Prognostic value of brain perfusion by MRI in the initial study of high grade gliomas.

OBJECTIVES: To evaluate if the tumour perfusion at the initial MRI scan is a marker of prognosis for survival in patients diagnosed with High Grade Gliomas (HGG). To analyse the risk factors which influence on the mortality from HGG to quantify the overall survival to be expected in patients. PATIENTS AND METHODS: The patients diagnosed with HGG through a MRI scan in a third-level hospital between 2017 and 2019 were selected. Clinical and tumour variables were collected. The survival analysis was used to determine the association between the tumour perfusion and the survival time. The relation between the collected variables and the survival period was assessed through Wald's statistical method, measuring the relationship via Cox's regression model. Finally, the type of relationship that exists between the tumour perfusion and the survival was analysed through the Lineal Regression method.Those statistical analysis were carried out using the software SPSS v.17. RESULTS: 38 patients were included (average age: 61.1 years old). The general average survival period was 20.6 months. A relationship between the tumour perfusion at the MRI scan and the overall survival has been identified, in detail, a group with intratumor values of relative cerebral blood volume (rCBV)>3.0 has shown a significant decline in the average survival period with regard to the average survival period of the group with values <3.0 (14.6 months vs. 22.8 months, p = 0.046). It has also been proved that variables like Karnofsky's scale and the response time since the intervention significantly influence on the survival period. CONCLUSIONS: It has become evident that the tumour perfusion via MRI scan has a prognostic value in the initial analysis of HGG. The average survival period of patients with rCBV less than or equal to 3.0 is significantly higher than those patients whose values are higher, which allows to be more precise with the prognosis of each patient.

The Origin, Dynamic Morphology, and PI4P-Independent Formation of Encephalomyocarditis Virus Replication Organelles.

Picornaviruses induce dramatic rearrangements of endomembranes in the cells that they infect to produce dedicated platforms for viral replication. These structures, termed replication organelles (ROs), have been well characterized for the Enterovirus genus of the Picornaviridae However, it is unknown whether the diverse RO morphologies associated with enterovirus infection are conserved among other picornaviruses. Here, we use serial electron tomography at different stages of infection to assess the three-dimensional architecture of ROs induced by encephalomyocarditis virus (EMCV), a member of the Cardiovirus genus of the family of picornaviruses that is distantly related. Ultrastructural analyses revealed connections between early single-membrane EMCV ROs and the endoplasmic reticulum (ER), establishing the ER as a likely donor organelle for their formation. These early single-membrane ROs appear to transform into double-membrane vesicles (DMVs) as infection progresses. Both single- and double-membrane structures were found to support viral RNA synthesis, and progeny viruses accumulated in close proximity, suggesting a spatial association between RNA synthesis and virus assembly. Further, we explored the role of phosphatidylinositol 4-phosphate (PI4P), a critical host factor for both enterovirus and cardiovirus replication that has been recently found to expedite enterovirus RO formation rather than being strictly required. By exploiting an EMCV escape mutant, we found that low-PI4P conditions could also be overcome for the formation of cardiovirus ROs. Collectively, our data show that despite differences in the membrane source, there are striking similarities in the biogenesis, morphology, and transformation of cardiovirus and enterovirus ROs, which may well extend to other picornaviruses.IMPORTANCE Like all positive-sense RNA viruses, picornaviruses induce the rearrangement of host cell membranes to form unique structures, or replication organelles (ROs), that support viral RNA synthesis. Here, we investigate the architecture and biogenesis of cardiovirus ROs and compare them with those induced by enteroviruses, members of the well-characterized picornavirus genus Enterovirus The origins and dynamic morphologies of cardiovirus ROs are revealed using electron tomography, which points to the endoplasmic reticulum as the donor organelle usurped to produce single-membrane tubules and vesicles that transform into double-membrane vesicles. We show that PI4P, a critical lipid for cardiovirus and enterovirus replication, is not strictly required for the formation of cardiovirus ROs, as functional ROs with typical morphologies are formed under phosphatidylinositol 4-kinase type III alpha (PI4KA) inhibition in cells infected with an escape mutant. Our data show that the transformation from single-membrane structures to double-membrane vesicles is a conserved feature of cardiovirus and enterovirus infections that likely extends to other picornavirus genera.

Studying the complications of bariatric surgery with intravenous contrast-enhanced multidetector computed tomography. / Estudio de las complicaciones de la cirugía bariátrica por tomografía computarizada multidetector con contraste intravenoso.

OBJECTIVE: To review the complications of bariatric surgery and their diagnosis with intravenous contrast-enhanced multidetector computed tomography (MDCT). MATERIAL AND METHODS: We retrospectively studied all patients who underwent gastric bypass or sleeve gastrectomy at our center during 2013 or 2014. We classified complications into early complications (appearing within 30 days of the intervention) and late complications. RESULTS: We reviewed 155 cases and found 24 complications in 22 patients: 16 early complications (7 intraperitoneal hematomas, 5 anastomotic dehiscences, 2 intestinal obstructions, and 2 external hernias) and 8 late complications (3 internal hernias, 3 intestinal perforations, and 2 marginal ulcers). Two patients died. All of these complications were diagnosed with intravenous contrast-enhanced MDCT, except one, which required a barium transit study. CONCLUSION: The rate of complications in bariatric surgery is high and the associated mortality is not negligible. Radiologists need to know the normal findings in these patients so they can quickly identify possible complications, most of which can be diagnosed with intravenous contrast-enhanced MDCT.

Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein.

Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues. The GFP(S11) (GFP with S11 fragment) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. Using correlative light electron microscopy (CLEM), we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. These tools will have multiple applications in future studies on the origin, location, and function of enterovirus ROs. IMPORTANCE Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for genome replication. Electron microscopy has revealed the morphology of enterovirus ROs, and immunofluorescence studies have been conducted to investigate their origin and formation. Yet, immunofluorescence analysis of fixed cells results in a rather static view of RO formation, and the results may be compromised by immunolabeling artifacts. While live-cell imaging of ROs would be preferred, enteroviruses encoding a membrane-anchored viral protein fused to a large fluorescent reporter have thus far not been described. Here, we tackled this constraint by introducing a small tag from a split-GFP system into an RO-resident enterovirus protein. This new tool bridges a methodological gap by circumventing the need for immunolabeling fixed cells and allows the study of the dynamics and formation of enterovirus ROs in living cells.

Mammalian orthoreovirus T3D infects U-118 MG cell spheroids independent of junction adhesion molecule-A.

In the canonical pathway, infection of cells by the wild-type mammalian orthoreovirus Type 3 Dearing (T3D) is dependent on the interaction of the viral spike protein σ1 with the high-affinity cellular receptor junction adhesion molecule-A (JAM-A). We previously demonstrated that the human glioblastoma cell line U-118 MG does not express JAM-A and resists reovirus T3D infection in standard cell culture conditions (SCCC). Heterologous JAM-A expression sensitises U-118 MG cells to reovirus T3D. Here we studied reovirus infection in U-118 MG cells grown in spheroid cultures with the premise that cells in such cultures resemble cells in tumours more than those grown under standard adherent cell culture conditions on a plastic surface. Although the U-118 MG cells in spheroids do not express JAM-A, they are susceptible to reovirus T3D infection. We show that this can be attributed to factors secreted by cells in the spheroids. The concentration of active extracellular proteases cathepsin B and L in the medium of spheroid cultures was increased 19- and 24-fold, respectively, as compared with SCCC. These enzymes can convert the reovirus particles into a form that can infect the U-118 MG cells independent of JAM-A. Taken together, these data demonstrate that infection of tumour cells by wild-type reovirus T3D is not strictly dependent on the expression of JAM-A on the cell surface.

Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy.

Correlative light and electron microscopy is an increasingly popular technique to study complex biological systems at various levels of resolution. Fluorescence microscopy can be employed to scan large areas to localize regions of interest which are then analyzed by electron microscopy to obtain morphological and structural information from a selected field of view at nm-scale resolution. Previously, an integrated approach to room temperature correlative microscopy was described. Combined use of light and electron microscopy within one instrument greatly simplifies sample handling, avoids cumbersome experimental overheads, simplifies navigation between the two modalities, and improves the success rate of image correlation. Here, an integrated approach for correlative microscopy under cryogenic conditions is presented. Its advantages over the room temperature approach include safeguarding the native hydrated state of the biological specimen, preservation of the fluorescence signal without risk of quenching due to heavy atom stains, and reduced photo bleaching. The potential of cryo integrated light and electron microscopy is demonstrated for the detection of viable bacteria, the study of in vitro polymerized microtubules, the localization of mitochondria in mouse embryonic fibroblasts, and for a search into virus-induced intracellular membrane modifications within mammalian cells.

Single stimulation of the posterior cord is superior to dual nerve stimulation in a coracoid block.

BACKGROUND: Both multiple injection and single posterior cord injection techniques are associated with extensive anesthesia of the upper limb after an infraclavicular coracoid block (ICB). The main objective of this study was to directly compare the efficacy of both techniques in terms of the rates of completely anesthetizing cutaneous nerves below the elbow. METHODS: Seventy patients undergoing surgery at or below the elbow were randomly assigned to receive an ICB after the elicitation of either a single radial nerve-type response (Radial group) or of two different main nerve-type responses of the upper limb, except for the radial nerve (Dual group). Forty milliliters of 1.5% mepivacaine was given in a single or a dual dose, according to group assignment. The sensory block was assessed in each of the cutaneous nerves at 10, 20 and 30 min. Block performance times and the rates of complete anesthesia below the elbow were also noted. RESULTS: Higher rates of sensory block of the radial nerve were found in the Radial group at 10, 20 and 30 min (P<0.05). The rates of sensory block of the ulnar nerve at 30 min were 97% and 75% in the Radial and in the Dual groups, respectively (P<0.05). The rate of complete anesthesia below the elbow was also higher in the Radial group at 30 min (P<0.05). CONCLUSIONS: Injection of a local anesthetic after a single stimulation of the radial nerve fibers produced more extensive anesthesia than using a dual stimulation technique under the conditions of our study.

[Meningioangiomatosis: report of two cases and literature review]. / Meningioangiomatosis. Descripción de dos casos y revisión de la literatura.

Meningioangiomatosis (MA) is a rare benign intracraneal lesion. The majority of cases are sporadic although the association of this lesion with familial neurofibromatosis (NF) type 2 is well known. NF-associated MA may be multifocal and is often asymptomatic and diagnosed at autopsy. Non-associated cases are usually symptomatic, occurs in children and young adults and frequently arise in leptomeninges and underlying cerebral cortex. In the present work, we describe two new non-associated cases of MA in two boys, seven and one year old with seizures that disappeared after surgical excision. Histopathologically, the lesion was predominantly cellular in one case and more fibrous in the other. From the literature review we concluded that sporadic cases present as single lesions which manifest by seizures or persistent headaches. Rarely MA has been described to coexist with meningiomas. Histopathologically, MA is characterized by a plaque-like proliferation of meningothelial and fibroblast- like cells surrounding small vessels and trapping islands of gliotic cortical tissue. The lesion does not show significant atypia, mitosis or necrosis. Although all cases of MA share unifying features, there are different degrees of histological presentation with cases predominantly cellular and others more fibrous and calcified. This could correspond to different stages in the evolution of the MA. Symptoms disappear with the complete excision of the lesion.

A comparison of single versus multiple injections on the extent of anesthesia with coracoid infraclavicular brachial plexus block.

Single-injection coracoid infraclavicular brachial plexus block produces inconsistent anesthesia of the upper limb. In this study, we sought to determine the number of injections needed to provide a reasonably complete anesthesia of the upper limb with this approach. Seventy-five patients were randomly assigned to receive a coracoid block guided by nerve stimulator with 42 mL of 1.5% mepivacaine with a single-injection (Group 1), dual-injection (Group 2), or triple-injection (Group 3) technique. No search for a specific motor response was performed in any group. Sensory and motor block was assessed 5 and 20 min after the end of the injection of local anesthetic. Significantly less complete anesthesia to pinprick in the distributions of the axillary, musculocutaneous, radial, ulnar, and medial cutaneous forearm nerves was found in Group 1 at 20 min. Significantly less complete paralysis for arm, wrist, and hand movements was found in Group 1 at 20 min. No significant difference was found between Groups 2 and 3. We conclude that dual and triple injection of local anesthetic guided by nerve stimulator increases the efficacy of coracoid block when compared with a single-injection technique.

Increased success rate with infraclavicular brachial plexus block using a dual-injection technique.

STUDY OBJECTIVE: To test the hypothesis that dual injection of the local anesthetic in coracoid infraclavicular brachial plexus block might enhance both sensory and motor block for anesthesia of the upper limb when compared with a single-injection technique. DESIGN: Comparative, prospective, randomized, investigator-blinded study. SETTING: University-affiliated hospital. PATIENTS: 60 patients scheduled for surgery of the hand, forearm, or elbow. INTERVENTIONS: Patients were randomly assigned to receive either a coracoid infraclavicular block guided by nerve stimulator with a single injection technique (Group 1) or a dual-injection technique (Group 2). Injection of 40 mL 1.5% mepivacaine was made after eliciting one evoked motor response in the upper limb with a nerve stimulator for coracoid infraclavicular block in Group 1 and injection of two separate doses of 20 mL 1.5% mepivacaine after elicitation of two motor responses in Group 2. MEASUREMENTS: Assessment of sensory and motor block in the upper limb 5 and 20 minutes after the end of the injection of the local anesthetic. The time needed to elicit the second response and to inject the second dose of local anesthetic was also recorded. MAIN RESULTS: Significantly higher rates of sensory block to pinprick on the distributions of axillary, musculocutaneous, radial, ulnar, and medial cutaneous of the forearm nerves were found in Group 2 at 20 minutes. Significantly higher rates of motor block for arm, wrist, and hand movements were found in Group 2 at 20 minutes. CONCLUSIONS: Dual injection of local anesthetic guided by nerve stimulator increases the efficacy of coracoid block when compared with a single injection of the same dose of local anesthetic.

Effect of highly active antiretroviral therapy on the incidence and clinical manifestations of visceral leishmaniasis in human immunodeficiency virus-infected patients.

This study analyzes the effect of highly active antiretroviral therapy (HAART) on visceral leishmaniasis in human immunodeficiency virus-infected individuals. We describe 66 episodes diagnosed in our hospital from 1994 through 2000 (35 episodes in patients who were not receiving HAART and 31 episodes in patients who were receiving HAART). Since HAART became available, the incidence of visceral leishmaniasis has significantly decreased and the number of first episodes has decreased, although the number of relapses is increasing.

[Acute contralateral hemothorax after cannulation of the left subclavian vein for hemodialysis]. / Hemotórax agudo contralateral tras la canulación de la vena subclavia izquierda para hemodiálisis.

Contralateral hemothorax after subclavian vein cannulation is a rare complication of this procedure. We report one case of acute contralateral hemothorax due to perforation of the superior vena cava during left subclavian vein cannulation for hemodialysis. Perforation was attributed to the forced insertion of the skin dilator, which was accidentally pushed too far. Chest pain began immediately after perforation and was followed, a few minutes later, by hemodynamic instability. The signs and symptoms described in this report may serve to warn of the possibility of this complication.

A novel neural network technique for analysis and classification of EM single-particle images.

We propose a novel self-organizing neural network for the unsupervised classification of electron microscopy (EM) images of biological macromolecules. The radical novelty of the algorithm lies in its rigorous mathematical formulation that, starting from a large set of possibly very noisy input data, finds a set of "representative" data items, organized onto an ordered output map, such that the probability density of this set of representative items resembles at its possible best the probability density of the input data. In a way, it summarizes large amounts of information into a concise description that rigorously keeps the basic pattern of the input data distribution. In this application to the field of three-dimensional EM of single particles, two different data sets have been used; one comprised 2458 rotational power spectra of individual negative stain images of the G40P helicase of Bacillus subtilis bacteriophage SPP1, and the other contained 2822 cryoelectron images of SV40 large T-antigen. Our experimental results prove that this technique is indeed very successful, providing the user with the capability of exploring complex patterns in a succinct, informative, and objective manner. The above facts, together with the consideration that the integration of this new algorithm with commonly used software packages is immediate, prompt us to propose it as a valuable new tool in the analysis of large collections of noisy data.

Epidural washout with high volumes of saline to accelerate recovery from epidural anaesthesia.

BACKGROUND: Prolonged postoperative blockade can follow neuraxial blocks for short surgical procedures. We investigated whether washout with a high volume of saline through an epidural catheter could provide a faster recovery after epidural anaesthesia. METHODS: Thirty patients were randomly assigned to a control group (no washout), to group 2x (epidural washout with twice the volume of 2% mepivacaine) and group 4x (epidural washout with four times that volume). RESULTS: Recovery times from sensory blockade at L2 were 151+/-24, 122+/-29 and 116+/-24 min for control, 2x and 4x groups respectively. Significant differences were found in both saline groups when compared with control group, but not between group 2x and group 4x. No differences were found concerning motor blockade. One patient in group 4x demonstrated signs of intracranial hypertension. Mepivacaine plasma concentrations were increased by saline washout in group 4x. CONCLUSIONS: Epidural washout with a high volume of saline can not be recommended since no clinically significant reduction in the recovery time can be achieved without risk.

High doses of mepivacaine for brachial plexus block in patients with end-stage chronic renal failure. A pilot study.

BACKGROUND AND OBJECTIVE: Patients with end-stage chronic renal failure are at risk of developing several serious postanaesthetic complications. Many anaesthesiologists perform brachial plexus anaesthesia with high doses of local anaesthetic in order to achieve an extensive blockade of the upper limb. Brachial plexus block is a suitable technique for anaesthesia for creation, repair or removal of vascular access for haemodialysis. The aim of this study was to measure mepivacaine plasma concentrations after axillary block with 650 mg plain mepivacaine in patients with end-stage chronic renal failure. METHODS: Mepivacaine plasma concentrations were assessed throughout a 150-min period, in 10 patients after axillary block with 650 mg plain mepivacaine (600 mg for axillary block and 50 mg for supplementation). RESULTS: Mepivacaine plasma concentrations expressed in microg mL(-1) as medians and their ranges were: 1.69 (1.23--7.78) at 5 min, 5.61 (4.36--8.19) at 30 min, 8.28 (3.83--11.21) at 60 min, 7.93 (5.63--11.1) at 90 min and 6.49 (5.56--8.35) at 150 min without any symptoms of toxicity. CONCLUSIONS: Brachial plexus anaesthesia with 650 mg plain mepivacaine did not result in serious systemic toxicity in these patients despite the high mepivacaine plasma concentrations found.

The DnaB.DnaC complex: a structure based on dimers assembled around an occluded channel.

Replicative helicases are motor proteins that unwind DNA at replication forks. Escherichia coli DnaB is the best characterized member of this family of enzymes. We present the 26 A resolution three-dimensional structure of the DnaB hexamer in complex with its loading partner, DnaC, obtained from cryo-electron microscopy. Analysis of the volume brings insight into the elaborate way the two proteins interact, and provides a structural basis for control of the symmetry state and inactivation of the helicase by DnaC. The complex is arranged on the basis of interactions among DnaC and DnaB dimers. DnaC monomers are observed for the first time to arrange as three dumb-bell-shaped dimers that interlock into one of the faces of the helicase. This could be responsible for the freezing of DnaB in a C(3) architecture by its loading partner. The central channel of the helicase is almost occluded near the end opposite to DnaC, such that even single-stranded DNA could not pass through. We propose that the DnaB N-terminal domain is located at this face.

pH-controlled quaternary states of hexameric DnaB helicase.

DnaB is the major helicase in the Escherichia coli replisome. It is a homohexameric enzyme that interacts with many other replisomal proteins and cofactors. It is usually loaded onto a single strand of DNA at origins of replication from its complex with its loading partner DnaC, then translocates in the 5' to 3' direction, unwinding duplex DNA in an NTP-driven process. Quaternary polymorphism has been described for the DnaB oligomer, a feature it has in common with some other hexameric helicases. In the present work, electron microscopy and in- depth rotational analysis studies of negatively stained specimens has allowed the establishment of conditions that govern the transition between the two different rotational symmetry states (C(3) and C(6)) of DnaB. It is shown: (a) that the pH value of the sample buffer, within the physiological range, dictates the quaternary organisation of the DnaB oligomer; (b) that the pH-induced transition is fully reversible; (c) that the type of adenine nucleotide complexed to DnaB, whether hydrolysable or not, does not affect its quaternary architecture; (d) that the DnaB.DnaC complex exists only as particles with C(3) symmetry; and (e) that DnaC interacts only with DnaB particles that have C(3) symmetry. Structural consequences of this quaternary polymorphism, as well as its functional implications for helicase activity, are discussed.

Mapping and fuzzy classification of macromolecular images using self-organizing neural networks.

In this work the effectiveness of the fuzzy kohonen clustering network (FKCN) in the unsupervised classification of electron microscopic images of biological macromolecules is studied. The algorithm combines Kohonen's self-organizing feature maps (SOFM) and Fuzzy c-means (FCM) in order to obtain a powerful clustering technique with the best properties inherited from both. Exploratory data analysis using SOFM is also presented as a step previous to final clustering. Two different data sets obtained from the G40P helicase from B. Subtilis bacteriophage SPP1 have been used for testing the proposed method, one composed of 2458 rotational power spectra of individual images and the other composed by 338 images from the same macromolecule. Results of FKCN are compared with self-organizing feature maps (SOFM) and manual classification. Experimental results prove that this new technique is suitable for working with large, high-dimensional and noisy data sets and, thus, it is proposed to be used as a classification tool in electron microscopy.

Premedication with EMLA cream for ambultory surgery in children.

Objectives: The objective of this study was to confirm the ability of EMLA(R) cream (Eutectic Mixture of Local Anaesthetics, Astra, Sweden) to provide effective dermal analgesia after topical application on the skin of the dorsum of the hand 1 h before venous cannulation for anaesthetic induction. Material and Methods: Prospective, randomized, double blind study. We included 100 children, ASA I-III, distributed into three groups: Group EMLA (E, n=34), Placebo (P, n=33) and Control (C, n=33). Results: The EMLA group of patients (E) refered an evaluation of pain (visual analogical scale (mean=2.34+/-2.41), significantly smaller than the other groups (Placebo=5.54+/-3.40, Control=6.03+/-2.77). Conclusion: EMLA cream, when topically applied 1 h before venous cannulation, provides effective dermal analgesia for venous cannulation. No general or local adverse reactions were observed.

Sequence-related protein export NTPases encoded by the conjugative transfer region of RP4 and by the cag pathogenicity island of Helicobacter pylori share similar hexameric ring structures.

RP4 TrbB, an essential component of the conjugative transfer apparatus of the broad-host-range plasmid RP4, is a member of the PulE protein superfamily involved in multicomponent machineries transporting macromolecules across the bacterial envelope. PulE-like proteins share several well conserved motifs, most notable a nucleoside triphosphate binding motif (P-loop). Helicobacter pylori HP0525 also belongs to the PulE superfamily and is encoded by the pathogenicity island cag, involved in the inflammatory response of infected gastric epithelial cells in mammals. The native molecular masses of TrbB and HP0525 as determined by gel filtration and glycerol gradient centrifugation suggested a homohexameric structure in the presence of ATP and Mg(2+). In the absence of nucleotides and bivalent cations, TrbB behaved as a tetramer whereas the hexameric state of HP0525 remained unaffected. Electron microscopy and image processing demonstrated that TrbB and HP0525 form ring-shaped complexes (diameter: 12 nm) with a central region (diameter: 3 nm) of low electron density when incubated in the presence of ATP and Mg(2+). However, the TrbB average image appeared to be more elliptical with strong twofold rotational symmetry whereas HP0525 complexes are regular hexagons. Six well defined triangle-shaped areas of high electron density were distinguishable in both cases. Covalent crosslinking of TrbB suggests that the hexameric ring is composed from a trimer of dimers, because only dimeric, tetrameric, and hexameric species were detectable. The toroidal structure of TrbB and HP0525 suggests that both proteins catalyze a repetitive process, most probably translocating a cognate substrate across the inner membrane.