Relationship between the honeydew of mealy bugs and the growth of Phlebopus portentosus.

BACKGROUND: Phlebopus portentosus and mealy bugs form a fungus-insect gall on the roots of host plants. The fungus and mealy bugs benefit mutually through the gall, which is the key link in the nutritional mechanism of P. portentosus. The cavity of the fungus-insect gall provides an ideal shelter for mealy bugs survival and reproduction, but how does P. portentosus benefit from this symbiotic relationship? METHODOLOGY AND RESULTS: Anatomical examination of fungus-insect galls revealed that one or more mealy bugs of different generations were living inside the galls. The mealy bug's mouthpart could penetrate through the mycelium layer of the inside of the gall and suck plant juice from the host plant root. Mealy bugs excreted honeydew inside or outside the galls. The results of both honeydew agar medium and quartz tests showed that the honeydew can attract and promote the mycelial growth of P. portentosus. A test of the relationship between the honeydew and the formation of the fungus-insect gall showed that honeydew promoted gall formation. CONCLUSIONS: All experimental results in this study show that the honeydew secreted by mealy bugs can attract and promote the mycelial growth of P. portentosus, forming a fungus-insect gall, because mealy bugs' honeydew is rich in amino acids and sugars.

Obstructive hydrocephalus as a result of giant cell tumor of the thoracic spine: A case report.

Giant cell tumors (GCTs) are rare bone tumors that account for ~5% of all primary bone tumors. When GCTs occur in the spine, patients usually present with localized pain and neurological symptoms, such as radiating pain or hyperesthesia. In the current report, an unusual case of a GCT of the thoracic spine associated with hydrocephalus is described. A 48-year-old male presented with urinary retention, loss of sensation in the lower limbs and inability to walk. The patient eventually developed hydrocephalus combined with altered consciousness, indicated by an inability to follow simple commands. Magnetic resonance (MR) imaging demonstrated the presence of a soft tissue mass at the T2 level, and biopsy examination of the tissue confirmed that it was a GCT. The patient experienced a sudden loss of consciousness due to an acute episode of obstructive hydrocephalus. A ventriculoperitoneal shunting procedure was performed to treat the hydrocephalus, and the patient regained normal consciousness, although the paraplegia persisted. An MR examination performed 30 months following surgery demonstrated that the tumor size was stable, consistent with the slow growth that is characteristic of GCTs. Diagnosis of GCTs may be challenging, and relies on radiographic and histopathologic findings. Although rare, acute hydrocephalus as a result of GCTs should not be excluded from a differential diagnosis.

Crystal structure of poly[bis-(µ2-5-hydroxy-nicotinato-κ(2) N:O (3))zinc].

The title coordination polymer, [Zn(C6H4NO3)2] n , was prepared under hydro-thermal conditions by the reaction of zinc nitrate with 5-hy-droxy-nicotinic acid in the presence of malonic acid. In the structure, the Zn(II) ion is coordinated by two carboxyl-ate O atoms and two pyridine N atoms of four 5-hy-droxy-nicotinate ligands in a distorted tetra-hedral coordin-ation environment. The µ2-bridging mode of each anion leads to the formation of a three-dimensional framework structure. Inter-molecular hydrogen bonds between the hy-droxy groups of one anion and the non-coordinating carboxyl-ate O atoms of neighbouring anions consolidate the crystal packing.

Fungus-insect gall of Phlebopus portentosus.

Phlebopus portentosus is a popular edible wild mushroom found in the tropical Yunnan, China, and northern Thailand. In its natural habitats, a gall often has been found on some plant roots, around which fungal fruiting bodies are produced. The galls are different from common insect galls in that their cavity walls are not made from plant tissue but rather from the hyphae of P. portentosus. Therefore we have termed this phenomenon "fungus-insect gall". Thus far six root mealy bug species in the family Pseudococcidae that form fungus-insect galls with P. portentosus have been identified: Formicococcus polysperes, Geococcus satellitum, Planococcus minor, Pseudococcus cryptus, Paraputo banzigeri and Rastrococcus invadens. Fungus-insect galls were found on the roots of more than 21 plant species, including Delonix regia, Citrus maxima, Coffea arabica and Artocarpus heterophyllus. Greenhouse inoculation trials showed that fungus-insect galls were found on the roots of A. heterophyllus 1 mo after inoculation. The galls were subglobose to globose, fulvous when young and became dark brown at maturation. Each gall harbored one or more mealy bugs and had a chimney-like vent for ventilation and access to the gall. The cavity wall had three layers. Various shaped mealy bug wax deposits were found inside the wall. Fungal hyphae invaded the epidermis of plant roots and sometimes even the cortical cells during the late stage of gall development. The identity of the fungus inside the cavity was confirmed by molecular methods.

A baculovirus isolated from wild silkworm encompasses the host ranges of Bombyx mori nucleopolyhedrosis virus and Autographa californica multiple nucleopolyhedrovirus in cultured cells.

A baculovirus, named BomaNPV S2, was isolated from a diseased larva of the wild silkworm, Bombyx mandarina. Notably, BomaNPV S2 exhibited a distinguishing feature in that its host range covered that of both Bombyx mori nucleopolyhedrosis virus (BmNPV) and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in cultured cells. It could replicate in cells of B. mori (Bm5 and BmN), Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn-5B1-4). However, occlusion-derived virions of BomaNPV S2 in B. mori cells contained only a single nucleocapsid, whereas they contained multiple nucleocapsids in Tn-5B1-4 cells. The complete genome sequence of BomaNPV S2, including predicted ORFs, was determined and compared with the genome sequence of its close relatives. The comparison results showed that most of the BomaNPV S2 genome sequence was shared with BmNPV (BmNPV T3) or BomaNPV S1, but several regions seemed more similar to regions of AcMNPV. This observation might explain why BomaNPV S2 covers the host ranges of BmNPV and AcMNPV. Further recombinant virus infection experiments demonstrated that GP64 plays an important role in BomaNPV S2 host-range determination.

[Flow cytometry for evaluation of sperm plasma membrane integrity in varicocele patients].

OBJECTIVE: To detect the sperm plasma membrane integrity (PMI) of varicocele (VC) patients using SYBR-14/PI fluorescent staining and flow cytometry, and investigate its clinical significance. METHODS: We collected semen samples from 120 men, including 30 grade-1 varicocele patients (VC1), 30 grade-2 (VC2), 30 grade-3 (VC3), and 30 normal fertile volunteer controls. Conventional semen analyses were performed by computer-assisted semen analysis (CASA). All the semen samples were washed with PBS and then subjected to SYBR-14/PI staining for the detection of sperm PMI by flow cytometry. The proportion of normal sperm with PMI was indicated as the percentage of sperm emitting green fluorescence (SYBR-14+/PI- %), sperm PMI was determined and sperm fertilization capacity predicted. RESULTS: Significant differences were detected in SYBR-14+/PI- and SYBR-14-/PI+ between the normal men and varicocele male patients (P < 0.01). The percentages of the sperm with PMI (SYBR-14+/PI- %) were remarkably lower in the VC1, VC2 and VC3 groups ([54.85 +/- 3.78]%, [45.37 +/- 4.12]% and [35.14 +/- 4.91]%) than in the normal controls ([70.79 +/- 6.71]%). SYBR-14+/PI-% was correlated positively with sperm motility (r=0.965, P < 0.01) and the percentage of grade a + b sperm (r = 0.874, P < 0.01), negatively with the percentage of grade d sperm (r = -0.965, P <0.01), but not significantly with pH, semen volume and liquefaction time (P > 0.05). CONCLUSION: SYBR-14/PI fluorescent staining and flow cytometry can quickly and exactly detect sperm PMI. Varicocele decreases sperm PMI, which might be an important cause of male infertility.

Co-expression of tetanus toxin fragment C in Escherichia coli with thioredoxin and its evaluation as an effective subunit vaccine candidate.

The receptor-binding domain of tetanus toxin (THc), which mediates the binding of the toxin to the nerve cells, is a candidate subunit vaccine against tetanus. In this study one synthetic gene encoding the THc was constructed and highly expressed in Escherichia coli by co-expression with thioredoxin (Trx). The purified THc-vaccinated mice were completely protected against an active toxin challenge in mouse models of disease and the potency of two doses of THc was comparable to that of three doses of toxoid vaccine. And a solid-phase assay showed that the anti-THc sera inhibited the binding of THc or toxoid to the ganglioside GT1b as the anti-tetanus toxoid sera. Furthermore, mice were vaccinated once or twice at four different dosages of THc and a dose-response was observed in both the antibody titer and protective efficacy with increasing dosage of THc and number of vaccinations. The data presented in the report showed that the recombinant THc expressed in E. coli is efficacious in protecting mice against challenge with tetanus toxin suggesting that the THc protein may be developed into a human subunit vaccine candidate designed for the prevention of tetanus.

Expression of a neurotoxin gene improves the insecticidal activity of Spodoptera litura nucleopolyhedrovirus (SpltNPV).

SpltNPV-C3 is a novel nucleopolyhedrovirus (NPV) screened from 189 wild Spodoptera litura nucleopolyhedrovirus (SpltNPV) clones collected throughout Japan and has high potential insecticidal activity against S. litura. In this study, we constructed two recombinant SpltNPVs, SpltNPV-Δegt and SpltNPV-Δegt-BmK, which were characterized by the disruption of the UDP-glucosyltransferase gene (egt) only and by replacing the egt locus with the Buthus martensi insect-specific toxin gene (BmK ITa1), respectively. The insecticidal activity of these two recombinant viruses was evaluated by 50% lethal time (LT(50)) and 50% lethal concentration (LC(50)) using third instar S. litura larvae. Bioassays showed that the LT(50) of SpltNPV-Δegt was reduced by 25% at a dose of 10(9) Occlusion bodies (OBs)/ml compared to the wild-type SpltNPV. However, the LC(50) values did not change. The SpltNPV-Δegt-BmK reduced LT(50) by 28% at the highest dose of OBs (10(9) OBs/ml) compared to the wild-type SpltNPV, and the LC(50) was nearly an order of magnitude lower than those of the wild-type SpltNPV and SpltNPV-Δegt. However, there was no discernable difference in the LT(50) between SpltNPV-Δegt-BmK and SpltNPV-Δegt when the 3rd instars of S. litura were fed a dose of 10(8) or 10(9) OBs/ml. The results suggest that these two recombinant forms of SpltNPV provide further opportunities to develop these viruses into commercially viable products to control S. litura populations.

Development and evaluation of candidate vaccine and antitoxin against botulinum neurotoxin serotype F.

To produce a vaccine suitable for human use, a recombinant non His-tagged isoform of the Hc domain of botulinum neurotoxin serotype F (rFHc) was expressed in Escherichia coli and purified by sequential chromatography. The rFHc was evaluated as a subunit vaccine candidate in mouse model of botulism. A dose-response was observed in both antibody titer and protective efficacy with increasing dosage of rFHc and number of vaccinations. These findings suggest that the rFHc is an effective botulism vaccine candidate. Further, we developed a new antitoxin against botulinum neurotoxin serotype F (BoNT/F) by purifying F(ab')(2) fragments from pepsin digested serum IgGs of horses inoculated with rFHc. The protective effect of the F(ab')(2) antitoxin against BoNT/F was determined both in vitro and in vivo. The results showed that the F(ab')(2) antitoxin could prevent botulism in mice challenged with BoNT/F and effectively delayed progression of paralysis from botulism in the therapeutic setting. Thus, our results provide valuable experimental data for this new antitoxin as a potential candidate for treatment of botulism caused by BoNT/F.

Development and preclinical evaluation of a new F(ab')2 antitoxin against botulinum neurotoxin serotype A.

Concern about the malicious applications of botulinum neurotoxin has highlighted the need for a new generation of safe and highly potent antitoxins. In this study, we developed and evaluated the preclinical pharmacology and safety of a new F(ab')2 antitoxin against botulinum neurotoxin serotype A (BoNT/A). As an alternative to formalin-inactivated toxoid, the recombinant Hc domain of botulinum neurotoxin serotype A (rAHc) was used to immunize horses, and the IgGs from the hyperimmune sera were digested to obtain F(ab')2 antitoxin. The protective effect of the new F(ab')2 antitoxin against BoNT/A was determined both in vitro and in vivo. The results showed that the F(ab')2 antitoxin could prevent botulism in mice challenged with BoNT/A and effectively delayed progression of paralysis from botulism in the therapeutic setting. The preclinical safety of the new F(ab')2 antitoxin was also evaluated, and it showed neither harmful effects on vital functions nor adverse effects such as acute toxicity, or immunological reactions in mice and dogs. Thus, our results provide valuable experimental data for this new antitoxin as a potential candidate for treatment of botulism caused by BoNT/A, and our findings support the safety of the new F(ab')2 antitoxin for clinical use. Our study further demonstrates the proof of concept for development of a similar strategy for obtaining potent antitoxin against other BoNT serotypes.

A new report of a mosaic dwarf viroid-like disease on mulberry trees in China.

A viroid-like disease causing mosaic leaves and dwarfism was found on mulberry plants in Zhejiang, China. Grafting of stems from infected plants onto healthy plants resulted in the same symptoms on the healthy plants. A circular small RNA (Mmd-v RNA1) was isolated from the infected plant leaves and caused identical symptoms after more than two years. Nucleotide sequencing indicated that the Mmd-v RNA1 contains 356 nt (GenBank accession no. NC_011362) without viroid characteristic regions, only sharing 30 nt sequence identity with that of the Cherry small circular viroid-like RNA 1 (isolate cscRNA1.150, GenBank accession no. DQ357628). This description is the first of viroid-like RNA infection of mulberry trees.

Comparative analysis of the genomes of Bombyx mandarina and Bombyx mori nucleopolyhedroviruses.

The Bombyx mandarina nucleopolyhedrovirus (BomaNPV) S1 strain can infect the silkworm, Bombyx mori, but is significantly less virulent than B. mori nucleopolyhedrovirus (BmNPV) T3 strain. The complete nucleotide sequence of the S1 strain of BomaNPV was determined and compared with the BmNPV T3 strain. The circular, double stranded DNA genome of the S1 strain was 126,770 nucleotides long (GenBank accession no. FJ882854), with a G+C content of 40.23%. The genome contained 133 potential ORFs. Most of the putative proteins were more than 96% identical to homologs in the BmNPV T3 strain, except for bro-a, lef-12, bro-c, and bro-d. Compared with the BmNPV T3 strain, however, this genome did not encode the bro-b and bro-e genes. In addition, hr1 lacked two repeat units, while hr2L, hr2R, hr3, hr4L, hr4R, and hr5 were similar to the corresponding hrs in the T3 strain. The sequence strongly suggested that BomaNPV and BmNPV are variants with each other, and supported the idea that baculovirus strain heterogeneity may often be caused by variation in the hrs and bro genes.

Genomic sequence, organization and characteristics of a new nucleopolyhedrovirus isolated from Clanis bilineata larva.

BACKGROUND: Baculoviruses are well known for their potential as biological agents for controlling agricultural and forest pests. They are also widely used as expression vectors in molecular cloning studies. The genome sequences of 48 baculoviruses are currently available in NCBI databases. As the number of sequenced viral genomes increases, it is important for the authors to present sufficiently detailed analyses and annotations to advance understanding of them. In this study, the complete genome of Clanis bilineata nucleopolyhedrovirus (ClbiNPV) has been sequenced and analyzed in order to understand this virus better. RESULTS: The genome of ClbiNPV contains 135,454 base pairs (bp) with a G+C content of 37%, and 139 putative open reading frames (ORFs) of at least 150 nucleotides. One hundred and twenty-six of these ORFs have homologues with other baculovirus genes while the other 13 are unique to ClbiNPV. The 30 baculovirus core genes are all present in ClbiNPV. Phylogenetic analysis based on the combined pif-2 and lef-8 sequences places ClbiNPV in the Group II Alphabaculoviruses. This result is consistent with the absence of gp64 from the ClbiNPV genome and the presence instead of a fusion protein gene, characteristic of Group II. Blast searches revealed that ClbiNPV encodes a photolyase-like gene sequence, which has a 1-bp deletion when compared with photolyases of other baculoviruses. This deletion disrupts the sequence into two small photolyase ORFs, designated Clbiphr-1 and Clbiphr-2, which correspond to the CPD-DNA photolyase and FAD-binding domains of photolyases, respectively. CONCLUSION: ClbiNPV belongs to the Group II Alphabaculoviruses and is most closely related to OrleNPV, LdMNPV, TnSNPV, EcobNPV and ChchNPV. It contains a variant DNA photolyase gene, which only exists in ChchNPV, TnSNPV and SpltGV among the baculoviruses.

A novel way to purify recombinant baculoviruses by using bacmid.

In the present study, we studied the feasibility of deleting essential genes in insect cells by using bacmid and purifying recombinant bacmid in Escherichia coli DH10B cells. To disrupt the orf4 (open reading frame 4) gene of BmNPV [Bm (Bombyx mori) nuclear polyhedrosis virus], a transfer vector was constructed and co-transfected with BmNPV bacmid into Bm cells. Three passages of viruses were carried out in Bm cells, followed by one round of purification. Subsequently, bacmid DNA was extracted and transformed into competent DH10B cells. A colony harbouring only orf4-disrupted bacmid DNA was identified by PCR. A mixture of recombinant (white colonies) and non-recombinant (blue colonies) bacmids were also transformed into DH10B cells. PCR with M13 primers showed that the recombinant and non-recombinant bacmids were separated after transformation. The result confirmed that purification of recombinant viruses could be carried out simply by transformation and indicated that this method could be used to delete essential genes. Orf4-disrupted bacmid DNA was extracted and transfected into Bm cells. Viable viruses were produced, showing that orf4 was not an essential gene.

Expression of Bombyx mori nucleopolyhedrovirus ORF76 in permissive and non-permissive cell lines by a novel Bac-to-Bac/BmNPV baculovirus expression system.

Open reading frame 76 of Bombyx mori nucleopolyhedrovirus (BmNPV), designated as Bm76, is a gene whose function is completely unknown. With EGFP fused to the 3' terminal of Bm76 as the reporter gene and BmNPV bacmid as the expression vector, a recombinant bacmid was successfully constructed expressing Bm76-EGFP fusion protein under the control of polyhedrin promoter in Bombyx mori cells (Bm cells), BmNPV's permissive cell line, laying the foundation for rescue experiment of Bm76 deletion mutant. Moreover, the supernatant from Bm cells transfected with the recombinant bacmid was used to infect Trichoplusia Ni cells (Tn cells), BmNPV's non-permissive cell line. Unexpectedly, the expression of Bm76-EGFP fusion protein in some Tn cells was detected, implying that viral DNA was replicated in these cells. The causes are being studied for the inability of BmNPV to produce enough viable budded viruses in Tn cells despite of viral DNA replication.

[Expression of human phosphodiesterase 3A gene using baculovirus expression system in insect cell].

AIM: To investigate the expression of recombinant human phosphodiesterase 3A (HPDE3A) using baculovirus expression system in Tn cell line. METHODS: The HPDE3A cDNA was recombined with baculovirus, and then the recombinant was transfected into Tn cell line. The expression of HPDE3A in Tn cell line was detected and identified by the RT-PCR, SDS-PAGE and Western blotting. RESULTS: The recombinant HPDE3A protein was stably expressed in Tn cell line and detected by the distinct morphological changes of Tn cell, RT-PCR, SDS-PAGE and Western blotting using polyclonal antibody. The M(w) of the recombinant protein was about 120 kD. CONCLUSION: Recombinant HPDE3A can be expressed in Tn cell line using the baculovirus expression system, and thus provided the basic material for studying its bioactivity and application in screening for HPDE3A inhibitor.

[Construction of a baculovirus transfer vector and expression of baculovirus-mediated gfp gene in larvae of Spodoptera litura].

To construct a novel baculovirus expression system of Spodoptera litura multicapsid nucleopolyhedrovirus, the 5' end and 3' end-flanking fragments of ph gene were amplified from the genome DNA of SpltMNPV, Japan-C3 strain using two pairs of primers synthesized according to SpltMNPV China-G2 strain genome DNA sequence published in GenBank. To obtain the transfer vector pSplt-gfp, the fragment of gfp gene was inserted into this vector between two fragments tandem linked into pUC18. The spli cells were cotransfected with pSplt-gfp and the wild SpltMNPV genome DNA. The recombinant virus containing gfp was selected with the limited dilution method. The fluorescence can be observed in the spli cells and the 3rd instar larvae after 24 and 48 hours by infection of the recombinant virus, respectively. The result showed that the recombinant virus was obtained successfully. It will be helpful to establish Spodoptera litura multicapsid nucleopolyhedrovirus expression system and more effective pesticide for Spodoptera litura.