Unlocking the mystery of the feeder organelle and versatile energy metabolism in Cryptosporidium parvum.

Xu and colleagues recently revealed the critical role of Cryptosporidium's feeder organelle in nutrient uptake, showcasing the parasite's ability to harness glucose and glucose-6-phosphate from host cells. This illuminates the sophisticated energy metabolism and survival strategies of the parasite, highlighting potential therapeutic targets.

Requirement of microtubules for secretion of a micronemal protein CpTSP4 in the invasive stage of the apicomplexan Cryptosporidium parvum.

The zoonotic Cryptosporidium parvum is a global contributor to infantile diarrheal diseases and opportunistic infections in immunocompromised or weakened individuals. Like other apicomplexans, it possesses several specialized secretory organelles, including micronemes, rhoptry, and dense granules. However, the understanding of cryptosporidial micronemal composition and secretory pathway remains limited. Here, we report a new micronemal protein in C. parvum, namely, thrombospondin (TSP)-repeat domain-containing protein-4 (CpTSP4), providing insights into these ambiguities. Immunostaining and enzyme-linked assays show that CpTSP4 is prestored in the micronemes of unexcysted sporozoites but secreted during sporozoite excystation, gliding, and invasion. In excysted sporozoites, CpTSP4 is also distributed on the two central microtubules unique to Cryptosporidium. The secretion and microtubular distribution could be completely blocked by the selective kinesin-5 inhibitors SB-743921 and SB-715992, resulting in the accumulation of CpTSP4 in micronemes. These support the kinesin-dependent microtubular trafficking of CpTSP4 for secretion. We also localize γ-tubulin, consistent with kinesin-dependent anterograde trafficking. Additionally, recombinant CpTSP4 displays nanomolar binding affinity to the host cell surface, for which heparin acts as one of the host ligands. A novel heparin-binding motif is identified and validated biochemically for its contribution to the adhesive property of CpTSP4 by peptide competition assays and site-directed mutagenesis. These findings shed light on the mechanisms of intracellular trafficking and secretion of a cryptosporidial micronemal protein and the interaction of a TSP-family protein with host cells.IMPORTANCECryptosporidium parvum is a globally distributed apicomplexan parasite infecting humans and/or animals. Like other apicomplexans, it possesses specialized secretory organelles in the zoites, in which micronemes discharge molecules to facilitate the movement and invasion of zoites. Although past and recent studies have identified several proteins in cryptosporidial micronemes, our understanding of the composition, secretory pathways, and domain-ligand interactions of micronemal proteins remains limited. This study identifies a new micronemal protein, namely, CpTSP4, that is discharged during excystation, gliding, and invasion of C. parvum sporozoites. The CpTSP4 secretion depends on the intracellular trafficking on the two Cryptosporidium-unique microtubes that could be blocked by kinesin-5/Eg5 inhibitors. Additionally, a novel heparin-binding motif is identified and biochemically validated, which contributes to the nanomolar binding affinity of CpTSP4 to host cells. These findings indicate that kinesin-dependent microtubular trafficking is critical to CpTSP4 secretion, and heparin/heparan sulfate is one of the ligands for this micronemal protein.

YiQiFuMai Injection Ameliorated Sepsis-induced Cardiomyopathy by Inhibition of Ferroptosis via xCT/GPX4 Axis.

ABSTRACT: Sepsis-induced cardiomyopathy (SIC) is a distinct form of myocardial injury that disrupts tissue perfusion and stands as the significant cause of mortality among sepsis patients. Currently, effective preventive or treatment strategies for SIC are lacking. YiQiFuMai injection (YQFM), composed of Panax ginseng C.A. Mey., Ophiopogon japonicus (Thunb.) Ker Gawl., and Schisandra chinensis (Turcz.) Baill., is widely used in China to treat cardiovascular diseases, such as coronary heart disease, heart failure and SIC. Research has shown that YQFM can improve cardiac function and alleviate heart failure through multiple pathways. Nevertheless, the mechanisms through which YQFM exerts its effects on SIC remain to be fully elucidated. In this study, we firstly investigated the therapeutic effects of YQFM on a SIC rat model and explored its effects on myocardial ferroptosis in vivo. Then, LPS-induced myocardial cell death model was used to evaluate the effects of YQFM on ferroptosis and xCT/GPX4 axis in vitro. Furthermore, using GPX4 inhibitors, we aimed to verify whether YQFM improved cardiomyocyte ferroptosis through the xCT/GPX4 axis. The results showed that YQFM was effective in alleviating myocardial injury in septic model rats. Besides, the concentrations of iron and the levels of lipid peroxidation-related factors (ROS, MDA and 4-HNE) were significantly decreased and the expression of xCT/GPX4 axis was up-regulated in SIC rats after YQFM treatment. In vitro studies also showed that YQFM alleviated iron overload and lipid peroxidation and activated xCT/GPX4 axis in LPS-induced myocardial cell death model. Moreover, GPX4 inhibitor could abolish the effects above. In summary, the study highlights the regulatory effect of YQFM in mitigating myocardial injury. It probably achieves this ameliorative effect by enhancing xCT/GPX4 axis and further reducing ferroptosis.

Research on the descaling characteristics of a new electrochemical water treatment device.

In recent years, chemical water treatment equipment has gained significant attention due to its environmental-friendly features, multifunctionality, and broad applicability. Recognizing the limitations of existing chemical treatment equipment, such as challenges in scale removal and the high water content in scale deposits, we propose a novel drum design for both anode and cathode, enabling simultaneous scale suction and dehydration. We constructed a small experimental platform to validate the equipment's performance based on our model. Notably, under the optimal operating parameters, the hardness removal rate for circulating water falls within the range of 19.6-24.46%. Moreover, the scale accumulation rate per unit area and unit time reaches 13.7 g h-1 m-2. Additionally, the energy consumption per unit weight of the scale remains impressively low at 0.16 kWh g-1. Furthermore, the chemical oxygen demand (COD) concentration decreased from an initial 106.0 mg L-1 to a mere 18.8 mg L-1, resulting in a remarkable total removal rate of 82.26%. In conclusion, our innovative electrochemical water treatment equipment demonstrates exceptional performance in scale removal, organic matter degradation, and water resource conservation, offering valuable insights for future research and development in chemical treatment equipment and electrochemical theory.

Cost-effective In Vivo and In Vitro Mouse Models for Evaluating Anticryptosporidial Drug Efficacy: Assessing Vorinostat, Docetaxel, and Baicalein.

BACKGROUND: Cryptosporidiosis is a significant diarrheal disease in humans and animals. Immunodeficient mice are the primary small animal models, but their high costs and specialized breeding/housing requirements limit in vivo drug testing. Numerous anticryptosporidial lead compounds identified in vitro remain untested in vivo. METHODS: Cryptosporidium tyzzeri, a natural mouse parasite closely related to Cryptosporidium parvum and Cryptosporidium hominis, was isolated to establish an infection model in immunocompetent mice. The model was validated using classic anticryptosporidial drugs (paromomycin and nitazoxanide) and then employed to assess the efficacy of 3 new leads (vorinostat, docetaxel, and baicalein). An in vitro culture of C. tyzzeri was also developed to complement the animal model. RESULTS: Chronic C. tyzzeri infection was established in chemically immunosuppressed wild-type mice. Paromomycin (1000 mg/kg/d) and nitazoxanide (100 mg/kg/d) demonstrated efficacy against C. tyzzeri. Vorinostat (30 mg/kg/d), docetaxel (25 mg/kg/d), and baicalein (50 mg/kg/d) were highly effective against C. tyzzeri infection. In vitro, nitazoxanide, vorinostat, docetaxel, and baicalein exhibited low to submicromolar efficacy against C. tyzzeri. CONCLUSIONS: Novel in vivo and in vitro models have been developed for cost-effective anticryptosporidial drug testing. Vorinostat, docetaxel, and baicalein show potential for repurposing and/or optimization for developing new anticryptosporidial drugs.

Liang-Ge Decoction Ameliorates Coagulation Dysfunction in Cecal Ligation and Puncture-Induced Sepsis Model Rats through Inhibiting PAD4-Dependent Neutrophil Extracellular Trap Formation.

Liang-Ge (LG) decoction could ameliorate coagulation dysfunction in septic model rats. However, the mechanism of LG in treating sepsis still needs to be clarified. Our current study established a septic rat model to evaluate the effect of LG on coagulation dysfunction in septic rats first. Second, we investigated the effect of LG on NET formation in septic rats. Finally, NETs and PAD4 inhibitors were further used to clarify if LG could improve the mechanism of sepsis coagulation dysfunction by inhibiting NET formation. Our findings indicated that treatment with LG improved the survival rate, reduced inflammatory factor levels, enhanced hepatic and renal function, and reduced pathological changes in rats with sepsis. LG could also alleviate coagulation dysfunction in septic model rats. Besides, LG treatment reduced NETs formation and decreased PAD4 expression in neutrophiles. In addition, LG treatment showed a similar result in comparison to the treatment with either NET inhibitors or PAD4 inhibitors alone. In conclusion, this study confirmed that LG has therapeutic effects on septic rats. Furthermore, the improvement of coagulation dysfunction in septic rats by LG was achieved through inhibiting PAD4-mediated NET formation.

On-target inhibition of Cryptosporidium parvum by nitazoxanide (NTZ) and paclitaxel (PTX) validated using a novel MDR1-transgenic host cell model and algorithms to quantify the effect on the parasite target.

Cryptosporidium parvum is a globally distributed zoonotic protozoan parasite that causes moderate to severe, sometime deadly, watery diarrhea in humans and animals, for which fully effective treatments are yet unavailable. In studying the mechanism of action of drugs against intracellular pathogens, it is important to validate whether the observed anti-infective activity is attributed to the drug action on the pathogen or host target. For the epicellular parasite Cryptosporidium, we have previously developed a concept that the host cells with significantly increased drug tolerance by transient overexpression of the multidrug resistance protein-1 (MDR1) could be utilized to evaluate whether and how much the observed anti-cryptosporidial activity of an inhibitor was attributed to the inhibitor's action on the parasite target. However, the transient transfection model was only applicable to evaluating native MDR1 substrates. Here we report an advanced model using stable MDR1-transgenic HCT-8 cells that allows rapid development of novel resistance to non-MDR1 substrates by multiple rounds of drug selection. Using the new model, we successfully validated that nitazoxanide, a non-MDR1 substrate and the only FDA-approved drug to treat human cryptosporidiosis, killed C. parvum by fully (100%) acting on the parasite target. We also confirmed that paclitaxel acted fully on the parasite target, while several other inhibitors including mitoxantrone, doxorubicin, vincristine and ivermectin acted partially on the parasite targets. Additionally, we developed mathematical models to quantify the proportional contribution of the on-parasite-target effect to the observed anti-cryptosporidial activity and to evaluate the relationships between several in vitro parameters, including antiparasitic efficacy (ECi), cytotoxicity (TCi), selectivity index (SI) and Hill slope (h). Owning to the promiscuity of the MDR1 efflux pump, the MDR1-transgenic host cell model could be applied to assess the on-parasite-target effects of newly identified hits/leads, either substrates or non-substrates of MDR1, against Cryptosporidium or other epicellular pathogens.

Genomic reconstruction and features of glycosylation pathways in the apicomplexan Cryptosporidium parasites.

Cryptosporidium is a genus of apicomplexan parasites infecting humans or other vertebrates. The majority of the Cryptosporidium species live in host intestines (e.g., C. parvum, C. hominis and C. ubiquitum), but there are a few gastric species (e.g., C. muris and C. andersoni). Among them, C. parvum is the most important zoonotic species, for which a number of glycoproteins have been reported for being involved in the interacting with host cells. However, little is known on the cryptosporidium glycobiology. Information on the glycosylation pathways in Cryptosporidium parasites remains sketchy and only a few studies have truly determined the glycoforms in the parasites. Here we reanalyzed the Cryptosporidium genomes and reconstructed the glycosylation pathways, including the synthesis of N- and O-linked glycans and GPI-anchors. In N-glycosylation, intestinal Cryptosporidium possesses enzymes to make a simple precursor with two terminal glucoses on the long arm (i.e., Glc2Man5GlcNAc2 vs. Glc3Man9GlcNAc2 in humans), but gastric species only makes a simpler precursor containing only the "core" structure (i.e., Man3GlcNAc2). There is an ortholog of glucosidase II (GANAB) in all Cryptosporidium species, for which the authenticity is questioned because it contains no signal peptide and exist in gastric species lacking terminal glucoses for the enzyme to act on. In O-linked glycosylation, all Cryptosporidium species may attach one-unit HexNAc (GalNAc and GlcNAc) and two-unit Fuc-type (Man-Fuc) glycans to the target proteins. Cryptosporidium lacks enzymes to further process N- and O-glycans in the Golgi. The glycosylphosphatidylinositol (GPI)-anchor in Cryptosporidium is predicted to be unbranched and unprocessed further in the Golgi. Cryptosporidium can synthesize limited nucleotide sugars, but possesses at least 12 transporters to scavenge nucleotide sugars or transport them across the ER/Golgi membranes. Overall, Cryptosporidium makes much simpler glycans than the hosts, and the N-glycoforms further differ between intestinal and gastric species. The Cryptosporidium N- and O-glycans are neutrally charged and have limited capacity to absorb water molecules in comparison to the host intestinal mucins that are negatively charged and highly expandable in waters.

Forsythiaside B ameliorates coagulopathies in a rat model of sepsis through inhibition of the formation of PAD4-dependent neutrophil extracellular traps.

Forsythiaside B (FTB) is one of the main components of Forsythia suspensa (Thunb.) Vahl and exerts anti-inflammatory and anti-oxidative effects. However, its mechanism of action as a treatment for sepsis remains unclear. In this study, we developed a rat model of sepsis using cecal ligation and puncture (CLP) to investigate the effects of FTB on sepsis-associated coagulopathies. Using rats with sepsis, we investigated the effects of FTB on neutrophil extracellular trap (NETs) formation and peptidylarginine deiminase 4 (PAD4) expression in neutrophils. NET (DNase1) and PAD4 (Cl-amidine) inhibitors were used to further investigate whether FTB mitigates sepsis-associated coagulopathies by inhibiting PAD4-dependent NETs production. Our results showed that treatment with FTB increased the survival rate, ameliorated the CLP-induced inflammatory response and multiple organ dysfunction, and reduced CLP-induced pathological changes. FTB also alleviated the associated coagulopathies. Additionally, we demonstrated that treatment with FTB inhibited NETs formation and downregulated PAD4 expression in peripheral neutrophils. The effects of FTB on coagulopathies were similar to those of monotherapy with NET or PAD4 inhibitors. In conclusion, our study confirmed that FTB can alleviate coagulopathies in rats with sepsis. The underlying mechanism of FTB's effect consists in inhibition of PAD4-dependent NETs formation.

Liang-Ge decoction ameliorates acute lung injury in septic model rats through reducing inflammatory response, oxidative stress, apoptosis, and modulating host metabolism.

Liang-Ge decoction (LG) has been used in the treatment of early stage of spesis and can ameliorate sepsis-associated lung injury. However, the mechanism of LG on sepsis-associated lung injury remains unknown. In this study, we established a rat model of sepsis-associated lung injury using the cecal ligation and puncture (CLP) method, and investigated the therapeutic effects of LG on lung injury in rats with sepsis. In addition, the anti-inflammatory, anti-oxidative and anti-apoptotic effects of LG on sepsis-associated lung injury model rats were evaluated. Besides, untargeted metabolomics was used to investigate the regulation of metabolites in rats with sepsis-associated lung injury after LG treatment. Our results showed that LG could decrease the wet/dry (W/D) ratio in lung and the total cell count and total protein concentration in bronchoalveolar lavage fluid (BALF) in septic model rats. Hematoxylin and eosin (HE) staining showed that LG reduced the infiltration of pro-inflammatory cells in lung. In addition, LG treatmment down-regulated the gene and protein expression of pro-inflammatory cytokins in lung tissue and BALF. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were increased and the level of methane dicarboxylic aldehyde (MDA) was decreased in lung tissue homogenate in septic model rats after LG treament. Moreover, the numbers of apoptotic cells in lung were reduced and the activity of lactic dehydrogenase (LDH) in BALF was decreased in septic model rats after LG treament. Untargeted metabolomics analysis showed that LG treatment affected the levels of 23 metabolites in lung in septic model rats such as citric acid, methionine, threonine, alpha-ketoglutaric acid, and inositol, these metabolites were associated with the glycine, serine and threonine metabolism, cysteine and methionine metabolism, inositol phosphate metabolism and citrate cycle (TCA cycle) pathways. In conclusion, our study demonstrated the therapeutic effetcts of LG on sepsis-associated lung injury model rats. Moreover, LG could inhibit the inflammatory response, oxidative stress, apoptosis and regulate metabolites related to glycine, serine and threonine metabolism, cysteine and methionine metabolism, inositol phosphate metabolism and TCA cycle in lung in sepsis-associated lung injury model rats.

Jian-Ti-Kang-Yi decoction alleviates poly(I:C)-induced pneumonia by inhibiting inflammatory response, reducing oxidative stress, and modulating host metabolism.

Jian-Ti-Kang-Yi decoction (JTKY) is widely used in the treatment of COVID-19. However, the protective mechanisms of JTKY against pneumonia remain unknown. In this study, polyinosinic-polycytidylic acid (poly(I:C)), a mimic of viral dsRNA, was used to induce pneumonia in mice; the therapeutic effects of JTKY on poly(I:C)-induced pneumonia model mice were evaluated. In addition, the anti-inflammatory and anti-oxidative potentials of JTKY were also investigated. Lastly, the metabolic regulatory effects of JTKY in poly(I:C)-induced pneumonia model mice were studied using untargeted metabolomics. Our results showed that JTKY treatment decreased the wet-to-dry ratio in the lung tissue, total protein concentration, and total cell count of the bronchoalveolar lavage fluid (BALF). Hematoxylin and Eosin (HE) and Masson staining indicated that the JTKY treatment alleviated the pathological changes and decreased the fibrotic contents in the lungs. JTKY treatment also decreased the expression of pro-inflammatory cytokines [interleukin (IL)-1ß, IL-6, and tumor necrosis factor-alpha (TNF-α)] and increased the levels of immunomodulatory cytokines (IL-4 and IL-10) in the BALF and serum. Flow cytometry analysis showed that the JTKY treatment lowered the ratio of CD86+/CD206+ macrophages in the BALF, decreased inducible nitric oxide synthase (iNOS) level, and increased arginase 1 (Arg-1) level in lung. JTKY also lowered CD11b+Ly6G+ neutrophils in BALF and decreased myeloperoxidase (MPO) activity in lung. Moreover, it also elevated superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and decreased methane dicarboxylic aldehyde (MDA) level in lung. Untargeted metabolomic analysis showed that the JTKY treatment could affect 19 metabolites in lung, such as L-adrenaline, L-asparagine, ornithine, and alpha-ketoglutaric acid. These metabolites are associated with the synthesis and degradation of ketone bodies, butanoate, alanine, aspartate, and glutamate metabolism, and tricarboxylic acid (TCA) cycle processes. In conclusion, our study demonstrated that treatment with JTKY ameliorated poly(I:C)-induced pneumonia. The mechanism of action of JTKY may be associated with the inhibition of the inflammatory response, the reduction of oxidative stress, and the regulation of the synthesis and degradation of ketone bodies, TCA cycle, and metabolism of alanine, aspartate, glutamate, and butanoate processes in lung.

The mucin-like, secretory type-I transmembrane glycoprotein GP900 in the apicomplexan Cryptosporidium parvum is cleaved in the secretory pathway and likely plays a lubrication role.

BACKGROUND: Cryptosporidium parvum is a zoonotic parasite and member of the phylum Apicomplexa with unique secretory organelles, including a rhoptry, micronemes and dense granules that discharge their contents during parasite invasion. The mucin-like glycoprotein GP900 with a single transmembrane domain is an immunodominant antigen and micronemal protein. It is relocated to the surface of excysted sporozoites and shed to form trails by sporozoites exhibiting gliding motility (gliding sporozoites). However, the biological process underlying its relocation and shedding remains unclear. The primary aim of this study was to determine whether GP900 is present as a transmembrane protein anchored to the plasma membrane on the surface of sporozoites and whether it is cleaved before being shed from the sporozoites. METHODS: Two anti-GP900 antibodies, a mouse monoclonal antibody (mAb) to the long N-terminal domain (GP900-N) and a rabbit polyclonal antibody (pAb) to the short C-terminal domain (GP900-C), were produced for the detection of intact and cleaved GP900 proteins in sporozoites and other parasite developmental stages by microscopic immunofluorescence assay and in discharged molecules by enzyme-linked immunosorbent assay. RESULTS: Both anti-GP900 antibodies recognized the apical region of unexcysted and excysted sporozoites. However, anti-GP900-N (but not anti-GP900-C) also stained both the pellicles/surface of excysted sporozoites and the trails of gliding sporozoites. Both antibodies stained the intracellular meronts, both developing and developed, but not the macro- and microgamonts. Additionally, the epitope was recognized by anti-GP900-N (but not anti-GP900-C) and detected in the secretions of excysted sporozoites and intracellular parasites. CONCLUSIONS: GP900 is present in sporozoites and intracellular meronts, but absent in sexual stages. It is stored in the micronemes of sporozoites, but enters the secretory pathway during excystation and invasion. The short cytoplasmic domain of GP900 is cleaved in the secretory pathway before it reaches the extracellular space. The molecular features and behavior of GP900 imply that it plays mainly a lubrication role.

Discovery of New Microneme Proteins in Cryptosporidium parvum and Implication of the Roles of a Rhomboid Membrane Protein (CpROM1) in Host-Parasite Interaction.

Apicomplexan parasites possess several unique secretory organelles, including rhoptries, micronemes, and dense granules, which play critical roles in the invasion of host cells. The molecular content of these organelles and their biological roles have been well-studied in Toxoplasma and Plasmodium, but are underappreciated in Cryptosporidium, which contains many parasites of medical and veterinary importance. Only four proteins have previously been identified or proposed to be located in micronemes, one of which, GP900, was confirmed using immunogold electron microscopy (IEM) to be present in the micronemes of intracellular merozoites. Here, we report on the discovery of four new microneme proteins (MICs) in the sporozoites of the zoonotic species C. parvum, identified using immunofluorescence assay (IFA). These proteins are encoded by cgd3_980, cgd1_3550, cgd1_3680, and cgd2_1590. The presence of the protein encoded by cgd3_980 in sporozoite micronemes was further confirmed using IEM. Cgd3_980 encodes one of the three C. parvum rhomboid peptidases (ROMs) and is, thus, designated CpROM1. IEM also confirmed the presence of CpROM1 in the micronemes of intracellular merozoites, parasitophorous vacuole membranes (PVM), and feeder organelles (FO). CpROM1 was enriched in the pellicles and concentrated at the host cell-parasite interface during the invasion of sporozoites and its subsequent transformation into trophozoites. CpROM1 transcript levels were also higher in oocysts and excysted sporozoites than in the intracellular parasite stages. These observations indicate that CpROM1, an intramembrane peptidase with membrane proteolytic activity, is involved in host-parasite interactions, including invasion and proteostasis of PVM and FO.

Host cells with transient overexpression of MDR1 as a novel in vitro model for evaluating on-target effect for activity against the epicellular Cryptosporidium parasite.

OBJECTIVES: To rapidly generate host cells with resistance to multiple compounds for differentiating drug action on parasite target or the host cell target (i.e. on-target or off-target effect) against the zoonotic enteric parasite Cryptosporidium parvum. METHODS: Transient overexpression of a multidrug resistance protein 1 (MDR1) gene in host cells (HCT-8 cell line) was explored to increase drug tolerance of the host cells to selected anti-cryptosporidial leads. In vitro cytotoxicity and anti-cryptosporidial efficacy of selected compounds were evaluated on the parasite grown in WT parental and transiently transfected HCT-8 cells. The approach was based on the theory that, for an epicellular parasite receiving consistent exposure to compounds in culture medium, overexpressing MDR1 in HCT-8 cells would increase drug tolerance of host cells to selected compounds but would not affect the anti-cryptosporidial efficacy if the compounds acted solely on the parasite target and the drug action on host cell target played no role on the antiparasitic efficacy. RESULTS: Six known anti-cryptosporidial leads were tested. Transient overexpression of MDR1 increased drug tolerance of HCT-8 cells on paclitaxel, doxorubicin HCl and vincristine sulphate (2.11- to 2.27-fold increase), but not on cyclosporin A, daunorubicin HCl and nitazoxanide. Increased drug tolerance in host cells had no effect on antiparasitic efficacy of paclitaxel, but affected that of doxorubicin HCl. CONCLUSIONS: Data confirmed that, at efficacious concentrations, paclitaxel acted mainly on the parasite target, while doxorubicin might act on both parasite and host cell targets. This model can be employed for studying the action of additional anti-cryptosporidial leads, and adapted to studying drug action in other epicellular pathogens. The limitation of the model is that the anti-cryptosporidial leads/hits need to be MDR1 substrates.

Promotion of chemerin in rat diabetic kidney disease through enhancement of TGF-ß1/Smads/CTGF pathway.

OBJECTIVE: Although increasing evidence shows that the adipokine chemerin is involved in diabetic kidney disease (DKD), it is still unclear whether the chemerin acts as a critical element in renal function through the signaling pathways of transforming growth factor ß1/Smads/connective tissue growth factor (TGF-ß1/Smads/CTGF) in the context of DKD. Therefore, we sought to determine the role of chemerin and TGF-ß1/Smads/CTGF signaling pathway in the development and/or progression of DKD. METHODS: We used rat renal mesangial cells (RMCs) and a DKD rat model as study subjects. RMCs and rats were randomly separated into different groups and transfected with the constructed chemerin expression vector pcDNA™ 3.1 (+)-chemerin. Rat renal function and inflammatory cytokines were assessed after treatment with chemerin or CCX832 (ChemR23 antagonist). Real time polymerase chain reverse transcription (RT-QPCR) was used to detect the mRNA expressions of TGF-ß1, Smad2, Smad4, and CTGF. Western blot was performed to determine protein expression for semiquantitative analysis. RESULTS: In in vitro studies, the mRNA and protein levels of TGF-ß1, Smad2, Smad4, and CTGF were significantly increased in the groups of high glucose and chemerin as compared to the normal control and normal glucose groups, most notably in the high glucose chemerin group (all P<0.05). In vivo studies revealed that the mRNA and protein levels of TGF-ß1, Smad2, Smad4, and CTGF were higher in the DKD group and the normal chemerin group than in the normal control group and the blocking receptor group, while appearing the highest in the DKD chemerin group (all P<0.05). Moreover, kidney/body weight ratio, urea, creatinine, and urine protein were increased, and the weight and endogenous creatinine clearance rate decreased in the DKD group and the normal chemerin group (all P<0.05). These changes were more pronounced in the DKD chemerin group. At the same time, blood glucose, triglycerides (TGs), and total cholesterol (TC) in the blocked receptor group was lower than those in the DKD group and the DKD chemerin group (all P<0.05). In contrast to those in the normal control group and blocked receptor group, tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1 showed higher concentrations in the DKD group and the normal chemerin group. This result was more pronounced in the DKD chemerin group (all P<0.05). CONCLUSION: Chemerin may play a role in DKD by enhancing the signaling pathways of TGF-ß1/Smads/CTGF transduction either in vitro or in vivo. Moreover, high glucose accelerates kidney injury by activating fibrotic pathways.

Unique Tubulin-Based Structures in the Zoonotic Apicomplexan Parasite Cryptosporidium parvum.

Cryptosporidium parasites are known to be highly divergent from other apicomplexan species at evolutionary and biological levels. Here we provide evidence showing that the zoonotic Cryptosporidium parvum also differs from other apicomplexans, such as Toxoplasma gondii, by possessing only two tubulin-based filamentous structures, rather than an array of subpellicular microtubules. Using an affinity-purified polyclonal antibody against C. parvum ß-tubulin (CpTubB), we observed a long and a short microtubule that are rigid and stable in the sporozoites and restructured during the intracellular parasite development. In asexual development (merogony), the two restructuring microtubules are present in pairs (one pair per nucleus or merozoites). In sexual developmental stages, tubulin-based structures are detectable only in microgametes, but undetectable in macrogametes. These observations indicate that C. parvum parasites use unique microtubule structures that differ from other apicomplexans as part of their cytoskeletal elements.

Effect of Electroacupuncture Combined with Guizhi Gegen Decoction on Cervical Vertigo and Its Influence on TCD of Vertebrobasilar Artery, Blood Rheology Indexes, and Quality of Life.

Cervical vertigo is a common clinical disease. In this study, we investigated the clinical efficacy of adding the treatment protocol of Gui Zhi Ge Gen Tang and electroacupuncture to the Western medical treatment in cervical vertigo. The results showed that the total effective rate of the study group was higher than that of the control group (P < 0.05). After treatment, the vertigo, headache, neck and shoulder pain, daily life and work, and psychological and social adjustment scores were higher in the study group than in the control group (P < 0.05). LVA, RVA, and VB were higher in the study group than in the control group after treatment (P < 0.05). The whole blood viscosity high cut, whole blood viscosity low cut, and plasma viscosity levels were lower in the study group than in the control group after treatment (P < 0.05). After treatment, PF, RF, RE, BP, GH, VT, SF, MH, and HT were higher in the study group than those before treatment (P < 0.05). The incidence of adverse reactions was not statistically significant in the study group compared with the control group (P > 0.05). This means that electroacupuncture combined with Gui Zhi Ge Gen Tang can effectively increase the blood flow velocity of the vertebrobasilar artery in patients with cervical vertigo and improve their blood rheology and quality of life with definite efficacy and high safety.

A Single-Pass Type I Membrane Protein from the Apicomplexan Parasite Cryptosporidium parvum with Nanomolar Binding Affinity to Host Cell Surface.

Cryptosporidium parvum is a globally recognized zoonotic parasite of medical and veterinary importance. This parasite mainly infects intestinal epithelial cells and causes mild to severe watery diarrhea that could be deadly in patients with weakened or defect immunity. However, its molecular interactions with hosts and pathogenesis, an important part in adaptation of parasitic lifestyle, remain poorly understood. Here we report the identification and characterization of a C. parvum T-cell immunomodulatory protein homolog (CpTIPH). CpTIPH is a 901-aa single-pass type I membrane protein encoded by cgd5_830 gene that also contains a short Vibrio, Colwellia, Bradyrhizobium and Shewanella (VCBS) repeat and relatively long integrin alpha (ITGA) N-terminus domain. Immunofluorescence assay confirmed the location of CpTIPH on the cell surface of C. parvum sporozoites. In congruence with the presence of VCBS repeat and ITGA domain, CpTIPH displayed high, nanomolar binding affinity to host cell surface (i.e., Kd(App) at 16.2 to 44.7 nM on fixed HCT-8 and CHO-K1 cells, respectively). The involvement of CpTIPH in the parasite invasion is partly supported by experiments showing that an anti-CpTIPH antibody could partially block the invasion of C. parvum sporozoites into host cells. These observations provide a strong basis for further investigation of the roles of CpTIPH in parasite-host cell interactions.

Implication of Potential Differential Roles of the Two Phosphoglucomutase Isoforms in the Protozoan Parasite Cryptosporidium parvum.

Phosphoglucomutase 1 (PGM1) catalyzes the conversion between glucose-1-phosphate and glucose-6-phosphate in the glycolysis/glucogenesis pathway. PGM1s are typically cytosolic enzymes in organisms lacking chloroplasts. However, the protozoan Cryptosporidium parasites possess two tandemly duplicated PGM1 genes evolved by a gene duplication after their split from other apicomplexans. Moreover, the downstream PGM1 isoform contains an N-terminal signal peptide, predicting a non-cytosolic location. Here we expressed recombinant proteins of the two PGM1 isoforms from the zoonotic Cryptosporidium parvum, namely CpPGM1A and CpPGM1B, and confirmed their enzyme activity. Both isoforms followed Michaelis-Menten kinetics towards glucose-1-phosphate (Km = 0.17 and 0.13 mM, Vmax = 7.30 and 2.76 µmol/min/mg, respectively). CpPGM1A and CpPGM1B genes were expressed in oocysts, sporozoites and intracellular parasites at a similar pattern of expression, however CpPGM1A was expressed at much higher levels than CpPGM1B. Immunofluorescence assay showed that CpPGM1A was present in the cytosol of sporozoites, however this was enriched towards the plasma membranes in the intracellular parasites; whereas CpPGM1B was mainly present under sporozoite pellicle, although relocated to the parasitophorous vacuole membrane in the intracellular development. These observations indicated that CpPGM1A played a house-keeping function, while CpPGM1B played a different biological role that remains to be defined by future investigations.

[Expert consensus on diagnosis and treatment of septic shock with integrated traditional Chinese and Western medicine].

OBJECTIVE: Septic shock is a pathological stage during the progress of sepsis. The mortality rate is high, and timely, standardized and comprehensive treatment is of great significance in reducing the mortality rate. At present, there are many domestic and international guidelines for the diagnosis and treatment of sepsis and septic shock. On this basis, China Association of Integrative Medicine Emergency Medicine Committee organized experts to collect evidence-based medical evidence and expert experience in the diagnosis and treatment by integrated Chinese and Western medicine, and referred to the Expert consensus on diagnosis and treatment of sepsis with integrated traditional Chinese and Western medicine published by China Association of Integrative Medicine Emergency Medicine Committee in 2013, developed Expert consensus on diagnosis and treatment of septic shock with integrated traditional Chinese and Western medicine. The consensus provided a "China approach" for the resolution of this worldwide problem. In the "China approach", the staging of septic shock is "longitude", and the "four syndromes and four methods" treatment thought is "latitude". Under the framework of interlaced latitude and longitude, we recommend a series of effective integrated Chinese and Western medicine treatment. The method embodies the wisdom of China and has certain guiding significance for the clinical treatment of septic shock.