Biological diversity of prokaryotic type IV secretion systems.

Type IV secretion systems (T4SS) translocate DNA and protein substrates across prokaryotic cell envelopes generally by a mechanism requiring direct contact with a target cell. Three types of T4SS have been described: (i) conjugation systems, operationally defined as machines that translocate DNA substrates intercellularly by a contact-dependent process; (ii) effector translocator systems, functioning to deliver proteins or other macromolecules to eukaryotic target cells; and (iii) DNA release/uptake systems, which translocate DNA to or from the extracellular milieu. Studies of a few paradigmatic systems, notably the conjugation systems of plasmids F, R388, RP4, and pKM101 and the Agrobacterium tumefaciens VirB/VirD4 system, have supplied important insights into the structure, function, and mechanism of action of type IV secretion machines. Information on these systems is updated, with emphasis on recent exciting structural advances. An underappreciated feature of T4SS, most notably of the conjugation subfamily, is that they are widely distributed among many species of gram-negative and -positive bacteria, wall-less bacteria, and the Archaea. Conjugation-mediated lateral gene transfer has shaped the genomes of most if not all prokaryotes over evolutionary time and also contributed in the short term to the dissemination of antibiotic resistance and other virulence traits among medically important pathogens. How have these machines adapted to function across envelopes of distantly related microorganisms? A survey of T4SS functioning in phylogenetically diverse species highlights the biological complexity of these translocation systems and identifies common mechanistic themes as well as novel adaptations for specialized purposes relating to the modulation of the donor-target cell interaction.

Enterococcus faecalis PcfC, a spatially localized substrate receptor for type IV secretion of the pCF10 transfer intermediate.

Upon sensing of peptide pheromone, Enterococcus faecalis efficiently transfers plasmid pCF10 through a type IV secretion (T4S) system to recipient cells. The PcfF accessory factor and PcfG relaxase initiate transfer by catalyzing strand-specific nicking at the pCF10 origin of transfer sequence (oriT). Here, we present evidence that PcfF and PcfG spatially coordinate docking of the pCF10 transfer intermediate with PcfC, a membrane-bound putative ATPase related to the coupling proteins of gram-negative T4S machines. PcfC and PcfG fractionated with the membrane and PcfF with the cytoplasm, yet all three proteins formed several punctate foci at the peripheries of pheromone-induced cells as monitored by immunofluorescence microscopy. A PcfC Walker A nucleoside triphosphate (NTP) binding site mutant (K156T) fractionated with the E. faecalis membrane and also formed foci, whereas PcfC deleted of its N-terminal putative transmembrane domain (PcfCDelta N103) distributed uniformly throughout the cytoplasm. Native PcfC and mutant proteins PcfCK156T and PcfCDelta N103 bound pCF10 but not pcfG or Delta oriT mutant plasmids as shown by transfer DNA immunoprecipitation, indicating that PcfC binds only the processed form of pCF10 in vivo. Finally, purified PcfCDelta N103 bound DNA substrates and interacted with purified PcfF and PcfG in vitro. Our findings support a model in which (i) PcfF recruits PcfG to oriT to catalyze T-strand nicking, (ii) PcfF and PcfG spatially position the relaxosome at the cell membrane to stimulate substrate docking with PcfC, and (iii) PcfC initiates substrate transfer through the pCF10 T4S channel by an NTP-dependent mechanism.

A patient-specific study of type-B aortic dissection: evaluation of true-false lumen blood exchange

BACKGROUND Aortic dissection is a severe pathological condition in which blood penetrates between layers of the aortic wall and creates a duplicate channel - the false lumen. This considerable change on the aortic morphology alters hemodynamic features dramatically and, in the case of rupture, induces markedly high rates of morbidity and mortality. METHODS In this study, we establish a patient-specific computational model and simulate the pulsatile blood flow within the dissected aorta. The k-ω SST turbulence model is employed to represent the flow and finite volume method is applied for numerical solutions. Our emphasis is on flow exchange between true and false lumen during the cardiac cycle and on quantifying the flow across specific passages. Loading distributions including pressure and wall shear stress have also been investigated and results of direct simulations are compared with solutions employing appropriate turbulence models. RESULTS Our results indicate that (i) high velocities occur at the periphery of the entries; (ii) for the case studied, approximately 40% of the blood flow passes the false lumen during a heartbeat cycle; (iii) higher pressures are found at the outer wall of the dissection, which may induce further dilation of the pseudo-lumen; (iv) highest wall shear stresses occur around the entries, perhaps indicating the vulnerability of this region to further splitting; and (v) laminar simulations with adequately fine mesh resolutions, especially refined near the walls, can capture similar flow patterns to the (coarser mesh) turbulent results, although the absolute magnitudes computed are in general smaller. CONCLUSIONS The patient-specific model of aortic dissection provides detailed flow information of blood transport within the true and false lumen and quantifies the loading distributions over the aorta and dissection walls. This contributes to evaluating potential thrombotic behavior in the false lumen and is pivotal in guiding endovascular intervention. Moreover, as a computational study, mesh requirements to successfully evaluate the hemodynamic parameters have been proposed.

How often parametrial involvement leads to post-operative adjuvant treatment in locally advanced cervical cancer after neoadjuvant chemotherapy and type C radical hysterectomy?

OBJECTIVE Parametrial involvement (PMI) is one of the most important factors influencing prognosis in locally advanced stage cervical cancer (LACC) patients. We aimed to evaluate PMI rate among LACC patients undergoing neoadjuvant chemotherapy (NACT), thus evaluating the utility of parametrectomy in tailor adjuvant treatments. METHODS Retrospective evaluation of consecutive 275 patients affected by LACC (IB2-IIB), undergoing NACT followed by type C/class III radical hysterectomy. Basic descriptive statistics, univariate and multivariate analyses were applied in order to identify factors predicting PMI. Survival outcomes were assessed using Kaplan-Meier and Cox models. RESULTS PMI was detected in 37 (13%) patients: it was associated with vaginal involvement, lymph node positivity and both in 10 (4%), 5 (2%) and 12 (4%) patients, respectively; while PMI alone was observed in only 10 (4%) patients. Among this latter group, adjuvant treatment was delivered in 3 (1%) patients on the basis of pure PMI; while the remaining patients had other characteristics driving adjuvant treatment. Considering factors predicting PMI we observed that only suboptimal pathological responses (OR: 1.11; 95% CI: 1.01, 1.22) and vaginal involvement (OR: 1.29 (95%) CI: 1.17, 1.44) were independently associated with PMI. PMI did not correlate with survival (HR: 2.0; 95% CI: 0.82, 4.89); while clinical response to NACT (HR: 3.35; 95% CI: 1.59, 7.04), vaginal involvement (HR: 2.38; 95% CI: 1.12, 5.02) and lymph nodes positivity (HR: 3.47; 95% CI: 1.62, 7.41), independently correlated with worse survival outcomes. CONCLUSIONS Our data suggest that PMI had a limited role on the choice to administer adjuvant treatment, thus supporting the potential embrace of less radical surgery in LACC patients undergoing NACT. Further prospective studies are warranted.

THE MOLECULAR INTERACTION BETWEEN TYPE II DIABETES AND ALZHEIMER’S DISEASE THROUGH CROSS-SEEDING OF PROTEIN MISFOLDING

With the population of the world aging, the prominence of diseases such as Type II Diabetes (T2D) and Alzheimer’s disease (AD) are on the rise. In addition, patients with T2D have an increased risk of developing AD compared to age-matched individuals, and the number of AD patients with T2D is higher than among aged-matched non-AD patients. AD is a chronic and progressive dementia characterized by amyloid-beta (Aβ) plaques, neurofibrillary tangles (NFTs), neuronal loss, brain inflammation, and cognitive impairment. T2D involves the dysfunctional use of pancreatic insulin by the body resulting in insulin resistance, hyperglycemia, hyperinsulinemia, pancreatic beta cell (β-cell) death, and other complications. T2D and AD are considered protein misfolding disorders (PMDs). PMDs are characterized by the presence of misfolded protein aggregates, such as in T2D pancreas (islet amyloid polypeptide - IAPP) and in AD brain (amyloid– Aβ) of affected individuals. The misfolding and accumulation of these proteins follows a seeding-nucleation model where misfolded soluble oligomers act as nuclei to propagate misfolding by recruiting other native proteins. Cross-seeding occurs when oligomers composed by one protein seed the aggregation of a different protein. Our hypothesis is that the pathological interactions between T2D and AD may in part occur through cross-seeding of protein misfolding. To test this hypothesis, we examined how each respective aggregate (Aβ or IAPP) affects the disparate disease pathology through in vitro and in vivo studies. Assaying Aβ aggregates influence on T2D pathology, IAPP+/+/APPSwe+/- double transgenic (DTg) mice exhibited exacerbated T2D-like pathology as seen in elevated hyperglycemia compared to controls; in addition, IAPP levels in the pancreas are highest compared to controls. Moreover, IAPP+/+/APPSwe+/- animals demonstrate abundant plaque formation and greater plaque density in cortical and hippocampal areas in comparison to controls. Indeed, IAPP+/+/APPSwe+/- exhibit a colocalization of both misfolded proteins in cerebral plaques suggesting IAPP may directly interact with Aβ and aggravate AD pathology. In conclusion, these studies suggest that cross-seeding between IAPP and Aβ may occur, and that these protein aggregates exacerbate and accelerate disease pathology, respectively. Further mechanistic studies are necessary to determine how these two proteins interact and aggravate both pancreatic and brain pathologies.

Analysis of Agrobacterium tumefaciens VirB6 and VirB9 of the VirB /D4 type IV secretion system

Agrobacterium tumefaciens uses the VirB/D4 type IV secretion system (T4SS) to translocate oncogenic DNA (T-DNA) and protein substrates to plant cells. Independent of VirD4, the eleven VirB proteins are also essential for elaboration of a conjugative pilus termed the T pilus. The focus of this thesis is the characterization and analysis of two VirB proteins, VirB6 and VirB9, with respect to substrate translocation and T pilus biogenesis. Observed stabilizing effects of VirB6 on other VirB subunits and results of protein-protein interaction studies suggest that VirB6 mediates assembly of the secretion machine and T pilus through interactions with VirB7 and VirB9. Topology studies support a model for VirB6 as a polytopic membrane protein with a periplasmic N terminus, a large internal periplasmic loop, five transmembrane segments, and a cytoplasmic C terminus. Topology studies and Transfer DNA immunoprecipitation (TrIP) assays identified several important VirB6 functional domains: (i) the large internal periplasmic loop mediates interaction of VirB6 with the T-DNA, (ii) the membrane spanning region carboxyl-terminal to the large periplasmic loop mediates substrate transfer from VirB6 to VirB8, and (iii) the terminal regions of VirB6 are required for substrate transfer to VirB2 and VirB9. To analyze structure-function relationships of VirB9, the phenotypic consequences of dipeptide insertion mutations were characterized. Substrate discriminating mutations were shown to selectively export the oncogenic T-DNA and VirE2 to plant cells or a mobilizable IncQ plasmid to bacterial cells. Mutations affecting VirB9 interactions with VirB7 and VirB10 were localized to the C- and N- terminal regions respectively. Additionally, “uncoupling” mutations identified in VirB11 and VirB6 that block T pilus assembly, but not substrate transfer to recipient cells, were also identified in VirB9. These results in conjunction with computer analysis establish that VirB9, like VirB6, is also composed of distinct regions or domains that contribute in various ways to secretion channel activity and T pilus assembly. Lastly, in vivo immunofluorescent studies suggest that VirB9 localizes to the outer membrane and may play a role similar to that of secretion/ushers of types II and III secretion systems to facilitate substrate translocation across this final bacterial barrier. ^

Petrology and geochemistry of sedimentary Unit III of ODP Hole 135-840B

The sedimentary sequence recovered at Site 840, on the Tonga frontal-arc platform, is 597.3 m thick and is subdivided into three lithostratigraphic units. The lowermost, late Miocene Unit III is 336.8 m thick and consists of a sequence of volcaniclastic mass-flow deposits (predominantly turbidites) interbedded with pelagic/hemipelagic deposits. Unit III was deposited in the forearc basin of the Lau volcanic arc, probably on a slope dominated by mass flows that built eastward from the ridge front and across the forearc. Upward through the unit a thinning and fining of individual turbidites takes place, interpreted to reflect a reduced sediment supply and a change from large to smaller flows. Decreasing volcanic activity with time is inferred from a decrease in coarse-grained volcaniclastic content in the upper part of the unit. The majority of the turbidites show the typical Bouma-type divisions, although both high- and low-density turbidity currents are inferred. High-density turbidity currents were especially common in the lower part of the unit. Geochemical analyses of detrital glass lie mainly in the low-K tholeiite field with a compositional range from basalt to rhyolite. A coherent igneous trend indicates derivation from a single volcanic source. This source was probably situated on the rifted part of the Lau-Tonga Ridge, within the present Lau backarc basin. The initial opening of the Lau Basin may have been around 6.0 m.y. ago. The onset of more extensive rifting, approximately 5.6 m.y. ago, is reflected in an increase in the silica content of volcanic glass. At the boundary toward Unit II, at approximately 5.25 Ma, an influx of thicker bedded and coarser grained volcaniclastic material is interpreted to reflect increasing volcanism and tectonism during the final breakup of the Lau-Tonga Ridge.

Isopoda E(S100) and surface sediment characteristics of ANDEEP I-III stations in the Southern Ocean deep sea

Shallow marine benthic communities around Antarctica show high levels of endemism, gigantism, slow growth, longevity and late maturity, as well as adaptive radiations that have generated considerable biodiversity in some taxa1. The deeper parts of the Southern Ocean exhibit some unique environmental features, including a very deep continental shelf2 and a weakly stratified water column, and are the source for much of the deep water in the world ocean. These features suggest that deep-sea faunas around the Antarctic may be related both to adjacent shelf communities and to those in other oceans. Unlike shallow-water Antarctic benthic communities, however, little is known about life in this vast deep-sea region2, 3. Here, we report new data from recent sampling expeditions in the deep Weddell Sea and adjacent areas (748-6,348 m water depth) that reveal high levels of new biodiversity; for example, 674 isopods species, of which 585 were new to science. Bathymetric and biogeographic trends varied between taxa. In groups such as the isopods and polychaetes, slope assemblages included species that have invaded from the shelf. In other taxa, the shelf and slope assemblages were more distinct. Abyssal faunas tended to have stronger links to other oceans, particularly the Atlantic, but mainly in taxa with good dispersal capabilities, such as the Foraminifera. The isopods, ostracods and nematodes, which are poor dispersers, include many species currently known only from the Southern Ocean. Our findings challenge suggestions that deep-sea diversity is depressed in the Southern Ocean and provide a basis for exploring the evolutionary significance of the varied biogeographic patterns observed in this remote environment.