Phytic acid is a new substitutable plant-derived antifungal agent for the seedling blight of Pinus sylvestris var. mongolica caused by Fusarium oxysporum.

Phytic acid (PA) is a new substitutable plant-derived antifungal agent; however, few reports have been published regarding its antifungal effects on pathogenic fungi. The present study explored the in vitro antifungal activity of PA against four phytopathogenic fungi and found that PA was the most effective at inhibiting the growth of Fusarium oxysporum. This study aimed to investigate the in vivo and in vitro antifungal activities of PA against the seedling blight of Pinus sylvestris var. mongolica caused by F. oxysporum and to determine its possible mechanism of action. The results showed that PA inhibited spore germination and mycelial growth of F. oxysporum in a concentration-dependent manner and exhibited strong inhibition when its concentration exceeded 1000 mg/L. It mainly destroyed the integrity of the cell membrane, increasing its cell membrane permeability, causing the cell contents to spill out, and impairing fungal growth. In addition, the leakage of intercellular electrolytes and soluble proteins indicated that PA used at its EC20 and EC50 increased the membrane permeability of F. oxysporum. The increase in malondialdehyde and hydrogen peroxide content confirmed that PA treatment at its EC20 and EC50 damaged the cell membrane of the pathogen. Scanning electron microscopy revealed that PA affected the morphology of mycelia, causing them to shrivel, distort, and break. Furthermore, PA significantly reduced the activities of the antioxidant-related enzymes superoxide dismutase and catalase, as well as that of the pathogenicity-related enzymes polygalacturonase, pectin lyase, and endoglucanase (EG) in F. oxysporum (P < 0.05). In particular, EG enzyme activity was maximally inhibited in F. oxysporum treated with PA at its EC50. Moreover, PA significantly inhibited the incidence of disease, and growth indices in Pinus sylvestris var. mongolica seedling blight was determined. In summary, PA has a substantial inhibitory effect on F. oxysporum. Therefore, PA could serve as a new substitutable plant-derived antifungal agent for the seedling blight of P. sylvestris var. mongolica caused by F. oxysporum.

Steroid hormone 20-hydroxyecdysone disturbs fat body lipid metabolism and negatively regulates gluconeogenesis in Hyphantria cunea larvae.

The steroid hormone 20-hydroxyecdysone (20E) has been described to regulate fat body lipid metabolism in insects, but its accurate regulatory mechanism, especially the crosstalk between 20E-induced lipid metabolism and gluconeogenesis remains largely unclear. Here, we specially investigated the effect of 20E on lipid metabolism and gluconeogenesis in the fat body of Hyphantria cunea larvae, a notorious pest in forestry. Lipidomics analysis showed that a total of 1 907 lipid species were identified in the fat body of H. cunea larvae assigned to 6 groups and 48 lipid classes. The differentially abundant lipids analysis showed a significant difference between 20E-treated and control samples, indicating that 20E caused a remarkable alteration of lipidomics profiles in the fat body of H. cunea larvae. Further studies demonstrated that 20E accelerated fatty acid ß-oxidation, inhibited lipid synthesis, and promoted lipolysis. Meanwhile, the activities of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase, and glucose-6-phosphatase were dramatically suppressed by 20E in the fat body of H. cunea larvae. As well, the transcriptions of genes encoding these 4 rate-limiting gluconeogenic enzymes were significantly downregulated in the fat body of H. cunea larvae after treatment with 20E. Taken together, our results revealed that 20E disturbed fat body lipid homeostasis, accelerated fatty acid ß-oxidation and promoted lipolysis, but negatively regulated gluconeogenesis in H. cunea larvae. The findings might provide a new insight into hormonal regulation of glucose and lipid metabolism in insect fat body.

Inhibitory effect and mechanism of action of juniper essential oil on gray mold in cherry tomatoes.

Juniper essential oil (JEO), which is mostly known as an immune system booster and effective detoxifier, has substantial antimicrobial activity. A comparison of the inhibitory effects of three plant essential oils from juniper (Juniperus rigida), cedarwood (Juniperus virginiana), and cypress (Crupressus sempervirens) on four plant pathogenic fungi indicated that JEO was the most effective at inhibiting the growth of gray mold (Botrytis cinerea). Additional studies were subsequently conducted to explore the in vivo and in vitro antifungal activity and possible mechanism of JEO against B. cinerea. The results show that JEO inhibited the germination of spores and mycelial growth of B. cinerea in a concentration-dependent manner and exhibited strong inhibition when its concentration exceeded 10 µL/mL. JEO also significantly inhibited the incidence of disease and diameters of gray mold lesions on cherry tomato fruit (Solanum lycopersicum). After 12 h of treatment with JEO, the extracellular conductivity, and the contents of soluble protein, malondialdehyde, and hydrogen peroxide were 3.1, 1.2, 7.2, and 4.7 folds higher than those of the control group, respectively (P < 0.05), which indicated that JEO can damage membranes. Scanning electron microscopy observations revealed that JEO affected the morphology of mycelia, causing them to shrivel, twist and distort. Furthermore, JEO significantly improved the activities of the antioxidant-related enzymes superoxide dismutase and catalase but reduced the pathogenicity-related enzymes polygalacturonase (PG), pectin lyase and endoglucanase of B. cinerea (P < 0.05). In particular, PG was reduced by 93% after treatment with JEO for 12 h. Moreover, the 18 constituents of JEO were identified by gas chromatography/mass spectrometry (GC-MS) analysis, mainly limonene (15.17%), γ-terpinene (8.3%), ß-myrcene (4.56%), terpinen-4-ol (24.26%), linalool (8.73%), α-terpineol (1.03%), o-cymene (8.35%) and other substances with antimicrobial activity. Therefore, JEO can be an effective alternative to prevent and control gray mold on cherry tomato fruit.

Transcriptomic analysis of interactions between Lymantria dispar larvae and carvacrol.

Due to its biological activity, carvacrol (CAR) is widely used in medicine, agriculture, and forestry. Our previous studies showed that in Lymantria dispar larvae, CAR treatment can induce the production of antifeedants and lead to growth inhibition and death of larvae. However, the effect CAR exerts on RNA levels in L. dispar larvae remains unclear. In this study, the Illumina HiSeq4000 sequencing platform was used to sequence the total RNA of L. dispar larvae. A total of six cDNA libraries (three treatments and three controls) were established and 39,807 genes were generated. Compared with the control group, 296 differentially expressed genes (DEGs) (142 up-regulated and 154 down-regulated) were identified after CAR treatment. GO and KEGG enrichment analyses showed that these DEGs mainly clustered in the metabolism of xenobiotics, carbohydrates, and lipids. Furthermore, 12 DEGs were found to be involved in detoxification, including six cytochrome P450s, two esterases, one glutathione peroxidase, one UDP-glycosyltransferase gene, and two genes encoding heat shock proteins. The expression levels of detoxification genes changed under CAR treatment (especially P450s), which further yielded candidate genes for explorations of the insecticidal mechanism of CAR. The reliability of transcriptome data was verified by qRT-PCR. The enzyme activities of CYP450 and acid phosphatase significantly increased (by 38.52 U/mg·prot and 0.12 µmol/min·mg, respectively) 72 h after CAR treatment. However, the activity of alkaline phosphatase did not change significantly. These changes in enzyme activity corroborated the reliability of the transcriptome data at the protein level. The results of GO enrichment analysis of DEGs indicated that CAR influenced the oxidation-reduction process in L. dispar larvae. Furthermore, CAR can cause oxidative stress in L. dispar larvae, identified through the determination of peroxidase and polyphenol oxidase activities, total antioxidant capacity, and hydrogen peroxide content. This study provides useful insight into the insecticidal mechanism of CAR.

Antifungal Activity of 6-Methylcoumarin against Valsa mali and Its Possible Mechanism of Action.

Valsa canker of apple (VCA) caused by Valsa mali severely affected apple production in east Asia. With the increase in drug resistance, there is an urgent need for efficient and environmentally friendly antifungal agents. Coumarins have attracted much attention due to their excellent antimicrobial activity against plant pathogens. In this study, the antifungal activity of several coumarins against phytopathogenic fungi was evaluated, and then the antifungal activity of the screened 6-MCM against V. mali and its underlying mechanism was further investigated. The results of the in vitro antifungal activity assay showed that some coumarins had significant inhibitory effects on V. mali. Notably, 400 mg/L of 6-MCM had the best antifungal activity of 94.6%. Further experiments showed that 6-MCM slowed down the growth of V. mali mycelia and the germination of spores in a concentration-dependent manner, with EC50 of 185.49 and 54.62 mg/L, respectively. In addition, 6-MCM treatment increased mycelial conductivity, extracellular protein leakage, and MDA content, resulting in damage to the cell membrane. Moreover, 6-MCM significantly reduced the cell wall degrading enzymes secreted by V. mali, including EG, PG and PL, thereby limiting its pathogenic capacity. SEM and TEM results showed that 6-MCM treatment had a significant effect on the morphology and ultrastructure of mycelial cells. Inoculation of isolated apple branches found that the application of 6-MCM effectively inhibited the development of VCA and significantly reduced the incidence. All these results suggest that 6-MCM has the potential as a green substitute for VCA control.

Inhibitory effects and mechanisms of vanillin on gray mold and black rot of cherry tomatoes.

Vanillin is a natural antimicrobial agent; however, there are few reports on its antifungal effect on postharvest pathogenic fungi. This study aimed to investigate the in vivo and in vitro antifungal activities of vanillin against gray mold (caused by B. cinerea) and black rot (caused by A. alternata) of cherry tomato fruit and to explain its possible mechanism of action. Vanillin strongly inhibits Botrytis cinerea and Alternaria alternata mycelial growth, spore germination, and germ tube elongation in a concentration-dependent manner (P<0.05). In vivo experiments showed that 4000 mg L-1 vanillin treatment inhibited cherry tomato gray mold and black rot occurrence. Besides, intercellular electrolytes, soluble proteins, and soluble sugars leakage indicated that 50 or 100 mg L-1 vanillin treatment increased Botrytis cinerea and Alternaria alternata membrane permeability. The increase of malondialdehyde and hydrogen peroxide contents confirmed that 50 or 100 mg L-1 vanillin treatment damages the pathogen membranes. Importantly, vanillin treatment inhibited the pathogenicity-related enzyme activities of the two pathogens to reduce their infection ability, among them PL enzyme activity in A. alternata was most inhibited, reducing by 94.7 % at 6 h treated with 100 mg L-1 vanillin. The hyphae morphology of the two pathogens changed, the mycelia were severely damaged, and the hyphae surface was deformed, shrunk, or even broken after 100 mg L-1 vanillin treatment. In summary, vanillin had a substantial inhibitory effect on postharvest gray mold and black rot in cherry tomato fruit. Therefore, vanillin can be an effective alternative to prevent and control cherry tomato postharvest diseases.

Transcriptome and cell wall degrading enzyme-related gene analysis of Pestalotiopsis neglecta in response to sodium pheophorbide a.

Sodium pheophorbide a (SPA) is a new alternative fungicide with low toxicity and high efficiency, which has high fungicidal activity against Pestalotiopsis neglecta, a pathogen that causes black spot needle blight of Pinus sylvestris var. mongolica. To utilize SPA for plant disease control, understanding its antifungal mechanism is essential. Six cDNA libraries were constructed from 3 d-old P. neglecta mycelia (three SPA-infected and three untreated groups) and 29,850 expressed genes were obtained by Illumina HiSeq4000 sequencing. Compared with controls, 3268 differentially expressed genes (DEGs) were identified in SPA-treated groups, including 1879 upregulated and 1389 downregulated genes. Most DEGs were involved in the metabolism of amino acids, carbohydrates, and lipids, as well as cell structure and genetic information processing. These findings were further confirmed by decreased conductivity, RNA and protein content, and activities of nicotinamide adenine dinucleotide-dependent malate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and succinate dehydrogenase. Moreover, qRT-PCR verified the reliability of the transcriptome results. After treatment with SPA at different concentrations for 60 min, the expressions of three cell wall degrading enzyme-related genes (PnEG, PnBG, and PnPG) were all suppressed. Overall, this study provided insights into the molecular mechanisms through which SPA inhibits P. neglecta, increasing the possibility of developing SPA into an effective fungicide in the future.

Key factors affecting photoactivated fungicidal activity of sodium pheophorbide a against Pestalotiopsis neglecta.

Recently, photodynamic therapy (PDT) and photoactivated pesticides have attracted considerable research attention. In the present study, we aimed to investigate the photodynamic activity of a chlorophyllous derivative, sodium pheophorbide a (SPA), and to evaluate its potential as a photoactivated fungicide. The singlet oxygen quantum yield, the photoreaction process, the anti-photobleaching ability in sterile water (H2O), the effect of light conditions on its antifungal activity, and its stability were all investigated. SPA showed significant fungicidal activity and photostability, during which Type I and Type II photodynamic reactions occurred simultaneously on Pestalotiopsis neglecta, and the influence of Type I was slightly larger than that of Type II. In addition, light promoted the antifungal activity of SPA. In particular, the antifungal activity was enhanced with increasing light intensity, and was strongest under 8000 lx conditions. Under monochromatic light sources, antifungal activity was strongest under green light s; however, the effect of monochromatic light was not as good as that of white light. From 0 to 24 h, the antifungal effect of the SPA solution was enhanced; however, the activity of the solution began to weaken after 24 h. Furthermore, our study confirmed that the antifungal activity of SPA was stable under different temperatures, pH values, and UV irradiation durations.

Diel pattern in the structure and function of the gut microbial community in Lymantria dispar asiatica (Lepidoptera: Lymantriidae) larvae.

In the present study, diel pattern in gut microbial communities in insects were evaluated. Lymantria dispar asiatica fourth instar larvae (72 ± 2 hr after molting) at noon (LdD) and midnight (LdN) were used for a comparative analysis of the gut microbial community. Ten bacterial operational taxonomic units (OTUs) were shared between LdD and LdN samples. One bacterial OTU was specific to LdD. The dominant gut microbes were OTU72 in LdD and OTU75 in LdN. A linear discriminant analysis effect size cladogram suggested that ten bacterial OTUs maintain significant differences in relative abundances between LdD and LdN. These results agreed with the discrete ellipses between LdD and LdN in principal coordinates analysis plots. Additionally, using phylogenetic investigation of communities by reconstruction of unobserved states, the gut microbial community was assigned to 23 functional terms, among which 22 exhibited significant differences between LdD and LdN. To conclude, the present study documented a diel pattern in the gut microbial community of L. dispar asiatica larvae.

Sodium pheophorbide a controls cherry tomato gray mold (Botrytis cinerea) by destroying fungal cell structure and enhancing disease resistance-related enzyme activities in fruit.

Sodium pheophorbide a (SPA) is a natural photosensitizer. The present study investigated the antifungal activity and mechanism of SPA against Botrytis cinerea in vitro and in vivo. Its inhibitory effect was studied on the spore germination and mycelial growth of B. cinerea. The effects of SPA on cell wall integrity, cell membrane permeability, and mycelial morphology of B. cinerea were also determined. Additionally, how SPA effected B. cinerea in vivo was evaluated using cherry tomato fruit. The results showed that SPA effectively inhibited the spore germination and mycelial growth of B. cinerea under light conditions (4000 lx). SPA significantly affected both cell wall integrity and cell membrane permeability (P < .05). In addition, SEM analysis suggested that B. cinerea treated with SPA (12.134 mg/mL) showed abnormal mycelial morphology, including atrophy, collapse, flattening, and mycelial wall dissolution. In vivo tests showed that SPA could increase the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) significantly (P < .05); however, SPA had no significant effect on phenylalanine ammonia lyase (PAL) activity. In short, SPA could destroy the fungal cell structure and enhance disease resistance-related enzyme activity in cherry tomatoes, thereby controlling cherry tomato gray mold.

Influence of dietary aconitine and nicotine on the gut microbiota of two lepidopteran herbivores.

The gut microbiota plays an important role in pheromone production, pesticide degradation, vitamin synthesis, and pathogen prevention in the host animal. Therefore, similar to gut morphology and digestive enzyme activity, the gut microbiota may also get altered under plant defensive compound-induced stress. To test this hypothesis, Dendrolimus superans larvae were fed either aconitine- or nicotine-treated fresh leaves of Larix gmelinii, and Lymantria dispar larvae were fed either aconitine- or nicotine-treated fresh leaves of Salix matsudana. Subsequently, the larvae were sampled 72hr after diet administration and DNA extracted from larval enteric canals were employed for gut microbial 16S ribosomal RNA gene sequencing (338 F and 806 R primers). The sequence analysis revealed that dietary nicotine and aconitine influenced the dominant bacteria in the larval gut and determined their abundance. Moreover, the effect of either aconitine or nicotine on D. superans and L. dispar larvae had a greater dependence on insect species than on secondary plant metabolites. These findings further our understanding of the interaction between herbivores and host plants and the coevolution of plants and insects.

Avermectin stress varied structure and function of gut microbial community in Lymantria dispar asiatica (Lepidoptera: Lymantriidae) larvae.

Lymantria dispar asiatica is a globally distributed herbivorous pest. Avermectin is a highly effective, broad-spectrum insecticide. In this study, fourth instar L. dispar asiatica larvae were exposed to a LC30 dose of avermectin. The structure and function of larval gut microbial community was analyzed to examine how gut microbiota in L. dispar asiatica larvae responded to avermectin stress. Results showed that the structure and function of gut microbial community in L. dispar asiatica larvae were varied by avermectin stress. To be precise, more than half quantity of the observed Optical Taxonomic Units (OTUs) showed significantly different abundances under avermectin stress. Linear discriminant analysis effect size (LEfSe) suggested nine bacterial genera and 12 fungal genera contributed to the different gut microbial community structure in L. dispar asiatica larvae. Gut microbial function classification (PICRUSt and FUNGuild) suggested that three bacterial function categories and a fungal function guild were significantly increased, and two fungal function guilds were significantly decreased by avermectin stress. This study furthers our understanding of the physiology of L. dispar asiatica larvae under avermectin stress, and is an essential step towards future development of potential pesticide targets.

Variation in the pH of experimental diets affects the performance of Lymantria dispar asiatica larvae and its gut microbiota.

To study dietary pH effects on Lymantria dispar asiatica larvae and provide a theoretical basis for its control in different forests, phosphate buffers (PBs) of pH 6, 7, and 8 were used to prepare experimental diets. The diet prepared with pH 6 PB was named as DPB6, with pH 8 PB as DPB8, and with pH 7 PB as DPB7 (control). The dietary pH was 5.00 in DPB6, 6.05 in control, and 6.50 in DPB8. After feeding on the diets with different pH values for 84 hr, fourth-instar caterpillars were randomly collected. Growth and various physiological traits were determined and 16S recombinant DNA sequencing was performed using the intestinal microflora of surviving larvae. Results showed that the mortality was 30% in DPB6, and 10% in DPB8, while no mortality was observed in control. The partial least squares discriminant analyses suggested that diets prepared with PB of different pH resulted in different food intake, amount of produced feces, weight gain, digestive enzyme activities, and antioxidant enzyme activities in larvae. Interestingly, both the highest weight gain and the lowest total antioxidant capacities were seen in control larvae. Results also showed that the larval gut microbiota community structure was significantly affected by dietary pH. Moreover, linear discriminant analysis effect size suggested that the family Acetobacteraceae in control, genus Prevotella in DPB8, and genus Lactococcus, family Flavobacteriaceae, family Mitochondria, and family Burkholderiaceae in DPB6 contributed to the diversity of the larval gut microbial community.

Purification, characterization, and sensitivity to pesticides of carboxylesterase from Dendrolimus superans (Lepidoptera: Lasiocampidae).

Through a combination of steps including centrifugation, ammonium sulfate gradient precipitation, sephadex G-25 gel chromatography, diethylaminoethyl cellulose 52 ion-exchange chromatography and hydroxyapatite affinity chromatography, carboxylesterase (CarE, EC3.1.1.1) from sixth instar larch caterpillar moth, Dendrolimus superans (Lepidoptera: Lasiocampidae) larvae was purified and its biochemical properties were compared between crude homogenate and purified CarE. The final purified CarE after hydroxyapatite chromatography had a specific activity of 52.019 µmol/(min·mg protein), 138.348-fold of crude homogenate, and the yield of 2.782%. The molecular weight of the purified CarE was approximately 84.78 kDa by SDS-PAGE. Three pesticides (dichlorvos, lambda-cyhalothrin, and avermectins) showed different inhibition to crude CarE and purified CarE, respectively. In vitro median inhibitory concentration indicated that the sensitivity of CarE (both crude homogenate and final purified CarE) to pesticides was in decreasing order of dichlorvos > avermectins > lambda-cyhalothrin. By the kinetic analysis, the substrates alpha-naphthyl acetate (α-NA) and beta-naphthyl acetate (ß-NA) showed lesser affinity to crude extract than purified CarE. The results also indicated that both crude homogenate and purified CarE had more affinity to α-NA than to ß-NA, and the Kcat and Vmax values of crude extract were lower than purified CarE using α-NA or ß-NA as substrate.

Pentatrichomonas hominis: first isolation from the feces of a dog with diarrhea in China.

A trichomonad-like parasite isolated from canine fecal samples in Changchun, China was successfully cultivated in vitro using RPMI1640 medium supplemented with 10% heat-inactivated calf serum and antibiotics. These were then subjected to scanning and transmission electron microscopy for ultrastructural study. This parasite has four anterior flagella of unequal length, one independent flagellum, and one recurrent flagellum. It exhibits an anterior nucleus, a Golgi complex, an axostyle, food vacuoles, and hydrogenosomes. These features are consistent with the ultrastructural characteristics of previously described Pentatrichomonas hominis. Polymerase chain reaction and sequence analysis of three genetic loci, including ITS1-5.8S rRNA-ITS2, 18S rRNA, and EF-1α, were also used to compare these samples with other trichomonad species. Molecular identification was also consistent with P. hominis. This is the first time that isolation of P. hominis has been isolated from dog in China, although several other strains of P. hominis have been isolated from human samples.

[Cloning and prokaryotic expression of casein kinase II subunit beta gene fragment of Dirofilaria immitis].

OBJECTIVE: To clone and express the partial fragment of Csnk2b gene of Dirofilaria immitis in prokaryotic cells, and analyze the immunoreactivity. METHODS: The partial fragment of Csnk2b gene was amplified by PCR with a pair of specific primers. The PCR product was cloned into pMD18-T, and then sub-cloned to pGEX-4T-1 expression vector. The constructed plasmid pGEX-4T-1-Csnk2b was transformed into E. coli Rosetta (DE3) and followed by expression of the protein induced by IPTG. The recombinant protein was analyzed by SDS-PAGE and identified by Western blotting. RESULTS: The PCR product was about 700 bp. Enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pGEX-4T-1-Csnk2b was constructed. SDS-PAGE results showed that the relative molecular weight (M(r)) of the fusion protein (GST-Csnk2b) was about 45 000. GST-Csnk2b reacted positively with mouse anti-D. immitis serum. CONCLUSION: The partial Csnk2b gene has been expressed in prokaryotic expression system and shows immunoreactivity.

Giardia intestinalis: effects of Pulsatilla chinensis extracts on trophozoites.

Pulsatilla chinensis is a medicinal root plant that has been used to treat a wide range of disease conditions. Our study determined the antiprotozoal activity of various P. chinensis extracts and fractions against Giardia intestinalis including their effects on parasite growth, cell viability, adherence, and morphology. Ethyl acetate extracts (IC50 = 257.081 µg/ml) were the most active to inhibit the growth of G. intestinalis followed by aqueous extract (PWE), saponins, and n-butanol extract. The PWE and ethyl acetate extract inhibited G. intestinalis trophozoites adherence after 3 h of incubation and killed almost 50 % of the parasite population in a time-dependent manner. Changes in morphology, presence of precipitates in the cytoplasm, dissolved cytoplasm with large vacuole, break of flagella and ventral disk, membrane blebs, and intracellular and nuclear clearance of the treated trophozoites were observed by scanning and transmission electron microscopy. We demonstrated that P. chinensis induced these changes in G. intestinalis morphology and consequently has potential therapeutic use against giardiasis.

Selection and identification of a new adhesion protein of Cryptosporidium parvum from a cDNA library by ribosome display.

In this study, we described a novel display method to identify surface adhesion proteins of Cryptosporidium parvum. A cDNA library of the sporozoite and oocyst stages of C. parvum was expressed on ribosome and selectively and specifically screened with intestinal epithelial cells (IECs) from newborn Cryptosporidium-free Holstein calves. Proteins were then enriched using a multi-step panning procedure. A new surface adherence protein of C. parvum was selected, named Cp20. Sequence analyses showed that Cp20 has a N-terminal signal peptide and four transmembrane regions. Indirect immunofluorescence assay (IFA) using an antibody specific for rCp20 demonstrated that the antibody specifically bound to the surface of sporozoites and oocysts. The recombinant plasmid pVAX1-Cp20 was constructed to examine the potential of the Cp20 gene as a target for specific preventive and therapeutic measures for cryptosporidiosis. The in vivo efficacies of the DNA vaccine was tested in BALB/c mice. The results indicated that the DNA vaccine elicited significant antibody responses and specific cellular responses when compared to control mice that received vector only or PBS. The DNA vaccine induced strong protective immune response against C. parvum and lower level of the oocysts shedding after challenge infection. This study suggested that Cp20 could serve as an effective target for specific preventive and therapeutic measures for cryptosporidiosis.

[The clinical features, chemotherapy responses and survival of 223 patients with newly diagnosed multiple myeloma].

OBJECTIVE: To explore the proportion, clinical and laboratory features, chemotherapy responses and long term survival of different kinds of newly diagnosed multiple myeloma (MM) in China. METHODS: The clinical data of 223 cases of newly diagnosed MM patients were gathered in the First Affiliated Hospital of Sun Yat-sen University from Jan, 2000 to Seb, 2007 were retrospectively analyzed. RESULTS: The proportions of each kind of MM including IgG, IgA, light chain, IgD, IgM and biclonal MM were 48.0%, 20.6%, 25.6%, 4.0%, 0.9% and 0.9% respectively. No IgE and nonsecretory myeloma was found. The median age of onset was 58 years, of which that of the IgA type was the oldest one and the light chain type was the youngest (P = 0.004). Bone pain, renal insufficiency, and anemia were the most common symptoms which accounted for 67.7%, 61.0% and 45.3% respectively. The incidences of renal inadequacy, hypercalcemia and pathological fracture in light chain type were higher than those in IgG and IgA types. Besides, no M protein were found in serum protein electrophoresis and no elevation of total globulin. The clinical features of IgD type were similar to that of the light-chain type. The total chemotherapy efficacy rate of 89 patients who were treated with more than 3 cycles in our hospital is 61.8%, which has no difference in all types. Median overall survival of the 89 patients was 33.0 months. CONCLUSION: IgG is the most common type in MM. Bone pain, anemia, hypercalcemia and renal insufficiency are common symptoms. Immunofixation electrophoresis should be performed routinely to avoid missed diagnosis of light-chain and IgD types of MM.

[Efficacy and toxicity of intravenous busulfan-based conditioning before allogeneic peripheral blood stem cell transplantation for leukemia].

BACKGROUND & OBJECTIVE: Busulfan (Bu) is commonly used as a component of conditioning regimens for hematopoietic stem cell transplantation. Erratic gastrointestinal absorption as a result of oral administration of Bu not only affects the efficacy, but also increases the risk of toxicity. This study was to analyze the efficacy and toxicity of intravenous Bu and cyclophosphamide (Cy) conditioning before allogeneic peripheral blood stem cell transplantation (allo-PBSCT) for leukemia. METHODS: Fifteen leukemia patients received intravenous Bu/Cy conditioning before allo-PBSCT, while 20 patients received oral Bu/Cy conditioning. The responses and adverse events of the 2 groups were assessed. RESULTS: All 15 patients in intravenous Bu/Cy group had hematopoietic engraftment. The median time of engraftment was 12 (9-15) days for neutrophils and 15 (11-24) days for platelets. Of the 15 patients, 6 (40.0%) developed acute graft-versus-host disease (aGVHD), including 4 cases of grade I-II aGVHD and 2 cases of grade III-IV aGVHD; during conditioning, 7 (46.6%) had vomiting, 1 (6.7%) had oral mucositis, 1 (6.7%) had hemorrhagic cystitis, 2 (13.3%) had hepatic damage, none developed seizure. With a median follow-up of 180 days (range, 35-420 days), 14 (93.3%) patients were alive, 1 died of severe aGVHD accompanied fungal infection of the lung and central nerve system. The occurrence rates of hepatic damage and oral mucositis were significantly lower in intravenous Bu/Cy group than in oral Bu/Cy group (13.3% vs. 60.0%, 6.7% vs. 80.0%, P<0.01). There were no significant differences in hematopoietic reconstruction, aGVHD, stomatitis, gastrointestinal reaction, and hemorrhagic cystitis between the 2 groups (P>0.05). CONCLUSION: The intravenous Bu/Cy conditioning before allo-PBSCT for leukemia has clear efficacy with low extramedullary toxicity.