demonstrated the highest activity with IC50 at 20.38 nM and Ki at 11.8 ± 1.78 nM, which was better than that of AZA (IC50 = 24.16 nM and Ki = 28.93 ± 0.05 nM). The second-best compound according to the constant of inhibition was found to be 181d (Ki = 18.96 ± 4.26). From the obtained results, it seems that these compounds can be lead compounds for the development of more active CA inhibitors.Kocyigit et al.  synthesiund 181a  with IC5ectively . Compoun182a–l, 50 in range of 0.35–1.34 μΜ against hCA I and 0.35–1.56 μΜ against hCA II. 4-Nitro derivative 182a exhibited the best activity among all compounds tested against both isoforms (IC50 of 0.35 μΜ), followed by 182b (4-F-derivative) with an IC50 of 0.37 μΜ, indicating the positive role of these groups for CA inhibitory activity. While 4-nitro derivative was the most potent, 2-nitro (182c) was the less potent, which was probably due to sterical impairment for the binding of the compounds to the Zn(II) ion in the enzyme active site. It should be mentioned that the compounds are non-competitive inhibitors with Ki values for hCA I (Ki = 0.33–1.21 μΜ) being close to the clinically used sulfonamide AZA , whereas Ki = 0.11–1.99 μM for hCA II were higher than AZA (Ki = 0.012 μM).Kılıcaslan et al.  reported183b (183a (Ki of 18.25 nM) compared to AZA (Ki of 12 nM). Although all compounds demonstrated quite a good inhibitory profile against both CA isoforms, their selectivity was low.Arslan et al.  synthesi183b  was foun184a–d  (184c (Ki = 0.8 nM), while for the hCA IX isoform, the inhibition constant ranged from 3.6 to 1000 nM. The best activity against this isoform was exhibited by compounds 184a and 184d with Ki values of 3.7 and 10 nM respectively better than that of both reference drugs AZA (Ki = 25.8 nM) and EZA (Ki = 34.2 nM). Thus, the most active compounds against four isoforms were compounds 184a–d, which can be a starting point for the further development of more effective agents.Abdoli et al.  reported184a–d  and evalse assay . All com12.1 nM) . As far 185–188a–c (185b and 186b were the most potent inhibitors of CA I isoform with dissociation constants (Kd) of 25 and 16.7 nM and intrinsing dissociation constants (Kdint) of 0.03 and 0.06 nM, respectively. Kd and Kdint of AZA were 1800 and 810 nM, respectively. Additionally, compound 185b was the most potent against CA II isoform (Kd = 71.4 nM) but less active than AZA (Kd = 46.98 nM). None of the compounds tested showed good activity against CA III, CA VA, CA VB, CA VI, and CA XII isoforms, while compounds 188b (Kd = 200 nM) and 188c (Kd = 417 nM) were among the most active ones against CA IV isoform compared to AZA (Kd 87 nM). Compound 186c showed the highest activity (Kd = 33.3 nM) against CAV isoform compared with AZA (Kd = 840 nM), while compound 186b was the most active among all tested compounds against CAVII isoform (Kd = 154 nm) but less active than AZA (Kd = 13 nM). The highest activity against CA IX isoform was shown by compounds 185a and 185c with Kd of 66.7 nM, which does not exceed the activity of AZA (Kd = 13 nM). Compound 186c exhibited excellent activity against CA X III isoform (Kd = 33.3 nM), which was almost 4-fold higher than that of AZA (Kd = 120 nM), whereas compound 185b was the best (Kd = 40 nM) against CA XIV isoform, which was better than AZA (Kd = 63 nM). The general observation of this study is that the para-substituted benzenesulfonamides (compounds 185–186(a–c)) exhibited higher binding affinity than their meta-substituted benzenesulfonamide analogs (compounds 187–188(a–c).Capkauskaite et al.  designed5–188a–c  and eval189a–d (189a) 5.3 nM < 4-Cl 13.1 nM < 4-Br 45.8 nM. It is interesting to mention that the unsubstituted derivative 189d exhibited the same activity with 4-F derivative 189a. In case of hCA IX isoform, the highest activity was achieved for compounds 189b (Ki = 23.3 nM) and 189c (Ki =25.4 nM), which demonstrated activity lower or equal with reference drug (Ki = 25 nM). In this instance, the role of halogen was the opposite. The larger the atomic radius, the larger the activity (4Br > 4-Cl > 4-F). Compounds 189b and 189c were also found to exhibit very good activity with KI values of 3.1 and 4.6 nM against hCA XII isoform being more potent than AAZ (Ki = 5.7 nM). In case of hCA XII, the presence of dihalogens was beneficial, following the order 2,4-di-F > 2.4-di-Cl. Finally, the introduction of 4-Me and 4-OMe substituents at the position 4 of the main core seems to be beneficial for inhibitory activity against hCA II, IX, and XII isoforms.Based on their previous observations ,280,281,189a–d  showed g190a–f, 2 hydrase assay -N-(2-trifluoromethylphenyl) acetamide), and 197c -thiazolidin-3-yl)acetic acid ethyl ester)   exhibite198  exhibiting MIC in range of 0.7–2.8 μg/mL comparable with vancomycin -2-(1-(2-(4-(hex-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)ethylidene)hydrazinecarboximidamid -4-methylthiazol-5-yl)pyrimidin-2-amine compound with 2-(4-butylphenyl)-5-(2-hydrazinylpyrimidin-4-yl)-4-methylthiazole (18) (MIC 0.4 μg/mL) and its analogue 19 (MIC 0.78 μg/mL) were found to be more potent than vancomycin.Hydrazide and 37b) was superior to ampicillin (MIC 0.03 µg mL−1) against S. pneumoniae. On the other hand, functionalized 2,3-dihydrothiazoles tagged with sulfisoxazole moiety (15a–e) demonstrated very good, better than amphotericin B  against A. flavus) and A. fumigatus with MIC being 0.4 μg/mLThe thiazolyl-hydrazothiazole (27e) displayed better activity (MIC at 7.81 μg/mL) compared to fluconazole (MIC 15.62 μg/mL) against Candida albicans ATCC 10,231 and Candida krusei ATCC 6258.2-Hydroxy-5-(2-(4-(2-(phenylamino) thiazol-4-yl)phenyl)thiazol-4-yl) benzamide ((E)-2-(2-(Cyclohex-3-en-1-ylmethylene) hydrazinyl)thiazole derivatives showed very good activity with MIC 0.015–3.91 μg/mL against C.species being equipotent with nystatin.As far as antitubercular activity is concerned, thiazole derivatives in general showed moderate activity. No one derivative exceeded the activity of reference drugs.H-pyranothiazol-5-amine showed excellent activity, which was 35 times higher than the reference drug against HA22T, while 2--4-oxothiazol-5(4H)-ylidene)-2-phenylhydrazin-2-ium-1-ide (67a) demonstrated the best activity against the NUGC cell line with an IC50 value of 23 ± 80 nmol/L higher than the reference drug CHS-828 (IC50 of 25 ± 10 nmol/L). Arylazothiazole derivative 72c (IC50 of 4.9 ± 0.5 μM) against hepatocellular (HepG2) carcinoma cell was more potent than cisplatin (IC50 = 6.9 ± 0.7 μM). (Z)-4--2-((E)-(4-(trifluoromethyl)benzylidene)hydrazono)-2,3-dihydrothiazole (74d) displayed the best activity against four tumor cell lines .Thiazole derivatives displayed different activity against different cancer cell lines. Thus, 2--7-imino-6,7-dihydro-591a demonstrated good activity against HCV virus of EC50 0.56 μM, while 2H,5H-chromeno thiopyranothiazole-5-carboxylic acids  were found to be the most active against influenza virus type A , exhibiting high activity with EC50 = 0.6 ÷ 2.5 μg/mL and SI = 40.0 ÷ > 170.0. Spirothiazolidinone derivatives of imidazo thiazole (98d) exhibited very good activity against Feline corona virus in CRFK cells, with an EC50 value of 4.8 μM and SI more than 20, being much more potent than ganciclovir. Pyrazolo  thiazole derivatives (101a and 101b) were the most potent inhibitors of HIV-1 replication against HIV-1 IIIB with EC50 = 0.74 and 1.08 μg/mL respectively while benzothiazole-based thiazolidin-4-ones (102a–102c) displayed excellent activity against HI1RT with IC50 value lower than 5 nm.Among antiviral thiazole derivatives, bis-thiazole derivative 104c, 104e, and 105f) with IC50 values of 1.6, 1.5, and 1.9 μΜ appeared to be good PTP1b inhibitors as well as 4-oxo-2-phenylthiazolidin-3-yl)methyl) benzoate (109e) with IC50 = 0.92 μΜ. On the other hand, thiazole-based quinoline salts (113) was found to be a good DPP4 inhibitor with an IC50 value of 0.38 nm, while coumarin–thiazoles (114a–114c) as well as 1,3-diarylpyrazol-rhodanine-3-hippuric acid derivatives were good inhibitors of ALR2 with IC50 values in range of 0.11–0.12 μΜ and 0.60 nM, respectively.The derivatives of {[5-arylidene-2-(4-fluorophenylimino)-4-oxothiazolidin-3-yl]methyl}benzoic acids and 2-thioxo-4-thiazolidinone analogues (phenyl)methyl)thiazol-2-amine (125a) could be useful in the treatment of cutaneous leishmaniasis, while the SI of compound 127b was found to be 7-fold higher than the minimum recognized for a promising drug against trypanosomatid parasites, considering this compound as a promising antileishmanial agent.Among compounds with antimalarial activity, the derivative of nocathiacin 129c–e with IC50 values of 12, 9.9, and 9.7 nM respectively and SI 10200, 9960, and 11700 respectively appeared to be good agents against trypanosomiasis being more potent than reference drugs pentamidine and Melarsoprol (IC50 2.8 and 4.0 μM). However, they lack in vitro results. 2,4-Disubstituted arylthiazole derivative 134α showed higher selectivity, indicating a promising perspective for the design of novel trypanocidals, whereas among isothiochromeno thieno  pyrimidine derivative 150 exhibited 96% inhibition of edema. On the other hand, benzimidazothiazole derivative 159a (IC50 4.040 nΜ and 4.520 nM respectively) was the most active COX-1/COX-2 inhibitor, being more than 10 times higher than celecoxib and 115 times more potent than naproxen.Among compounds with anti-inflammatory activity, pyrazolylthiazolecarboxylates 170a and 171 exhibited very good antioxidant activity  according to TBARS assay, while purine–pyrazole hybrids incorporating thiazole, thiazolidinone, or rhodanine moiety compounds 173a, 173b, and 174a–b exhibited good DPPH free radical scavenging potential (IC50 from 4.89 to 14.43 μg/mL) higher than ascorbic acid with an IC50 of 15.34 μg/mLIt was observed that thiazolidinone derivatives of 1,3-thiazole 180a inhibited hCA II isoform in the nanomolar range (Ki = 0.07 nM) compared to AZZ (Ki = 12 nM). On the other hand, benzo[d]thiazole-5- and 6-sulfonamide 184c inhibited hCA VII with Ki = 0.8 nM, while compounds 189b and 189c were very good inhibitors against hCA XII isoform with KI values of 3.1 and 4.6 nM against hCA XII isoform being more potent than AAZ (Ki = 5.7 nM).It should be mentioned that among thiazole derivatives with CA inhibitory activity, 4-[ amino]benzene-1-sulfonamide derivative 197b -thiazolidin-3-yl]-N-(2-trifluoromethylphenyl) acetamide) 197b and 197c -thiazolidin-3-yl)acetic acid ethyl ester)   demonstrThe general observation is that hybrid derivatives of thiazole with different heterocyclic rings such as piperazine, pyridine, thiophen, imidazole, triazine, coumarin etc. as well as hydrazonyl thiazole derivatives are responsible for activities discussed in this review, while for PTP1b inhibition, thiazolidinone and heteroarylidene 1,3-thiazolidin-4—one derivatives are promising scaffolds.


Mycobacterium tuberculosis. BlaC was the major inactivator of the cephalosporin subclass of β-lactams. However, the PBPs PonA1 and PonA2 inactivated penicillins and carbapenems more effectively than BlaC. These findings demonstrate that select M. tuberculosis PBPs are effective at inactivating several β-lactams. Lesser-known PBPs, DacB, DacB1, DacB2, and Rv2864c, a putative PBP, were comparably more resistant to inhibition by all β-lactam subclasses. Additionally, Rv1730c exhibited low affinity to most β-lactams. Based on these findings, we conclude that in M. tuberculosis, BlaC is not the only source of inactivation of β-lactams. Therefore, the historical paradigm does not accurately describe the relationship between β-lactams and M. tuberculosis.Penicillin binding proteins (PBPs) have been extensively studied due to their importance to the physiology of bacterial cell wall peptidoglycan and as targets of the most widely used class of antibiotics, the β-lactams. The existing paradigm asserts that PBPs catalyze the final step of peptidoglycan biosynthesis, and β-lactams inhibit their activities. According to this paradigm, a distinct enzyme class, β-lactamases, exists to inactivate β-lactams. This paradigm has been the basis for how bacterial diseases are treated with β-lactams. We tested whether this historical view accurately reflects the relationship between β-lactams and the PBPs and the β-lactamase, BlaC, of IMPORTANCEM. tuberculosis, the causative agent of tuberculosis, kills more humans than any other bacterium. β-lactams are the most widely used class of antibiotics to treat bacterial infections. Unlike in the historical model that describes the relationship between β-lactams and M. tuberculosis, we find that M. tuberculosis penicillin binding proteins are able to inactivate select β-lactams with high efficiency. Staphylococcus aureus, Bacillus subtilis, and Escherichia coli. Since similar observations were made in these organisms, a paradigm that describes the mechanistic basis of β-lactam activity was developed and assumed to be generally applicable to a wide range of bacteria  are a class of enzymes that are present in virtually all bacteria , 2. Thei3–bacteria –14. Accopical PG , 16.M. tuberculosis, the causative agent of tuberculosis, kills more humans than any other bacterium . Download Table S2, TIF file, 1.6 MB.Amount of each β-lactam hydrolyzed by Copyright © 2022 Kumar et al.2022Kumar et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/msphere.00039-22.5FIG S1M. tuberculosis PBPs. The amount of each β-lactam hydrolyzed by a PBP was measured for 120 min. Reaction conditions for all PBP-β-lactam pairs were identical. Each assay was performed in triplicate under identical reaction conditions. The mean and standard error are reported. Download FIG S1, TIF file, 1.3 MB.Hydrolysis of β-lactams by Copyright © 2022 Kumar et al.2022Kumar et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/msphere.00039-22.6FIG S2M. tuberculosis PBPs in the presence of a β-lactam. Each panel shows nitrocefin hydrolyzed by a specific M. tuberculosis protein. Each M. tuberculosis PBP or BlaC was incubated with a β-lactam for 30 min, following which, nitrocefin was added. Hydrolysis of nitrocefin was monitored for 120 min. Each assay was performed in triplicate under identical reaction conditions. The mean and standard error are reported. Download FIG S2, TIF file, 1.1 MB.Hydrolysis of nitrocefin by Copyright © 2022 Kumar et al.2022Kumar et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the PonA2 exhibited a dominant inactivation profile against penicillins, as 24% of total hydrolytic activity against this β-lactam subclass was exhibited by this protein. PonA1, with 12% of the total hydrolytic activity against penicillins, was also more potent than BlaC (10%) , Table 1BlaC exhibited the most potent hydrolyzing activity (25%) against the cephalosporins. The levels of inactivation of cephalosporins by all other proteins were similar to each other with less than 10% each, while PonA1 and PonA2 were the least effective at inactivating this β-lactam subclass. The less-well-known putative PBPs Rv1367, Rv1730, and Rv2864 hydrolyzed ceftriaxone at a higher rate than other proteins, including BlaC .We extended these data to determine the overall susceptibility of each β-lactam subclass to the PBPs , Fig. S1M. tuberculosis PBPs.Inactivation of a β-lactam reporter by  In the above-described assay, one of the surprising findings was that PonA1 and PonA2 were more effective than BlaC at inactivating most penicillins and carbapenems. Based on these findings, we hypothesized the following: exposure of a protein to a β-lactam that it hydrolyzed at a high rate will not inhibit its β-lactamase activity. To test this hypothesis, we used on orthogonal approach based on determination of the amount of nitrocefin hydrolyzed by each PBP in the presence of each β-lactam. Nitrocefin is a commonly used chromogenic reporter for monitoring PBP hydrolytic activity , is then determined  with BlaC, Rv1367c, and Rv2864c could not be detected. Download FIG S3, TIF file, 0.4 MB.Verification of BOCILLIN FL binding to Copyright © 2022 Kumar et al.2022Kumar et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/msphere.00039-22.4TABLE S4M. tuberculosis PBP gene locus IDs and sequences of DNA oligomers used to amplify DNA of each gene. Download Table S4, TIF file, 0.6 MB.List of Copyright © 2022 Kumar et al.2022Kumar et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 50 of each β-lactam and PBP pair is shown in 50 of most penicillins were among the highest observed. This outcome is expected as BOCILLIN belongs to the penicillin subclass, and therefore, it is unlikely that another penicillin is preferably bound by the proteins. However, an important exception was observed. The FIC50 of all β-lactams (except for faropenem) was consistently lowest for PbpB compared to other PBPs, indicating that this PBP has the highest binding affinity for penicillins, cephalosporins, and carbapenems. These data are in full accord with the inactivation results  was used for protein overexpression. Luria-Bertani broth, Tris-Cl, sodium chloride salts, and imidazole salts were purchased from Sigma-Aldrich. Powder forms of all β-lactams . The amino acid sequence of each protein was analyzed using TMHMM (E. coli BL21(DE3) to generate expression clones. Each protein was overexpressed by inducing BL21(DE3) culture in LB broth with 0.25 mM IPTG  overnight at 16°C with constant shaking at 150 rpm (6 tag were purified by Ni-nitrilotriacetic acid (NTA)-based affinity chromatography with minor protein-specific optimizations as necessary , PbpB(Rv2163c), PonA1(Rv0050), PonA2Rv3682), DacB(Rv3627c), DacB1(Rv3330), DacB2(Rv2911), Rv1367c, Rv1730c, Rv2864c, and BlaC(Rv2068c) were PCR amplified using genomic DNA (gDNA) of 2, DacB(Rng TMHMM  to identng TMHMM  and clonng TMHMM . All resecessary  70–72).–72.M. tu10.1128/msphere.00039-22.8FIG S4M. tuberculosis genes encoding PBPs and BlaC were cloned, overexpressed, and purified from E. coli. This SDS-PAGE shows the purified proteins. Download FIG S4, TIF file, 0.1 MB.Proteins used in the study. Copyright © 2022 Kumar et al.2022Kumar et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Each β-lactam was used at a final concentration of 1 mM, which was subsequently mixed with each PBP or BlaC each at 10 to 15 μM for a 100-μL final reaction volume in 10 mM Tris-Cl buffer, pH 7.4. The β-lactam ring opening reaction was monitored for 120 min, 25°C, using a SpectraMax 250 spectrophotometer. Each assay was performed in triplicate under identical reaction conditions. For each protein and β-lactam combination, the rate of β-lactam hydrolysis was determined, and the composite hydrolysis was computed for each β-lactam class by each PBP and BlaC , Fig. S1Nitrocefin  is a chromogenic β-lactam whose hydrolysis is quantified by measuring the transition from the substrate form (390 nm) to the product (496 nm) . Nitrocemax = 504 nm)  is a fluorescent penicillin (λ 504 nm) . BOCILLI 504 nm) . Each pr


Streptomyces isolates, namely, UNOB3_S3 and UNOC14_S4, obtained from volcanic soils in Albay, Philippines. The genome assemblies comprised 7.63 Mb and 8.24 Mb, respectively. Genome mining revealed genes and biosynthetic gene clusters that encode putative insecticidal products.We report here the draft genome sequences of two  Streptomyces produces secondary metabolites encoded by a group of genes known as biosynthetic gene clusters (BGCs). Some of these bioactive metabolites could be important sources of environmentally friendly insecticides  consisting of the following: 4 g/L yeast extract, 4 g/L dextrose, 10 g/L malt extract, and 20 g/L agar. Colonies were picked after 5 days of incubation at 28°C.Isolates UNOB3_S3 and UNOC14_S4 were obtained from volcanic soils in Uno, Malilipot, Albay, Philippines using the serial dilution method (g) were subjected to genomic DNA extraction using the Zymo fungal/bacterial DNA mini prep DNA kit. Libraries were prepared using the Illumina Nextera DNA XT library preparation kit and sequenced using the MiSeq sequencing kit v3, 600 cycles (2 × 301 bp), on the Illumina MiSeq system RUO.Isolates UNOB3_S3 and UNOC14_S4 maintained at 4°C in YMA slants were cultivated for 3 days with continuous shaking (250 rpm at room temperature [RT]) in 50 mL yeast malt broth , consisting of the following: 4 g/L yeast extract, 4 g/L dextrose, and 10 g/L malt extract. The final fermentation was performed in 450 mL YMB (pH 7.0) for 5 days with continuous shaking (250 rpm at RT). Bacterial cells pelleted by centrifugation  database showed 96.9% and 97.1% completeness for UNOB3_S3 and UNOC14_S4, respectively .Streptomyces luteoverticillatus CGMCC 15060 (accession number GCA_003970715.1) was the closest to UNOB_S3, while Streptomyces olivoreticuli subsp. olivoreticuli ATCC 31159 (accession number GCA_003391135.1) was closest to UNOC14_S4, with 99.28% and 98.8% similarity between their 16S rRNA genes, respectively. A subsequent average nucleotide identity based on BLAST (ANIb) analysis using the Kostas Lab ANI/amino acid identity (AAI) matrix  database. Annotation using NCBI PGAP version 2021-07-01.build5508  predicteJAILXP000000000 and JAINFC000000000, respectively. The versions described in this paper are the first versions, JAILXP010000000 and JAINFC010000000. Their respective SRA accession numbers are SRR15561083 and SRR15561183.The genome sequences of UNOB3_S3 and UNOC14_S4 have been deposited in DDBJ/ENA/GenBank under the accession numbers


Arthrinium phaeospermum can cause branch wilting of Bambusa pervariabilis × Dendrocalamopsis grandis, causing great economic losses and ecological damage. A. phaeospermum was sequenced in sterile deionized water (CK), rice tissue (T1) and B. pervariabilis × D. grandis (T2) fluid by RNA-Seq, and the function of Ctf1β 1 and Ctf1β 2 was verified by gene knockout. There were 424, 471 and 396 differentially expressed genes between the T2 and CK, T2 and T1, and CK and T1 groups, respectively. Thirty DEGs had verified the change in expression by fluorescent quantitative PCR. Twenty-nine DEGs were the same as the expression level in RNA-Seq. In addition, ΔApCtf1β 1 and ΔApCtf1β 2 showed weaker virulence by gene knockout, and the complementary strains Ctf1β 1 and Ctf1β 2 showed the same virulence as the wild-type strains. Relative growth inhibition of ΔApCtf1β 1 and ΔApCtf1β was significantly decreased by 21.4% and 19.2%, respectively, by adding H2O2 compared to the estimates from the wild-type strain and decreased by 25% and 19.4%, respectively, by adding Congo red. The disease index of B. pervariabilis × D. grandis infected by two mutants was significantly lower than that of wild type. This suggested that Ctf1β genes are required for the stress response and virulence of A. phaeospermum. Bambusa pervariabilis × Dendrocalamopsisgrandis (hybrid bamboo), a kind of hybrid bamboo, is a vital renewable resource and is known as the “second forest” [Arthrinium phaeospermum has occurred in a large area of the Yangtze River basin, resulting in plant death and huge economic losses, threatening the construction of ecological barriers. A. phaeospermum is a plant pathogenic fungus distributed all over the world and a generalist with a broad range of host species [B. pervariabilis × D.grandis [Phyllostachys prominens [Phyllostachys viridis [ forest” . This hy forest” . However species . In addigrandis , its hosgrandis , sugarcagrandis , Phyllosrominens ,8, new orominens , Phyllos viridis , etc.B. pervariabilis × D. grandis caused by A. phaeospermum indicated that the AP protein toxin can damage the mitochondrial membrane of host B. pervariabilis × D. grandis shoots and inhibit respiration [B. pervariabilis × D. grandis [A. phaeospermum by column chromatography and anion exchange chromatography showed strong virulence, different from the previously known pathogenic factors, such as arthrichitin and arthrinic acid, isolated from human skin diseases by Vijayakumar et al. [Pseudomonas aeruginosa ZB27 had a favorable effect on B. pervariabilis × D. grandis blight [A. phaeospermum and the defense pathway of B. pervariabilis × D. grandis at the molecular level. Based on the whole genome information of A. phaeospermum [B. pervariabilis × D. grandis after pathogen infection, among which the differential proteins were enriched mainly in the biological process and cell components [B. pervariabilis × D. grandis induced by inactivated protein toxin, among which the differentially abundant proteins were mainly enriched in biological processes and cell components. At the same time, the differentially expressed genes were involved mainly in lignin and phytoprotein synthesis, tetrapyrrole synthesis, redox reactions, photosynthesis, and other processes [Previous research on the shoot blight of piration . At the  grandis , and itsr et al.  and Bloor et al. . Previour et al. ,16. In ts blight . In addiospermum , signifimponents . There wrocesses ,20.However, the interaction between host plants and fungal pathogens is well known to be essentially a coevolutionary mechanism. Pathogens invade host plants and then activate plant pathogen-associated molecular patterns-triggered immunity (PTI) due to the recognition of PAMPs of pathogenic fungi, including local cell wall reinforcement, induction of defense genes, secretion of chitinase, production of reactive oxygen species and production and release of antimicrobial compounds. To inhibit PTI, pathogens produce effectors to escape the recognition of immune receptors and induce the production of resistant proteins in plants to trigger ETI. Therefore, when pathogens interact with host plants, in addition to the metabolic changes in the host plants, the gene expression in the pathogens will also be significantly different ,22,23,24Arabidopsis thaliana [A. phaeospermum and B. pervariabilis × D. grandis has not been reported during host infection. It is necessary to screen some genes related to A. phaeospermum infection and pathogenicity by transcriptome analysis, but the specific functions of these candidate genes during infection are not clear. Gene knockout is an important means to study gene function. At present, the function of key genes in many fungi has been confirmed, including Beauveria bassiana [Aspergillus niger [Trichoderma harzianum [B. pervariabilis × D. grandis tissue and verified the genes with significant differences by fluorescent quantitative PCR. Finally, we explored gene function by gene knockout. The purpose of this study was to provide reliable reference data for further study on the pathogenic mechanism of A. phaeospermum and the disease resistance mechanism of B. pervariabilis × D. grandis.In recent years, with the in-depth study of the interaction between pathogens and host plants and the rapid development of sequencing technology, RNA-seq has been widely used to study the mechanism of plant pathogen interactions. At present, RNA-seq in the study of plant pathogen interactions focuses mainly on screening host plant disease resistance genes and disease resistance metabolic pathways, which provides a theoretical basis for plant disease resistance mechanisms. For example, RNA-seq can be used in rice , Arabidothaliana , tomato thaliana  and othebassiana , Aspergius niger , and Triarzianum . This paA. phaeospermum was isolated by tissue isolation method [B. pervariabilis × D. grandis, the accession number of A. phaeospermum ITS in NCBI database is OK626768. The isolate was stored in China Forestry Culture Collection Center, numbered cfcc 86860 .Microorganism: n method  from disB. pervariabilis × D. grandis  were planted in the bamboo-growing areas of reclaimed farmland  in Sichuan, China. The study area was at an altitude of 515.98 m with annual temperatures of 6.8 °C to 26.1 °C and annual precipitation of 1300–1700 mm. Pathogenicity test sample: One-year-old B. pervariabilis × D. grandis were planted in greenhouse of Chengdu, Sichuan, China at a temperature of 25 °C.Plant tissue sample: One-year-old B. pervariabilis × D. grandis plant tissue fluid. Young shoots of B. pervariabilis × D. grandis were cut to 20 g, disinfected with 1.5% sodium hypochlorite for 1 min, disinfected with 75% alcohol for 3 min, washed with sterile deionized water three times, ground with 20 mL sterile deionized water and filtered with sterile gauze. The filtrate was centrifuged in a 20 mL centrifugal tube at 4 °C and 7000 rpm for 10 min. The supernatant was transferred to a new 20 mL centrifugal tube and centrifuged four times. Finally, the supernatant was removed by using a sterile 0.22-μm bacterial filter. The tissue liquid of B. pervariabilis × D. grandis was obtained and stored at −20 °C for subsequent experiments. The method of Andrade et al. [The method of Andrade et al.  and Jiane et al.  was alsoB. pervariabilis × D. grandis were placed on PDA medium , spread evenly with a coater and placed on a clean worktable for future use. A. phaeospermum was inoculated into PDA medium coated with 2 mL sterile deionized water (CK), PDRA medium coated with 2 mL sterile rice tissue fluid (T1) and PDHA medium coated with 2 mL sterile B. pervariabilis × D. grandis tissue fluid (T2). Each treatment was repeated 3 times, cultured in a 25 °C incubator for 7 days and preserved.Mycelium treatment. Two milliliters of sterile deionized water, sterile rice tissue fluid and sterile A. phaeospermum grown in sterile deionized water, sterile rice tissue fluid and sterile B. pervariabilis × D. grandis tissue fluid. Then, the genomic DNA was removed by DNase, RNase inhibitor  and other reagents. Agarose gel electrophoresis, a Nanodrop microspectrophotometer  and an Agilent 2100 bioanalyzer were used to test the total RNA integrity, purity and quality [The phenol chloroform method  was used quality , respectA. phaeospermum. Poly(A) + RNA was isolated from aggregated RNA samples by using oligonucleotide (dT) beads [PrimeScriptTM Double Strand cDNA Synthesis Kit (TaKaRa) was used to construct the cDNA library of T) beads . FragmenT) beads . The cDNp = 94 (minimum percent identity for overlaps), –l = 40 (minimum overlap length), and –v = 30 (maximum length of unmatched overhangs). We assembled nine samples  to obtain contigs and then used TGICL to cluster the contigs to remove redundancy. All annotations were performed on the merged contig set. To obtain GO information for the contig, first, the contig sequence was aligned to the protein database Nr  by blastx, and the protein with the highest sequence similarity to a given contig was obtained; thus, the Nr annotation information of the contig was obtained. Subsequently, according to Nr annotation information, contig GO annotation information was obtained using Blast2GO software [−5. http://www.ncbi.nlm.nih.gov/BLAST/, accessed on 10 November 2018) [http://www.geneontology.org/, accessed on 10 November 2018); the Kyoto Encyclopedia of Genes and Genomes (KEGG) [http://www.genome.jp/kegg/, accessed on 10 November 2018); the Clusters of Protein Homology (KOG) [http://www.ncbi.nlm.nih.gov/KOG/, accessed on 10 November 2018); the NCBI Non-Redundant Protein Sequence database (Nr) [ftp://ftp.ncbi.nih.gov/blast/db/, accessed on 10 November 2018); and a manually annotated, nonredundant protein sequence database (Swiss-Prot) [http://www.uniprot.org/, accessed on 10 November 2018). After obtaining GO annotations for each contig, we used WEGO software [Before assembly, the low-quality sequences from raw reads were removed, including sequences with ambiguous bases (denoted with >10% ‘N’ in the sequence trace), low-quality reads  and reads with adaptors. Transcriptome de novo assembly was carried out with the short read assembly program Trinity  . TGICL ssoftware . The merer 2018)  (http://s (KEGG)  (http://gy (KOG)  (http://ase (Nr)  (ftp://fss-Prot)  (http://software  to classB. pervariabilis × D. grandis tissue fluid (T2) was analyzed. First, RSEM software [http://www.bioconductor.org/packages/release/bioc/html/edgeR.html, accessed on 10 November 2018) based on R language was used for different analyses. Genes with significant differences in expression were defined as having a false discovery rate (FDR) < 0.05 and 2 log FC > 2. GO  [The differential expression of pathogenic fungi in sterile deionized water (CK), sterile rice tissue fluid (T1) and sterile software    function., 2000)  pathway B. pervariabilis × D. grandis tissue fluid. The qPCR system consisted of the following: 10 μL Mix , 7.4 μL ddH2O, F/R 0.8 μL, and 1 μL cDNA (CK/T2). The qPCR was as follows: 95 °C for 1 min, 95 °C for 30 s and 60 °C for 15 s. Forty cycles were repeated from the second step to the third step, and the dissolution curve was added at the final step. Each group of qPCR was repeated three times, and the average value was calculated. GAPDH was used as an internal reference gene to detect the expression changes of 30 candidate pathogenic genes in 3 different treatment groups. Data were analyzed by the 2−△△Ct method [Firstly, we selected the genes with high differential multiples in several treatment groups, including up-regulated differential genes and down-regulated differential genes. In addition, 30 candidate genes were screened by combining the gene function annotation information in the database. We selected GAPDH and Actin as internal reference genes in RT-PCR analysis to determine whether RT-PCR with GAPDH and Actin as reference genes is consistent with transcriptional sequencing results, so as to verify the reliability of transcriptome sequencing data. Primer Premier 5.0 software was used to design fluorescence quantitative PCR primers using CDS regions of the internal reference gene GAPDH and 30 candidate genes  and cutinase transcription Factor 1 beta 2 (Ctf1β 2) gene as the center, 1300 bp upstream and 1300 bp downstream were selected as homologous arms to design primers to amplify homologous arms of Ctf1β from A. phaeospermum DNA. Meanwhile, primers were designed to amplify the hygromycin phosphotransferase gene (hph) from the pSilent-1 vector. (The primer sequence is shown in Ctf1β-5-F/R (Ctf1β-3-F/R and hph-F/hph-R) 1/1 µL, 2 × TransTaq HiFi PCR SuperMix 25 µL, nuclease-free water 22 µL. Reaction procedure: 94 °C 5 min  34 cycles, 72 °C 10 min. Ctf1β-5, Ctf1β-3 and hph gene fragments were obtained. The fusion fragments were obtained by fusion PCR using LA Taq (Takara). The first round PCR amplification system: Ctf1β-5 3 µL, Ctf1β-3 3 µL, hph 6 µL, 10 × LA buffer 3 µL, dNTP Mix 1 µL, LA Taq 0.3 µL, ddH2O 13.7 µL. Reaction procedure: 94 °C for 3 min, , 34 cycles, 72 °C for 7 min. Second round PCR amplification system: The first round PCR product 2 µL, 10 × LA buffer 5 µL, dNTP mix 2 µL, LA Taq 0.5 µL, ddH2O 37.5 µL, Ctf1β-5-F/Ctf1β-3-R 1/1 µL. Reaction procedure: 94 °C 3 min  34 cycles, 72 °C 10 min. The fusion fragment Ctf1β-5-hph-Ctf1β-3 was obtained. Enzyme digestion system: 10 × QuickCut Green Buffer 5 μL, pCAMBIA0380 DNA 7 μL, ApaI 1 μL, Hind III 1 μL, ddH2O 36 μL. Procedure reaction: 37 °C for 5 min. We obtained vector pCAMBIA0380 linearized by ApaI and HindIII the fusion fragment containing the complementary sequences at ApaIand HindIII of vector pCAMBIA0380. The vector pCAMBIA0380 and fusion fragment Ctf1β-5-hph-Ctf1β-3 were ligated by clonExpress II one step cloning kit . Linked reaction system: linearized pCAMBIA0380 5 μL. Fusion fragment 3 μL, 5 × CEIIBuffer 4 μL, Exnase 2 μL and ddH2O 6 μL. Procedure reaction: 37 °C for 30 min, and cooling at 4 °C for 5 min. The recombinant plasmid was transformed with DH5α , the plasmid was added to 50 µL DH5α, then ice bathed 25 min, 42 °C 30 s, ice bathed 2 min, 500 uL SOC liquid medium was been added in it, and shaken 200 rmp at 37 °C for 1 h. and detected by colony PCR.We used an improved split-marker method to identify gene function . Taking Protoplast preparation: Preparation of enzymatic hydrolysate: 0.2 g lysine, 0.5 g driselease, 20 mL 1.2 M KCl, magnetic stirring beads, stirring for 15 min, then 4 °C, 3500 rmp, acceleration of both up and down, centrifugation for 10 min. The supernatant was filtered through a bacterial filter to obtain the enzymatic hydrolysate. The mycelia were cultured for 2 days at 28 °C, and 180 rpm were filtered with single layer microcloth, washed with 25 mL 1.2 M KCl, added to the enzymatic hydrolysate, and cultured at 70 rpm and 30 °C for 9 h. The protoplasts were examined by microscopy, filtered by double-layer microscopy, washed with 25 mL of 1.2 M KCl, and centrifuged, and the supernatant was discarded. Then, 1 mL STC was added to the suspension for standby.Ctf1β-5-hph-Ctf1β-3 was added to the 5 × 106 protoplast and incubated at room temperature for 20 min. PEG was added three times, with an interval of 10 min each time and 400 μL was added each time. After standing at room temperature for 20 min, 10 mL TB3 liquid medium was added. The components of TB3 medium included yeast extract 3 g, acid hydrolyzed casein 3 g, sucrose 200 g and distilled water 1 L. Then, after culturing at 90 RMP and 25 °C for 10 h, 45 mL TB3 medium containing 350 µg/mL hyg antibiotic was added to the protoplast. The plate was poured after mixing, the culture was inverted at 25 °C for 3 days.Genetic transformation: Then, 30 μL of the fusion fragment Ctf1β-3-hph)-F/R, (Ctf1β-5-hph)-F/R and Ctf1β-F/Ctf1β-R were used for internal detection.Transformant detection: Lysis buffer for microorganization to direct PCR (TaKaRa) was used to lyse the mycelium, and Ex Taq was used for detection. Hph-F/hph-R, , 2 mol/L NaCl, and 40 mmol/L HB. pervariabilis × D. grandis with uniform growth were selected. Five plants were randomly selected and sprayed on the upper eight shoots of each plant with the mycelial suspension of the wild-type strain. The remaining 15 plants were treated with the mycelial suspension of mutant ΔAPCtf1β 1, the mycelial suspension of mutant ΔAPCtf1β 2 and sterile water, with five plants per treatment. The samples were subjected to bagging moisturizing and sprayed once every 12 h, 3 times in total, using 3 independent replicates. The incidence was investigated 20 days after inoculation. The disease index was calculated as follows [Twenty plants of two-year-old  follows . Disease6 protoplasts according to the method in 2O2 at 25 °C for 5 days to analyze the differences in vegetative growth among the strains. For the pathogenicity determination, twenty plants of two-year-old B. pervariabilis × D. grandis with uniform growth were selected. Five plants were randomly selected and sprayed on the upper eight shoots of each plant with the mycelial suspension of the wild-type strain. The remaining 15 plants were treated with the mycelial suspension of Ctf1β 1 complemented strain and Ctf1β 2 complemented strain, and sterile water, with five plants per treatment. The samples were subjected to bagging moisturizing and sprayed once every 12 h, 3 times in total, using 3 independent replicates. The incidence was investigated 20 days after inoculation. Refer to Taking the CDS region of hph gene as the center, 1300 bp upstream and 1300 bp down-stream were selected as homologous arms to design primers  were 85.00%, 86.08%, 85.11%, 84.81%, 84.17%, 79.07%, 83.10%, 81.61% and 83.97%, respectively  analysis results of sequence assembly quality are shown in A. phaeospermum were deposited in the NCBI Sequence Reads Archive (SRA) under the accession numbers SRR9278662, SRR9278661, SRR9278664, SRR9278663, SRR9278658, SRR9278657, SRR9278660, SRR9278659 and SRR9278665. The assembled contigs have been published in the NCBI Transcriptome Shotgun Assembly (TSA) under the accession number GHWG00000000 , including 12,861 contigs that came from the filters applied by TSA.The base percentage of Q30 in each sample after filtering was not less than 94.76%. The percentage of GC content was more than 56.46%. The reads’ lengths of 9 samples are 150 bp. The transcriptome sequencing results met the quality requirements of subsequent assembly analysis. The sequencing information of samples CK-1, CK-2, CK-3, T1-1, T1-2, T1-3, T2-1, T2-2 and T2-3 is shown in ectively . By IlluA. phaeospermum under the three different treatment conditions were annotated and compared with SwissProt databases, Kyoto Encyclopedia of Genes and Genomes (KEGG) the Clusters of Protein Homology (KOG) and NCBI nonredundant (Nr) protein databases. The additional remonal redundant sequences obtained a total of 13,077 contigs. There were 10,155 contigs with annotated information in four major databases  was inconsistent with the result of transcriptome sequencing, and the consistency rate was 90%. Among the 10 differentially expressed genes selected from the CK and T2 treatment groups, 10 genes showed the same change trend as transcriptome sequencing, with a consistent rate of 100%. Among the 10 differentially expressed genes selected from the CK and T1 treatment groups, all genes showed the same change trend as the result of transcriptome sequencing, with a consistent rate of 100%. Among these genes, the expression of Ctf1β in the T2 group was significantly higher than the expression of Ctf1β in the CK group, which may be related to A. phaeospermum infection of B. pervariabilis × D. grandis. needing to produce a large amount of cutinase to degrade cutin. In this case, Ctf1β is considered a gene related to A. phaeospermum virulence, it has further functional research value.To verify the reliability of the RNA sequencing data, qRT–PCR was used to evaluate the expression under different culture conditions. When housekeeping genes GAPDH and Actin were used as internal reference genes, the expression trend of differential genes was the same and consistent with the results of transcriptome sequencing. We selected 10 DEGs from three different comparison groups to verify the change in expression . Among tA. phaeospermum was inhibited by hygromycin. The higher the concentration of hygromycin, the stronger the inhibition. When the concentration of hygromycin was 350 µg/mL, the A. phaeospermum did not grow at all. Similarly, the growth of A. phaeospermum was inhibited by genomycin. The higher the concentration of genomycin, the stronger the inhibition. When the concentration of genomycin was 100 µg/mL, the A. phaeospermum did not grow at all. The hph, Ctf1β1-5′, Ctf1β1-3′, Ctf1β2-5′ and Ctf1β2-3′ fragments were obtained by PCR amplification. The knockout vectors of the Ctf1β1 and Ctf1β2 genes were constructed successfully by the improved split-marker method. From left to right were 15 K DNA marker (TransGen Biotech), recombinant plasmid DNA, empty plasmid DNA, recombinant plasmid double digested product and Ctf1β 1 and Ctf1β 2 knockout vector fusion fragments , while obvious symptoms on twigs and leaves of B. pervariabilis × D. grandis. were found in the wild-type strains . Ctf1β 1 and Ctf1β 2 have been proven to play an important role in the detoxification of reactive oxygen species (ROS). This result is consistent with the conclusion that the BbCtf1 α and BbCtf1 β deletion mutants of entomopathogenic Beauveria bassiana and the ccsge1 deletion mutants of the plant pathogen Cytospora chrysosperma are more sensitive to hydrogen peroxide [Ctf1β 1 and Ctf1β 2 were essential for oxidative stress and could inhibit the plant immune response. In addition, the loss of Ctf1β 1 and Ctf1β 2 gene function leads to the increased sensitivity of pathogens to cell wall disturbance stress (exposure to Congo red), consistent with the change in stress response sensitivity caused by the deletion of the BbCtf1 α and BbCtf1 β genes in B. bassiana [Ctf1β is crucial in maintaining cell wall integrity.In this study, we observed that the symptoms of plant disease were different between wild-type and mutant strains by knocking out the  surface . However and wax . In addi and wax ,76,77. Tthe host ,79. Cutithe host ,81. Ctf1genicity . From thtabolism . The outtabolism ,86,87,88peroxide ,89. The bassiana . These r


OXH-S was found to be an addition product of sulfite and oxypeucedanin hydrate (OXH-N). Then, the cytotoxicity and anti-inflammatory activity of OXH-N, OXH-S, and water extracts of sulfured (extraction-S), and unsulfured Baizhi (extraction-N) were evaluated. OXH-S and extraction-S were less toxic than OXH-N and extraction-N, respectively. A comparison of OXH-N with OXH-S and extraction-N with extraction-S showed no significant differences in anti-inflammatory activity. These results suggest that sulfur fumigation can reduce toxicity and does not influence the anti-inflammatory activity of Baizhi, even after chemical composition changes. The proposed protocol based on marker screening, pharmacology, and safety evaluation provides a scientific basis for the standardization and regulation of sulfured Baizhi and other medical materials.Sulfur Angelicae Dahuricae Radix (Baizhi) is a common medicinal herb in Asian countries. A practical protocol combining metabolomics, pharmacology, and cytotoxicity was developed to comprehensively evaluate the influence of sulfur-fumigation on the quality of Baizhi. Furocoumarins could be transformed into sulfur-containing compounds during the sulfuring process, among which  In traditional Chinese medicine (TCM), sulfur fumigation is an extensively used processing method that has been employed for over 100 years. The method allows good moisture-retention, while inhibiting insect and mold growth, as well as preserving the color and freshness of the medicinal products . Sulfur p-hydroxybenzyl hydrogen sulfite in sulfured Gastrodia Rhizoma (Sulfur fumigated TCM contains many sulfur-containing compounds . The pre Rhizoma . StudiesAngelica dahurica (Hoffm.) Benth. & Hook. f. ex Franch. & Sav. (Apiaceae), is an important medicinal plant that has been widely cultivated across several Asian countries, particularly China, for thousands of years  is the most commonly available TCM on the market. Baizhi, the dried root of of years . It is cof years . All theof years  of Baizhof years . Howeverof years  and antiof years  activitiof years . Howeverd = 0.5 cm) on the surface. Next, 20 g of sulfur was then lighted with 12 ml alcohol in the box for sulfured groups. 12 ml alcohol was lighted in the box of unsulfured group. Twenty-4 hours later, all samples were dried at 40°C, and ground to a fine powder (50 mesh) using a pulverizer , and were stored at 4°C for further analysis. Sulfured Baizhi decoction pieces (10 kg) for the purification of markers of sulfured Baizhi were purchased at the Bozhou TCM trading market  on October 23, 2019. All samples were identified as A. dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. by Professor Lan-Ping Guo.Three fresh Baizhi samples (unsulfured Baizhi 1–3) were collected from Bozhou, Anhui Province, China  on August 13, 2018. For ultra-performance LC quadrupole time-of-flight MS (UPLC-Q-TOF-MS) analysis, sulfured Baizhi 1–3 samples were prepared as reported previously to obtain the material with sulfur dioxide residue at approximately 750 mg/kg . The freAnalytical-grade methyl red, acetate, alizarin red S, and sodium sulfate were obtained from Shanghai Macklin Biochemical Co., Ltd. . Analytical-grade hydrogen peroxide (30%) was obtained from Beijing Chemical Works  and 0.01 mol/L sodium hydroxide solution for volumetric analysis was obtained from the National Institute of Metrology . Deionized water (18.2 MΩ cm) was prepared using a Milli-Q system . Liquid chromatography (LC)-grade acetonitrile , LC/mass spectrometry (MS)-grade formic acid , analytical-grade methanol , and lipopolysaccharide  were also used. Oxypeucedanin hydrate (OXH-N) with high purity (95%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. .1 and compound 3. A dissecting microscope , charge-coupled device camera , MULTIZOOM AZ100 microscope , microinjector , micropipette puller , 6-well plate , and methylcellulose  were used to evaluate anti-inflammatory activity and cytotoxicity.A BSA224S analytical balance , centrifuge 5415D , SB-800DTD ultrasonic processor , and 0.22 µm polytetrafluoroethylene filter  were used for UPLC-Q-TOF-MS analysis. An EYELA N-3010 rotary evaporator  and SP825L macroporous resin  were used to purify compound g at 25°C for 10 min, each sample extract was filtered through a 0.22 µm membrane filter prior to UPLC-Q-TOF-MS analyses, and 50 µL of each extract was mixed to prepare a quality control sample.The powdered sulfured and unsulfured samples were ultrasonically extracted with 70% ethanol for 90 min at 25°C and 40 kHz using an ultrasonic processor to prepare samples with a concentration of 200 mg/ml. After centrifugation at 13,800 × UPLC separation was performed on an Acquity UPLC I-Class system . A Waters UPLC Acquity HSS T3 column  was used for chromatographic separation. The mobile phase consisted of (A) 0.1% (v/v) formic acid in deionized water and (B) acetonitrile containing 0.1% (v/v) formic acid. The linear elution gradient was optimized as follows: 5% B (0–2 min), 5→30% B (2–7 min), 30→45% B (7–12 min), 45→60% B (12–25 min), 60→90% B (25–30 min), 90→98% B (30–30.05 min), and 98% B (30.05–33 min). The parameters were set as follows: flow rate, 0.5 ml/min; injection volume, 1 µL; column temperature, 40°C; UV, 200–400 nm.Chemical profile analysis was performed on a Waters Xevo G2-S Q-TOF-MS system (coupled with electron spray ionization) in both negative and positive electrospray ionization modes. The Q-TOF-MS parameters were set as follows: acquisition mass range, 50–1,500 Da; scan time, 0.5 s; capillary voltage, 2.0 kV; cone voltage, 40 V; source temperature, 100°C; desolvation gas temperature, 280°C; cone gas flow rate, 50 L/h; and desolvation flow rate, 650 L/h. The collision energy was set to 6 eV (trap) for low-energy scans and 30–60 eV for high-energy scans, and all MS data were acquired using leucine-enkephalin at a concentration of 200 ng/ml to ensure accuracy and reproducibility. The accurate mass weights were 554.2615 and 556.2771 in negative and positive ion modes, respectively.UPLC-QTOF-MS-guided isolation has been described previously for sulfur-containing derivatives . Sulfure1 and 324 mg of compound 3 were obtained. The purity of compounds 1 and 3 were found to be more than 80 and 95%, respectively. The purity was determined by UPLC.The water fraction was subjected to SP825L macroporous resin column chromatography and eluted with aqueous ethanol  of each solvent was used). The purification procedures were guided by UPLC-Q-TOF-MS. Then, 30% ethanol was purified by 1,260 Infinity HPLC . Finally, 2 mg of compound 1 and 3 were prepared at a concentration of 100 μg/ml in 70% methyl. Next, 1 µL of each compound was injected for UPLC-Q-TOF-MS to obtain high-resolution MS data. Compounds 1 (2 mg) and 3 (5 mg) were dissolved in 1 ml D2O and transferred to a nuclear tube (d = 2 mm) for 1H nuclear magnetic resonance (NMR), 13C NMR, heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond correlation (HMBC), the spectra of which were recorded on a Bruker Avance III 600MH spectrometer  operating at 600 MHz for 1H and 150 MHz for 13C, with tetramethylsilane used as an internal standard.Compounds R)- and (S)-(3)-α-methyl-α-trifluoromethylphenyl lactic acid (MTPA) esters of compound 3, a solution of compound 3 (4 mg) in pyridine-d5 (0.5 ml) and (R)-(-)-MTPA Cl (4 µL) was left to stand overnight at 20–30°C with stirring under argon protection to obtain (R)-(3)-MTPA ester. Similarly, the (S)-(3)-MTPA ester was obtained from compound 3 using (S)-(-)-MTPA Cl.Mosher’s method  was usedR,12S)-3 and five conformers were selected for -3 based on their Boltzmann distribution (>1%). The theoretical calculation of electronic circular dichroism was conducted in methanol using the time-dependent density functional theory at the CAM-B3LYP/6-31 + g  level for the optimized conformers of 3. The rotational strengths for 50 excited states were calculated.For electronic circular dichroism calculations, a conformation search of compound 3 was performed in Molclus (version 1.9.9.4) based on the xtb program using the GFN-xTB method . Conformg at 25°C for 10 min. The supernatant was concentrated and freeze-dried to obtain 3.0 g extractions of unsulfured (extraction-N) and 3.1 g of sulfured (extraction-S) Baizhi. The drug extract ratios were 0.15 g/g. OXH-N is oxypeucedanin hydrate. OXH-S was obtained as described in UPLC-QTOF-MS-Guided Marker Isolation and Purification.Sulfured and unsulfured Baizhi (20.02 g) were extracted for 1 h by soaking in water (20× amount of unsulfured and sulfured Baizhi) for 1 h, followed by centrifugation at 13,800 ×Evaluated Using MTT Assays and Determination of MTC). This preparation was filtered through a sterile filter membrane and stored at 4°C.OXH-N, OXH-S, extraction-N, and extraction-S (40 mg) were dissolved in dimethyl sulfoxide . RPMI-1640 culture medium  was added to the target concentration (described in Cell Viability 2 in a humidified incubator. Each of the cell strains were divided into 13 groups: one of the groups served as the control (no treatment with drugs), whereas the other 12 groups were treated with three different concentrations of OXH-N, XOH-S, extraction-N, or extraction-S.PC12, L02, and HK2 cells, which were kindly provided by the Stem Cell Bank of the Chinese Academy of Sciences, Shanghai, China, were cultured as previously reported . All cel2) for 12 h. Then different concentrations of drugs  were added. After 24 h, the medium was discarded. CCK-8 solution (10 µL) and culture medium (10 µL) were added to each well of the plate. The plate was incubated for an additional 2 h in an incubator , after which, the absorbance was measured at 450 nm using a microplate reader . Cell viability was expressed as a percentage of the control. The growth inhibition rate was calculated as follows:Cell viability was determined using the Cell Counting Kit (CCK)-8 assay . Cell suTo determine the maximum tolerated concentration (MTC) of each drug, cells were treated with 1, 50, 100, 200, 400, 800, and 1,000 mg/L of each drug. At least three independent experiments were performed.Preparation of Experimental Samples) were dissolved in water to prepare the mother liquor at a concentration of 2000 μg/ml. These samples were diluted with water to the target concentrations.Extraction-N and extraction-S  2012-0171. All experiments were performed in accordance with the requirements of the International Association for Assessment and Accreditation of Laboratory Animal Care (certification number: 001458). All experiments were approved by the Institutional Animal Care and Use Committee at Hunter Biotechnology, Inc. In total, 810 transgenic Tg (corola: MPX) zebrafish larvae at 3 days post-fertilization (dpf) were used for the experiments, which are described in Determination of MTC and Establishment of Inflammation Model and Drug Administration.Transgenic Tg (MPX: EGFP) zebrafish  were maiIn total, 360 zebrafish were randomly divided into 12 groups with 30 zebrafish in each group including a blank control group, modal control group, and 10 experiment groups (treated with different drugs at different dosages). Zebrafish larvae (3 dpf) were exposed to different concentrations  of extraction-N and OXH-N for 1 h in the test solution. A microinjector  was used to inject LPS into the yolk sac of each zebrafish, except for those in the blank control group. After 2 h, the phenotype and mortality of zebrafish were observed and recorded to determine the MTC.The proportions of normal, malformed, and dead zebrafish larvae were also recorded at 24 and 48 h using an SZ780 stereo microscope .Determination of MTC. The doses of each drug were 55.6, 167, and 500 μg/mL. A microinjector  was used to inject LPS into the yolk sac of each zebrafish, except for those in the blank control group. Two hours after administration, 10 zebrafish were randomly chosen from each group and photographed under a continuous zoom fluorescent microscope with electric focusing . The number of fluorescent neutrophils at the site of inflammation was counted by eye. The anti-inflammatory effects of each drug on LPS-induced inflammation in zebrafish were evaluated by analyzing the number of neutrophils. The inflammatory regression effect  was calculated based on the number of neutrophils. The formula used for calculation was as follows:The inflammation model was established according to standard protocols . In totap < 0.05 was considered to indicate statistically significant results.The cytotoxicity and anti-inflammatory results are presented as the means ± standard deviation . One-wayThe zebrafish facility and laboratory at Hunter Biotechnology, Inc. are accredited by the AAALAC. After the experiments, all zebrafish were anesthetized and euthanized with 0.25 g/L tricaine methane sulfonate according to the American Veterinary Medical Association requirements for euthanasia using anesthetic. This study was approved by the Institutional Animal Care and Use Committee at Hunter Biotechnology, Inc. with approval number 001458.3) in water was added to three clean round-bottomed flasks and refluxed and decocted at 100°C for 1 h. Water was added to replace that lost before boiling. The sample solutions were then filtered through a 0.22 µm membrane filter prior to analysis, and 1 µL was injected into the UPLC-PDA system to detect concentrations of OXH-N and OXH-S before and after decoction.Twenty-five microliters of 0.011 mg/ml OXH-S (compound The content of sulfur dioxide residue in the non-boiled and boiled samples was determined using a standard method documented in the Chinese Pharmacopeia 2020 version (Part four) Appendix 2331 . The nonThe chemical constituents of Baizhi were separated within 23 min in both the positive and negative ion modes. Significant differences were observed between unsulfured and sulfured Baizhi in the negative mode. Therefore, the experiment was conducted in the negative mode. Some components of Baizhi were notably affected by sulfur fumigation, such as compounds with retention times between 9 and 21 min .−, [M-H2O-H]−, [M + HCOO]−, [2M-H]−, [M + Na-2H]−, [M + Cl]−, [M + K-2H]−, and [2M + HCOO]− were selected as addition ions. The samples were categorized into sulfured group, unsulfured group, and quality control group, and all data peaks were aligned with that of the quality control group. A total of 6,002 peaks were obtained, and the compounds with p value ≤0.01, minimum coefficient of variation ≥30, and max fold-change ≥2 were selected for principal component analysis. The quality control samples in the PCA score plot suggested good stability and repeatability of LC–MS data. The sulfured and unsulfured samples were well-separated and divided into two groups as shown in 1–6, 10, and 12. The compounds decreased significantly were compounds 7, 9, and 11.For screening the markers of sulfured and unsulfured Baizhi, raw data obtained using Masslynx V4.1 were imported into QI software (Waters Corp.), and [M-H]1 (adduction product of byakangelicin and sulfurous acid) and compound 3 (adduction product of oxypeucedanin hydrate and sulfurous acid) are described in Structural Analysis of Compounds 1 and 3. Based on ion fragment pathway of compound 1 and 3, the structures of other sulfur-containing derivatives were proposed. The ion fragment pathway of compound 1 and 3 are show in 4 had the same molecular and fragment pathway of compound 3, so 4 was regarded as the other addition product of oxypeucedanin hydrate and sulfurous acid, and was named 3,4-dihydrooxypeucedanin-3 hydrate sulfonic acid. The fragment pathway of compound 4 is shown in 2 had one more H2SO3 compared with imperatorin, so it was speculated to be 3,4-dihydroimperatorin-4-sulfonic acid; the fragment pathway is shown in 10 and 12 were identified as dihydrobyakangelicol sulfonic acid and 3,4-dihydrophellopterin-4-sulfonic acid, respectively. The fragments of 10 and 12 are shown in 6 had similar fragmentation as compound 1 -6-hydroxy-5-benzofuranyl)-3-sulfopropanoic acid. Similarly, compound 5 was identified as 3-(7-(2-en-3-methylbutoxyl)-6-hydroxy-5-benzofuranyl)-3-sulfopropanoic acid. The fragment pathway is shown in The configuration of compound mpound 1 , but had3 showed a relatively high response in mass spectrometer. Therefore, we next purified compound 3 and obtained its structural information.The structures of the markers are shown in 3 was obtained as a yellow powder. Its molecular formula, C16H18O9S, was established using UPLC-Q-TOF-MS with m/z 385.0591 [M-H]− , m/z 771.1204 [2M-H]−, and m/z 793.1116 [2M-Na+2H]− . Compared with (R)-heraclenol and OXH-N (C16H16O6) in unsulfured Baizhi, there was one additional H2SO3 in compound 3. Compound 3 was predicted to be a sulfite derivative of (R)-heraclenol or OXH-N. In the 1H NMR  spectrum of compound 3, 1H NMR  values comprising δ 7.75 , 7.10 , 4.92 , 4.69 , 4.45 , 3.84 , 3.34 , 3.23  and 1.28  were observed. These data were consistent with the spectrum of oxypeucedanin hydrate  and 3.29  were lower in the spectrum of compound 3 than in that of oxypeucedanin hydrate because the single bond between C-3 and C-4 changed into a double bond and the sulfur atom was directly linked to C-4. Additionally, δH 4.92  suggested a sulfonate group connected to C-4. 13C NMR  values are listed in 3 are shown in 1H NMR spectra, and 13C NMR spectra of compound 3 are shown in Compound  hydrate . However3, the absolute configuration at the C-12 position was confirmed using Mosher’s method as described in Structural Analysis of Compounds 1 and 3. Comparison of the 1H NMR data for the R-(3)- and S-(3)-MTPA esters of 3 in CDCl3 (S)-absolute configuration. -3 and -3 are presented in 3 was named -3,4-dihydrooxypeucedanin hydrate-4-sulfonic acid (OXH-S).To determine the stereostructure of compound in CDCl3  showed turation. . The ele1 was obtained as a brown powder. Its molecular formula, C17H20O10S, was established using UPLC-Q-TOF-MS with m/z 415.0691 [M-H]- , m/z 831.1523 [2M-H]- , and m/z 853.1298 [2M-Na+2H]- . Compared with byakangelicin (C17H18O7) in unsulfured Baizhi, there was one additional H2SO3 in compound 1. Compound 3 was predicted to be a sulfite derivative of byakangelicin. In the NMR spectrum of compound 1, 1H NMR  values of δ 7.59 , 6.96 , 4.66 , 4.36 , 4.06 , 3.69 , 3.18 , 3.08  and 1.06  were observed. These data were in agreement with the spectrum of compound 3. However, compound 1 had δH 3.96 , which was not present in compound 3, indicating an extra methyl group in compound 1. Compared with compound 3, δH 7.10  was missing, indicating that the extra methyl group was connected to C-5. 1H NMR and 13C NMR of compound 1 and compound 3 are shown in 3. Detailed high-resolution electrospray ionization MS spectra and related fragments, related HMBC spectra, HSQC spectra, 1H NMR spectra, and 13C NMR spectra of compound 1 are shown in 1 was named 3,4-dihydrobyakangelicin-4-sulfonic acid.Compound These data demonstrated that transformation of coumarins into dihydrocoumarin sulfonic acid was a reliable result . The mait-test are shown in The most important concern during sulfuring is safety, as many researchers have suggested that the sulfuring process leads to health risks . TherefoExtraction-N and extraction-S had similar chemical compositions and OXH-N and OXH-S had similar structures. Therefore, prior to testing the anti-inflammatory activity of extraction-N, extraction-S, OXH-N, and OXH-S, the toxicity of extraction-N and OXH-N was examined. At 2 h after exposure to extraction-N and OXH-N, no zebrafish died with a concentration range of 125–500 μg/ml. However, at concentrations of 1,000 and 2000 μg/ml, the drugs precipitated. Accordingly, the MTC was 500 μg/ml. Based on these results, 55.6, 167, and 500 μg/ml were selected for subsequent experiments.p < 0.01). Therefore, this model was deemed suitable for evaluating zebrafish inflammation.An LPS-induced zebrafish inflammation model was used to evaluate the anti-inflammatory effects of the drugs. Many fluorescent neutrophils were recruited at the inflammation site in the LPS-induced zebrafish. The average number of fluorescent neutrophils at the inflammatory site of the LPS-induced zebrafish model was 17.9 ± 1.26, whereas that in the blank control group was 4.8 ± 0.66. Compared with that in the blank control group, inflammatory regression was 45% in the model group -3,4-dihydrooxypeucedanin hydrate-4-sulfonic acid into oxypeucedanin hydrate during boiling. Additionally, sulfur fumigation (with a sulfur dioxide residue of 767 mg/kg) was found to decrease cytotoxicity without adversely affecting the anti-inflammatory properties of Baizhi. However, further studies are required to verify these results and conclusively depict the effects of sulfur fumigation on Baizhi. Interestingly, we also observed that combining sulfite with other compounds might improve their water solubility. For example, andrographolide total ester sulfonate injection (Xiyanping Zhusheye) and menadione sodium bisulfite, which are treated with sulfite, have been used clinically and are recorded in the Chinese Pharmacopeia (This study demonstrated that sulfur fumigation of Baizhi influences its chemical composition, leading to transformation of the coumarins into dihydrocoumarin sulfonic acids, and conversion of (4macopeia . Thus, sThis study had some limitations. Although the anti-inflammatory effect of Baizhi is not the only pharmacological effect of this TCM, it is considered the most important attribute . Nonethe


Thermoprotei Thermofilum sp. ex4484_79 and subjected to enzymatic and structural characterization. The purified β-glucosidase (TsBGL) exhibited maximum activity at 90°C and pH 5.0 and displayed maximum specific activity of 139.2μmol/min/mgzne against p-nitrophenyl β-D-glucopyranoside (pNPGlc) and 24.3μmol/min/mgzen against cellobiose. Furthermore, TsBGL exhibited a relatively high thermostability, retaining 84 and 47% of its activity after incubation at 85°C for 1.5h and 90°C for 1.5h, respectively. The crystal structure of TsBGL was resolved at a resolution of 2.14Å, which revealed a classical (α/β)8-barrel catalytic domain. A structural comparison of TsBGL with other homologous proteins revealed that its catalytic sites included Glu210 and Glu414. We provide the molecular structure of TsBGL and the possibility of improving its characteristics for potential applications in industries.Beta-glucosidase is an enzyme that catalyzes the hydrolysis of the glycosidic bonds of cellobiose, resulting in the production of glucose, which is an important step for the effective utilization of cellulose. In the present study, a thermostable β-glucosidase was isolated and purified from the  The β-glucosidase (BGL) family (EC 3.2.1.21) comprise a wide range of enzymes that catalyzes the hydrolysis of the glycosidic bond to a nonreducing terminal beta-D-glucosyl residue, releasing glucose . BGLs pl8, β-jelly roll, and (α/α)6 d (α/α)6 . Membersn donors . Thermosn donors .Thermofilum sp. ex4484_79 is a Thermoprotei isolated from hydrothermal deep-sea sediments (Thermoprotei Thermofilum sp. ex4484_79 (TsBGL), characterized its catalytic properties, and explored its crystal structure. Our results provide further insights into the potential industrial use of the key proteins involved in the enzymatic hydrolysis of cellulose.ediments . Most enE. coli strain DH5α  was used as the host for cloning, and E. coli strain BL21 (DE3) plysS  was used as protein expression. E. coli cells were grown at 37°C in LB medium containing 10g NaCl, 10g tryptone, and 5g yeast extract  per liter at pH 7.0, and LB agar medium was added with 1.5–2.0% (w/v) agar.Plasmid pET22 was used as the vector for gene cloning and expression. Multiple alignment of amino acid sequences of homologs was performed using ClustalX v.2 program . Second In vitro site-directed mutagenesis of the TsBGL gene on plasmid pET22 was performed using KOD-Plus-Neo . The primers used are listed (e listed . All theNdeI and XhoI sites to generate the pET22-SUMO-TsBGL expression plasmids (E. coli BL21 (DE3) plysS. Bacteria were grown at 37°C in LB medium containing 34mg·mL−1 chloramphenicol and 50mg·mL−1 ampicillin until the OD600 reached 0.6–0.8. Isopropyl-β-D-thiogalactopyranoside was added at a final concentration of 0.5mm to induce the expression of the recombinant protein. After induction at 25°C for 10h, the cells were collected by centrifugation at 5000×g for 5min at 4°C.The open reading frame (ORF) of TsBGL (GenBank: WP_010868057.1) and His6-SUMO tag was synthesized by GENEWIZ Company and cloned into pET22 plasmid between plasmids . The wilvia two cycles of lysis in an ultra-low temperature cell sandblasting machine at a pressure of 1,200bar, and the cell debris were removed by centrifugation at 13000×g for 40min at 4°C. The supernatant was heated at 65°C for 30min and then centrifuged at 13000×g for 10min to remove the precipitate. After passing the supernatant through a HiTrapTM Ni-NTA column  equilibrated with lysate, the resin was washed with a buffer , and then, a linear elution gradient  was used to elute TsBGL containing six His and SUMO tags. The tags were cleaved by incubating overnight with the Ulpl enzyme at 4°C as described in previous study , disrupted us study . After tp-nitrophenyl-β-D-glucopyranoside. For all enzymatic assays, the N-terminal His6-SUMO tag was removed using Ulp1 to prevent any interference with the enzymatic activity. The reaction mixture (100μl) contained 5μl of appropriately diluted enzyme and 95μl of 0.5mm p-nitrophenyl β-D-glucose (pNPGlc) in sodium acetate buffer (HOAc-NaOAc) pH 5.0. The reaction was stopped by addition of 100μl of 1M Na2CO3 after being incubated at 90°C for 10min. The nitrophenol released was quantified by measuring the absorbance at 410nm (The enzymatic activity of wild-type TsBGL and mutants was determined using a microtiter plate method that measures the hydrolysis of at 410nm . One uni2HPO4 (pH 6.0–8.0), and HEPES-NaOH (pH 8.0–10.0), followed by measuring the activity under conditions of optimal reaction temperature (determined in preliminary experiments). The effect of temperature on enzymatic activity was evaluated by incubating the reaction at a temperature range of 55 to 100°C, and the activity was measured at the optimum pH.The behavior of the enzyme activity as a function of pH was studied by incubating the enzyme and substrate in several suitable buffer systems, i.e., HOAc-NaOAc (pH 3.0–6.0), citric acid-Nap-nitrophenol and GOD-POD (glucose oxidase-peroxidase) methods  and pNPGlc as substrates, respectively.The substrate specificity of TsBGL was determined using different cellulosic polysaccharides, oligosaccharides, and synthetic substrates and tested using the  methods . The enzpNPGlc were determined by varying the concentration (0.1–6.0mm), and those for the natural substrate cellobiose were determined by varying the concentration (1–150mm) under optimal reaction conditions. The Km and Vmax values were calculated by nonlinear regression of the Michaelis–Menten plots with Graphpad Prism 8 , and the apparent kcat values were calculated by assuming all protein was active enzyme.Kinetic parameters using 2, MnCl2, FeCl3, CuCl2, CoCl2, NiCl2, BaCl2, SrCl2, ZnCl2, LiCl2, and EDTA  and in response to n-dodecyl-N, N-dimethylamine-N-oxide (DDAO), n-octyl-β-D-glucoside, n-dodecyl-β-D-maltoside (DDM), trimethylamine N-oxide dihydrate (TMANO), Triton X-100, Tween 20, and SDS . All of the compounds were added to the buffer and substrate; then, the enzyme was added. Results were compared with those of the control sample that was not exposed to these chemicals (with a reference of 100%).The influence of various metal ions and reagents on TsBGL-catalyzed reactions was evaluated by measuring the enzyme activity in the presence of KCl, MgClTo evaluate thermal stability, the enzyme was incubated at different temperatures  for different times  at the optimal pH. The residual activity of the enzyme was determined, considering the activity of the enzyme without pre-incubation was defined as 100%, and used to calculate the enzyme activity, expressed as a percentage of the enzyme activity without pre-incubation during incubation.pNPGlc as the substrate at the optimal pH and temperature, while adding glucose solutions of different concentrations to the reaction system, so that the final concentration in the system was 0.1, 0.25, 0.5, 1, 1.5, 2, and 2.5M, reacted for 10min, and the residual enzyme activity was measured after terminating the reaction.The product inhibition analysis test used Commercial crystallization kits  were used to screen the preliminary results by sitting-drop vapor diffusion in 4×96 conditions at 16°C. The optimal crystallization conditions were 30% (v/v) 2-methyl-2,4-pentanediol, 100mm imidazole/hydrochloric pH 6.5, 200mm ammonium sulfate, and 10% (w/v) PEG 3350. The crystals were quickly immersed in an antifreeze solution composed of 80% stock solution and 20% glycerin and then immediately transferred to a nitrogen-cooled puck. Diffraction data were collected at a wavelength of 0.9792Å at-196°C. The MAR DTB detector was used to image the BL17U beamline of the Shanghai Synchrotron Radiation Facility SSRF; . The HKLThermosphaera aggregans  cells. The target protein TsBGL was purified to 95% homogeneity by Ni-NTA and DEAE columns. The protein concentration was approximately 15mg/ml  showed that TsBGL exhibited a typical Michaelis–Menten behavior with half-saturation constant (Km), maximum velocity (Vmax), and catalytic constant (kcat) values of 0.617mm, 139.2μmol/min/mgenz and 136.05s−1, respectively, for pNPGlc, 6.24mm, 24.3μmol/min/mgenz and 23.8s−1, respectively, for cellobiose, 1.05mm, 127.4μmol/min/mgenz and 124.5s−1, respectively, for pNPGal, and 38.1mm, 7.59μmol/min/mgenz and 7.46s−1, respectively, for lactose 8 barrels were first discovered in triose phosphate isomerase in 1975 and are therefore referred to as a TIM barrel , β2 (Asn77-Ile83), β3 (Lys146-Asn152), β4 (Asp203-Asn209), β5 (Tyr271-Ile278), β6 (Trp335-Tyr340), β7 (Pro409-Glu414), and β8 (Asn445-His451), surrounded by eight α-helices, namely, α1 (Asn59-Ile74), α2 (Asn126-Arg143), α3 (Lys181-Gly199), α4 (Asn232-Asp258), α5 (Arg287-Asn302), α6 (Pro393-Glu405), α7 (Ile424-Lys444), and α8  separated by approximately 5Å, which function as a nucleophile and a proton donor, respectively (TsBGL had the same (β/α)ectively . The preectively . Structuectively ,B. Accorectively , in the ectively . These rectively .Hungateiclostridium thermocellum exhibits maximum activity at 65°C and retain 50% of its activity after incubation at 60°C for 1h, that from Thermoanaerobacterium thermosaccharolyticum DMS 571 exhibits maximum activity at 70°C and retain 50% of its activity after incubation at 60°C for 2h, and that from Caldicellulosiruptor saccharolyticus DSM 8903 exhibits maximum activity at 70°C and retain 50% of its activity after incubation at 70°C for 24h.  of TsBGL was 139.2μmol/min/mgenz for pNPGlc and only 24.3μmol/min/mgenz for cellobiose. We found that cellobiose had a lower Vmax than pNPGlc, which conformed to a model for substrate preference . The PDB ID is 7F1N.The datasets generated for this study can be found in the structure of TsBGL was deposited in Protein data bank  and Science and Technology Research Program of Shanghai (no. 19DZ2282100).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.