Here, we propose the marine unicellular diatom Phaeodactylum tricornutum as a model organism for testing the membrane penetrability of CA inhibitors (CAIs). Seven inhibitors belonging to the sulphonamide type and possessing a diverse scaffold have been explored for their in vitro inhibition of the whole diatom CAs and the in vivo inhibitory effect on the growth of P. tricornutum. Interesting, inhibition of growth was observed, in vivo, demonstrating that this diatom is a good model for testing the cell wall penetrability of this class of pharmacological agents. Considering that many pathogens are difficult and dangerous to grow in the laboratory, the growth inhibition of P. tricornutum with different such CAIs may be subsequently used to design inhibition studies of CAs from pathogenic organisms.Carbonic anhydrases (CAs) are ubiquitous metalloenzymes, which started to be investigated in detail in pathogenic, as well as non-pathogenic species since their pivotal role is to accelerate the physiological CO P. tricornutum is a eukaryotic organism characterised by fusiform cells with a cell wall poor in silica,P. tricornutum encodes for nine CAs: five α-CAs confined in the matrices of the four-layered plastid membranes, two β-CAs (PtCA1 and PtCA2) located in the pyrenoid and two mitochondrial γ-CAsθ-CA, has been identifiedi) to RuBisCO and phosphoenolpyruvate carboxylase,The physiologic/biosynthetic processes requiring COAAZ, MZA, which are clinically used agents, and compounds 1–5 have been explored for their in vitro inhibition of the diatom CAs and in vivo inhibitory effect on the growth of the P. tricornutum cell. Our results demonstrate that the growth of the P. tricornutum cells is affected by the CAIs and the unicellular diatom represents a good model for verifying the CAIs membrane penetrability.Here, seven inhibitors belonging to the sulphonamide types, such as the 2.2.1.3–5 used in the work were reported earlier by our groups,AAZ and MZA were commercially available from Sigma-Aldrich . All the chemicals and solvents were purchased from Sigma-Aldrich . All reactions involving air- or moisture-sensitive compounds were performed under a nitrogen atmosphere using dried glassware and syringes techniques to transfer solutions. Nuclear magnetic resonance  spectra were recorded using a Bruker Advance III 400 MHz spectrometer in DMSO-6d. Chemical shifts are reported in parts per million (ppm) and the coupling constants (J) are expressed in Hertz (Hz). Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet; bs, broad singlet; dd, double of doubles. The assignment of exchangeable protons was confirmed by the addition of D2O. Analytical thin-layer chromatography (TLC) was carried out on Sigma Aldrich silica gel F-254 plates. Flash chromatography purifications were performed on Sigma Aldrich Silica gel 60 (230–400 mesh ASTM) as the stationary phase and ethyl acetate/n-hexane or MeOH/DCM were used as eluents. Melting points (mp) were measured in open capillary tubes with a Gallenkamp MPD350.BM3.5 apparatus and are uncorrected. The solvents used in MS measures were acetone, acetonitrile (Chromasolv grade), purchased from Sigma-Aldrich , and mQ water 18 MΩ, obtained from Millipore's Simplicity system . The mass spectra were obtained using a Varian 1200 L triple quadrupole system  equipped by Electrospray Source (ESI) operating in both positive and negative ions. Stock solutions of analytes were prepared in acetone at 1.0 mg mL−1 and stored at 4 °C. Working solutions of each analyte were freshly prepared by diluting stock solutions in a mixture of mQ water:acetonitrile 1:1 (v/v) up to a concentration of 1.0 µg mL−1 The mass spectra of each analyte were acquired by introducing, via syringe pump at 10 µL min−1, of the its working solution. Raw-data were collected and processed by Varian Workstation Vers. 6.8 software.Compounds 2.1.1.B (1.2 eq) was added to a solution of 5-imino-4-methyl-4,5-dihydro-1,3,4-thiadiazole-2-sulphonamide A  and pyridine (3.0 eq) in dry acetonitrile (5 ml) at 0 °C under a nitrogen atmosphere. The solution was stirred at r.t. until the starting material was consumed (TLC monitoring), then quenched with HCl 1 M (15 ml). The formed precipitate was filtered-off and purified by silica gel column chromatography eluting with 40% EtOAc in n-hexane to afford the title compound 1 as a white solid. 52% yield; m.p. 295–6 °C; silica gel TLC fR0.47 (EtOAc/n-hexane 60% v/v); δH : 1.32 , 4.08 , 7.56 , 8.20 , 8.46 ; δC : 31.6, 35.8, 39.3, 126.3, 130.1, 133.3, 156.7, 159.0, 166.3, 174.3; m/z (ESI negative) 353.0 [M-H]−.4-t-Butylbenzyl chloride 2.1.2.C  and N-boc-ethylenediamine D (1.2 eq) in dry DMF (3 ml) under a nitrogen atmosphere, followed by DMAP (0.03 eq) and EDCI (1.2 eq). The solution was stirred at r.t. until the starting material was consumed (TLC monitoring), then quenched with slush (15 ml) and extracted with EtOAc (2 × 20 ml). The organic layers were washed with HCl 0.5 M (2 × 15 ml) and brine (2 × 15 ml), dried over Na2SO4, filtered-off and concentrated under vacuo. The obtained residue was purified by silica gel column chromatography eluting with 10% MeOH in DCM to afford the title compound 2 as a white solid. 73% yield; m.p. 198–199 °C; silica gel TLC fR0.35 (MeOH/DCM 10% v/v); δH : 1.41 , 3.17 , 3.34 , 6.95 , 7.50 , 7.93 , 8.02 , 8.66 ; δC : 29.2, 40.4, 40.4, 78.7, 126.5, 128.8, 138.4, 147.2, 156.7, 166.3; m/z (ESI negative) 341.9 [M-H]−.HOAt (1.2 eq) was added to a solution of 4-sulphamoylbenzoic acid 2.2.P. tricornutum (Pt1) Bohlin was obtained from the Provasoli-Guillard National Centre for Culture of Marine Phytoplankton. Cultures were grown in f/2-Si medium−1 s−1), 12 h:12 h dark–light cycle as described by De Riso and co-workersThe CCMP632 strain of 2.3.7; 0.75 × 107; 1 × 107; 1.5 × 107 and 2 × 107 cells) were spotted on f/2-Si agar plates (volume of the spot 5µl). Cells were inoculated with CAIs  diluted with 10% DMSO at two different concentrations: 0.4 and 1.0 mM. Plates inoculated in an identical manner but only with 10% DMSO and plates inoculated only with f2/-Si were used as control. Cell survival was monitored after 5 days of growth at 18 °C under white fluorescent lights (70 μmol m–1 s–1), 12 h:12 h dark–light cycle.Different dilutions of wild-type Pt1 cells . 1 ml of pAMBS resin was applied to an empty column (BioRad) and equilibrated with 0.1 M Tris-HCl, pH 7.5 buffer containing 0.2 M K2SO4, 0.5 mM EDTA. The sample containing about 1 mg of total protein was loaded on the p-AMBS column equilibrated as aforementioned. Unbound proteins were removed by washing extensively with the same buffer. The bound carbonic anhydrase was eluted using 0.4 M KSCN dissolved in 0.1 M Tris–HCl, pH 7.5 buffer. The CA-containing fractions were pooled, dialysed and concentrated by ultrafiltration. The CA-containing sample was subject to SDS-PAGE.All the purification steps were carried out at a temperature of 4 °C. Approximately, 10 g of pelleted diatom culture were homogenised in 20 ml of 20 mM Tris-HCl buffer pH 8.3 containing 102.5.Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) was carried out according to Laemmli2.6.2 as substrate following the pH variation due to the catalysed conversion of CO2 to bicarbonate. Bromothymol blue was used as pH indicator. The production of hydrogen ions during the CO2 hydration reaction lowers the pH of the solution leading to a colour transition of the dye. The time required for the colour change is inversely proportional to the amount of CA present in the sample. The Wilbur-Anderson units (WAU) were calculated according to the following definition: one WAU of CA activity is defined as the ratio (T0 − T)/T, where T0  and T  are recorded as the time (in seconds) required for the pH to drop from 8.3 to the transition point of the dye (pH 6.8) in a control buffer and the presence of enzyme, respectively.CA activity assay was a modification of the procedure described by Capasso et al.2.7.2 hydration activity of the algal homogenates. Phenol red was used as pH indicator (0.2 mM) in 20 mM Hepes as buffer (pH 7.5) working at the absorbance maximum of 557 nm or, alternatively, bromothymol blue  working at an absorbance maximum of 602 nm. 20 mM Na2SO4 was added to maintain constant the ionic strength in both media. The initial rates of the CA-catalysed CO2 hydration reaction were followed for a period of 10–100 s. For each inhibitor at least six traces of the initial 5–10% of the reaction are used for determining the initial velocity. The uncatalysed rates are determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionised water and dilutions up to 0.01 nM were done thereafter with the assay buffer. The inhibitor and enzyme solutions were pre-incubated together at room temperature prior to assay, in order to allow for the formation of the E-I complex. The rates of the CA-catalysed reaction were followed by measuring the absorbance at the λmax of the proper pH indicator, with the inhibition constants obtained by non-linear least-squares methods using PRISM 3 and the Cheng–Prusoff equation as the mean from at least three different determinations.An Applied Photophysics stopped-flow instrument was used for assaying the total CAs catalysed CO2.8.http://www.swissadme.ch/) was used to calculate the logP of the sulphonamide compoundsPrediction of logP is a method for obtaining information on the partition coefficient of a compound. The platform SwissADME in vivo activity, a balanced hydrosolubility and liposolubility of the sulphonamide inhibitors are necessary, even if some sulphonamides characterised by low lipid solubility, such as acetazolamide (AAZ), are used as effective drugs for a long period. In the present paper, seven sulphonamides inhibitors were investigated for their lipophilicity and effects on the growth of P. tricornutum: acetazolamide (AAZ), methazolamide (MZA), and compounds 1–5 , which all incorporate predominantly hydrophilic moieties. The remaining sulphonamides also possess various highly lipophilic moieties  which lead to positive LogP values and thus an enhanced liposolubility over AAZ and MZA  were extracted and purified from 10 g of pelleted diatom culture. Using CO2 as a substrate, the activity of PtrCAs in the extract was determined. Most of the CA activity was recovered in the soluble fraction of cellular extract after centrifugation and the calculated specific activity was 200 Wilbur-Anderson units (WAU) mg−1 of protein. The extract containing the diatom hydratase activity was further purified by p-aminomethylbenzenesulphonamide (pAMBS) affinity chromatography because most of the diatom CA-classes show affinity for this resin as described in the literatureThe genome of 3.3.AAZ) and methazolamide (MZA), which are clinically used agents, and compounds 1–5, were used for determining the IC50 values relative to the inhibition of the PtrCAs purified from the marine diatom P. tricornutum as described above . These inhibitors were less efficient for the human isoform hCA I, and two of them resulted in inefficient inhibitors . It is interesting to note that among the three CAIs with a low lipophilicity  and the four inhibitors with a good lipophilicity   .3.4.in vivo cell wall penetrability of the sulphonamide inhibitors AAZ, MZA, 1–5 cannot be easily quantified, and thus we tested their effects on the cellular growth of P. tricornutum. Different numbers of diatom cells were spotted on the agar plates containing two different concentrations of each inhibitor (0.4 and 1 mM)   and 5. Tt PtrCAs , impaireof cells . At highted here . Howevert 1.0 mM  and 5. Tt 1.0 mM  and 5. Bt 0.4 mM .4.Phaeodactylum tricornutum as a model organism for testing the membrane penetrability of CAIs and their effects on the growth of the organism. P. tricornutum is an eukaryotic organism characterised by fusiform cells with a cell wall poor in silica,AAZ, MZA, and compounds 1–5, resulted to be effective inhibitors of the diatom CAs when tested in vitro (IC50–s in the range of 8.6–96 nM). Some of these inhibitors probably can cross the membrane of P. tricornutum possessing both hydrophobic and hydrophilic moieties in their molecule. The most efficient in vivo inhibitors were compounds 4, 3, 1 and 5 with a noticeable inhibitory growth effect at a low number of cells. Furthermore, these sulphonamides inhibitors showed a different behaviour when tested at the concentrations of 0.4 and 1.0 mM, which is difficult to rationalise. For example, MZA was not efficient at 0.4 mM, but it interfered efficiently with the diatom growth when used at 1.0 mM. Probably this effect might be due to the off-targeting of other proteins than the CAs considered here. In general, these results demonstrate that P. tricornutum might be considered as an excellent and rather simple model for testing the membrane penetrability of new CAIs and their effects on the growth of the organism. Considering that many pathogens are difficult and dangerous to grow in the laboratory, the preliminary results obtained for the growth inhibition of P. tricornutum with different such CAIs may be subsequently used to design inhibition studies of CAs from pathogenic organisms, which may pave the way to novel anti-infectives with a diverse mechanism of action.In this study, we proposed the marine unicellular diatom


Babesia bovis, Babesia bigemina and Theileria equi are worldwide tick-borne hemoprotozoan that cause diseases characterized by fever, anemia, weight loss and abortion. A common feature of these diseases are transition from acute to chronic phases, in which parasites may persist in the host for life, and becoming a reservoir for tick transmission. The live-attenuated vaccines for B. bovis and B. bigemina are not available for worldwide use due to legal restrictions and other concerns such as potential erythrocyte antigen and pathogen contamination, and a vaccine for T. equi is not available. The use of chemotherapeutics is essential to treat and control these diseases, but several studies have shown the development of drug-resistance by these parasites, and safe and effective alternative drugs are needed. Tulathromycin, a macrolide antibiotic, has proven to be effective against a vast range of bacteria and Plasmodium yoelli, a Babesia and Theileria related intra-erythrocytic apicomplexan. Draxxin® (tulathromycin) is currently licensed to treat infections that cause respiratory diseases in cattle in several countries. In this study, the activity of Draxxin® was tested in vitro on cultured B. bovis, B. bigemina and T. equi. Addition of the drug to in vitro cultures resulted in cessation of parasite replication of the three species tested, B. bovis, B. bigemina and T. equi, with estimated IC50 of 16.7 ± 0.6 nM; 6.2 ± 0.2 nM and 2.4 ± 0.1 nM, respectively, at 72 h. Furthermore, neither parasites nor parasite DNA were detectable in cultures treated with IC100, suggesting Draxxin® is a highly effective anti-Babesia/Theileria drug. Importantly, the IC50 calculated for Draxxin® for the Babesia/Theileria parasites tested is lower that the IC50 calculated for some drugs currently in use to control these parasites. Collectively, the data strongly support in vivo testing of Draxxin® for the treatment of bovine babesiosis and equine piroplasmosis. ® (tulathromycin) in in vitro growth of B. bovis, B. bigemina and T. equi parasites at 72 h after drug addition to the culture.The effect of different concentrations of Draxxin •Chemotherapeutics are critical to treat bovine babesiosis and equine theileriosis.•® in vitro B. bovis, B. bigemina, T. equi cultures.Study of the activity of Draxxin•® to the cultures resulted in cessation of parasite replication.Addition of Draxxin•50 (nM) of 16.7; 6.2 and 2.4 to B. bovis, B. bigemina and T. equi, respectively.IC•® is a highly effective anti-Babesia/Theileria drug.Results suggests that Draxxin The related tick-borne apicomplexan Theileria equi has significant economic consequences, especially in equine sport activities and the racing industry through restricting movement of infected horses  and diminazene aceturate . However, imidocarb dipropionate may have negative secondary effects in treated animals, and may result in salivation, vomiting, diarrhea and injection site inflammation . Furthermore, long term use of these treatments invariably results in the emergence of drug-resistant parasites . Importantly, a specific formulation  of this drug has been already approved for the treatment of respiratory disease, keratoconjunctivitis and foot rot in cattle in several countries including the US. Therefore, this drug is currently approved by the FDA and widely commercialized, greatly facilitating the process for new potential applications. A previous study demonstrated that tulathromycin has antimalarial activity in in vivo mice infected with Plasmodium yoelli  inhibits the development of Babesia and Theileria parasites in in vitro cultures. The study presented here evaluates the use of a commercial available chemotherapeutic, Draxxin® (tulathromycin) as a drug that inhibits the in vitro growth of B. bovis, B. bigemina and T. equi.Therefore, taking these considerations together, the diseases caused by these apicomplexan parasites may prove to become intractable, and new anti-parasitic drugs are urgently needed. Tulathromycin is a semi-synthetic macrolide antibiotic of the subclass triamilide  or with 10% hematocrit equine erythrocytes (T. equi). Cultures were maintained at 37 °C in an atmosphere of 5% CO2, 5% O2 and 90% N2 and media was replaced daily.2.2B. bovis, B. bigemina and T. equi parasites were performed to evaluate the effect of the Draxxin® (tulathromycin). This study was performed in 96-well plates using 180 μl cultures per well containing the different Draxxin® concentration tested  diluted in culture medium, with an initial percentage of parasitized erythrocytes (PPE) of 0.2% in all subcultures. The first screening was to identify if Draxxin® has an inhibitory effect on Babesia spp. and T. equi strains and calculate the 50% inhibitory concentrations (IC50). Cultures were grown in the presence and absence of Draxxin®, and non-infected erythrocytes were also kept in culture media as baseline data for flow cytometry analysis. The culture medium was replaced daily with 150 μl medium per well containing the respective Draxxin® concentration. Cultures were also monitored daily up to 72hr by flow cytometry to measure the PPE and by Giemsa-stained slides for any morphological changes.In vitro inhibition assays for B. bovis and B. bigemina and 9% for T. equi. This assay was performed using of Draxxin® at IC100: B. bovis (37.2 nM), B. bigemina (12.4 nM) and T. equi (18.6 nM). Cultures grown in the absence of Draxxin® were used as control for no parasite growth inhibition, and culture of non-infected erythrocytes maintained in medium was used as negative control. The culture medium was replaced daily with 150 μl medium per well containing the respective Draxxin® concentration for a period of 72 h. After 72 h, parasites were cultivated in media (in the absence of Draxxin®), with splits every 48 h for a period of 8 days. The PPE was evaluated at 72 h and 8 days by flow cytometry.A new set of in vitro growth inhibition assays was performed targeting a starting PPE of 1% for ® (imidocarb dipropionate) was used as a positive control for the in vitro inhibition assays for B. bovis, B. bigemina and T. equi parasites, using an identical protocol as described above. The drug concentrations used were: 1 μM for B. bovis and for T. equi, and a set of different concentrations  for the calculation of the IC50 of Imizol® for B. bigemina. The initial PPE used was 0.2% in all subcultures. Cultures were monitored daily at 72 h by flow cytometry to measure the PPE.The effect of the Imizol50 values were calculated using GraphPad Prism 7 software by fitting the PPE values (wells treated with Draxxin® at different concentration compared to the positive control well) in a nonlinear regression with a confidence interval of 95% (95% CI). Total inhibitory concentrations (IC100) was calculated as the doses of drug required to inhibit the parasite growth to an identical level as found for non-infected erythrocytes (approximately 0.1%).All experiments were carried out in triplicate for each concentration tested. The IC2.3g for 5 min at 4 °C. The supernatant was discarded, the cell pellet was washed twice with 150 μl of phosphate buffer saline (PBS) pH 7.2 and then, the cell pellet was resuspended in 200 μl of 25 μg/μl Hydroethidine (HE) (Invitrogen) and incubated in 5% CO2 incubator at 37 °C for 20 min in the dark. After incubation, excess of HE was washed from the cells by the addition of 200 μl of PBS followed by centrifugation at 450 × g for 5 min at 4 °C. The supernatant was discarded and the cell pellet was resuspended in 200 μl of fresh PBS. Then, resuspended cells were transferred to samples tube containing approximately 3 ml of PBS containing 0.2% sodium azide for analysis by flow cytometry using a FACSCaliber (Becton Dickinson) at a flow rate of approximately 2500 events/s with 50,000 events collected. Data was analyzed by FCS Express 3 software (De Novo Software). Stained non-infected erythrocytes were used as a negative control and gated to determine the percentage between infected and non-infected erythrocytes.Flow cytometric assay was performed as described by 2.4B. bovis merozoite surface antigen (msa)-1 gene, B. bigemina limulus coagulation factor C domain protein (ccp)-3 and T. equi erythrocyte merozoite antigen (ema)-1 genes in parasites treated with IC50 and IC100 concentrations and non-treated parasites (control well) at 72 h post-addition of Draxxin®. For B. bovis the IC50 and IC100 concentrations are 16.7 nM and 37.2 nM, respectively; for B. bigemina the IC50 and IC100 concentrations are 6.2 nM and 12.4 nM, respectively; and for T. equi the IC50 and IC100 concentrations are 2.4 nM and 18.6 nM, respectively. The set of primers, PCR cycling and reaction were performed as described by Bastos et al. and Silva et al. . P < 0.05 were considered statistically significant. The area under the concentration (AUC) values was calculated using GraphPad Prism 7 software.Statistical significance was determined using a two-sample 33.1® to inhibit the in vitro growth of B. bovis, B. bigemina and T. equi parasites. The in vitro growth of those parasites was inversely related to the concentrations of Draxxin® applied, until no growth of parasites was reached , and B. bovis (IC50 of 16.7 nM). In addition, the calculated IC100 values were 37.2 nM, 12.4 nM, and 18.6 nM for B. bovis, B. bigemina and T. equi parasites, respectively  were also tested.The performance of the drug might be affected by the existing parasite load in a given infected animal. The effects of the addition of the calculated IC® in B. bovis, B. bigemina and T. equi cultures, respectively (P < 0.05) . No parasite growth was evident in the following 72 h, which was the full duration of the study  but the parasite growth was significantly decreased at 24 h and 48 h after treatment (P < 0.05).Parasites were not able to growth in the cultures with the addition of 37.2 nM, 12.4 nM and 18.6 nM of Draxxinectively A–C regar < 0.05) A–C, sugghe study A–C. In cB. bovis, B. bigemina and T. equi cultures at IC100 values and after 72 h with Draxxin® was 720 ng*hr/μl, 1680 ng*hr/μl and 1080 ng*hr/μl, respectively.The AUC estimated values for 3.350 and IC100 doses of Draxxin® for 72 h. Total gDNA was extracted from all cultures at 72 h, and analyzed by qPCR. While quantitative PCR detected parasite DNA in all samples treated with IC50, no gDNA was detected from the samples collected from cultures treated with IC100 doses for B. bovis and T. equi. However although qPCR detected 0.17% of total parasites in B. bigemina gDNA samples derived from parasites treated with IC100, no parasites were detected by FACS. The PPE of B. bovis, B. bigemina and T. equi cultures in the absence of Draxxin®, IC50 and IC100, respectively, are shown in 100 for the three parasites tested.Cultured parasites were treated with either IC4B. bovis, B. bigemina and T. equi parasites by the commercially available antibiotic Draxxin® at relatively low concentrations (nM). Interestingly, B. bovis parasites require 3 times more Draxxin® to fully inhibit the parasite growth, with an IC50 which is approximately 3 times higher than the IC50 of B. bigemina, and approximately 7 times higher than the IC50 of T. equi. The reasons for increased IC50 in B. bovis remain unknown, but could be related to the mechanism(s) of drug action. It was shown that macrolide antibiotics binds with the 23S prokaryotic rRNA  than the IC50 calculated for clofazimine, a drug that demonstrated inhibitory effect in in vitro, but remains unlicensed for in vivo use  for treating pneumonia  would also be active against B. bovis, B. bigemina and T. equi.To our knowledge, this study showed for the first time the inhibitory effects of Draxxin® showed a full inhibitory effect on the in vitro growth of B. bovis, B. bigemina and T. equi. Draxxin® is commercially available currently, and approved for use in beef and dairy cattle. Future studies will evaluate the in vivo efficacy of the FDA-approved dosage regimen for the treatment of B. bovis, B. bigemina infection in cattle and T. equi in horses. The process for its worldwide approval for the treatment of these parasitic diseases can be highly facilitated, considering that the human, food and animal safety of the FDA-approved dosage regimen is well established.In conclusion, DraxxinMGS, NV and CES designed the study. MGS performed the in vitro studies and statistical analysis. MGS, NV, DK and CES wrote the manuscript. All authors read and approved the final version of the manuscript.The authors declare no competing interests.


Paratylencholaimus sanshaensis gen. nov. sp. nov. from Hainan is proposed as a new member of the family Tylencholaimellidae Jairajpuri, 1964. Paratylencholaimus gen. nov. is close to Phellonema Thorne, 1964 and Goferus Jairajpuri & Ahmad, 1992 but can be differentiated mainly by having basal part of odontophore rod-like and without knobs, and basal part of pharynx expanded gradually. Tylencholaimus zhongshanensis sp. nov. from Guangdong and Dorylaimoides shapotouensis sp. nov. from the Ningxia Hui Autonomous Region are also described herein. Phylogenetic analyses based on the 18S rDNA and the D2–D3 region of the 28S rDNA support that the three new species are valid. The classifications of the families Tylencholaimellidae and Mydonomidae Thorne, 1964 are revised mainly based on the analysis of the morphology of odontostyle and odontophore. After these revisions, Paratylencholaiminae subfam. nov. including Paratylencholaimus gen. nov. and Goferus is proposed. Athernema and Agmodorus of Tylencholaimellidae are transferred into Mydonomidae, and the subfamily Athernematinae of Tylencholaimellidae is dismissed. The main characteristics of the family Mydonomidae and Tylencholaimellidae are revised. Keys to the genera of Mydomonidae and Tylencholaimellidae are included.Three new species of the order Dorylaimida  Pearse, 1942 were identified and described.  In the classification proposed by Tylencholaimus de Man, 1876 (Tylencholaimidae), and the second one from the Ningxia Hui Autonomous Region is a new member of the genus Dorylaimoides Thorne & Swanger, 1936 (Mydonomidae). These species are herein described as Tylencholaimus zhongshanensis sp. nov. and Dorylaimoides shapotouensis sp. nov. The third one from Hainan is interesting. It equips with dorylaimoid cuticle that is different from Tylencholaimidae (equips with tylencholaimoid cuticle), but other characteristics are highly similar to those of Tylencholaimidae. And later, three more populations of this species from Guangdong were collected. With further examinations, this species was suggested to be a member of a new genus of the family Tylencholaimellidae, herein described as Paratylencholaimus sanshaensis gen. nov. sp. nov. Detailed descriptions based on microscopy and phylogenetic analysis based on the 18S rDNA and the D2–D3 region of the 28S rDNA of the three new species were presented. In addition, the classification of Tylencholaimellidae and Mydonomidae was discussed, one new subfamily of Tylencholaimellidae was proposed, and keys to the genera of the revised Tylencholaimellidae and Mydonomidae were provided.During nematode investigations in China, three new species of Dorylaimina were identified. One from Guangdong belongs to the genus n > 30. Nematodes were prepared for SEM observations as described by Soil samples were collected from the rhizosphere soil of some plants from Hainan, Guangdong and Ningxia, respectively. Nematode populations were extracted from samples using the modified Baermann funnel method . Then, sA single nematode was placed into 10 µL mixed solution (distilled water: 2 ×buffer for KOD FX = 1:1) and cut using a sterilized needle. The genomic DNA was extracted by adding 1 µL 20 µg/mL proteinase K and then reacting at 65 °C for 1 h and 95 °C for 15 min. PCR reaction systems were performed in a 10 µL reaction mixture containing 5 µL of 2 ×buffer for KOD FX, 0.3 µL of each primer (10 µM), 2 µL of dNTPs (200 µM), 1 µL of DNA, 1.2 µL of distilled water and 0.2 µL of KOD FX (1 U/µL). Two overlapping fragments of the 18S rDNA were amplified using primer set 988F (5′–CTCAAAGATTAAGCCATGC–3′) and 1912R (5′–TTTACGGTCAGAACTAGGG–3′) for the first fragment, and 1813F (5′–CTGCGTGAGAGGTGAAAT–3′) and 2646R (5′–GCTACCTTGTTACGACTTTT–3′) for the second fragment . For theThe sequences of the three new species were respectively compared with sequences in GenBank using BLAST. Sequences of species of Tylencholaimidae, Leptonchidae, Mydonomidae, Tylencholaimellidae, Mononchida Jairajpuri, 1969 and Nygolaimina Ahmad & Jairajpuri, 1979 were aligned along with the sequences of the three new species. The sequence alignments were performed, and conservative regions were selected using MEGA v6. For the Bayesian inference (BI) analysis, the substitution saturation was tested by DAMBE. The best-fit models were selected by AIC (Akaike Information Criterion) in MrModeltest v2.3. Bayesian trees were constructed by using MrBayes v3.1.2 running the chain for 1,000,000 generations with a sample frequency of 1,000 generations, and setting the ‘burnin’ at 2500. The topologies were used to generate a 50% majority rule consensus tree. Posterior probabilities (PP) were given for appropriate clades.http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:427B5E52-23B0-4474-B179-4FA9FC5E7C9C. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix Pars proximalis vaginae with conoid walls, 5–8 µm long and 6–7.5 µm wide, pars refringens lacking, pars distalis vaginae 3–4 µm long. Prerectum 3.2–4.8 times and rectum 0.6–0.9 times anal body diameter long. Tail hemispheroid to elongate-hemispheroid, 1.1–1.4 times the anal body diameter long.Female. Measurements are listed in Male. Unknown. All soil samples were processed, but no males were found.Phalaenopsis sp. from Zhongshan, Guangdong, China.Rhizosphere soil of Female holotype and six female paratype specimens (slide numbers: 0422627.A and 0422627.B) are deposited in the Lab of Plant Nematology/Research Center of Nematodes of Plant Quarantine, South China Agricultural University, Guangzhou, Guangdong, China, and three paratype specimens (slide numbers: 0422627.C) are deposited in the Key Laboratory of Vegetation Restoration and Management of Degraded Ecosystems, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, Guangdong, China.The new species is named after its type locality, Zhongshan City.Tylencholaimus zhongshanensis sp. nov. is characterized by having a body length of 473–605 µm; lip region offset and approximately one-third of the body diameter at posterior end of the neck region; amphid aperture 30% as wide as the lip region; odontostyle 5–6 µm, 0.7–0.9 times the lip region width long; odontophore rod-like with small basal knobs, 5–7 µm long, 0.8–1.3 times as long as the odontostyle; basal expansion of pharynx occupying 33–38% of the total pharyngeal length; female genital system prodelphic; postvulval sac completely absent; vulva transverse; prerectum 3.2–4.8 times anal body diameter long; tail 16–21 µm, hemispheroid to elongate-hemispheroid, 1.1–1.4 times the anal body diameter long.Tylencholaimus were compared with Tylencholaimus zhongshanensis sp. nov. mainly based on T. proximus Thorne, 1939 (c = 24 or more), pharynx expanded behind middle, inner part of lips not offset sharply, but it can be differentiated from T. proximus (pars dilatata), posterior genital branch completely absent (vs. absent or reduced to a rudimentary sac less than one-third of the corresponding body width long) and vagina slightly directed forward (vs. transverse). The new species is close to T. ibericus Peña-Santiago & Coomans, 1994  and three sequences for the D2–D3 region of 28S rDNA  were deposited in GenBank. The BLAST search for the 18S rDNA showed the highest similarity (96%) to the sequences of T. helanensis (KU992903 and KU992904). For the D2–D3 region of 28S rDNA, both sequences showed the highest similarity (86%) to the sequences of T. helanensis (KU992905 and KU992906). In Bayesian trees for both the 18S rDNA and D2–D3 region of 28S rDNA , and female prodelphic (vs. didelphic-amphidelphic)   althoughpars dilatata and a slender part, anterior one 32–63 µm long and posterior one 46–53 µm long. Sphincter present at the junction of oviduct and uterus. Uterus simple and with a wide lumen, anterior 53–65 µm long and posterior 45–75 µm long. A lot of sperm in the uterus and a few cells in the pars dilatate of oviduct serving as spermatheca. Vulva transverse. Vagina extending 45–56.5% inwards the corresponding body width. Pars proximalis vaginae with thick walls, 12–14 µm long and 11–13 µm wide, pars refringens lacking. Prerectum 3.3–5.9 times and rectum 0.9–1.5 times the body diameter at anus long. Tail elongate conoid with rounded terminus, posterior region bent dorsally, 2.8–3.9 times the anal body diameter long.Female. Measurements are listed in Male. Unknown. All soil samples were processed, but no males were found.Rhizosphere soil of apple trees from Shapotou Region, Zhongwei City, the Ningxia Hui Autonomous Region, China; GPS coordinate 104°59.723′E, 37°28.153′N.Female holotype and four female paratype specimens (slide numbers: M72.A and M72.B) are deposited in the Lab of Plant Nematology/Research Center of Nematodes of Plant Quarantine, South China Agricultural University, Guangzhou, Guangdong 510642, China, and two female paratype specimens (slide numbers: M72. C) are deposited in the Key Laboratory of Vegetation Restoration and Management of Degraded Ecosystems, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, Guangdong 510642, China.The new species is named after Shapotou Region, a successful soil restoration area in China.Dorylaimoides shapotouensis sp. nov. is characterized by having a body length of 1.02–1.13 mm; lip region rounded and offset by a constriction, lips practically amalgamated, labial papillae protruding; odontostyle 9–13 µm, asymmetrical with a distinct lumen, odontophore arcuated and narrowing posteriorly, 8–9.5 µm; basal expanded part of pharynx occupying 21–28% of the total pharyngeal length; genital system didelphic-amphidelphic; prerectum 3.3–5.9 times and rectum 0.9–1.5 times the body diameter at anus long; tail elongate conoid with rounded terminus and the posterior region bent dorsally, 53–73 µm, 2.8–3.9 times the anal body diameter long.D. leptusD. siddiqiiD. micoletzkyi  Thorne & Swanger, 1936 and D. punctatus Khan & Park, 1999 in having a didelphic genital system and a conoid elongate tail dorsally bent at the end and longer than 45 µm based on the key provided by D. leptus (D. siddiqii (D. micoletzkyi (D. punctatus (L = 1.02–1.13 mm vs. 1.3–1.4 mm), lower ‘a’ value (a = 32.6–36.9 vs. 39.5–45.7), odontostyle longer (9–13 µm vs. 7.3–8.0 µm), tail shorter (53–73 µm vs. 77–83 µm) and cuticle with fine transverse striations (vs. cuticle marked with zig-zag lines throughout the body).The new species is close to . leptus  by odontsiddiqii  with lonoletzkyi , the newoletzkyi ; vs. 17.oletzkyi , pharynxDorylaimoides shapotouensis sp. nov.  was obtained and deposited in GenBank (accession numbers: KU662325 for the 18S rDNA and KU662324 for the D2–D3 region of 28S rDNA). The BLAST search for the 18S rDNA showed the highest similarity (99%) to the sequence of D. micoletzkyi (AY284830) and showed 8 nucleotide differences. The D2–D3 region of 28S rDNA showed the highest similarity (95%) to the sequences of D. micoletzkyi (AY593004) with 40 nucleotide and 4 gaps differences. In the 18S rDNA phylogenetic reconstructions (Dorylaimoides with 84% support. In the D2–D3 region of 28S rDNA phylogenetic reconstructions (D. micoletzkyi and D. limnophilus (an opisthodelphic species).Each sequence of 18S rDNA and D2–D3 region of 28S rDNA of ructions , the newructions , the newDorylaimoides shapotouensis sp. nov. overlap those of eight documentary populations of D. micoletzkyi . In the 28S rDNA Bayesian tree, Dorylaimoides shapotouensis sp. nov. showed a closer relationship with another didelphic species, D. micoletzkyi rather than with the opisthodelphic species D. limnophilus. However, the inner relationships of Dorylaimoides remain unclear in the 18S rDNA Bayesian tree. Thus, to clarify the evolutionary relationships among the three groups with different genital system types, more molecular data of Dorylaimoides are needed.Most measurements of oletzkyi , but DorTylencholaimellidae. Paratylencholaiminae subfam. nov. Cuticle dorylaimoid with fine transverse striations. Lip region cap-shape, offset from the body. Amphidial fovea not sclerotized. Odontostyle straight, tubular with small aperture, without dorsal accessory pieces. Odontophore rod-like and basal part slightly expanded. Guiding ring simple. Pharynx slender in anterior part, the basal part expanded occupying one-third of the total pharyngeal length. Female genital system didelphic. Vulval lips not sclerotized. Tail short, rounded to conoid-round. Males unknown.Paratylencholaimus gen. nov. is close to Goferus , as its inner characteristics are similar to those of the genus Tylencholaimus, but it has a different type of cuticle which is dorylaimoid.The new genus is named as Pars proximalis vaginae 5–8 µm long and 5–9 µm wide, pars refringens lacking, pars distalis vaginae 2–4 µm long. Prerectum 1.2–4.4 times and rectum 0.8–1.7 times anal body diameter long. Anal region ventrally flattened to distinctly bulge. Tail rounded to conoid-round, 0.8–1.4 times anal body diameter long.Female. Measurements are listed in Male. Unknown. All soil samples were processed, but no males were found.The new species is named after the Sansha City, which is its type locality.Female holotype, twelve female paratype specimens  and 44 female from Huadu and Zhongshan  are deposited in the Lab of Plant Nematology/Research Center of Nematodes of Plant Quarantine, South China Agricultural University, Guangzhou, Guangdong 510642, China, and five female from Boluo (slide numbers: BoLuo.A and Boluo.B) are deposited in the Key Laboratory of Vegetation Restoration and Management of Degraded Ecosystems, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, Guangdong 510642, China.Euphorbia sp. from Yongxing Island, Sansha City, Hainan, China.Rhizosphere soil of Scindapsus sp. from Huadu District, Guangzhou, Guangdong, China; culture medium of Phalaenopsis sp. from Zhongshan, Guangdong, China; rhizosphere soil of Citrus sp. from Boluo County, Huizhou, Guangdong, China.Culture medium of Paratylencholaimus sanshaensis gen. nov. sp. nov. is characterized by having body 581–772 mm long; lip region cap-shaped and offset from the body; amphid apertures quite small; odontostyle straight, 7–11 µm long and 0.8–1.2 times the lip region width long; odontophore rod-like, 6–11 µm, 0.8–1.4 times as long as the odontostyle; hemizonid occurs at the level of nerve ring; pharyngeal basal bulb occupying 34–45% of the total pharyngeal length; oviduct and uterus slender without differentiations, junction of oviduct and uterus indistinct; vulva transverse; pars refringens lacking; prerectum 1.2–4.4 times and rectum 0.8–1.7 times anal body diameter long; tail rounded to conoid-round, 15–21 µm long, 0.8–1.4 times the anal body diameter long.Paratylencholaimus sanshaensis gen. nov. sp. nov. The anus of this population showed morphological diversity: nematodes with normal color intestines have a flat anal region, while the nematodes with a black mass in intestines have bulge anus. The culture medium of Phalaenopsis sp., which this population is collected from, consists of three levels: sphagnum on the top, coco coir in the middle and barks at the bottom. It can be suggested that the nematodes with a black mass in intestines may feed on the barks. This finding indicates that the food source can influence the morphology of nematodes, and the feeding type of Paratylencholaimus sanshaensis gen. nov. sp. nov. is omnivorous.One noticed phenomenon was observed in the Zhongshan population of Paratylencholaimus sanshaensis gen. nov. sp. nov. were obtained. The interindividual variabilities were observed both in the 18S rDNA and D2–D3 region of 28S rDNA. Seventeen sequences for 18S rDNA  and fourteen sequences for 28S rDNA (827 bp) were deposited in GenBank . 1 bp interindividual variability was observed only in 18S rDNA. The BLAST search for the 18S rDNA sequences showed the highest similarity (98%) to the sequence of an unidentified species (EF024986). The D2–D3 region of 28S rDNA showed the highest similarity (85%) to the sequences of Dorylaimus sp. (KP954677). In both the 18S rDNA and D2–D3 region of 28S rDNA Bayesian trees  dorylaimoid: inner layer not loose and without radial elements; (b) tylencholaimoid: inner layer loose with irregular radial elements. Among all the families, only Leptonchidae and Tylencholaimidae had tylencholaimoid cuticle, whereas the others had dorylaimoid cuticle. The morphology of the basal expansion of pharynx of the new genus is similar to Paratylencholaimus gen. nov. should be placed under the subfamily Tylencholaimellinae due to its amphideal fovea that is not sclerotized. However, Paratylencolaimus gen. nov. and its close relative genus Goferus has odontophore without distinct basal knobs and basal expansion occupying greater than one-fifth of the total pharyngeal length. In contrast, the remainder genera of Tylencholaimellinae  except Agmodorus Thorne, 1964 (see further) have distinct basal knobs and basal expansion about or less than one-fifth of the total pharyngeal length. To adjust this, we propose to place the new genus and Goferus under a new subfamily, namely, Paratylencholaiminae subfam. nov.According to the latest classifications of Tylencholaimellidea , ParatylDorylaimida, Dorylaimina, Tylencholaimellidae. Cuticle dorylaimoid without radial refractive elements. Lip region continuous or offset from the body. Amphideal fovea not sclerotized. Odontostyle straight without pieces, odontophore without basal knobs. Expanded part of pharynx pyriform and unconstructed. Female genital system didelphic. Tail elongate to conoid-rounded. Two genera.Paratylencholaimus gen. nov.GoferusJairajpuri & Ahmad, 1992According to Athernema and Agmodorus has not the typical tube shaped. We also found that these two genera with the arched odontophore, conoid to filiform tail and the opisthodelphic female genital system are more closely related to the family Mydonomidae Thorne, 1964 which is mainly characterized by having odontostyle asymmetry, odontophore straight or arched, basal expansion cylindrical and no longer than one-third of the pharynx length, female didelphic or opisthodelphic. Thus, we propose to transfer Athernema and Agmodorus into the family Mydonomidae Thorne, 1964 according to the morphology mentioned above, and under the subfamily Mydonominae Thorne, 1964 according to the body length less than three mm, and cancel the subfamily Athernematinae.The odontostyle of The main characteristics of the families Mydonomidae, Tylencholaimellidae and the subfamily Tylencholaimellinae should be revised as follow:Mydonomidae: cuticle dorylaimoid; lip region continuous or slightly offset, occasionally cap like; lips rounded, usually amalgamated; odontostyle short, asymmetry or not typical tubular, with distinct lumen; odontophore straight or arched; basal expansion cylindrical and no longer than one-third of the pharynx length, occasionally offset; female genital system didelphic-amphidelphic or mono-opisthodelphic; spicula dorylaimoid; ventromedial supplements spaced, 1–20; tail variable, short and rounded to elongate or filiform, similar or dissimilar in sexes.Tylencholaimellidae: cuticle dorylaimoid; lip region cap-like, more or less offset, lips amalgamated; odontostyle short, tubular, occasionally with accessory stiffening piece; odontophore with or without basal knobs or flanges; basal expansion short pyriform, usually offset, occupying one-fifth to one-third of the pharynx length; female amphidelphic or opisthodelphic, exceptionally prodelphic; vulva transverse; spicula dorylaimoid; ventromedial supplements none to two, spaced; tail short and rounded to filiform, similar in sexes.Tylencholaimellinae: amphideal fovea not sclerotized; odontostyle straight without accessory stiffening piece; odontophore with distinct basal knobs or flanges, basal expansion occupying about one-fifth of the total pharyngeal length, female didelphic or monodelphic and tail long and filiform to short and hemispheroid.The new classifications of Mydonomidae and Tylencholaimellidae are:Paratylencholaimus gen. nov. In the new classifications, Athernema and Agmodorus of Tylencholaimellidae are transferred into Mydonomidae, and the subfamily Athernematinae of Tylencholaimellidae was canceled. Keys to the genera of these two families are also provided. However, deeper inner relationships of the genera of Tylencholaimellidae and Mydonomidae remain unclear and more information including morphology and phylogeny of these two families are needed.Both the morphology and phylogenetic analysis results support that the three new species and the new genus are valid. The classifications and the main characteristics of the families Tylencholaimellidae and Mydonomidae are revised due to the propositions of Paratylencholaiminae subfam. nov. and 10.7717/peerj.7541/supp-1File S1Click here for additional data file.10.7717/peerj.7541/supp-2File S2Click here for additional data file.


Candida auris is an emerging, often multi-resistant, yeast that causes invasive infections in healthcare settings. Patients may be colonized for months and C. auris has been shown to remain viable on surfaces for at least 14 days. It is widely considered that the environment may be a reservoir for transmission of C. auris. The efficacy of pulsed-xenon ultraviolet (PX-UV) mobile devices on C. auris has not been tested previously. In a laboratory setting, we tested efficacy of a PX-UV system on C. auris and C. parapsilosis, another candida known to be responsible for outbreaks in healthcare settings and survive for at least 28 days in the environment.C. parapsilosis and C. auris was carried out in a broth liquid culture medium at 37 °C until concentration ranges 10 5–10 6 colony-forming units (CFUs) per millilitre were obtained. Glass slides were inoculated with 10 μl of C. auris stock culture and allowed to dry. Slides were positioned perpendicular to the floor at a distance of 1.25 m from the floor. Exposure time were run uninterrupted for 5-, 10- and 15-min cycles at 1- and 2-m distance.Cultures and growth of clinical strains of C. auris CFU after a 5-min cycle at 1-m distance, and 99.6% reduction after a 10-min cycle at 2-m distance. There was a 98.5% reduction in C. parapsilosis CFU after a 5-min cycle at 1-m distance, and 95.2% reduction after a 10-min cycle at 2-m distance.There was a 99.4% reduction in The PX-UV mobile device is easy to use and has short cycle times that makes it easier to disinfect all areas outside the room where the patient received care. Further studies are needed in hospital environment, to assess the cumulative impact of repeated sessions. Candida auris is an emerging, often multi-resistant, yeast that causes invasive infections in healthcare settings [C. auris has the ability to cause large healthcare outbreaks [C. auris has been shown to remain viable on surfaces for at least 14 days [C. auris. The Centre for Disease Control and prevention (CDC) recommends post discharge terminal cleaning and disinfection of patients’ rooms and cleaning and disinfection of areas outside the rooms where patients received care [C. auris and other candida species to UV-C and that extended exposure time (20 to 30 min) might be needed [C. auris. As a comparison, we also tested the PX-UV system on C. parapsilosis, another candida known to be responsible for outbreaks in healthcare settings and survive for at least 28 days in the environment [settings , 2. C. autbreaks , 4. Patiutbreaks , 5 and C 14 days , 7. It ived care . It has ved care . Howevere needed . In theiC. parapsilosis and C. auris was carried out in a broth liquid culture medium at 37 °C until concentration ranges 105–106 colony-forming units (CFUs) per millilitre were obtained. Glass slides were inoculated with 10 μl of C. auris stock culture and allowed to dry.Prior cultures and growth of clinical strains of C. parapsilosis.Slides were positioned perpendicular to the floor at a distance of 1.25 m from the floor. Exposure time were run uninterrupted for 5-, 10- and 15-min cycles. The experiment was conducted at 1-m distance from the centre of the robot and repeated at a 2-m distance. Tests were conducted in triplicate. Three controls treated in identical conditions were prepared with each organisms. The controls remained covered during the experiment. After exposure, the control and PX-UV exposed slides were incubated in 10 ml phosphate-buffered saline (PBS) and agitated for 30 s to extract CFUs. Serial dilutions were plated onto Chromagar Candida and incubated at 36*for 48 h. The total viable count was quantified into number of colony forming unit. The percent reduction in the pathogens were calculated in comparison with unexposed control slides. The experiment was repeated for C. auris are detailed in Table C. auris CFU observed after a 5-min cycle, while no growth was observed at 10 and 15 min. At 2-m distance from the light bulb, the C. auris CFU reduction was 90.2% after a five minutes cycle, and 99.6% after a 10-min cycle. There was no growth at 15 min.C. parapsilosis CFU was 98.5% after a 5-min cycle. There was no growth at 10 and 15 min. At 2-m distance, the average efficacy was only 15.7% after a 5-min cycle while it was 95.2% after a 10-min cycle. There was no growth after a 15-min cycle.The laboratory testing results for C. auris after a 5-min cycle and no growth after 10- and 15-min cycles. Similarly, for C. parapsilosis there was a 98.5% reduction after a 5-min cycle and no growth after 10- and 15- min cycles. We found that at 2-m distance, there was a 15.7% reduction in C. auris after a 5-min cycle, a 95.2% reduction after a 10-min cycle, and no growth after a 15-min cycle. For C. parapsilosis, there was a 90.2% reduction after a 5-min cycle, a 99.6% reduction after a 10-min cycle, and no growth after a 15-min cycle.We found that at 1-m distance from the PX-UV light bulb, there was a 99.4% reduction in C. auris. The strengths of this study include the inclusion of two common Candida species with varying distances from the PX-UV bulb. Previous studies have not tested the efficacy of PX-UV for Candida spp. Further studies are needed in hospital environment, to assess the cumulative impact of repeated sessions.The limitations of this study include a relative small study numbers and only one strain of Candida auris and Candida parapsilosis are two pathogens commonly involved in outbreaks in healthcare settings outbreaks. They are often recovered from the hospital environment, where they can survive for a long time. Additional disinfection with hydrogen peroxide vapour is effective on C. auris and C. parapsilosis. However, this method is labour intensive as it requires sealing of the bed space, or doors and vents in case of a single-bed room, and monitoring of the concentration of H2O2 inside the enclosure before permitting patients or staff to enter into the room. The cycle time can last 2 to 8 h according to the H2O2 vapour system used. By comparison, the PX-UV technology, is easy to use and has significantly shorter cycle times that makes it easier to disinfect all areas outside the room where the patient received care, as recommended by the CDC.We found a 99.6% reduction after a 10-min cycle at 2-m distance, and a 95.2% reduction after a 10-min cycle at 2-m distance. The PX-UV mobile device is easy to use and has short cycle times compared to other no-touch room disinfection technologies. Further studies are needed in hospital environment, to assess the cumulative impact of repeated sessions.


Diabetes during pregnancy is an increasingly common metabolic disorder, associated with significantly increased risks for both mother and child. Aim of this study was to compare maternal and perinatal outcomes in women with pregestational (PDM) type 1 (T1DM), type 2 diabetes (T2DM), gestational diabetes mellitus (GDM) and compare these to pregnancies not complicated with diabetes. This study also evaluated a specifically organized care-model mostly involving specialist diabetes nurses.Retrospective population-based records review 2009–2012. Rates of maternal ) and fetal outcomes , macrosomia, congenital malformations/intrauterine death) were assessed and potential predisposing or contributing factors as maternal age, ethnicity, obesity, weight gain, parity, HbA1c levels, insulin types and doses.p = 0.0001), pregnancy weight gain lower , more delivered preterm (p = 0.0001), by CS (p = 0.05), and had more LGA neonates (p = 0.06) than the group without diabetes. Among pregnancies with diabetes, GDM mothers gained less weight (9.9 kg vs 13.5 kg) (p = 0.006), and rates of CS (p = 0.03), preterm deliveries (p = 0.001) and LGA (p = 0.0001) were not increased compared to PDM; More T1DM infants were LGA, 60% vs. 27% in T2DM. In pregnancies with diabetes obesity, excessive weight gain and multiparity were associated with increased risk of LGA neonates, and mother’s type of diabetes and gestational week were associated with higher rates of CS.Among 280 pregnancies 48 were PDM, 97 GDM and 135 without diabetes. Within the group with diabetes, early-pregnancy BMI was higher (Weight gain during pregnancy was lower in pregnancies with diabetes and prevalence of LGA, CS and preterm deliveries in GDM was not elevated, also for T2DM, except increased prevalence of LGA in T1DM that warrants increased clinical attention, indicating that this model of antenatal diabetes care may have contributed to improved maternal and fetal outcomes. Diabetes is a common medical complication of pregnancy, affecting at least 10% of all pregnancies globally . GDM usuPregnancies complicated by diabetes are associated with significantly increased risks for both mother and child , 6. ConcAll this makes it crucial to examine the results of current clinical care and assess if the care provided is sufficient or needs to be changed. We aimed to examine the outcome of maternity diabetes care in the Kronoberg Health Care Region, in southeastern Sweden  . In KronThe primary aim was to evaluate the proportion of perinatal outcomes, as rates of LGA, CS and malformations/intrauterine death, among pregnancies complicated by T1DM or T2DM, and compare the outcome with the GDM group, and with pregnancies not complicated by diabetes.Secondarily, we aimed for the groups described above to also assess maternal outcomes as the proportion that delivered prematurely, were affected by preeclampsia or neonatal hypoglycemia. All outcomes were examined for potential predisposing or contributing factors as maternal age, parity, marital status, obesity, weight gain, glycated hemoglobin (HbA1c) level during the three trimesters of pregnancy and where applicable, daily doses of long- and short-acting insulin at gestational week 26–28 and at delivery.This is a retrospective population based observational study of all pregnancies complicated by diabetes, PDM, type 1 or type 2, or GDM, who were admitted to the obstetrical ward at Växjö Central Hospital, Region Kronoberg, for antenatal care and delivery from January 1st, 2009 to December 31st, 2012. Data was collected by review of electronic medical records from the Departments of Internal Medicine and Obstetrics and Gynecology  at Växjö Central Hospital and Ljungby Hospital, the only two hospitals in Kronoberg. Data for the GDM population was partly collected during a previously conducted pilot study by midwifery students at Linnaeus University [The study protocol was approved by the Regional Ethical Review Board at Linköping University, to which Kronoberg is referred for Ethical Reviewing.Pregnant women with PDM were managed according to routine procedures. As soon as pregnancy was confirmed, they were referred to a specialist team with combined experience, where specialist diabetes nurses, midwives, obstetricians and endocrinologists collaborate. The care was delivered within Maternity Health Care/Department of Obstetrics and the Specialist Outpatient Diabetes Clinic of the Department of Internal Medicine at Växjö Central Hospital. The patients from Western Kronoberg start at Ljungby local Hospital and are referred for delivery at the secondary hospital in Växjö. The care model which is publicly evaluated for the first time here was developed over many years in Kronoberg and differs from usual care in the rest of Sweden. The main difference is that pregnant women with diabetes did not routinely have contact with diabetes specialist physicians. Contact was almost exclusively (95%) performed through the diabetes department by specialist diabetes nurses. The obstetrics department did not participate in the diabetes care.All women with T2DM, who used oral hypoglycemic agents (OHA) before pregnancy were treated with diet alone or shifted to insulin immediately upon contact with the hospital specialist diabetes clinic or later when needed. The use of Metformin during pregnancy was not yet established in the region during the study period. All pregnant women taking insulin were asked to perform blood glucose tests before and after meals and at bedtime daily (7 times) and to adjust the insulin doses accordingly.The patients were in contact with the diabetes nurses every 2–3 weeks, more frequently during the last trimester of the pregnancy period, and if needed, every week. In Sweden, all women attend prenatal care. Non-attendance is very rare. Glucose goals for the first half of pregnancy were fasting values between 4.5–6.0 mmol/l, post-prandial levels between 6.5–7.5 mmol/l and HbA1c < 52 mmol/mol. For the second half of pregnancy target fasting value was between 4.0–5.5 mmol/l, post-prandial levels < 6.5 mmol/l and HbA1c 36–42 mmol/mol. HbA1c values were measured at 3–4 week intervals  with the2, and 30 kg/m2; and gestational weight gain (GWG) (kg), dichotomized </≥8 kg. This and the debate and recommendations of recent years of limited weight gain in pregnancies affected by overweight and especially obesity, and three groups of women with diabetes in our study had mean BMI above the range of obesity (< 30 kg/m2), are the reasons for using > 8 kg. The recommendation for gestational weight gain for women with these ranges of BMI has been suggested to 5–9 kg, or less if more obese, which has even been described to reduce perinatal complications [Maternal characteristics assessed included age at conception , type and duration of diabetes , ethnic/social background , employment was defined as full-time, part-time or other when it included other activities such as being student, on maternity leave or unemployed. Marital status was defined as married/living together vs living alone, previous number of pregnancies. Exercise equivalent to brisk walking 30 min ≥3 times/week were reference to 1-3 times/week or 0 = inactive and miscarriages, previous episodes of GDM, hypertension, smoking (yes/no), type of insulin used , insulin quantity required, , mean HbA1c (mmol/mol) during the three trimesters of pregnancy, BMI at first visit (early pregnancy BMI), also dichotomized </≥ 25 kg/mications , 15.The maternal/fetal outcomes investigated were frequency of spontaneous abortions, preeclampsia, perinatal/intrauterine mortality, congenital malformations , pre-term delivery (< 37 weeks), vaginal/cesarean delivery (CS), macrosomia (birth weight > 4500 g), large-for-gestational-age (LGA), birth weight (g), Apgar score at 5 min after birth (< 7/≥7), neonatal hypoglycemia, and prevalence of shoulder dystocia.Preeclampsia was defined as development of hypertension and proteinuria ≥0.3 g/24 h after 20 weeks’ gestation. LGA was defined as birth weight greater than the 90th percentile for gestational age. Neonatal hypoglycemia was defined as blood glucose level < 2.6 mmol/l during the first 24 h of life. Perinatal mortality was defined as death up to 30 days after childbirth. Multiparity was defined as parity of ≥2 deliveries vs first. LADA (Latent Autoimmune Diabetes in Adults) is by WHO defined as a variant of autoimmune type 1 diabetes, which is islet autoantibody positive, but not insulin requiring at the time of diagnosis .P-value ≤0.05 was considered significant. All tests were two-sided. Binary logistic regression (forward enter) was performed with the variables CS, LGA, GWG and congenital malformations/intrauterine mortality (CMD) as dependent variables, yielding Crude Odds Ratios (COR) with 95% CI. All variables were transformed to binary and tried in single regression analysis. Variables that had p-values < 0.25 for the specific outcome were included in multiple regression models , with dependent variables LGA, CS, GWG and CMD for adjusted odds ratios (AOR). Independent variables in each multiple regression can be derived from the tables, or the descriptions in the results section. The statistical analyses were carried out with SPSS .Descriptive data if normally distributed were described by mean and standard deviation, if not normally distributed by median and range (min and max). The t-test and chi 2-test, including Fisher’s exact test, were used for comparison between groups. The non-parametric Mann-Whitney U-test, was used for not normally distributed data. ANOVA was used for comparison of levels of HbA1c during three trimesters. There were 145 pregnancies complicated by diabetes and 135 pregnancies without diabetes, 280 in total. Of the pregnancies complicated by diabetes, 48 (33%) had PDM and 97 had (67%) GDM. Within the PDM group 37 women (77.1%) had T1DM and 11 (22.9%) had T2DM.Baseline data are displayed in Table p = 0.006) in comparison to the GDM group. Within the PDM group, T2DM mothers had higher BMI at admission, 34.0 vs 26.7 kg/m2, (p = 0.005) compared to T1DM mothers. PDM mothers were more physically active (65%) compared to GDM (37%). More T2DM mothers were unemployed compared to those with T1DM (p = 0.04), or GDM, or without diabetes. Median duration of diabetes was higher among the women with T1DM compared to T2DM, 15 ± 8.1 vs 6.0 ± 6.3 years. The incidence of previous GDM was higher in the T2DM  [p = 0.002). As expected, a significantly higher number of patients in the PDM group used insulin during pregnancy while for the GDM treatment with diet-only was sufficient for almost 50% to reach the HbA1c goal. There were no maternal deaths, neither among mothers with or without diabetes.Mean maternal age for women with PDM was 30.7 ± 6 and for the GDM group 32.0 ± 5 years (ns). The T2DM mothers tended to be older, 33.5 ± 6, than those with T1DM, 29.8 ± 5 years. The PDM group gained more weight during pregnancy (as LADA) . More wo2), a larger proportion were smokers (17% vs. 4.4%) and a greater proportion were of Asian origin . The women without diabetes gained more weight during pregnancy (p = 0.005).Mothers with any diabetes type, compared to the group without diabetes, had higher early-pregnancy BMI . There was a higher proportion of caesarian deliveries in the PDM compared to the GDM group (42% vs. 25%) (p = 0.03). Of all the mothers 25% (65/262) delivered by CS, 19% (24/127) of those without diabetes. Mother’s type of diabetes  and gestational week (p = 0.01) were associated with CS in women with any type of diabetes, (Table 2 0.08) 3.4 (1.4–7.9) p = 0.005; while multiparity, early BMI > 30 kg/m2, ethnicity, birth week, smoking or LGA did not (p = 0.18–0.97). Among those without diabetes, for delivery by CS, AOR (R2 0.29) remained significant for early pregnancy BMI > 30 kg/m2 6.2 (1.8–21.5) p = 0.004 and multiparity 0.28 (0.10–0.78) p = 0.014, but not for ethnicity, birth week, smoking or LGA (p = 0.29–1.0) Very few pregnancies,  with T2DM and  of control pregnancies, were complicated by congenital malformations. There was one cardiac malformation (Ventricle Septum Defect) in the T1DM group  and two, one Morbus Down and one retentio testis in the T2DM group . Additionally, in the T2DM group, one intrauterine death was observed in week 37 + 3 days. This was the only case of intrauterine death, 1/48, 2% of PDM vs none in the GDM group, and none among the 135 control pregnancies. Early pregnancy BMI > 30 kg/m2 was a significant risk factor for congenital malformations and/or intrauterine mortality (p = 0.04) and gestational weight gain > 8 kg tended to reach significance as a risk factor (p = 0.07). Multiple regression analysis for congenital malformations/intrauterine death among the PDM mothers revealed that only early pregnancy BMI > 30 kg/m2 remained associated with the outcome, AOR 0.85 (0.73–0.99), p = 0.03; while GWG, ethnicity, duration of diabetes and mean dose of long acting insulin at gestational week 26–28 lost their significances (p = 0.4–1.0).The proportion of live born infants born preterm due to either spontaneous onset of labor or termination of pregnancy due to obstetrical reasons was higher in the PDM group,  and they had a lower APGAR score 5 min after birth  compared to the GDM. Among children of women with PDM 53% (25/48) were LGA compared to only 13% (13/97) in the GDM group (p = 0.0001) and by CS  compared to those not complicated by diabetes. There was a tendency towards more LGA neonates among the pregnancies with diabetes .Among pregnant women with any diabetes type more delivered preterm  and among those without diabetes 17.0% (23/135).p = 0.15). Only two children, in the PDM group, 4.3% (2/48), were macrosomic at birth. There was a significant association between type of diabetes and risk for LGA newborns, (p = 0.0001). Women with T1DM had a 9.5 times higher risk for having LGA children compared to women with GDM (Table p = 0.04) as were gestational weight gain ≥8 kg (p = 0.01). Women with diabetes treated with insulin were also found to have significantly higher risk for LGA children (p = 0.002). In multiple regression analysis, among the mothers with any type of diabetes during pregnancy, the following factors remained significant for LGA after adjustment (R2 0.49) (AOR): diabetes type T1DM 31.3 (7.8–125), P < 0.001; multiparity 6.2 (1.8–21), p = 0.003: early pregnancy BMI > 30 kg/m2 7.2 (2.0–26), p = 0.003; GWG 3.8 (1.0–14.1), p = 0.047 and marital status  18.4 (1.7–206), p = 0.02; while mother’s age, birth week, smoking and physical activity lost their significance (p = 0.10–0.97). In multiple regression analysis the adjusted odds ratio (AOR) for marital status  tended to be 5.0 (0.92–26), p = 0.061, for LGA in the controls, all other factors in the equation  vs. 27% (3/11) among T2DM, the difference did not reach significance . Early pregnancy BMI > 25 kg/m2 and > 30 kg/m2 were protective of GWG ≥ 8 kg among mothers with diabetes, OR 0.27–0.43, especially GDM while diabetes type was associated with increased OR of GWG < 8 kg, in T1DM 3.0 (1.4–6.6) p = 0.006; and T2DM 4.4 (1.2–16.3) p = 0.03, Table 2 0.25) for both early pregnancy BMI > 30 kg/m2 0.34 (0.14–0.82) p = 0.015; ethnicity (Asian) 0.11 (0.20–0.73) p = 0.02; and LGA 3.8 (1.1–12.8) p = 0.03 remained significant, and diabetes type T1DM tended to be 0.30 (0.08–1.1) p = 0.06.For all mothers, with or without diabetes, GWG was associated with the outcome of LGA, OR 3.2 1.1–9.5)  p = 0.05p = 0.05) and showed a tendency to be lower during the third trimester (p = 0.09), in the T2DM pregnancies compared to the T1DM pregnancies, while there was no significant difference during the first trimester . Our findings are in agreement with a review that included papers of both T1DM and T2DM pregnancies 1987–2008 from many countries globally, that also found that women with T2DM were older (33.9 vs. 28.8 years) and heavier (30.2 vs. 24.2 kg/m2) compared to women with T1DM [In this population based study we examined maternal and fetal outcomes and predisposing factors in all pregnancies complicated by diabetes during 2009–2012 within the whole Health Care Region Kronoberg in southern Sweden. For comparison, a group of pregnancies not complicated by diabetes matched for age, parity and date of delivery, was investigated. We found high mean early pregnancy BMI, as expected in the GDM and T2DM mothers, but also in the T1DM group. GDM has previously been observed to be linked with high maternal BMI . A Frencications , 18. TheThe incidence of previous GDM, as expected, was significantly higher among T2DM. Women that have had GDM are more likely to develop T2DM than those with normal glucose tolerance during pregnancy, but studies vary in their estimates of risk . A largeOur study, in line with other studies worldwide, showed an association between the demographic characteristics of pregnant women and development of GDM. Ethnic origin has been demonstrated to have a large influence on the prevalence of gestational diabetes with mothers of Asian or African origin having respectively, four and two times higher risk to develop GDM, compared to mothers of Caucasian origin , 26. In In the present study the mode of delivery was CS in 42% of the women with PDM and 25% of the women with GDM in comparison to only 19% of the women without any diabetes. This was also higher than the average in in Sweden, 16.6% according to the Swedish National Board of Health and Welfare 2012 . As all . In the current study and in accordance with some recent studies [.Obesity and excessive weight gain during pregnancy have been associated with increased risk of LGA children . This is studies , 31, we  studies . The eti studies , so in T studies .2 and two of them had a weight gain ≥8 kg during pregnancy (p = 0.07) while no other variables reached significance. One might also speculate, however, if the low proportion of adverse outcomes, except for LGA, in our study, is a result of the care providing model with extra involvement of specialized diabetes nurses.The T2DM group of women had a milder glycemic disturbance compared to the T1DM group, with lower HbA1c at the first visit and throughout pregnancy. This might be attributed to a better β-cell function due to intrinsic characteristics and shorter duration of diabetes , 36. TheObesity in pregnancies complicated by diabetes, especially type 1 diabetes, where obesity is now more prevalent, was found to be associated with increased risk of LGA neonates. That weight gain during pregnancy was lower among the pregnancies complicated by diabetes, and that the frequency of LGA, or other complications, except for delivery by Cesarean Section, was not elevated in the group with gestational diabetes, indicated that this model of antenatal diabetes care delivered mainly by specialist diabetes nurses may have contributed to the improved outcomes for GDM, and for pregestational type 1 and type 2 diabetes, except for level of HbA1c in the last trimester, which could be lower in women with T1DM, as could the prevalence of LGA children. The increased prevalence of LGA in T1DM despite better maternal BMI compared to T2DM, and mostly good glycemic control warrants increased clinical attention and further investigation.


Camellia sinensis) flowers, which are an abundant and waste resource, contained enzymes that could transform acetophenone to 1PE. In the present study, we extracted crude enzymes from tea flowers and optimized the production conditions of 1PE using response surface methodology. The optimized conditions were an extraction pH of 7.0, a reaction pH of 5.3, a reaction temperature of 55 °C, a reaction time of 100 min, a coenzyme NADPH concentration of 3.75 μmol/mL in the reaction assay, and a substrate acetophenone concentration of 1.25 μmol/mL in the reaction assay. The results provide essential information for future industrial 1PE production using plant-derived enzymes.1-Phenylethanol (1PE) can be used as a fragrance in food flavoring and cosmetic industries and as an intermediate in the pharmaceutical industry. 1PE can be synthesized from acetophenone, and the cost of 1PE is higher than the cost of acetophenone. Therefore, it is important to establish an effective and low-cost approach for producing 1PE. Our previous studies found that tea ( So far, more than 1700 volatiles, which contribute to approximately 1% of all plant-specialized (secondary) metabolites currently known, have been identified in more than 90 plant families ,3. On thCamellia sinensis) is an important crop and cultivated in more than 30 countries including China, Japan, India, and Kenya. The most utilized part of tea plants is the leaf, which is generally used to make the most widely consumed beverage aside from water. However, less attention has been paid to tea flowers. Since asexual propagation was applied to tea plants, tea flowers became a “waste resource”, that competes with tea leaves for water and nutrients. In China, over 4.0 billion kilograms of tea flowers are available annually  ring-acetophenone), 50 μL of coenzyme (NADPH), and 100 μL of a 100 mM potassium phosphate buffer. The different reaction conditions  and centrifuged . The supernatant was dried over anhydrous sodium sulfate. Samples were then analyzed by GC-MS, which was same as the previous study [m/z 70–200). Characteristic ions of [2H5]ring-1PE were m/z 84, m/z 112, and m/z 127.To exclude the possible effects from acetophenone and 1PE that occur in a crude enzyme solution, the substrate in the enzyme assay used labeled [nditions  were invR)-1PE and (S)-1PE. The injector temperature was 180 °C, splitless mode was used with a splitless time of 1 min, and helium was the carrier gas with a velocity of 1.0 mL/min. The GC temperatures were as follows: 60 °C for 2 min, a ramp of 40 °C/min to 105 °C, followed by 2 °C/min to 137 °C, then 80 °C/min to 180 °C, and 180 °C for 10 min.GC-MS equipped with an InertCap CHIRAMIX column  was employed to determine .Our present study characterized the in vitro properties of 1PE synthetic enzymes from tea flowers, suggesting that the enzymes were thermostable, and the enzyme extraction pH and the reaction pH were different at the highest enzyme activity. Furthermore, this study established an effective and low-cost system for producing 1PE, with the following optimized conditions: an extraction pH of 7.0, a reaction pH of 5.3, a reaction temperature of 55 °C, a reaction time of 100 min, a coenzyme NADPH concentration of 3.75 μmol/mL in the reaction assay, and a substrate acetophenone concentration of 1.25 μmol/mL in the reaction assay. These results provide essential information for future industrial 1PE production using plant-derived enzymes. In addition, this will contribute to future applications of functional proteins from abundant and waste tea flower resources.


Entamoeba histolytica, EhiCA. This enzyme has a high catalytic activity for the physiologic CO2 hydration reaction, with a kcat of 6.7 × 105 s−1 and a kcat/Km of 8.9 × 107 M−1 × s−1. An anion inhibition study of EhiCA with inorganic/organic anions and small molecules revealed that fluoride, chloride, cyanide, azide, pyrodiphosphate, perchlorate, tetrafluoroborate and sulfamic acid did not inhibit the enzyme activity, whereas pseudohalides (cyanate and thiocyanate), bicarbonate, nitrate, nitrite, diethyldithiocarbamate, and many complex inorganic anions showed inhibition in the millimolar range (KIs of 0.51–8.4 mM). The best EhiCA inhibitors were fluorosulfonate, sulfamide, phenylboronic acid and phenylarsonic acid (KIs in the range of 28–86 μM). Since β-CAs are not present in vertebrates, the present study may be useful for detecting lead compounds for the design of effective enzyme inhibitors, with potential to develop anti-infectives with alternative mechanisms of action.We report the cloning and catalytic activity of a β-carbonic anhydrase , isolated from the pathogenic protozoan  They are among the fastest catalysts known in nature [The carbonic anhydrases CAs, EC 4.2.1.1) are enzymes that effectively catalyze the reaction between COonate HCO− and pro.2.1.1 arn nature ,7,8,9,10onate HCO− and pro2 for the photosynthetic enzyme Rubisco in the chloroplasts of many plants/algae [Helicobacter pylori contains only one α- and one β-CA, whose inhibition with sulfonamides impairs the growth of the pathogen in vitro and in vivo [Leishmania spp., a β-class CA enzyme (LdcCA) was recently shown to be a potential drug target [L. amazonensis and L. infantum, two species which provoke serious disease in the tropical and subtropical countries [The first β-CA was discovered in 1939, but it took several decades until it was recognized as evolutionarily and structurally distinct from the previously studied CAs, those belonging to the α-class . After tts/algae ,17,18).  in vivo ,15. The g target ,21. IndeEntamoeba histolytica is a pathogenic protozoan human parasite causing amebiasis, which can be expressed as colitis or abscess of intestines or liver [E. histolytica is ingested with contaminated food or water as mature cysts, which excystate in the small intestine. The released trophozoites will then invade the large intestine [E. histolytica is capable of lysing human tissues, killing immune effector cells by contact-dependent cytolysis [E. histolytica, therapies for the invasive forms produce many adverse side effects [E. histolytica, denominated EhiCA.or liver ,23. The or liver ,23. E. hntestine . E. histytolysis ,24. The ytolysis . The invytolysis ,26. Rareytolysis ,23. Alth effects ,23, and  effects . Thus, wE. histolytica, EhiCA, in the E. coli production system  as reported earlier for other CAs, such as hCA VII [We produced the β-CA of  hCA VII . As a re2 hydration reaction) [2 hydration to bicarbonate and protons), with a kcat of 6.7 × 105 s−1 and a kcat/Km of 8.9 × 107 M−1 × s−1, being thus 1.8 times more effective as a catalyst, compared to the slow human isoform hCA I . Furthermore, like most enzymes belonging to the CA superfamily, EhiCA was inhibited by acetazolamide : A standard, clinically used sulfonamide CA inhibitor [Is of 509 nM  , comparif 509 nM .Haemophilus influenzae, Vibrio cholerae, Escherichia coli, Salmonella typhimurium, two isoforms from Mycobacterium tuberculosis [Synechocystis sp. PCC 6803 [In order to rationalize the effective catalytic activity of EhiCA, we aligned the amino acid sequence of this protein with that of other β-CAs, such as those from the pathogenic bacteria rculosis ,27,28,29PCC 6803   The anions which did not show inhibitory activity against EhiCA were fluoride, chloride, and, surprisingly, cyanide and azide, which are highly effective inhibitors of α-CAs such as hCA I and II ; pyrodipIs in the range of 28–86 µM ((ii) The most effective EhiCA inhibitors were sulfamide  ,34 and f28–86 µM . As seenN,N-diethyl-dithiocarbamate were also sub-millimolar EhiCA inhibitors, with KIs in the range of 0.28–0.87 mM. Some of these compounds are typical metal complexing agents , and their propensity to bind the zinc ion in this β-CA explains these inhibitory activities. However, others,  show less affinity to act as metal complexing anions [2 hydratase activity might be crucial during the life cycle of this protozoan. However, this speculation needs careful validation.(iii) Several anions, such as cyanate, selenocyanate, bicarbonate, stannate, tellurate, tetraborate, and g anions . The inhIs in the range of 1.7–8.4 mM).(iv) Many anions acted as low millimolar EhiCA inhibitors. They include iodide, thiocyanate, carbonate, nitrate, nitrite, hydrogensulfide, selenite, perrhenate, perruthenate, peroxydisulfate, trithiocarbonate, and imidosulfonate (KIs in the range of 11.5–365.8 mM ((v) Anions with a less effective inhibitory action against EhiCA were bromide, bisulfite, and sulfate, with K365.8 mM .E. coli. The DNA sequence was retrieved from UniProt, and modified for recombinant protein production. We provided the sequence of the insert, and the actual construction of the plasmid vector was performed by GeneArt . The structure of the insert was specifically modified for production in E. coli. The insert was ligated into a modified plasmid vector, pBVboost.We produced the EhiCA as a recombinant protein in 600). The cells were collected by centrifugation, and the wet weight of the cell pellet was 303 g. The fermentation was performed by the Tampere facility of Protein Services (PS). The cell pellet (approximately 35 g) was suspended in 150 mL of binding buffer containing 50 mM Na2HPO4, 0.5 M NaCl, 50 mM imidazole, and 10% glycerol (pH 8.0), and the suspension was homogenized with an EmulsiFlex-C3  homogenizer. The lysate was centrifuged at 13,000× g for 15 min at 4 °C, and the clear supernatant was mixed with HisPur™ Ni-NTA Resin  and bound to the resin for 2 h at room temperature on the magnetic stirrer. Then resin was washed with the binding buffer and collected onto an empty column with an EMD Millipore™ vacuum filtering flask  and filter paper. The protein was eluted from the resin with 50 mM Na2HPO4, 0.5 M NaCl, 350 mM imidazole, and 10% glycerol (pH 7.0). The protein was re-purified with TALON® Superflow™ cobalt resin . The eluted protein fractions were diluted with binding buffer , so that the imidazole concentration was under 10 mM. The protein binding and elution was performed as described above. The purity of the protein was determined with gel electrophoresis (SDS-PAGE), and visualized with PageBlue Protein staining solution . Protein fractions were pooled and concentrated with 10 kDa Vivaspin® Turbo 15 centrifugal concentrators  at 4000× g at 4 °C. Buffer exchange in 50 mM TRIS (pH 7.5) was done using the same centrifugal concentrators. His-tag was cleaved from the purified protein by Thrombin CleanCleave Kit , according to manufacturer’s manual.The freeze-dried plasmid was prepared, according to manufacturer’s manual. Deep-frozen BL21 Star™ (DE3) cells  were slowly melted on ice. Once melted, 25 µL of the cell suspension and 1 µL of the plasmid solution were combined. The suspension was kept on ice for 30 min. Heat shock was performed by submerging the suspension-containing tube into 42 °C water for 30 s, and was then incubated on ice for 2 min. To the tube 125 µL of S.O.C Medium  was added, and the tube was incubated for 1 h with constant shaking (200 rpm) at 37 °C. Growth plates  were prewarmed at 37 °C for 40 min. Then, 20 µL and 50 µL of suspension was spread on two plates which were incubated overnight at 37 °C. A volume of 5 mL preculture was prepared by inoculating single colonies from growth plates onto an LB medium with gentamycin (ratio 1:1000). It was then incubated overnight at 37 °C with constant shaking (200 rpm). The production was executed according to pO-stat fed batch protocol, which is essentially as described in Määttä et al. . There w2 hydration reaction [2 hydration reaction, 10 mM Hepes  or TRIS  as buffers, and 0.1 M NaClO4 (for maintaining constant ionic strength), were used for a period of 10 s at 25 °C. The CO2 concentrations ranged from 1.7 to 17 mM, for the determination of the kinetic parameters and inhibition constants. For each inhibitor, at least six traces of the initial 5–10% of the reaction were used for determining the initial velocity. The uncatalyzed rates were determined in the same manner, and subtracted from the total observed rates. Stock solutions of inhibitors (10 mM) were prepared in distilled and deionized water, and dilutions of up to 1 µM were done thereafter with the assay buffer. Enzyme and inhibitor solutions were pre-incubated together for 15 min (standard assay at room temperature) prior to assay, in order to allow for the formation of the enzyme–inhibitor complex. The inhibition constants were obtained by non-linear least-squares methods, using PRISM 3 and the Cheng–Prusoff equation [An Sx.18Mv-R Applied Photophysics  stopped-flow instrument has been used to assay the catalytic activity of various CA isozymes for the COreaction . Phenol equation ,37,38.Entamoeba histolytica, EhiCA, the etiological agent of diarrhea and amebic liver abscesses. This new enzyme has a high catalytic activity for the physiologic CO2 hydration reaction, with a kcat of 6.7 × 105 s−1 and a kcat/Km of 8.9 × 107 M−1 × s−1. An anion inhibition study of EhiCA with inorganic/organic anions and small molecules was performed, in order to detect interesting leads for effective inhibitors. Fluoride, chloride, cyanide, azide, pyrodiphosphate, perchlorate, tetrafluoroborate, and sulfamic acid did not inhibit the enzyme activity, whereas pseudohalides (cyanate and thiocyanate), bicarbonate, nitrate, nitrite, diethyldithiocarbamate, and many complex inorganic anions showed inhibition in the millimolar range (KIs of 0.51–8.4 mM). The best EhiCA inhibitors were fluorosulfonate, sulfamide, phenylboronic acid, and phenylarsonic acid (KIs in the range of 28–86 μM). Since β-CAs are not present in vertebrates, the present study may be useful for detecting lead compounds for the design of effective enzyme inhibitors, with potential to develop anti-infectives with alternative mechanisms of action.In the search for alternative drug targets against anti-protozoan agents, we report the cloning and catalytic activity of a β-CA from