In 2009, the Side-Row Continuous Canopy Shaking Harvester project was set to develop such technology. The prototype comprises two symmetrical harvesters trailed by a farm tractor. Each harvester has a vibratory rotor with flexible rods, a catching platform with conveyors belts delivering fruits to a temporary storage bag. The removal efficiency of canopy shakers are influenced by factors like shaking frequency, ground speed as well as the dimension and shape of olive canopy. In 2014 authors started a trial to evaluate the influence of pruning in olive yield and in the performance of the Side-Row Continuous Canopy Shaking Harvester. The trial was established in an irrigated olive orchard of Picual cultivar planted in 1996 with the array 7 m x 3.5 m. In a randomised complete block design with three replications, four treatments are being compared leading to 12 plots with 30 trees/plot. The treatments under study are: T1—manual pruning using chain saws, in 2014 and 2017; T2—mechanical pruning: topping and hedging the two sides of the canopy, followed by manual pruning complement to remove wood suckers inside the canopy, in 2014 and 2017; T3—mechanical pruning: topping the canopy parallel to the ground and hedging southeast side of the canopy in 2014 and 2017; topping the canopy in July 2015 (summer pruning); hedging northwest side in winter 2016; T4—mechanical pruning: topping and hedging the two sides of the canopy in 2014 and 2017; topping the canopy in July 2015 (summer pruning). Regarding to olive yield per tree, significant differences were found among treatments on different years. However, no significant differences were found regarding the average olive yield per tree, over the period of 2014–2017. Regarding to the olive removal efficiency, only in 2016, significant differences were found among treatments on different years. No significant differences were found regarding the average of the olive removal efficiency, over the period of 2014–2017. In Portugal there are currently 40,000 ha of high density olive groves (200 to 500 trees per hectare), mostly irrigated. Despite the recent diffusion of the super high density olive grove, still there will be more than 1.5 million ha of high density olive groves worldwide.Olive harvest in high density olive orchards is usually performed by a tractor mounted trunk shaker and a canvas manually placed on the ground under the tree. Less labor demanding solutions based on inverted umbrellas linked to the trunk shaker have limited use since trees are very closely spaced to allow the umbrella to open.Only changing from a discrete trunk shaking to a continuous canopy shaking principle will improve working capacity and will reduce the dependency over scarce and expensive labor. Grape and coffee over-the-row canopy harvesters could be used with good results in young intensive olive orchards not higher than 2.5 to 3.5 m or wider than 2 m . The samCultivar, tree shape, canopy density, and pruning affect mechanical harvesting efficiency with canopy shakers .It has been reported  that witThis paper presents and discusses the results of research conducted on a 20-year-old intensive olive grove to evaluate the influence of mechanical pruning on olive production and the performance of the SRCCSH.The high density olive orchard (HD) used in the trial was established in 1996 in Herdade da Torre das Figueiras in the Alentejo region of southern Portugal . This drip irrigated HD olive orchard of Picual cultivar was installed in an array of 7 m x 3.5 m.The orchard was planted on Chromic Luvisol soil (FAO). This region is semi-arid with strong continental influence and an annual rain mean of 500 mm concentrated in the winter.3/ha.The orchard is drip irrigated twice a week, from May till October, receiving annually an estimated volume of 1,500–2,000 mFlusicladium oleaginum (Castagne) Ritschel & U. Braun], olive moth (Prays oleae Bernard), olive fly (Bactrocera oleae Gmelin.) and olive anthracnose (Colletotrichum acutatum Simmons or Colletotrichum gloeosporioides Penz.). Weed control was done spraying glyphosate in the rows and with a shredder between rows. About 80 units of nitrogen, 30 units of phosphorus, and 50 units of potassium were applied to the soil and by drip irrigation in average by year.The HD olive orchard was sprayed to control olive leaf spot [kW (DIN) 4WD agricultural tractor.Mechanical pruning was performed using an R&O (Reynolds & Oliveira Ltd.) disk-saw pruning machine Figure 1The manual pruning complement to the mechanical pruning was executed by telescopic chain saws.The SRCCSH is a prototype Figure 2The SRCCSH is based on two symmetrical machines, each one trailed by a farm tractor, moving alongside a same tree row, harvesting both sides of the trees. Fruit removal is made by a vibratory rotor with flexible rods for engaging and shaking the olive bearing branches. Vibration frequency of the vibratory rotor can be altered adjusting the tractor power-take-off speed. Removed olives are collected on a platform and conveyed to a temporary storage bag.Operational parameters of SRCCSH were adjusted at the beginning of the harvesting season Table 1.Four treatments , shown in Treatment 1 (T1)—manual pruning performed in 2014 and 2017. One man per tree performed selective cuts with a chainsaw to control canopy dimension, removing branches with excessive growth, either overhanging the canopy toward the space between rows or taller than the vibratory mast of the SRCCSH.Treatment 2 (T2)—mechanical pruning followed by a manual pruning complement was made in 2014 and 2017. Mechanical pruning consisted on a horizontal cut (topping) at the uppermost part of the canopy and a vertical cut on each side of the tree (hedging). Topping was done at approximately 3.6 m (2014) and 3.3 m (2017) in height, from the ground; hedging was done at approximately 1.8 m from the tree trunk. Manual pruning complement was performed to remove wood suckers inside the canopy and also wood stumps on each side of canopy.Treatment 3 (T3)—mechanical pruning performed each year. In 2014, trees were topped (as in T2) followed by a vertical cut (hedging) of the southeastern side of the canopy. In July 2015 a summer topping at 3.6 m height (from the ground) was done to control growth suckers developed in uppermost part of the canopy after 2014 topping. In March 2016 the northwestern side of the canopy was hedged at a distance of 2.0 m from the tree trunk. In March 2017, trees were topped at 3.3 m height (from the ground) and hedged on the southeastern side of the canopy at a distance of 1.8 m from the tree trunk.Treatment 4 (T4)—mechanical pruning performed in 2014, 2015, and 2017. In 2014, trees were topped (as in T2) and hedging each side of the canopy. In July 2015, a summer topping at 3.6 m height was done to control growth suckers developed at the uppermost part of the canopy, after 2014 topping. In March 2017 a horizontal cut was made at 3.3 m height followed by hedging both sides of the canopy at a distance of 1.8 m from the tree trunk.Pruning operations were timed to calculate the work rates. Tree measurements of the height of the tree from the ground, width of the canopy and the distance from the base of the canopy to the ground were recorded in five trees randomly selected in each plot. Measurements were done in the same trees in the entire period of tests (2014 to 2017) and taken before and after pruning interventions or in early spring for the non-pruning years.The mass of olives caught by the SRCCSH was measured weighing the bags from each plot.The evaluation of the mass of olive removed but not caught by the harvester was done weighing the fruits collected on canvas placed under a group of three olive trees at three locations randomly selected in each plot.To quantify the mass of olives not removed by the harvester, all trees in each plot were vibrated by a trunk shaker complemented by manual harvest with poles.Total yield per tree was obtained adding the mass of olives caught by the harvester (in the bags) to the mass of olives dropped to the ground (on the canvas) plus the mass left on the treeHarvest efficiency was calculated as follows:One-way analysis of variance were performed to annual data and general linear model univariate analysis for average data, using IBM SPSS version 24 software. Mean separation was performed by Multiple Range Duncan test at 5 and 10% significance level.To avoid low work rates of manual pruning complement reported in previous research work  a clarifIn Lateral cuts of the canopy allow the tractor to move faster since the volume of leaf mass that is eliminated is smaller than in the horizontal cuts of the upper part of the canopy. In the horizontal cut the tractor has to move slower to reduce the risk of leaf and branches mass accumulating in front of the cutter bar, locking the cutter disks.Summer cuts tend to require the tractor to move even slower, since the branches to be cut are highly flexible and tend to bend in front of the disk saw, aggravated when disks are not conveniently sharped.Topping and hedging on both faces corresponds to a working capacity of 155 to 180 trees per hour.Topping and hedging of only one side did not show a higher work rate (186 to 192 trees per hour) because of the non-productive return path required to restart the work in the same direction.Summer topping despite being carried out at a lower working speed than the winter topping, registered a greater working capacity, since only one machine pass was necessary for each row of trees, whereas two passes were required in winter topping due to wider canopies and limited cutter bar width.Based on the working capacities presented in In 2014, the interventions with the disk pruning machine, comprising topping and hedging of both sides (T4) had a cost similar to that of strictly manual pruning (T1).When, in addition to topping and hedging, a manual complement (T2) is added, pruning became more expensive than strictly manual pruning (T1).In 2017, as a result of a more severe pruning intervention, the work rate was lower compared to what had occurred in 2014. As a consequence, the cost weight of manual pruning gave rise to higher pruning costs in T1 and T2 treatments than in T3 and T4 treatments.Concerning total values, the cost of strictly mechanical pruning was lower than pruning with manual interventions.The height of the trees is a relevant dimension in that the highest rods of the vibratory mast of the SRCCSH are at approximately 3.6 m above the ground.This dimension is relevant in that, once the interface of the SRCCSH has been positioned in relation to the trunk of the olive tree, the rods of the vibratory mast must penetrate the canopy, leaving the steel guiding tubes of the rods outside the canopy. Since the SRCCSH allows lateral placement of the vibratory mast, In 2015 significant differences in olive yield were registered between treatments (P < 0.05). Treatment 3 obtained a significantly lower yield (P ≤ 0.05) than the other treatments as a consequence of the higher yield obtained in the previous year. Given that the olive tree is characterized by an alternate bearing, a year with low production allows more vegetative growth. The greater leaf mass developed this year will boost higher yield the following year. This characteristic explains the considerable increase in production that occurred in treatments T1, T2, and T3 from 2014 to 2015. In the case of treatment T3 this increase was not so pronounced, since in 2014 the vegetative growth was conditioned by the existing tree production.In 2016 significant differences (P < 0.05) were found in olive yield between treatments. Treatment 3 and treatment 4 show significantly (P ≤ 0.05) lower yield than treatments 1 and 2. The fall in production of treatment 3 compared to treatments 1 and 2 is associated with a reduction in canopy volume due to topping in summer 2015 and cutting in the northwest side in winter 2016, which left these trees with lower leaf mass and consequently with lower fruiting potential. High production in a smaller tree canopy (T4 in 2015) will tend to penalize the release of productive branches and consequently the production of 2016. In 2017 production shows the opposite trend to 2016, although without significant differences between treatments (P > 0.1). On average, no significant differences between treatments were found (P > 0.1)These results show the potential of mechanical pruning as a method for reducing labor dependence, without significant negative influence in olive production, in line with the results obtained in traditional olive orchards by The present work also seems to reveal that frequent mechanical pruning interventions tend to penalize olive yield. It appears desirable to perform a more intense mechanical pruning spaced in time, as recommended by Manual pruning complement to mechanical pruning did not increase olive yield in comparison to the obtained in trees with the absence of manual pruning complement.Manual pruning complement to the mechanical pruning (T2), particularly in following years to the mechanical pruning, should be regarded as a potentially important technique, since it may contributed to higher yields as verified by - low level of production;- olives with anthracnose disease;- olives at an advanced maturity stage .In 2017, In 2015 significant differences (P < 0.05) were found between treatments in harvest efficiency. At the 2015 harvest, and assuming that the size of the trees is revealed by their dimensions in 2016 Figure 7At harvest olives were below the 4 m height, whereas in treatments T1 and T2 production was above the 4 m mark. However, the results of harvest efficiency in 2015 reveal that more factors not counted for at the present work have to be considered. Fruit detachment force and fruit weight presented a low correlation with harvest efficiency . The spaOn average (period 2015 to 2017), no significant differences (P > 0.05) among treatments in harvest efficiency were found.In the period of 4 years, on average, there were no significant differences (P ≤ 0.05) between treatments in olive production.- the cost with only mechanical pruning is lower than pruning with manual interventions.- manual complement pruning was not relevant since it did not lead to increased production and led to an increase in pruning costs, as had already occurred in the traditional olive grove trial. Complementary manual pruning interventions only make sense to eliminate excess wood accumulated over a relatively long period of time and should be carried out sporadically. Two supplementary annual pruning interventions over a 4-year period are not advisable.- the option for pruning with a disk machine on the side faces in alternate years has also not been interesting since it does not reduce the pruning costs, it does not enhance production of olives. Carrying out the cuts on the sides of the canopy more frequently, while allowing the canopy volume to be controlled, does not allow for the full production potential of the regrowth that appear after the cuts on the side faces.In view of this fact:In terms of the influence of pruning on the performance of the SRCCSH, there were no significant differences between treatments.All datasets generated for this study are included in the article/supplementary material.AD conceived and designed the experiment. AD, JF, AP, and JP performed the evaluation. AD and JP analyzed the data and wrote the paper.This work is funded by National Funds through FCT—Foundation for Science and Technology under the Project UID/AGR/00115/2013 and by Portuguese Agriculture Ministry research program PRODER under the project 55344.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


HLA‐A2  mRNA with a novel mechanism. However, the structural basis for the ubiquitination catalyzed by hMEX‐3C Ring finger domain remains evasive. In this study, we solved the crystal structure of dimeric Ring finger domain of hMEX‐3C and compared it with the complex structure of MDM2/MDMX–UbcH5b–Ub. Our ubiquitination assay demonstrated that the Ring finger domain of hMEX‐3C acts as a ubiquitin E3 ligase in vitro, cooperating with specific E2 to mediate ubiquitination. Then, we identified several key residues in Ring finger domain of hMEX‐3C possibly involved in the interaction with E2–Ub conjugate and analyzed the E3 ligase activities of wild type and mutants at key sites. Additionally, zinc chelation experiments indicated that the intact structural stability is essential for the self‐ubiquitination activity of the Ring finger domain of hMEX‐3C. Taken together, our studies provided new insight into the mechanism of the Ring finger domain of hMEX‐3C that may play an important role in eliciting antiviral immune responses and therapeutic interventions.MEX‐3C, a novel RNA binding E3 ubiquitin ligases, contains two N‐terminal heterogeneous nuclear ribonucleoprotein K homology (KH) domains and C‐terminal Ring finger domain. Recent evidence has suggested that human MEX‐3C has a strong bondage with carcinogenesis and the MEX‐3C‐mediated ubiquitination of RIG‐I is essential for the antiviral innate immune response. Moreover, the Ring finger domain of MEX‐3C could regulate the degradation of  Ubiquitination is the process of attaching ubiquitin (Ub), a small functional regulatory protein, to another targeted molecule. It plays very important roles in many biological processes including protein cytosis, proteasomal degradation, cell‐cycle control, protein trafficking, DNA damage repair, and gene transcription.Caenorhabditis elegans MEX‐3 and involved in the post‐transcriptional regulation.HLA‐A2) allotypes. It binds the 3′UTR of HLA‐A2 mRNA, which induces Ring finger domain dependent degradation.Human MEX‐3 protein, belonging to a novel subfamily of evolutionarily conserved RNA binding proteins, is the human homologs of Ring finger domain is responsible for the ubiquitination reaction and defines the largest family of E3 ligases. Among more than 600 human E3 ligases, there are at least 15 which contain an RNA‐binding domain,in vitro, we performed ubiquitination assays and found that it functions as an E3 ubiquitin ligase and catalyzes self‐ubiquitination. Furthermore, mutations at the possible interaction interface of hMEX‐3C with E2 mostly decreased the ubiquitination activities. Taken together, the intact hMEX‐3C Ring finger domain is responsible for the interaction with E2 enzyme and required for the ubiquitin ligase activity.In this study, we solved the crystal structure of the dimeric Ring finger domain of hMEX‐3C which employs a typical Ring fold and is stabilized by the coordination of two zinc ions. In addition, by superimposing the structure of the hMEX‐3C Ring homodimer with that of MDM2–MDMX–UbcH5b–Ub (PDB ID 5MNJ), a model of hMEX‐3C Ring–E2–Ub complex was proposed and several interface residues of hMEX‐3C Ring finger domain required for recruitment of E2 were, therefore, identified. To characterize the E3 ubiquitin ligase activity of hMEX‐3C Ring finger domain 2CX(9–39)CX(1–3)CX(2–3)C/HX2CX(4–48)CX2C, in which Cys and His are Zinc‐binding residues. By sequence alignment, we found that hMEX‐3C Ring finger domain has seven conserved Cys residues  and one His residue (H625) which are likely involved in zinc binding [Fig. The sequence alignment showed that the hMEX‐3C Ring finger domain shares significant homology with several known Ring finger domains [Fig. X(9–39)CX–3CX(2–3)Rwork and Rfree 20.7% and 24.3%, respectively. The Ring finger domain structure was refined in space group P1, with eight molecules per asymmetric unit. Data collection and refinement statistics are summarized in Table 2+) are coordinated within a C3HC4 “cross brace” manner  by seven Cys side chains and one His side chain, which enables the correct folding and biological activity of the Ring finger domain [Fig. 2+ ions are very important to maintain the stability of the intact Ring finger domain structure and contribute to its E3 ligase activity.To understand the molecular details of the Ring finger domain of hMEX‐3C, we determined the crystal structure of Ring finger domain homodimer (Residues 605–659) at a resolution of 2.2 Å with Z‐score of 9.1, PDB code 2vje), MYLIP , and XIAP . The superimposition indicated that the Ring finger domain structure of hMEX‐3C closely resembles that of MDM2 (PDB 2vje) [Fig. Using Dali search for structural homologs of hMEX‐3C Ring finger domain,in vitro ubiquitination assay to determine the activity of hMEX‐3C Ring finger domain. In the assay, GST‐fused hMEX‐3C Ring finger domain, E1 and various E2s  were incubated together with ubiquitin. After incubation, the reactants were determined by western blot using anti‐GST and anti‐Ub antibodies. In the presence of both E2 of UbcH5b and UbcH5c, the Ring finger domain of hMEX‐3C was multi‐ubiquitinylated [Fig. The sequence conservation and structural homology of hMEX‐3C Ring finger domain with other different E3 ubiquitin ligases indicate its significant membership to E3 ubiquitin ligase category [Fig. To further determine the minimum requirement of the component in ubiquitination assays, we fixed UbcH5b as the E2 enzyme. Then each component in the assay including Ring finger domain of hMEX‐3C, E1, ubiquitin, and E2 (UbcH5b) was removed from the mixture, respectively. We found that, for all incomplete mixtures, there was no observed band compared with the complete reaction mixture [Fig. in vitro ubiquitination assay results indicated that hMEX‐3C Ring finger domain functions as an E3 ligase in combination with specific E2s, such as UbcH5b. We reasoned that the hMEX‐3C Ring finger domain contained a crucial recognition site for the E2. Given that the overall structure of hMEX‐3C Ring finger domain closely resembles the Ring finger domain of MDM2, we speculated that the Ring finger domain of hMEX‐3C may interact with UbcH5b via the same binding site MDM2 used. The interaction between MDM2 Ring finger domain and UbcH5b–Ub showed that several key residues of MDM2/MDMX play a crucial role in stabilizing the closed UbcH5b–Ub conformation.As shown in Figure 2+ binding in the ubiquitination of hMEX‐3C Ring finger domain, we made mutations of zinc‐coordinating cysteine to serine. The two adjacent cysteine residues (C608 and C611) and the distant C632 were mutated to serine, respectively [Fig. Since zinc coordination is essential to the structure and function of zinc finger domains, we speculated that zinc binding of hMEX‐3C Ring finger domain is needed to mediate ubiquitylation. To evaluate the role for zinc coordination in the ubiquitination of hMEX‐3C Ring finger domain, we used metal‐chelating reagents, such as EDTA and DTPA, to treat the Ring finger domain and observed a loss of activity [Fig. Caenorhabditis elegans. C. elegans MEX‐3 protein is a regulator of translation that specifies the posterior blastomere identity during early embryogenesis and contributes to the maintenance of germline totipotency, immune responses, differentiation, and cancer.C. elegans MEX‐3.HLA‐A2 mRNA, inducing its Ring‐dependent degradation. The Ring finger domain of MEX‐3C has a central role for RNA degradation of HLA‐A2 mRNA, therefore, modulating natural killer cell activity.in vivo Lys‐63 linked ubiquitination of RIG‐I and mutation of one cysteine in the Ring finger domain leads to the abolishment of RIG‐I ubiquitination.MEX‐3 was first identified as a translational repressor in nematode 2CX(9–39)CX(1–3)HX(2–3)C/HX2CX(4–48)CX2C.3HC4 (RING‐HC) or C3H2C3 RING (RING‐H2). The cysteines/histidine  coordinate one cation and others  coordinate the second cation.Ring finger domains, differing considerably in composition and length, are characterized by seven cysteines and histidine which coordinate two zinc ions. The consensus sequence is CXX(9–39)CX–3HX(2–3)in vivo that localizes to stress granules (SG) and triggers retinoic acid‐inducible gene‐1 (RIG‐I)‐dependent antiviral responses by RIG‐I ubiquitination.in vitro’ E3 ligase activity of the Ring finger domain of hMEX‐3C with three different E2s  [Fig. The Ring finger domain is known for its potential E3 ubiquitin activity, cooperating with E2 to generate polyUb chains.It has been reported that the residues in the zinc chelating loops and α helix of Ring finger domain are needed for their interaction with E2.Our results provided first structural insight into the Ring finger domain of hMEX‐3C. Furthermore, our entire experiments showed strong evidence that the Ring finger domain, in the specific molecular context, is a module that cooperates with specific E2s and facilitates ubiquitination. We hope this evidence will help to further clarify the biological function of hMEX‐3C.The gene encoding the Ring finger domain of hMEX‐3C (Residues 603–659), was amplified by PCR with corresponding primers. The PCR products were purified and digested with NdeI and XhoI. Then, the digested fragment was cloned into pGEX‐TEV vector, a modified vector for the expression of Glutathione S‐transferase (GST) fusion proteins with a proteolytic site. Mutants of Ring finger domain of hMEX‐3C were obtained using Quick Change II Site‐Directed Mutagenesis kit (Agilent).E. coli. BL21 (DE3) competent cells. The single colony was inoculated into 5 mL LB medium and incubated at 37°C overnight. The following day, 5 mL strains were then transferred to a 100 mL LB medium for approximately 3 h. The 100 mL strains were finally added to 1 L LB medium with 20 μg/mL ampicillin. Protein expression was induced by 0.4 mM isopropyl‐b‐d‐thiogalactopyranoside (IPTG) at 16°C for 20 h. The cells were harvested by centrifugation at 5000 rpm for 10 min and the pelleted cells were resuspended in a lysis buffer . The cells were lysed by sonication at 4°C followed by centrifugation at 13,000 rpm for 30 min. The protein lysates were purified on a GST column  using 1 M NaCl, 20 mM Tris–HCl (pH 8.0). The eluted proteins were further purified using Highload 16/60 Superdex 75 size exclusion column  equilibrated with 500 mM NaCl, 20 mM Tris–HCl (pH 8.0). The target proteins were analyzed and confirmed by SDS‐PAGE. The SDS‐PAGE analysis showed the presence of a high molecular protein band of about 33 kDa. The mutants were purified by the same procedure as described above.Plasmids of GST‐hMEX‐3C Ring finger domain and the mutants were transformed into M NaCl, 20 mM Tris (pH 8.0). The crystals of hMEX‐3C Ring finger domain were obtained by sitting drop vapor diffusion against 0.2 M ammonium acetate, 0.1 M Bis–Tris (pH 6.5) and 25% w/v polyethylene glycol 3350 at 18°C with a protein concentration of 15 mg/mL. For the crystals, the mother liquid supplemented with 30% (v/v) glycerol was used as cryoprotectant. Crystals were soaked with the cryoprotectant and then flash‐frozen in liquid nitrogen.Prior to crystallization, the Ring finger domain of hMEX‐3C was dialyzed against a buffer containing 200 mhttp://www.pymol.org).Diffraction data from crystals of hMEX‐3C Ring finger domain were collected on beamline BL19U at Shanghai Synchrotron Radiation Facility (SSRF) at the SSRF and processed with HKL2000. Processing statistics are shown in Table M E1 (Sigma‐Aldrich), 1 μM each of the different E2s , 5 μM His6 ubiquitin (Sigma‐Aldrich), 5 mM ATP, 5 mM MgCl2, 5 mM DTT, and 50 mM Tris–HCl (pH 7.5) and ddH2O to a final volume of 10 μL. After dissolved the reactions with 10 μL of 2 × SDS sample loading buffer, products were analyzed by SDS‐PAGE. The reactions were analyzed by western blotting using anti‐Ub and anti‐GST antibodies (Sigma‐Aldrich).For this assay, 1 μg of bacterially produced GST‐hMEX‐3C Ring finger domain WT and mutants were, respectively, incubated at 37°C for 90 min in a reaction mixture with 100 nM of ethylenediaminetetraacetic acid (EDTA) and disodiumtriaminopentacetic acid (DTPA) potent chelators.To analysis the chelation experiment, GS‐bound proteins were incubated for 18 h at 4°C with three changes of PBS buffer plus 5 m−1 at 25°C. The proteins were concentrated to 0.10 mg/mL in 30 mM phosphate buffer (pH 8.0). A buffer sample was used as a control.The CD measurements of the Ring finger domain of hMEX‐3C and mutants were implemented on a Jasco‐810 spectropolarimeter. The CD spectra were recorded at wavelengths from 200 to 260 nm using 0.1 cm path length cell and the scan rate is 100 nm minThe coordinates for the structure described in this work have been deposited in the Protein Data Bank (PDB) under the Accession code 5ZI6.Fig S1. Size exclusion chromatography (SEC) of hMEX‐3C Ring finger domain. GST fused MEX‐3C Ring finger domain digested by TEV protease and then loaded into S75 column. The molecular weight of hMEX‐3C Ring finger domain calculated from its elution volume.Click here for additional data file.


Pyrroloquinoline quinone (PQQ) is known as the “third coenzyme” following nicotinamide and flavin. PQQ-dependent enzymes have previously been found only in prokaryotes, and the existence of a eukaryotic PQQ-dependent enzyme was in doubt. In 2014, we found an enzyme in mushrooms that catalyzes the oxidation of various sugars in a PQQ-dependent manner and that was a PQQ-dependent enzyme found in eukaryotes. This paper presents the X-ray crystal structures of this eukaryotic PQQ-dependent quinohemoprotein, which show the active site, and identifies the amino acid residues involved in the binding of the cofactor PQQ. The presented X-ray structures reveal that the AA12 domain is in a binary complex with the coenzyme, clearly proving that PQQ-dependent enzymes exist in eukaryotes as well as prokaryotes. Because no biosynthetic system for PQQ has been reported in eukaryotes, future research on the symbiotic systems is expected. Coprinopsis cinerea (CcPDH) of the Basidiomycete division, is composed of a catalytic PQQ-dependent domain classified as a member of the novel auxiliary activity family 12 (AA12), an AA8 cytochrome b domain, and a family 1 carbohydrate-binding module (CBM1), as defined by the Carbohydrate-Active Enzymes (CAZy) database. Here, we present the crystal structures of the AA12 domain in its apo- and holo-forms and the AA8 domain of this enzyme. The crystal structures of the holo-AA12 domain bound to PQQ provide direct evidence that eukaryotes have PQQ-dependent enzymes. The AA12 domain exhibits a six-blade β-propeller fold that is also present in other known PQQ-dependent glucose dehydrogenases in bacteria. A loop structure around the active site and a calcium ion binding site are unique among the known structures of bacterial quinoproteins. The AA8 cytochrome domain has a positively charged area on its molecular surface, which is partly due to the propionate group of the heme interacting with Arg181; this feature differs from the characteristics of cytochrome b in the AA8 domain of the fungal cellobiose dehydrogenase and suggests that this difference may affect the pH dependence of electron transfer.Pyrroloquinoline quinone (PQQ) was discovered as a redox cofactor of prokaryotic glucose dehydrogenases in the 1960s, and subsequent studies have demonstrated its importance not only in bacterial systems but also in higher organisms. We have previously reported a novel eukaryotic quinohemoprotein that exhibited PQQ-dependent catalytic activity in a eukaryote. The enzyme, pyranose dehydrogenase (PDH), from the filamentous fungus IMPORTANCE Pyrroloquinoline quinone (PQQ) is known as the “third coenzyme” following nicotinamide and flavin. PQQ-dependent enzymes have previously been found only in prokaryotes, and the existence of a eukaryotic PQQ-dependent enzyme was in doubt. In 2014, we found an enzyme in mushrooms that catalyzes the oxidation of various sugars in a PQQ-dependent manner and that was a PQQ-dependent enzyme found in eukaryotes. This paper presents the X-ray crystal structures of this eukaryotic PQQ-dependent quinohemoprotein, which show the active site, and identifies the amino acid residues involved in the binding of the cofactor PQQ. The presented X-ray structures reveal that the AA12 domain is in a binary complex with the coenzyme, clearly proving that PQQ-dependent enzymes exist in eukaryotes as well as prokaryotes. Because no biosynthetic system for PQQ has been reported in eukaryotes, future research on the symbiotic systems is expected. Escherichia coli, rely on the environment to supply the needed PQQ (Pyrroloquinoline quinone (PQQ) was discovered in 1964  was subseded PQQ . There heded PQQ , 11, theCoprinopsis cinerea  of 1.1 nM. Phylogenetic analyses of the amino acid sequence of CcPDH revealed the existence of a new category of PQQ quinoproteins that are present not only in fungi but also in bacteria, archaea, and amoebozoa; these proteins are significantly different from known quinoproteins. In a previous study, a protein homologous to CcPDH exhibited 2-keto-d-glucose dehydrogenase (2KGDH) activity, which is a novel catalytic activity for a PQQ-dependent enzyme (b (protoheme IX) prosthetic groups, it can be referred to as a quinohemoprotein. Some bacterial PQQ-dependent enzymes contain one or several c-type hemes and, hence, are also called quinohemoproteins , a PQQ-dependent dehydrogenase domain (AA12) in the middle of the sequence, and a C-terminal cellulose-binding domain classified as a member of the family 1 carbohydrate-binding module (CBM1). CBM1 domains are often found as molecules attached to fungal cellulolytic enzymes and are known to have the ability to adhere to crystalline cellulose in plant cell walls (We previously found a eukaryotic PQQ-dependent pyranose dehydrogenase (PDH) from the filamentous fungus us paper ), which t enzyme . Our finproteins . As showll walls .CcPDH was modeled using the Phyre2 protein fold recognition server, and the predicted structure was compared with that of other quinoproteins and structurally similar proteins. Because the AA12 domain showed low homology to the six-blade quinoproteins but no similarity to the eight-blade quinoproteins, the AA12 domain was predicted to have a six-blade, instead of an eight-blade, quinoprotein structure.Known prokaryotic PQQ-dependent enzymes have a propeller superbarrel fold in common. The PQQ molecule is bound in the middle of the catalytic domain together with a catalytically essential calcium or other divalent metal ion. The cofactor is bound mainly by hydrogen bonds through its carboxyl groups. There are two types of propeller superbarrel folds: an eight-blade β-propeller structure, with each blade consisting of a four-strand antiparallel β-sheet, and a six-blade β-propeller structure , 19. Thewww.cazy.org), the cytochrome domain of CcPDH is classified in subfamily 1 of auxiliary activity family 8 (AA8). AA8 domains feature an unusual fold that contains the highest β-structure content among known cytochrome structures. AA8 proteins fold into an immunoglobulin-like β-sandwich with a Met/His-coordinated b-type heme  cofactor, and CDH is a member of the AA3 subfamily 1 (AA3_1) family  and the AA8 domain (the cytochrome domain) of CcPDH. The crystal structure confirms that the enzyme is PQQ dependent by revealing an active site containing a PQQ and a calcium ion.According to the Carbohydrate-Active Enzymes (CAZy) database (ype heme . Most of) family . CDH is ) family . LPMO ox) family , 25. CDH) family . In the 8 domain . The AA8CcPDH and resolved their crystal structures individually because the crystallization of the full-length CcPDH was considered to be hindered by the peptide linker regions, which are interdomain connectors. The AA12 domain lacking the AA8 domain maintains the catalytic activity at the same level as the full-length enzyme  was used for the initial phase determination of the apo-AA12 domain crystals soaked in the mother liquor containing Pt, Au, or Hg ions. The apo-AA12 domain crystal used for the high-resolution data set diffracted to a 1.5-Å resolution, and the final model was refined to CcPDH) with 1 calcium ion, 1 formate ion, 1 sulfate ion, 2 glycerol molecules, and 778 water molecules. A polyethylene glycol molecule is partially modeled as a triethylene glycol moiety located close to Asp601 in the apo-AA12 domain. The AA12 molecule contains 3 disulfide bonds . The N-glycosylation site, Asn551, was clearly glycosylated, as predicted by the NetNGlyc server (http://www.cbs.dtu.dk/services/NetNGlyc/). The PQQ molecule was bound by the AA12 domain at the bottom of the active site, which is a small cavity near the pseudosymmetry axis of the propeller fold . This crystal packing arrangement appears to prevent PQQ binding, which explains why soaking PQQ to obtain the crystallized holostructure did not succeed.Similar to other PQQ-dependent enzymes, g pocket . The othg pocket ; howeverPhanerochaete chrysosporium CDH   (CcPDH) with a single b-type heme, 2 2-methyl-2,4-pentanediol molecules, 1 acetate ion, and 155 water molecules. One disulfide bond (Cys138-Cys141) that is conserved in the AA8 domain of CDHs from various filamentous fungi was observed. Asn140 is glycosylated with a single GlcNAc molecule, as predicted by the NetNGlyc (version 1.0) server.The AA8 domain takes the shape of an antiparallel β-sandwich fold that is very similar to that of the corresponding AA8 domain of CDHs from filamentous fungi, including that of the α = 179) . The asyCcPDH closely resemble those of the AA8 domain of PcCDH, whereas the amino acid residues surrounding the heme are different (PcCDH significantly lowered the rate of intramolecular electron transfer from FAD to heme b (CcPDH is Ala185. Other aromatic residues surrounding the heme in PcCDH (Phe160 and Phe168) are absent  in CcPDH. On the other hand, Tyr108 in CcPDH is conserved in CDHs and forms a hydrogen bond with the d-propionate side chain of the heme b. Interestingly, Glu162 in PcCDH corresponds to Arg181 in CcPDH, which interacts with the propionate group of the heme, which results in a local positive charge at the heme binding pocket of the AA8 domain  and the cytochrome domain (AA8). PQQ quinoproteins have been found to have a conserved propeller superbarrel fold structure. The crystal structure presented in this study revealed that the AA12 domain of CcPDH has a 6-blade β-propeller structure. A search for the structural homology with the Dali server  revealed that the AA12 domain exhibits a high degree of structural homology to bacterial PQQ quinoproteins in the Protein Data Bank (PDB), such as that of Asd in Pyrobaculum aerophilum , despite a relatively low (<20%) sequence identity  are not conserved in CcPDH . The details of substrate binding by the AA12 domain of CcPDH should be revealed by further analysis, e.g., using a substrate-bound structure.The structural alignment of the AA12 domain with the known bacterial PQQ-dependent dehydrogenase structures is shown in b in CcPDH is dependent on the pH and is the rate-limiting step under acidic to weakly basic conditions. For the flow of electrons to proceed in CDHs, the interdomain transfer of electrons from FAD to the heme has attracted much attention. Environmental pH affects the IET process in CDHs. We demonstrated that PcCDH exhibits optimal flavin reduction of a cellobiose substrate at pH 4.5. Alternatively, the interdomain transfer of electrons from the flavin to the heme proceeds effectively at pH 3.5 and does not occur at a pH above 6.0  is approximately pH 6.0, whereas that for the overall activity  is 8.5 . This mebove 6.0 . In mostbove 6.0 , 33. Thebove 6.0 , 35. Witbove 6.0 . Using hociation . IET shoCcPDH, providing new structural insight into the electron transfer process during plant cell wall degradation by cellulolytic fungi. This research will be advanced in various fields such that the entire picture of the decomposition of woody biomass in nature will become clear.In this study, we have succeeded in determining the X-ray structures of a binary complex of the AA12 domain with the cofactor to prove clearly that PQQ-dependent enzymes exist in eukaryotes as well as prokaryotes. This finding also reveals the active site and the amino acid residues involved in binding the cofactor PQQ. In conjunction with prior biochemical findings, this structural analysis effectively ends the controversy surrounding the existence of PQQ-dependent enzymes in eukaryotes. This paper presents the X-ray crystal structures of the AA8 cytochrome domain of CcPDH was heterologously expressed and purified as described previously  of CcPDH was obtained by site-directed mutagenesis using a pPICZα vector harboring the gene coding for the full-length CcPDH template. PCR was carried out with KOD Plus (version 2) DNA polymerase  using the following oligonucleotide pair: 5′-CCTCCGCTCTCTTGAGGTGCCCCGACC-3′ and 5′-GGTCGGGGCACCTCAAGAGAGCGGAGG-3′. The underlined residues represent the targets for substitutions that introduce a stop codon. The mutated sequence was verified by DNA sequencing with a model 3130 genetic analyzer , and the recombinant plasmid was transformed into Pichia pastoris for subsequent protein expression according to the same protocol used for the wild-type recombinant enzyme. The recombinant AA8 domain was purified from the culture filtrate using ultrafiltration and three-step column chromatography. The culture was centrifuged , and the supernatant was filtered through a 100-kDa-cutoff membrane filter on a QuixStand system . After concentrating the supernatant solution with a 5-kDa-cutoff membrane filter on the QuixStand system, ammonium sulfate was added to the concentrated solution to a final concentration of 1 M, and the solution was then applied to a Toyopearl Phenyl-650S column (75-ml bed volume) equilibrated with 20 mM Tris-HCl buffer containing 1 M ammonium sulfate (pH 8.5). The proteins were eluted with a linear reverse gradient of 20 mM Tris-HCl buffer (pH 8.5) without ammonium sulfate, and the fraction exhibiting an absorbance at 430 nm was collected, desalted, and loaded on a Toyopearl DEAE-650S column (180-ml bed volume) equilibrated with 20 mM Tris-HCl (pH 8.5). The protein was eluted from the column with a linear gradient of 0 to 400 mM NaCl in the same buffer. The fraction containing the recombinant protein was collected and deglycosylated using endo-β-N-acetylglucosaminidase H . After changing the buffer to a 20 mM Tris-HCl solution (pH 8.5) using a Vivaspin 20 filter with a 5-kDa cutoff , the sample was loaded on a Resource Q column (6-ml bed volume) equilibrated with 20 mM Tris-HCl buffer at pH 8.5 and eluted from the column with a linear gradient of 0 to 500 mM NaCl in the same buffer. The purity of the recombinant AA8 domain was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by its absorption spectrum. The protein concentration of the AA8 domain was determined by setting the absorbance to 280 nm and using an extinction coefficient of 162.1 mM−1 cm−1. The purified AA12 and AA8 domains were dissolved in 5 mM sodium acetate buffer at pH 5.0 and 5 mM Tris-HCl at pH 8.0, respectively. The apo-AA12 domain crystals were obtained with a 17.9-mg/ml protein solution by the sitting drop vapor diffusion method using a reservoir solution composed of 0.2 M sodium formate, 20% polyethylene glycol 3350, and 20 mM CaCl2. The sodium formate was replaced with the same concentration of potassium formate to promote crystal growth, and the crystals obtained from both conditions were used for data collection. Crystals of the AA8 domain grew in a drop composed of 4.5-mg/ml AA8 domain and 40% 2-methyl-2,4-pentanediol (MPD) that was incubated for 1 week at 20°C. The crystals of the binary complex comprising the AA12 domain with PQQ were obtained from 166-mg/ml holo-AA12 domain in solution with 50 mM sodium acetate buffer, pH 6.0, containing 1 mM CaCl2, which was incubated for 10 weeks at 4°C.The AA12 domain  of 2PtCl4, HgCl2, or HAuCl4 for 4 h. The diffraction images were processed using XDS  Ring and PF Advanced Ring (PF-AR) at the High Energy Accelerator Research Organization . X-ray diffraction data sets for the holo-AA12 crystals were collected at beam line BL41XU at Spring-8 . Crystals were flash cooled with a nitrogen stream at 95 K prior to data collection. The apo-AA12 crystals were transferred to a reservoir solution containing 25% glycerol as a cryoprotectant. To obtain the heavy-atom-derivative data sets used for phase determination, the apo-AA12 crystals were soaked in reservoir solution containing 2 mM Ksing XDS . Determising XDS , followesing XDS . The holsing XDS , using tber 1D7D ) as the http://www.rcsb.org/), under accession numbers 6JWF, 6JT5, and 6JT6 for the holo-AA12, the apo-AA12, and the AA8 domain of CcPDH, respectively.The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (


Metallic glass composites with shape memory crystals show enhanced plasticity and work-hardening capability. We investigate the influence of various critical structural aspects such as, the density of crystalline precipitates, their distribution and size, and the structural features and intrinsic properties of the phase on the deformation behavior of metallic amorphous Cu Improving the plastic deformability of metallic glasses (MGs) is the key to the extensive use of these attractive materials in structural and functional applications. Hence, understanding and controlling the deformation mechanisms of MGs is a hot research topic and remains a major ongoing issue. The most common strategy to improve the ductility of MGs is the fabrication of MG composites with a crystalline phase ,4,5,6,7.nd twins . This hand twins ,23,24. AMolecular dynamics (MD) simulations have provided an atomistic description of the deformation mechanism in MG composites with a B2 shape memory phase ,26. An aIn this paper, we present MD computer simulations of the deformation processes in MG composites with B2 CuZr crystalline precipitates. In our previous work, the deformation behavior and the underlying deformation mechanism of only one type of MG composite have been investigated . Here, wFor studying the mechanical properties of MG composites with shape memory crystals, classical MD simulations were performed using the program package LAMMPS . A glassv et al. . The potv et al. ,32. Althv et al. ), our siv et al. , we usedv et al.  to invesFor simulating the deformation mechanism, slab-shaped Cus strain , calculas strain . The marTo investigate the effect the density of inclusions on the deformation behavior of CuUpon tensile load, the MG composite with 15 inclusions (14.1 vol.%) shows homogeneous deformation. Initially, STZs mostly nucleate at the corners of the B2 inclusions, where the stress is concentrated, and around the soft amorphous-crystalline interface ,37. OnceWhen the number of B2 precipitates decreases to only 9 (8.4 vol.%) the response of the MG composite becomes extremely brittle and plastic deformation is highly localized in one dominant shear band  the 9n18uc composite shows homogeneous plastic deformation. Although the free path for shearing in this MG composite is long enough to form critical shear bands (≈23 nm) these planar defects are confined by the crystalline precipitates and the strain is homogeneously distributed in the whole composite on different slip planes carrying plastic deformation. Even at a strain level of 16% none of these shear bands becomes critical and there is a strong competition between the glassy and crystalline phases to accommodate the elastic energy. Hence, together with the formation of an organized pattern of multiple shear bands, the B2 inclusions are severely deformed and transform into the R and BCT phases. Both the distribution and the volume fraction of the B2 inclusions have a strong impact on the deformation mechanism of MG composites.A quantitative interpretation of the aforementioned deformation mechanisms in MG composites can be derived by considering the strain localization parameter , ψ=1N∑i=ions see . Hence, To emphasize the superior mechanical properties of MG composites with shape memory crystals we compare their deformation mechanism to the one of composites with nanocrystalline inclusions which deform via dislocation activity. For this, we directed our attention to the two ductile MG composites, 15n10uc and 9n18uc, respectively, and replaced the B2 CuZr phase with pure face-centered cubic (fcc) Cu. Under loading, the composites with fcc inclusions show a more localized plastic deformation than the composites with shape memory phase. The fcc inclusions in the 15n10uc composite are too soft to block the shear band propagation cutting through the precipitates and slip transfer into the crystalline phase takes place. Initially, STZs are activated at the corners of the precipitates as well as by interactions with dislocations in the fcc inclusions ,42,43 anThe ld first . Neverthases see . AdditioIn this paper, we have presented molecular dynamics simulations of the nanostructure–property relationships in shape memory metallic glass composites. Our results reveal that MG composites with a relatively low density of small B2 inclusions (14.1 vol.%) show homogeneous plastic deformation. The narrow spacing between the inclusions does not allow the STZs to percolate and form major shear bands. Hence, the plastic deformation in 15n10uc composite is mediated by the formation of embryonic shear bands and the martensitic phase transformation of B2 shape memory inclusions. Reducing the number (volume fraction) of B2 inclusions in the MG composites yields the ductile-to-brittle transition. The distance between inclusions in 9n10uc composites is within the critical shear band length which in turn leads to a highly localized deformation process. Otherwise, to ensure the arrest of shear bands and hence, homogeneous plastic deformation, a larger fraction of B2 inclusions beyond 28% is required. Substituting the shape memory inclusions with soft fcc Cu crystals renders more localized plastic deformation in the MG composites. Shear bands can cut through the Cu precipitates and a critical shear front occurs. Thus, by carefully choosing the density, size and spatial distribution of shape memory crystals, one can obtain MG composites that exhibit large tensile ductility for even a low-volume fraction of the shape memory phase.


Understanding the functions and mechanisms of biological systems is an outstanding challenge. One way to overcome it is to combine together several approaches such as molecular modeling and experimental structural biology techniques. Indeed, the interplay between structural and dynamical properties of the system is crucial to unravel the function of molecular machinery’s. In this review, we focus on how molecular simulations along with structural information can aid in interpreting biological data. Here, we examine two different cases: (i) the endosomal translocation toxins  and (ii) the activation of adenylyl cyclase inside the cytoplasm . Structural biology evolved from the determination of the structure of small metalloproteins  that areforce-field [Molecular modeling approaches face other issues. One long-lasting problem is the size of the conformational space that must be explored, which is still a challenge despite the development of high-power computing and of enhanced sampling techniques ,4,5. Anoce-field ,7. Neverce-field ,9,10.In the present review, we present how the cooperative use of in silico and experimental structural biology approaches improve our knowledge of specific steps of the insertion of bacterial toxins and virulence factors into the host cells. We focus the review on two events, widely recognized as essential in the toxicology field: (i) the translocation of diphtheria, botulinic and tetanus toxins through the membrane of endosome vesicles, (ii) the activation of adenylyl cyclases in the cytoplasm of host cells. The choice of these biological topics was motivated by the existence of several X-ray crystallographic structures of these toxins, as well as by the presence of large conformational transitions investigated by molecular modeling studies.Diphtheria toxin (DT) was recognized as a major disease-causing agent at the end of 19th century, when filtered bacterial culture was shown to reproduce the disease in animal models. This 58 kDa protein includes three domains: the catalytic (C) domain (residues 1–193), the middle translocation (T) domain (residues 205–378) and the receptor-binding (R) domain (residues 386–535) . The monDT is endocytosed into the cells by binding its fragment B to the heparin-binding epidermal growth factor-like precursor (HB-EGF). Under acidic conditions of the late endosome, the domain C is cleaved and translocated into the cytosol. Once in the cytoplasm the C domain then catalyzes the transfer of the ADP-ribose moiety of NAD onto the elongation factor-2 (EF-2), rendering EF-2 inactive and consequently causing protein synthesis to stop in the host cell.Several structures of DT ,13,14,15The membrane translocation and the effect of acidic pH were further studied by a large set of biophysical methods: fluorescence ,21,22,23Using fluorescence techniques, several pieces of information were obtained on the behavior of the T domain within DT: (i) the hydrophobic helices TH5-TH9 tend to be more exposed to aqueous solution in the isolated T domain ; (ii) thThe effect of pH was also studied on the isolated T domain. Using hydrogen/deuterium exchange experiments coupled to mass spectrometry , the strdent HDX  kinetic Neutron reflectivity experiments  providedSeveral results have been reported for the oligomeric states of DT and of the domain DT in the membrane. It was hypothesized ,33 that To investigate the extensive but contradictory experimental information on the T and DT interaction with the membrane, several molecular modeling studies have been realized. Flores et al.  developef lipids  revealedThe initial models of the protein conformations and protein-membrane association  were furμs  demonstreriments . ThermodThe overall view of DT translocation through the endosome membrane is that this process follows a very complex mechanism. The numerous experiments performed on this system have provided some insights, but several contradicting models have been proposed. MD simulations provide a conformational view with respect to the experimental observations, highlighting the great heterogeneity of conformation observed for DT in the different environments studied. The role of some His residues was clarified, but, due to the enormous complexity of the conformational landscape of DT during the translocation, up to now only a superficial exploration was possible, despite the large computational time invested.Clostridium botulinum, and cause botulism mostly through food poisoning. Among the BoNTs, various types (from A to G) exist, from which the serotypes A, B and E are lethal. The typical symptom of botulism is flaccid paralysis, which is the inability to contract skeletal muscles, inducing impaired vision, followed by paralysis of facial muscles and ultimately respiratory failure due to diaphragm paralysis. The TeNT is produced by Clostridium tetani and causes the disease tetanus, which is drastically different from botulism and is characterized by periodic hypercontraction of skeletal muscles called spastic paralysis.The botulinum toxins (BoNTs) and the tetanus toxin (TeNT) are the most potent toxins known, they target the nervous system and are consequently named neurotoxins . The BoNIn agreement with the different symptoms associated to the poisoning, BoNT and TeNT target neuromuscular junction but with distinct destinations, as the catalytic domain of BoNTs is released into the cytosol of motor neurons, whereas the catalytic domain of TeNT is finally released into the cytosol of inhibitory neurons. Both toxin families enter into the synaptic recycling vesicle system, through binding to gangliosides (a class of glycosphingolipids) and to synaptic vesicle membrane proteins. Once the neurotoxin is embarked into a synaptic vesicle, a change of pH toward acidic values induces a conformational change of the toxin, and consequently the translocation of the catalytic domain through the vesicle membrane to the synapse cytosol. This catalytic domain is then separated from BoNT or TeNT and expelled from the vesicle by the cleavage of a very conserved disulfide bridge. The catalytic domain, a zinc-dependent protease, cleaves various SNARE proteins depending on the type of the toxin, thus generating the toxic effect.Clostridium botulinum and Clostridium tetani, a unique chain is present. Depending on the toxin types, this chain is cleaved in two peptidic chains, either during secretion from the bacterial pathogen or later within the host. The mature toxins are thus formed by two protein chains (light chain: Lc and heavy chain: Hc) covalently connected through a disulfide bridge. All structures  their size, about 500 residues for DT, about 1300 residues for BoNT and TeNT; (ii) a quite intricate topology for BoNT/TeNT with a belt region extending from the HN domain to grip the catalytic LC domain, whereas the topology of DT is based on more short-range interactions. The BoNT/E structure  displaysBacillus anthracis, CyaA from Bordetella pertussis and ExoY from Pseudomonas aeruginosa [The adenylyl cyclase toxins are present in several pathogens. Up to now, three of these toxins have been studied at the molecular level: the edema factor (EF) from ruginosa ,64. X-raruginosa , CyaA [6ruginosa  and ExoYruginosa . EF and ruginosa ,69.CyaA revealed itself as a very good prototype for developing various biotechnological applications ,71 whichThe interactions of AC and EF with calmodulin are different, although they share similar global features . The bareraction C, is morObservation of AC/CaM interaction in the X-ray crystallographic structure  Figure C suggestic patch , stronglic patch . Furthersolution ,86.However, available MD trajectories allowed to enlarge this point of view. Indeed, the removal of Caulations  the brea studies : mutatio studies  reveal tIn the complex AC/C-CaM, the loop containing residues 226–232, located at the extremity of SA region which is not visible in the crystal structure, but has been reconstructed using Modeller , displayas shown  that, inIn the structures of AC, the C terminal lobe of CaM is complexed with two Caa2+ ions . For MD ions [2+ , CaM disf AC/CaM  and EF/Cf AC/CaM ,92,93, rf AC/CaM ,95, the Along the course of MD simulations , free AC (N-CaM) .Bordetella pertussis, remains predominantly sensitive to antibiotic treatment, yet resistant strains are evolving [Whooping cough, caused by evolving ,101 makievolving ,105,106.evolving ,113,114 evolving ,116. Monevolving .Bacillus anthracis led [A virtual screening study of the adenylyl cyclase EF from acis led  to the dacis led , which wacis led , includiThe examples described here illustrate the importance of the combined use of molecular modeling and experimental structural biology approaches. Indeed, from the initially determined structures of EF and AC, it would have been impossible to guess that the same family of compounds, the thiophen ureidoacids, could inhibit the enzymatic activity of both toxins. The use of molecular modeling can thus make possible to rationalize structural properties. Similarly, the comparison of the DT and BoNT/TeNT structures enabled detecting common physico-chemical properties, such as the open/closed conformation transition or the partial unfolding at low pH. Therefore, it could be expected that the description of the DT already obtained is qualitatively transferable to toxins BoNT and TeNT.


N-acetyltransferase (GNAT) domain organization have been identified in actinobacteria. The ACT domain is fused to the GNAT domain, conferring amino acid-induced allosteric regulation to these protein acetyltransferases (Pat) ). Members of the AAPatA family share similar secondary structure and are divided into two groups based on the allosteric ligands of the ACT domain: the asparagine (Asn)-activated PatA and the cysteine (Cys)-activated PatA. The former are mainly found in Streptomyces; the latter are distributed in other actinobacteria. We investigated the effect of Asn and Cys on the acetylation activity of Sven_0867  and Amir_5672 , respectively, as well as the relationship between the structure and function of these enzymes. These findings indicate that the activity of PatA and acetylation level of proteins may be closely correlated with intracellular concentrations of Asn and Cys in actinobacteria. Amino acid-sensing signal transduction in acetyltransferases may be a mechanism that regulates protein acetylation in response to nutrient availability. Future work examining the relationship between protein acetylation and amino acid metabolism will broaden our understanding of post-translational modifications (PTMs) in feedback regulation.One hundred and fifty GCN5-like acetyltransferases with amino acid-binding (ACT)-GCN5-related  Bacillus subtilis, regulating the expression of the acuA gene, which encodes an acetyltransferase, is under the control of catabolite control protein A (CcpA), a global regulatory protein affected by the quality of the carbon source available to the cell [Escherichia coli, two acetylation-signaling pathways mediated by cAMP have been identified. The carbon regulating CRP–cAMP complex induces the expression of yfiQ, which encodes the Gcn5-like acetyltransferase YfiQ  [Salmonella enterica, the expression of the genes encoding the acetyltransferase Pat and deacetylase CobB is activated by IolR, a repressor of myo-inositol catabolism involved in the utilization of cyclohexane-1,2,3,4,5,6-hexol, a cyclic polyol abundant in soil, as a carbon source [Protein lysine acetylation has emerged as an important metabolic regulatory mechanism in bacteria, since the discovery of reversible acetylation of acetyl–CoA (Ac–CoA) synthetase via enzymatic acetylation, such as acetyltransferase  and deacthe cell . In Eschand Pka) ,8, thus and Pka) . Furtherand Pka) . In Salmn source .Mycobacterium tuberculosis and Mycobacterium smegmatis, cAMP directly activates the Pat, MtPat (Rv0998) and MsPat (MSMEG_5458) by binding to a cyclic nt-binding domain that is fused to the N-terminus of the catalytic GCN5-related N-acetyltransferase (GNAT) domain [Micromonospora aurantiaca (MaKat), is used for amino acid-induced allosteric regulation of the enzyme [Streptomyces; the latter are distributed in other actinobacteria. Our results present an example of a novel signal transduction mechanism for regulating protein acetylation in actinobacteria via amino acid-sensing acetyltransferases in response to intracellular concentrations of Asn and Cys.Allosteric effectors also control the activity of protein acetyltransferases (Pat). In ) domain . Recentle enzyme . In the All strains were purchased from the China General Microbiological Culture Collection Center (CGMCC). All the reagents, purchased from Sigma, were of the highest grade available commercially.patA and homologous acs genes coding for Ac–CoA synthetase were amplified by PCR using genomic DNA from bacterial strains. After digestion with restriction enzymes, genes coding for PatA and Ac–CoA synthetase (Acs) proteins were ligated into pET-28a, which was predigested with EcoRI and HindIII, generating pET28a-PatA and pET28a-Acs. The overexpression and purification of proteins in the E. coli BL21-DE3 was performed as described previously [All eviously . Protein+ to NADH, resulting in an increase in absorbance at 340 nm [Acetylation activity of acetyltransferases was measured continuously by a coupled enzymatic assay using a fluorescence plate reader . The production of CoA is measured using pyruvate dehydrogenase mediated reduction of NADt 340 nm ,14. The t 340 nm .Protein samples were electrophoresed on an SDS/PAGE and then transferred on to a PVDF membrane for 1 h at 100 V. The membrane was blocked in BSA blocking buffer for 2 h at 25°C. We used an acetyl-lysine antibody  diluted to 1:15000. After incubation at 4°C for 10–12 h, the blot was washed with TBS and Tween 20 (TBST) three-times at ambient temperature. Chemiluminescence was detected using the ECL enhanced system and a luminescent image analyzer . To continuously monitor the acetyl-lysine sites, the acetylated Acs were isolated using the SDS/PAGE and proteins were solubilized according to previously described filter-aided sample preparation (FASP) procedures [+, 10 mM MgCl2, 3 units of malate dehydrogenase, 0.4 units of citrate synthase, and 0.3 μM SveAcs or AmiAcs. The reaction was initiated with 100 mM potassium acetate and the rate was determined continuously for 5 min by measuring NADH production at 340 nm. One unit was defined as the conversion of 1.0 μmol of NAD+ to NADH per min in the presence of saturating levels of acetate.The specific activity of Acs was determined at 30°C in a transparent 384-well microplate reader  at 340 nm . The sta2HPO4, and 6 mM KH2PO4, pH 8.0). Samples were diluted to 0.25 μg/μl and incubated with and without 2 mM Asn or 2 mM Cys. The CD spectra were collected between 190 and 260 nm, and three repeat scans were averaged. Parameters of the secondary structure were analyzed using CDNN 2.1 .CD spectra were prepared using a CD spectrophotometer  and 1 mm cuvette equilibrated at 25°C. All samples were desalted and dialyzed against buffer P  according to previously described procedure ,17. The AmiPatA was built by homology modeling from Swiss Model server (https://swissmodel.expasy.org/) using four ACT domains with the greatest similarity to that of AmiPatA  according to the previously described procedure [AmiPatA. LibDock was used to perform molecular docking. The conserved binding site was selected as docking center, and a sphere with a radius of 10 Å were considered as binding region for docking studies.The structure of the ACT domain of rocedure . DiscoveMultiple alignments of the ACT and GNAT domains, as well as a phylogenetic analysis of full-length sequences of all ACT-GNAT proteins, were performed using ClustalW 2.1 and MEGA 5.10 software.SveAcs, and AmiAcs. GraphPad Prism 5.0 was used for statistical analyses, preparation of graphs, and data analysis. Data are presented as means ± S.E.M. for three independent experiments (n=3); statistical significance was accepted at P≤0.05.Microsoft Excel was used to quantitate protein concentrations and the activities of PatA, MaKat  containing an ACT-GNAT domain organization is an amino acid-regulated Pat that can acetylate Ac–CoA synthetase [Streptomyces. Some AAPatAs were found in Amycolatopsis (10), Actinoplanes (5), and Micromonospora (5). We performed a phylogenetic tree analysis using the protein sequences of all AAPatAs , Micromonosporales , Pseudonocardiales , and Streptosporangiales . Exceptions included A4FK80 from Saccharopolyspora erythraea DSM 40517 (Pseudonocardiaceae), which was between Streptomycetales and Micromonosporales, and A0A0N0T6Y4 from Nocardia sp. NRRL S-836 , which was dispersed amongst the Pseudonocardiales. The results revealed the sequence evolution of AAPatA acetyltransferases in actinobacteria, which can be used as a molecular marker for speciation of actinobacteria.Previous work has shown that nthetase . One hun AAPatAs . Figure MaKat from M. aurantiaca and other Pat , the most highly conserved motif QXXGX (G/A) for Ac–CoA recognition and binding, and a glutamic acid residue provides as an active site to deprotonate the lysine residue of the acetyl acceptor, were observed in GNAT domains of all 24 AAPatAs . The experimental data showed that all AAPatAs can directly acetylate the putative AMP-forming Ac–CoA synthetase (AmiAcs from Actinosynnema mirum strain DSM 43827) in vitro . These results demonstrate that AAPatA is a novel type of Pat.We overexpressed and purified 24 AAPatA acetyltransferases (indicated by the red dot in L-amino acids. Acetylation was measured continuously using a coupled enzymatic assay to monitor increase in absorbance at 340 nm (ΔA340). These increases resulted from the formation of NADH by pyruvate dehydrogenase because of AAPatA-dependent CoA production [340, while no change in the values of ΔA340 were observed after the addition of other amino acids. These increases in the values of ΔA340 suggest that the acetylation activity of AAPatAs is enhanced by the conformational changes resulting from Cys or Asn binding the ACT domain. The amino acid ligands of AAPatAs are listed in Streptomyces; the latter are distributed in other actinobacteria. Furthermore, we performed a phylogenetic analysis of the sequences that make up the GNAT and ACT domains in the AAPatA proteins. As shown in The ACT domain binds to more than one amino acid, by inducing conformational changes, it can generate variation in the activity of the catalytic domain. To identify the amino acid ligands of ACT domains in AAPatA acetyltransferases, we investigated the activities of all AAPatAs acetylating putative Ac–CoA synthetases with and without the addition of the 20 oduction –14. The GXL, at the interface of β1 and α1, may play an important role in the binding of specific amino acids. Bioinformatics analysis and coupled enzymatic assay showed that AAPatAs, the protein lysine acetyltransferases (KATs) that acetylate the Ac–CoA synthetase, are divided into two groups: Cys_AAPatA and Asn_PatA. Next, we characterized AmiPatA (Cys_AAPatA) from A. mirum DSM 43827 and SvePatA (Asn_PatA) from S. venezuelae DSM 40230.Multiple alignments of ACT domains in the 24 PatA proteins revealed that all ACT domains contain the conserved βαββαβ-fold and the highly conserved glycine residue between the first β1 strand and the first α1 helix; Asn-binding ACT domains have higher sequence conservation than the Cys-binding ACT domains . The β1 A. mirum-derived AmiPatA, we used a spectrophotometric assay to assess the acetylation of AmiAcs by AmiPatA in the presence and absence of Cys. As shown in AmiAcs. The initial rate of acetylation of AmiAcs by AmiPatA (indicated by the formation of NADH) was estimated to be 3.8 μM/min, which increased to 6.8 μM/min (an increase by 179%) in the presence of Cys. The acetylation level of AmiAcs was assessed by Western blotting using an anti-acetyl-lysine antibody (Anti-AcK) in the presence or absence of Cys. Cys increased the acetylation level of AmiAcs  IMR) was identified in the conserved PXXXXGK motif of the AMP-forming acyl–CoA synthetases; this motif is recognized by the previously reported bacterial acetyltransferases [AmiPatA catalyzed acetylation reaction of AmiAcs in the absence and presence of Cys using a coupled enzymatic assay previously described [Km and kcat values of AmiPatA for AmiAcs are 45.5 μM and 0.03 s−1, respectively. Cys was able to decrease the Km value of AmiPatA for AmiAcs by three-fold, and increase kcat value by 1.5-fold.To examine whether Cys regulates the enzymatic activity of f AmiAcs B; furthen of Cys C. When tli cells , indicatf AmiAcs D. The acescribed . The datE. coli was the first enzyme found to contain the ACT domain [AmiPatA, the structure of the ACT domain in AmiPatA was modeled using four ACT domains with the greatest similarity  to that of AmiPatA . The docking result showed that the residues involved in hydrogen bonding with Cys were Asp16, Ser17, Gly19, Ala20, and Leu21; in particular, Asp16 and Ser17 were located in the loop between the first strand (β1) and the first helix (α1) of the AmiPatA ACT domain  in T domain  and foldT domain . To inveT domain A. The chn of Cys B. CD speffectors ,23.16 and the hydrophilic amino acid Ser17 to a neutral and hydrophobic amino acid alanine. The CD spectra of D16A and S17A mutant proteins showed that Cys does not induce changes in the secondary structures of AmiPatA mutants , and the rate of acetylation was increased by 176% in the presence of Asn (3.92 nmol of NADH formed per min/ml). Western blotting was conducted to detect the level of SveAcs acetylation with an anti-Ack antibody in the presence and absence of Asn. Acetylation of SveAcs was clearly observed after incubation with SvePatA and Ac–CoA; acetylation was significantly increased in the presence of Asn  used to modulate enzyme activity in prokaryotes. Accumulating evidence suggests that metabolic networks are co-ordinated via reversible acetylation of enzymes that regulate cellular metabolism to maintain homeostasis in the rapidly changing microenvironment. The present study examined the archetypical acetyltransferases AAPatAs possessing GNAT and ACT domains, and shows a novel signaling pathway for regulating the acetylation of cellular proteins. These findings indicate that in actinobacteria, acetyltransferase activity and protein acetylation may be tightly correlated with intracellular amino acid metabolism.Nε-acetylation of proteins is enzyme-catalyzed acetylation and deacetylation, which relies on KATs and deacetylases (HDACs). Reversible enzyme-catalyzed acetylation is regulated at different levels, such as transcriptionally and allosterically, to adjust the acetylation of specific proteins responding to intracellular nutritional signals. In B. subtilis, the acuA gene encoding an acetyltransferase is controlled by the carbon catabolite control protein, CcpA [E. coli, the cAMP receptor protein CRP induces the expression of yfiQ, which encodes the acetyltransferase YfiQ, thus increasing the acetylation level of proteins in response to the intracellular cAMP signal [S. enterica, lolR, the repressor of myo-inositol catabolism, activates the transcription of pat and cobB [The conventional mechanism for reversible in, CcpA . In E. cP signal . In S. eand cobB .+-dependent sirtuin deacetylases, or 1 NAD+-independent protein deacetylase. The genome of S. coelicolor encodes 77 putative GNAT acetyltransferases (Pfam00583), 2 sirtuin-type deacetylases , and an AcuC-like NAD+-independent deacetylase (SCO3330). The genome of S. avermitilis encodes 43 putative GNAT acetyltransferases, 1 sirtuin-type deacetylase (SAV_537), and 1 AcuC-like deacetylase (SAV_4729). The genome of S. lividans encodes 72 putative GNAT acetyltransferases, 2 sirtuin-type protein deacetylases , and 1 NAD+-independent deacetylase (EFD68590). The genome of Saccharopolyspora erythraea encodes 42 putative GNAT acetyltransferases, 1 NAD+-dependent deacetylase (SACE_3798), and 1 NAD+-independent deacetylase (SACE_1779). Thus, the number of GNAT acetyltransferases in a bacterial species may reflect the metabolic complexity of the species. Thus far, all the known bacterial Pat identified contain the GNAT domain having low sequence homology, and sharing a conserved motif A, sequence (R/Q)XXGX(G/A) for Ac–CoA recognition and binding, and a glutamate residue providing as an active site. It is worth noting that there has been considerable debate about the function of the conserved glutamate as a catalytic base [BsAcuA from B. subtilis [RpKatA from Rhodopseudomonas palustris [SacAcuA from Saccharopolyspora erythraea [There are three classes of KATs that catalyze the donation of an acetyl group from acetyl-coenzyme A (Ac-CoA) to an ε-amino group of a deprotonated lysine. These KATs include the GNAT family, the MYST family, and the p300/CBP family . The gentic base ,27. In psubtilis , RpKatA se SAV_47. The genase SACE_79. Thus,SePat in S. enterica [EcPatZ from E. coli and RpPat from R. palustris) and variants (SlPatA from Streptomyces lividans) of SePat [+-forming CoA ligase domains; the function of the latter is undetermined, but it has homology with ADP-forming acyl–CoA synthetases. The catalytic mechanism and domains function of SePat and EcPatZ have been investigated [SePat revealed that SePat has two distinct sites for Ac–CoA binding, and shows the positive co-operativity (with a Hill coefficient of 2.2 ± 0.2) where binding of Ac–CoA to one site increases the affinity of a second binding site [EcPatZ for Ac–CoA display atypical sigmoidal activity, suggesting that EcPatZ also reveals the positive co-operativity with a Hill coefficient of 7.91 ± 0.22 [SeAcs, and inhibits its activity through competitive binding to the ATP/AMP pocket, whose binding with cAMP promotes SeAcs acetylation, and further resulting in inhibition of SeAcs activity [Class Iα Pat, such as the first discovered enterica , the homof SePat ,29,30, astigated ,32. Biocing site . Recent activity .MtPatA in Mycobacterium tuberculosis and MsPatA in M. smegmatis) [MxKat in Myxococcus xanthus) [MaKat in Micromonospora aurantiaca) [M. xanthus. In the present study, we found that the amino acid-sensing AAPatA acetyltransferases are divided into two groups based on allosteric effectors: the Asn-regulated PatA and Cys-regulated PatA. The former are found only in Streptomycetaceae; the latter are distributed in other actinobacteria . Class Iβ Pat with different regulatory domains use diverse metabolic signals to regulate lysine acetylation in response to varying physiological conditions and environmental changes. To investigate the function of Class Iβ enzymes, it is necessary to identify their physiological substrates. A universal stress protein USP (MSMEG_4207), Acs, and eight additional acyl-CoA synthetases may be the possible acetylated protein targets of MsPatA, and their acetylation may be dependent upon the levels of intracellular cAMP [Class Iβ Pat are allosteric enzymes with two domains in which an effector-binding regulatory domain is fused to the GNAT catalytic domain. The cAMP-sensing acetyltransferases (egmatis) ,35, NADPxanthus) , and amiantiaca)  are examlar cAMP ,12,34. NClick here for additional data file.


Flour dust in the respiratory tract affects lung function. Flour dust is a heterogeneous organic substance which can have a tendency to cause respiratory ailments. There is growing consensus on the deleterious effects of flour dust on respiratory symptoms and lung performance of flour mill workers.1, FEV1/FVC, PEFR, and FEF25%  −75% were measured. Productive cough, dry cough, wheeze, and breathlessness were evaluated using BMRC questionnaire guidelines, administered through face-to-face interview. The study design was comparative cross-sectional. A total of 54 flour mill workers who work for more than eight-hour shift per day and 54 control subjects matched for sex, age, weight, height, and area of residence were enrolled. Anthropometric measurement was done. Lung function was measured by using a digital portable spirometer (Spiro Pro) based on the ATS guidelines. FVC, FEV1 , PEFR , and FEF25%  −75%, , but not significant for FEV1/FVC . Flour mill workers developed 27.7% of restrictive type and 11.1% of obstructive type of lung disorders. Percentage prevalence of respiratory symptoms was evaluated as dry cough (27.7% vs. 9.3%), productive cough (11.1% vs. 5.6%), wheeze (14.8% vs. 3.8%), and breathlessness (16.6% vs.7.4%) in flour mill workers and controls, respectively. This study showed statistically significant reduction in the mean values of pulmonary function tests in flour mill workers as compared to their matched controls. Reduction of pulmonary function indices in study subjects was significant for FVC , FEV Based on the results of the present study, occupational exposure to flour dust could cause respiratory dysfunction, thereby reducing lung efficiency. Industrialization has been established to optimally fulfill various human needs. Over the course of time, some industries exert hazardous effect on the health of human beings . Those hOccupational lung diseases are a broad group of diagnosis caused by the inhalation of dusts and chemicals. As a result, improper control of these substances can result in a severe threat to site workers because most of the workers are unaware of this hazardous effect on health . AccordiFlour dust is a heterogeneous substance with respiratory sensitizing properties and long-term exposure to it may cause acute or chronic respiratory disease . SeveralThe study aims to investigate the effect of flour dust on the pulmonary function by analyzing the pulmonary function tests that will estimate the effectiveness of the lung. This study is therefore undertaken to assess the respiratory symptoms and pulmonary function problems related to flour mill workers. It will also encourage communication between various concerned groups and organizations and foster an improved understanding of the potential problems of flour dust in the country.Comparative cross-sectional study design was employed to assess respiratory symptoms and pulmonary function parameters in workers of flour mills. The study was conducted in Lideta subcity, Addis Ababa, Ethiopia, from August to September 2016. Addis Ababa encompasses 10 subcities and 116 woredas. It is located at altitude of 2,400 meters above sea level. Based on the data from Lideta subcity trade and development bureau and urban agriculture corporation, ten of 116 woredas are located in Lideta subcity containing a total of 76 flour mills during a time of data collection.The source populations were all flour mill workers in Addis Ababa city, Ethiopia. The study population were all flour mill workers and controls in Lideta subcity in the age range between 18 and 43 years. Study group: it consisted of flour mill workers in Lideta subcity in the age range between 18 and 43 years. All subjects were male as there were no female workers in the flour mill industry in the selected sites during the study period.Control group: non-flour mill workers in Lideta subcity anthropometrically matched to study group were selected as control group.Double proportion formula was used for the determination of sample size. The proportions of the sample size were calculated from previous study on association of flour dust on PFTs and prevalence of respiratory symptoms of chronic cough for flour mill workers and control groups  . Then, tMillers who were available in the workplace between the age range of 18 and 64 years and working in the flour mill for more than one year were included.Subjects with gross clinical abnormalities of the vertebral column, thoracic cage, neuromuscular diseases, known cases of gross anemia, diabetes mellitus, pulmonary tuberculosis, hypertension, drug addicts, known cardio pulmonary disease, cigarette smokers, tobacco and khat chewers, bakery workers, and those who underwent vigorous exercise or abdominal or chest surgery were excluded from the study.The sample consisted of all flour mill workers in the mill and matched non-flour mill workers in Addis Ababa, Lideta subcity. Lideta subcity was selected randomly by lottery method of the ten subcities. Lideta subcity contains 10 woredas and 76 flour mills. Among 10 woredas 4 woredas were selected by random sampling. There are 30 flour mills in the 4 woredas and 24 flour mills in the 4 woredas were selected for this study until sample size was saturated. Non-flour mill workers in Lideta subcity matched for age, sex, height, and weight to flour mill workers were selected as controls. Flour mill workers and controls age was 18-64 years selected by convenient sampling technique. Selected flour mill workers and controls fulfilling eligibility criteria were interviewed and anthropometry was measured and finally spirometry was done.Each participant was informed about the objective of the study and the benefit associated with the study immediately before sample collection. Millers who volunteered to participate in the study have answered questions in the questionnaire, which were relevant to their sociodemographic information. A structured questionnaire was prepared in English and translated into Amharic language and was retranslated back to English by a linguist to ensure consistency. Training was given to the data collectors regarding the objectives of the study and ways of administering the questionnaire by the principal investigator. The prepared questionnaire was pretested prior to the actual data collection on seven (7) informants that were not included in the main survey.2)Height and weight of the study participants were measured. The height was measured without shoes by the use of a meter rule and approximated to the nearest one cm. Weight was also measured using weighing scale (nearest to one kg), with light clothing and without phones or any encumbrance that could alter their appropriate weight. Body mass index (BMI) was calculated through this formula: weight/squared height  was used to measure pulmonary function indices. Spirometric tests in both exposed and control subjects were done based on ATS guideline. Before every test, the mouthpiece was disinfected in standard solution and then attached to the spirometer. Subjects were told to put the nosepiece on the nose to prevent air blowout. Then, they were also instructed to breathe in fully until their lung filled maximally. Precaution was taken to avoid leakage from mouth pieces. The subjects expire forcefully and as fast and complete as possible until there is no more air left to expel. Spirometric measurements were performed in sitting position and at a fixed time of the day at room temperature (20-25°C), to minimize diurnal variations. The measurements were done in three trials, and one of the three trials, which was the best, was taken and interpreted. Prior to the actual tests, calibration with one-liter syringe and familiarization steps were done.1, FEV1/FVC, PEFR, and FEF25% −75% and duration of exposure. Binary logistic regression and odds ratio were used to analyze and estimate the prevalence of respiratory symptoms. Variables having P value <0.05 are considered as statistically significant.Data was edited, coded, entered, and analyzed by SPSS version 20. Independent sample t-test was used for the comparison of the actual value mean respiratory score of flour mill workers and controls. Bivariate analyses were computed to test whether there was association between FVC, FEVThe study was carried out after ethical clearance and approval was obtained from research committee of the Department of Physiology, Addis Ababa University. The study participants were briefed about the objective and procedures of the study. Thereafter, both verbal and written informed consents were obtained from those volunteers and were selected for the study. Confidentiality of information was maintained by excluding personal identifiers.All workers included in the study were males. The age of the flour mill workers ranged between 18 and 43 years with a mean of 27.37±6.71 years while the age of the comparison group ranged between 18 and 43 years with a mean of 28.00±5.33 years .Personal protective devices used by the workers were investigated in the study area. Among workers, 57.4% (n= 31) do not wear any equipment, 9.3% (n=5) wear safety shoes, masks, and gloves, and 33.3% (n=18) wear only masks . We obseAnthropometric parameters of the study subjects in terms of demographic variables demonstrate the comparison between the flour mill workers and their matched control subjects. There were no significant differences between the means of anthropometric parameters: in terms of age, weight, height, and BMI between the groups. The statistical comparison of the matching variables  shows no difference between the two groups .The percentage prevalence of dry cough, productive cough, wheeze, and breathlessness was 27.7%, 11.1%, 14.8%, and 16.6% for exposed informants, respectively, and 9.3%, 5.6%, 3.7%, and 7.4% for control subjects, respectively . These vBinary logistic regression was used to compare the respiratory symptoms and measure the strength of the association. The odds of dry cough in flour mill workers exposed to flour dust association were found to be statistically significant (p<0.05).1/FVC difference is not significant (p>0.05).Percentage predicted values of exposed and control groups were indicated . There w1, and PEFR are highly significant (P<0.001) and FEF25% −75% are significant (P<0.01). The forced expiratory flow rate at the middle fifty percent (FEF25%  −75%) of the exposed group was lower (3.87L/s) than that of the controls (4.60 L/s). This reduction in flow rate was statistically significant (P <0.01). The PEFR in flour mill workers of the exposed group was lower (5.43 L/s) than that of the controls (7.87 L/s); this reduction was statistically significant (P<0.001).The independent sample t-test  for equa1, and FEV1/FVC, PEFR and FEF25%  −75%) as the duration of exposure increases. Both pulmonary function indices and duration of exposure move in opposite direction. Based on their service years (1-12 years) duration of exposure and pulmonary function parameters relationship were determined. Bivariate correlation result showed negative correlation between FVC and duration of exposure which was found to be statistically significant . Pearson‘s correlation coefficient was used to quantify the degree of linear relationship between pulmonary function indices  and duration of exposure , 27.7% developed restrictive and (n=6) 11.1% developed obstructive type of lung disorder. The rest of the study subjects 61.1% (n=33) had normal pulmonary function. Two subjects (3.7%) in the control group developed obstructive type of lung disorder and the rest of the subjects had normal pulmonary function status .The present study revealed the prevalence of respiratory symptoms of dry cough (27.7% vs. 9.3%), productive cough (11.1% vs. 5.6%), wheeze (14.8% vs. 3.7%), and breathlessness (16.6% vs. 7.4%) in flour mill workers and controls, respectively. In this study, the higher prevalence of respiratory symptoms in flour mill workers may be due to prolonged exposure of flour dust, unhygienic conditions, and poorly ventilated workplaces of the study areas as compared to the controls. A study by Wagh and his colleagues on the influence of flour dust on flour mill workers compared to the nonexposed controls found that flour mill workers had three times shortness of breath and developed four times frequent coughing compared to controls [Similar findings were reported in Egypt  on the eThe present study investigated the prevalence and the type of pulmonary impairment observed among the flour mill workers. We found 27.7% (n=15) of the workers develop restrictive type of lung disorder while 11.1% (n=6) develop obstructive type of lung disease. On the other hand, there were only two control subjects (3.7%) that showed an obstructive type of lung disorder. Hence, the prevalence of obstructive and restrictive lung diseases was higher in exposed group than in control group. The plausible explanation for the increased prevalence of restrictive lung impairment in exposed group is mainly due to flour dust that reacts with lymphoid and connective tissue in the terminal and respiratory bronchioles and interstitial inflammatory cells.1, FEV1 /FVC, FEF25% −75%, and PEFR, showed statistically significant reductions in the mean values in the flour mill workers as compared to their matched controls.The results of spirometry done by  showed tThe findings of the present study were in line with others  who repo1, in workers exposed to flour dust compared to the control group, suggesting that flour dust persistently affects the pulmonary function parameters. The present study is also consistent with another investigation that showed flour dust can adversely affect FVC, FEV1, and FEV1/FVC [1, PEFR, and FEF25% −75% and duration of flour dust exposure. This is in line with other findings that long-term exposure to flour dust decreased FVC, FEV1, PEFR, and FEF25%  −75% values [25% −75% value in exposed workers may be due to the obstructive pattern of the medium and smaller airways in the flour mill workers.A study by  also obsFEV1/FVC . We foun% values , 17, 21.Similar studies conducted in Nigeria  showed tWe found that majority of the workers do not wear any equipment, some workers wear safety shoe, masks, and gloves, and 33.3% wear only masks. Another similar study found that some workers used a mask .This study failed to estimate the concentration of organic flour dust in the working area and was unable to determine subclinical cardiopulmonary and other systemic diseases that may affect the result of the study in both cases and controls.1, FEV1 /FVC, PEFR, and FEF25% −75%) and lung efficiency due to excessive exposure to fine organic flour dust as compared to the controls. There is an inverse relationship between duration of exposure and respiratory function which means that when the duration of exposure increases, there is a deterioration of pulmonary function indices. Flour mill workers develop both restrictive and obstructive types of lung disorder. Personal protective devices were not available at workplaces and the workers usually do not wear the materials.Percentage prevalence of respiratory symptoms was higher among exposed workers as compared to control groups and the difference was more vivid for dry cough. Flour mill workers showed significantly reduced values in the pulmonary function tests (FVC, FEV