BackgroundAlthough a variety of methods and expensive kits are available, molecular cloning can be a time-consuming and frustrating process.ResultsHere 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.ConclusionOur 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.

Over the past decades, 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 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 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.

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 ␣7 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 ␣7 nAChR.The Cys-loop receptor family of ligand-gated ion channels are allosteric proteins (1-4). Binding of neurotransmitter to the receptor binding site located in the N-terminal domain induces conformational changes (5), which propagate to the transmembrane domain (M1-M4) 3 through the interface between these two domains to open the gate, which is formed by M2 domains (see Fig. 1A). Coupling through this interface is mainly mediated by noncovalent interactions between loops 2 and 7 (Cysloop) from N-terminal domain and M2-M3 linker from the transmembrane domain (see Fig. 1B) (6, 7). In addition, pre-M1 and M1 is the only covalent linkage between N-terminal domain and transmembrane domain, which also play a role in controlling channel gating (7). However, agonist binding does not guarantee channel opening. With prolonged exposure to neurotransmitters, most receptors are driven to a refractory state, termed desensitization (8). At the single channel level, desensitization appears as long-lasting nonconducting states (9). Desensitization is a widespread phenomenon in most ligand-gated ion channels. It plays an important role in shaping synaptic transmission (10, 11). Desensitization of the Cys-loop receptor family has been well characterized kinetically. It involves dramatic increase in binding affinity to agonists (12, 13) and closure of the ion conducting pathway (9) with little structural change in transmembrane domain (14). It is an intrinsic property of the receptors (15).Previous studies have identified numerous factors that can influence desensitization. For example, desensitization is dependent on agonists (16) and receptor subtypes (17, 18). Mutations of a residue in the binding pocket (19) or between two binding loops...