Penis size differences (or, more precisely, perceived differences) are the basis of an enormous amount of male anxiety. It is true that there are differences in the flaccid size of the penis, but that has little to do with penis size or sexual functioning in the erect state. The average penis is from two and a half to four inches in the flaccid state and from five and a half to six and a half inches when erect. The diameter is about one inch flaccid and one and a half inches when erect. It is more meaningful to say a normal penis is of proper size to function during intercourse. This definition includes almost all men.

Interestingly, three out of four men believe that their penises are smaller than average, which illustrates how the performance machine model dominates male sexuality, leaving men to feel anxious and insecure. Psychological and relational health is promoted by adopting a positive body image, which includes accepting your penis. Remember, solid scientific evidence demonstrates that there is no relationship between penis size and sexual desire or response for either the man or the woman. Most women say it is not the size but how you make love that is important. Canadian pharmacy viagra

A related myth is that a large penis results in the woman being orgasmic during intercourse. This is based on the mistaken belief that the vagina is the woman’s major sex organ. In truth, the woman’s most sensitive genital organ is her clitoris—a small, cylindrical organ located at the top of the vaginal opening where it joins with the labia (“lips” of the vagina). The clitoris has a multitude of nerve endings—like the glans of your penis, only concentrated in a much smaller area. It is the focal point of her physical sexual pleasure. Most women prefer indirect clitoral stimulation, whether with your hand, tongue, or penis.

During intercourse, the clitoris is stimulated by the pulling and rubbing action caused by pelvic thrusting—stimulation that is independent of penis size. The vagina, which is in contact with the penis, has fewer nerve endings, most of which are in the outer third. Moreover, the vagina is an active rather than a passive organ, which means that the vagina swells and expands with a woman’s arousal to engage the penis and can adjust to the penis whatever its size. It usually takes 10 to 20 minutes of pleasuring (foreplay) for the vagina to fully expand. If a couple is rushing to intercourse, the man mistakenly thinks that his penis is too small because the vagina does not feel snug. The remedy is enjoying pleasurable touch and genital stimulation before intercourse to allow her body and vagina to reach the plateau stage of arousal. Sexual incompatibility based on the couple’s genitals is, with extremely rare exceptions, a myth.

It’s generally accepted that modification of a viral antigen by fusion to a cellular protein, like eCTLA-4, could improve the efficacy of DNA vaccine. Here we constructed DNA plasmids encoding HTNV N or GP fused to eCTLA-4 protein (pcDNA3/eCTLA4-S or M). Compared to DNA vaccine encoding HTNV N or GP alone, pcDNA3/eCTLA4-S (M) greatly improved the speed and magnitude of HTNV specific humoral immune response in mice. Lu et al. reported similar modulation effect of eCTLA4 on woodchuck hepatitis virus nucleoprotein in mice and woodchuck models. Nicholas and his colleagues also observed the enhancement of immune responses to pro cathepsin B antigen in sheep model by fusion to eCTLA4 [40] In addition, there is a higher frequency of CD8+/IFN+ T-cells in mice immunized with pcDNA3/eCTLA4-S (M) DNA plasmids than that of pcDNA3/S (M). As the high affinity of eCTLA4 to its B7 ligand of APCs, our results indicated that eCTLA4 targeting may facilitate the antigen intake and processing by APCs, which will possibly improve the efficacy of DNA vaccine.

Another interesting finding of our study is that the efficacy of HTNV DNA vaccine was augmented by CpG motifs. When co-administration with CpG motifs, HTNV DNA vaccine induced better immune responses in mice compared with immunization with HTVN DNA vaccine alone. Vaccination with pcDNA3/eCTLA4-S (M) DNA plasmids plus CpG motifs elicited the highest antibody and cellular immune responses compared to all the other groups. Mice receiving pcDNA3/S (M) plus CpG motifs, though showed lower antibody titer one week after first immunization than that of mice vaccinated with pcDNA3/eCTLA4-S (M) DNA plasmids alone, exhibited comparable antibody response after the second injection. The recognition of CpG motifs is through toll-like receptor 9 (TLR-9) and then induces a broad range of immunological effects on APCs. Adjuvant effect of CpG motifs have been demonstrated in mice, humans as well as other species. Thus, after co-delivery of CpG motifs with HTNV DNA vaccine, it’s conceivable that APCs may be activated firstly by CpG motifs, then display enriched costimulatory molecules on the surface. This early event may provide a more efficient intake of antigen mediated by eCTLA4 later on through binding with B7 ligand. This may, if any, at least partially explain the observed augmentation of humoral and cellular immune responses induced by HTNV DNA vaccine in combination with CpG motifs.

CD8+ T-cells play a vital role in protection against hantavirus infection by cell-mediated mechanisms. In order to evaluate the CD8+ T-cell response to vaccination, the splenocytes from mice vaccinated with DNA vaccine plasmids 1 week after each immunization were restimulated with HTNV N protein-specific peptides and analyzed by ELISPOT. The splenocytes from mice 1 week after third immunization were restimulated and analyzed by Intracellular Cytokine Staining assay. Number of CD8+IFN-γ-secreting splenocytes was significantly higher than other groups (p < 0.01) at 21 days after 1st immunization in mice receiving CpG+peCTLA4-M+S vaccine (Figure 3A). Consistently, mice vaccinated with pcDNA3/eCTLA4-S+M plasmids plus CpG1826 motif demonstrated higher frequencies of CD8+IFN+ T-cells to HTNV N protein-specific peptides compared with all the other groups in flow cytometery analysis. These results indicate that eCTLA4 fusion strategy could enhance the Th1-type cellular immune response. Discussion

DNA immunization with plasmids expressing hantavirus N protein and glycoprotein by intramuscular vaccination induced specific immune responses to the corresponding viral antigens in mice. In this study, we demonstrated that, a better magnitude of humoral and cellular immune responses could be generated in mice by DNA vaccine plasmids encoding HTNV N and GP fused to eCTLA4, a bioactive factor targeting to antigen presenting cells (APCs).

IFA was also used to verify the expression of eCTLA4-N and eCTLA4-GP fusion proteins as described in Methods. BHK cells were transiently transfected with pcDNA3/eCTLA4-M or pcDNA3/eCTLA4-S construct. The expression of eCTLA4-GP or eCTLA4-N fusion protein was detected with Gc- specific antibody (Y22) or N-specific antibody (L13F3) [28,29] respectively as demonstrated in (Figure 1C, a and 1c). Furthermore, eCTLA4-GP and eCTLA4-N fusion proteins could also be captured by monoclonal antibody of anti-mouse eCTLA-4.

Antibody responses to HTNV N and GP induced in mice following immunization with plasmids expressing eCTLA4-N/GP fusion protein

To evaluate whether eCTLA4 fusion strategy could enhance immunogenicity on HTNV DNA vaccine, C57 mice were immunized with DNA plasmids expressing HTNV N and GP, or eCTLA4-N and GP fusion proteins with or without 10 μg of CpG1826 motifs. The antibody immune response to HTNV N or GP was determined by N-specific ELISA or IFA assays. (Figure 2) The levels of N protein-specific IgG were found to be substantially induced one week after first immunization in mice that received pcDNA3/eCTLA4-S+M DNA plasmids alone or with CpG1826 (Figure 2A), and significantly higher than that of mice receiving pcDNA3/S+M DNA plasmids alone or with CpG1826. One week after second injection, mice immunized with pcDNA3/eCTLA4-S+M plasmids plus CpG1826 showed significantly higher N protein-specific IgG antibody titers compared to groups of mice that received pcDNA3/eCTLA4-S+M or pcDNA3/S+M DNA plasmids alone (p < 0.05), and about 3.5-fold higher than that of mice receiving pcDNA3/S+M DNA plasmids plus CpG1826 though not achieved statistic significance. After two boosts, all mice that received HTNV DNA vaccine plasmids had substantial increase of N protein-specific IgG antibody titers. DNA plasmids expressing eCTLA4-N and GP fusion proteins, combined with CpG1826, elicited the highest N protein-specific IgG antibody titers one week after third immunization compared to all the other groups (p < 0.05). In addition, we also observed that the magnitude of glycoprotein specific IgG antibody was significantly improved by vaccination with DNA plasmids expressing eCTLA4-N and GP fusion proteins, especially when combined with CpG1826. No eCTLA-4-specific antibodies were detected in sera of mice receiving DNA plasmids expressing fusion proteins (data not shown), which is consistent with the results of Lu et al. These results indicate that eCTLA4 fusion strategy and CpG motif could improve the immunogenicity of HTNV DNA vaccine.

During this process, the complementary sequences located at 3′ end of the paired oligo would form a dimer and in the presence of DNA polymerase and dNTP, each oligo would extend its 3′ end using the second oligo as a template.

Cloning of the double-stranded DNA

To ensure the purity of the standard template, the products were ligated into the pGEM-T Easy Vector System II (Promega, Madison, USA) and transformed into competent Escherichia coli JM109 cells, according to the manufacturer’s protocol. Screening for the gene insert was performed by quick lysis at 95°C of the E. coli cells for 5 minutes, followed by PCR using 5 μl of the cell lysate as previously described.

Sequencing of cloned dsDNA and sequence analysis

The plasmid DNA from positive clones was extracted using the QIAprep Spin Miniprep Kit according to the manufacturer’s protocol (Qiagen, Melbourne, Australia). The inserts were sequenced using Applied Biosystems BigDye terminator chemistry version 3.1 (Foster City, CA, USA), on an ABI Prism 373 DNA sequencer. Sequences were identified using the FastA program group accessed through Biomanager.

Ligase Chain Reaction (LCR) in the detection of templates containing resistance mutation

Liner templates containing resistance and wild type were generated by PCR amplification of plasmid DNA, followed by purification using Millipore PCR purification plate (Millipore, Billerica, MA, USA). The linear PCR products were quantitated using spectrophotometer and dsDNA DNA copy number were estimated by DNA calculator. 5 × 1011 copies of standard templates were used for testing the specificity of the LCR system. LCR probes targeting resistance template were designed. Each template was targeted by four LCR probes about 35-45nt with 2 of the probes having 5′ end phosphorylation. Ligation of LCR probes to standard templates was carried out by mixing the standard template with 1pmol of each LCR probes, 2U of pfu DNA ligase (Stratagene, Integrated Sciences, Cedar Creek, TX, USA) in 20 mM Tris-HCl (pH 7.5), 20 mM KCl, 10 mM MgCl2, 0.1% Igepal, 0.01 mM rATP, 1 mM DTT with total reaction volume of 25 μl. Multiple cycle ligation was conducted to validate the specificity of the probe in recognizing its corresponding template. The reaction condition include one cycle of 5 min at 94°C to denature the dsDNA followed 10 cycles of by 94°C 30 s and 4 min ligation at 65°C. The final product were run on 10% TBE gel (Invitrogen, Mount Waverley VIC Australia) and visualized under UV light with ethidium bromide staining.

The most significant advantage of using synthetic oligonucleotides is flexibility. Our approach has proven that the use of laboratory safe artificial templates can be a relatively cost effective, simple and efficient alternative to difficult to acquire material related to an infection or bioterrorism agent. The production of standard templates via the formation of oligo dimers and PCR using synthetic oligos does not require the use of viral or bacterial strains and oligos of any desired sequence can be made commercially. This approach may have the potential to produce a false-positive product due to contamination and therefore caution may be necessary. However, this problem can be solved by the introduction of exclusive restriction endonuclease digestion sites in the artificial template, which would allow for rapid confirmation of a false-positive result. Certain restriction endonuclease (SmaI and BamHI) can be used directly in the PCR buffer (without buffer exchange) and thus the PCR product from the control can be easily and rapidly distinguished from testing samples. In addition, the standard artificial template could be designed to be slightly smaller or larger in size, which would allow for direct visual identification of contamination with the synthetic control.

Overall, this technology may have wide ranging applications and may revolutionize molecular diagnostics and the use of multiplexing assays.

Design and synthesis of long oligos

Long single-stranded oligonucleotides of 89-98 bases were designed, each containing wild-type template or drug resistance mutations. After synthesis, each oligonucleotide was PAGE purified (Sigma-Aldrich, Sydney, Australia). Paired long oligos were designed carrying >20bp complementary sequences at their 3′ ends.

Further, in the event of an influenza pandemic, drug resistant strains and their transmission could be clinically highly significant, meaning that sensitive and specific techniques are required for their early and clinically relevant detection. However, due to the low frequency of naturally occurring resistance mutations in influenza infected patients receiving NA inhibitor treatment, the highly pathogenetic nature of influenza A H5N1 strains, and the technical complexity and time consuming nature of generating of NA resistant strains in vitro, collection of all known resistance mutations as positive controls is challenging. Therefore, our approach utilizing the formation of oligonucleotide dimers between two commercially synthesised long single stranded DNA templates (~95 bases each), to generate ~170 bp double-stranded artificial DNA templates, containing resistance mutations is not only innovative, but a more molecularly feasible and durable. In this way, it offers significant advantages in synthesizing even longer double stranded DNA templates, which can be used as positive control templates in molecular diagnostics. In the present study, we have used these double stranded artificial DNA templates with all known genetic mutations associated with influenza A drug resistance as a positive control in the development of a ligase chain reaction (LCR) for detecting NA inhibitor drug resistance mutations in patient samples.

Positive controls are an integral component of any sensitive molecular diagnostic tool, but this can be affected, if several mutations are being screened in a scenario of a pandemic or newly emerging disease where it can be difficult to acquire all the necessary positive controls from the host. This work describes the development of a synthetic oligo-cassette for positive controls for accurate and highly sensitive diagnosis of several mutations relevant to influenza virus drug resistance.

Results

Using influenza antiviral drug resistance mutations as an example by employing the utility of synthetic paired long oligonucleotides containing complementary sequences at their 3′ ends and utilizing the formation of oligonucleotide dimers and DNA polymerization, we generated ~170bp dsDNA containing several known specific neuraminidase inhibitor (NAI) resistance mutations. These templates were further cloned and successfully applied as positive controls in downstream assays.

Conclusion

This approach significantly improved the development of diagnosis of resistance mutations in terms of time, accuracy, efficiency and sensitivity, which are paramount to monitoring the emergence and spread of antiviral drug resistant influenza strains. Thus, this may have a significantly broader application in molecular diagnostics along with its application in rapid molecular testing of all relevant mutations in an event of pandemic.

Background

Recently, the occurrence in humans of infection with virulent avian influenza A H5N1 and the emergence of swine origin pandemic influenza A H1N1 2009 strain have sparked fear of an ongoing pandemic with novel genetic characters. While vaccines remain the most effective public health strategy for prevention, antiviral drugs such as neuraminidase inhibitors (NAIs), oseltamivir and zanamivir, could play an important role in the response to the early phases of a pandemic, if available in sufficient quantities. However, like other antiviral agents, the emergence of influenza viruses with reduced susceptibility to the NAI is inevitable during treatment. To date, strains with altered susceptibility to NAI have been recovered from approximately 1% of immunocompetent adult patients and up to 18% of pediatric patients. In addition, oseltamivir-resistant influenza A H5N1 and pandemic H1N1 2009 viruses with the H274Y mutation have been reported from patients during oseltamivir treatment.

Findings
The ability of NS1 protein from two avian influenza subtypes, H6N8 and H4N6, to inhibit NF-κB promoter activation was assessed. Further, efforts were made to characterize the genetic basis of this inhibition. We found that allele A NS1 proteins of H6N8 and H4N6 are significantly better in preventing dsRNA induced NF-κB promoter activation compared to allele B of corresponding subtypes, in a species independent manner. Furthermore, the ability to suppress NF-κB promoter activation was mapped to the effector domain while the RNA binding domain alone was unable to suppress this activation. Chimeric NS1 proteins containing either RNA binding domain of allele A and effector domain of allele B or vice versa, were equally potent in preventing NF-κB promoter activation compared to their wt. NS1 protein of allele A and B from both subtypes expressed efficiently as detected by Western blotting and predominantly localized in the nucleus in both A549 and MiLu cells as shown by in situ PLA.

Conclusions
Here, we present another aspect of NS1 protein in inhibiting dsRNA induced NF-κB activation in an allele dependent manner. This suggests a possible correlation with the virus’s pathogenic potential.

Introduction
Within hours of host-pathogen interaction, the type 1 interferons (IFNs), an essential arm of innate immune response, are induced to initiate a range of antiviral processes. The binding of dsRNA, produced as a viral by-product (or administered externally such as poly I:C) to helicases or toll-like receptors (TLR), initiates a series of events culminating in the activation of two kinase complexes: TANK-binding kinase 1-inhibitor of kappa B-kinase ε (TBK1-IKK-ε) and IKK-α/β/γ. TBK1-IKK-ε phosphorylates interferon regulatory factor 3 and 7 (IRF3 and IRF7) while IKK-α/β/γ phosphorylates and hence activates nuclear factor-κB (NF-κB) transcription factor. Activated NF-κB translocates to the nucleus where it induce the transcription of IFN-α and IFN-β as well as other pro-inflammatory cytokines together with ATF2/c-Jun (AP-1), p300 and CBP. NF-κB consists of a family of transcription factors that play indispensable roles in mediating inflammation, immune responses to pathogen infection, proliferation, apoptosis, and other cellular activities. Because of the essential role of NF-κB in stimulation of IFN-α/β synthesis, many viruses have evolved different strategies to subvert this system. The non-structural protein 1 (NS1) of influenza A viruses is one of best example having ability to prevent NF-κB activation.

Adsorption and receptor recognition

The process of virus attachment/absorption to the host cell is probably a multistep event. It is not a new proposal that virus infection begins with scanning of the cell surface that begins with a general “sticking” to the cell via one or more proteins (or the cell membrane itself) followed by “rolling” on the cell surface as it locates the proper receptor to initiate the penetration step of virus entry. This type of interaction has been identified recently using single particle fluorescence resonance energy transfer (FRET). SINV saturated with a membrane intercalated fluorescent self-quenching dye can be visualized when the dye is excited with a specific wavelength light. Fusion of virus with the cell membrane is detected when lipid mixing (fusion) releases the proteins and dequenching of the dye occurs causing it to fluoresce. Using this technique it has been shown that SINV moves on the cell surface in a neutral pH environment. If the pH of the medium bathing the cells is lowered the virus does not fuse with the cell surface as is the case with influenza, rather the virus “freezes” in place (K. Wenniger, unpublished observation). These observations indicate that viruses probe the cell surface for the proper receptor molecule.