SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. This protocol describes our fluorescence resonance energy transfer (FRET)-based single vesicle-vesicle fusion assays for SNAREs and accessory proteins. Both lipid-mixing (with FRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRET pairs present on a DNA hairpin that becomes linear via hybridization to a complementary DNA) are described. These assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. The details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. These assays can be used to study the roles of various SNARE proteins, accessory proteins and effects of different lipid compositions on specific fusion steps. The total time required to finish one round of this protocol is 3–6 d.

Sequence recognition through base-pairing is essential for DNA repair and gene regulation, but the basic rules governing this process remain elusive. In particular, the kinetics of annealing between two imperfectly matched strands is not well characterized, despite its potential importance in nucleic acid-based biotechnologies and gene silencing. Here we use single-molecule fluorescence to visualize the multiple annealing and melting reactions of two untethered strands inside a porous vesicle, allowing us to precisely quantify the annealing and melting rates. The data as a function of mismatch position suggest that seven contiguous base pairs are needed for rapid annealing of DNA and RNA. This phenomenological rule of seven may underlie the requirement for seven nucleotides of complementarity to seed gene silencing by small noncoding RNA and may help guide performance improvement in DNA- and RNA-based bio- and nanotechnologies, in which off-target effects can be detrimental.

ABSTRACT: Cardiovascular collapse is the major factor contributing to the mortality of trauma-hemorrhage (T-H) patients. Toll-like receptors (TLRs) play a critical role in T-H-induced cardiac dysfunction. This study evaluated the role of TLR9 agonist, CpG-ODN 1826, in cardiac functional recovery after T-H. Trauma-hemorrhage was induced in a murine model by soft tissue injury and blood withdrawals from the jugular vein to a mean arterial pressure of 35±5 mm Hg. Mice were treated with CpG-ODN 1826 (10 μg/30g body weight) by intra-peritoneal injection one hour prior to T-H (N=5-8/group). Hemodynamic parameters were measured before, during hemorrhage, and at 60 min after T-H. Trauma-hemorrhage significantly decreased the mean arterial pressure and left ventricular pressure compared with sham controls. In contrast, CpG-ODN administration significantly attenuated the decrease in arterial pressure and left ventricular pressure due to T-H. Trauma-hemorrhage markedly decreased myocardial levels of phosphorylated Akt by 57.9%. However, CpG-ODN treatment significantly blunted the decrement in phospho-Akt by activating the PI3K/Akt signaling pathway. The PI3K inhibitor LY294002 partially abolished CpG-induced cardioprotection, indicating that additional signaling pathways are involved in the protective effect of CpG-ODNafter T-H. We observed that CpG-ODN treatment also significantly attenuated the decrease in myocardial phospho-ERK levels after T-H. Inhibition of ERK by U0126 also partially abolished the cardioprotective effect of CpG-ODN after T-H. Our data suggests that CpG-ODN significantly attenuates T-H-induced cardiac dysfunction. The mechanisms involve activation of both PI3K/Akt and ERK signaling pathways. The TLR9 agonist, CpG-ODN 1826, may provide a novel treatment strategy for preventing or managing cardiac dysfunction and enhancing recovery in T-H patients.

Pulmonary hypertension (PH) is defined as a resting mean pulmonary artery pressure greater than 25 mmHg. The World Health Organization (WHO) classifies PH into five categories. The WHO nomenclature assumes shared histology and pathophysiology within categories and implies category-specific treatment. Imaging of the heart and pulmonary vasculature is critical to assigning a patient's PH syndrome to the correct WHO category and is also important in predicting outcomes. Imaging studies often reveal that the etiology of PH in a patient reflects contributions from several categories. Overlap between Categories 2 and 3 (left heart disease and lung disease) is particularly common, reflecting shared risk factors. Correct classification of PH patients requires the combination of standard imaging (chest roentgenograms, ventilation-perfusion scans, echocardiography, and the 12-lead electrocardiogram) and advanced imaging (computed tomography, cardiac magnetic resonance imaging, and positron emission tomography). Despite the value of imaging, cardiac catheterization remains the gold standard for quantification of hemodynamics and is required before initiation of PH-specific therapy. These cases illustrate the use of imaging in classifying patients into WHO PH Categories 1-5.

Abstract Background/aims: To determine the success of Mohs micrographic surgery (MMS) for periocular basal cell carcinoma (BCC) at a tertiary referral centre in the UK. Design: Retrospective interventional case series covering 5 years of practice. Methods: Review of medical records of 104 consecutive patients who underwent MMS for confirmed periocular BCC. The main outcome measure was biopsy-proven recurrence of BCC at the same anatomical location after MMS. Secondary outcome measures included tumour site, histological subtype and length of follow-up. Results: 104 patients underwent MMS for periocular BCC from January 2003 to July 2008. 63 (62%) of the surgeries were for primary BCC and 25 procedures (25%) were for recurrent or residual BCC. 64% of tumours were nodular BCC. The mean follow-up was 28 months (range 1-85 months). Six recurrences were identified in total. The recurrence rate following MMS for primary BCC was 1.6%(1 patient) compared to 20%(5 patients) in the patient group treated for residual or recurrent tumours. The mean time to recurrence was 39 months (range 16-71 months). Conclusions: MMS for primary BCC has a very high success rate but the recurrence rate increases significantly in cases of recurrent or residual tumours.

Influenza A virus possesses a segmented genome of eight negative-sense, single-stranded RNAs. The eight segments have been shown to be represented in approximately equal molar ratios in a virus population; however, the exact copy number of each viral RNA segment per individual virus particles has not been determined. We have established an experimental approach based on multicolor single-molecule fluorescent in situ hybridization (FISH) to study the composition of viral RNAs at single-virus particle resolution. Colocalization analysis showed that a high percentage of virus particles package all eight different segments of viral RNAs. To determine the copy number of each RNA segment within individual virus particles, we measured the photobleaching steps of individual virus particles hybridized with fluorescent probes targeting a specific viral RNA. By comparing the photobleaching profiles of probes against the HA RNA segment for the wild-type influenza A/Puerto Rico/8/34 (PR8) and a recombinant PR8 virus carrying two copies of the HA segment, we concluded that only one copy of HA segment is packaged into a wild type virus particle. Our results showed similar photobleaching behaviors for other RNA segments, suggesting that for the majority of the virus particles, only one copy of each RNA segment is packaged into one virus particle. Together, our results support that the packaging of influenza viral genome is a selective process.

The genomes of influenza A viruses consist of eight negative-strand RNA segments. Recent studies suggest that influenza viruses are able to specifically package their segmented genomes into the progeny virions. Segment-specific packaging signals of influenza viral RNAs (vRNAs) are located in the 5' and 3' noncoding regions as well as in the terminal regions of the open reading frames. To date, how these packaging signals function during genome packaging remains unclear. Previously, we generated a 7-segmented virus, in which the hemagglutinin (HA) and neuraminidase (NA) segments of the influenza A/Puerto Rico/8/34 virus were replaced by a chimeric influenza C hemagglutinin/esterase/fusion (HEF) segment carrying the HA packaging sequences. The robust growth of the HEF virus suggested that the NA segment is not required for the packaging of other segments. In this study, in order to determine the roles of the other seven segments during the influenza A virus genome assembly, we continued to use this HEF virus as a tool, and analyzed the effect of substituting the packaging sequences of other segments with those of the NA segment. Our results showed that deleting the packaging signals of the PB1, HA, or NS segments had no effect on the growth of the HEF virus, while the growth was greatly impaired when the packaging sequences of the PB2, PA, NP or M segment were removed. These results indicate that the PB2, PA, NP and M segments play a more important role than the remaining four vRNAs during the genome packaging process.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. This protocol describes our fluorescence resonance energy transfer (FRET)-based single vesicle-vesicle fusion assays for SNAREs and accessory proteins. Both lipid-mixing (with FRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRET pairs present on a DNA hairpin that becomes linear via hybridization to a complementary DNA) are described. These assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. The details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. These assays can be used to study the roles of various SNARE proteins, accessory proteins and effects of different lipid compositions on specific fusion steps. The total time required to finish one round of this protocol is 3-6 d.

A major hurdle for molecular mechanistic studies of many proteins is the lack of a general method for fluorescence labeling with high efficiency, specificity and speed. By incorporating an aldehyde motif genetically into a protein and improving the labeling kinetics substantially under mild conditions, we achieved fast, site-specific labeling of a protein with ∼100% efficiency while maintaining the biological function. We show that an aldehyde-tagged protein can be specifically labeled in cell extracts without protein purification and then can be used in single-molecule pull-down analysis. We also show the unique power of our method in single-molecule studies on the transient interactions and switching between two quantitatively labeled DNA polymerases on their processivity factor.

The assembly of the bacterial ribosomal small subunit (SSU) begins with the folding of the five-way junction upon interaction with the primary binding protein S4. This complex contains the largest contiguous molecular signature, which is a conserved feature in all bacterial 16S rRNAs. In a previous study, we used all-atom molecular dynamics simulations to demonstrate that the coevolving signature in the N-terminus of S4 is intrinsically disordered and capable of accelerating the binding process through a fly-casting mechanism. In this paper, we capture the simultaneous folding and binding of the five-way junction and r-protein S4, by use of a structure-based Go potential implemented within the framework of the all-atom molecular dynamics CHARMM force field. Comparisons between the all-atom MD simulations and FRET experiments identify multiple metastable conformations of the naked five-way junction without the presence of S4. Using the hybrid MD-Go simulations, different folding pathways are observed for the refolding of the five-way junction upon partial binding of S4. Our simulations illustrate the complex nature of RNA folding in the presence of a protein binding partner and provide insight into the role of population shift and the induced fit mechanisms in the protein:RNA folding and binding process.