Dynamics of the Interaction between Cotton Bollworm Helicoverpa armigera and Nucleopolyhedrovirus as Revealed by Integrated Transcriptomic and Proteomic Analyses.

Over the past decades, Helicoverpa armigera nucleopolyhedrovirus (HearNPV) has been widely used for biocontrol of cotton bollworm, which is one of the most destructive pest insects in agriculture worldwide. However, the molecular mechanism underlying the interaction between HearNPV and host insects remains poorly understood. In this study, high-throughput RNA-sequencing was integrated with label-free quantitative proteomics analysis to examine the dynamics of gene expression in the fat body of H. armigera larvae in response to challenge with HearNPV. RNA sequencing-based transcriptomic analysis indicated that host gene expression was substantially altered, yielding 3,850 differentially expressed genes (DEGs), whereas no global transcriptional shut-off effects were observed in the fat body. Among the DEGs, 60 immunity-related genes were down-regulated after baculovirus infection, a finding that was consistent with the results of quantitative real-time RT-PCR. Gene ontology and functional classification demonstrated that the majority of down-regulated genes were enriched in gene cohorts involved in energy, carbohydrate, and amino acid metabolic pathways. Proteomics analysis identified differentially expressed proteins in the fat body, among which 76 were up-regulated, whereas 373 were significantly down-regulated upon infection. The down-regulated proteins are involved in metabolic pathways such as energy metabolism, carbohydrate metabolism (CM), and amino acid metabolism, in agreement with the RNA-sequence data. Furthermore, correlation analysis suggested a strong association between the mRNA level and protein abundance in the H. armigera fat body. More importantly, the predicted gene interaction network indicated that a large subset of metabolic networks was significantly negatively regulated by viral infection, including CM-related enzymes such as aldolase, enolase, malate dehydrogenase, and triose-phosphate isomerase. Taken together, transcriptomic data combined with proteomic data elucidated that baculovirus established systemic infection of host larvae and manipulated the host mainly by suppressing the host immune response and down-regulating metabolism to allow viral self-replication and proliferation. Therefore, this study provided important insights into the mechanism of host-baculovirus interaction.

FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method.

BACKGROUND: Although a variety of methods and expensive kits are available, molecular cloning can be a time-consuming and frustrating process. RESULTS: Here we report a highly simplified, reliable, and efficient PCR-based cloning technique to insert any DNA fragment into a plasmid vector or into a gene (cDNA) in a vector at any desired position. With this method, the vector and insert are PCR amplified separately, with only 18 cycles, using a high fidelity DNA polymerase. The amplified insert has the ends with ~16-base overlapping with the ends of the amplified vector. After DpnI digestion of the mixture of the amplified vector and insert to eliminate the DNA templates used in PCR reactions, the mixture is directly transformed into competent E. coli cells to obtain the desired clones. This technique has many advantages over other cloning methods. First, it does not need gel purification of the PCR product or linearized vector. Second, there is no need of any cloning kit or specialized enzyme for cloning. Furthermore, with reduced number of PCR cycles, it also decreases the chance of random mutations. In addition, this method is highly effective and reproducible. Finally, since this cloning method is also sequence independent, we demonstrated that it can be used for chimera construction, insertion, and multiple mutations spanning a stretch of DNA up to 120 bp. CONCLUSION: Our FastCloning technique provides a very simple, effective, reliable, and versatile tool for molecular cloning, chimera construction, insertion of any DNA sequences of interest and also for multiple mutations in a short stretch of a cDNA.

Desensitization of alpha7 nicotinic receptor is governed by coupling strength relative to gate tightness.

Binding of a neurotransmitter to its membrane receptor opens an integral ion conducting pore. However, prolonged exposure to the neurotransmitter drives the receptor to a refractory state termed desensitization, which plays an important role in shaping synaptic transmission. Despite intensive research in the past, the structural mechanism of desensitization is still elusive. Using mutagenesis and voltage clamp in an oocyte expression system, we provide several lines of evidence supporting a novel hypothesis that uncoupling between binding and gating machinery is the underlying mechanism for α7 nicotinic receptor (nAChR) desensitization. First, the decrease in gate tightness was highly correlated to the reduced desensitization. Second, nonfunctional mutants in three important coupling loops (loop 2, loop 7, and the M2-M3 linker) could be rescued by a gating mutant. Furthermore, the decrease in coupling strength in these rescued coupling loop mutants reversed the gating effect on desensitization. Finally, coupling between M1 and hinge region of the M2-M3 linker also influenced the receptor desensitization. Thus, the uncoupling between N-terminal domain and transmembrane domain, governed by the balance of coupling strength and gate tightness, underlies the mechanism of desensitization for the α7 nAChR.

[Detection of genomic duplications and deletions of the DMD gene in affected males and female carriers by using mutiplex ligation-dependent probe amplification].

OBJECTIVE: To detect genomic deletion and duplication mutations in the dystrophin gene of the Duchenne muscular dystrophy (DMD) patients and their potential female carriers. METHODS: Genomic deletions and duplications of the DMD gene in 32 affected males and 27 potential female carriers were screened by mutiplex ligation-dependent probe amplification (MLPA). RESULTS: Of the 32 investigated affected males, 24 were detected to have deletions of one or more exons of the DMD gene, 1 patient had a duplication from exon 5 to 55, 1 patient had a nonsense point mutation (R768X) in exon 19, the other 6 affected males were predicted to have possible disease-causing point mutations. MLPA analysis showed a DMD deletion or duplication in 18 female relatives, and the female carriers had the same deletion or duplication as their probands, respectively. CONCLUSION: MLPA analysis is proven to be an efficient tool for identification of both affected males and female carriers of DMD rearrangements in cases in which the disease-causing mutation in the affected male was not known. It could provide useful information for the genetic counseling of the family involved.

[Identification of deletion/duplication mutations in DMD gene by multiple ligation probe amplification and denaturing high-performance liquid chromatography].

OBJECTIVE: To compare the effectiveness of using multiple ligation probe amplification (MLPA) and denaturing high-performance liquid chromatography (DHPLC) in screening the exon deletions and duplications of the DMD gene. METHODS: MLPA technique was applied to detect exon deletions and duplications previously confirmed by denaturing high-performance liquid chromatography (DHPLC). RESULTS: From October 2004 to October 2005, 22 unrelated DMD probands and their possible female relatives with clinical diagnosis with dystrophinopathy at our hospital entered this study. Both DHPLC and MPLA detected DMD gene depletion in 11 probands and DMD duplications in 3 probands. MLPA detected deletions and duplications in 2 probands, which were not detected by DHPLC. MLPA also successfully identified the carriage status of the potential female carriers of the probands. CONCLUSION: Compared with DHPLC and traditional PCR techniques, MLPA is a superior tool to analyze the deletions and duplications in affected males as well as in the identification of the carriage status of potential females carriers.

[Sibling brother and sister both with Duchenne muscular dystrophy].

OBJECTIVE: To investigate the clinical and lab features of sibling brother and sister both with Duchenne muscular dystrophy (DMD). METHODS: We conducted comprehensive clinical and lab investigations including the test of serum enzymes, electromyography (EMG), electrocardiography, color Doppler echocardiography, HE staining of skeletal muscles, immunohistochemical study of dystrophin and utrophin, multiple ligation probe amplification (MLPA) on exon 1-79 of dystrophin gene, and short tandem repeat-poly- merase chain reaction of CA repeats located in dystrophin gene. RESULTS: These two patients were confirmed to suffer from DMD. They were characterized by typical features of DMD including typical clinical manifestations, increased serum enzymes, EMG presenting myogenic impairment, HE staining presentation belonging to DMD, negative dystrophin in brother, and inconstantly positive on the sarcolemma of sister. Furthermore, no deletion or duplication was found in the 1-79 exons of dystrophin gene. The suffering brother and sister carried the same maternal X chromosome. CONCLUSIONS: Carriers of DMD gene show typical clinical and laboratory manifestations of DMD. Comprehensive examinations should be performed for such carriers.

[Identification of disease-causing point mutations in DMD patients' dystrophin gene without large deletions/duplications].

OBJECTIVE: To detect the disease-causing point mutations in the dystrophin gene of Duchenne muscular dystrophy (DMD) patients. METHODS: The approach of denaturing high performance liquid chromatography (DHPLC) coupling with sequencing was used to screen the point mutations of 79 exons and the untranslated regions of dystrophin gene without large deletions/duplications, which was in 6 unrelated DMD probands from 6 DMD families. RESULTS: Five disease-causing mutations, 697-698insGT, C616T, G1255T, C4279T, and C2302T, were ides created the new stop codons in downstream sites of mutations, respectively. In addition to the disease-causing point mutations, a point mutation T5586+61A in intron 39 was also found at patient 3, and a missense mutation A694T in exon 8 was detected at patient 5. Four point mutations, C2168+13T, 5740-13dupG, G5234A and C5280T, were also detected at patient 6 whose causative point mutation was unavailable. Seven point mutations have not been reported previously. Bi-directional PCR amplification of specific alleles (Bi-PASA) method was established to distinguish the haplotypes of heterozygote or homozygote in a single PCR reaction. CONCLUSION: Via automated DHPLC screening or detecting the subexonic mutations in dystrophin gene is feasible to clinical laboratories, and also is a superior method in terms of sensitivity and efficiency.

[Transfection and in vitro expression of human microdystrophin gene in rat mesenchymal stem cells].

OBJECTIVE: To construct the eukaryotic expression vector of human microdystrophin gene and observe its expression in rat mesenchymal stem cells (rMSCs) in vitro. METHODS: The plasmid PBSK-MICRO containing human microdystrophin cDNA was digested by restriction endonuclease, and the resultant microdystrophin fragment was inserted into the NotI site of pcDNA3.1(+) to prepare the eukaryotic expression vector-pcDNA3.1(+)/ microdystrophin, which was identified by endonuclease digestion and sequencing. The recombinant plasmid was transfected into rMSCs via lipofectamine, and after G418 selection, the expression of microdystrophin was detected by RT-PCR and indirect immunofluorescence assay. RESULTS: Microdystrophin gene fragment was correctly inserted into the plasmid pcDNA3.1(+), as conformed by sequencing and digestion with Not I and Hind III. The total mRNA of the transfected rMSCs was extracted and microdystrophin mRNA expression was found in the cells by RT-PCR. Indirect immunofluorescence assay for the protein expression of microdystrophin showed bright red fluorescence in the transfected rMSCs. CONCLUSION: Eukaryotic expression plasmid pcDNA3.1(+)/microdystrophin has been constructed successfully and microdystrophin can be expressed in transfected rMSCs in vitro, which may facilitate further research of Duchenne muscular dystrophy treatment by genetically modified allogeneic stem cell transplantation.

Cloning of CYP9G2 from the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae).

Cytochrome P450s constitute a superfamily of hemoproteins, important in the metabolism of endogenous and xenobiotic compounds. The full-length cDNA of a novel cytochrome P450, CYP9G2, was isolated from a cDNA library. The cDNA is 2143 bp in length and contains an open reading frame from 50 to 1615 bp, encoding a protein of 521 amino acid residues. The putative P450 protein contains a highly hydrophobic N terminus and a P450 protein signature motif, FG/S*G*R*C*G***A/G, known as the important ligand for heme binding, analysis of the NH2-terminal sequence indicated that CYP9G2 is a microsomal P450. Using polymerase chain reaction with primers specific to CYP9G2, the genomic structure of CYP9G2 was analyzed, and it was found that the gene contains seven introns and eight exons within the coding region, all the sequences of the exon-intron junctions are consistent with the AG-GT rule. Multiple alignment indicated that CYP9G2 is most similar to CYP9E2 from the Blattella germanica (42.7% identity), it is also similar to the insect P450s in family 9, including CYP9L1 from Anopheles gambiae (38.7%) and CYP9A1 from Heliothis virescens (39.5%).