Microsporidia infection upregulates host energy metabolism but maintains ATP homeostasis.

Microsporidia are a group of obligate intracellular parasites which lack mitochondria and have highly reduced genomes. Therefore, they are unable to produce ATP via the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Instead, they have evolved strategies to obtain and manipulate host metabolism to acquire nutrients. However, little is known about how microsporidia modulate host energy metabolisms. Here, we present the first targeted metabolomics study to investigate changes in host energy metabolism as a result of infection by a microsporidian. Metabolites of silkworm embryo cell (BmE) were measured 48 h post infection by Nosema bombycis. Thirty metabolites were detected, nine of which were upregulated and mainly involved in glycolysis (glucose 6-phosphate, fructose 1,6-bisphosphate) and the TCA cycle (succinate, α-ketoglutarate, cis-aconitate, isocitrate, citrate, fumarate). Pathway enrichment analysis suggested that the upregulated metabolites could promote the synthesization of nucleotides, fatty acids, and amino acids by the host. ATP concentration in host cells, however, was not significantly changed by the infection. This ATP homeostasis was also found in Encephalitozoon hellem infected mouse macrophage RAW264.7, human monocytic leukemia THP-1, human embryonic kidney 293, and human foreskin fibroblast cells. These findings suggest that microsporidia have evolved strategies to maintain levels of ATP in the host while stimulating metabolic pathways to provide additional nutrients for the parasite.

In-vitro cultivation of Nosema bombycis sporoplasms: A method for potential genetic engineering of microsporidia.

Microsporidia are obligate intracellular parasites and cannot be cultured in vitro, which limits the use of current genetic engineering technologies on this pathogen. We isolated sporoplasms of Nosema bombycis to attempt to culture the pathogen in vitro. Cell-free medium was designed and successfully maintained the sporoplasms for 5 days. The sporoplasms were able to absorb ATP from the medium and DNA replicated during cultivation, although there was not a significant change in morphology and number of sporoplasms. Our study provides a strategy for in vitro cultivation and genetic manipulation of microsporidia. .

Characterization of Hsp70 gene family provides insight into its functions related to microsporidian proliferation.

Heat shock protein 70 (Hsp70), a highly conserved protein family, is widely distributed in organisms and plays fundamental roles in biotic and abiotic stress responses. However, reports on Hsp70 genes are scarce in microsporidia, a very large group of obligate intracellular parasites that can infect nearly all animals, including humans. In this study, we identified 37 Hsp70 proteins from eight microsporidian genomes and classified them into four subfamilies (A-D). The number of Hsp70 genes in these microsporidia was significantly fewer than in Rozella allomycis and yeast. All microsporidian species contained genes from each subfamily and similar subcellular locations (mitochondria, endoplasmic reticulum, cytosol, and cytosol and/or nucleus), indicating that each Hsp70 member may fulfil distinct functions. The conserved structures and motifs of the Hsp70 proteins in the same subfamily were highly similar. Expression analysis indicated that the subfamily C cytosol (cyto)-associated Hsp70s is functional during microsporidia development. Immunofluorescence assays revealed that Cyto-NbHsp70 was cytoplasmically located in the proliferation-stage of Nosema bombycis. Cyto-NbHsp70 antiserum also labeled Encephalitozoon hellem within infected cells, suggesting that this antiserum is a potential molecular marker for labeling the proliferative phases of different microsporidian species. The propagation of N. bombycis was significantly inhibited following RNAi of Cyto-NbHsp70, indicating that Cyto-NbHsp70 is important for pathogen proliferation. Our phylogenetic data suggest that Hsp70 proteins evolved during microsporidia adaption to intracellular parasitism, and they play important roles in pathogen development.

Morphology and Transcriptome Analysis of Nosema bombycis Sporoplasm and Insights into the Initial Infection of Microsporidia.

Microsporidia are obligate intracellular parasites that infect a wide variety of host organisms, including humans. The sporoplasm is the initial stage of microsporidian infection and proliferation, but its morphological and molecular characteristics are poorly understood. In this study, the sporoplasm of Nosema bombycis was successfully isolated and characterized after the induction of spore germination in vitro The sporoplasm was spherical, 3.64 ± 0.41 µm in diameter, had the typical two nuclei, and was nonrefractive. Scanning and transmission electron microscopy analyses revealed that the sporoplasm was surrounded by a single membrane, and the cytoplasm was usually filled with relatively homogeneous granules, possibly ribosomes, and contained a vesicular structure comprising a concentric ring and coiled tubules. Propidium iodide staining revealed that the sporoplasm membrane showed stronger membrane permeability than did the cell plasma membrane. Transmission electron microscopy (TEM) revealed that the sporoplasm can gain entry to the host cell by phagocytosis. Transcriptome analysis of mature spores and sporoplasms showed that 541 significantly differentially expressed genes were screened (adjusted P value [Padj] < 0.05), of which 302 genes were upregulated and 239 genes were downregulated in the sporoplasm. The majority of the genes involved in trehalose synthesis metabolism, glycolysis, and the pentose phosphate pathway were downregulated, whereas 10 transporter genes were upregulated, suggesting that the sporoplasm may inhibit its own carbon metabolic activity and obtain the substances required for proliferation through transporter proteins. This study represents the first comprehensive and in-depth investigation of the sporoplasm at the morphological and molecular levels and provides novel insights into the biology of microsporidia and their infection mechanism.IMPORTANCE Once awoken from dormancy, the cellular matter of microsporidia is delivered directly into the host cell cytoplasm through the polar tube. This means that the microsporidia are difficult to study biologically in their active state without a contaminating signal from the host cell. Sporoplasm is a cell type of microsporidia in vitro, but relatively little attention has been paid to the sporoplasm in the past 150 years due to a lack of an effective separation method. Nosema bombycis, the first reported microsporidium, is a type of obligate intracellular parasite that infects silkworms and can be induced to germinate in alkaline solution in vitro We successfully separated the N. bombycis sporoplasm in vitro, and the morphological and structural characteristics were investigated. These results provide important insight into the biology and pathogenesis of microsporidia and potentially provide a possible strategy for genetic manipulation of microsporidia targeting the sporoplasm.

Evolutionary and functional studies on microsporidian ATP-binding cassettes: Insights into the adaptation of microsporidia to obligated intracellular parasitism.

ATP-binding cassette (ABC) transporters comprise the largest family of transmembrane proteins and are found in all domains of life. The ABCs are involved in a variety of biological processes and as exporters play important roles in multidrug resistance. However, the ABC transporters have not been addressed in microsporidia, which are a very large group of obligate intracellular parasites that can infect nearly all animals, including humans. Here, a total of 234 ABC transporters were identified from 18 microsporidian genomes and classified into five subfamilies, including 74 ABCBs, 2 ABCCs, 18 ABCEs, 15 ABCFs, 102 ABCGs and 23 uncategorized members. Two subfamilies, ABCA and ABCD, are found in most organisms, but lost in microsporidia. Phylogenetic analysis indicated that microsporidian ABCB and ABCG subfamilies expanded by recent gene duplications, which resulted in the two largest subfamilies in microsporidia. Functional analysis via qRT-PCR and Western blotting revealed that NoboABCG1.1, an ABCG member of Nosema bombycis, is expressed in mature spores and up-regulated from 1 dpi to 6 dpi in infected silkworm midgut. IFA and IEM analysis showed that NoboABCG1.1 is localized on the plasma membrane of the sporoplasm, meront and mature spore. The propagation of N. bombycis was significantly inhibited after the RNAi of NoboABCG1.1 expression, indicating that NoboABCG1.1 is important to the pathogen proliferation. In conclusion, our study uncovered that the ABCs evolved during microsporidia adaption to intracellular parasitism and play important roles in the pathogen development.

Pathological analysis of silkworm infected by two microsporidia Nosema bombycis CQ1 and Vairimorpha necatrix BM.

Microsporidia Nosema bombycis CQ1 can be vertically transmitted in silkworm Bombyx mori but Vairimorpha necatrix BM cannot. Therefore, the pathological differences in silkworm infected with these two microsporidia required clarification. Here, we compared the virulence of N. bombycis CQ1 and V. necatrix BM against silkworm. The pathological characteristics in intestine, testis and ovary were surveyed using paraffin sections, scanning electron microscopy and transmission electron microscopy. Our data firstly showed that the virulence of V. necatrix BM was weaker than that of N. bombycis CQ1. Secondly, the typical symptom of V. necatrix BM infection is making xenomas, which are full of pathogens in different stages, at the posterior of intestine. However, no xenomas were formed surrounding intestines infected with N. bombycis CQ1. Thirdly, N. bombycis CQ1 can cluster spores near the trachea while infecting ovaries. It is worth noting that N. bombycis CQ1 infected epithelial cells and connective tissues of ovaries, while V. necatrix BM did not. Although silkworm ovaries can not be infected by V. necatrix BM in vivo, it can infect embryonic and ovarian cell lines in vitro. This study is the first report about comparing infection features of N. bombycis CQ1 and V. necatrix BM in silkworm tissues and it provided elaborate and visual information of pathological characteristics which can help to explain the different transmission strategies of these two microsporidia.

Precise hepatectomy guided by minimally invasive surgery: a novel strategy for liver resection.

Liver resection has been established currently as an effective and standard treatment for patients suffering from both benign and malignant hepatobiliary diseases. Although substantial improvement in perioperative mortality rate and morbidity resulting from appropriate candidates selection, advanced surgical techniques and enhanced perioperative care, hepatectomy is still burdened by about 5% mortality rate and some lethal postoperative complications, especially postoperative liver insufficiency and failure. Various approaches have been advocated to minimize stress and insult on patients due to operative procedures. It becomes important to preserve remnant hepatic function as much as possible to improve the outcome of hepatectomy. Minimally invasive concept and fast track surgery are crucial breakthrough in the natural history of surgery and have been employed in liver resection. To safely and accurately perform hepatic resection, owing to our experiences with recent advances in surgical techniques and perioperative administration for liver resection, a novel strategy, "precise hepatectomy" originating from minimally invasive surgery has been developed, which includes precise preoperative planning, sophisticated intraoperative techniques and careful postoperative management. This strategy is characteristic by involvement of minimally invasive concept in overall therapy, from preoperative assessment to postoperative care, optimization of a series of advanced techniques and proper employment of surgical instruments in light of actual individual information. However, further prospective studies, especially randomized controlled trials in high volume centers, remain essential to compare the safety and therapeutic efficacies between precise hepatectomy and conventional surgical procedures.

microRNA expression alteration after arsenic trioxide treatment in HepG-2 cells.

BACKGROUND AND AIM: More and more microRNA (miRNA) are found to be involved in tumor genesis and progress. Arsenic trioxide has been an effective chemotherapeutic drug in cancer therapy for many years. In this study, we aimed to find the miRNA involved in the mechanisms of arsenic trioxide treatment in cancer therapy. METHODS: We detected the expression profile of miRNA by miRNA microarray and quantitative real-time polymerase chain reaction. Cell viability assay, flow cytometry analysis, prediction of miRNA targets, Western blot analysis and luciferase reporter assay were carried out to determine the role of one selected miRNA, namely mir-29a, in affecting the biological behaviors of HepG-2 cells. RESULTS: Among the 677 human miRNA in the microarray, five miRNA were upregulated and four were downregulated in HepG-2 cells treated with arsenic trioxide compared to their controls. If only changes above two folds were considered, four miRNA were identified, namely miR-24, miR-29a, miR-30a and miR-210, which were all upregulated. Among them, miR-29a showed a positive therapeutic effect in liver cancer cells by inhibiting cell growth and inducing cell apoptosis, and PPM1D was confirmed to be the target gene of miR-29a. Furthermore, a synergy effect was detected between miR-29a and arsenic trioxide. CONCLUSIONS: Arsenic trioxide altered miRNA expression profile in HepG-2 cells. Among the altered miRNA, miR-29a seemed to take a role in the mechanism of arsenic trioxide in liver cancer therapy. The synergy effect between miR-29a and arsenic trioxide may offer this drug a new chance in cancer therapy by decreasing its dose and toxic side-effects.

[Comparative study of mannitol-induced acute kidney impairments in patients of different ages suffering from subarachnoid hemorrhage].

OBJECTIVE: To analyze and compare the clinical characteristics of mannitol-induced acute renal function impairment in treatment of patients of different ages suffering from subarachnoid hemorrhage (SAH) for the first time. METHODS: This study was a review of 1 361 patients with available hospital records admitted to the division of neurology due to SAH from February 1989 to December 2005. Among them, 94 patients had acute renal function impairment as a result of mannitol administration. Of these patients, 35 patients were middle-aged (<60 years old) and 59 patients of old age (>60 years old). The study included the time of appearance of renal function impairment after mannitol medication, its prognosis, the administration of nephrotoxic drugs, and the dose and duration of mannitol therapy. RESULTS: In old age group, abnormal renal function and urine routine appeared in 5 days (median), and 4 days (median) respectively, and acute renal failure (ARF) was diagnosed in 5 days, and the incidence rate of ARF was 20.3% after treatment with mannitol. The respective event appeared 7 days, 11 days, 9 days and 2.8% (P<0.05 or P<0.01) respectively, in middle-aged group. The data indicated the elderly patients had poorer tolerance to mannitol, with earlier occurrence of mannitol nephrosis, poorer outcome of kidney impairment and worse prognosis. Forty-three patients (72.9%) were treated with katlex (with a median of total dosage of 400 mg) in old-aged group, 35 patients (100.0%) were treated with katlex (with a median of total dosage of 800 mg) in middle-aged group, and there was a significant difference between two groups (P<0.01). The data indicated the incidence of mannitol nephrosis was lower in patients treated with large dosage of mannitol and katlex. The mortality was 3.4% (2/59 cases) and 0% (0/35 cases) in old-aged and middle-aged groups respectively (P=0.528). CONCLUSION: The elderly patients have higher rate of mannitol-induced impairment of renal function after SAH than middle-aged patients. The data emphasize that proper combination therapy of mannitol with katlex is an effective measure in preventing renal failure.

[The importance of nonrecurrent laryngeal nerve in thyroid surgery].

OBJECTIVE: To investigate the anatomic variation of nonrecurrent laryngeal nerve (NRLN) and its surgical identification and prevention during thyroidectomy. METHODS: The database of 5 NRLN cases was analyzed to investigate the difference of operative maneuvers and procedures. RESULTS: All 5 NRLN were located in the right side. Two cases were found have vocal cord paralysis and 1 case recovered in 3 cases who have NRLN injures. CONCLUSIONS: Any transverse bond should not be cut between vascular and laryngeal except middle thyroid vein. Recurrent laryngeal nerve (RLN) should be dissected during thyroid excision. Cervical pneumogastric nerve should be systematic dissected to detect whether RNLN is exist, if RLN is not exist in the same side.