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Human lice have always been a major public health concern due to their vector capacity for louse-borne infectious diseases, like trench fever, louse-borne relapsing fever, and epidemic fever, which are caused by Bartonella quintana , Borrelia recurrentis , and Rickettsia prowazekii , respectively. Those diseases are currently re-emerging in the regions of poor hygiene, social poverty, or wars with life-threatening consequences. These louse-borne diseases have also caused outbreaks among populations in jails and refugee camps. In addition, antibodies and DNAs to those pathogens have been steadily detected in homeless populations. Importantly, more bacterial pathogens have been detected in human lice, and some have been transmitted by human lice in laboratories. Here, we provide a comprehensive review and update on louse-borne infectious diseases/bacterial pathogens. [ABSTRACT FROM AUTHOR]

Microreaction technology is an important technology for process intensification and high efficient chemical synthesis. The use of ionic liquids (ILs) as novel reaction media is another rapidly developing orientation for process intensification considering their strong dissolubility, low vapor pressure and adaptable physicochemical properties. The combination of the two technologies has been emerging fast in various applications, due to the ability to intensify the utility of ILs in microreactors. This review presents a summary of the recent progress on the transport and reaction processes with ILs in microreactors. A typical characteristic of ILs is their higher viscosity, so the effects of fluid viscosity on the hydrodynamics and mass transfer are highlighted. In addition, new correlations considering both shear and inertial forces are proposed to predict the flow regime transitions in a wide range of fluid viscosity. With respect to mass transfer, the unit cell model for Taylor flow is modified to represent the effect of viscosity on flow topology inside droplets/slugs. [ABSTRACT FROM AUTHOR]

The synergistic effects of gas agitation and ultrasound on mass transfer between immiscible liquids were investigated in an in-house made ultrasonic microreactor. With the introduction of inert gas (N 2 ), a three-phase slug flow with slug bubbles either dispersed in continuous aqueous phase or encapsulated in oil plugs was observed. Under ultrasound irradiation, slug bubbles underwent surface wave oscillation and induced agitation in microchannel. In addition, microbubbles were generated by acoustic cavitation, oscillating intensely and resulting in the formation of O/W emulsion. Bubble oscillation (i.e., slug bubbles and microbubbles) as well as emulsification promoted liquid-liquid mass transfer significantly. Extraction of vanillin from aqueous solution to toluene was employed to demonstrate the mass transfer enhancement. Compared with silent operation, both mass transfer coefficient and extraction efficiency were largely improved by the combined use of gas agitation and ultrasound. With gas flow velocity being 0.005–0.083 m/s at fixed ultrasound power of 30 W, the overall mass transfer coefficients ranged from 0.047 s −1 to 0.429 s −1 , which was 2.33–17.20 times larger than the corresponding liquid-liquid two-phase process without ultrasound irradiation. [ABSTRACT FROM AUTHOR]

The present work focuses on the hydrodynamics variation and mass transfer characteristics of Taylor flow along long serpentine microchannels with a square cross-section. The volumetric mass transfer coefficient ( k L a ) is regarded as the transient change value to characterize the gas-liquid mass transfer process of CO 2 in water. All experimental data of Taylor bubble are obtained from 1000 continuously captured images. An online high-speed imaging method and the unit cell model are adopted in this study. The effects of gas and liquid flow rates, together with microchannel geometry are investigated on Taylor bubble characteristics in terms of length, velocity and the mass transfer performance. Taylor bubble length shrinks and subsequently plateaus out along the flow direction from the T-junction, resulting in the decrease in Taylor bubble velocity. k L a in a unit cell gradually decreases along the serpentine microchannel, and increases as the channel cross-sectional area decreases. As the gas flow rate increases under a given liquid flow rate, a critical point is found for the evolution of k L a and k L (that is the liquid phase mass transfer coefficient). The results indicate that the contribution of the circulation in the liquid slug to k L is dominant before the critical point compared to the leakage flow in the liquid film. All these findings in this work give important information to understand the dynamic change in gas-liquid Taylor flow mass transfer within microchannels. They will serve as basis for designing and optimizing gas-liquid multiphase microreactors in the future. [ABSTRACT FROM AUTHOR]

This paper presents a study on the hydrodynamics and mass transfer performance of absorption of CO 2 into deionized water and DEA solutions in a microreactor under elevated pressures up to 4.0 MPa. It is demonstrated that increasing gas density (system pressure) leads to increased pressure drop. Considering such effect, a modified Lockhart-Martinelli model is developed, showing good prediction over the two-phase pressure drop. For the mass transfer performance, parametric studies by varying system pressure, temperature of feeding fluids, molar ratio of DEA/CO 2 and CO 2 -loaded DEA solutions are presented. Two empirical correlations based on the power consumption are developed for the prediction of mass transfer coefficients in physical and chemical absorptions, respectively. The results suggest significant intensification is achieved in the microreactor. Besides, dramatic increase of CO 2 loading in the rich absorbents is also obtained, which is very attractive for solvent regeneration. The findings in this paper can serve as guidance in the design of processes with high-pressure operations, such as natural gas purification or biogas recovery. [ABSTRACT FROM AUTHOR]

CO 2 desorption from N -methyldiethanolamine (MDEA) rich solutions was investigated experimentally in a highly efficient microchannel reactor. The effects of various parameters including MDEA concentration, desorption temperature, solution flow rate and CO 2 loading on the CO 2 desorption rate were studied. It was found that the CO 2 desorption rate was most sensitive to the desorption temperature. The CO 2 desorption almost reached equilibrium when the solution residence time was about 7 s, indicating the advantages of using microchannel reactors for rapid CO 2 desorption. The value of k L a was in the range of 0.36–2.68 s −1 in the experiments, which is comparable to that of absorption in microchannels and much larger than in conventional equipment. For the design of micro-desorbers, empirical correlations were proposed to predict the mass transfer coefficient with good prediction performance. [ABSTRACT FROM AUTHOR]

Flow and mass transfer of gas-liquid slug flow under elevated pressures up to 3.0 MPa in a microchannel are investigated with CO 2 -water system. The results show that the ratio of the initial bubble length to the unit cell length is linear with the injection gas volume fraction under each pressure condition, but the slope decreases with an increase in the system pressure. The mass transfer coefficients are calculated with a unit cell model based on the dissolution rate of gas bubbles. Increasing pressure leads to larger mass transfer coefficients, as well as higher amount of gas absorption during the bubble formation. But the fraction of gas absorption during the bubble formation stage is only about 1.5~4.0% of feeding gas. For the bubble dissolution in the main channel, the dissolution rates at different flow rates differ very little for short contact distances from the T-junction, whereas the balance limitation of dissolution at large contact distances only depends on the amount of liquid in a unit cell. [ABSTRACT FROM AUTHOR]

Abstract: Physical absorption of CO2 bubbles under slug flow has been studied in a T-junction microchannel. Based on the absorption rate of gas bubbles, an online photographic method is proposed to determine the mass transfer coefficients. Validity of this method is verified by evaluating bubble expansion due to pressure drop and by comparing the results with literature. The effect of fluid properties on the mass transfer has also been studied with ethanol solutions, which shows that mass transfer coefficient increases with the increase of ethanol concentration. The amount of gas absorbed during the bubble formation process has been measured to be about 2–10% of the inlet gas phase, and is found to linearly scale with the maximum mass transfer rate. For each fluid system, the initial dissolution rates of bubbles differ very little for short contact distance, whereas, the final amount of dissolution only depends on the residence time. [Copyright &y& Elsevier]

Abstract: The main purpose of this mini-review is to provide information on opportunistic human infections caused by Tritrichomonas spp. Rare cases of human trichomoniasis, including fatal meningoencephalitis and peritonitis, have been reported exclusively in immunosuppressed or immunocompromised individuals, suggesting the protozoa are human opportunistic pathogens. These infections are readily diagnosed by the characteristic morphology of the protozoa. Species identification is augmented by testing with molecular methods, such as PCR and DNA sequencing. Risk factors for infection and treatment are also discussed. [Copyright &y& Elsevier]

Abstract: The Leishmania spp. protozoa have an abundant surface metalloprotease called MSP (major surface protease), which in Leishmania chagasi is encoded by three distinct gene classes (MSPS, MSPL, MSPC). Although MSP has been characterized primarily in extracellular promastigotes, it also facilitates survival of intracellular amastigotes. Promastigotes express MSPS, MSPL, and two forms of MSPC RNAs, whereas amastigotes express only MSPL RNA and one MSPC transcript. We confirmed the presence of MSPC protein in both promastigotes and amastigotes by liquid chromatography–tandem mass spectrometry (LC–MS/MS). More than 10 MSP isoforms were visualized in both amastigotes and promastigotes using two-dimensional immunoblots, but amastigote MSPs migrated at a more acidic pI. Promastigote MSPs were N-glycosylated, whereas most amastigote MSPs were not. Immuno-electron microscopy showed that two-thirds of the promastigote MSP is distributed along the cell surface. In contrast, most amastigote MSP localized at the flagellar pocket, the major site of leishmania endocytosis/exocytosis. Biochemical analyses indicated that most amastigote MSP is soluble in the cytosol, vesicles or organelles, whereas most promastigote MSP is membrane-associated and GPI anchored. Activity gels and immunoblots confirmed the presence of a novel proteolytically active amastigote MSP of higher Mr than the promastigote MSPs. Furthermore, promastigote MSP is shed extracellularly whereas MSP is not shed from axenic amastigotes. We conclude that amastigotes and promastigotes both express multiple MSP isoforms, but these MSPs differ biochemically and localize differently in the two parasite stages. We hypothesize that MSP plays different roles in the extracellular versus intracellular forms of Leishmania spp. [Copyright &y& Elsevier]

Abstract: Major surface protease (MSP) facilitates Leishmania promastigote evasion of complement-mediated lysis in the mammalian host and enhances host macrophage phagocytosis of the promastigotes. We previously showed that the steady-state abundance of MSP protein increases 14-fold during in vitro cultivation of L. chagasi promastigotes from logarithmic to stationary phase, despite the fact that the total amount of MSP mRNA does not increase. Furthermore, 10 major MSP isoforms are differentially expressed in different promastigote growth phases, and attenuation of parasites by long-term in vitro cultivation influences MSP isoform expression. Herein, we report that although about two-thirds of newly synthesized MSP becomes surface localized, the rest of the MSP does not reach the promastigote surface. This internal MSP is stable without detectable decrease in abundance up to 6 days after biosynthesis. Furthermore, surface-localized MSP is released at different rates from logarithmic and stationary phase virulent Leishmania promastigotes. These data are consistent with the hypothesis that the major mechanism regulating MSP abundance is the rate of loss of surface-localized MSP from the promastigote surface, and that internally localized MSP is very stable. [Copyright &y& Elsevier]

The major surface protease (MSP or GP63) of the Leishmania spp. protozoa facilitates parasite evasion of complement-mediated killing, phagocytosis by macrophages, and intracellular survival in macrophage phagolysosomes. Immunoblots of several Leishmania species have shown there are distinct MSP isoforms, but the biochemical bases for these differences are unknown. Northern blots show that transcripts of the three tandem gene classes encoding Leishmania chagasi MSP (MSPS, MSPL, MSPC) are differentially expressed during parasite growth in vitro. Cell-associated MSPs increase in abundance during growth, correlating directly with parasite virulence. We examined whether distinct products of these >18 MSP genes are either differentially expressed or differentially processed during parasite growth. Two-dimensional gel electrophoresis and immunoblots delineated more than 10 MSP isoforms in stationary phase L. chagasi, distributed between pIs of 5.2–6.1 and masses of 58–63 kDa. Post-translational modifications including N-glycosylation, GPI anchor addition and phosphorylation did not account for all differences among the isoforms. MALDI-TOF mass spectrometry demonstrated that at least some L. chagasi MSPs were the products of different MSP genes. One isoform was not available for surface biotinylation, suggesting it could be located internally. Parasites in logarithmic growth expressed only four MSP isoforms, and an attenuated strain of L. chagasi (L5) did not express one of the MSP classes (MSPS). These data demonstrate that the products of individual MSP genes are differentially expressed during Leishmania development. We hypothesize they may play different roles during parasite migration through its two hosts. [Copyright &y& Elsevier]

Leishmania sp. are digenetic protozoa that cause an estimated 1.5–2 million new cases of leishmaniasis per year worldwide. Among the molecular factors that contribute to Leishmania sp. virulence and pathogenesis is the major surface protease, alternately called MSP, GP63, leishmanolysin, EC3.4.24.36, and PSP, which is the most abundant surface protein of leishmania promastigotes. Recent studies using gene knockout, antisense RNA and overexpression mutants have demonstrated a role for MSP in resistance of promastigotes to complement-mediated lysis and either a direct or indirect role in receptor-mediated uptake of leishmania. The MSP gene clusters in different Leishmania sp. include multiple distinct MSPs that tend to fall into three classes, which can be distinguished by their sequences and by their differential expression in parasite life stages. Regulated expression of MSP class gene products during the parasite life cycle occurs at several levels involving both mRNA and protein metabolism. In this review we summarize advances in MSP research over the past decade, including organization of the gene families, crystal structure of the protein, regulation of mRNA and protein expression, biosynthesis and possible functions. The MSPs exquisitely demonstrate the multiple levels of post-transcriptional gene regulation that occur in Leishmania sp. and other trypanosomatid protozoa. [Copyright &y& Elsevier]

The protozoan Leishmania chagasi expresses a surface metalloprotease, GP63, whose abundance increases 14-fold as parasites grow from logarithmic to stationary phase. L. chagasi GP63 is encoded by three classes of MSP genes that are differentially expressed during parasite growth. Using metabolic labeling and immunoprecipitation, we found L. chagasi GP63 first appeared as a 66-kDa band that was replaced by a 63-kDa protein. This pattern also occurred in transfected L. donovani harboring detectable products of only one MSP gene, suggesting a precursor–product relationship. The half-life of GP63 increased from 29 h in logarithmic phase to >72 h in stationary phase promastigotes. GP63 loss from the cell was complemented by the appearance of a 63-kDa GP63 in extracellular medium in both membrane-associated and -free forms. Calculations suggested that the long and lengthening T1/2 of cell-associated GP63 accounts in part for its progressive accumulation in the cell during promastigote growth. The current findings add yet another level of complexity to post-transcriptionally regulated expression of an abundant surface molecule in a trypanosomatid protozoan. [Copyright &y& Elsevier]

• Clogging of microchannel was investigated with NaCl salting-out process. • The respective anti-clogging effect of ultrasound and segmented flow was compared. • A novel strategy utilizing ultrasound-driven slug dredge microchannel was proposed. • Three oscillating slug-induced dredging modes was proposed. • An efficient aspirin recrystallization process was realized. A novel anti-clogging strategy by combining ultrasound and segmented flow was proposed and successfully applied to continuous crystallization process. The individual anti-clogging effect of ultrasound and segmented flow was first investigated, finding that both strategies only slow down the fouling process, but not capable of breaking the clogged solid sediment. As for the combining strategy, segmented slug/bubble was driven to oscillate intensely with ultrasound and dredge the fouled microchannel through three different mechanisms. Not only the long-term operation of microreactor system was guaranteed, but also the necessary energy input was reduced by 1–2 orders of magnitude compared to individual ultrasound. Finally, the ultrasound-driven oscillating slug/bubble was utilized for recrystallization of aspirin. Under E v 0.54–2.60 J/mL, a product yield of 53 %-72 % (equilibrium yield 72 %) was obtained with aspirin average size in the range of 7.84–32.01 μm. The synergistic strategy can be well generalized to various crystallization process once properly designed. [ABSTRACT FROM AUTHOR]

• The hydrodynamics, mixing and mass transfer of two-phase processes are reviewed. • Bubble/droplet formation mechanism and scaling models are summarized. • The dynamic behavior during formation stage is highlighted. • The circulation topology and mixing inside both droplets and slugs are discussed. • The interfacial mass transfer regimes and simulative models are presented. Microreaction technology is preferential in process intensification and chemical synthesis, especially in multiphase applications. A thorough understanding of the hydrodynamics and mass transfer is the pre-requisite for implementing such applications. This review discusses the recent progress on the flow and mass transfer of two-phase systems in microchannels, in a multi-scale view from local mechanism to global behavior. The flow patterns, the formation of bubbles/droplets, and the manipulation of bubbles/droplets are presented and discussed in detail. The mass transfer aspects include the velocity profile and mixing inside droplets/slugs, as well as the interfacial mass transfer mechanism and simulative models. The aim of the review is to show directly the physical ingredients which determine the transport phenomena, help explain the observed behavior, and guide reactor design. To simplify the physical ingredients, the attention is focused on straight channels while phenomena in other channels (e.g. , meandering channel) are neglected. [ABSTRACT FROM AUTHOR]

• The effect of fluid viscosity on liquid-liquid mass transfer was studied. • Several local transfer patterns were observed at both straight channels and bends. • Ka and K can increase with the increase in μ C at large flow rates. • Three mass transfer regimes were identified and correlated to local transfer patterns. Mass transfer characteristics of liquid-liquid Taylor flow with various fluid pairs are investigated, in order to reveal the viscous effects of the fluids. The concentration pattern in continuous slugs, mass transfer time and mass transfer coefficient are discussed and analyzed. Several different concentration patterns originated from recirculation are observed at both straight channels and curved bends, respectively. It is shown that the mass transfer coefficient (Ka and K) can increase with the increase in the viscosity of the continuous phase at large flow rates, which is explained by the increased contribution of the film at the lateral droplet side and its exchange with the bulk slug. Based on the dominance of different parts (i.e. , film, cap), three mass transfer regimes are distinguished. Correlations for each regime, which covers a wide range of fluid viscosity of 0.89–45.6 mPa·s, are developed to predict the mass transfer coefficient. [ABSTRACT FROM AUTHOR]

[Display omitted] • Multiphase processes containing solid in microchannels stably manipulated. • Hydrodynamics of Pickering emulsion systems in microchannels characterised. • A scaling law of droplets size as a function of Q d / Q c , μ d / μ c and Ca established. In this work, a method based on Pickering emulsion systems (PES) was proposed to manipulate multiphase processes containing solid particles in microchannels without adhesion and/or accumulation. The flow hydrodynamics of PES in a microchannel were systematically characterized. For comparison, a suspension system (SS, ethyl acetate with suspended SiO 2 nanoparticles-water) and a viscous fluid system (VFS, ethyl acetate with methyl-silicone oil–water) were examined. It was found liquid–liquid two-phase characteristics of PES, SS and VFS around T-junction within the main microchannel depended on surface hydrophobicity of stabilizing SiO 2 nanoparticles, amount of SiO 2 particles added, viscosity and flow rate. Four flow patterns were observed under these operating conditions for PES and VFS, where superficial velocity was the key factor affecting the flow patterns transition. Finally, a scaling law of the droplet size was established as a function of Q d / Q c , μ d / μ c and Ca. [ABSTRACT FROM AUTHOR]

[Display omitted] • Haemonchus contortus protein Hc -SPI-I8A functions as an inhibitor of blood clotting contributed by the TIL domain. • Hc -SPI-I8 is highly expressed in the parasitic stages and at the hypodermis, intestine and gonads of H. contortus. • Hc -SPI-I8 was in the same clade as the homologues, which could inhibit coagulation by interacting with clotting factors. • Hc -SPI-I8A is most likely disturbing the extrinsic coagulation cascade by interacting with Oa TSP1CP through its TIL domain. • Hc -SPI-I8A might also interfering in the intrinsic coagulation cascade by an unknown mechanism. Haemonchus contortus , a blood-feeding nematode, inhibits blood coagulation at the site of infection to facilitate blood-sucking and digesting for successful parasitism. However, the mechanism underlying anti-coagulation at the host-parasite interface is largely unknown. In the current study, Hc-spi-i8 , which has two greatly different transcripts named Hc-spi-i8a and Hc-spi-i8b , respectively, was described. Hc -SPI-I8A was a serine protease inhibitor containing a trypsin inhibitor-like cysteine rich (TIL) domain, while Hc -SPI-I8B was not. Hc -SPI-I8A/B were primarily expressed in the hypodermis, intestines and gonads in the parasitic stages of H. contortus. Hc -SPI-I8A interacted with Ovis aries TSP1-containing protein (Oa TSP1CP), which was determined by yeast two-hybrid, co-immunoprecipitation (Co-IP), pull down and co-localization experiments. The blood clotting time contributed by the TIL domain was prolonged by Hc -SPI-I8A. Hc -SPI-I8A is most likely interfering in the extrinsic coagulation cascade by interacting with Oa TSP1CP through its TIL domain and intrinsic coagulation cascade by an unknown mechanism. These findings depict a crucial point in the host-parasite interaction during H. contortus colonization, which should contribute to drug discovery and vaccine development in fighting against this important parasite worldwide. [ABSTRACT FROM AUTHOR]

Highlights: [•] Process of CO2 absorption by MEA solutions was analyzed in microchannel reactors. [•] Effects of relevant parameters on the process were evaluated. [•] k 1 for CO2 reaction with MEA was successfully determined under ambient pressure. [•] An Arrhenius-type equation was obtained for k 1 for CO2 absorption in microchannel. [Copyright &y& Elsevier]

• The effects of both μ c and μ d on slug flow are investigated. • The slug flow occurrence is predicted based on composite dimensionless numbers. • For squeezing regime, the droplet scaling law is different from the classical theory. • The film thickness increased more rapidly with Ca D than in gas–liquid slug flow. • Correlations were proposed to reflect the effect of recirculations in both phases on the pressure drop. The hydrodynamics of liquid–liquid slug flow with various fluid systems were investigated, aiming at revealing the viscous effects of both phases. The operation range, droplet formation, film thickness and pressure drop of slug flow were studied and discussed. The results reveal the importance of inertia and viscous forces of both phases in the transition of flow patterns. Accordingly, a universal flow map based on composite terms of Ca c Re c 0.5 and Ca d Re d 0.5 was adopted, which worked well for a wide viscosity range. For droplet formation in the present system, both the squeezing and shearing regimes deviated from the classical ideal models. Under higher capillary number, the film was accumulated at the corners and lateral planes, leading to distinct droplet shapes from literature. For the two-phase pressure drop, new empirical correlations were proposed to describe the recirculation inside both the droplets and slugs. The findings can provide better guidance for optimized reactor design. [ABSTRACT FROM AUTHOR]

Both Fasciola hepatica and Clonorchis sinensis are endemic in China, South Korea, Japan and other Southeast Asian countries. Reliable and sensitive diagnostic methods are needed for detecting their infections in humans and animals. Differential simplex and duplex polymerase chain reaction (PCR) methods were developed. The PCRs targeted the second internal transcribed spacer ( its 2) (408 bp) of F. hepatica , and NADH dehydrogenase subunit 2 gene ( nad 2) (527 bp) of C. sinensis . Both simplex PCRs detected as little as 2 pg genomic DNA in one microliter in a 25 μL PCR reaction system. The duplex PCR had similar detection limit as well, and detected as low as one egg in 200 mg feces. These methods were analytical specific with no amplification being observed from the gemonic DNA of Fasciolopsis buski , Haemonchus contortus , Ascaris ovis or Eimeri ahsata . Of 158 sheep fecal samples collected from various farms, four and one samples were PCR-positive for F. hepatica and C. sinensis , respectively. The duplex PCR method described here is time-saving and convenient, and may prove to be an invaluable tool for molecular detection and epidemiological investigation of F. hepatica and C. sinensis in endemic area. [ABSTRACT FROM AUTHOR]

The immunochromatographic test (ICT) is a convenient and low-cost method that can rapidly obtain results (10 min) under normal conditions. In this study, we established an ICT assay with two monoclonal antibodies: TgSAG3-3A7 and TgSAG3-4D5 based on the conserved protein of TgSAG3 that can be expressed in all the infective stages of T. gondii. 20 nm gold particles were prepared and conjugated with TgSAG3-3A7 MAb at the concentration of 12.5 μg/mL. TgSAG3-4D5 MAb were used as the capture antibody because of its higher affinity tested by ELISA. The detection limit of the ICT assay was 100 ng with visual detection under optimal conditions of analysis. Positive porcine serum of Cryptosporidium suis , Mycoplasma suis , Streptococcus suis , Salmonella choleraesuis , Cysticercus cellulosae , Isospora suis , and Trichinella spiralis were used to evaluate the specificity of this ICT and no cross-reactivity was observed. 310 porcine serum samples obtained from pig farms in Zhejiang Province, China were detected by this ICT and ELISA kit, 23 positive samples were found by the developed strip with the rate of 7.42% comparing with 22 positive samples detected by the commercially ELISA kit which the positive rate was 7.1%, the relative sensitivity and specificity of this ICT are 100% and 99.65%. Therefore, the ICT established in this study is proved effective, simple, specific and sensitive of T. gondii detection. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii ( T. gondii ) is one of the most common parasite that can infect almost any warm-blooded animals including humans. The cyclic nucleotide-dependent protein kinase (PKA) regulates a spectrum of intracellular signal pathways in many organisms. Protein kinase catalytic subunit (PKAC) is the core of the whole protein, and plays an important role in the life cycle of T.gondii . Here, T.gondii PKAC ( Tg PKAC) overexpression strain ( Tg PKAC-OE) was constructed. The growth of the Tg PKAC-OE, RH △Ku80 , and Tg PKAC inhibition strains ( Tg PKAC-H89) were analysed by SYBR-green real-time PCR, and the ultrastructure was observed by transmission electron microscopy. The survival rate in mice was also recorded to analyse the virulence of the parasites. We also investigated the subcellular localization of Tg PKAC in Vero cells by laser scanning microscope. We found that Tg PKAC-OE strain exhibited obviously increased growth rate in Vero cells in vitro , and infected mice survived for a shorter time compared to wild type strain. Ultrastructural analysis found more autophagosomes-like structures in Tg PKAC-H89 parasite compared to RH △Ku80 strain, and the relative expression level of Toxoplasma gondii autophagy-related protein (ATG8) in Tg PKAC-H89 parasite was higher than wild type parasite. Laser confocal results showed that Tg PKAC was mainly expressed in the cytoplasm of Vero cells. In conclusion, we hypothesized that inhibition of Tg PKAC could cause autophagy of Toxoplasma gondii and then influence the replication of the parasite. Tg PKAC plays an important role in parasite virulence in vivo , and the subcellular localization was successfully detected in Vero cells. Our data will provide a basis for further study of Tg PKAC function and help screen drug targets of T. gondii . [ABSTRACT FROM AUTHOR]

The apicoplast, which is the result of secondary endosymbiosis, is a distinctive subcellular organelle and a crucial therapeutic target for apicomplexan parasites. The majority of apicoplast-resident proteins are encoded by the nuclear genome and target the apicoplast via bipartite targeting signals consisting of a signal peptide and a transit peptide. The properties and functions of these peptides are poorly understood, which hinders the identification of apicoplast proteins and the study for plastid evolution. Here, the targeting signals of the recently discovered apicoplast tRNA thiouridylase Tg MnmA of Toxoplasma gondii were analyzed. Our data using a reporter (the enhanced green fluorescent protein) fused with individual fragments containing various numbers of its N-terminal amino acids unequivocally revealed that the first 28 amino acids of Tg MnmA functioned as a signal peptide for cellular secretion. The N-terminal 150 amino acids were sufficient to direct the fusion protein to the apicoplast, whereas its deletion caused the fusion protein to be localized to the mitochondrion. Our data further demonstrated that the apicoplast, rhoptry, and mitochondrion shared similar targeting signals, indicating that the apicoplast localization peptide was trans-organellar in function. In addition, the apicoplast localization peptide was important for the healthy proliferation of tachyzoites. In conclusion, the targeting signals of the nucleus-encoded apicoplast-targeted protein Tg MnmA have been mapped out and the importance of this localization peptide has been elucidated in the current study. • The first N-terminal 150 AA of Tg MnmA target the EGFP to the apicoplast. • Without the localization peptide, Tg MnmAΔ1−150-EGFP localizes to the mitochondrion. • Mis-localization of Tg MnmA greatly affects the proliferation of T. gondii in vitro. [ABSTRACT FROM AUTHOR]