Craniodental ecomorphology of the large Jurassic ichthyosaurian Temnodontosaurus.

Marine amniotes have played many crucial roles in ocean ecosystems since the Triassic, including predation at the highest trophic levels. One genus often placed into this guild is the large Early Jurassic neoichthyosaurian Temnodontosaurus, the only post-Triassic ichthyosaurian known with teeth which bear a distinct cutting edge or carina. This taxonomically problematic genus is currently composed of seven species which show a wide variety of skull and tooth morphologies. Here we assess the craniodental disparity in Temnodontosaurus using a series of functionally informative traits. We describe the range of tooth morphologies in the genus in detail, including the first examples of serrated carinae in ichthyosaurians. These consist of false denticles created by the interaction of enamel ridgelets with the carinal keel, as well as possible cryptic true denticles only visible using scanning electron microscopy. We also find evidence for heterodonty in the species T. platyodon, with unicarinate mesial teeth likely playing a role in prey capture and labiolingually compressed, bicarinate distal teeth likely involved in prey processing. This type of heterodonty appears to be convergent with a series of other marine amniotes including early cetaceans. Overall, the species currently referred to as the genus Temnodontosaurus show a range of craniodental configurations allowing prey to be captured and processed in different ways - for example, T. eurycephalus has a deep snout and relatively small bicarinate teeth likely specialised for increased wound infliction and grip-and-tear feeding, whereas T. platyodon has a more elongate yet robust snout and larger teeth and may be more adapted for grip-and-shear feeding. These results suggest the existence of niche partitioning at higher trophic levels in Early Jurassic ichthyosaurians and have implications for future work on the taxonomy of this wastebasket genus, as well as for research into the ecology of other extinct megapredatory marine tetrapods.

Aetiology and pathogenesis of hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a chronic inflammatory disorder. Patients develop inflamed nodules and abscesses and, at later stages of disease, epithelialized tunnels and scars in skinfolds of axillary, inguinal, gluteal and perianal areas. Quality of life is affected due to severe pain, purulent secretion, restricted mobility and systemic involvement. Genetics and lifestyle factors including smoking and obesity contribute to the development of HS. These factors lead to microbiome alteration, subclinical inflammation around the terminal hair follicles, and infundibular hyperkeratosis, resulting in plugging and rupture of the follicles. Cell-damage-associated molecules and propagating bacteria trigger inflammation and lead to massive immune cell infiltration that clinically manifests as inflamed nodules and abscesses. The immune system plays a key role also in the progression and chronification of skin alterations. Innate proinflammatory cytokines (e.g. interleukin-1ß and tumour necrosis factor-α), mediators of activated T helper (Th)1 and Th17 cells (e.g. interleukin-17 and interferon-γ), and effector mechanisms of neutrophilic granulocytes, macrophages and plasma cells are involved. Simultaneously, skin lesions contain anti-inflammatory mediators (e.g. interleukin-10) and show limited activity of Th22 and regulatory T cells. The inflammatory vicious circle finally results in pain, purulence, tissue destruction and scarring. Chronic inflammation in patients with HS is also frequently detected in organs other than the skin, as indicated by their comorbidities. All these aspects represent a challenge for the development of therapeutic approaches, which are urgently needed for this debilitating disease. This scholarly review focuses on the causes and pathogenetic mechanisms of HS and the potential therapeutic value of this knowledge.

Pharmacological study of cefoxitin as an alternative antibiotic therapy to carbapenems in treatment of urinary tract infections due to extended-spectrum-ß-lactamase-producing Escherichia coli.

Cefoxitin could be an alternative to carbapenems in extended-spectrum-beta-lactamase-producing Escherichia coli (ESBL-EC) infections. However, pharmacological and clinical data regarding cefoxitin are limited. Using a recent pharmacological model and the MICs of ESBL-EC collected from pyelonephritis, we determined the probabilities to reach four pharmacological targets: free cefoxitin concentrations above the MIC during 50% and 100% of the administration interval (T>MIC = 50% and T>MIC = 100%, respectively) and free cefoxitin concentrations above 4× MIC during 50% and 100% of the administration interval (T>4MIC = 50% and T>4MIC = 100%, respectively). Cefoxitin could be used to treat ESBL-EC pyelonephritis, but administration modalities should be optimized according to MICs in order to reach pharmacological targets.

Evaluation of the Andromas matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of aerobically growing Gram-positive bacilli.

Matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapid and simple microbial identification method. Previous reports using the Biotyper system suggested that this technique requires a preliminary extraction step to identify Gram-positive rods (GPRs), a technical issue that may limit the routine use of this technique to identify pathogenic GPRs in the clinical setting. We tested the accuracy of the MALDI-TOF MS Andromas strategy to identify a set of 659 GPR isolates representing 16 bacterial genera and 72 species by the direct colony method. This bacterial collection included 40 C. diphtheriae, 13 C. pseudotuberculosis, 19 C. ulcerans, and 270 other Corynebacterium isolates, 32 L. monocytogenes and 24 other Listeria isolates, 46 Nocardia, 75 Actinomyces, 18 Actinobaculum, 11 Propionibacterium acnes, 18 Propionibacterium avidum, 30 Lactobacillus, 21 Bacillus, 2 Rhodococcus equi, 2 Erysipelothrix rhusiopathiae, and 38 other GPR isolates, all identified by reference techniques. Totals of 98.5% and 1.2% of non-Listeria GPR isolates were identified to the species or genus level, respectively. Except for L. grayi isolates that were identified to the species level, all other Listeria isolates were identified to the genus level because of highly similar spectra. These data demonstrate that rapid identification of pathogenic GPRs can be obtained without an extraction step by MALDI-TOF mass spectrometry.

Who is to blame? Perspectives of caregivers on barriers to accessing healthcare for the under-fives in Butere District, Western Kenya.

BACKGROUND: Kenya, like many developing nations, continues to experience high childhood mortality in spite of the many efforts put in place by governments and international bodies to curb it. This study sought to investigate the barriers to accessing healthcare services for children aged less than five years in Butere District, a rural district experiencing high rates of mortality and morbidity despite having relatively better conditions for child survival. METHODS: Exit interviews were conducted among caregivers seeking healthcare for their children in mid 2007 in all the 6 public health facilities. Additionally, views from caregivers in the community, health workers and district health managers were sought through focus group discussions (FGDs) and key informant interviews (KIs). RESULTS: Three hundred and ninety-seven respondents were surveyed in exit interviews while 45 respondents participated in FGDs and KIs. Some practices by caregivers including early onset of child bearing, early supplementation, and utilization of traditional healers were thought to increase the risk of mortality and morbidity, although reported rates of mosquito net utilization and immunization coverage were high. The healthcare system posed barriers to access of healthcare for the under fives, through long waiting time, lack of drugs and poor services, incompetence and perceived poor attitudes of the health workers. FGDs also revealed wide-spread concerns and misconceptions about health care among the caregivers. CONCLUSION: Caregivers' actions were thought to influence children's progression to illness or health while the healthcare delivery system posed recurrent barriers to the accessing of healthcare for the under-fives. Actions on both fronts are necessary to reduce childhood mortality.

Rothia spp. infective endocarditis: A systematic literature review.

OBJECTIVE: To describe the epidemiological, clinical, microbiological, and therapeutic features and outcomes of Rothia infective endocarditis (RIE) and extracardiac infections (ECRI). METHODS: We performed a systematic literature review of published cases of RIE and ECRI. RESULTS: After inclusion of a personal case report, 51 cases of RIE and 215 cases of ECRI were reported. Compared with ECRI patients, RIE patients were significantly more often males (80% versus 59%), intravenous drug users (IVDU) (20% versus 3%), immunocompetent (76% versus 31%), and infected with R. dentocariosa (55% versus 13%) but lacked significant differences with regard to median age (45 years [6-79]), rate of orodental abnormalities (33%), and six-month mortality (14%). Following microbiological documentation, RIE was most often treated with a beta-lactam antibiotic alone (39%) for a median duration of six weeks and required surgery in 39% of cases. CONCLUSION: RIE is rare and likely secondary to a dental portal of entry or cutaneous inoculation in IVDU. Its prognosis seems to be favorable.

Comparison of the in vitro efficacies of moxifloxacin and amoxicillin against Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular bacterium that causes severe infections associated with a high mortality rate. Moxifloxacin presents extended activity against gram-positive bacteria and has recently been suggested to be a potential alternative in the treatment of listeriosis. We evaluated the in vitro efficacy of moxifloxacin against L. monocytogenes using a combination of epidemiological and experimental approaches. The median MIC of moxifloxacin for a large collection of L. monocytogenes strains of various origins (human, food, and environment) was 0.5 microg/ml (MIC range, 0.064 to 1 microg/ml). No differences were observed, irrespective of the origin of the strains. Moreover, no cross-resistance with fluoroquinolones was detected in strains that have been reported to be resistant to ciprofloxacin. The in vitro activities of moxifloxacin and amoxicillin were compared by time-kill curve and inhibition of intracellular growth experiments by using a model of bone marrow-derived mouse macrophages infected by L. monocytogenes EGDe. Both moxifloxacin and amoxicillin were bactericidal in broth against extracellular forms of L. monocytogenes. However, moxifloxacin acted much more rapidly, beginning to exert its effects in the first 3 h and achieving complete broth sterilization within 24 h of incubation. Moxifloxacin has a rapid bactericidal effect against intracellular reservoirs of bacteria, whereas amoxicillin is only bacteriostatic and appears to prevent cellular lysis and the subsequent bacterial spreading to adjacent cells. No resistant bacteria were selected during the in vitro experiments. Taken together, our results suggest that moxifloxacin is an interesting alternative to the reference treatment, combining rapid and bactericidal activity, even against intracellular bacteria.

FhuA-mediated phage genome transfer into liposomes: a cryo-electron tomography study.

BACKGROUND: The transfer of phage genomes into host cells is a well established but only dimly understood process. Following the irreversible phage binding to a receptor in the bacterial outer membrane, the DNA is ejected from the viral capsid and transferred across the bacterial cell envelope. In Escherichia coli, the mere interaction of the phage T5 with its outer membrane receptor, the ferrichrome transporter FhuA, is sufficient to trigger the release of the DNA from the phage capsid. Although the structure of FhuA has been determined at atomic resolution, the understanding of the respective roles of phage and bacterial proteins in DNA channeling and the mechanisms by which the transfer of the DNA is mediated remains fragmentary. RESULTS: We report on the use of cryo-electron tomography to analyze, at a molecular level, the interactions of T5 phages bound to FhuA-containing proteoliposomes. The resolution of the three-dimensional reconstructions allowed us to visualize the phage-proteoliposome interaction before and after release of the genome into the vesicles. After binding to its receptor, the straight fiber of the phage T5 (the "tip" of the viral tail made of pb2 proteins) traverses the lipid bilayer, allowing the transfer of its double-stranded DNA (121,000 bp) into the proteoliposome. Concomitantly, the tip of the tail undergoes a major conformational change; it shrinks in length (from 50 to 23 nm), while its diameter increases (from 2 to 4 nm). CONCLUSIONS: Taking into account the crystal structure of FhuA, we conclude that FhuA is only used as a docking site for the phage. The tip of the phage tail acts like an "injection needle," creating a passageway at the periphery of FhuA, through which the DNA crosses the membrane. A possible mechanistic scenario for the transfer of the viral genome into bacteria is discussed.

Untargeted next-generation sequencing-based first-line diagnosis of infection in immunocompromised adults: a multicentre, blinded, prospective study.

OBJECTIVE: Infections are the major cause of morbidity and mortality in immunocompromised patients. Improving microbiological diagnosis in these patients is of paramount clinical importance. METHODS: We performed this multicentre, blinded, prospective, proof-of-concept study, to compare untargeted next-generation sequencing with conventional microbiological methods for first-line diagnosis of infection in 101 immunocompromised adults. Patients were followed for 30 days and their blood samples, and in some cases nasopharyngeal swabs and/or biological fluids, were analysed. At the end of the study, expert clinicians evaluated the results of both methods. The primary outcome measure was the detection rate of clinically relevant viruses and bacteria at inclusion. RESULTS: Clinically relevant viruses and bacteria identified by untargeted next-generation sequencing and conventional methods were concordant for 72 of 101 patients in samples taken at inclusion (κ test=0.2, 95% CI 0.03-0.48). However, clinically relevant viruses and bacteria were detected in a significantly higher proportion of patients with untargeted next-generation sequencing than conventional methods at inclusion (36/101 (36%) vs. 11/101 (11%), respectively, p <0.001), and even when the latter were continued over 30 days (19/101 (19%), p 0.003). Untargeted next-generation sequencing had a high negative predictive value compared with conventional methods (64/65, 95% CI 0.95-1). CONCLUSIONS: Untargeted next-generation sequencing has a high negative predictive value and detects more clinically relevant viruses and bacteria than conventional microbiological methods. Untargeted next-generation sequencing is therefore a promising method for microbiological diagnosis in immunocompromised adults.

Use of detergents in two-dimensional crystallization of membrane proteins.

Structure determination at high resolution is actually a difficult challenge for membrane proteins and the number of membrane proteins that have been crystallized is still small and far behind that of soluble proteins. Because of their amphiphilic character, membrane proteins need to be isolated, purified and crystallized in detergent solutions. This makes it difficult to grow the well-ordered three-dimensional crystals that are required for high resolution structure analysis by X-ray crystallography. In this difficult context, growing crystals confined to two dimensions (2D crystals) and their structural analysis by electron crystallography has opened a new way to solve the structure of membrane proteins. However, 2D crystallization is one of the major bottlenecks in the structural studies of membrane proteins. Advances in our understanding of the interaction between proteins, lipids and detergents as well as development and improvement of new strategies will facilitate the success rate of 2D crystallization. This review deals with the various available strategies for obtaining 2D crystals from detergent-solubilized intrinsic membrane proteins. It gives an overview of the methods that have been applied and gives details and suggestions of the physical processes leading to the formation of the ordered arrays which may be of help for getting more proteins crystallized in a form suitable for high resolution structural analysis by electron crystallography.

Three-dimensional reconstruction from a frozen-hydrated specimen of the chiton Lepidochiton sp. hemocyanin.

A frozen-hydrated specimen of the hemocyanin of the chiton Lepidochiton sp. has been subjected to a three-dimensional reconstruction by the random conical tilt- series m wall and a collar complex. The wall is composed of five oblique wall units, disposed as a five-stranded, right-handed helix, separated by five clefts. The oblique wall unit is composed of two strings of functional units separated by a groove parallel with the cleft. The collar complex is a crown-like structure composed of five collar complex units, located at one end of the molecule and slightly protruding outside the cylinder wall. The collar complex unit comprises a collar unit probably composed of two functional units, one of which is connected to the wall, and an arch composed of two additional functional units, each connected to the wall by a narrow bridge. Each arch crosses a cleft between adjacent oblique wall units. The indentations present on both circular faces of the molecule and the dispositions of the masses resemble those of cephalopod hemocyanins.

Structural analysis of junctions formed between lipid membranes and several annexins by cryo-electron microscopy.

The (annexin II-p11)2 tetramer has been proposed to participate in exocytosis and several other members of the annexin superfamily have been reported to aggregate liposomes in vitro. In this context, the Ca2+-dependent binding of several annexins to chromaffin granules and liposomes was investigated by cryo-electron microscopy. The Ca2+-dependent aggregation of lipid membranes by (annexin II-p11)2 results from the spontaneous self-organization of the protein into two-dimensional plaques, which are visualized in projection as characteristic junctions. The junctions have a constant thickness of 210(+/-10) A and present a symmetrical distribution of electron-dense material arranged into seven stripes. They were observed over a wide range of Ca2+ concentrations, down to 2 microM. The molecular components corresponding to the seven electron-dense stripes were assigned as follows: the two associated membranes give rise to two outer stripes each and the three central stripes correspond to the (annexin II-p11)2 tetramer. Each annexin II molecule interacts with the outer lipid leaflet of one membrane, giving rise to one stripe, while the central stripe is due to the (p11)2 dimer with which both annexin II molecules interact. Both annexin II and annexin I also induced the Ca2+-dependent aggregation of liposomes via junctions that lack the central (p11)2 moiety and present only six high-density stripes. As expected, both annexin V and annexin III bind to liposomes without inducing their aggregation.

Three-dimensional reconstruction of the alpha D and beta C-hemocyanins of Helix pomatia from frozen-hydrated specimens.

The three-dimensional (3D) reconstructions of the di-decameric forms of alpha D and beta C-hemocyanins of the Roman snail Helix pomatia and of the decameric half molecules of alpha D-hemocyanin were carried out on frozen-hydrated specimens observed in the electron microscope by using the random conical tilt series method. The three 3D volumes were examined by computing solid-body surface representations and slices through the volume and by eroding the structure progressively through raising of the threshold. The di-decameric molecule of alpha D and beta C-hemocyanins, reconstructed from side views, are very similar and are composed of a cylindrical wall, comprising ten oblique wall units, and of two collar complexes located at both ends of the cylinder, comprising each five arches and an annular collar made up of five collar units. Erosion of the structure reveals that the wall looks like a segment of a five-stranded right-handed helix and that each oblique wall unit resembles a figure 8 inclined to the right. The decameric half molecule of alpha D-hemocyanin, reconstructed from end-on views, resembles the whole molecule, except that the collar is thinner and appears composed of five independent collar complex units. It is suggested that the difference in structural appearance of the collar complex between the whole and the half alpha D-hemocyanin may be due to the missing cone artifact, induced by the angular limitations imposed by the goniometer of the electron microscope. The comparison between the alpha D-hemocyanin and the beta C-di-decameric hemocyanin at high thresholds suggests that in the beta C-hemocyanin the oblique wall units of each half molecule may be linked by two connections, whereas in alpha D-hemocyanin there may be only one. This difference in the number of connections may be responsible for the lower stability of the alpha D molecule at high salt concentration.

Three-dimensional reconstruction of the chlorocruorin of the polychaete annelid Eudistylia vancouverii.

The chlorocruorin of the polychaete Eudistylia vancouverii observed in the electron microscope in vitreous ice, was subjected to a three-dimensional (3D) reconstruction by the random conical tilt series method. The 3D volume with a resolution of 35 A reconstructed from 1062 images in top, side and intermediate view orientations has a D6 point-group symmetry. It possesses the characteristic hexagonal bilayer (HBL) appearance. Each hexagonal half-molecule comprises size hollow globular substructures (HGS) presumed to correspond to the dodecameric subunits. In projection, when the molecule is viewed along its 6-fold axis, the two halves are not perfectly eclipsed. The vertices of the upper hexagonal layer are 14 degrees rotated clockwise compared with those of the lower half. At a threshold displaying 100% of the expected molecular volume, the 3D volume contains in its center a flat hexagonal central mass disconnected from the rest of the volume. Several types of connections, termed c1 through c4, are visible between the HGSs. The c1 and c2 connections link the HGSs of the same hexagonal half-molecule. The c3 connections make a hexagonal inner bracelet linking the HGSs of each half-molecule. The c4 connections link pairs of HGSs superposed in the two hexagonal layers. Because of the half-molecules rotation around the 6-fold axis, the two HGSs linked by a c4 connection are not exactly superposed. It is proposed that the c3 and c4-connection bodies and less probably the flat central hexagonal mass are composed of chloroheme-deficient linker chains. When eroding the 3D volume by raising the threshold, the HGS appears composed of three elongated structures likely containing four globin chains. In addition, they show an approximate 3-fold symmetry. At high thresholds, two of these masses, dumbbell-shaped, separate into globular masses while the third structure remains compact as long as 1% of the molecular volume is displayed.

Quaternary structure of Octopus vulgaris hemocyanin. Three-dimensional reconstruction from frozen-hydrated specimens and intramolecular location of functional units Ove and Ovb.

A frozen-hydrated sample of Octopus vulgaris hemocyanin was imaged at 0 degree and 40 degrees tilt angle under low dose conditions by transmission electron microscopy. A three-dimensional reconstruction by the method of random conical tilt series produced a three-dimensional volume to which a D5 symmetry was applied. Examination of serial sections in the volume and surface representation at various thresholds allowed the five arches containing functional unit Ovg to be localized at the interdimeric subunit groove. In another set of experiments specific polyclonal antibodies were used to label functional units Ovb and Ove in the cylinder wall. The observation of the negatively stained immunocomplexes showed that Ovb is located in the external tiers of functional units and Ove in the internal tier. These results suggest that the direction of the polypeptide chains in the cylinder wall may be only partially antiparallel. A model of the quaternary structure is proposed with the following features: (1) the external tiers of functional units comprise four units each (Ova-d) coming from a single polypeptide chain; (2) the internal tier comprises two functional units from each polypeptide chain (Ove-f); (3) the interdimeric subunit arches connect the two copies of a single functional unit (Ovg) located in each polypeptide chain.

Three-dimensional reconstruction of Limulus polyphemus hemocyanin from cryoelectron microscopy.

Hemocyanin (Hc) the respiratory pigment of the horseshoe crab Limulus polyphemus (Lp) is composed of 48 approximately 75 kDa copper-containing subunits arranged in eight hexameric groups. In this study, we used the random conical tilt series method to do a three-dimensional (3D) reconstruction of Lp Hc observed in vitreous ice. This approach allowed the unambiguous determination of the handedness of the molecule. Lp Hc contains two superimposed 4 x 6mer structures possessing the same structural features as the other 4 x 6meric Hcs, namely flip and flop views and a rocking effect. Moreover, 3D fitting of the X-ray structure of subunit LpII with the reconstruction volume shows that the intra4 x 6meric contacts described in arthropod Hcs also occur within Limulus Hc. The two half-molecules composing the 8 x 6mer have their flop faces in contact (flop/flop association), the main links being formed by subunits LpIV. Model building shows that the flop/flop association is the only possible arrangement which allows the assembly of the whole particle. The two alternate constructions (flip/flop and flip/flip) are forbidden because of steric hindrance.