Klebsiella pneumoniae at 2.3 Å resolution is presented. Mutational experiments and cell-growth assays confirm Arg28 and Thr51 as critical residues for MazF ribonuclease activity.The first crystal structure of a type II toxin–antitoxin complex structure of  Klebsiella pneumoniae at 2.3 Å resolution is presented. The K. pneumoniae MazEF complex consists of two MazEs and four MazFs in a heterohexameric assembly. It was estimated that MazEF forms a dodecamer with two heterohexameric MazEF complexes in solution, and a truncated complex exists in heterohexameric form. The MazE antitoxin interacts with the MazF toxin via two binding modes, namely, hydrophobic and hydrophilic interactions. Compared with structural homologs, K. pneumoniae MazF shows distinct features in loops β1–β2, β3–β4 and β4–β5. It can be inferred that these three loops have the potential to represent the unique characteristics of MazF, especially various substrate recognition sites. In addition, K. pneumoniae MazF shows ribonuclease activity and the catalytic core of MazF lies in an RNA-binding pocket. Mutation experiments and cell-growth assays confirm Arg28 and Thr51 as critical residues for MazF ribonuclease activity. The findings shown here may contribute to the understanding of the bacterial MazEF TA system and the exploration of antimicrobial candidates to treat drug-resistant K. pneumoniae.Bacterial toxin–antitoxin (TA) systems correlate strongly with physiological processes in bacteria, such as growth arrest, survival and apoptosis. Here, the first crystal structure of a type II TA complex structure of  Two kinds of MazE antitoxins were used in the following experiments. To obtain high-resolution diffracted crystals, MazE truncated from Lys45 to Leu76 was used for crystallography. Thus, the primers used for PCR were as follows: truncated MazE, forward, 5′-G GAA TTC CAT ATG AAA GCT GGC CCG ACG C-3′, and reverse, 5′-CCG CTC GAG TTA CAG CAT CTC CTT ACC-3′; MazF, forward, 5′-G GAA TTC CAT ATG ACG ACA TAT TGT CCA G-3′, and reverse, 5′-CCG CTC GAG TTA TGC TTT AAT AAT TTT TG-3′. Nde1 and Xho1 were used as restriction enzymes for cloning, and the restriction sites are underlined above. The PCR products of MazE and MazF were doubly cleaved by Nde1 and Xho1 and ligated into vectors that had been cleaved by the same enzymes. For structure determination, MazE and MazF were ligated into pET28a and pET21a with no tags, respectively. For the biological assay, MazF was ligated into pET28a with an additional residual tag whose amino-acid sequence was MGSSHHHHHHSSGLVPRGSH. Each plasmid was then transformed into Escherichia coli DH5α competent cells.The genes encoding 2.2.K. pneumoniae MazEF complex, plasmids containing the MazE antitoxin and MazF toxin genes were cotransformed into E. coli Rosetta2 (DE3) pLysS competent cells. The cells were grown in Luria broth at 37°C until the OD600 of the cell culture reached 0.6. Isopropyl 1-thio-β-d-galactopyranoside  was added for target-protein overexpression, and the cells were further incubated at 37°C for 4 h. The cultured cells were harvested by centrifugation at 11 355g at 10°C and stored in a −82°C deep freezer. The harvested cells were then suspended in buffer A  containing 10%(v/v) glycerol and lysed by ultrasonication. After centrifugation at 28 306g for 1 h, the supernatant containing soluble proteins was filtered using a 0.45 µM syringe filter (Sartorius) and loaded onto an affinity chromatography column of nickel-nitrilotriacetic acid-agarose (Bio-Rad) that had been equilibrated with buffer A. The column was washed with buffer A containing 50 mM imidazole, and the target protein MazEF bound to Ni2+ resin was eluted using an imidazole gradient (100–700 mM). For further purification of MazEF using ion-exchange chromatography, the elution buffer was exchanged with buffer B , and the eluted protein was loaded onto a HiTrap Q column  pre-equilibrated with the same buffer. The protein was then eluted with a NaCl gradient (0–1000 mM). As the final purification step, size-exclusion chromatography (SEC) on a HiLoad 16/600 Superdex 200 prep-grade column was used for buffer exchange to final buffer  and for conducting the final MazEF purification. In the above experimental steps, the target protein was identified using SDS–PAGE and concentrated using an Amicon Ultra centrifugal filter unit (Millipore). Finally, 7.5 mg ml−1 MazEF was used for crystallization.For the structure determination of the E. coli Rosetta2 (DE3) pLysS competent cells. However, the cells were incubated for only 1 h after IPTG induction because of the toxicity of MazF to E. coli. The purification procedures were the same as for the MazEF complex, except that ion-exchange chromatography was omitted for MazF toxin since no contamination appeared in the purification of MazEF. The final buffer containing MazF was 20 mM Tris, pH 8.0, 200 mM NaCl.For the MazF ribonuclease-activity assay, the MazF toxin was overexpressed in 2.3.−1 in 20 mM Tris, pH 8.0, 50 mM NaCl with 0.5 µl of reservoir solution. Crystals of the MazEF complex were grown using the sitting-drop vapor-diffusion method at 20°C. The crystallization solution for the MazEF complex was 5%(w/v) PEG 1000, 100 mM sodium phosphate dibasic/citric acid at pH 4.2 and 40%(v/v) reagent alcohol. The crystal was transferred to cryoprotectant solution containing 20%(v/v) glycerol in the reservoir prior to mounting. Furthermore, the crystals were cooled using liquid nitrogen immediately prior to data collection. The diffraction data were collected using the ADSC Quantum Q270r CCD detector at the 5C and 11C beamlines of Pohang Light Source, Republic of Korea. The crystals of the MazEF complex belong to the triclinic P1 space group, with unit-cell parameters of a = 41.558, b = 45.501, c = 69.952 Å, and α = 102.06, β = 93.60, γ = 116.36°. The calculated total mass of the MazEF complex, including the N-terminal (His6) tag, was 29.8 kDa. All raw data were scaled and processed using HKL2000 ; Kamada et al., 20031/2 > 0.9 for the resolution cutoff. Therefore, the resolution of data used in this study is 2.3 Å. However, since several residues from the MazE antitoxin were not correctly matched in the electron-density map, the subsequent manual model building of MazEF was conducted using Coot  was used to generate all of the structural figures  and Index 1 and 2 (Hampton Research) kits, by mixing 0.5 µl of protein solution at 7.5 mg ml2.4.M. Metal-dependent ribonuclease activity was also investigated using different kinds of metal ions, such as Mg2+, Ca2+, Mn2+ and Zn2+. The concentrations of MazF toxin and metal ions were fixed at 4 and 50 µM, respectively. All experiments were performed in triplicate.To validate the ribonuclease activity of MazF toxin, an RNase alert kit (IDT) was used. The principle is as follows. In the normal state, a fluorophore is covalently attached to one end of a synthetic RNA strand and is quenched by a quencher group at the other end of the RNA strand. However, the synthetic RNA is digested and the quencher is released when the ribonuclease interacts with substrates. Then, the released fluorophore emits fluorescence at 520 nm upon excitation at 490 nm. The resulting relative fluorescence units (RFU) were detected using a SPECTRAmax GEMINI XS spectrofluorometer (Molecular Devices). We first investigated the concentration-dependent ribonuclease activity of MazF, whose concentration was gradually increased with values of 0.5, 1, 2, 4 and 10 µ2.5.For the mutational study of the key catalytic residues of MazF, Arg28, Thr51, Lys79 and Arg86 were mutated to alanine. The resulting mutant proteins were designated R28A, T51A, K79A and R86A. Mutation was conducted using the EZchange site-directed mutagenesis kit  according to the manufacturer’s protocol. Mutated MazF proteins were expressed and purified using the same procedures as were used for the native proteins.2.6.E. coli strain Rosetta (DE3) pLysS. Transformed cells from single colonies grown on 0.1% glucose-containing M9 medium plates were grown overnight, and the overnight cultures were diluted to an OD600 of 0.1. The diluted cells were further grown until the OD600 of the cell suspension reached 0.3, at which time 0.5 mM IPTG was added to induce protein expression. The cells were incubated at 37°C for 6 h after induction by IPTG and monitored at 1 h intervals.For the cell-growth assay, the plasmids expressing MazF, R28A, T51A, K79A and R86A, were transformed into 3.3.1.K. pneumoniae was determined at a resolution of 2.3 Å. The asymmetric unit of the MazEF complex is composed of two MazE antitoxins and four MazF toxins in a heterohexameric assembly [Fig. 1a)]. By comparison with reference proteins in gel-filtration calibration kits  using SEC, the predicted molecular weights of both truncated and untruncated MazEF complexes in solution were obtained. In SEC, untruncated MazEF elutes at ∼12.97 ml, while truncated MazEF elutes at ∼15.11 ml [Fig. 1b)]. These values correspond to molecular masses of 150.878 and 55.91 kDa, respectively, using the calibration curve for several standard proteins [Fig. 1c)]. Because the theoretical molecular weights of the untruncated and truncated heterohexameric MazEF complexes are 73.9 and 59.5 kDa, respectively, it can be estimated that MazEF forms a dodecamer with two heterohexameric MazEF complexes in solution, and the truncated complex exists in heterohexameric form.The crystal structure of the MazEF complex from a)]. The MazE monomer interacts with the MazF homodimer via its long C-terminus loop, which wraps around the MazF dimer and extends further towards the empty edge between MazF monomers.In the heterotrimeric subunit of MazEF, the MazE monomer was positioned between symmetrically arrayed MazF dimers [Fig. 2PISA ]. Furthermore, the residues Leu14, Ala30, Val47, Pro49 and Leu81 in MazF (chain F) make hydrophobic interactions with Leu76 (chain D) in MazE by forming a hydrophobic core, which could only be observed between chain D and chain F owing to the lack of electron density for Leu76 in chain A [Fig. 2b)].The MazE antitoxin interacts with the MazF toxin via two binding modes, namely, hydrophobic and hydrophilic interactions. The calculation of surface area and analysis of interactions between MazE and MazF were conducted using a)]. In addition, 13 salt bridges are formed between MazE (chain A) and MazF (chain B) and three salt bridges are formed between MazE (chain A) and MazF (chain C). Furthermore, the interface area of chain A and chain B is 1123.8 Å2 and the interface area of chain A and chain C is only 549.8 Å2. Therefore, it can be inferred that chain A and chain B have stronger binding capacity than chain A and chain C.Additionally, the C terminus of MazE contributes to the hydrophilic interaction between MazE and MazF, with several residues involved in hydrogen bonding or salt bridges. In detail, the following residues are necessary for forming hydrogen bonds: Glu65, Tyr66, Asp69, Ser70, Lys73, Glu74 and Met75 of MazE (chain A); Asn17, Pro18, Gly43, Thr51, Gln77, Lys79, Asp82 and Arg86 of MazF (chain B); and Asn17, Lys79, Asp82 and Arg86 of MazF (chain C) .The structure of 2. Although hydrogen bonds produce a certain influence on the formation of the dimeric interface, the main driving forces of MazF dimerization are hydrophobic interactions. In detail, a hydrophobic core is formed around the α3-helices, which are located facing each other. In this hydrophobic core, the α3-helix of one MazF monomer contacts the α3-helix, α1-helix and β3-strand of the other proximal monomer. Nine residues are involved in this interaction between two MazF monomers, among which Val46 and Ile110 are the key residues necessary for the dimerization of MazF. Ile110 in the α3-helix is involved in hydrophobic interactions with Val33, Val46, Val103 and Val106. In addition, Ile46 in the β3-strand participates in hydrophobic interactions with Val78, Ile109 and Ile110 [Fig. 3b)].Overall, more than 15 residues are involved in the dimerization of MazF. The homodimeric interface of MazF buries an area of 911 ÅDALI      , with an r.m.s. deviation of 2.5 Å, a Z score of 12.9 and a sequence identity of 28%].To compare the tertiary structures of MazF toxin and its homologs, the Z scores with its structural homologs. The structural-alignment results show that the tertiary structures of the homologs approximately overlap, with the exception of loops β1–β2, β3–β4 and β4–β5 [Fig. 3c)]. Thus, it can be inferred that these three loops have the potential to represent unique characteristics of MazF, especially various substrate recognition sites.Interestingly, although they are from different kinds of bacterial strains, MazF toxin shows high et al., 2017a)]. In the closed state, loop β1–β2 extends to the adjacent toxin monomer to conceal the adjacent region where the MazE antitoxin binds. In contrast, binding of the MazE antitoxin not only displaces the β1–β2 linker to induce an open state but also occupies ‘Site A’ of the MazF homodimer. As a result, the interactions between MazF monomers are disrupted, especially in the ‘TA interface pocket’  ]. Thus, the basic principles by which these structural homologs recognize specific RNA via loop β1–β2 are somewhat different. In detail, when MazF homologs with a long loop β1–β2, such as K. pneumoniae MazF, E. coli MazF and B. subtilis MazF in ‘closed’ states, the TA interface pocket is concealed owing to the extended interactions of the relatively long loop β1–β2, which was mentioned above. As a result, the RBP exists only on the left ‘hemisphere’ of the MazF homodimer [Fig. 4a)]. In contrast, loop β1–β2 of M. tuberculosis MazF4 is too short to close the corresponding pocket completely and hence exposes negatively charged binding pockets ]. The sequence of K. pneumoniae MazF was aligned with those of five structural homologs from different types of bacterial strains, which were mentioned in the previous subsection. The results revealed several highly conserved residues: Gly9, Gly26, Arg28, Pro29, Asn39, Thr51, Asp76 and Gln77. These residues can be divided into three categories via their mechanism of action in MazF: (i) Gly9, Gly26 and Asn39; (ii) Pro29, Asp76 and Gln77; and (iii) Arg28 and Thr51. In detail, residues in category (i) are related to the formation of the tertiary structure of MazF, while those in categories (ii) and (iii) are involved in interacting with RNA substrates. For example, the roles of glycines correspond to hydrogen-bonded turns of the loops and asparagines improve the stability of α1-helices. Since the K. pneumoniae MazEF crystal structure was not determined with the RNA substrate bound, it was easier to illustrate the roles of the residues in categories (ii) and (iii) by superimposing K. pneumoniae MazF with the RNA substrate-bound structures of E. coli MazF (PDB code 5cr2)    [Fig. 5b)]. Although different constructs of RNA substrates were used for crystallization, each of them contained the common RNA sequence (UACAU). Among them, Pro29 makes hydrophobic contact with the uracil base (1U) of the substrate through interaction with its side chain. Therefore, it is the most important residue defining substrate specificity  of the specific RNA sequence via hydrophilic interactions. A previous study reported that MazF became inactive in vivo when aspartate and glutamine were mutated to alanine. Additionally, isothermal-titration-calorimetry results revealed that these mutations resulted in weaker RNA-binding affinity compared with wild-type (WT) protein ].Furthermore, Arg28 and Thr51 are highly conserved in several structural homologs and are located on either side of the substrate in the RBP [Fig. 53.4.K. pneumoniae MazF was confirmed by the increase in the resulting fluorescence (RFU) with increasing concentration of the MazF monomer protein from 0.5 to 10 µM. The reaction was saturated at concentrations greater than 4 µM [Fig. 6a)]; thus, the concentration of MazF was fixed at 4 µM in all of the following RNase-activity assays. Furthermore, different kinds of divalent metal ions had no great effect on the RNase activity of MazF [Fig. 6b)]. This means that the catalytic process of MazF is independent of metal ions, whereas the catalytic processes of other ribonuclease proteins, such as VapC 26 and VapC 30, in M. tuberculosis are highly mediated by divalent metal cations  were designed. Among them, Lys79 and Arg86, which are far from the active site, are only involved in hydrophilic interaction between MazE and MazF. As a result, two mutants of K. pneumoniae MazF (R28A and T51A) showed a significant reduction in ribonuclease activity in vitro compared with mutants (K79A and R86A) of MazF and WT MazF [Fig. 6c)]. In addition, the results of the bacterial-growth assay revealed that expression of R28A and T51A MazF mutants in E. coli Rosetta (DE3) pLysS results in normal growth after IPTG induction, whereas K79A and R86A MazF mutants and WT MazF inhibit cell growth [Fig. 6d)]. This demonstrates that Arg28 and Thr51 play a critical role in K. pneumoniae MazF ribonuclease activity.To demonstrate that Arg28 and Thr51 play a critical role in et al., 2017K. pneumoniae MazF could be identified by comparison with its structural homologs. Among them, Arg28 and Thr51 are the key residues involved in RNA catalysis. As a result, the mechanism of action of K. pneumoniae MazF is different from that of other ribonucleases, such as RNase H and VapC toxin, which are magnesium-dependent ribonucleases. For RNase H, a water molecule coordinated by Mg2+ begins a nucleophilic attack on a scissile phosphate of the RNA strand, forming a pentacovalent intermediate. After that, another water molecule reprotonates the 3′-OH group, resulting in the formation of 5′ phosphate and 3′-OH groups (Yang et al., 2006K. pneumoniae MazF, following the buildup of the negatively charged phosphate on the cleavage site stabilized by Thr51, the product, namely, 2′,3′-cyclic phosphate, is formed via a Grotthuss-like mechanism (Zorzini et al., 2016K. pneumoniae MazF indeed affected its ribonuclease activity, while the alanine mutant of the corresponding threonine showed only a small decrease in ribonuclease activity in some other MazFs (Ahn et al., 2017et al., 2017K. pneumoniae MazF contributes to catalysis.In general, MazF homologs adopt a PemK-like fold that cleaves single-stranded mRNA transcripts (Hoffer 4.K. pneumoniae MazEF, we were able to both analyze the tertiary structure of the full-length MazF toxin and clearly identify the binding interface of MazEF. Loop β1–β2 was found to play the most important role in MazF, as it functions as a gate to modulate an open to closed state. When MazE binds with MazF, it not only displaces the β1–β2 linker to induce an open state but also occupies Site A of the MazF homodimer, resulting in blockage of the downstream region of the RNA-binding site. In addition, Arg28 and Thr51, which lie in the RBP, are the key residues for the catalytic reaction. The results for the binding interface provide valuable information for designing peptides or small molecules to artificially disrupt the MazEF complex. If we could design substances that display high affinity for the TA interface pocket, free MazF toxin that possesses a naked site A and an active site will be produced, resulting in ribonuclease activity. In conclusion, the tertiary structural information reported here will contribute to the exploration of antimicrobial candidates to treat drug-resistant K. pneumoniae.Based on the high-resolution crystal structure of Klebsiella pneumoniae MazEF, 7byePDB reference:


Syzygium nervosum (Ma-Kiang in Thai). Microwave-assisted extraction (MAE), which utilizes microwave radiation to heat the extraction solvent quickly and effectively, was used to recover DMC-rich extract from Syzygium nervosum fruit. To determine the DMC content, a highly accurate and precise HPLC technique was developed. The influences of MAE conditions, including the solid–liquid ratio, microwave power, and microwave duration on the content of DMC, were sequentially employed by a single factor investigation and response surface methodology (RSM) exploratory design. The predicted quadratic models were fitted due to their highly significant (p < 0.0001) and excellent determination coefficient (R2 = 0.9944). The optimal conditions for producing DMC-rich extract were a ratio of sample to solvent of 1:35 g/mL, a microwave power of 350 W, and a microwave time of 38 min. Under the optimal MAE setting, the DMC content reached 1409 ± 24 µg/g dry sample, which was greater than that of the conventional heat reflux extraction (HRE) (1337 ± 37 µg/g dry sample) and maceration (1225 ± 81 µg/g dry sample). The DMC-rich extract obtained from MAE showed stronger anticancer activities against A549 (human lung cancer cells) and HepG2 (human liver cancer cells) than the individual DMC substance, which makes MAE an effective method for extracting essential phytochemicals from plants in the nature.2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethyl chalcone (DMC) is a biological flavonoid that is present in the fruits of  Syzygium is a genus in the Myrtaceae family with over 1200 species worldwide. It is widely distributed in tropical Africa, Asia, Australia, New Caledonia, New Zealand, and the Pacific Islands, among other places .The precision method was assessed using QC sample of DMC standard . Assay of five samples of each concentration was used for assay intra-day precision. Daily assays of the samples for five days were examined for inter-day precision. The relative standard deviation (RSD) values of DMC content in five replicates were determined. The accuracy of method was investigated by spiking standards of DMC in extract solution at three different concentration levels  and calculating the percent recovery .S. nervosum fruits to ethanol was fixed at 1:35 (g/mL), and samples were taken at 20 h at room temperature (25 °C). HRE was also conducted under reflux, the solid-to-liquid ratio was 1:35 g/mL, and the temperature was fixed during the extraction at the ethanol boiling point for 2 h [Maceration was performed in a 500 mL Erlenmeyer flask covered with aluminum foil to minimize evaporation and degradation. Ethanol was chosen as the solvent, the ratio of  for 2 h . The extp < 0.05) with SPSS software, version 17.0 .Each DMC extraction conditions was performed three times, and the measured DMC contents were represented as their mean ± SD. Statistical variations among DMC extraction conditions were estimated using one-way analysis of variance (ANOVA), and significant differences were determined using post hoc testing  and HepG2 (human liver cancer cells) cells were generously supported by Dr. Ratchada Cressey and Dr. Preeyanat Vongchan ), respectively. A549 and HepG2 cells were grown in Dulbecco’s modified Eagle medium (DMEM) containing 10% of fetal bovine serum (FBS) and antibiotics (100 µg/mL of streptomycin and 100 U/mL of penicillin) until reaching 80% confluence. This stage of cells was used for cytotoxicity testing by MTT assay. Besides cancer cells, human peripheral blood mononuclear cells (PBMCs) were also used as normal control cells. PBMCs were prepared from 3 healthy donors by Ficoll-Hypaque gradient centrifugation and cultured in RPMI medium containing 10% of FBS and antibiotics (100 µg/mL of streptomycin and 100 U/mL of penicillin).4 cells/well) or human PBMCs (1 × 105 cells/well) were introduced to a 96-well tissue culture plate. DMC-rich extract and individual DMC were dissolved in dimethyl sulfoxide (DMSO) and diluted to desire concentrations. Different concentrations of extracts were added into each well. Cells were then incubated at 37 °C in a 5% CO2 incubator for 24 or 48 h. After incubation, 20 µL of MTT solution  was added into each well. The plate was then further incubated at 37 °C for another 4 h. Culture medium was drained out and the formazan dye crystal was dissolved with 100 µL of DMSO. Absorbance was determined at 540 nm by using microplate reader. Three independent measurements were used to determine each result. The cell viability data were represented as a percentage when compared to the control group (100%).The 3--2,5-diphenyltetrazolium bromide (MTT) assay was applied to investigate the cytotoxic effect of DMC-rich extract and individual DMC on cancer (A549 and HepG2) and normal (human PBMCs) cells. Cancer (1 × 10Syzygium nervosum fruits using ethanol as the solvent. The highest extraction efficiency was obtained (1409 ± 24 µg/g dry sample) when the sample-to-solvent ratio, microwave power, and microwave time were 1:35 g/mL, 350 W, and 38 min, respectively. The acquired extraction DMC content, according to the current MAE approach, was greater than HRE (1337 ± 37 µg/g dry sample) and maceration (1225 ± 81 µg/g dry sample). The DMC-rich extract obtained from MAE shows stronger anti-cancer activity than the individual DMC compound; hence, the combined actions of the phytochemicals may be partly responsible for the activities. Both compounds were not toxic to normal human PBMCs, indicating that they were specific to A549 and HepG2 cells. Due to its reduction in the process time and solvent waste, the MAE approach is considered as an efficient alternative procedure for the extraction of DMC when compared to its traditional maceration method.Microwave-assisted extraction (MAE) is successfully applied for the separation of DMC-rich extract from


Some superfamily members can also act as esterases using p-nitrophenyl esters as substrates. The ALDHTt from Thermus thermophilus was recombinantly expressed in E. coli and purified to obtain high yields (approximately 15–20 mg/L) and purity utilising an efficient heat treatment step coupled with IMAC and gel filtration chromatography. The use of the heat treatment step proved critical, in its absence decreased yield of 40% was observed. Characterisation of the thermophilic ALDHTt led to optimum enzymatic working conditions of 50 °C, and a pH of 8. ALDHTt possesses dual enzymatic activity, with the ability to act as a dehydrogenase and an esterase. ALDHTt possesses broad substrate specificity, displaying activity for a range of aldehydes, most notably hexanal and the synthetic dialdehyde, terephthalaldehyde. Interestingly, para-substituted benzaldehydes could be processed efficiently, but ortho-substitution resulted in no catalytic activity. Similarly, ALDHTt displayed activity for two different esterase substrates, p-nitrophenyl acetate and p-nitrophenyl butyrate, but with activities of 22.9% and 8.9%, respectively, compared to the activity towards hexanal.Aldehyde dehydrogenases (ALDH), found in all kingdoms of life, form a superfamily of enzymes that primarily catalyse the oxidation of aldehydes to form carboxylic acid products, while utilising the cofactor NAD(P) ALDHs exist as dimers [+ cofactor to form the corresponding carboxylic acid and NAD(P)H. Additionally, some ALDHs can act as esterases [+; (4) hydrolysis of the resulting thioester; and (5) dissociation of the reduced cofactor with concomitant regeneration of the enzyme via NAD(P)+ binding [p-nitrophenyl ester substrate creating an oxyanion intermediate; (3) the intermediate rearranges and the nitrophenol group leaves; (4) Glu268 abstracts hydrogen from an ordered water molecule making it a nucleophile and attacks the carbonyl in the thioacyl enzyme complex; and (5) formation of the tetrahedral intermediate and release of the carboxylic acid and free enzyme [Found in all kingdoms of life, aldehyde dehydrogenases (ALDH) EC;1.2.1.3) constitute a large family of NAD(P).2.1.3 cos dimers ,8. Theirsterases ,10 or casterases , indicatsterases ,13. The + + . PreviouALDHs are often not substrate specific, and can oxidise a wide range of aliphatic and aromatic, endogenous and exogenous aldehydes ,17,18, wE. coli enzymes phenylacetaldehyde dehydrogenase and lactaldehyde dehydrogenase were characterised after the ALDH1, 2, and 3 from humans [Geobacillus thermoleovorans (ALDHGtv) and betaine biosynthesis in E. coli [While ALDHs have been well-characterised in humans, emerging research places a primary focus on their prokaryotic counterpart’s structure and function, which has been less explored until recently ,30,31. Tm humans , and are E. coli ,35. Rece E. coli . Interes E. coli ,37, high E. coli . Geotrichum candidum (ALDHGc) displayed catalytic activity with an extensive range of aldehyde substrates [Gc demonstrated conserved or increased specific activity for a spectrum of para-substituted benzaldehydes, short to medium chain aliphatics , and dialdehydes, including terephthalaldehyde with respect to benzaldehyde. Thermophilic ALDHs have also proven to have broad substrate specificity. ALDHGtv can process medium to long chain aldehydes, while ALDH from Geobacillus thermodenitrificans (ALDHGtd) and Pseudomonas putida (ALDHPp) exhibited activity for 15 and 21 different aromatic and aliphatic substrates respectively, working optimally at 60 °C [Pp is highly hydrophobic, linked to the enzyme preferring non-polar substrates and converting them more efficiently, e.g., acetaldehyde is the most inefficiently processed. ALDHPp could even process the pyrene ring derived pyrene-1-carboxaldehyde. Many ALDHs possess broad substrate specificity, and, in some cases, the true biological substrate(s) has not been identified. Human ALDH1 family members are primarily characterised as retinaldehyde oxidising isozymes. A recent study demonstrated that ALDH1A members could process medium chain aliphatic compounds as well as retinaldehyde . The ALDbstrates . ALDHGc at 60 °C ,39. The 2O in combination with sodium cyanide, besides complicated reaction protocols. Biocatalytic methods employing enzymes are attractive due to the use of mild reaction conditions coupled with the regio- and chemo-selectivity. Investigation of ALDHs in biocatalysis has only been marginally investigated, but their exquisite chemoselectivity and broad substrate scope makes them attractive candidates [ALDH enzymes have the potential to act as a biocatalytic route for carboxylic acid synthesis . Traditindidates ,41,42. Andidates . Tt was resolved revealing a tetrameric organisation  and aligned in multiple sequence format using the Clustal Omega multiple sequence alignment tool . The protein sequence alignments were visualised using GeneDoc software. The basic local alignment search tool (BLAST) was carried out for ALDHTt utilising the NCBI’s protein BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi accessed on 9 December 2021). Sequences for alignment were obtained from NCBI for ALDHTt (6FJX), human ALDH1A3 (5FHZ) and succinate semialdehyde dehydrogenase (SS-ALDH) from E. coli (3JZ4).Protein sequences were retrieved from the NCBI database   competent cells (Invitrogen). Expression of ALDHTt was performed in ZYP-5052 auto induction media, supplemented with ampicillin (50 μg/mL), and inoculated with 1% (v/v) of overnight culture of transformed cells in LB broth grown at 37 °C. Expression was carried out for 48 h at 25 °C, 200 rpm, with subsequent cell collection at 6000× g, 4 °C for 15 min. The cell pellet was resuspended in lysis buffer containing 20 mM Tris-HCl pH 7.5, 5 mM β-mercaptoethanol, 10 mM imidazole, and 500 mM NaCl, with 5 mL added per g of cells. The lysis buffer was supplemented with 0.25 mg/mL lysozyme, 20 μg/mL DNase I, and 200 mM MgCl2, and frozen at −80 °C overnight. Following overnight freezing, the cells were thawed, sonicated, heat treated at 65 °C for 15 min, and collected by centrifugation at 17,000 rpm, 4 °C for 30 min. The soluble fraction was filtered through a 0.45 μm nylon filter and loaded onto an XK 16/20 column containing chelating Sepharose fast flow activated with 0.2 M NiSO4, and pre-equilibrated with 20 mM Tris-HCl pH 7.5, 5 mM β-mercaptoethanol, 10 mM imidazole, and 200 mM NaCl for affinity chromatography. Bound proteins were eluted using a step gradient of imidazole at concentrations of 50, 100, 200, and 500 mM in 20 mM Tris-HCl, 5 mM β-mercaptoethanol and 150 mM NaCl, pH 7.5. Fractions containing the ALDHTt were dialysed overnight at 4 °C against 50 mM Tris-HCl, 5 mM β-mercaptoethanol and 250 mM NaCl, pH 7.5, and concentrated using Amicon Ultra-15 centrifugal filters, 50 kDa MWCO (Merck Millipore). Approximately 1 mL of concentrated protein was loaded onto a HiLoad 16/60 Superdex 200 pg column, pre-equilibrated with 50 mM Tris-HCl pH 7.5, 5 mM β-mercaptoethanol and 150 mM NaCl. The eluted fraction was concentrated to a desired concentration (25–30 mg/mL), snap-frozen in liquid nitrogen, and stored at −80 °C until further use. The construct DNA pET-22b(+)-ALDHTt  was tranTt was coupled with an anti-6xhis-HRP antibody, followed by colorimetric detection utilising the substrate 5-Tetramethylbenzidine (TMB). Protein samples were run on a 12% SDS-PAGE gel, stained using Instant Blue (Sigma) and destained in deionised water overnight. For Western blot, proteins were transferred from an unstained gel to nitrocellulose membrane at 80 mAmp for 90 min, followed by subsequent blocking and washing steps. The ALDHTt was confirmed by matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry using α-cyano-4-hydroxycinnamic acid (HCCA) as the matrix. The ALDHTt (31.12 mg/mL) was diluted 1 in 30 in water to minimise the presence of salts before analysis. A 1 µL aliquot of protein solution was added to 1 µL of the HCCA matrix, with 1 µl of sample then added to the steel sample plate. The sample plate was inserted into the MALDI-TOF Ultraflex mass spectrometer (Bruker) and run in linear cationic 5–60 kDa mode, using 2500 shots. The theoretical molecular mass was determined using Expasy online molecular weight calculation tools (https://web.expasy.org accessed on 1 November 2021) and amino acid sequencing of ALDHTt (6FJX) from NCBI.The molecular mass of the ALDHTt was determined spectrophotometrically at 340 nm by measuring the increase in absorbance due to the production of NADH by the enzyme. The reaction mixture was analysed in a plastic cuvette at 50 °C and consisted of 2 mM NAD+, 40 µL of 0.38 mg/mL ALDHTt, 10 mM potassium phosphate at a pH of 8, and 2 mM hexanal in a final reaction volume of 1.8 mL, unless otherwise stated. All ALDHTt reactions were continuously monitored for 2 min at 340 nm. The activity of ALDHTt was examined over a temperature range from 20 to 50 °C; higher temperatures were not utilised due to the volatility and flammability of the aldehyde substrates. All solutions (with the exception of the enzyme) were heated to the appropriate temperature in a water bath before use. Measurements were taken utilising a Cary60 UV-vis spectrophotometer equipped with a temperature controller. The activity of ALDHTt was also examined over a pH range of 2–10 using various buffers; pH 2 (10 mM potassium chloride-HCl buffer); 3−5 (10 mM citrate buffer); 6–8 (10 mM potassium phosphate); and 9–10 (10 mM Tris-HCl buffer).The catalytic activity of ALDHTt to first identify the substrate scope of the enzyme. Once conversion of both substrates was identified, in silico screening of aldehyde substrates was carried out, resulting in a library of 12 aliphatic, cyclic and aromatic aldehydes  in a final reaction volume of 1.8 mL. All enzymatic assays were continuously monitored for 2 min.Model aliphatic   and aromldehydes  for use Tt for the reduction of carboxylic acids to aldehydes utilising NADH was investigated. The reduction reaction was determined spectrophotometrically by monitoring the decrease in absorbance at 340 nm due to the conversion of NADH to NAD+. The reaction mixture consisted of 10 mM potassium phosphate pH 8, 40 µL of 0.38 mg/mL ALDHTt, 150 µM NADH and either hexanoic acid (2 mM) or terephthalic acid (0.12 mM), with the reaction monitored for 5 min at 50 °C.The action of ALDHMichaelis-Menten kinetics analysis was carried out for hexanal, benzaldehyde, and terephthalaldehyde, using the assay conditions outlined above at 25 and 50 °C varying substrate concentration. The ranges of concentrations used were as follows: 0.1–2 mM hexanal, 0.2–3.5 mM benzaldehyde, and 0.025–1 mM terephthalaldehyde.+ was carried out using 2 mM hexanal at 25 and 50 °C. The NAD+ concentration varied from 1–200 µM.Michaelis–Menten kinetics analysis for NADTt, the model substrate hexanal was utilised for conversion to hexanoic acid with direct detection of the carboxylic acid product via HPLC. An Agilent 1260 Infinity Series  was used for HPLC analysis and the acquired data was processed with the Agilent OpenLAB CDS software. Chromatographic separations for the detection of hexanoic acid from ALDHTt enzymatic assays were carried out using an Agilent Microsorb-MV 100-5 C18 column (250 × 4.6 mm). The system was maintained at 30 °C with a run time of 10 min. The mobile phase which comprised of A: 10 mM potassium phosphate, phosphoric acid at a pH of 2.4, and B: HPLC gradient grade acetonitrile   was delivered to the column at a flow rate of 1 mL/min, which yielded a column back pressure of ~190 bar. Samples were filtered through 0.45 μm nylon filters and 60 μL injections were made. UV detection was conducted at a wavelength of 210 nm.To further demonstrate the catalytic mechanism of the ALDHTt was purified in the absence of β-mercaptoethanol for esterase activity assessment in order to prevent artefactual catalysis of the ester. Following the nickel affinity chromatography step, the fractions containing ALDHTt were dialysed overnight against 50 mM Tris-HCl, 250 mM NaCl, pH 7.5, and subsequently loaded onto a HiLoad 16/60 Superdex 200 pg column pre-equilibrated with 50 mM Tris-HCl with a pH of 7.5, and 150 mM NaCl, snap frozen in liquid nitrogen and stored at −80 °C. To facilitate direct comparison of the esterase and dehydrogenase reaction rates, both activities were determined using protein samples purified without β-mercaptoethanol. The reactions were conducted in 10 mM potassium phosphate pH 8.0 at 25 °C unless otherwise stated. Two ester substrates were examined with ALDHTt at concentrations of 1 mM for p-nitrophenyl acetate (PNP-acetate) and 500 µM of p-nitrophenyl butyrate (PNP-butyrate). Standard solutions of PNP-acetate were prepared with acetone as a solvent to minimise spontaneous hydrolysis. Stock concentrations were selected so that the acetone concentration in the assay solutions did not exceed 0.1%. When direct comparison between the two activities was desired, a comparable acetone concentration was added to the standard assay system for dehydrogenase activity using hexanal. Esterase activity was monitored spectrophotometrically at 400 nm by measuring the increase in absorbance due to the production of the p-nitrophenoxide ion by the enzyme. All esterase data was corrected for the minor rate of spontaneous hydrolysis of substrate in the absence of enzyme (blank rate). A pH dependent extinction coefficient of 17 × 103 M−1cm−1 was calculated via a calibration curve of p-nitrophenol in 10 mM potassium phosphate pH 8, 0.1% acetone (pKa = 7.1) (ALDHa = 7.1) .Tt was overexpressed in E. coli BL21(DE3), and purified to apparent homogeneity using IMAC and gel filtration chromatography. Enzymatic activity assessments were carried out to determine the enzyme’s optimum pH and temperature for catalysis. In addition, 12 aldehyde substrates were tested for efficient conversion by the ALDHTt, with eight aliphatic, aromatic, and dialdehydes demonstrating catalytic activity. This sample pool of substrates demonstrated that the ALDHTt is not substrate specific, and indicates the capabilities of ALDHTt in terms of its substrates scope. ALDHTt was also examined for esterase activity demonstrating conversion of PNP-acetate and PNP-butyrate reliant on the absence of cofactor. Esterase activity occurred at a much lower rate than dehydrogenase activity, a common characteristic of ALDHs.The 59 kDa his-tagged ALDHTt allowed for determination of its closest related homolog based on sequence identity. The putative ALDH from the thermophile Thermus parvatiensis with 529 amino acids resulted in a sequence identity of 97.92%, and is proposed to display a C-terminal extension characteristic of ALDHTt. The highest related homologs (86–97% sequence identity) were all members of the Thermus genus, but none have been characterised to date. The well-characterised human ALDH1A3 returned a sequence identity of 39.03% in relation to ALDHTt  at 25 °C for 48 h , and gel filtration chromatography yielding approximately 15–20 mg/L of culture. During Ni affinity chromatography, ALDHTt eluted at 200 mM imidazole demonstrating substantial purity and efficient separation from host cell proteins  and amino acid sequence (6FJX) from NCBI. MALDI-TOF analysis of the ALDHTt returned a molecular mass of 59.393 kDa  (The optimum operation conditions of the ALDHctively) a. The opctively) b. The acTt has recently been reported [Tt can oxidise aliphatic, aromatic, and dialdehydes, specifically, hexanal, propanal, acetaldehyde, benzaldehyde, p-tolualdehyde, trans-cinnamaldehyde, and terephthalaldehyde, at reasonable rates of oxidation . The sTt follows Michaelis–Menten type mechanism, and the kinetic parameters  for hexanal, benzaldehyde, and terephthalaldehyde were calculated using NAD+ as cofactor at both 25 and 50 °C. , followed by hexanal (1.08 and 0.99 mM) and benzaldehyde (1.41 and 1.52 mM). The Kcat values follow a similar trend with the fastest rate demonstrated by terephthalaldehyde (1.05 and 12.08 s−1), followed by hexanal (0.51 and 5.71 s−1), with benzaldehyde displaying the slowest turnover number (0.19 and 1.46 s−1) . The KM for the cofactor, NAD+, of 4.7 µM (25 °C) and 9.2 µM (50 °C), was significantly lower than the substrates trialled, demonstrating increased affinity for the cofactor  was examined. The rate of oxidation of hexanal by ALDHTt was conserved in 0.1% acetone; however, a decrease of approx. 23% was observed in 5% acetone , esterase activity for PNP-acetate was greatly diminished. At levels of 100 µM NAD+ no esterase activity was evident  than NAD+ (130 µM) [Tt displays the inverse. ALDH1A3 has a much higher affinity for the hexanal substrate than ALDHTt, whereas ALDHTt shows higher affinity for the cofactor. ALDH1A3 also demonstrated esterase activity for PNP-acetate, alike to ALDHTt.In 1999, when the focus of ALDH studies was primarily eukaryotic models, a systematic classification system based on protein sequence identity was adopted . This st classes . BLAST seristics . Both ALficiency . However(130 µM) , ALDHTt Tt possess a conserved Thr at the “bottom” residue, and show a conservative substitution of hydrophobic residues at the “neck” (Leu to Ala respectively). This conservative substitution is demonstrated across human ALDH1 family members [Tt is evident. The mouth residue typically carries out a size selection function, for example human ALDH2 with the preferred substrate acetaldehyde possesses a bulky Met at this position, while ALDH1A3 using retinaldehyde harbours a Gly [Tt with Glu at the “mouth” position might relate to optimal substrates being smaller than retinaldehyde, but larger than acetaldehyde, a trend which was adhered to experimentally with the trialled substrates, e.g., hexanal, trans-cinnamaldehyde, and terephthalaldehyde.Across ALDHs, substrate specificity has been related to the shape and geometry of the substrate entry channel of the enzyme, linked to three key amino acids at distinct positions, termed the “mouth” (136), “neck” (471), and “bottom” 315) (human ALDH1A3 numbering) [  and ALDH from Thermoproteus tenax (ALDHTtx) (PDB: 1UXT), acquire 30.75 and 29.23% sequence identity, respectively, compared to ALDHTt [+ favouring enzymes, with ALDHPp acting as a dimer and ALDHTtx as a tetramer. ALDHPp displays broad substrate specificity with the ability to process aliphatic and aromatic aldehydes at high catalytic efficiencies, including hexanal and benzaldehyde. ALDHTtx, however, is specific for glyceraldehyde-3-phosphate. Through analysis of a range of thermophilic ALDHs, it was found that they are quite diverse in oligomeric state and substrate specificity. ALDHGtv exists as an octamer, with ALDHGtd displaying a tetrameric formation with the possibility of octamers formed via dimerization of tetramers [+ [Sulfolobus solfataricus (ALDHSs) favouring NADP+ [Sulfolobus tokodaii (ALDHSt) also demonstrated esterase activity for PNP-acetate [Two thermophilic ALDHs with resolved structures, ALDHo ALDHTt ,51. Bothetramers ,39. Overamers [+ ,39,51,52ng NADP+ . The ALDTt was first co-purified as a contaminant during native 3caa-type cytochrome oxidase isolation from T. thermophilus [T. thermophilus. Additionally, the sequence relation between ALDH1A3 and ALDHTt further highlights this possibility as ALDH1A3′s primary substrate is retinaldehyde. However, retinoid signalling mechanisms are generally considered to be of animal origin, and to date it has only been characterised in animals. More recently, retinoic acid was identified within prokaryotic models, including Bacillus cereus [Chlorogloeopsis fritschii PCC 6912 possesses three orthologs of the enzymes responsible for the conversion of β-carotene to retinoic acid in the signalling pathway recognised in animals. Interestingly, the ALDH was recognised to convert retinaldehyde to retinoic acid suggesting evidence of the use of the pathway in prokaryotic models, and one of the possible functional roles of ALDHTt.Interestingly, ALDHmophilus . This iss cereus  and cyans cereus , exhibits cereus ,54, whics cereus ,56,57. TTt proved an efficient method for the production of the full-length enzyme displaying high purity, while also obtaining high production yields. Heat precipitation protocols have been regularly incorporated as a selective purification step for thermostable proteins expressed in mesophilic hosts [T. thermophilus proteins expressed in E. coli, including other dehydrogenases and the ALDH family member 1-pyrroline-5-carboxylate dehydrogenase (P5CDHTt) [T. thermophilus, at 75 °C for 15 min was deemed the most efficient step, enriching the enzyme 11-fold, and resulting in 47% yield of a homogeneous protein [T. thermophilus. Heat treatment is a promising step to include in a purification protocol, as it does not involve any chemical reagents while also denaturing and aggregating host proteins. In addition, cellular proteases are often denatured, removing their threat to the desired protein product, and thus increasing product yield. Previous studies have demonstrated that addition of heat treatment steps to purification protocols can allow for increased yield and purity [E. coli cells increasing the overall yield of the process [Tt purification, the heat treatment step occurs before the removal of cell debris, and acts not only as a method for inactivation of host cell proteins, but also as an additional cell disrupting mechanism. A previous report outlined how increased purity and yield of recombinant thermophilic catalase from Bacillus sp. expressed in E. coli was achieved via heat treatment purification at 65 °C for 2 h, resulting in a three-fold increase in specific activity of the cell supernatant [Thermotoga maritima where buffer choice, temperature, and treatment time were extensively optimised to achieve high purity (95%) and high yields (44.8 mg/L) [Recombinant expression and purification of ALDHic hosts ,59,60,61P5CDHTt) ,63. The d purity ,65. Heat process . However.8 mg/L) . Previou.8 mg/L) ,65. Tt, is strongly affected by temperature. It is expected that the specific activity of ALDHTt would increase further at elevated temperatures up to 80 °C, given the optimum growth conditions of T. thermophilus [T. thermophilus demonstrated similar trends with respect to effect of temperature and pH [Tt was also assayed at 50 °C using the substrate Δ1-pyrroline-5-carboxylate with enzymatic activity observed in the pH range of 6–10, with a broad optimum from 7–10 [T. thermophilus showed highest catalytic activity at a pH between 9–10, with little or no activity present in acidic conditions. The enzymes demonstrated their highest specific activity at 73 and 85 °C, respectively. Thermophilic ALDHs tend to work best under neutral to basic conditions at temperatures from 50–80 °C, as demonstrated by P5CDHTt above. Other examples include, ALDHGtv, which works optimally at 55 °C, pH 10 [Pp at 60 °C, pH 8.5 [Gtd at 60 °C, pH 8 [Gtd displaying 56% residual activity following incubation at 70 °C for 1 h. In comparison ALDHSs was assayed at 70 °C, pH 7 [St at 80 °C, pH 8 [As a thermophilic enzyme, the specific activity of ALDHmophilus ,68. Howee and pH ,69. P5CDrom 7–10  and glutC, pH 10 , ALDHPp , pH 8.5 , and ALD°C, pH 8 . Interes°C, pH 7  and ALDH°C, pH 8 . Tt could convert aliphatic and aromatic aldehydes, para-substituted benzaldehydes could be efficiently converted by ALDHTt, while ortho-substituted benzaldehydes demonstrated no catalytic activity. Previous reports outline that ortho-substituted benzaldehydes can result in reduced ALDH activity due to steric hindrance effects around the aldehyde group, particularly when the associated substitution group is bulky [Gtd could process 2-chlorobenzaldehyde and o-phthalaldehyde, but at low residual activities, compared to formaldehyde of 28.4 and 10.8%, respectively [Pp could also convert ortho-substituted benzaldehydes [Tt at 50 °C was maintained with the selection of substrates, highlighting that substrate specificity is not dependent on temperature.Many ALDH enzymes are reported as being non-specific for substrate, demonstrating catalytic activity with a range of natural and synthetic aldehydes, including thermophilic members ,52,70,71is bulky ,40. Therectively , while Aldehydes . The theTt’s broad substrate specificity coupled with its increased specific activity at elevated temperatures makes it an attractive target for industrial synthesis of carboxylic acids via biocatalytic mechanisms. Its thermostability will allow for its use in biocatalysis at temperatures where mesophilic counterparts may be unstable or become denatured. The substrate scope of ALDHTt provides a range of interesting potential carboxylic acid products, most notably terephthalic acid and p-toluic acid, which are used as a precursor in polyethylene terephthalate (PET) plastic manufacture and as an intermediate in terephthalic acid and polymer synthesis, respectively [ALDHectively ,73.Tt has a significant preference for NAD+ cofactor rather than NADP+, displaying 33.4 % residual activity when NADP+ is used with hexanal at 50 °C [M for NAD+ is significantly lower than that of the substrates analysed. The KM for terephthalaldehyde was significantly lower than the other two substrates, possibly indicative of its dialdehyde nature and the availability of extra aldehyde groups for binding to the ALDHTt. In contrast, benzaldehyde with only one aldehyde group but a similar structure has a higher KM than terephthalaldehyde. Similar results were obtained using ALDHGc [Gc, it decreased by 14.3- and 51.0-fold with respect to benzaldehyde and terephthalaldehyde. This further highlights the use of the two aldehyde groups on dialdehydes by ALDHs for decreased KM, and increased specific activity. Furthermore, when ALDHGc utilised dialdehydes with ortho-substituted Br, its specific activity was comparable to benzaldehyde  due to the availability of the second aldehyde group for processing, whereas o-bromobenzaldehyde only demonstrated 8% residual activity compared to benzaldehyde. However, ALDHGc demonstrated miniscule residual activity (˂1%) for o-phthalaldehyde, constructed by a benzaldehyde with an ortho-substitution of the second aldehyde group. Here, the availability of both aldehyde groups is occluded due to steric hindrance, resulting in minor specific activity. It was previously demonstrated that ALDHat 50 °C , a trendat 50 °C ,53,74 thg ALDHGc , where tTt has dual dehydrogenase and esterase activity and can act independently on aldehyde or p-nitrophenyl ester substrates. The esterase activity of ALDHs was first realised in human [p-nitrophenol, including prokaryotic models [Tt displayed esterase activity for the conversion of two ester substrates, PNP-acetate and PNP-butyrate, without requiring the addition of NAD+ cofactor. Throughout the literature, it is highlighted that the addition of NAD+ can either accelerate the ALDH esterase reaction or inhibit it, depending on the isozyme in question [+ (2.5 fold with 200 µM), whereas the same concentrations (50–200 µM) nearly abolished human ALDH3 esterase activity [Tt acquires a rate-limiting step characteristic of human ALDH3 enzymes for the esterase reaction; however, its closest related homolog by sequence identity was the human ALDH1A3. In the presence of 500 µM NAD+ ALDH1A3 exhibited esterase activity for PNP-acetate [St displayed esterase activity for PNP-acetate, and was also inhibited by NAD+ [+ and activity was abolished at a concentration of 1 mM. ALDHin human  and horsin human  ALDH, anin human . An arrac models ,52,76,77c models . ALDHTt question ,15,78,79-acetate . Again, Tt from T. thermophilus is a bifunctional enzyme with inherent dehydrogenase and esterase activity, while being non-substrate specific in relation to both reactions. The ALDHTt can catalyse the oxidation of aliphatic, aromatic, and dialdehydes, most notably hexanal and terephthalaldehyde. Biochemical characterisation of the enzyme in terms of substrate scope helps understand possible biological mechanisms, such as the detoxification of certain aldehyde substrates or retinoic acid biosynthesis via association with cytochrome oxidase. Characterisation of the lesser studied prokaryotic ALDH models is important to broaden the understanding of this ever-growing superfamily. Additionally, the thermophilic nature of the ALDHTt, working in the range of 20–50 °C, coupled with its broad substrate scope, allows for application of this enzyme in biocatalytic mechanisms. Possible applications relate to the oxidation of aldehydes to synthesise carboxylic acids chemoselectively, or even the hydrolysis of esters for production of desirable alcohols. However, the reduced catalytic rate of the latter needs to be considered.In summary, it can be concluded that the tetrameric ALDH


Dermatophagoides pteronyssinus which should be included in vaccines for treatment of house dust mite (HDM) allergy. Aim of this study was to characterize the IgE epitopes of Der p 7.Several studies indicate that Der p 7 is an important and clinically relevant allergen of Recombinant Der p 7 was expressed and purified, analyzed for fold by circular dichroism and tested for its allergenic activity by basophil activation. Seven overlapping, surface-exposed peptides (P1–P7) with a length of 27 to 37 amino acids, which spanned the Der p 7 sequence, were synthesized and tested for IgE reactivity and allergenic activity by basophil activation assay. Carrier-bound peptides were studied for their ability to induce allergen-specific IgG antibodies in rabbits. Peptide-specific antibodies were used to inhibit allergic patients` IgE binding to Der p 7 by ELISA for mapping of IgE epitopes.rDer p 7 showed high allergenic activity comparable with Der p 5, Der p 21, and Der p 23. None of the seven tested peptides showed any IgE reactivity or allergenic activity when tested with HDM- allergic patients indicating lack of sequential IgE epitopes on Der p 7. IgE inhibition experiments using anti-peptide specific IgGs and molecular modeling enabled us to identify discontinuous, conformational IgE epitopes of Der p 7.IgE epitopes of Der p 7 belong to the conformational and discontinuous type whereas sequential Der p 7 peptides lack IgE reactivity. It should thus be possible to construct hypoallergenic vaccines for Der p 7 based on carrier-bound allergen peptides. House dust mites (HDM) are one of the most relevant allergen sources worldwide . ContactIn this study we focused on Der p 7 which according to recent studies seems to be a very important HDM allergen. Originally, Der p 7 was categorized as mid-tier group of mite allergens with a frequency of IgE binding 17% to 52% depending on the tested population –8. A verAllergen-specific immunotherapy (AIT) is the only allergen-specific form of treatment with disease-modifying and long-lasting effects –15. HoweThe crystal structure of Der p 7 has been solved but little information is available regarding the binding of patient`s IgE to Der p 7 . In thisTo study the clinical relevance of Der p 7, sera from 100 clinically-well characterized HDM-allergic patients had been tested for IgE reactivity to a comprehensive panel of HDM allergen molecules including Der p 7 by ImmunoCAP ISAC technology as described . Detailehttps://www.meduniwien.ac.at/web/rechtliches/good-scientific-practice/).Serum samples from non-allergic subjects were used as negative controls. All sera samples were analyzed in an anonymous manner after the study with approval of the ethics committee of the Medical University of Vienna, Austria (EK 641/2014). All experiments were performed in accordance with guidelines for good scientific practice of the Medical University of Vienna   and purified using a conjugation kit according to manufacturer’s instructions . E. coli was cloned into pET-17b , and expression was performed in E. coli BL21 (DE3)  by adding 0.5 mM IPTG to the bacterial cultures and incubation in a GFL 3033 incubator  for further 4 h at 37°C. Bacterial pellets were collected by centrifugation for 15 min, at 3,000g and frozen at −20°C. Frozen pellets were re-suspended in 25 mM Imidazole, pH 7.4, 0.1% Triton X-100, lysozyme (500 µg) was added, and cells were stirred for 20 min at RT. Three cycles of freezing and thawing (in liquid nitrogen and 50° water bath) were applied to open the cells, followed by addition of 1 µl DNAse (stock: 1 mg/ml) and stirring for 10 min at RT. NaCl was added to a final concentration of 100 mM, and debris was removed by centrifugation at 19,900g for 20 min at 4°C. Proteins other than Der p 7 were precipitated from the supernatant by increasing the concentration of ammonium sulfate (0 - 40%: 226 g/L) and stirring the mixture at 4°C for 2 h. Precipitated proteins were removed by centrifugation , and additional amount of ammonium sulfate was added (40–60%: 120 g/L) to the supernatant while stirring the mixture at 4°C for 2 h. Precipitated proteins were removed by centrifugation for 30 min, 19,900g, at 4°C. Der p 7 accumulated in the supernatant and was further polished by hydrophobic interaction chromatography using a HiTrap™ Phenyl FF column, 5 ml,  with solutions A , and elution solution B . rDer p 7-containing fractions were combined and dialyzed against solution C  to continue purification by hydroxypatite chromatography using a Bio-Gel HT Hydroxyapatite 5 ml column . Solution D  was used for elution of Der p 7. Fractions containing Der p 7 were combined, dialyzed against 10 mM Na2HPO4, pH 7, and stored at −20°C. Purity and migration pattern of Der p 7 were analyzed by SDS-PAGE.Seven overlapping peptides with a length between 27 and 37 amino acids were identified based on the prediction of surface exposure of amino acids as determined by the ProtScale bioinformatics tool from the ExPASY server  . Cysteintscale/) . The ideThe secondary structure of rDer p 7 was studied by circular dichroism (CD) spectroscopy using a Jasco J-810 spectropolarimeter . Measurements were performed between 250 and 190 nm with a resolution of 0.5 nm at a scanning speed of 50 nm/min. Final spectra were normalized by subtracting the buffer spectrum. Results are shown as mean residue ellipticities at a given wave length. The melting point was determined as described . The secRecombinant Der p 5, Der p 21, and Der p 23 were purified as described –29.http://www.rcsb.org/pdb/home/home.do; PDB: 3H4Z)  . The solB: 3H4Z) . SAS is 125I-labeled anti-human IgE Abs  and visualized by autoradiography (Kodak XOMAT film).Aliquots containing 0.5 µg of purified rDer p 7, each of the Der p 7-derived peptides and for control purposes BSA were dotted onto a nitrocellulose membrane . Membranes were blocked with gold buffer , three times for 20 min and then incubated with HDM-allergic patients’ sera (diluted 1:10 in gold buffer) and with a serum from a non-allergic person (1:10 in gold buffer) overnight at 4°C. Bound IgE was detected with 1:10 diluted 5/well) were loaded overnight with sera from the house dust mite allergic patients and, for control purposes, with the serum from one non-allergic individual at a dilution of 1:20 as described  expressing human high-affinity IgE receptor FcϵRI , and cells plus allergen without serum (marked as buffer) were used to determine the cut-off. Supernatants were analyzed for β-hexosaminidase activity as described previously . ExperimPeptide-specific IgG antibodies were obtained by immunizing rabbits three times (first booster injection after 4 weeks and a second booster injection after 7 weeks) with each of the KLH-conjugated peptides (200 µg/injection) and, for control purposes, with recombinant Der p 7 protein (200 µg/injection) . In total, four rabbits were immunized per peptide/protein, two using Freund’s adjuvant (1× complete and 2× incomplete Freund’s adjuvant) and two rabbits using aluminium hydroxide as adjuvant . Pre-immune sera were obtained from the rabbits before immunization. Rabbit immune responses were analyzed by ELISA. For the measurement of specific rabbit IgG antibodies, ELISA plates  were coated overnight at 4°C with 1 µg/ml of rDer p 7. After blocking, the plates were incubated overnight with serial dilutions of the corresponding rabbit antisera , or the corresponding pre-immune sera (1: 2.000). Bound rabbit IgG antibodies were detected with a 1:2.500 diluted horseradish peroxidase-labeled donkey anti-rabbit IgG antiserum . The optical density (OD) values corresponding to bound antibodies were measured at 405 and 492 nm. All determinations were conducted as duplicates, and results were expressed as mean values with deviations of less than 10%.3, anti-sera from two rabbits immunized with the same adjuvant were combined in ratio 1:1 to obtain a total sera dilution of 1:50 for both, CFA-immunized rabbits and Al(OH)3-immunized rabbits, respectively. Plates were then washed, and after overnight incubation with sera from HDM-allergic patients (dilution between 1:5 and 1:10), bound IgE antibodies were detected with horseradish peroxidase-labeled goat anti-human IgE antibodies (KPL). The percentage reduction in IgE binding achieved by pre-incubation with rabbit antisera was calculated as follows: 100 − (ODI/ODP) × 100) where ODI and ODP represent optical density values after pre-incubation with the rabbit immune serum or pre-immune serum, respectively ectively , 33.In this study we used sera from clinically well characterized HDM allergic patients  to compaTo study the allergenic activity of Der p 7 we investigated its ability to activate basophils by testing a series of allergen concentrations and sera from HDM allergic patients. We found that Der p 7 could activate basophils at similar low concentrations  like Der p 5, Der p 21, and Der p 23 which have been identified as clinically relevant allergens molecules , having a length from 27 to 37 amino acids S1. The Recombinant Der p 7 could be expressed and purified as a folded and monomeric protein. Next, we investigated by basophil activation if Der p 7-derived peptides may exhibit some allergenic activity, in particular when they are mixed Figure 4Immunization of rabbits with each of the seven KLH-coupled peptides (P1-P7) induced IgG antibodies that recognized folded rDer p 7 at least up to a dilution of 1:2.000 . When CFA was used as adjuvant, the mean inhibition obtained with anti-Der p 7 was 83%, the highest inhibitions with anti-peptide antisera were obtained with anti-P1 and anti-P2 antibodies  followed by anti-P7, anti-P4, and anti-P3 . The patients/participants provided their written informed consent to participate in this study.MC and RV designed the study, interpreted the results, and wrote the manuscript. MC, H-JH, TG, SG, K-WC, YR-M, and KF conducted experiments, analyzed the data, and prepared the figures. WH, PZ, RZ, PL, and FH collected allergic patients’ sera and performed clinical characterization of patients. MF-T, SV, SF, and WK analyzed and interpreted the results. RV supervised and coordinated the study. All authors contributed to the article and approved the submitted version.This study was supported by grants F4602, F4604, F4605, and F4607 of the Austrian Science Fund (FWF), by the Danube Allergy Research Cluster, Country of Lower Austria, and by a research grant from WORG Pharmaceuticals, Hangzhou, China. The funder, WORG Pharmaceuticals, was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.RV has received research grants from Viravaxx, Vienna, Austria, HVD Biotech, Vienna, Austria and WORG Pharmaceuticals, Hangzhou, China and serves as consultant for Viravaxx and WORG.The remaining 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.


Western Blot analysis indicated a significant increase in Drp1 expression in the diabetic retinas compared to those of WT mice; retinas of diabetic Drp1+/− mice showed reduced Drp1 level compared to those of diabetic mice. A significant increase in the number of acellular capillaries (AC) and pericyte loss (PL) was observed in the retinas of diabetic mice compared to those of the WT control mice. Importantly, a significant decrease in the number of AC and PL was observed in retinas of diabetic Drp1+/− mice compared to those of diabetic mice concomitant with increased expression of pro-apoptotic genes, Bax, cleaved PARP, and increased cleaved caspase-3 activity. Preventing diabetes-induced Drp1 overexpression may have protective effects against the development of vascular lesions, characteristic of diabetic retinopathy.High glucose (HG)-induced Drp1 overexpression contributes to mitochondrial dysfunction and promotes apoptosis in retinal endothelial cells. However, it is unknown whether inhibiting Drp1 overexpression protects against the development of retinal vascular cell loss in diabetes. To investigate whether reduced Drp1 level is protective against diabetes-induced retinal vascular lesions, four groups of mice: wild type (WT) control mice, streptozotocin (STZ)-induced diabetic mice, Drp1 Diabetic retinopathy is the leading cause of blindness in the working-age population and, unfortunately, there is no cure for this ocular complication . DiabetiDrp1 is a GTPase that is considered to be a principal regulator of mitochondrial fission ,19. It iDrp1 overexpression has been widely reported in HG and diabetic conditions. Podocytes and glomerular mesangial cells grown in HG medium exhibit significant Drp1 upregulation and increased mitochondrial fission, which promote podocyte loss and compromise glomerular function, suggesting that elevated Drp1 plays a role in the pathogenesis of diabetic nephropathy ,26,27. I+/− mouse and investigated whether reduced levels of Drp1 in these mice were protective against the development of acellular capillaries and pericyte loss. Specifically, proapoptotic genes Bax, cleaved PARP, and caspase-3 activity were examined in addition to TUNEL assays, which were performed to identify vascular cells undergoing apoptosis in retinal capillaries.To determine whether increased levels of Drp1 contribute to the development of apoptotic death of vascular cells in the diabetic retina, in the present study, we induced diabetes in the Drp1+/− mice bred into the C57/BL6J strain background provided by Dr. Hiromi Sesaki [+/− mouse model was generated can be referred to in a previous study [+/− allele is represented as a 539-bp fragment amplified by primers 1 and 2. Drp1+/+ allele is represented as a 315-bp fragment amplified by primers 2 and 3. In the present study, Drp1−/− mice were not used as this genotype is embryonically lethal [Studies involving animals were carried out following the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the IACUC Committee of Boston University . Male and female mice were used in the present study to address any sex-related differences. A total of 12 male and 12 female WT C57BL/6J mice , as well as 12 male and 12 female Drp1i Sesaki  were useus study . Polymery lethal ,34,35.+/− mice were randomly assigned to receive 5 consecutive STZ injections intraperitoneally to induce diabetes at a concentration of 40 mg/kg body weight. Additionally, 12 WT mice and 12 Drp1+/− mice were randomly assigned to receive 5 consecutive citrate buffer injections intraperitoneally as vehicle, representing non-diabetic control groups. To verify the diabetes status in the animals, blood and urine glucose levels were measured 3 days post-STZ injection. Routine blood glucose assessment was performed 3 times per week. Depending on the hyperglycemic status, NPH insulin injections were administered to achieve a level of ~350 mg/dL. A total of 16 weeks after the onset of diabetes, animals were sacrificed, blood was collected from each animal and blood glucose and HbA1c levels were measured. Following sacrifice, retinas from each animal were isolated, and total protein extracted from all samples.12 WT mice and 12 Drp1To study the expression and distribution of Drp1 in the retinal capillary networks, retinal trypsin digestion (RTD) preparations  were subWB analysis was carried out as described . Briefly+/− animals, fluorometric analysis was carried out using a commercially available caspase-3 assay kit . Lysates from retinal tissues were isolated using the kit’s proprietary lysis buffer, incubated on ice for 10 min, and homogenized. Following BCA assay of the retinal tissue lysates, 20 μg of protein from each sample was used to perform the fluorometric evaluation of caspase-3 activity. The retinal lysates were mixed with reaction buffer containing DTT  and Acetyl-Asp-Glu-Val-Asp-7-amino-4 trifluoromethylcoumarin (DEVD-AFC) , a fluorogenic substrate specific to caspase-3. The reaction mixture representing each sample was transferred to corresponding wells in a 96-well plate, incubated at 37 °C for 2 h, and subjected to fluorescent excitation and emission at 400 nm and 505 nm, respectively. Specifically, cleavage of the DEVD-AFC substrate is carried out by activated caspase-3 resulting in the formation of free AFC molecules, which can be detected at 400 nm excitation and 505 nm emission [To evaluate caspase-3 activity in retinas of diabetic animals and Drp1After animals were sacrificed, eyes were enucleated and placed in 10% formalin, and retinas isolated and exposed to 0.5 M glycine for 24 h. To isolate retinal capillaries, RTD was performed as described . BrieflyTo detect cells undergoing apoptosis in retinal capillaries, terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL) assay was performed using a kit  as described previously . Brieflyt-test for comparisons between two groups, or one-way ANOVA followed by Bonferroni’s post-hoc test for comparisons between multiple groups. Statistical significance was considered at p < 0.05.Data are shown as mean ± standard deviation. Values representing experimental groups are shown as percentages of the control. The normalized values were subjected to Student’s +/+) exhibited a band as expected at 0.54 kb whereas Drp1 heterozygous knockout (Drp1+/−) mice exhibited a band as expected at 0.32 kb  mice (Drp1 0.32 kb .+/− animals, Drp1 immunostaining was performed in RTDs from each experimental group. Interestingly, Drp1 immunostaining was significantly increased in RTDs of diabetic mice compared to that of non-diabetic WT mice (151 ± 14% of WT vs. 100 ± 15% of WT, p < 0.01; n = 6; +/− mice compared to that of non-diabetic WT mice (73 ± 7% of WT vs. 100 ± 15% of WT, p < 0.05; n = 6; +/− mice compared to that of diabetic mice (111 ± 16% of WT vs. 151 ± 14% of WT, p < 0.01; n = 6; +/− mice (111 ± 16% of WT vs. 73 ± 7% of WT, p < 0.01; n = 6; To determine whether the distribution of Drp1 is altered in retinal capillary networks of diabetic animals and Drp1p < 0.01; n = 12; +/− exhibited a significant decrease in retinal Drp1 expression compared to that of WT mice (63 ± 10% of WT, p < 0.01; n = 6; +/− mice (80 ± 16% of WT, p < 0.01; n = 12; Data from Western blot analysis showed that Drp1 expression level is significantly upregulated in diabetic mouse retinas compared to that of WT mouse retinas (133 ± 13% of WT, p < 0.01; n = 12; p < 0.01; n = 12; p < 0.01; n = 12; +/− mice showed a decrease in diabetes-induced Bax expression (113 ± 19% of WT, p < 0.01; n = 12; p < 0.01; n = 12; p < 0.01; n = 12; To assess whether reduced Drp1 level is protective against diabetes-induced pro-apoptotic genes, expression levels of Bax and cleaved PARP, as well as caspase-3 activity were monitored in retinal tissues. In retinas of diabetic mice, gene expression levels of pro-apoptotic Bax and cleaved PARP were significantly increased (Bax: 152 ± 23% of WT, p < 0.01; n = 6; +/− mice compared to that of diabetic mice (180 ± 29% of WT vs. 284 ± 28% of WT, p < 0.05; n = 6; To assess whether downregulating Drp1 expression protects apoptotic cell death in retinal vascular cells, TUNEL assay was performed in RTDs from each experimental group. Data indicate that there is a significant increase in the number of TUNEL-positive cells in RTDs of diabetic mice compared to that of non-diabetic WT mice (284 ± 28% of WT vs. 100 ± 33% of WT, p < 0.01; n = 12; p < 0.01; n = 12; +/− mice, the numbers of AC and PL significantly decreased compared to those of diabetic mice (AC: 111 ± 7% of WT vs. 181 ± 23% of WT, p < 0.01; n = 12; p < 0.01; n = 12; +/− mice exhibited no significant difference in the numbers of AC and PL compared to those in non-diabetic WT mice (AC: 93 ± 13% of WT vs. 100 ± 17% of WT, p > 0.05; n = 12; p > 0.05; n = 12; To evaluate the effects of diabetes and reduced Drp1 levels in the development of AC and PL, retinal trypsin digestion was carried out and the numbers of AC and PL between the experimental groups were analyzed. RTD data indicate a significant increase in the numbers of AC and PL in retinal capillary networks of diabetic mice compared to those of non-diabetic WT mice (AC: 181 ± 23% of WT vs. 100 ± 17% of WT, +/− animals exhibiting ~40% reduction in Drp1 levels. Similarly, data from the present study showed no significant sex-related differences in neither WT or Drp1+/− animals. These findings indicate that increased Drp1 level is closely associated with the development of retinal vascular lesions, and that reducing it could prevent apoptotic vascular cell death associated with diabetic retinopathy.Present study provides novel evidence that Drp1 expression is significantly increased in retinas of diabetic mice, and that reduced levels of Drp1 provide beneficial effects in preventing apoptotic death of retinal vascular cells and subsequent development of acellular capillaries and pericyte loss characteristic of diabetic retinopathy. Of note, no significant adverse effects were observed in the retina and other tissues of Drp1Mitochondrial morphology and functionality are intrinsically linked . Breakdo−/− cells are protected against apoptosis [While studies have established that participation of Drp1 is essential in regulating mitochondrial fission, mechanisms underlying Drp1 upregulation and its role in inducing apoptosis remain unclear. Increased Drp1 activation triggers apoptosis by promoting translocation of Bax to mitochondria and ultimately activating caspase-3 signaling . In addipoptosis  and Drp1poptosis , suggestGrowing evidence shows that blocking Drp1 overexpression could be effective in preventing mitochondrial fission and protecting against apoptotic cell death ,51,52,53


Aflatoxin B1 (AFB1)-contaminated paddy samples (n = 3) exceeded the regulatory threshold (5 µg·kg−1). The contamination of paddy with AFs and FBs was not significantly different by growing seasons and cultivation localities. Evidently, in the winter–spring season, fumonisins frequently occurred in paddy planted in Can Tho, while AFs were found in paddy planted in regions Dong Thap and An Giang, and such toxins were absent in Can Tho. Furthermore, the selection of paddy varieties strongly impacted the occurrence of these toxins, especially AFs, for example, line DT8 and Jasmine were susceptible to AFs and FBs. In addition, poor pre- and post-harvest practices  had an impact on the AFs and FBs contamination. Our findings can help to understand the dynamics of AFs and FBs in the rice chain in the Vietnamese Mekong Delta, leading to the mitigation of the contamination of AFs and FBs in rice.The current study aimed to evaluate the impact of the crop season, cultivation region, and traditional pre- and post-harvest agricultural practices on mycotoxin contamination in the Mekong Delta rice chain of Vietnam. The results showed that aflatoxins (AFs) and fumonisins (FBs) were predominantly detected in both paddy ( Oryza sativa L.) is one of the most important staple foods besides wheat and maize. Rice is also a vital food and a pivotal export product for Vietnamese people [Rice . Therefore, the purpose of this complementary study is to evaluate the impact of (i) crop seasons, (ii) cultivation regions, and (iii) traditional farming practices on aflatoxin and fumonisin contamination in the rice chain in the Mekong Delta, Vietnam.Despite the fact that Vietnamese traditional pre-harvest and post-harvest practices have been demonstrated to be associated with increased contamination of maize with mycotoxins ,16,17, tn = 103/230), with median levels ranging from 2.3 µg·kg−1 to 547 µg·kg−1. Out of twenty-three analyzed mycotoxins, using the LC-MS/MS technique, fifteen were found  and white rice . In particular, aflatoxin B1 (AFB1)-contaminated paddy samples (n = 3) exceeded the regulatory threshold (5 µg·kg−1) [Forty-five percent of the paddy and white rice samples were contaminated with mycotoxins , SA18 (n = 30), AW18 (n = 15), WS18-19 (n = 50), and AW19 (n = 60).−1, 30–70% for FB1, FB2, and FB3), followed by WS 17-18 , and AW19 . The median contents, however, were not clearly different among these seasons for both toxins   a,b.p-value > 0.05) , while the incidence of these toxins was lower in WS17-18 (27 to 53%) .At the transport stage, the mycotoxin analysis in paddy showed that the AFs appearance was more frequently in the SA18 than in the WS17-18, commonly AFB2 (50% vs. 27%). FB contamination in paddy was similar to that on the field. It means the FB incidence was slightly different among the seasons, but the median concentration was not significantly different among these seasons ( > 0.05) b. For inp-value > 0.05)  in all seasons  a. During seasons b. Interep-value > 0.05), whereas the appearance of FBs was higher in this season in the former stages , compared to further stages .Overall, paddy collected in the winter–spring seasons was contaminated more frequently by AFs throughout the rice chain  (−1), An Giang (med-pos: 97± 4575–1236 ± 1029 µg·kg−1), and Dong Thap (med-pos: 53 ± 1427–876 ± 858 µg·kg−1)   a. AFG1 w > 0.05) b.−1 vs. 0.0 µg·kg−1)  (−1)  (−1 vs. 0.0 µg·kg−1)  .Regarding mycotoxin contamination at drying, storing in the warehouse, and milling, the data stated that Dong Thap and An Giang paddy still contained high levels of AFs, while FBs were found to be lower or absent at these stages in three provinces a,b. In d > 0.05) a. Simila > 0.05) a. In con > 0.05) a. Remarkp-value >0.05), whereas FBs appeared higher in the field and transportation stages, compared to further stages throughout the chain in all regions .Generally, AFs were detected in An Giang and Dong Thap at most stages (n = 10), IR50404 (n = 15), DT8 (n = 6), Jasmine (n = 7), and other paddy lines (n = 8) were planted by Mekong Delta farmers. Of those, DT8, IR50404, and OM5451 were prone to the contamination of AFB2 and AFG2. −1) was contaminated more frequently with AFB2 than line OM5451  and line IR50404 . Similarly, AFG2 was predominantly found in DT8 (50%) rather than the other paddy varieties   was higher than that in IR50404  and other paddy varieties   (OM5451 (arieties . However > 0.05) . Interes > 0.05) .®) after harvesting crops were not contaminated by AFB2 and AFG2, while the incidence of such toxins on paddy in the residue-free fields was 33% and 21% (removing off), and 5% and 0% (burning), respectively. The median levels were not significantly different among the three methods    . Similar > 0.05) . As a map-value > 0.05)  (Fields where a mixed fertilizer (inorganic and organic) was used were contaminated with higher median levels of AFB2 and AFG2 than the fields where only inorganic fertilizer was applied ( > 0.05) . On the  > 0.05) .n = 23), compared to a combination of vehicles  (14%). AFB2 was absent in paddy transported by boats (n = 16)  (−1), followed by trucks (med-pos: 267 −1), and finally combined trucks-boats (med-pos: 63 −1)   . On the  > 0.05) . Similar > 0.05) .Drying of rice crops is a traditional and common practice used to minimize water activity or moisture content in grains by rice processing companies in the Mekong Delta, Vietnam. The drying system that is applied commonly in the Mekong Delta, is cheap, simple, and easy to conduct. It consists of a cement or iron sheet covered by a plastic sheet containing paddy above and a furnace used to supply hot air to dry paddy by the fans .p-value > 0.05)  and 12–28 h, with median of positives and incidence ranging from 65 −1 and 9–63%, respectively. > 0.05) . SimilarRice is one of the most popular commodities around the world . HoweverIn this study, the incidence of AFs in rice was lower than reported in five provinces in central Vietnam , Northern = 51/100) [AFB2 was detected more commonly than AFB1 throughout the rice chain in this work. This is different from other reports in some Asian countries, which indicated that the prevalence of AFB1 contamination in rice was more frequent than other aflatoxins  such as in Pakistan ,32, Thai 51/100) . Such a A. flavus [F. proliferatum in rice grains [Results of our study indicated that AFs and FBs usually occurred in the winter–spring season. This could be associated with temperature, humidity or rainfall, and drought . Indeed,. flavus ,28,29 at. flavus . For FBse grains , producie grains . Moreovee grains . Howevere grains . These cTrichoderma-based fertilizer to improve soils before culturing the rice crop, which also inhibits aflatoxin production in paddy of this province. Such result is in agreement with many studies; for instance, A. flavus was inhibited by Trichoderma sp., leading to a reduction in AFB1 concentration (39%) on sorghum grains [Trichoderma-containing fertilizer for their crops.Furthermore, the results showed that Can Tho paddy was not contaminated by AFs throughout the rice chain. This could be explained as rice companies and farmers in Can Tho used a m grains , and 30%m grains . Regardim grains . In factntration % on sorgm grains . Therefo®)   to decomv/v), resulting in an increase in AF contamination, as organic fertilizers contain high levels of carbon organic matter, leading to stimulating fungal proliferation [A. flavus and F. proliferatum in the Mekong Delta rice chain, the prevalence of both strains (positive with AFB1 and FB1) were found to be higher in the paddy collected in the fields using a combined fertilizer (inorganic and organic) than that in the inorganic applying fields. Hence, to reduce mycotoxigenic fungal infection as well as mycotoxin contamination, farmers should use inorganic fertilizers for their paddy crops.Fertilizers have been used to improve soils to increase the yield of crops. In the present work, 10% of the farmers  used a mferation ,44 and Aferation , such asBased on the survey, rice processing companies in the Mekong Delta utilized boats to transport paddy from the fields to the factories . The datPaddy was dried after harvesting by drying systems, operated with the following principles. The ventilators collect the hot air from the furnace and blow it into the drying chamber containing the rice above. Afterward, hot air or smoke flows through narrow gaps (iron sheet) to contact with the paddy or heats cement sheet, causing moisture or water in the grains to escape, resulting in water loss and rice drying . Althougw of paddy grains on-field and transport stages (>0.95) were observed to be higher when compared to samples at the drying, storage, and milling stage (>0.69) (p < 0.05)  , leadingAflatoxins and fumonisins were predominant contaminants in the rice chain in the Mekong Delta. Crop seasons, regions, and traditional agricultural practices  were main drivers for an increase in mycotoxin contamination throughout the rice chain. Our findings can be considered as advice on good agricultural practices, to reduce mycotoxins pre- and post-harvest in the Mekong Delta rice chain.n = 184) and white rice (n = 46) were collected from farmers and rice companies or cooperatives belonging to contract farming systems, characterized by a high yield production of rice and representative in three regions, Can Tho, An Giang, and Dong Thap (n = 46), after transporting to the factory (n = 46), dried paddy (n = 46), stored paddy (three months in the warehouse) (n = 46), and polished rice (milled from stored paddy) (n = 46). Regarding sampling in both stages , each sample was taken at nine points, whereas samples from transport as well as storage stages were randomly taken in 10 different bags. Polished rice samples were conducted at different time points during the milling (5–20 min). Afterward, each sample that was collected at different points or bags or during different milling stages was mechanically pooled into one composite sample (2 kg), and then conveyed to a polyethylene zipper bag. Samples were transported to Ghent University, Belgium, and stored at −20 °C for mycotoxin analysis .A parallel study of fungi in the rice chain was performed on the same samples of this study, evaluating contamination of mycotoxin in rice samples collected in the Mekong Delta, Vietnam. In detail, a total of 230 samples consisting of paddy (ong Thap  in 2017–w) levels of 230 samples were determined by the EZ-200 . The instrument was calibrated based on four calibration standards , Mg(NO3)2 (0.529 aw), NaCl (0.753 aw), and NH4H2PO4 (0.93 aw)) supplied by the manufacturer. Each grain sample was carried out three times. The results are shown in Water activity (a−1). Twenty mL of extraction solvent  was added to the samples, agitated for 1 h and then centrifuged at 4000 rpm for 15 min. The supernatant was transferred to a pre-conditioned C18 solid-phase extraction (SPE) column . The eluate was collected in a volumetric flask of 25 mL. The samples were re-extracted with 5 mL hexane. The defatted extract was divided into two fractions, the first fraction (6 mL) was filtered through a Whatman glass microfilter , and 20 mL of acetonitrile/acetic acid  was added to the second fraction (10 mL)). This mixture was purified by a MultiSep® 226 AflaZon column . After washing the column with 5 mL acetonitrile/acetic acid , the total MultiSep 226 eluate was mixed with 2 mL of the filtered fraction. The mixture was dried by a nitrogen flow in a water bath at 40 °C/1 atm. The residue was dissolved in 150 µL after washing the column with 5 mL acetonitrile/acetic acid . After centrifugation at 4000 rpm at 4 °C for 5 min, the supernatant was transferred in an Ul-trafree® centrifuge filter , and centrifuged at 10,000 rpm for 5 min. Finally, the filtrate was brought into a vial to analyze mycotoxins by LC-MS/MS [Each ground paddy or white rice sample (5 g) was spiked with de-epoxy-deoxynivalenol (DOM)  as an internal standard ). The sample was analyzed for 23 different mycotoxins, using tandem mass spectrometry (MS/MS) with a Quattro Premier™ XE tandem quadrupole mass spectrometer . The matrix-matched calibration curves fitted by linear regression showed a coefficient of determination (R2), ranging from 0.951 to 0.999. The average recoveries for all the mycotoxins ranged from 80% to 111%. The intraday and interday precision ranged from 3–14% and 6–20%, respectively. The LOD values were 0.5 µg·kg−1 , 13 µg·kg−1 (FB1), 17 µg·kg−1 (FB2), and 53 µg·kg−1 (FB3). The LOQs were 1.5 µg·kg−1 for each AF, whereas the values were 26, 35, and 105 µg·kg−1 for FB1, FB2, and FB3, respectively. The detailed analytical conditions were described by [Liquid chromatography-tandem quadrupole mass spectrometer (LC-MS/MS) analysis was conducted, based on an ultra-high performance liquid chromatography (UHPLC) system , equipped with a symmetry guard column   and a symmetry Cribed by ,53.Aspergillus flavus and Fusarium proliferatum in the rice chain linked to seasons, regions and traditional agricultural practices in Mekong Delta, Vietnam”, status: under revision).A survey was operated using a face-to-face interview with a questionnaire to understand Mekong Delta pre-and post-harvest traditional agricultural practices . Forty-sAll the descriptive statistics were conducted using the R software version 3.5.1, SPSS version 20 and Microsoft excel version 13. A non-parametric Kruskal–Wallis test and a post hoc Games–Howell and Dunn’s test for pairwise comparisons at a significance level of α = 0.05 were performed. Mycotoxin levels were displayed in median ± SE (standard errors). The water activity of 230 samples was analyzed using one-way ANOVA in IBM SPSS Statistic version 20.0. Data of the interview were analyzed by Microsoft Excel 2013.


Haemonchus contortus—an economically important parasitic nematode of livestock. This panel was screened in vitro against exsheathed third-stage larvae (xL3) of H. contortus using an established phenotypic assay, and the potency of select compounds to inhibit larval motility and development assessed in dose-response assays. Of the 245 compounds screened, three—designated MPK18, MPK334 and YAK308—induced non-wildtype larval phenotypes and repeatedly inhibited xL3-motility, with IC50 values of 45.2 µM, 17.1 µM and 52.7 µM, respectively; two also inhibited larval development, with IC50 values of 12.3 µM (MPK334) and 6.5 µM (YAK308), and none of the three was toxic to human liver cells (HepG2). These findings suggest that these compounds deserve further evaluation as nematocidal candidates. Future work should focus on structure–activity relationship (SAR) studies of these chemical scaffolds, and assess the in vitro and in vivo efficacies and safety of optimised compounds against adults of H. contortus.Due to widespread multi-drug resistance in parasitic nematodes of livestock animals, there is an urgent need to discover new anthelmintics with distinct mechanisms of action. Extending previous work, here we screened a panel of 245 chemically-diverse small molecules for anti-parasitic activity against  Haemonchus, Ostertagia, Teladorsagia, Trichostrongylus, Cooperia, Nematodirus and Oesophagostomum, cause some of the most important diseases, affecting the health of hundreds of millions of animals , with very substantial economic losses every year  to test the active compounds  for their effect on the larval development over 7 days. Subsequently, worms were fixed in 1% iodine, and the rate of larval development was assessed microscopically appraising the presence (L4) or absence (xL3) of mouth and pharynx [50 values, compound concentrations were log10-transformed, fitted using a variable slope four-parameter equation with constraining the top value to 100% using a least squares (ordinary) fit model using GraphPad Prism (v. 9.0.2) software.Following the measurement of larval motility at 72 h, plates containing xL3s were incubated for four more days at 38 °C [10%  CO2 at >90% humidity, and incubated with individual, serially-diluted compounds  in a final volume of 100 µL. Doxorubicin (10 µM) and DMEM* + 0.25% DMSO (vehicle), serving as positive and negative controls, respectively, were added to the individual 96-well plates. Monepantel and moxidectin were also serially diluted—prepared in the same manner as active compounds—and used as ‘negative’ reference controls. After 48 h of incubation, cellular viability was determined by the crystal violet staining assay [50) values, compound concentrations were log10-transformed, fitted using a variable slope four-parameter equation with a least squares (ordinary) fit model employing GraphPad Prism (v. 9.0.2) software.HepG2 human hepatoma cells were seeded into wells of a 96-well microtitre plate in 80 µL of Dulbecco’s modified eagle medium (DMEM), supplemented with 10% foetal bovine serum, 100 U/mL of penicillin, 100 µg/mL of streptomycin and 0.25 µg/mL of amphotericin B (DMEM*) at a density of 1 × 10ng assay . The absH. contortus larvae encourage future SAR studies of analogues of MPK18, MPK334 and YAK308, with a future focus on developing an anthelmintic against strongylid nematodes of animals.The findings for the three new chemical entities with in vitro-anthelmintic activities against