Growth-phase-dependent synthesis of histones in the archaeon Methanothermus fervidus.

Histone preparations from Methanothermus fervidus (HMf) contain two small polypeptides, HMfA and HMfB, which in solution are dimers and compact DNA to form nucleosome-like structures. These archaeal nucleosome-like structures constrain positive DNA supercoils, in contrast to the negatively supercoiled DNA in eukaryal nucleosomes. HMfA has been found to make up as much as 80% of HMf preparations synthesized by M. fervidus cells during the exponential growth phase of batch cultures but to decrease to approximately 50% as cultures enter the stationary phase. By using a nondenaturing polyacrylamide gel system at pH 6.1, we have demonstrated that HMf preparations contain HMfA homodimers, HMfB homodimers, and HMfA-HMfB heterodimers and that heating a mixture of recombinant HMfA and HMfB homodimers at 95 degrees C for 5 min generates HMfA-HMfB heterodimers. Circular dichroism spectroscopy indicates that HMfA and HMfB have very similar secondary structures, but based on agarose gel electrophoretic mobility shifts, DNA topology assays, and electron microscopy, they have different DNA binding properties. HMfA binding to DNA could be detected at lower protein/DNA ratios than HMfB, but HMfB binding resulted in more extensive DNA compaction. The increased HMfB synthesized in cells approaching the stationary phase and the highly compacted state of HMfB-bound DNA are consistent with preparations for the impending period of limited genome activity.     

Molecular and biological properties of MH2D12, a spontaneous mil deletion mutant of avian oncovirus MH2

Avian oncogenic retrovirus MH2 carries two cell-derived oncogenes, v-mil and v-myc. From an infectious stock of MH2 a spontaneous deletion mutant, MH2D12, that has lost most of the v-mil gene but has retained a complete and functional v-myc gene, has been isolated. Nonproducer quail embryo cells transformed by MH2D12 in the absence of helper virus contain two virus-specific proteins: a gag-related protein of 53,000 Da (p53gag), and a v-myc gene product of 59,000/61,000 Da (p59/61v-myc) indistinguishable from the v-myc protein encoded by MH2. MH2D12 viral RNA contains all T1-oligonucleotides specific for the MH2 v-myc gene but none of those characteristic for the v-mil gene. The genetic structure of molecularly cloned proviral DNA of MH2D12 was revealed by restriction mapping, blot hybridization, heteroduplex analysis, and nucleotide sequencing. The MH2D12 provirus is homologous to the MH2 genome but has suffered a deletion of 1271 nucleotides from the central region encompassing the 3' end of delta gag and all of v-mil except the very 3' 31 nucleotides directly adjacent to the v-myc gene. A nine-nucleotide overlap of homology to gag or mil at the delta gag/delta mil junction suggests that recombination between homologous sequence elements of the delta gag and v-mil domains of MH2 was involved in the genesis of MH2D12. The nucleotide sequence analysis predicts that the carboxyterminal 17 amino acids of p53gag are encoded by the residual v-mil sequences and by intron-derived v-myc sequences. Transformation of quail embryo cells by MH2D12 can be assayed by focus and colony formation of transformed cells. This indicates that the v-mil gene is not essential for these activities. However, size and morphology of foci and colonies, and cellular morphology of cultured MH2D12-transformed cell lines can easily be distinguished from those observed in cell transformation by MH2 and resemble more those seen in cell transformation by viruses containing the myc oncogene only.     

Transcatheter Edge-to-Edge Repair for Treatment of Tricuspid Regurgitation

BackgroundTricuspid regurgitation (TR) is a frequent disease with a progressive increase in mortality as disease severity increases. Transcatheter therapies for treatment of TR may offer a safe and effective alternative to surgery in this high-risk population.ObjectivesThe purpose of this report was to study the 1-year outcomes with the TriClip transcatheter tricuspid valve repair system, including repair durability, clinical benefit and safety.MethodsThe TRILUMINATE trial (n = 85) is an international, prospective, single arm, multicenter study investigating safety and performance of the TriClip Tricuspid Valve Repair System in patients with moderate or greater TR. Echocardiographic assessment was performed by a core laboratory.ResultsAt 1 year, TR was reduced to moderate or less in 71% of subjects compared with 8% at baseline (p < 0.0001). Patients experienced significant clinical improvements in New York Heart Association (NYHA) functional class I/II (31% to 83%, p < 0.0001), 6-minute walk test (272.3 ± 15.6 to 303.2 ± 15.6 meters, p = 0.0023) and Kansas City Cardiomyopathy Questionnaire (KCCQ) score (improvement of 20 ± 2.61 points, p < 0.0001). Significant reverse right ventricular remodeling was observed in terms of size and function. The overall major adverse event rate and all-cause mortality were both 7.1% at 1 year.ConclusionTranscatheter tricuspid valve repair using the TriClip device was found to be safe and effective in patients with moderate or greater TR. The repair itself was durable at reducing TR at 1 year and was associated with a sustained and marked clinical benefit with low mortality after 1 year in a fragile population that was at high surgical risk. (TRILUMINATE Study With Abbott Transcatheter Clip Repair System in Patients With Moderate or Greater TR; NCT03227757)     

Structural rearrangements in the phage head-to-tail interface during assembly and infection

Many icosahedral viruses use a specialized portal vertex to control genome encapsidation and release from the viral capsid. In tailed bacteriophages, the portal system is connected to a tail structure that provides the pipeline for genome delivery to the host cell. We report the first, to our knowledge, subnanometer structures of the complete portal–phage tail interface that mimic the states before and after DNA release during phage infection. They uncover structural rearrangements associated with intimate protein–DNA interactions. The portal protein gp6 of bacteriophage SPP1 undergoes a concerted reorganization of the structural elements of its central channel during interaction with DNA. A network of protein–protein interactions primes consecutive binding of proteins gp15 and gp16 to extend and close the channel. This critical step that prevents genome leakage from the capsid is achieved by a previously unidentified allosteric mechanism: gp16 binding to two different regions of gp15 drives correct positioning and folding of an inner gp16 loop to interact with equivalent loops of the other gp16 subunits. Together, these loops build a plug that closes the channel. Gp16 then fastens the tail to yield the infectious virion. The gatekeeper system opens for viral genome exit at the beginning of infection but recloses afterward, suggesting a molecular diaphragm-like mechanism to control DNA efflux. The mechanisms described here, controlling the essential steps of phage genome movements during virus assembly and infection, are likely to be conserved among long-tailed phages, the largest group of viruses in the Biosphere.The dsDNA bacterial viruses (phages or bacteriophages) and herpes viruses keep their genetic information packed at high pressure inside an icosahedral protein capsid. During virus particle assembly the genome is translocated into a prebuilt procapsid through a specialized portal vertex of the capsid (1, 2). Termination of the DNA packaging reaction is coordinated with closure of the portal system to avoid leakage of the viral genome. The outflow of DNA is prevented by conformational changes in the portal protein and binding of head completion proteins building the viral genome gatekeeper (3). In bacteriophages, the resultant complex [connector (4)] provides the connection point for the tail. The head-to-tail interface (HTI), or neck, is composed of the connector and of the tail-completion protein(s) found between the connector and the helical tail tube (Fig. S1A) (3, 5). Phage tails are responsible for host cell recognition and delivery of the viral genome to the host cytoplasm (6). At the beginning of viral infection the phage adsorption apparatus, located at the tail end distal from the capsid, binds to the host receptor, generating a signal that triggers opening of the neck (7). DNA then moves through the tail tube to enter the host cell. That tailed bacteriophages are the most abundant biological entities on Earth indicates the evolutionary advantage of this strategy for infecting bacterial cells. Infection by these viruses plays a central role in microbial ecosystems dynamics and in the horizontal transmission of genetic information within the bacterial world (8).Bacillus subtilis tailed bacteriophage SPP1 is a paradigm for viruses with a portal system (9). The viral particle is composed of an isometric icosahedral capsid ∼60 nm in diameter, shielding the 45.9-kbp-long viral chromosome (10). The portal protein gp6 (57.3 kDa subunit mass) is incorporated at a single vertex of the procapsid as a circular oligomer with a central channel that serves as a conduit for DNA passage (11). The portal vertex acts as a platform for the assembly of the viral DNA-translocating motor (12). Termination of DNA packaging is coordinated with disassembly of the motor and binding of gp15 subunits (11.6 kDa) to gp6, extending the portal channel that is closed underneath by the gp16 protein (12.5 kDa) (Fig. S1A) (13). The assembled complex represents the 180-Å-high connector that consists of three stacked cyclical homo-oligomers, each composed of 12 subunits of the portal protein gp6, of the adaptor gp15, and of the stopper gp16 (4, 13). Gp16 operates as a docking platform for the SPP1 preassembled tail tapered by the tail-to-head joining protein gp17 (15 kDa) (14, 15). Binding of the flexible 1,600-Å-long helical tail to the connector completes the formation of the HTI (7, 16). The capsid-distal region of the tail features an adsorption apparatus. Binding of this apparatus to the host cell receptor YueB (17, 18) triggers a domino-like cascade of conformational changes within the gp17.1/gp17.1* tail tube (7, 16, 19), signaling for opening of the gp16 stopper to initiate delivery of the SPP1 genome to the host cell.We report here subnanometer structures of the SPP1 HTI before and after DNA release obtained by cryoEM and single-particle analysis. The EM structures were used for flexible docking of X-ray and NMR atomic models of protein components of the HTI, allowing the uncovering the network of protein–protein and protein–DNA interactions in the complete HTI. The follow-up structure-driven functional analysis unraveled the allosteric mechanism by which the gatekeeper system assembles to lock DNA inside the virion after the genome-packaging reaction. It also provided experimental evidence supporting a model in which reversible diaphragm-like motion is the mechanism that controls viral genome release from the HTI for delivery to the host cell.     

Therapeutical potential of blood-derived progenitor cells in patients with peripheral arterial occlusive disease and critical limb ischaemia.

AIMS: Despite considerable advances in the therapy of patients with peripheral arterial occlusive disease (PAOD) and critical limb ischaemia (CLI), a substantial number remain, in whom amputation has to be considered the only and final option. Recent evidence from animal models of hind limb ischaemia suggests that neovascularization induced by circulating blood-derived progenitor cells (CPCs) may permit limb salvage. It remains unclear, however, whether an intra-arterial application of autologous CPCs in patients with infrapopliteal PAOD and CLI is safe, feasible, and of potentially beneficial effects. METHODS AND RESULTS: Seven patients with critical PAOD were treated with an intra-arterial infusion of autologous CPCs (39+/-24 x 10(6)) isolated from peripheral blood. Pre-interventional stimulation with G-CSF and CPC application was well tolerated. Twelve weeks after CPC administration, the pain-free walking distance increased from 6+/-13 to 195+/-196 m. A significant increase in the ankle-brachial index, transcutaneous O(2), flow-dependent vasodilation, flow reserve in response to adenosine, and endothelium-dependent vasodilation was observed. CONCLUSION: These preliminary data in a small series of patients with CLI without surgical or interventional options indicate that CPC application is safe, feasible, and may improve both functional and clinical indices.     

Current approaches to pulmonary regurgitation

Objective: To evaluate the effects on ventricular function and volumes following right ventricular outflow tract reconstruction (RVOTR) with pulmonary homograft replacement (PVR) and percutaneous pulmonary valve implantation (PPVI) for predominant pulmonary regurgitation. This study was not intended to compare the two approaches. Methods: We prospectively examined 25 patients (mean age 21+/-13 years, 96% tetralogy of Fallot, 1/25 with conduit dysfunction) who had PVR with RVOTR for severe pulmonary regurgitation (PR), and 11 patients (mean age 20+/-9 years, 64% tetralogy of Fallot, 9/11 with conduit dysfunction) who underwent PPVI for predominant PR. Mean age at primary repair in both groups was 4.3+/-6.6 years. Magnetic resonance imaging was performed prior to, and 1 year following, interventions. Results: Before procedure, NYHA classification was similar in both groups 2.1+/-0.5. Following interventions, there was a significant reduction in RV volumes in both groups. In the surgical (PVR) group, RV end-diastolic volume (EDV) decreased from 151+/-49 to 97+/-32ml/m(2) (p<0.0001) whereas end-systolic volume (ESV) decreased from 80+/-43 to 46+/-23ml/m(2) (p<0.0001). In the PPVI group, RV EDV decreased from 106+/-27 to 89+/-25ml/m(2) (p=0.002) and RV ESV from 49+/-20 to 40+/-16ml/m(2) (p=0.034). Both groups had a significant improvement in RV (63+/-20 to 72+/-16ml/beat, p=0.003 (PVR group), 53+/-14 to 67+/-16ml/beat, p=0.030 (PPVI group)) and LV effective stroke volume (61+/-18 to 73+/-16ml/beat, p=0.001 (PVR group); 59+/-24 to 75+/-16ml/beat, p=0.009 (PPVI group)). Conclusions: Following either PVR with RVOTR or PPVI, there was a significant reduction in RV volumes and an improvement in RV function. Importantly, in both groups, LV effective SV increased, and this may be the parameter to judge the benefit of the procedure. These results also support PPVI as an extra dimension in complex RVOT management.     

Disease threats posed by alien species: the role of a poxvirus in the decline of the native red squirrel in Britain

Red squirrels are declining in the United Kingdom. Competition from, and squirrel poxvirus (SQPV) disease carried by, grey squirrels are assumed to be determining the decline. We analyse the incidence of disease and changes in distribution of the two species in Cumbria, from 1993 to 2003 and compare these to the predictions of an individual-based (IB) spatially explicit disease model simulating the dynamics of both squirrel species and SQPV in the landscape. Grey squirrels increased whilst red squirrels declined over 10 years. The incidence of disease in red squirrels was related to the time since grey squirrels arrived in the landscape. Analysis of rates of decline in red squirrel populations in other areas showed that declines are 17-25 times higher in regions where SQPV is present in grey squirrel populations than in those where it is not. The IB model predicted spatial overlap of 3-4 years between the species that was also observed in the field. The model predictions matched the observed data best when contact rates and rates of infection between the two species were low. The model predicted that a grey squirrel population control of >60% effective kill was needed to stop the decline in red squirrel populations in Cumbria.