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Toxoplasma, an important intracellular parasite of humans and animals, causes life-threatening toxoplasmosis in immunocompromised individuals. Although Toxoplasma secretory proteins during acute infection (tachyzoite, which divides rapidly and causes inflammation) have been extensively characterized, those involved in chronic infection (bradyzoite, which divides slowly and is surrounded by a cyst wall) remain uncertain. Regulation of the cyst wall is essential to the parasite life cycle, and polysaccharides, such as chitin, in the cyst wall are necessary to sustain latent infection. Toxoplasma secretory proteins during the bradyzoite stage may have important roles in regulating the cyst wall via polysaccharides. Here, we focused on characterizing the hypothetical T. gondii chitinase, chitinase-like protein 1 (TgCLP1). We found that the chitinase-like domain containing TgCLP1 is partially present in the bradyzoite microneme and confirmed, albeit partially, its previous identification in the tachyzoite microneme. Furthermore, although parasites lacking TgCLP1 could convert from tachyzoites to bradyzoites and make an intact cyst wall, they failed to convert from bradyzoites to tachyzoites, indicating that TgCLP1 is necessary for bradyzoite reactivation. Taken together, our findings deepen our understanding of the molecular basis of recrudescence and could contribute to the development of novel strategies for the control of toxoplasmosis.

Abstract Background The surgical strategy for thoracic esophageal cancer that invades the lungs is controversial. In particular, invasion of the pulmonary vein is often regarded unresectable. We successfully applied bilateral video-assisted thoracic surgery (VATS) in esophagectomy for esophageal cancer with left inferior pulmonary vein invasion following induction chemoradiotherapy (CRT), with a favorable response. Case presentation A 64-year-old woman was diagnosed with squamous cell carcinoma of the lower third of the esophagus. Computed tomography (CT) revealed that the tumor was suspected to be invading the main trunk of the left lower pulmonary vein and left lower lung. We initiated induction CRT comprising 5-fluorouracil, cisplatin, and concurrent radiotherapy at 50.4 Gy/28Fr. CT revealed shrinkage of the tumor, and the main trunk of the left inferior pulmonary vein was released from the tumor invasion. We considered the tumor to be completely resectable. VATS esophagectomy is usually performed using a right-sided approach. However, the right-sided approach is inappropriate for evaluating tumors around the left inferior pulmonary vein. We started with left-sided VATS to determine tumor resectability and dissected between the esophagus and the main trunk of the left inferior pulmonary vein. We only needed to perform partial resection of the left lower lobe. We then performed a right-sided VATS esophagectomy and lymphadenectomy with partial en bloc resection of the left lower lobe. Following this, we performed hand-assisted laparoscopic proximal gastrectomy and reconstruction using the gastric remnant. The postoperative course was uneventful. The patient was discharged on postoperative day 14. Histopathological examination of the surgical specimen revealed a complete pathological response without any remnant tumor or lymph node metastasis. There were no signs of recurrence or metastasis at the 1-year follow-up. Conclusions Curative resection for thoracic esophageal cancer that invades the pulmonary vein could be possible via the bilateral VATS approach following induction CRT with a favorable response.

Abstract Background After esophagectomy for esophageal and esophagogastric cancer, more than half of patients have lost > 10% of their body weight at 12 months. In most cases, the gastric remnant is used for reconstruction after esophagectomy. One of the most serious nutritional complications of this technique is delayed gastric emptying caused by gastric remnant mobilization and denervation of the vagus nerve. The aim of the PYloroplasty versus No Intervention in GAstric REmnant REconstruction after Oesophagectomy (PYNI-GAREREO) trial is to analyze the clinical outcome of modified Horsley pyloroplasty (mH-P) as a method of preventing delayed gastric emptying. Methods The PYNI-GAREREO trial is designed as an open randomized, single-center superiority trial. Patients will be randomly allocated to undergo gastric remnant reconstruction with mH-P (intervention group) or no intervention (control group) in parallel groups. All patients with esophageal cancer or esophagogastric cancer planning to undergo curative minimally invasive esophagectomy will be considered for inclusion. A total of 140 patients will be included in the study and randomized between the groups in a 1:1 ratio. The primary outcome is the body weight change at 6 months postoperatively, and the secondary outcomes are the nutritional status, postoperative complications, functional outcome, and quality of life until 1 year postoperatively. Discussion We hypothesize that mH-P after minimally invasive esophagectomy more effectively maintains patients’ nutritional status than no pyloroplasty. Trial registration UMIN Clinical Trials Registry UMIN000045104. Registered on 25 August 2021. https://center6.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000051346 .

Entamoeba histolytica is a parasitic protozoan with roles in pathogenicity of intestinal amoebiasis. E. histolytica trophozoites lack functional mitochondria and their energy production depends mostly on glycolysis. D-Glucose has a pivotal role in this process and trophozoites store this sugar as glycogen in glycogen granules. Rare sugars, which are defined as sugars present in nature in limited amounts, are of interest as natural low-calorie sweeteners for improving physical conditions of humans. One such rare sugar, D-allose, can be absorbed by a sodium-dependent glucose cotransporter as a substitute for D-glucose, and some rare sugars are known to inhibit growth of cancer cells, Caenorhabditis elegans and Tritrichomonas foetus. Based on these observations, we examined the effects of rare sugars on growth of E. histolytica trophozoites, together with those of D-galactose and D-fructose. The results indicate that treatment with D-allose or D-psicose (D-allulose) alone inhibits proliferation of E. histolytica trophozoites, but that these sugars enhance proliferation of trophozoites in the presence of D-glucose or D-galactose. The trophozoites could take up D-glucose and D-galactose, but not D-fructose, D-allose or D-psicose. Cell sizes of the trophozoites also differed depending on the culture medium.

Abstract Background Spontaneous retroperitoneal hematoma is defined as bleeding in the retroperitoneal space without any triggers such as trauma, invasive procedures, and abdominal aortic aneurysm. Case presentation A 48-year-old Japanese man who experienced sudden abdominal pain, severe hypotension, and decreased hemoglobin was diagnosed with spontaneous retroperitoneal hematoma. Contrast-enhanced computed tomography revealed massive left retroperitoneal hematoma; however, neither extravasation nor causative aneurysm was noted. Through conservative management with close monitoring, he was treated and discharged on the tenth hospital day without any morbidity. Conclusions Spontaneous retroperitoneal hematoma treatment comprises conservative management, transcatheter arterial embolization, and surgical intervention. The mortality rate of spontaneous retroperitoneal hematoma is so high that the optimal treatment timing needs to be carefully judged on the basis of detailed evaluation, and management algorithm with clear criteria.

Abstract Cryptosporidium parvum is an apicomplexan parasite that causes severe zoonotic diarrhea in humans and calves. Since there are no effective treatments or vaccines for infants or immunocompromised patients, it is important to understand the molecular mechanisms of the parasite–host interaction for novel drug discovery. Mitogen-activated protein kinase (MAP kinase) is a key host factor in interactions between host and various pathogens, including parasites. Although the function of conventional MAP kinases against parasite infection has been investigated, that of atypical MAP kinases remains largely unknown. Therefore, we focused on one of the atypical MAP kinases, MAPK4, and its effect on C. parvum infection in human intestinal cells. Here, we report that MAPK4-deficient intestinal cells showed a significant reduction in C. parvum infection. We also show that host MAPK4 has a role in host cell survival from C. parvum infection. In addition, we show that C. parvum requires host MAPK4 for its successful invasion and asexual reproduction. Taken together, our data suggest that MAPK4 is an important host factor contributing to C. parvum infection in human intestinal cells.

Toxoplasma gondii is an intracellular parasitic protozoan with a high infection rate in mammals, including humans, and birds. There is no effective vaccine, and treatment relies on antiparasitic drugs. However, existing antiprotozoal drugs have strong side effects and other problems; therefore, new treatment approaches are needed. Metal nanoparticles have attracted increased interest in the biomedical community in recent years because of their extremely high surface area to volume ratio and their unique reactivity that could be exploited for medicinal purposes. Previously, we confirmed the anti-Toxoplasma effects of gold, silver, and platinum nanoparticles, in a growth inhibition test. Here, we asked whether the anti-Toxoplasma effect could be confirmed with less expensive metal nanoparticles, specifically iron oxide nanoparticles (goethite and hematite). To improve the selective action of the nanoparticles, we modified the surface with l-tryptophan as our previous findings showed that the bio-modification of nanoparticles enhances their selectivity against T. gondii. Fourier-Transform Infrared Spectroscopy (FTIR) analysis confirmed the successful coating of the iron oxide nanoparticles with l-tryptophan. Subsequently, cytotoxicity and growth inhibition assays were performed. L-tryptophan-modified nanoparticles showed superior anti-Toxoplasma action compared to their naked nanoparticle counterparts. L-tryptophan enhanced the selective toxicity of the iron oxide nanoparticles toward T. gondii. The bio-modified nanoparticles did not exhibit detectable host cell toxicity in the effective anti-Toxoplasma doses. To elucidate whether reactive oxygen species contribute to the anti-Toxoplasma action of the bio-modified nanoparticles, we added Trolox antioxidant to the assay medium and found that Trolox appreciably reduced the nanoparticle-induced growth inhibition.

Transcriptional variation has been studied but post-transcriptional modification due to RNA editing has not been investigated in Plasmodium. We investigated developmental stage-specific RNA editing in selected genes in Plasmodium falciparum 3D7. We detected extensive amination- and deamination-type RNA editing at 8, 16, 24, 32, 40, and 46 h in tightly synchronized Plasmodium. Most of the editing events were observed in 8 and 16 h ring-stage parasites. Extensive A-to-G deamination-type editing was detected more during the 16 h ring stage (25%) than the 8 h ring stage (20%). Extensive U-to-C amination-type editing was detected more during the 16 h ring stage (31%) than the 8 h ring stage (22%). In 28S, rRNA editing converted the loop structure to the stem structure. The hemoglobin binding activity of PF3D7_0216900 was also altered due to RNA editing. Among the expressed 28S rRNA genes, PF3D7_0532000 and PF3D7_0726000 expression was higher. Increased amounts of the transcripts of these two genes were found, particularly PF3D7_0726000 in the ring stage and PF3D7_0532000 in the trophozoite and schizont stages. Adenosine deaminase (ADA) expression did not correlate with the editing level. This first experimental report of RNA editing will help to identify the editing machinery that might be useful for antimalarial drug discovery and malaria control.

The effect of polymer addition on transition to turbulence in a two-dimensional water-flow channel was experimentally investigated by flow visualization using reflective flakes. The flow entering the channel test section maintains a high disturbance level by expanding laterally after reaching a high Reynolds number upstream the test section. In order to obtain the intermittency factor (turbulence fraction), the visualized images were classified into non-turbulent and turbulent regions, and the streamwise scale of the streaks appearing in the non-turbulent region was estimated from the autocorrelation coefficient computed by shifting the images in the streamwise direction. The visualization results show that similar to the pure water case, intermittent flow with a patch-like distribution of turbulent and non-turbulent areas clustered by streamwise streaks is observed. The Reynolds number at which the intermittency increases shifts toward higher Reynolds numbers with increasing polymer concentration, indicating a delay of transition. The streaks appearing in the non-turbulent region elongate with increasing polymer concentration. At high concentrations, straight elongated streaks penetrate through the turbulent regions, suggesting that the polymer addition affects the stability of the streaks. These changes of the streak behavior indicate that the polymer affects not only the transition Reynolds number but also the flow structure during the transition process.

It is now more than 100 years since the work of von Kármán (1921) on the boundary-layer flow over a rotating disk was published in the first volume of Zeitschrift für Angewandte Mathematik und Mechanik (ZAMM, Vol. 1(4), pp 233-252). Recently, there has been a large amount of work undertaken addressing the instability and transition of the boundary-layer flows over rotating disks and cones using theoretical, numerical and experimental techniques. Here we will discuss some different methods to analyze experimental data that can give insight into the instability and transition to turbulence of boundary-layer flows over rotating slender and broad cones (including the disk). At first, we discuss the pdf-method (probability density function) that allows a simple way to determine regions of instability growth, transition and fully developed turbulence. Secondly, we look at various ways to use spectral information to investigate the boundary layers giving a deeper understanding of the transition process. Finally, a method to determine the most probable flow structure leading up to fully developed turbulence is discussed. We envisage that some of these methods can be useful in analyzing instability and transition also in other flow cases.

Curcumin (CUR) is known for its wide folkloric effects on various infections; however, its solubility status has remained a hindrance to its bioavailability in the host. This study evaluated the comparative effects of CUR and CUR-nanoparticle in vitro on T. congolense, T. b. brucei, and T. evansi. Additionally, CUR and CUR-nanoparticle anti-Trypanosoma efficacy were assessed in vivo against T. congolense. All the CUR-nanoparticles were two folds more effective on the T. congolense as compared to CUR in vitro, with recorded efficacy of 3.67 ± 0.31; 7.61 ± 1.22; and 6.40 ± 3.07 μM, while the CUR-nanoparticles efficacy was 1.56 ± 0.50; 28.16 ± 9.43 and 13.12 ± 0.13 μM on T. congolense, T. b. brucei, and T. evansi, respectively. Both CUR and CUR-nanoparticles displayed moderate efficacy orally. The efficacy of CUR and CUR-nanoparticles in vivo was influenced by solubility, presence of food, and treatment period. CUR-treated mice were not cured of the infection; however, the survival rate of the orally treated mice was significantly prolonged as compared with intraperitoneal-treated mice. CUR-nanoparticles resulted in significant suppression of parasitemia even though relapsed was observed. In conclusion, CUR and CUR-nanoparticles possess moderate efficacy orally on the trypanosomes as compared to the intraperitoneal treatment.

Abstract Background Replaced right hepatic artery (rRHA) is a common vascular variation, and combined resection of this vessel is sometimes needed for the curative resection of pancreatic head malignancy. Safe surgical management has not been established, and there is a small number of reported cases. Here, we reported five cases, wherein preoperative embolization of rRHA was performed for combined resection. Case presentation All patients had pancreatic head malignancies that were in contact with rRHA. We performed a preoperative embolization of the rRHA before the scheduled pancreaticoduodenectomy for the combined resection. Arterial embolization was safely accomplished, and the communicating arcade from the left hepatic artery via the hilar plate was clearly revealed in all cases. Four patients underwent the operative procedure, except for one patient who had liver metastasis at laparotomy. No patient suffered from a severe abnormal liver function during the management; however, one patient had multiple liver infarctions during the postoperative course. Conclusions Preoperative embolization for the combined resection of rRHA in pancreaticoduodenectomy can be a management option for the precise evaluation of hemodynamics after sacrificing rRHA. In our cases, arterial flow to the right liver lobe was supplied by the left hepatic artery via the bypass route, including the communicating arcade of the hilar plate.

Aim Non‐occlusive mesenteric ischemia (NOMI) is a fatal condition with a low survival rate in most cases. The risk factors for perioperative mortality in NOMI cases are unclear. The purpose of this study was to define the risk factors for mortality in patients with NOMI undergoing surgery. Methods Thirty‐eight consecutive patients who underwent surgery for NOMI at Teine Keijinkai Hospital between 2012 and 2020 were included in the study. Patient information, including age, sex, physical findings, comorbidities, laboratory data, and computed tomography and surgical findings were retrospectively analyzed. Results Of the 38 patients, 18 (47%) died before discharge. Significant univariate predictors of mortality were a high Sequential Organ Failure Assessment (SOFA) score, high lactate level, low blood pH, and short intestinal length after surgery. In the multivariate analysis, a high SOFA score (odds ratio 1.33, P = 0.036) and short intestine length after surgery (odds ratio 34.7, P = 0.003) were identified as independent risk factors for perioperative mortality. Conclusion The preoperative SOFA score and postoperative residual intestinal length may be predictors of death in NOMI surgical patients, not age and the content of comorbidities.

Cryptosporidium spp. are gastrointestinal opportunistic protozoan parasites that infect humans, domestic animals, and wild animals all over the world. Cryptosporidiosis is the second leading infectious diarrheal disease in infants less than 5 years old. Cryptosporidiosis is a common zoonotic disease associated with diarrhea in infants and immunocompromised individuals. Consequently, cryptosporidiosis is considered a serious economic, veterinary, and medical concern. The treatment options for cryptosporidiosis are limited. To address this problem, we screened a natural product library containing 87 compounds of Traditional Chinese Medicines for anti-Cryptosporidium compounds that could serve as novel drug leads and therapeutic targets against C. parvum. To examine the anti-Cryptosporidium activity and half-maximal inhibitory doses (EC50) of these compounds, we performed in vitro assays (Cryptosporidium growth inhibition assay and host cell viability assay) and in vivo experiments in mice. In these assays, the C. parvum HNJ-1 strain was used. Four of the 87 compounds (alisol-A, alisol-B, atropine sulfate, and bufotalin) showed strong anti-Cryptosporidium activity in vitro (EC50 values = 122.9±6.7, 79.58±13.8, 253.5±30.3, and 63.43±18.7 nM, respectively), and minimum host cell cytotoxicity (cell survival > 95%). Furthermore, atropine sulfate (200 mg/kg) and bufotalin (0.1 mg/kg) also showed in vivo inhibitory effects. Our findings demonstrate that atropine sulfate and bufotalin are effective against C. parvum infection both in vitro and in vivo. These compounds may, therefore, represent promising novel anti-Cryptosporidium drug leads for future medications against cryptosporidiosis.

Cryptosporidium and Toxoplasma are parasites that have caused problems worldwide. Cryptosporidium causes severe watery diarrhoea and may be fatal in immunocompromised patients and in infants. Nitazoxanide is the only agent currently approved by the FDA, but its efficacy is limited. Toxoplasmosis is also a problem in the immunocompromised, as currently available treatment options have limited efficacy and patient tolerance can be poor. In the present investigation, we screened libraries of epigenetic compounds to identify those that inhibited C. parvum growth. Nullscript was identified as a compound with an inhibitory effect on C. parvum and T. gondii growth, and was less toxic to host cells. Nullscript was also able to significantly decrease oocyst excretion in C. parvum-infected SCID mice.

Abstract Background Reports on the antischistosomal effect of several antimalarial drugs such as artesunate, mefloquine, and amodiaquine suggest that febrifugine, which exerts an antimalarial effect, can also be expected to possess antischistosomal potential. The present study investigates the antischistosomal effects of febrifugine. Methods In experiment 1, Schistosoma mansoni adult worm pairs were incubated in a medium alone as a control or supplemented with febrifugine at 0.05, 0.1, 0.2, and 0.5 μg/ml for 14 days. The morphology of the worms and the egg production of the female worms were observed simultaneously. In experiment 2, the incubation was conducted as in experiment 1, except that the febrifugine concentrations were reduced to 0.005, 0.01, and 0.02 μg/ml. In addition, S. mansoni adult worms were incubated with either 0.5 μg/ml febrifugine or none as a control for 5 days and stained with neutral red dye. Results Febrifugine significantly reduced the survival of S. mansoni male and female worms at concentrations of 0.02–0.5 μg/ml following incubation for 14 days and remarkably inhibited the daily egg output of the female worms. The non-treated male and female worms remained morphologically normal within the period of 14 days, whereas male and female worms treated with febrifugine at different concentrations gradually twisted and subsequently died. In addition, S. mansoni adult worms were incubated with either 0.5 μg/ml febrifugine or none as a control for 5 days and stained with neutral red dye. Non-treated male worms were morphologically normal and stained dark red with neutral red, while febrifugine-treated male worms appeared similar to those in the control group and were stained at a slightly lower level of dark red than the non-treated male worms. Non-treated female worms were morphologically normal, and their intestinal tract and vitellaria were stained deep red and dark red, respectively. In contrast, febrifugine-treated female worms were morphologically damaged, and their intestinal tract and vitellaria remained mostly unstained and stained dark red, respectively. Conclusion Febrifugine exerts potent antischistosomal effects and can be expected to contribute to the development of a novel antischistosomal drug.

Toxoplasma gondii chronically infects the brain as latent cysts containing bradyzoites and causes various effects in the host. Recently, the molecular mechanisms of cyst formation in the mouse brain have been elucidated, but those in the human brain remain largely unknown. Here, we show that abnormal glutamine metabolism caused by both interferon-γ (IFN-γ) stimulation and T. gondii infection induce cyst formation in human neuroblastoma cells regardless of the anti-T. gondii host factor nitric oxide (NO) level or Indoleamine 2,3-dioxygenase-1 (IDO1) expression. IFN-γ stimulation promoted intracellular glutamine degradation in human neuronal cells. Additionally, T. gondii infection inhibited the mRNA expression of the host glutamine transporters SLC38A1 and SLC38A2. These dual effects led to glutamine starvation and triggered T. gondii stage conversion in human neuronal cells. Furthermore, these mechanisms are conserved in human iPSC-derived glutamatergic neurons. Taken together, our data suggest that glutamine starvation in host cells is an important trigger of T. gondii stage conversion in human neurons.

Hepatic manifestations of Epstein-Barr virus (EBV) infection are relatively common, mild, and self-limiting. Although fulminant hepatic failure has been reported in a few cases, the contributing factors are unclear. This report discusses a pediatric case of EBV-associated acute liver failure that required urgent liver transplantation; however, liver damage continued to progress post-liver replacement. Monoclonal CD8+ T cells that preferentially infiltrated the native and transplanted liver were positive for EBV-encoded small RNA, suggesting a pathophysiology similar to that of EBV-associated hemophagocytic lymphohistiocytosis and chronic active EBV infection. Therefore, subsequent chemotherapy and hematopoietic cell transplantation was conducted, which led to cure. This is the first case of EBV-associated acute liver failure that relapsed post-liver transplant. As such, it sheds light on an under-recognized clinical entity: liver-restricted hyperinflammation caused by EBV-infected monoclonal CD8+ T cells. This phenomenon needs to be recognized and differentiated from hepatitis/hepatic failure caused by EBV-infected B cells, which has a relatively benign clinical course.

Currently, toxoplasmosis affects nearly one-third of the world’s population, but the available treatments have several limitations. This factor underscores the search for better therapy for toxoplasmosis. Therefore, in the current investigation, we investigated the potential of emodin as a new anti-Toxoplasma gondii while exploring its anti-parasitic mechanism of action. We explored the mechanisms of action of emodin in the presence and absence of an in vitro model of experimental toxoplasmosis. Emodin showed strong anti-T. gondii action with an EC50 value of 0.03 µg/mL; at this same effective anti-parasite concentration, emodin showed no appreciable host cytotoxicity. Likewise, emodin showed a promising anti-T. gondii specificity with a selectivity index (SI) of 276. Pyrimethamine, a standard drug for toxoplasmosis, had an SI of 2.3. The results collectively imply that parasite damage was selective rather than as a result of a broad cytotoxic effect. Furthermore, our data confirm that emodin-induced parasite growth suppression stems from parasite targets and not host targets, and indicate that the anti-parasite action of emodin precludes oxidative stress and ROS production. Emodin likely mediates parasite growth suppression through means other than oxidative stress, ROS production, or mitochondrial toxicity. Collectively, our findings support the potential of emodin as a promising and novel anti-parasitic agent that warrants further investigation.

During sexual reproduction/conjugation of the ciliate Tetrahymena thermophila, the germinal micronucleus undergoes meiosis resulting in four haploid micronuclei (hMICs). All hMICs undergo post-meiotic DNA double-strand break (PM-DSB) formation, cleaving their genome. DNA lesions are subsequently repaired in only one ‘selected’ hMIC, which eventually produces gametic pronuclei. DNA repair in the selected hMIC involves chromatin remodeling by switching from the heterochromatic to the euchromatic state of its genome. Here, we demonstrate that, among the 15 Tetrahymena Snf2 family proteins, a core of the ATP-dependent chromatin remodeling complex in Tetrahymena, the germline nucleus specific Iswi in Tetrahymena IswiGTt and Rad5Tt is crucial for the generation of gametic pronuclei. In either gene knockout, the selected hMIC which shows euchromatin markers such as lysine-acetylated histone H3 does not appear, but all hMICs in which markers for DNA lesions persist are degraded, indicating that both IswiGTt and Rad5Tt have important roles in repairing PM-DSB DNA lesions and remodeling chromatin for the euchromatic state in the selected hMIC.

Summary: The pathogenesis of malaria parasites depends on host erythrocyte modifications that are facilitated by parasite proteins exported to the host cytoplasm. These exported proteins form a trafficking complex in the host cytoplasm that transports virulence determinants to the erythrocyte surface; this complex is thus essential for malaria virulence. Here, we report a comprehensive interaction network map of this complex. We developed authentic, unbiased, highly sensitive proteomic approaches to determine the proteins that interact with a core component of the complex, SBP1 (skeleton-binding protein 1). SBP1 interactomes revealed numerous exported proteins and potential interactors associated with SBP1 intracellular trafficking. We identified several host-parasite protein interactions and linked the exported protein MAL8P1.4 to Plasmodium falciparum virulence in infected erythrocytes. Our study highlights the complicated interplay between parasite and host proteins in the host cytoplasm and provides an interaction dataset connecting dozens of exported proteins required for P. falciparum virulence. : Microbiology; Parasitology; Proteomics Subject Areas: Microbiology, Parasitology, Proteomics

Inosine 5′-monophosphate dehydrogenase (IMPDH) is an essential enzyme for the production of guanine nucleotides. Disruption of IMPDH activity has been explored as a therapeutic strategy for numerous purposes, such as for anticancer, immunosuppression, antiviral, and antimicrobial therapy. In the present study, we established a luciferase-based high-throughput screening system to identify IMPDH inhibitors from our chemical library of known bioactive small molecules. The screening of 1400 compounds resulted in the discovery of three irreversible inhibitors: disulfiram, bronopol, and ebselen. Each compound has a distinct chemical moiety that differs from other reported IMPDH inhibitors. Further evaluation revealed that these compounds are potent inhibitors of IMPDHs with kon values of 0.7 × 104 to 9.3 × 104 M−1·s−1. Both disulfiram and bronopol exerted similar degree of inhibition to protozoan and mammalian IMPDHs. Ebselen showed an intriguing difference in mode of inhibition for different IMPDHs, with reversible and irreversible inhibition to each Cryptosporidium parvum IMPDH and human IMPDH type II, respectively. In the preliminary efficacy experiment against cryptosporidiosis in severe combined immunodeficiency (SCID) mouse, a decrease in the number of oocyst shed was observed upon the oral administration of disulfiram and bronopol, providing an early clinical proof-of-concept for further utilization of these compounds as IMPDH inhibitors.

Modern optical communication technology can realize a large-scale multilevel (or M-ary) optical signal. Investigating the quantum mechanical nature of such a large-scale M-ary optical signal is essential for a unified understanding of quantum information science and optical communication technology. This article focuses on the quantum-mechanical non-orthogonality for a collection of pure quantum states and proposes a non-orthogonality index based on the least squares error criterion in quantum detection theory. First, we define the index for linearly independent signals, and the proposed index is analyzed through numerical simulations. Next, the index is applied to a highly large-scale M-ary phase-shift keying (PSK) coherent state signal. Furthermore, the index is compared with the capacity of the pure state channel with the PSK signal. As a result, it is shown that a highly large-scale M-ary PSK coherent state signal exhibits a quantum nature even when the signal transmission power is very high. Thus, the theoretical characterization of a highly large-scale M-ary coherent state signal based on the proposed index will be the first step toward a better understanding of cutting-edge optical communication technologies such as the quantum stream cipher Y00.

Cryptosporidium is an opportunistic parasite that has been reported in >30 avian hosts worldwide, however, there is no information regarding Cryptosporidium spp. in poultry in Bangladesh. Accordingly, we investigated the prevalence of Cryptosporidium spp. in poultry at open live bird markets in Bangladesh. A total of 197 samples were randomly collected from poultry at open live bird markets in Bangladesh and screened for the detection of Cryptosporidium. Initial microscopic examination revealed Cryptosporidium spp. was observed in 19.8% (39/197) of the poultry specimens. Subsequent nested PCR targeting the 18S rRNA gene revealed that 15.7% (31/197) of the samples were Cryptosporidium positive. Of these 31 samples, 17 were Cryptosporidium baileyi (8.7%), 12 were Cryptosporidium meleagridis (6.0%), and 2 were Cryptosporidium parvum (1.0%). Nucleotide sequence analysis of the GP60 gene of the C. meleagridis revealed that two subtypes (IIIbA21G1R1 and IIIbA23G1R1), which were found in broiler, native and sonali chickens and a pigeon, matched those previously reported in humans and poultry. We identified two novel subtypes (IIIbA21G2R1 and IIIbA20G2R1) in sonali chickens, a broiler chicken and a layer chicken. We also amplified the GP60 gene of C. parvum and found two subtypes (IIaA11G2R1 and IIaA13G2R1) in a sonali and a broiler chicken that were previously reported in calf. These findings suggest that poultry can be a source of cryptosporidial infections for humans and animals in Bangladesh. This is the first molecular investigation of Cryptosporidium genotypes and subtypes in poultry at open live bird markets in Bangladesh. Keywords: Bangladesh, Cryptosporidium, Live bird markets, PCR-based sequencing, Phylogenetic analyses, Poultry

Abstract Background Malaria is a major infectious disease in the world. In 2015, approximately 212 million people were infected and 429,000 people were killed by this disease. Plasmodium falciparum, which causes falciparum malaria, is becoming resistant to artemisinin (ART) in Southeast Asia; therefore, new anti-malarial drugs are urgently needed. Some excellent anti-malarial drugs, such as quinine or ART, were originally obtained from natural plants. Hence, the authors screened a natural product library comprising traditional Chinese medicines (TCMs) to identify compounds/extracts with anti-malarial effects. Methods The authors performed three assays: a malaria growth inhibition assay (GIA), a cytotoxicity assay, and a malaria stage-specific GIA. The malaria GIA revealed the anti-malarial ability and half-maximal inhibitory concentrations (IC50) of the natural products, whereas the malaria stage-specific GIA revealed the point in the malaria life cycle where the products exerted their anti-malarial effects. The toxicity of the products to the host cells was evaluated with the cytotoxicity assay. Results Four natural compounds (berberine chloride, coptisine chloride, palmatine chloride, and dehydrocorydaline nitrate) showed strong anti-malarial effects (IC50 90%) using P. falciparum 3D7 strain. Two natural extracts (Phellodendri cortex and Coptidis rhizoma) also showed strong antiplasmodial effects (IC50 80%). These natural products also demonstrated anti-malarial capability during the trophozoite and schizont stages of the malaria life cycle. Conclusions The authors identified four compounds (berberine chloride, coptisine chloride, palmatine chloride, and dehydrocorydaline nitrate) and two extracts (Phellodendri cortex and Coptidis rhizoma) with anti-malarial activity, neither of which had previously been described. The IC50 values of the compounds were comparable to that of chloroquine and better than that of pyrimethamine. These compounds and extracts derived from TCMs thus show promise as potential future anti-malarial drugs.

Mucin-type O-glycosylation involves the attachment of glycans to an initial O-linked N-acetylgalactosamine (GalNAc) on serine and threonine residues on proteins. This process in mammals is initiated and regulated by a large family of 20 UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) (EC 2.4.1.41). The enzymes are encoded by a large gene family (GALNTs). Two of these genes, GALNT2 and GALNT3, are known as monogenic autosomal recessive inherited disease genes with well characterized phenotypes, whereas a broad spectrum of phenotypes is associated with the remaining 18 genes. Until recently, the overlapping functionality of the 20 members of the enzyme family has hindered characterizing the specific biological roles of individual enzymes. However, recent evidence suggests that these enzymes do not have full functional redundancy and may serve specific purposes that are found in the different phenotypes described. Here, we summarize the current knowledge of GALNT and associated phenotypes.

Background: Toxoplasmosis constitutes a large global burden that is further exacerbated by the shortcomings of available therapeutic options, thus underscoring the urgent need for better anti-Toxoplasma gondii therapy or strategies. Recently, we showed that the anti-parasitic action of inorganic nanoparticles (NPs) could, in part, be due to changes in redox status as well as in the parasite mitochondrial membrane potential. Methods: In the present study, we explored the in vitro mode of action of the anti-T. gondii effect of NPs by evaluating the contributions of host cellular processes, including the tryptophan pathway and hypoxia-inducing factor activity. NPs, at concentrations ranging from 0.01 to 200 µg/ml were screened for anti-parasitic activity. Sulfadiazine and/or pyrimethamine served as positive controls. Results: We found that interplay among multiple host cellular processes, including HIF-1α activity, indoleamine 2,3-dioxygenase activity, and to a larger extent the tryptophan pathway, contribute to the anti-parasitic action of NPs. Conclusion: To our knowledge, this is the first study to demonstrate an effect of NPs on the tryptophan and/or kynurenine pathway. General significance: Our findings deepen our understanding of the mechanism of action of NPs and suggest that modulation of the host nutrient pool may represent a viable approach to the development of new and effective anti-parasitic agents.

Neutrophils (PMNs) are the most abundant cellular component of our innate immune system, where they play central roles in the pathogenesis of and resistance to a broad range of diseases. However, their roles in malarial infection remain poorly understood. Therefore, we examined the transcriptional gene profile of human PMNs in response to Plasmodium falciparum-parasitized erythrocytes (iRBCs) by using oligonucleotide microarrays. Results revealed that PMNs induced a broad and vigorous set of changes in gene expression in response to malarial parasites, represented by 118 upregulated and 216 downregulated genes. The transcriptional response was characterized by the upregulation of numerous genes encoding multiple surface receptors, proteins involved in signal transduction pathways, and defense response proteins. This response included a number of genes which are known to be involved in the pathogenesis of malaria and other inflammatory diseases. Gene enrichment analysis suggested that the biological pathways involved in the PMN responses to the iRBCs included insulin receptor, Jak-STAT signaling pathway, mitogen-activated protein kinase (MAPK), and interleukin and interferon-gamma (IFN-γ) signaling pathways. The current study provides fundamental knowledge on the molecular responses of neutrophils to malarial parasites, which may aid in the discovery of novel therapeutic interventions.

Toxoplasma gondii is the causative pathogen for Toxoplasmosis. Bumped kinase inhibitor 1NM-PP1 inhibits the growth of T. gondii by targeting TgCDPK1. However, we recently reported that resistance to 1NM-PP1 can be acquired via a mutation in T. gondii mitogen-activated protein kinase like 1 (TgMAPKL-1). Further characterization of how this TgMAPKL-1 mutation restores the inhibitory effect of 1NM-PP1 would shed further light on the function of TgMAPKL-1 in the parasite life cycle. Therefore, we made parasite clones with TgMAPKL-1 mutated at the gatekeeper residue Ser 191, which is critical for 1NM-PP1 susceptibility. Host cell lysis of RH/ku80-/HA-TgMAPKL-1S191A was completely inhibited at 250 nM 1NM-PP1, whereas that of RH/ku80-/HA-TgMAPKL-1S191Y was not. By comparing 1NM-PP1-sensitive (RH/ku80-/HA-TgMAPKL-1S191A) and -resistant (RH/ku80-/HA-TgMAPKL-1S191Y) clones, we observed that inhibition of TgMAPKL-1 blocked cell cycle progression after DNA duplication. Morphological analysis revealed that TgMAPKL-1 inhibition caused enlarged parasite cells with many daughter cell scaffolds and imcomplete cytokinesis. We conclude that the mutation in TgMAPKL-1 restored the cell cycle-arresting effect of 1NM-PP1 on T. gondii endodyogeny. Given that endodyogeny is the primary mechanism of cell division for both the tachyzoite and bradyzoite stages of this parasite, TgMAPKL-1 may be a promising target for drug development. Exploration of the signals that regulate TgMAPKL-1 will provide further insights into the unique mode of T. gondii cell division.

Drug treatment for toxoplasmosis is problematic, because current drugs cannot eradicate latent infection with Toxoplasma gondii and can cause bone marrow toxicity. Because latent infection remains after treatment, relapse of infection is a problem in both infections in immunocompromised patients and in congenitally infected patients. To identify lead compounds for novel drugs against Toxoplasma gondii, we screened a chemical compound library for anti-Toxoplasma activity, host cell cytotoxicity, and effect on bradyzoites. Of 878 compounds screened, 83 demonstrated >90% parasite growth inhibition. After excluding compounds that affected host cell viability, we further characterized two compounds, tanshinone IIA and hydroxyzine, which had IC50 values for parasite growth of 2.5 μM and 1.0 μM, respectively, and had no effect on host cell viability at 25 μM. Both tanshinone IIA and hydroxyzine inhibited parasite replication after invasion and both reduced the number of in vitro-induced bradyzoites, whereas, pyrimethamine, the current therapy, had no effect on bradyzoites. Both tanshinone IIA and hydroxyzine are potent lead compounds for further medicinal chemistry. The method presented for evaluating compounds for bradyzoite efficacy represents a new approach to the development of anti-Toxoplasma drugs to eliminate latency and treat acute infection.

Galactose and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica has roles in pathogenicity and induction of protective immunity in rodent models of amoebiasis. Recently, the intermediate subunit of the lectin, Igl1, of E. histolytica has been shown to have hemolytic activity. However, the corresponding lectin is also expressed in a non-virulent species, Entamoeba dispar, and another subunit, Igl2, is expressed in the protozoa. Therefore, in this study, we compared the activities of Igl1 and Igl2 subunits from E. histolytica and E. dispar using various regions of recombinant Igl proteins expressed in Escherichia coli. The recombinant E. dispar Igl proteins had comparable hemolytic activities with those of E. histolytica Igl proteins. Furthermore, Igl1 gene-silenced E. histolytica trophozoites showed less hemolytic activity compared with vector-transfected trophozoites, indicating that the expression level of Igl1 protein influences the activity. These results suggest that the lower hemolytic activity in E. dispar compared with E. histolytica reflects the lower expression level of Igl1 in the E. dispar parasite.

In this study, the in vitro effects of amodiaquine (AQ) monotherapy on the egg output of paired adult Schistosoma mansoni worms and their survival during in vitro culture were assessed. In addition, the gross morphological alterations of male and female worms caused by AQ were visually observed under a dissecting microscope. AQ significantly reduced the daily egg output of paired adult S. mansoni worms following incubation for 14 days at 1-5 µg/mL, but not at 0.5 µg/mL, compared with the control group. AQ also reduced the survival of male and female worms at concentrations of 2 and 5 µg/mL, respectively. Moreover, exposure to 5 µg/mL AQ caused severe swelling and/or localisation of black content in the body of all male and female worms within one or two days of incubation; subsequently, shrinkage in the male worms and elongation in the female worms were observed. The initial morphological alterations caused by AQ occurred along the intestinal tract of the male and female worms. To our knowledge, this is the first study to report not only the efficacy of AQ at concentrations lower than 5 µg/mL on paired adult S. mansoni worms, but also the effects of AQ on the intestinal tracts of worms in in vitro culture.

In this study, the in vitro effects of amodiaquine (AQ) monotherapy on the egg output of paired adult Schistosoma mansoni worms and their survival during in vitro culture were assessed. In addition, the gross morphological alterations of male and female worms caused by AQ were visually observed under a dissecting microscope. AQ significantly reduced the daily egg output of paired adult S. mansoni worms following incubation for 14 days at 1-5 µg/mL, but not at 0.5 µg/mL, compared with the control group. AQ also reduced the survival of male and female worms at concentrations of 2 and 5 µg/mL, respectively. Moreover, exposure to 5 µg/mL AQ caused severe swelling and/or localisation of black content in the body of all male and female worms within one or two days of incubation; subsequently, shrinkage in the male worms and elongation in the female worms were observed. The initial morphological alterations caused by AQ occurred along the intestinal tract of the male and female worms. To our knowledge, this is the first study to report not only the efficacy of AQ at concentrations lower than 5 µg/mL on paired adult S. mansoni worms, but also the effects of AQ on the intestinal tracts of worms in in vitro culture.

ABSTRACT Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects warm-blooded vertebrates, including humans. Asexual reproduction in T. gondii allows it to switch between the rapidly replicating tachyzoite and quiescent bradyzoite life cycle stages. A transient cyclic AMP (cAMP) pulse promotes bradyzoite differentiation, whereas a prolonged elevation of cAMP inhibits this process. We investigated the mechanism(s) by which differential modulation of cAMP exerts a bidirectional effect on parasite differentiation. There are three protein kinase A (PKA) catalytic subunits (TgPKAc1 to -3) expressed in T. gondii. Unlike TgPKAc1 and TgPKAc2, which are conserved in the phylum Apicomplexa, TgPKAc3 appears evolutionarily divergent and specific to coccidian parasites. TgPKAc1 and TgPKAc2 are distributed in the cytomembranes, whereas TgPKAc3 resides in the cytosol. TgPKAc3 was genetically ablated in a type II cyst-forming strain of T. gondii (PruΔku80Δhxgprt) and in a type I strain (RHΔku80Δhxgprt), which typically does not form cysts. The Δpkac3 mutant exhibited slower growth than the parental and complemented strains, which correlated with a higher basal rate of tachyzoite-to-bradyzoite differentiation. 3-Isobutyl-1-methylxanthine (IBMX) treatment, which elevates cAMP levels, maintained wild-type parasites as tachyzoites under bradyzoite induction culture conditions (pH 8.2/low CO2), whereas the Δpkac3 mutant failed to respond to the treatment. This suggests that TgPKAc3 is the factor responsible for the cAMP-dependent tachyzoite maintenance. In addition, the Δpkac3 mutant had a defect in the production of brain cysts in vivo, suggesting that a substrate of TgPKAc3 is probably involved in the persistence of this parasite in the intermediate host animals. IMPORTANCE Toxoplasma gondii is one of the most prevalent eukaryotic parasites in mammals, including humans. Parasites can switch from rapidly replicating tachyzoites responsible for acute infection to slowly replicating bradyzoites that persist as a latent infection. Previous studies have demonstrated that T. gondii cAMP signaling can induce or suppress bradyzoite differentiation, depending on the strength and duration of cAMP signal. Here, we report that TgPKAc3 is responsible for cAMP-dependent tachyzoite maintenance while suppressing differentiation into bradyzoites, revealing one mechanism underlying how this parasite transduces cAMP signals during differentiation.

Fifty-two bats captured during July 2008 in the Philippines were tested by reverse transcription–PCR to detect bat coronavirus (CoV) RNA. The overall prevalence of virus RNA was 55.8%. We found 2 groups of sequences that belonged to group 1 (genus Alphacoronavirus) and group 2 (genus Betacoronavirus) CoVs. Phylogenetic analysis of the RNA-dependent RNA polymerase gene showed that groups 1 and 2 CoVs were similar to Bat-CoV/China/A515/2005 (95% nt sequence identity) and Bat-CoV/HKU9–1/China/2007 (83% identity), respectively. To propagate group 2 CoVs obtained from a lesser dog-faced fruit bat (Cynopterus brachyotis), we administered intestine samples orally to Leschenault rousette bats (Rousettus leschenaulti) maintained in our laboratory. After virus replication in the bats was confirmed, an additional passage of the virus was made in Leschenault rousette bats, and bat pathogenesis was investigated. Fruit bats infected with virus did not show clinical signs of infection.

Central nervous system injury induces a regenerative response in ensheathing glial cells comprising cell proliferation, spontaneous axonal remyelination, and limited functional recovery, but the molecular mechanisms are not fully understood. In Drosophila, this involves the genes prospero and Notch controlling the balance between glial proliferation and differentiation, and manipulating their levels in glia can switch the response to injury from prevention to promotion of repair. In the mouse, Notch1 maintains NG2 oligodendrocyte progenitor cells (OPCs) in a progenitor state, but what factor may enable oligodendrocyte (OL) differentiation and functional remyelination is not understood. Here, we asked whether the mammalian homologue of prospero, Prox1, is involved. Our data show that Prox1 is distributed in NG2+ OPCs and in OLs in primary cultured cells, and in the mouse spinal cord in vivo. siRNA prox1 knockdown in primary OPCs increased cell proliferation, increased NG2+ OPC cell number and decreased CC1+ OL number. Prox1 conditional knockout in the OL cell lineage in mice increased NG2+ OPC cell number, and decreased CC1+ OL number. Lysolecithin-induced demyelination injury caused a reduction in CC1+ OLs in homozygous Prox1-/- conditional knockout mice compared to controls. Remarkably, Prox1-/- conditional knockout mice had smaller lesions than controls. Altogether, these data show that Prox1 is required to inhibit OPC proliferation and for OL differentiation, and could be a relevant component of the regenerative glial response. Therapeutic uses of glia and stem cells to promote regeneration and repair after central nervous system injury would benefit from manipulating Prox1.

Because bats are associated with emerging zoonoses, identification and characterization of novel viruses from bats is needed. Using a modified rapid determination system for viral RNA/DNA sequences, we identified a novel bat betaherpesvirus 2 not detected by herpesvirus consensus PCR. This modified system is useful for detecting unknown viruses.

Toxoplasma gondii is an intracellular parasite that invades nucleated cells, causing toxoplasmosis in humans and animals worldwide. The extremely wide range of hosts susceptible to T. gondii is thought to be the result of interactions between T. gondii ligands and receptors on its target cells. In this study, a host cell-binding protein from T. gondii was characterized, and one of its receptors was identified. P104 (GenBank Access. No. CAJ20677) is 991 amino acids in length, containing a putative 26 amino acid signal peptide and 10 PAN/apple domains, and shows low homology to other identified PAN/apple domain-containing molecules. A 104-kDa host cell-binding protein was detected in the T. gondii lysate. Immunofluorescence assays detected P104 at the apical end of extracellular T. gondii. An Fc-fusion protein of the P104 N-terminus, which contains two PAN/apple domains, showed strong affinity for the mammalian and insect cells evaluated. This binding was not related to protein-protein or protein-lipid interactions, but to a protein-glycosaminoglycan (GAG) interaction. Chondroitin sulfate (CS), a kind of GAG, was shown to be involved in adhesion of the Fc-P104 N-terminus fusion protein to host cells. These results suggest that P104, expressed at the apical end of the extracellular parasite, may function as a ligand in the attachment of T. gondii to CS or other receptors on the host cell, facilitating invasion by the parasite.

Organisms are structurally robust, as cells accommodate changes preserving structural integrity and function. The molecular mechanisms underlying structural robustness and plasticity are poorly understood, but can be investigated by probing how cells respond to injury. Injury to the CNS induces proliferation of enwrapping glia, leading to axonal re-enwrapment and partial functional recovery. This glial regenerative response is found across species, and may reflect a common underlying genetic mechanism. Here, we show that injury to the Drosophila larval CNS induces glial proliferation, and we uncover a gene network controlling this response. It consists of the mutual maintenance between the cell cycle inhibitor Prospero (Pros) and the cell cycle activators Notch and NFκB. Together they maintain glia in the brink of dividing, they enable glial proliferation following injury, and subsequently they exert negative feedback on cell division restoring cell cycle arrest. Pros also promotes glial differentiation, resolving vacuolization, enabling debris clearance and axonal enwrapment. Disruption of this gene network prevents repair and induces tumourigenesis. Using wound area measurements across genotypes and time-lapse recordings we show that when glial proliferation and glial differentiation are abolished, both the size of the glial wound and neuropile vacuolization increase. When glial proliferation and differentiation are enabled, glial wound size decreases and injury-induced apoptosis and vacuolization are prevented. The uncovered gene network promotes regeneration of the glial lesion and neuropile repair. In the unharmed animal, it is most likely a homeostatic mechanism for structural robustness. This gene network may be of relevance to mammalian glia to promote repair upon CNS injury or disease.

cAMP-dependent protein kinase (PKA) has been implicated in the asexual stage of the Toxoplasma gondii life cycle through assaying the effect of a PKA-specific inhibitor on its growth rate. Since inhibition of the host cell PKA cannot be ruled out, a more precise evaluation of the role of PKA, as well as characterization of the kinase itself, is necessary.The inhibitory effects of two PKA inhibitors, H89, an ATP-competitive chemical inhibitor, and PKI, a substrate-competitive mammalian natural peptide inhibitor, were estimated. In the in vitro kinase assay, the inhibitory effect of PKI on a recombinant T. gondii PKA catalytic subunit (TgPKA-C) was weaker compared to that on mammalian PKA-C. In a tachyzoite growth assay, PKI had little effect on the growth of tachyzoites, whereas H89 strongly inhibited it. Moreover, T. gondii PKA regulatory subunit (TgPKA-R)-overexpressing tachyzoites showed a significant growth defect.Our data suggest that PKA plays an important role in the growth of tachyzoites, and the inhibitory effect of substrate-competitive inhibitor PKI on T. gondii PKA was low compared to that of the ATP competitive inhibitor H89.