Aim. To assess memory improvement and neuroprotective and antioxidant effects of Mitragyna inermis (M. inermis) leaf decoction on the central nervous system. Methodology. Leaf decoction of M. inermis was tested on learning and memory in normal and scopolamine-induced cognitive impairment in mice using memory behavioral tests such as the Morris water maze, object recognition task, and elevated plus maze. Oxidative stress enzymes—catalase, superoxide dismutase, and the thiobarbituric acid reactive substance, a product of lipid peroxidation—were quantified. In each test, mice 18 to 25 g were divided into groups of 5. Results. The extract reversed the effects of scopolamine in mice. The extract significantly increased discrimination index in the object recognition task test and inflexion ratio in the elevated plus maze test. The times spent in target quadrant in MWM increased while the transfer latency decreased in mice treated by M. inermis at the dose of 196.5 mg/kg. The activity levels of superoxide dismutase and catalase were significantly increased, whereas the thiobarbituric acid reactive substance was significantly decreased after 8 consecutive days of treatment with M. inermis at the dose of 393 mg/kg. Conclusion. These results suggest that M. inermis leaf extract possess potential antiamnesic effects. M. inermis are used in Cameroonian traditional medicine to treat mental diseases. M. inermis is a shrub which grows on low alluvial plains and swampy savannah of many countries in West Africa [M. inermis is also found in the sub-Saharan Africa [M. inermis showed considerable antiplasmodial activity [M. inermis leaves phytochemical analysis reveals the presence of alkaloids, catechic tannins, flavonoids, polyphenols, saponosides, sterols, and triterpenes [M. inermis in normal as well as in scopolamine-induced cognitive deficit in mice. We used the elevated plus-maze test, the object recognition test, and the Morris water maze test to investigate the integrity of the memory, as well as the quantification of oxidative stress enzymes  and the thiobarbituric acid reactive substance activity (TBARS), a lipid peroxidation product in mice brain.Dementia is a syndrome of gradual onset and continuing decline of higher cognitive functioning. It is a common disorder in older persons and becomes more prevalent in each decade of life . The most Africa . M. inern Africa , 17. Thin Africa , 19. M. activity , 21. M. terpenes . The aimAdult Swiss albino mice of both sexes weighing 18–25 g obtained from the National Veterinary Laboratory of Cameroon  were used. The animals were housed in standard polypropylene cages, at 25°C, on a 12/12 h light-dark cycle. Animals received food and water ad libitum. Mice were divided in 7 groups of 5 mice for each test with amnesic induction and 6 groups of 5 mice in each test without amnesic induction. All animal experiments were carried out in accordance with the national principles of laboratory animal care (number FWA-IRB00001954).M. inermis were collected in the morning during dry season, at Yagoua in the Far North Region of Cameroon. The botanical identification was performed at the National Herbarium of Yaoundé. A voucher specimen was deposited at the Yaoundé Herbarium on the number 8886/SRFcam. The leaves were dried in the shade and were powdered. 10 g of the powder was boiled in 50 mL of distilled water for 20 min. The supernatant was collected, cooled, and filtered (15 mL). After evaporation, 0.59 g of dry extract was obtained (yield: 5.9%).Fresh leaves of The doses are obtained from the dose used by the traditional practitioner and the number is obtained from calculation, knowing that the dose of administration is 10 mL/kg.V = 15 mL; mass after evaporation: m = 0.59.Volume after filtration: Initial concentration: Ci = m/V.Ci = 0.0393 g/mL = 39.3 mg/mL.Ci = dose/dose of administration.Volume of administration = 10 mL/kg.Dose = Ci × dose of administration.Dose = 393 mg/kg = D1; D1 is the dose used by the traditional practitioner.D2 = D1/2 = 196.5 mg/kg, D2 is obtained after dilution at 1/2.D3 = D1/4 = 98.25 mg/kg, D3 is obtained after dilution at 1/4.D4 = D1/10 = 39.3 mg/kg, D4 is obtained after dilution at 1/10.Piracetam (Nootropil (R), UCB S.A., Bruxelles, Belgique) and scopolamine (Pemason Hyoscine(R), Jiangsu Huayang Pharmaceutical Co., China) were procured from the local pharmacies of Ngaoundere, Cameroon.n = 5) and treated each day for 8 days as follows: group I (scopolamine-treated group) received distilled water  + scopolamine ; group II received M. inermis  + scopolamine ; group III received M. inermis  + scopolamine ; group IV received M. inermis  + scopolamine ; and group V received M. inermis  + scopolamine . Group VI was treated with piracetam  + scopolamine  and group VII  received distilled water  + distilled water .Swiss albino mice (18–25 g) of both sexes were used. In each scopolamine-induced amnesia test, mice were divided into 7 groups  was given to the different groups only on the 8th day, 30 minutes after the respective treatments, to induce cognitive deficit in mice.M. inermis ; group III received M. inermis ; group IV received M. inermis ; group V received M. inermis ; group VI was treated with piracetam ; and group VII  received distilled water . 30 minutes after the different administration, scopolamine  was given to each group. Only group VII  did not receive scopolamine.In the Morris water maze test, all the administrations were done on the 4th day of the test as follows: group I (scopolamine-treated group) received distilled water ; group II received n = 5) and treated each day for 8 days as follows: group 1  received distilled water ; groups 2, 3, 4, and 5 received M. inermis at the doses of 39.3, 98.25, 196.5, and 393 mg/kg, p.o., respectively; and group 6 received piracetam .In each test without scopolamine, mice were divided into 6 groups  to evaluate learning and memory in mice .IR = (L0 − L1)/L1 [The apparatus consisted of two open arms (25 cm × 5 cm) and two enclosed arms (25 cm × 5 cm × 12 cm). The arms extended from a central platform (5 cm × 5 cm) and the maze was elevated to a height of 40 cm from the floor. On the 8th day, after the last treatment, each mouse was placed at the end of an open arm, facing away from the central platform. The transfer latency (TL), the time taken by mouse with all its four legs to move into one of the enclosed arms, was recorded as L0. If the animal did not enter into one of the enclosed arms within 90 s, it was gently pushed into one of the two enclosed arms and the TL was assigned as 90 s. The mouse was allowed to explore the maze for another 2 minutes and then returned to its home cage. The retention of this learned task was examined 24 h after the 8th day trial (on 9th day) and the TL was recorded as L1. The effect on TL was expressed by inflexion ratio (IR). IR was calculated using the formula: − L1)/L1 . TL on tExploration of novelty in an open-field has been extensively exploited in neuroscience studies of behavior and brain functions in rats and mice, and it is used in the study of memory , 28.The apparatus consisted of a plywood box as used by De Lima et al. with modification (40 × 40 × 30 cm) . The objThe MWM is a white circular pool (100 cm in diameter and 45 cm in height) with a featureless inner surface. The circular pool was filled with water in which 500 mL of milk had been mixed to a height of 30 cm (20 ± 1°C). The pool was divided into four quadrants of equal area. A white platform (6 cm in diameter and 29 cm in height) was centered in one of the four quadrants of the pool and submerged 1 cm below the water surface so that it was invisible at water level. In the water maze experiments, the day prior to the experiment was dedicated to swim training for 60 s in the absence of the platform. In the following days, the mice were given two trial sessions each day for 4 consecutive days. During each trial, the time taken to swim to the platform (escape latency) was recorded. This parameter was averaged for each session of trials and for each mouse. Once the mouse located the platform, it was permitted to remain on it for 10 s. If the mouse did not locate the platform within 120 s, it was placed on the platform for 10 s and then removed from the pool by the experimenter . The mouse was given second trial  with an intertrial interval of 20 min for 4 consecutive days . In the Twenty-four hours after the last training session, a probe trial was performed, after removing the platform, to evaluate memory .Immediately after the test (8th day), the animals were sacrificed and the brain of each animal was carefully isolated. Whole brain samples were rinsed with ice cold normal saline (0.9% NaCl). The tissues were weighed and 20 mg tissue/mL homogenate of brain samples was prepared by homogenizing in phosphate buffer (pH 7.4). The homogenates were centrifuged at 10,000 rev for 10 min at 4°C, and the resulting supernatant was used for the estimation of nonenzymatic and enzymatic antioxidants.2−) was generated from the photoreduction of riboflavin and was deducted by nitro blue tetrazolium dye (NBT) reduction method. Measurement of superoxide anion scavenging activity was performed based on the method described by Winterbourne et al., [μL of supernatant, 700 μL of reactive was introduced. The reactive was composed of 1.5 mL of solution of 100 mM Tris/HCl (pH 7.8), 75 mM NBT, 2 μM riboflavin, and 6 mM EDTA. Absorbance of mixture was measured at 560 nm by a spectrometer. All the tests were performed in triplicate and the results averaged. The results were expressed as units/mg protein.Superoxide radical  and 200 μL of H2O2. The reaction was terminated by the addition of 200 μL of acid reagent (dichromate/acetic acid mixture). All the tubes were heated for 10 minutes and the absorbance was read spectrometrically at 620 nm. CAT activity was expressed in terms of mmol H2O2/mg protein.CAT activity was determined by the method of Sinha, . BrieflyμL of supernatant, 700 μL of 9% phosphoric acid and 250 μL of thiobarbituric acid were introduced. After agitation, the mixture was boiled in bain-marie for 60 min and cooled. Then, 1250 μL of butanol was added and the mixture was agitated by vortex for 20 s and centrifuged at 25°C for 20 min. The absorbance was spectrometrically read at 534 nm. The results were expressed as mmol/mg protein.The degree of lipid peroxidation was established by the estimation of the concentrations of TBARS in brain. The TBARS content was determined as described by Yagi, . In 250 M. inermis decoction were realized using qualitative and colorimetric methods , for thIn 1 mL of the plant extract, some drops of sulfuric acid (2%) were added. Then, some drops of Meyer's reagent were added to mixture. And white precipitate indicated the presence of alkaloids.M. inermis decoction was mixed to 5 mL of solution of DMSO 1,5%. The mixture was boiled in bain-marie at 70°C in 3 min and filtered. 2 mL of ferric chloride 3% was added to 3 mL of filtrate. The green dark coloration indicated the presence of catechotannins.3 mL of In 1 mL of the decoction, 2 mL of sodium hydroxide 1N was added. An intense yellow color was produced in the plant extract, which became colorless on addition of a few drops of dilute chlorhydric acid which indicates the presence of flavonoids.M. inermis decoction was mixed to 5 mL of diethyl ether. The mixture was agitated for homogenization and then let to rest. After addition of ammonia 10%, red or violet coloration indicated the effective presence of anthraquinones in the extract.0,5 mL of P < 0.05.The data were expressed as mean ± SD. Statistical significances of differences among treatments were determined by use of one-way analysis of variance (ANOVA), followed by Dunnett's bilateral comparisons or by Duncan's test. Differences were considered significant when M. inermis.Results of phytochemical characterization tests show the presence of flavonoids, alkaloids, tannins, and anthraquinones in the leaf decoction of M. inermis for 8 days significantly decreased the TL (P < 0.05) and significantly increased the IR compared to normal control group (P < 0.001)  .P < 0.001). These mice showed also a significant decrease of the IR compared to normal control group (P < 0.001) (P < 0.001). At the dose 196.5 mg/kg, the TL was 36.2 ± 2.16 and the IR 0.9. Mice treated with piracetam showed also a significant decrease of TL (P < 0.001) and a significant increase of IR (P < 0.001) compared to the scopolamine-treated group. The effects of the plant extract at the dose 196.5 mg/kg were greater than the effect of piracetam (Mice that received distilled water and scopolamine (57.2 s) showed a significant increase of TL compared to normal control group (40.2 s) (< 0.001) . On the iracetam .M. inermis, like piracetam, significantly increased the discrimination index and the exploration time of the novel object and decreased the exploration time of the familiar object (P < 0.01)  .M. inermis significantly increased the discrimination index from 0.08 in the scopolamine-treated group (group I) to 0.54 and 0.61 (P < 0.001) in mice treated with M. inermis at the doses 98.25 and 196.5 mg/kg, p.o., respectively. The exploration time of the novel object was also significantly increased by M. inermis from 21.2 ± 0.6 in the scopolamine-treated group to 38.0 ± 1.6 and 40.2 ± 3.0 in mice treated with M. inermis at the doses 98.25 and 196.5 mg/kg, p.o., respectively (P < 0.001). The two same doses of M. inermis significantly decreased the exploration time of the familiar object (P < 0.001). Piracetam significantly increased the discrimination index and the exploration time of the novel object and decreased the exploration time of the familiar object (P < 0.001). Here also, the effect of M. inermis at the dose 196.5 mg/kg is greater than the effect of piracetam (The extract of iracetam .P < 0.01). The different doses of M. inermis significantly decreased the escape latency induced by scopolamine from 48.2 ± 3.7 s in the scopolamine-treated group to 41.0 ± 1.6 and 32.2 ± 1.9 s when mice were treated with M. inermis at the doses 98.25 and 196.5 mg/kg, respectively (P < 0.01 and P < 0.001). Piracetam (200 mg/kg) also significantly decreased the escape latency to 41.6 ± 2.7 (P < 0.01)  to 48.2 ± 3.7 s in the scopolamine-treated group of mice (I) ( < 0.01) .P < 0.01). M. inermis significantly increased the time spent in target quadrant. This time increased from 50.6 ± 3.0 s in the mice scopolamine-treated group to 68.4 ± 2.7 in mice treated with M. inermis extract at the dose 196.5 mg/kg (P < 0.001). Piracetam (200 mg/kg) also significantly increased the time spent in target quadrant (P < 0.001)  significantly decreased the time spent by mice scopolamine-treated group  in the target quadrant compared to normal control group  (< 0.001) .P < 0.001) CAT and SOD activities of scopolamine-treated groups (group I) compared to normal control groups (group VII). Mice treated with M. inermis showed a significant increase in CAT activity. The CAT activity was increased from 58.58 ± 4.65 mmol H2O2/mg protein in the scopolamine-treated group to 251.44 ± 32.55, 274.36 ± 8.78, and 325 ± 18.89 mmol H2O2/mg protein in groups treated with M. inermis at the doses 98.25, 196.5, and 393 mg/kg, respectively (P < 0.001). SOD activity also significantly increased from 0.76 ± 0.23 units/mg protein in scopolamine-treated group to 5.05 ± 0.48, 4.86 ± 0.19, and 6.39 ± 0.42 units/mg protein in groups treated with the same doses of M. inermis (P < 0.001). CAT and SOD activities were significantly increased by piracetam (P < 0.001)  significantly decreased (< 0.001) .P < 0.01). The doses 98.25, 196.5, and 393 mg/kg of M. inermis significantly decreased the TBARS concentration to 0.64 ± 0.10, 0.62 ± 0.06, and 0.59 ± 0.01 mmol/mg protein, respectively (P < 0.01). Piracetam also induced a significant increase in the TBARS activity (P < 0.01)  compared to the normal group  ( < 0.01) .M. inermis for 8 days increased IR and decreased TL indicated the improvement in learning and memory in normal mice (without scopolamine). M. inermis also increased IR and decreased TL scopolamine-treated mice, indicating the learning and memory processes protection [Scopolamine significantly decreased IR and significantly increased TL in mice, indicating impairment of learning and memory in the EPM. That result was obtained by other authors –39, sincotection .M. inermis suggested improvement of learning and memory [M. inermis since M. inermis significantly increased DI and novel object exploration time, indicating that M. inermis possesses memory-enhancing activity in view of its facilitatory effect on remembering the original object [M. inermis may play a nootropic role in mice brain. Nootropic agents or cognitive enhancers are reported to improve mental functions such as cognition, memory, or attention [In ORT, the increase of the DI and novel object exploration time in mice treated with d memory –46. Scopl object . These rttention .M. inermis significantly reversed this effect by decreasing the escape latency time prolonged by scopolamine, suggesting also the remembering on the platform by these mice [MWM test  showed iese mice , 50. MWMese mice .M. inermis-treated mice showed a significant longer stay in the platform quadrant. Consequently, these results suggest that M. inermis improves long-term and reference memory impairments induced by scopolamine [In the probe trial, the polamine , 53, 54.M. inermis strongly reversed the decrease of CAT and SOD induced by scopolamine in the brain and increased the level of CAT and SOD in the brain, it was suggested that M. inermis possess antioxidant activities. Coupling to that, TBARS is an end product of lipid peroxidation [SOD is an important enzyme in living cells for maintaining normal physiological conditions and coping with oxidative stress . Decreasxidation . Increasxidation . In addixidation –62. Thes2O2, which in turn generate OH, resulting in initiation and propagation of lipid peroxydation [Restraint stress causes significant reduction in CAT, SOD, and GSH levels and increased lipid peroxidation in rat brain . Malondixydation . Reversixydation . Antioxixydation . Antioxixydation .M. inermis has clearly demonstrated neuroprotective properties. M. inermis inhibited brain TBARS levels and raised the endogenous antioxidants SOD and CAT suggesting that its neuroprotective effects may be due to antioxidant action and, therefore, might play an effective free radical scavenger and antioxidant.M. inermis leaf decoction showed the presence of flavonoids, alkaloids, saponosides, polyphenols, catechic tannins, sterols, and triterpenes [Phytochemical analysis of terpenes . Flavonoterpenes . Flavonoterpenes –71.M. inermis has learning- and memory-enhancing effect in normal mice and antiamnesic activity in a scopolamine mice model. It exhibited a cognitive-enhancing effect by reversing scopolamine-induced learning and memory deficits and neurotoxicity at least in part through improvement in brain levels of SOD, CAT, and MDA. These results confirm at least in part the common use of M. inermis in traditional medicine as a neuroprotective and antiamnesic plant. However, further studies are needed to understand the mechanisms used by M. inermis to exercise its effects.In conclusion,


By combining the recently reported repelling natural dihydrostilbene scaffold with an oxime moiety found in many marine antifoulants, a library of nine antifouling hybrid compounds was developed and biologically evaluated. The prepared compounds were shown to display a low antifouling effect against marine bacteria but a high potency against the attachment and growth of microalgae down to MIC values of 0.01 μg/mL for the most potent hybrid. The mode of action can be characterized as repelling via a reversible non-toxic biostatic mechanism. Barnacle cyprid larval settlement was also inhibited at low μg/mL concentrations with low levels or no toxicity observed. Several of the prepared compounds performed better than many reported antifouling marine natural products. While several of the prepared compounds are highly active as antifoulants, no apparent synergy is observed by incorporating the oxime functionality into the dihydrostilbene scaffold. This observation is discussed in light of recently reported literature data on related marine natural antifoulants and antifouling hybrids as a potentially general strategy for generation of improved antifoulants.The online version of this article (10.1007/s10126-018-9802-z) contains supplementary material, which is available to authorized users. Marine biofouling, the rapid colonization and growth of organisms on marine surfaces, commonly occurs on ships, buoys, cooling systems, and aquaculture equipment  , where it accumulates in high amounts (up to 6% of the dry leaf weight). Upon leaching into the surrounding soil, it imparts an allelopathic effect, suppressing seedling growth and germination 1) Fig. , b. Bata1) Fig. ,Balanus amphitrite cyprid larvae at lower concentrations (IC50 = 3–1.3 μg/mL) than the individual butenolide and geraniol components , psammaplin A, and debromohemibastadin-1 (4), contain the oxime functionality . Compound 2 also displayed low toxicity against the latter two fouling species and, in particular, effectively inhibited the settlement of C. savignyi even after 5 days . The oximes were obtained as single isomers, of which the geometry was not determined. General experimental procedures and compound characterization are provided in the A library of nine dibenzylic hybrid molecules based on both the 3,4-dimethoxy substituents, found in AF compound Representative example of oxime synthesis using method A.3•OEt2 (3 mL). The reaction was heated at 90 °C for 3 h, cooled to room temperature, quenched with aqueous sodium acetate , and extracted into ethyl acetate (3 × 10 mL). The combined organic extracts were washed with brine and dried over Na2SO4. The solvent was removed under reduced pressure and the resulting residue purified by column chromatography (petroleum ether–ethyl acetate) to afford the desired deoxybenzoin  as an amorphous solid. IR (neat) νmax 3495, 3293, 1610, 1510, 1432, 1304, 1276, 1232, 1179, 1022, 959, 865, 756 cm−1; 1H NMR  δ 7.14 , 7.09 , 6.95 , 6.77 , 6.69 , 4.03 , 3.72 ; 13C NMR  δ 159.5, 158.3, 147.5, 146.1, 130.7, 130.7, 129.4, 119.8, 115.9, 114.8, 114.7, 55.6, 31.7; HRMS m/z 274.1075 .Catechol  and 4-methoxyphenyl acetic acid  were dissolved in BFRepresentative example of oxime synthesis using method B.N,3,5-trimethoxy-N-methyl benzamide . The combined organic extracts were washed with water, brine, and dried over Na2SO4. The solvent was removed in vacuo, and the resulting reside was purified by column chromatography (petroleum ether:ethyl acetate) to afford the corresponding deoxybenzoin  δ 7.26–7.24 , 7.21–7.14 , 6.78 , 6.45 , 4.17 , 3.74 ; 13C NMR  δ 160.7, 157.5, 137.5, 136.5, 128.6, 128.6, 126.4, 104.8, 101.5, 55.4, 32.3; HRMS m/z 294.1106 .A solution of B. improvisus were reared in a laboratory cultivating system at Tjärnö Marine Biological Laboratory, University of Gothenburg, Sweden, as previously described by Berntsson et al.  were aseptically added to the compound containing microplate wells (10–0.01 μg/mL), and the plates were incubated for 48 h at 26 °C. Media only was used as a blank. Bacterial growth was monitored spectroscopically at 630 nm. The minimal inhibitory concentration (MIC) for bacterial growth was defined as the lowest concentration which results in a decrease in OD. After 48-h incubation time, the bacterial adhesion assay was conducted by emptying the wells and rinsing with sterile seawater (100 μL) to remove non-attached cells, and air-drying at room temperature. The residual bacterial biofilm was stained with aqueous crystal violet  and the OD measured at 595 nm . The plates were then incubated for 5 days at 20 °C under constant light exposure (140 μmol m−2 s−1). Both microalgal adhesion and growth inhibition were measured. Growth was determined by analysis of liberated chlorophyll a after centrifugation and methanol addition. Chlorophyll a was quantified fluorometrically. MIC value for algal growth was defined as the lowest concentration yielding a decrease in chlorophyll a content. Microalgal adhesion was determined in an analogous manner where the non-attached algal cells were removed prior to methanol addition (100 μL), releasing chlorophyll a from the remaining algal biofilms. The MIC for adhesion was defined as the lowest compound concentration causing a reduction in optical density. Toxicity experiments were performed in an analogous manner to those described for the bacterial assays.Microplates containing the compounds in ranging concentrations (10–0.01 μg/mL) were prepared from MeOH stock solutions as previously described , and dishes containing 0.1% of DMSO were used as negative controls. The commercial AF agent Sea-nine™ was employed as a positive control.Stock solutions of compounds in DMSO were serially diluted in untreated polystyrene Petri dishes containing 10 mL of filtered (0.2 μm) seawater, affording final concentrations ranging from 0.1 to 10 μg/mL. Freshly molted balanide cyprids (18–22) were added to each Petri dish and incubated at ambient temperature (20–25 °C) for 5 days. Cyprid metamorphosis was assessed using a dissecting microscope where the numbers of juvenile settled, free-swimming, and dead cyprids were noted. Initial compound screening was conducted at 5 μg/mL, and full IC7–15, bioassays were conducted that cover a spectrum of marine fouling organisms, including bacteria and microalgae  and the macrofouling barnacle, Balanus improvisus. As a relevant positive control, data for the commercial AF booster Sea-nine™ is included. Comparisons are also made with other relevant natural AF compounds.Marine biofouling is a highly complex and dynamic phenomenon, which is influenced by a range of processes at the physical, chemical, and biological levels , but was inactive in the adhesion assays, a similar antibacterial profile to the natural product ianthelline , the hybrid compounds displayed a similar activity against the investigated marine bacteria . H. coffeaeformis in particular is a commonly used model species for diatom adhesion and growth  resulted in reduced antimicroalgal activities. These antialgal activities are high and superior to several reported natural antifoulants, including ianthelline, which displays poor antialgal activity  and their harder calcareous counterparts  , but this activity was accompanied by high toxicity. The remaining compounds yielded IC50 values that ranged between 1.0 and 5.0 μg/mL and, importantly, displayed very low toxicity at 5.0 μg/mL, comparable to that of the negative control DMSO  in filtered seawater . Although weaker than the positive control Sea-nine™, these inhibitory activities are higher than a large number of reported AF natural products and comparable to well-studied potent natural antifoulants such as barettin, ianthelline, polygodial, synoxazolidinone A & C, oroidin, butenolide, and geraniol , advocating that, in this case, the addition of the oxime functionality did not provide improved activity. Examining the influence of structure  on settlement inhibition gave no clear relationships (data not included), suggesting that these compounds might exert their activity on multiple cellular targets.Considering activity and toxicity, the tetra-substituted Fusetani . Compouna et al. . CompounH. coffeaformis and C. closterium is promising as it has been shown that AF coatings can struggle to minimize the slime formation, which is influenced by these species .A number of the tested dihydrostilbene-oxime hybrids were effective antifoulants, in particular against microalgae and balanide larval settlement, where the activities were comparable or better than many previously reported antifoulants Fusetani . The genAs embodied by the hybrid approach of Takamura et al. , the cur7 and 15, and their non-oxime containing counterparts. Against balanide settlement inhibition, similar IC50 values were observed for the four compounds; however, the oxime motif of 15 significantly increased toxicity at 5 μg/mL in comparison to its dihydrostilbene analogue   vs 5 (inactive); Fig. 3 and 4 and numerous other structurally similar marine natural products  ion containing catalytic center . This is likely reflective of the breadth of studied species, and therefore of their diverse and evolutionary distinct cellular pathways. Whereas the potency of dihydrostilbenes against balanide cyprid inhibition could be linked to hydrophobicity, a similar link for the oxime hybrids was not noticed , which is particularly encouraging. A number of the compounds were also effective at inhibiting the settlement of balanide larvae at low concentrations, which demonstrated their potency against a macrofouling species.ESM 1(DOCX 1.47 mb)


Cancer registry data show that survival of colorectal cancer (CRC) in the United Kingdom is poor compared with other European countries and the United States, yet these data sources lack information on patient comorbidities and medication use, which could help explain these differences.Among individuals aged 40–89 years in The Health Improvement Network (2000–2014), we identified first ever cases of CRC and calculated incidence rates with 95% confidence intervals (CIs). For CRC cases and non-cases in two separate calendar years (2002 and 2014), we evaluated patient demographics, lifestyle factors, comorbidities and medication use and bowel screening.p = 0.064). Similarly, in 2002, 12.5% were diagnosed at age 50–59 years compared with 16.2% in 2014 (p = 0.002). Between 2002 and 2014, previous bowel screening increased in both CRC cases (+ 10.6%) and non-cases (+ 9.7%)(p < 0.001 for both groups). Greater rises in the following were seen among CRC cases compared with non-cases: diabetes , obesity , hypertension , atrial fibrillation (+ 2.6% [p < 0.01] vs. + 0.3% [p < 0.001]), and use of proton pump inhibitors (+ 11.5% vs. + 9.0%), anti-hypertensives (+ 9.9% vs. + 1.4%) and warfarin (+ 3.2% vs. + 0.4%); p < 0.001 for CRC cases and non-cases with respect to each medication.The incidence of CRC remained relatively constant across the study period; incidence rates per 10,000 person-years (95% CIs) were 9.27 (8.59–1.01) in 2000, 10.65 (10.15–11.18) in 2007 and 8.37 (7.93–8.83) in 2014. Incidence rates per 10,000 person-years were higher in men than women at 11.44 (95% CI: 10.35-12.66) vs. 7.40 (95% CI: 6.59–8.32) in 2000, and 9.39 (95% CI: 8.74–10.10) vs. 7.38 (95% CI: 6.81–8.00) in 2014. An increase was seen in the proportion of CRC cases diagnosed at age < 60 years. In 2002, 3.5% of CRC cases were diagnosed at age 40–49 compared with 5.1% in 2014  contains supplementary material, which is available to authorized users. In Europe, colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second most commonly diagnosed cancer in females . In the While cancer registries are suitable for determining population-level incidence and survival – including adjustment for age, stage at diagnosis and socio-economic status – data such as the prevalence of comorbidities, medication use before cancer onset and other potential confounders are not systematically recorded. This hinders adjustment for these factors and limits the robustness of comparisons that can be made when making comparisons of CRC survival between populations. In the UK, prospectively collected data on patient comorbidities, prescribed medications and lifestyle factors can be ascertained from primary care databases of electronic medical records (EMRs). Using a validated UK primary care database, we conducted a retrospective cohort study to evaluate trends in the contemporary incidence of CRC in the UK and characteristics preceding CRC diagnosis, including specific comorbidities and medication use. The study protocol was approved by an independent scientific review committee (reference number 14-088A1).We used data from The Health Improvement Network (THIN), a primary care database of anonymized EMRs in the UK covering approximately 6% of the UK population . The datAnnually, from 2000 to 2014, we identified all individuals in THIN aged 40–89 years with a registration status of permanent or died. To enter the study, individuals were required to have no previous record of any type of cancer and at least 1 years of enrolment with their primary care practitioner (PCP). All members of the study population were followed-up from the date of entry into the study year (start date) until a first recorded diagnosis for CRC, aged 90 years, death or the end of the calendar year , whichever came first. Individuals with a first recorded diagnosis for CRC during follow-up were deemed to be incident cases of CRC. No additional validation step of CRC cases, such as manual review of patient records or validation with the PCP via questionnaires, was performed because we have previously shown the recording of CRC in THIN to have a high level of validity and completeness – using linkage to hospitalization data, the positive predictive value for CRC in THIN was 97.9% (556/568) and the false negative rate was 6.1% (36/592) [N = 931] and 2014 [N = 1330]), we obtained data on patient demographics , lifestyle factors, healthcare use , gastrointestinal comorbidities and symptoms, bowel screening procedures , other comorbidities (with a focus on cardiovascular conditions) and medications. Lifestyle factors and BMI were ascertained any time before the start date, using the most recent value/record. Comorbidities  were ascertained within the 5 years before the start date. Healthcare use was ascertained in the year before the start date, and medication use was defined as use on the start date or within the previous 30 days. For comparison, we also ascertained these data for all non-CRC cases in 2002  and 2014 . For each study year this comprised all individuals who did not have a first recorded diagnosis for CRC during follow-up.For CRC cases in two separate calendar years approximately a decade apart  for each calendar year in the study period using Poisson regression, censoring at the occurrence of another type of cancer. Incidence rates were calculated as the number of first-ever cases of CRC per 10,000 person-years, for the total study population and stratified by sex and 10-year age group. For patient characteristics in 2002 and 2014, data were expressed as frequency counts and percentages and differences compared using Chi2 test for categorical variables, apart from the mean age at the start date, which was presented along with its standard deviation (SD).The annual incidence of CRC per 10,000 person-years remained relatively constant across the study period; incidence rates increased from 9.27 (95% CI: 8.59–1.01) in 2000 to 10.65 (95% CI: 10.15–11.18) in 2007, followed by a decreased trend during the later years in the study period, falling to 8.37 (95% CI: 7.93–8.83) in 2014 Fig. . In men,p = 0.711). A rise was seen in the proportion of CRC cases diagnosed at ages < 60 years; in 2002, 3.5% of CRC cases were diagnosed at age 40–49 compared with 5.1% in 2014 (p = 0.064). Similarly, in 2002, 12.5% were diagnosed at age 50–59 years compared with 16.2% in 2014 (p = 0.002). The mean age at diagnosis was 70.2 years (SD: 10.2) in 2002 and 69.4 years (SD: 11.2) in 2014. Corresponding data (age at start date) for non-cases was 58.6 (SD: 12.8) in 2002 and 57.6 (SD: 12.6) in 2014. Between 2002 and 2014, the proportion of current smokers among CRC cases decreased by 3.1%, with a similar decrease seen among non-cases (− 2.5%) (p < 0.001 for both). A decline in non-drinkers was seen in both cases and non-cases (p < 0.001). While obesity (BMI ≥30 kg/m2) was recorded more frequently in 2014 than in 2002 in both groups (p < 0.001), the increase was greater among CRC cases than for non-cases (+ 14.5% vs. + 10.1%).Demographics, lifestyle factors and healthcare use among CRC cases and non-cases in 2002 and 2014 are shown in Table p < 0.001 and + 9.7% in non-cases, p < 0.001). The proportion of cases with a record of colonoscopy also increased among CRC cases  and in non-cases . The prevalence of sigmoidoscopy was similar across the study years in cases (p = 0.412) but increased among non-cases (p < 0.001), while the prevalence of barium enema decreased among both groups (p < 0.001 for both).Gastrointestinal comorbidities, symptoms and investigative procedures among CRC cases and non-cases in 2002 and 2014 are shown in Table p < 0.001) vs. + 6.5% (p < 0.001) for diabetes, + 8.3% (p < 0.001) vs. + 3.6% (p < 0.001) for hypertension, and + 2.6% (p < 0.01) vs. + 0.3% (p < 0.001) for atrial fibrillation. This trend was also seen with use of proton pump inhibitors (PPIs), anti-hypertensives and warfarin use at the start date  (Table p < 0.001) and decreased slightly in non-cases (p < 0.001), while current use of non-steroidal anti-inflammatory drugs decreased in both CRC cases (p = 0.002) and non-cases (p < 0.001). Statin prescribing notably increased among cases  and increased but to a lesser extent among non-cases .As shown in Table In this large population-based study set in a representative primary care setting, we have described the contemporary epidemiology of CRC in the UK and characterized CRC patients at the time of diagnosis in two calendar years more than a decade apart. Few studies have described changes in the comorbidity profile of CRC patients over time  and we aWhile we have shown that the incidence of CRC has remained relatively stable in the UK over the last 15 years, incidence rates appear to have declined in more recent years, particularly in men aged ≥60 years. Our study also found increased uptake of the National Bowel Screening programme over the study period, which could explain the finding of a slightly earlier mean age at diagnosis in 2014 compared with 2002. The national faecal occult blood test screening programme was rolled out in England in June 2006 and in Scotland in June 2007 with the aim of reducing the number of incident cases through detecting pre-cancerous CRC adenomas. Primary care practitioners involved in the programme were informed of participants who failed to complete the programme, and this may have contributed to an increase in the detection or reporting of new cases that may have previously gone unnoticed – this could explain the peak in CRC incidence rates in this current study in 2007. The incidence rates of CRC in this present study are in line, albeit slightly higher, with those from UK Cancer Registry data  and simiSeveral factors should ideally be taken into account when analyzing trends in survival of patients with CRC across time and when making comparisons with other geographical populations, including the comorbidity profile and medication use of patients at the time of diagnosis. Several studies have shown that high comorbidity levels are associated with poorer survival , 16–22. Strengths of our study include the large sample size representative of the UK population as a whole; Blak et al.  have shounpublished data). It should be noted that 55.1% of the CRC cases in 2002 and 61.4% of CRC cases in 2011 did not have details on stage recorded in the free text.Advanced stage at CRC diagnosis is clearly associated with worse survival , 27, andIn conclusion, our findings suggest that the increased prevalence of obesity, hypertension, atrial fibrillation and the pill burden among CRC cases should be considered when evaluating and making comparisons in patterns of CRC survival.Additional file 1:Supplementary Methods. (DOCX 13 kb)


As data on the phenotype, characteristics and management of patients with Fragile X Syndrome (FXS) are limited, we aimed to collect such data in Germany in experienced centres involved in the treatment of such patients.EXPLAIN-FXS is a prospective observational  study (registry) performed between April 2013 and January 2016 at 18 sites in Germany. Requirements for patient participation included confirmed diagnosis of FXS by genetic testing (>200 CGG repeats) and written informed consent. Patients were followed for up to 2 years.Seventy-five patients  were analysed. The mean 6-item score, determined according to Giangreco , was 6.9 ± 2.5 points. At least one neurological finding each was noted in 53 patients (69.7 %). Specifically, ataxia was noted in 5 patients (6.6 %), lack of fine motor skills in 40 patients, (52.6 %), muscle tonus disorder in 4 patients (5.3 %), and other neurological disorders in 39 patients (51.3 %). Spasticity was not noted in any patient.Seizures were reported in 6 patients (8.1 %), anxiety disorders in 22 patients (30.1 %), depression in 7 patients (9.6 %), ADHD/ADD in 36 patients (49.3 %), impairment of social behavior in 39 patients (53.4 %), and other comorbidities in 23 patients (31.5 %). The mean Aberrant Behaviour Checklist Community Edition (ABC-C) score on behavioral symptoms, obtained in 71 patients at first documentation, was 48.4 ± 27.8 . The mean visual analogue scale (VAS) score, obtained in 59 patients at first documentation, was 84.9 ± 14.6 points .This report describes the largest cohort of patients with FXS in Europe. The reported observations indicate a substantial burden of disease for patients and their caregivers. Based on these observations, an early expert psychiatric diagnosis is recommended for suspected FXS patients. Further recommendations include multimodal and multi-professional management that is tailored to the individual patient’s needs.NCT01711606. Registered on 18 October 2012.The ClinTrials.gov identifier is  Fragile X syndrome (FXS) is among the most common inherited genetic disorders leading to intellectual disability and autism . It is cReduced intelligence is a major symptom of FXS, varying from learning difficulties to severe cognitive impairment . Speech,Therapeutic options are very limited . The fulFurther, non-pharmacological therapy such as speech-language therapy or occupational therapy is frequently indicated . In a reBased on their synaptic mechanisms, specific agents including arbaclofen, ganaxolone, acamprosate, minocycline, as well as the mGluR5 inhibitors AFQ056 (mavoglurant by Novartis) and RG7090 (basimglurant by Roche) have been investigated in FXS , 20. At In Germany and other European countries, data on the phenotype, characteristics, quality of life, caregiver burden and other aspects of the management of patients with FXS are limited. The current study aimed to fill these gaps. It was initiated as a longitudinal study to investigate changes in psychometric parameters over time.Patients  required confirmed diagnosis of FXS by genetic testing (>200 CGG repeats), as well as written informed consent, to participate. No additional genetic testing was required for this study.Patient data were collected at first documentation and approximately every 6 months thereafter. Patients were to be followed for at least 2 years. As many patients were included at a later stage, the duration of follow-up for these patients was limited.The present study is a prospective observational  study (registry) in Germany. Details of the study protocol have been reported earlier . The stuIn 20 % of participating centres, on-site monitoring of the centres was done with source data verification. Physicians received remuneration for participation.Physicians from about 50 centres , all experienced in the management of FXS, participated in the study. Specialists from other disciplines were also involved, such as child/adolescent psychiatrists, general psychiatrists, or physicians in social paediatric centres.Demographic data Availability of prior genetic testing results (to confirm FXS diagnosis)Status/medical history of relatives . No data on premutation in parents was collected.Insurance status FXS patient history Educational status or employment status, as appropriateComorbid disorders of the patient Neurologic/psychiatric statusPrevious and current medical and non-pharmacological therapy for FXSSymptoms Check List (SCL-27) , 26Economic parameters: contacts by physicians , days of hospitalisation since suspected FXS diagnosis, caregiver’s absence from work○ IQ testing: Test of adaptive intelligence  , Hamburg○ Aberrant Behavior Checklist Community Edition (ABC-C) , 32, a pResults of psychometric testing:○ EQ-5D (adults)  or EQ-5D○ Eltern Belastungs Inventar (EBI): German version of the Parenting Stress Index (PSI) questionnaire, if caregiver is a family member ○ Nurses’ Observation Scale for In-patient Evaluation (NOSIE), if caregiver is not a family member , 37○ Short version of the International Classification of Functioning, Disability, and Health (Mini-ICF) rating for psychiatric disorders , filled Quality of life questionnaires (available as paper forms in the study file)At the first documentation visit, data on the following parameters were documented:Noteworthy life events Educational status and employment status of the patientcurrent medical and non-pharmacological therapyEconomic parametersResults of psychometric testing (as at the first documentation visit)Quality of life questionnaires (as at the first documentation visit)At the follow-up visits (approximately every 6 months), information on the following parameters was documented, in the same manner as at first documentation:Data on patient history and characteristics were retrieved from medical records. Data on quality of life and psychometric assessments were collected on self-administered paper forms which were filled out by the patients and/or their caregivers.The sample size was based on feasibility aspects. The analysis set consisted of all patients with confirmed FXS diagnosis included in the study. Data were analysed descriptively, using established statistical and epidemiological methods. Continuous variables are reported as median with interquartiles and other percentiles, and as mean with standard deviation (SD), together with minimum and maximum values. Categorical variables were reported as absolute values and relative percentages. Statistical analyses were performed with SAS version 9.2 .The authors adhered to the STROBE methodology .Of the 76 patients who were screened and found eligible, 75 provided data and were included in the analysis. Their characteristics are shown in Table Of these 75 patients, 63 (84.0 %) were males and 12 (16.0 %) were females. The mean age was 16.7 ± 14.5 years, ranging from 2 to 82 years. Patients had siblings with FXS in 17 cases (22.7 %), indicating an extra disease burden on the afflicted families.The main caregivers were the parents (or a parent) for 62 patients (82.7 %). Notably, of these parents, 45 were married (61.6 %) and 6 were not married but living together (8.2 %).FXS patients were usually seen by a general practitioner or a paediatrician. .The mean 6-item score according to Giangreco  was 6.9 ± 2.5 points.Impaired intelligence was determined based on school performance, academic achievement, and IQ tests. Attendance at a school for children with special needs was a major criterion for impaired intelligence. Psychometric tests used at the first documentation were the Snijders Oomen-Test (10 patients); the HAWIK/HAWIE (8 patients); the Kaufman Test K-ABC (5 patients); other (not specified) tests (10 patients).n = 75 patients), 65 patients had an IQ value < 70 points; 8 patients an IQ value of 70 to 85 points; and 2 patients > 85 points. Overall, 73 patients (97.3 %) were deemed to have impaired intelligence.For the calculation of the Giangreco index , moving home , separation of parents , death of a close person , starting school/work , and other events . No such relevant life events occurred in 59 patients (77.6 %).Fifty one patients (77.3 %) had been attending school, and 33 of these patients were currently attending school. Mean duration of school education was 7.7 ± 3.6 years (range 1- 15). Of these, 32 children and adolescents (62.7 % of those attending a school) were in a school for the intellectually disabled, 14 (27.5 %) attended a similar school type for learning disabled students (“Förderschule”), 7 (13.7 %) attended a regular school with integration measures, 2 (3.9 %) attended a regular school, and 3 (4.9 %) attended a different school type (none of the above).The 27-item symptom checklist mean score, calculated in 54 patients at first documentation, was 4.7 ± 3.1 points.The patients had taken various IQ tests prior to first documentation or took these during the course of the study.The Aberrant Behaviour Checklist Community Edition (ABC-C) score, obtained in 71 patients at first documentation, was 48.4 ± 27.8  total score, obtained in 64 patients, was 74.6 ± 12.1 . In the child domain, the mean score was 78.5 ± 10.0 and in the parent domain, it was 65.6 ± 13.2. Overall, patients reported a low/normal stress level in 6 cases (9.4 %), a high stress level in 21 cases (32.8 %), and a very high stress level in 37 cases (57.8 %). Children reported a low/normal stress level in 2 cases (3.1 %), a high stress level in 14 cases (21.9 %), and a very high stress level in 48 cases (75.0 %). In parents, the stress level was lower, and low/normal stress levels were reported by 25 respondents (39.1 %), high stress levels by 22 respondents (34.4 %), and very high stress levels by 50 respondents (78.1 %).The Nurses’ Observational Scale for Inpatient Evaluation (NOSIE) mean index value, based on answers from 28 patients at first documentation, was 151.5 ± 0.5 .On the mini-ICF, the average value at first documentation, obtained in 73 patients, was 2.2 ± 0.8 .The EQ-5D index in adults at first documentation, obtained in 41 patients, was 0.7 ± 0.3 points . The EQ-5D index in children at first documentation, obtained in 22 patients, was 0.7 ± 0.3 points . The EQ-5D time trade-off value in adults at first documentation, obtained in 22 patients, was 0.8 ± 0.3 points . The EQ-5D time trade-off value in children at first documentation, obtained in 41 patients, was 0.7 ± 0.3 points . On the EQ-5D visual analogue scale (VAS), the mean value at first documentation, obtained in 59 patients, was 84.9 ± 14.6 points .The EXPLAIN study is the largest and the only longitudinal European observational study investigating clinical characteristics and caregiver situation of patients with FXS. It documents 75 individuals in a wide age range, from 2 to 82 years. As expected, the prevalence of neurological findings and other comorbidities was very high. Almost every patient in our study had a relevant mental or neurological disorder which required pharmacological or non-pharmacological treatment. In younger patients, externalizing symptoms prevailed; in older patients, internalizing symptoms prevailed.The only systematic recording of data on patients with FXS is currently taking place in the USA, in the context of individual projects, namely by the Fragile X Clinical & Research Consortium (FXCRC), with its FORWARD Registry and Database, and by our Fragile X World Surveys  , 15, 40.The majority of documented patients in the present study were children and adolescents; only 29 were adults. Thus, it is understandable that most patients were still living with their parents, who mostly were the main caregivers. This indicates solid family structures, and might be a finding based on patient selection. The proxy information collected in this study can be considered to be “first-hand” and very reliable.In light of the common social and behavior problems linked with FXS, an important tool for our study was the ABC-C questionnaire. It is a sensitive instrument for rating maladaptive and inappropriate behaviors in patients with intellectual disabilities. In patients with FXS, it has also been used as an endpoint in clinical studies  –43. For The study shows a high burden of comorbidities as well as mental symptoms. The rates of mental health symptoms, at least in children and adolescents, were higher than the prevalence of such symptoms in the general population of individuals with intellectual disability in this age range. The prevalence of mental health symptoms in the general population of individuals with intellectual disability has been documented as follows: 24 % with impaired social behavior; 13 % with emotional problems; and 26 % with hyperactivity . In EXPLCompared with another study in a large US American cohort (89 women and 239 men aged between 22 and 64 years) , affectiIt was an unexpected finding that the comorbid mental health symptoms and diseases usually were treated with medications. Usually the prescription of psychopharmacological drugs in individuals with impaired intelligence is between 21 and 45 % . The higIn terms of quality of life assessment, as expected, patients with FXS had limitations in terms of self-care and usual activities, but also anxiety/depression. Compared with the German reference population , patientAs a limitation to this study, a number of instruments which were used in other FXS studies were not applied in our study. For example, the mavoglurant studies had applied, among others, the SRS scale to identify the presence of autistic social impairment; the TEA-Ch to test every-day attention and inhibition in children; or the widely used Clinical Global Impression (CGI) scale to assess treatment response in psychiatric patients . This stIn conclusion, the findings in EXPLAIN-FXS confirm the substantial burden of psychiatric and mental problems in patients with FXS in all age groups. In these patients, an early expert psychiatric diagnosis should be initiated. The resulting management should be multimodal and multi-professional and should be tailored to the individual patient’s needs.


Tm-shift method for detection of dog-derived Ancylostoma ceylanicum and A. caninum, three sets of primers were designed based on three SNPs  of their internal transcribed spacer 1 (ITS1) sequences. The detection effect of the Tm-shift was assessed through the stability, sensitivity, accuracy test, and clinical detection. The results showed that these three sets of primers could distinguish accurately between A. ceylanicum and A. caninum. The coefficient of variation in their Tm values on the three SNPs was 0.09% and 0.15% (ITS71), 0.18% and 0.14% (ITS197), and 0.13% and 0.07% (ITS296), respectively. The lowest detectable concentration of standard plasmids for A. ceylanicum and A. caninum was 5.33 × 10−6 ng/μL and 5.03 × 10−6 ng/μL. The Tm-shift results of ten DNA samples from the dog-derived hookworms were consistent with their known species. In the clinical detection of 50 fecal samples from stray dogs, the positive rate of hookworm detected by Tm-shift (42%) was significantly higher than that by microscopic examination (34%), and the former can identify the Ancylostoma species. It is concluded that the Tm-shift method is rapid, specific, sensitive, and suitable for the clinical detection and zoonotic risk assessment of the dog-derived hookworm.To develop a  Ancylostoma caninum, A. ceylanicum, A. braziliense, and Uncinaria stenocephala, that can infect dogs [ A. ceylanicum and A. caninum in China [ A. ceylanicum has become the second most common species of hookworm that can infect humans in Asia [ A. caninum can reach adulthood in people [Hookworms are common intestinal parasites distributed globally, which can cause serious harm to the health of dogs, cats, and human beings. There are four species of hookworms, that is,ect dogs . Accordiin China . It was  in Asia , 4, espe in Asia , 6, Japa in Asia , Malaysi in Asia , Laos [9 in Asia , and Ind in Asia . Occasion people . These tn people , 13. TheFecal examination is a traditional method for detecting hookworms, but it is easily missed under mild infection. Because of morphological similarities of different hookworm eggs, it is difficult to identify them only by their appearance. Presently, many molecular detection technologies have been applied to the identification of hookworms, such as specific polymerase chain reaction (PCR), multiplex polymerase chain reaction (multi-PCR), restriction fragment length polymorphism (RFLP), and high resolution melting (HRM) –16. HoweTm-shift based on the SNP is a new molecular detection method, where two specific primers and a reverse primer are designed as follows: the 3′-end of specific primer binds to SNP loci, and two GC-rich sequences with unequal length and no pairing with the template are added at the 5′-end, respectively. Then, PCR products of samples are detected according to different peaks of melting curve in different Tm values [Tm-shift method can satisfy the high-throughput research and has efficient, accurate, and inexpensive advantages [Tm-shift genotyping method for detection of cat-derived Giardia lamblia assemblages A and F [Single nucleotide polymorphism (SNP) has become a molecular genetic marker of the third generation after RFLP and microsatellite sequence (MS) , 18. Them values . The Tm-vantages . So far,vantages –23. Rece A and F , which hTm-shift method for detecting A. ceylanicum and A. caninum from dogs based on three SNP loci of rDNA ITS1 sequences of two hookworms, in order to establish a rapid and accurate method for identifying the species of dog-derived hookworms which could provide a new technical means for clinical detection of hookworms and zoonotic risk assessment.This study developed a  A. ceylanicum and A. caninum samples were isolated and identified by Liu et al. [Adultu et al. , 6, pres2O), and total genomic DNA was extracted using the Wizard® SV Genomic DNA Purification System  according to the manufacturer's protocols. All clinical fecal samples were dissolved in ddH2O and then pretreated with 5 cycles of heating at 100°C for 5 min, followed by immediate freezing at −80°C for 5 min. Genomic DNA of clinical fecal samples was extracted using Stool DNA extraction kit  according to the manufacturer's protocols. Extracted DNAs were then stored at −20°C for use.The adult hookworms preserved were repeatedly washed with double-distilled water (ddH A. ceylanicum and A. caninum were amplified using a forward primer AF (5′-CTTTGTCGGGAAGGTTGG-3′) and a reverse primer AR (5′-TTCACCACTCTAAGCGTCT-3′) designed by Liu et al. [μL, including 12.5 μL of Premix Ex-Taq polymerase , 9.5 μL of ddH2O, 0.5 μL of each primer AF/AR (50 μmol/L), and 2 μL of DNA sample. PCR cycling parameters were as follows: 1 cycle at 94°C for 5 min and then 35 cycles at 94°C for 30 sec, at 61.5°C for 30 sec, and at 72°C for 45 sec, followed by 1 cycle at 72°C for 7 min. The PCR products were analysed by gel electrophoresis in 1.5% agarose gels, stained with 0.2 mg/ml ethidium bromide, and visualized on a UV transilluminator.The ITS1 sequences ofu et al. . The pre Escherichia coli and connected with pMD18-T  and then transferred into DH5α Competent Cells . Positive clones were screened by bacterial PCR and sent to Shenggong Corporation  for sequencing. The plasmid DNAs were extracted using the Plasmid Kit . These plasmids containing ITS1 sequence of A. ceylanicum and A. caninum were named AceP and AcaP, respectively. The A260/A280 value and concentration of plasmids (1 μL) were measured using ultra-micro-spectrophotometer, where A260/A280 value and optimal concentration should be 1.8~2.0 and 50 ng/μL. These plasmids were stored at −20°C for use.The purified PCR products were cloned inTm-shift primers (including two forward specific primers and one common reverse primer) based on three SNPs  were designed by software Primer Premier 5.0, referencing to two sequences  downloaded from GenBank. Their sequence composition and predictive amplification fragment are shown in μL, and stored at −20°C for use. PCR amplification and Tm-shift reaction were performed once in Rotor-Gene Q. The reaction system was performed in 20 μL, including 10 μL of SYBR®Premix Ex-Taq™ II (2x), 0.4 μL of long-tail primer, 0.4 μL of short-tail primer, 0.8 μL of common reverse primer, 7.4 μL of ddH2O, and 1.0 μL of plasmid. PCR cycling parameters were as follows: initial denaturation at 95°C for 5 min and then 40 cycles at 95°C for 10 sec and 63°C for 30 sec. Melting process was from 70 to 95°C at the rate of 0.5°C/sec.Three sets of Tm-shift was assessed through the stability, sensitivity, and accuracy test. We tested the reproducibility of two known standard plasmids (AceP and AcaP). Intra-assays were tested seven times, and interassays were tested three times for each plasmid. For detection of the sensitivity of the Tm-shift method, two standard plasmids (AceP and AcaP) were diluted ten times and detected according to the concentration of 1 : 10~1 : 108. To evaluate the Tm-shift method's accuracy, we detected ten samples with known hookworm species. All reaction system and cycling parameters were the same as mentioned above.The detection effect of the Tm-shift method. Tm-shift classification results were further confirmed by DNA sequencing.Fifty fecal samples from the stray dogs were examined by the flotation technique with saturated zinc sulfate and the  A. ceylanicum and A. caninum was 404 bp long ( A. ceylanicum from Japan (LC036567) and the highest similarity (99%) with A. caninum from Australia (KP844730). Thus, two hookworms were identified as A. ceylanicum and A. caninum. The A260/A280 values of positive plasmids AceP and AcaP were between 1.8 and 2.0, respectively. Their concentrations were 53.3 ng/μL and 50.3 ng/μL, respectively.The amplified ITS1 fragment from two genomic DNAs of dog-derived bp long  and the Tm-shift based on ITS71, ITS197, and ITS296 for standard plasmid AceP and AcaP are shown in Tm-shift method based on three SNPs could distinguish between A. ceylanicum and A. caninum. Software analysis showed that the Tm values of AceP (A. ceylanicum) and AcaP (A. caninum) in three sets of primers were 88.0°C and 86.0°C (ITS71), 86.0°C and 85.0°C (ITS197), and 84.0°C and 85.0°C (ITS296), respectively.The standard curves of Tm) was 0.15 and 0.09%. In primer ITS197, the CV of AceP and AcaP melting temperature was 0.14 and 0.18%. And in primer ITS296, the CV of AceP and AcaP melting temperature was 0.07 and 0.13%. When AceP and AcaP samples were diluted to 1 : 107 (5.33 × 10−6 ng/μL and 5.03 × 10−6 ng/μL), the Tm-shift method was still able to identify both of them. However, when the samples were diluted to 1 : 108, all of them could not be determined , which thus can reduce pollution to the environment and ensure safety of the researchers. The Tm-shift method has clear price advantage compared with HRM, because of the use of instruments for ordinary fluorescence PCR and fluorescent dyes for ordinary SYBR Green I rather than saturation dyes Eva Green and LC Green. In addition, the detection efficiency is improved through amplification of the target fragment by two-step method.Currently, the species identification of hookworm is mainly dependent on molecular means such as specific PCR, multi-PCR, RFLP, and HRM –16. Howe failure . CompareTm-shift detection method has its unique advantages, it also has some limitations. It has higher demand of two specific primers, whose Tm value should be 59~62°C, and their length should be controlled in 15~22 bp. In order to give better amplification efficiency of Taq polymerase, the gap of the Tm values between the reverse primer and the specific primers (before joining at the 5′-end sequence) must be maintained between 4 and 5°C. It is due to the high screening conditions of specific primers that some SNP loci failed to design suitable primers. Among five SNP loci  could distinguish between two species of hookworms. The Tm value of the melting curve between A. ceylanicum and A. caninum differed at 2°C on ITS71 but 1°C on ITS197 and ITS296. The ITS71 locus identification had the best effect. Among 50 dog-derived fecal samples detected, 21 samples were positive for hookworms by Tm-shift method (14 A. ceylanicum and 7 A. caninum), which had 4 more positive samples than microscopic examination (17 positive samples), and can accurately identify the species of hookworms from stray dogs.Although the SNP loci  originalTm-shift method based on three SNP loci of ITS1 sequences of A. ceylanicum and A. caninum was used for the first time for their classification. The successful identification of two hookworms indicates that the method is specific, sensitive, economical, practical, and safe, which can provide a new technical means for clinical detection of hookworm and zoonotic risk assessment.In conclusion,


Centrocestus formosanus, Haplorchis pumilio, H. taichui, H. yokogawai, Procerovum varium and Stellantchasmus falcatus. For molecular phylogenetic and systematic studies on trematodes, we need more prospective markers for taxonomic identification and classification. This study provides near-complete ribosomal transcription units (rTU) from Haplorchis pumilio and H. taichui and demonstrates the use of 28S rDNA sequences for identification and phylogenetic analysis.Heterophyidiasis is now a major public health threat in many tropical countries. Species in the trematode family Heterophyidae infecting humans include H. pumilio and H. taichui from human hosts in Vietnam, were determined and annotated. Sequence analysis revealed tandem repetitive elements in ITS1 in H. pumilio and in ITS2 in H. taichui. A phylogenetic tree inferred from 28S rDNA sequences of 40 trematode strains/species, including 14 Vietnamese heterophyid individuals, clearly confirmed the status of each of the Vietnamese species: Centrocestus formosanus, Haplorchis pumilio, H. taichui, H. yokogawai, Procerovum varium and Stellantchasmus falcatus. However, the family Heterophyidae was clearly not monophyletic, with some genera apparently allied with other families within the superfamily Opisthorchioidea (i.e. Cryptogonimidae and Opisthorchiidae). These families and their constituent genera require substantial re-evaluation using a combination of morphological and molecular data. Our new molecular data will assist in such studies.The near-complete ribosomal transcription units (rTU), consisting of 18S, ITS1, 5.8S, ITS2 and 28S rRNA genes and spacers, from Alaria alata, family Diplostomidae), six common human pathogenic heterophyids were identified and clearly resolved. The phylogenetic tree inferred from these sequences again confirmed anomalies in molecular placement of some members of the family Heterophyidae and demonstrates the need for reappraisal of the entire superfamily Opisthorchioidea. The new sequences provided here supplement those already available in public databases and add to the array of molecular tools that can be used for the diagnosis of heterophyid species in human and animal infections.The 28S rDNA sequences are conserved among individuals within a species but varied between genera. Based on analysis of 40 28S rDNA sequences representing 19 species in the superfamily Opisthorchioidea and an outgroup taxon ( Centrocestus formosanus, Haplorchis pumilio, H. taichui, H. yokogawai, Procerovum varium and Stellantchasmus falcatus [Many members of the trematode family Heterophyidae Odhner, 1914, use fishes as intermediate hosts and humans as definitive hosts . Six spefalcatus , 3 are afalcatus . Heteropfalcatus , 12, 13.falcatus , 9, 14.DNA sequences are commonly used for molecular diagnosis and systematic/phylogenetic studies. Although markers are often chosen from the mitochondrial genome, sequences from the nuclear ribosomal transcription unit (rTU)  are particularly useful and reliable for this purpose , 15–22. Haplorchis pumilio and H. taichui, commonly found in humans. Portions of the 28S rRNA gene from other heterophyids infecting humans in Vietnam are also presented, i.e. Centrocestus formosanus, Haplorchis yokogawai, Procerovum varium and Stellantchasmus falcatus. The data will be used to explore the phylogenetic positions of these genera in the family Heterophyidae and in the class Trematoda.The aim of this paper is to present the sequence of near-complete ribosomal transcription units from Haplorchis spp. and Centrocestus spp. were collected from fish species  and cercariae from freshwater snails (Melanoides tuberculata) in Nam Dinh Province [Metacercariae of Province , 14  [Haplorchis taichui and HpDzH of H. pumilio, collected from people in Quang Tri and Nam Dinh Provinces, respectively, were chosen for amplification and sequencing of the rTU. Only the 28S region was amplified and sequenced from other species for molecular identification and phylogenetic analysis (Table Adults of  (MgSO4) , 14 (Tab (MgSO4) , 5, 14. Total genomic DNA was extracted from individual cercariae, metacercariae or adult specimens using the GeneJET™ Genomic DNA Purification Kit , according to the manufacturer’s instructions. Genomic DNA was eluted in 50 μl of the elution buffer provided in the kit and stored at -20 °C. The DNA concentration was estimated using a GBC UV/visible 911A spectrophotometer  and diluted to a working 50 ng/μl: 2 μl were used as template in a PCR of 50 μl volume.H. pumilio and H. taichui, are listed in Table All rTU-universal primers, used both for amplification and sequencing the rTU of 2O. All PCRs were performed in a MJ PTC-100 thermal cycler with initiation at 94 °C for 5 min, followed by 35 cycles consisting of denaturation for 30 s at 94 °C, annealing at 56 °C for 30 s, extension at 72 °C for 6 min; and a final extension at 72 °C for 10 min. The PCR products (10 μl of each) were examined on a 1% agarose gel, stained with ethidium bromide, and visualized under UV light .PCR reactions of 50 μl were prepared using 25 μl of DreamTaq PCR Master Mix (2×)  and 2 μl DNA template (50 ng/μl), 2 μl of each primer (10 pmol/μl), 2 μl DMSO (dimethyl sulfoxide) and 17 μl HThe amplicons were eluted from the gel and subjected to direct sequencing by primer-walking in both directions.Euryhelmis costaricensis (GenBank: AB521797); Isthmiophora hortensis (AB189982); Paragonimus kellicotti (HQ900670); Paramphistomum cervi [Centrocestus sp. (AY245699); and Haplorchis pumilio (AY245706) and Haplorchis taichui (AY245705) [Boundaries of ribosomal 18S, 5.8S and 28S genes were determined by alignment, using the Clustal X program , with knum cervi ; and somY245705) . For intY245705) .http://iubio.bio.indiana.edu/soft/molbio/ibmpc/genedoc-readme.html)  as an outgroup species. The alignment was trimmed to the length of the shortest sequence, saved in FASTA format and imported into the MEGA6.06 software. To examine the phylogenetic position of the Vietnamese heterophyids relative to other trematodes, a phylogenetic tree was reconstructed  of 14 Vietnamese heterophyids and 25 additional sequences, representing species of all three families of the superfamily Opisthorchioidea available in GenBank, and including another 17 sequences from members of the family Heterophyidae, were aligned using GENEDOC2.7 (available at: ) Tables . Also inH. pumilio and H. taichui were determined. The 28S rDNA sequences are conserved among individuals within a species but variable between species and genera. The near-complete rTU is 4,943 nucleotides in length for H. pumilio, and 4,796 nucleotides for H. taichui. These sequences have been deposited in GenBank under accession nos. KX815125 and KX815126, respectively. We did not sequence the IGS due to the highly repetitive sequences included in this region. The five regions of the rTU are: 18S, ITS1, 5.8S, ITS2 and 28S, structurally organized as usually seen in the ribosomal DNA operon of metazoans  from H. pumilio and H. taichui, the 18S gene was 1,992 bp in length, and the 5.8S gene was 160 bp long; however, the currently sequenced portion of the 28S gene obtained from H. pumilio is 1,397 bp, and that of H. taichui, 1,403 bp  contains five complete tandem repeats,  and TRB (TRB1-2 each of 123 bp) followed by a partial TRB3 of 84 bp (Table H. taichui (797 bp) lacks repeats. In contrast to ITS1, the ITS2 region (444 bp) in H. taichui from Vietnam (HTAQT3), possesses three tandem repeats, each of 83–85 bp, while in H. pumilio (HpDzH) this region lacks repeats  with members of the Cryptogonimidae, whereas in the ML tree it was depicted as basal to all other opisthorchioideans  or basal in the Opisthorchioidea , Sequences of two other heterophyids, Euryhelmis costaricensis from Japanese martens (Martes melampus) [Cryptocotyle lingua, fell into a strongly supported clade , all other members of which belonged to the family Opisthorchiidae   and CrypHaplorchis pumilio and H. taichui, which infect humans in Vietnam. The obtained sequences encompass virtually the complete 18S gene  and almost half of the 28S gene  [In this study, we have presented sequences of the near-complete ribosomal transcription units (rTUs) for two common species of the family Heterophyidae, –5.5 kb) , 17. AlsH. pumilio and in the ITS2 of H. taichui. ITS sequences of both species have been reported from Israel [H. pumilio possessed only two short tandem repeats (30 bp) in their ITS1, in strong contrast to the Vietnamese sequences, in which the ITS1 contained five complete repeats and one incomplete copy. The ITS1 sequences differed substantially in length between Vietnamese and Israeli individuals of the same species, 1,106 vs 640 bp in H. pumilio; and 797 vs 582 bp in H. taichui, due to differences in numbers of tandem repeats. These indicate intraspecific polymorphism as reported commonly in trematodes [We have found repetitive sequences tandemly arranged in the ITS1 of m Israel . Israeliematodes , 33. Likematodes , 33, 40.ematodes . At the ematodes , 41, 42.The 18S and 28S rDNA sequences, however, are of considerable value for species identification and phylogenetic analysis , 43, 44.Procerovum varium, which rendered Haplorchis paraphyletic. This relationship has also been noticed by others . Clearlm et al.  using 18m et al.  using 18dea Fig. . Such a H. pumilio and H. taichui from human hosts in Vietnam. The ITS1 in H. pumilio and ITS2 in H. taichui contained tandem repeats. The 28S rDNA sequences are conserved among individuals within a species but variable between species and genera. Based on 28S rDNA sequence analysis of 40 sequences representing 19 species in the superfamily Opisthorchioidea, six common human pathogenic heterophyids, Centrocestus formosanus, Haplorchis pumilio, H. taichui, H. yokogawai, Procerovum varium and Stellantchasmus falcatus were clearly resolved. In addition, the phylogenetic tree inferred from these sequences again confirmed anomalies in molecular placement of some members of the family Heterophyidae and demonstrates the need for reappraisal of the entire superfamily Opisthorchioidea. The new sequences provided here supplement those already available in public databases and add to the array of molecular tools that can be used for the diagnosis of heterophyid species in human and animal infections.In conclusion, the present study determined and annotated the near-complete ribosomal transcription unit (rTU), consisting of 18S, ITS1, 5,8S, ITS2 and 28S rRNA genes and spacers, from


Eupenicillium javanicum is a filamentous fungus that has been shown to express industrially applicable enzymes and chemical components, such as antifungal compounds. The biotechnological potential of E. javanicum and proteases made us search a novel protease from this microorganism. The macromolecule was isolated, the main biochemical properties was evaluated, and the specificity of the protease subsites was determined. The protease was produced under solid-state bioprocess with wheat bran and isolated by two chromatography steps with yield of 27.5% and 12.4-fold purification. The molecular mass was estimated at 30 kDa. The N-terminal sequence of the first 20 amino acid residues was AVGAGYNASVALALEKALNN. The enzyme presented higher proteolytic activity at pH 6.0 and 60°C. The protease is stable at wide range of pH values and temperatures and in the presence of surfactants. The “primed” side of the catalytic site showed the highest catalytic efficiency of the enzyme isolated from E. javanicum. The S′1 subsite is responsible for catalyzing the protease reaction with substrates with tyrosine in P′1. These findings provide important insights into the biochemical characterization of a highly active protease from E. javanicum and may facilitate the development of industrial processes involving this protease.Enzymes have important features that may facilitate their application in industrial processes and have been used as alternatives to chemical catalysts. In particular, proteases can be isolated from microorganisms, which provide important sources of advantageous enzymes for industrial processes. For example,  Enzymes present some advantages when compared with chemical catalysts. These macromolecules are able to catalyze a variety of chemical reactions, industrial processes have been shifting to the application of enzymes rather than chemicals as catalysts . In addiProteases are hydrolytic enzymes that have been used in industrial processes for decades. Microorganisms under bioprocesses can act as sources of various proteases to improve the protease production and activity owing to their rapid growth and potential for genetic modification, thereby allowing scientists to enhance the features of proteases for applications in industrial processes .The solid-state bioprocess present potential applications in the production of enzymes and other industrial products. It has been defined as bioprocess in the absence or near-absence of free water, with enough moisture to support the growth and metabolic activity of the microorganism. Other characteristic is the use of low-cost agro-industrial residues, it makes the solid-state fermentation attractive financially and sustainably .Eupenicillium javanicum has been shown to produce different products with industrial interests, such as antifungal compounds . The fungus could be maintained in Sabouraud medium at 4°C for up to 1 month.The fungus Eupenicillium javanicum was grown in 250-mL Erlenmeyer flasks with 30 mL Sabouraud culture medium. The inoculum was maintained for 7 days at 30°C, and the mycelial surface was then scraped in presence of 20 mL of saline solution composed by 0.1%[w/v] (NH4)2SO4 + 0.1% [w/v] NH4NO3 + 0.1% [w/v]MgSO4⋅7H2O.E. javanicum under SSB in 250-mL erlenmeyer flasks containing 5 g wheat bran and 9.0 mL saline solution. The medium was sterilized by autoclaving at 121°C for 40 min. One milliliter of the inoculum was added before incubation at 30°C. After 140 h, the bioprocess was stopped, and 40 mL distilled water (4°C) was added to each flask for extracellular enzyme solubilization. This process was aided by maceration with a plastic rod, and the flasks were then agitated in a shaker at 200 rpm for 30 min at 4°C. The material was filtered and centrifuged at 5,000 × g for 20 min at 4°C. The supernatant was collected as the enzymatic extract . One unit of activity was defined as the amount of the enzyme required to cause an increase of 0.001 of absorbance at 280 nm  using Sephadex G-50 resin. The equilibration buffer was 50 mM acetate (pH 5.0) with 50 mM NaCl, and the elution flow rate was 0.62 mL/min, regulated by a peristaltic pump . The resin was equilibrated with five column volumes (CV), and 5 mL of sample was then applied. The gradient was isocratic, and 5-mL fractions were collected.Enzyme fractions were subjected to dialysis with a 14-kDa membrane and 50 mM Tris-HCl buffer (pH 8.0) for 24 h at 4°C. The dialyzed samples (15 mL) were applied to Tricorn columns with 6 mL Resource-Q resin (anionic properties), pre-equilibrated with five CV of 50 mM Tris-HCl buffer (pH 8.0). After application, the resin was washed with the same buffer (two CV), and a linear salt gradient was then started from 0 to 500 mM NaCl using 20 CV of buffer at an elution flow rate of 2 mL/min. One-milliliter fractions were collected. The process was carried out using an ÄKTA Purifier chromatograph  at 25°C.Proteolytic activity assays and analysis by determination of absorbance at 280 nm were carried out to determine the enzymatic and protein profiles, respectively, and a NanoVue UV/Visible Spectrophotometer  was used to quantify total protein at 280 nm, after each chromatographic process.The enzyme fraction purity was evaluated by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) using 12% gels  stained The N-terminal sequence was determined by cleaving the N-terminal amino acids of proteins and peptides using Edman degradation on a Protein Sequencer PPSQ-33A instrument (Shimadzu Corporation). The PTH-amino acids were separated by high-performance liquid chromatography (HPLC), identified, and analyzed by comparing retention times and UV absorption with a previously quantified standard .-1 cm-1 in a spectrophotometer (Fluorescence resonance energy transfer (FRET) peptides were synthesized in an automatic solid-phase peptide synthesizer , responsible for the molecular fluorescence emission, was conjugated to the N-terminal amino group, and ethylenediamine (EDDnp), a quencher of fluorescence, was conjugated to the C-terminal carboxyl group . A fluorThe enzymatic reaction was carried out using a Lumina fluorescence spectrometer (Thermo Fischer Scientific Inc.) coupled with a Peltier system 4-Position Cell Holder Fluorescence device to control the agitation speed and assay temperature. Reactions with Abz-KLRSSKQ-EDDnp substrate were carried out in a quartz cuvette with an optical path length of 10 mm. The wavelengths were set to λex: 320 nm and λem: 420 nm. Data were collected and analyzed using Luminous software version 3.0.post hoc Tukey, results with p < 0.05 were considered significant.The optimum pH was determined by evaluating different pH values from 4.0 to 10.5 with intervals of 0.5 pH units at 40°C. The buffers used in this analysis were acetate (pH 4.0–5.0), MES (pH 5.5–6.0), HEPES (pH 7.0–8.0), BICINE (pH 8.5–9.0), and CAPS (pH 9.5–10.5). After determining the optimum pH, the optimum temperature was determined from 30 to 75°C, with intervals of 5°C, at pH 6.0. The statistical analysis was performed with one-way ANOVA  and The stability of the enzyme at different pH values was evaluated by incubating the pure enzyme at 25°C for 60 min at various pH values (4.0–10.5), with intervals of 0.5 pH units, followed by reaction at pH 6.0 and 45°C. The buffers used in this analysis were acetate (pH 4.5–5.0), MES (pH 5.5–6.0), HEPES (pH 7.0–8.0), BICINE (pH 8.5–9.0), and CAPS (pH 9.5–10.0).Thermal stability at different temperatures (30–60°C) and for different incubation times  was evaluated by proteases pre-incubation, followed by reaction at pH 6.0 and 45°C.2, CuCl2, CaCl2, MgCl2, BaCl2, and AlCl3. The inhibitors tested in this analysis were phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), iodoacetic acid (IAA), and pepstatin (100 mM stock solutions) according to the protocol described by post hoc Tukey, results with p < 0.05 were considered significant.The effects of ions on proteolytic activity were analyzed using NaCl, CoClThe effects of surfactants on proteolytic activity were analyzed using sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), TritonX-100, and Tween 20 solutions. Pure enzyme solution and surfactants were added to each reaction at final concentrations of 0.1, 0.2, 0.5, or 1.0% for all surfactants except SDS, which was analyzed at 0.02, 0.04, 0.06, 0.08, and 0.1%. The effects of these surfactants were then analyzed by reaction for 5 min at 45°C, followed by further incubation at pH 6.0 at 45°C.Pure enzyme solution and test agents were added to reaction tubes at final concentrations of 10, 25, 50, 100, or 150 mM for urea, guanidine, or DTT. The effects of these three components were then analyzed by reaction for 5 min at 45°C, followed by further incubation at pH 6.0 at 45°C.The molar concentration of the enzyme was determined by active site titration with the inhibitor phosphoramidon, as described by 3, P2, P1, P′1, P′2, and P′3, represented as follows in enzymes: S3, S2, S1, S′1, S′2, and S′3 .Kinetic assays were performed to study the effects of changes in the positions of the amino acids in the substrate and to determine the substrate preference of the anchoring enzyme according to the distribution of amino acids in the peptide sequence. These variations were defined as P and S′3 . The kinEnzymatic kinetic data were obtained by addition the substrate to the reaction cuvette with increasing concentrations. The experiments were performed at pH 6.0 and 45°C, and absorbance was measured using a Lumina fluorescence spectrometer (Thermo Scientific), with control of agitation and reaction temperature using a Fluorescence Peltier 4-Position Cell Holder (Thermo Fischer Scientific Inc.). The wavelengths were set to λex: 320 nm and λem: 420 nm.KM, kcat, and kcat/KM to determine the preference of the enzyme for different substrates.The kinetic parameters were obtained from the Michaelis-Menten equation calculated by non-linear regression of data from hydrolysis of the substrate using Grafit version 5.0. We analyzed the E. javanicum produced a protease that could be reproducibly purified using the steps described in Table 1. A final yield of 27.5% was obtained, with 12.4-fold purification. The elution profile from Sephadex G-50 resin chromatography showed two protein peaks, and peak II (fractions 70–90) exhibited proteolytic activity . These fractions were then subjected to anion exchange chromatography (Resource-Q resin), and the elution profile showed four protein peaks. Peaks II (fractions 61–63) and III (fractions 65–67) showed enzymatic activity . Peak III was used as the purified enzyme in subsequent steps as this peak showed higher specific activity, fold purification, and yield than peak II. Figure 3 shows the purity and estimated molecular weight of the enzyme (30 kDa).The fungus E. javanicum was AVGAGYNASVALALEKALNN. The N-terminal sequence was similar (query cover: 75% and identity: 73%) with other proteases secreted from the filamentous fungi Colletotrichum salicis (KXH28323.1), C. fioriniae PJ7 (XP 007596472.1), C. gloeosporioides Nara gc5 (XP 007281997.1), and C. Simmondsii (KXH42882.1), as shown in Figure 4.The N-terminal sequence of the first 20 amino acid residues of the protease from p < 0.05 . Proteolytic activity was lower at more extreme pH values. Enzymatic assays with incubation at different temperatures showed an increase from 30 to 60°C, followed by a drop in activity at temperatures over 60°C. The enzyme maintained and 80% or more activity from 50 to 70°C. The optimum temperature was 60°C, with statistical difference compared with all temperatures tested, p < 0.05 .For biochemical characterization of the enzyme, we first analyzed the optimum pH. Interestingly, the protease showed about 75% or more residual activity from pH 5.5–8.0, with 97% or more between pH 5.5 and 6.5, the optima pH was at 6.0, with The optimum temperature was 60°C, with statistical difference compared with all pH values except at 6.5, Figure 7). The protease showed great thermal stability during a 60-min incubation; from 30 to 55°C, the residual activity was about 80% or more, and at 60°C, the residual activity of the enzyme was 50% . Thus, these results suggested that the enzyme was thermostable despite being produced by a mesophilic fungus.Analysis of protease stability at different pH values showed that the enzyme had greater stability from pH 4.5–10.0 with maintenance of about 67% or more of it stability after 60 min of incubation at 25°C (p < 0.0001 (Table 2), respectively, while aluminum decreased the original proteolytic activity by 16%. Additionally, EDTA inhibited the proteolytic activity by about 69% with statistical difference, p < 0.0001, whereas PMSF, IAA, and pepstatin inhibited proteolytic activity by 30, 27, and 20%, respectively, (Table 3). These results suggested that the enzyme maybe a metalloprotease.Metal ions can either positively and negatively affect protease activity. In this study, we found that 10 mM sodium, barium, or cobalt increased proteolytic activity by 52, 24, and 24%, only sodium showed statistical difference compared with control, Figure 9 shows the residual activity of the protease isolated from E. javanicum in the presence of surfactants. The enzyme was stable at different concentrations of Triton X-100 and Tween 20; at the maximum concentration tested (1%), the protease maintained about 68 and 58% of its activity, respectively. Increases in CTAB and SDS concentrations decreased protease activity; specifically, the protease retained about 28% activity in the presence of 0.1% CTAB and about 30% activity in the presence of 0.02% SDS.Figure 10, urea, guanidine, and DTT also affected proteolytic activity. Increased concentrations of DTT resulted in decreased enzymatic activity, maintaining about 40% of enzyme activity when the concentration of DTT was 100 mM. In the presence of 150 mM urea, the enzyme maintained about 54% of its original activity. Interestingly, guanidine had a greater effect, reducing the activity of the enzyme by only 10% when used at a concentration of 150 mM.As shown in Table 4 shows the catalytic specificity of the protease for S1, S2, and S3 subsites in relation to replacement of amino acids at P1, P2, and P3 positions using a FRET peptide series based on the sequence Abz-KLRSSKQ-EDDnp (considering the cleavage between Arg and Ser). The position P1 provided higher catalytic efficiency with arginine, phenylalanine, and methionine . Moreover, the protease exhibited higher affinity when glutamine was in this amino acid position, with a KM of 0.004 mM. Most of the substrates were cleaved between P′1 and P′2; in the presence of arginine and glutamine, the substrate was cleaved as follows: P1↓P′1↓P′2. The best values for the S2 subsite were with substrates containing valine and arginine at the P2 substrate position; the catalytic efficiencies were 2,141 and 1,850 mM-1⋅seg-1. The enzyme showed high affinity for phenylalanine, with a KM of 0.012 mM. The substrates were cleaved between P′1 and P′2, except for phenylalanine and tyrosine, which were cleaved as follows: P1↓P′1↓P′2. The kinetic parameters of the protease at S3 showed that the presence of hydrophobic amino acids, such as valine, isoleucine, and alanine, led to the highest catalytic efficiencies . The enzyme showed high affinity for valine, with a KM of 0.010 mM. All substrates were cleaved at two positions, P1↓P′1↓P′2, except in the presence of serine, where the cleavage pattern was P′1↓P′2; that of arginine was not determined.Table 5 shows values of the protease kinetic parameters at S′1, S′2, and S′3 subsites related to substrate substitution at P′1, P′2, and P′3 positions. Analysis of the S′1 data showed that the substrate containing tyrosine was the most hydrolyzed, with a catalytic efficiency of 87,849 mM-1⋅seg-1, followed by arginine  and lysine . All substrates evaluated could be hydrolyzed by protease, particularly tyrosine, which resulted in the highest enzyme affinity (KM: 0.007 mM). The most frequently cleaved site was P′1↓P′2, except in the presence of valine (P2↓P1), lysine (P′1↓P′2↓P′3), and Arg or Met (P2↓P1P′1↓P′2). The protease kinetic parameters in isoleucine presence at S′2 subsite provided high catalytic efficiency with 13,700 mM-1⋅seg-1. The affinity of the enzyme was higher in the presence of isoleucine and tyrosine, KM 0.003. Additionally, the most frequently cleaved sites were P2↓P1↓P′1 for proline and tyrosine; P1↓P′1↓P′2 for alanine, arginine, glutamine, glutamic acid, and histidine; P2↓P1↓P′1↓P′2 for glycine; and P2↓P1↓P′1↓P′2↓P′3 for histidine. The S′3 subsite had higher catalytic efficiency in the presence of tyrosine and phenylalanine . The cleavage was different for all substrates.E. javanicum. According to the literature, the Penicillium genus has the potential to produce different proteases; for example, Penicillium waksmanii .Previous studies have shown that other metalloproteases exhibit variable molecular weights. For example, entation . Additiolypeatus  and Bacip TKU004  produce E. javanicum, many microorganisms are able to produce neutral proteases. For example, P. digitatum . Peptide substrate synthesis and cleavage site determination . Determination the N-terminal sequence (EA).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.