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Recently, it has been shown by overnight driving simulation studies that microsleep density is the only known sleepiness indicator which rapidly increases within a few seconds immediately before sleepiness related crashes. This indicator is based solely on EEG and EOG and subsequent adaptive pattern recognition. Accurate microsleep recognition is very important for the performance of this sleepiness indicator. The question is whether expensive evaluations of microsleep events by a) experts are necessary or b) non-experts provide sufficient evaluations. Based on 11,114 microsleep events in case a) and 12,787 in case b) recognition accuracies were investigated utilizing (i) artificial neural networks and (ii) support-vector machines. Cross validated classification accuracies ranged between 92.2 % for (i,b) and 99.3 % for (ii, a). It is concluded that expert evaluations are very important to provide independent information for detecting microsleep.

Abstract Background We have recently released a comprehensive, manually curated database of mammalian protein complexes called CORUM. Combining CORUM with other resources, we assembled a dataset of over 2700 mammalian complexes. The availability of a rich information resource allows us to search for organizational properties concerning these complexes. Results As the complexity of a protein complex in terms of the number of unique subunits increases, we observed that the number of such complexes and the mean non-synonymous to synonymous substitution ratio of associated genes tend to decrease. Similarly, as the number of different complexes a given protein participates in increases, the number of such proteins and the substitution ratio of the associated gene also tends to decrease. These observations provide evidence relating natural selection and the organization of mammalian complexes. We also observed greater homogeneity in terms of predicted protein isoelectric points, secondary structure and substitution ratio in annotated versus randomly generated complexes. A large proportion of the protein content and interactions in the complexes could be predicted from known binary protein-protein and domain-domain interactions. In particular, we found that large proteins interact preferentially with much smaller proteins. Conclusion We observed similar trends in yeast and other data. Our results support the existence of conserved relations associated with the mammalian protein complexes.

Background/aims: Endoscopic submucosal dissection (ESD) has been established as a treatment modality for superficial esophageal squamous cell carcinoma (ESCC). Long-term follow-up data are lacking in Western countries. The aim of this study was to analyze long-term survival in a Western center., Methods: Patients undergoing ESD for ESCC were included. The analysis was performed retrospectively using a prospectively collected database., Results: R0 resection rate was 96.7% (59/61 lesions in 58 patients). Twenty-seven patients (46.6%) fulfilled the curative resection criteria (M1/M2) (group A), 11 patients (19.0%) had M3 lesions without lymphovascular invasion (LVI) (group B), and 20 patients (34.5%) had lesions with submucosal invasion or LVI (group C). Additional treatment was recommended after non-curative resection. It was not performed in 20/31 patients (64.5%), mainly because of comorbidities (75%). Twenty-nine out of 58 (50.0%) patients died during a mean follow-up of 3.7 years. Death was related to ESCC in 17.2% (5/29) of patients. The disease-specific survival rate after curative resection was 100%. Overall survival rates after 5 years were 61.5%, 63.6% and 28.1% for groups A, B, and C, respectively. The overall survival was significantly worse after non-curative resection (p=0.038)., Conclusion: Non-curative resection is frequent after ESD for ESCC in Western patients. The long-term prognosis is limited and mainly determined by comorbidity. Early diagnosis and pre-interventional assessments need to be improved.

Background: Pylorus-preserving pancreaticoduodenectomy (ppPD) is a standard surgical procedure for the treatment of resectable neoplasms of the periampullary region. One of the most common postoperative complications after ppPD is delayed gastric emptying (DGE) which reduces quality of life, prevents a timely return to a solid oral diet and prolongs the length of hospital stay. In a retrospective analysis, intraoperative endoluminal pyloromyotomy was associated with a reduced rate of DGE. The aim of this study is to investigate the effect of intraoperative endoluminal pyloromyotomy on postoperative DGE after ppPD in a randomised and controlled setting., Methods: This randomised trial features parallel group design with a 1:1 allocation ratio and a superiority hypothesis. Patients with a minimum age of 18 years and an indication for ppPD are eligible to participate in this study and will be randomised intraoperatively to receive either endoluminal pyloromyotomy or atraumatic stretching of the pylorus. The sample size calculation (n=64 per study arm) is based on retrospective data. The primary endpoint is the rate of DGE within 30 days. Secondary endpoints are quality of life, operation time, estimated blood loss, length of hospital stay, morbidity and mortality., Discussion: DGE after ppPD is a common complication with an incomplete understood aetiology. Prevention of DGE could improve outcomes and enhance quality of life after one of the most common procedures in pancreatic surgery. This trial will expand the existing evidence on intraoperative pyloromyotomy, and the results will provide additional data on a simple surgical technique that could reduce the incidence of postoperative DGE., Trial Registration: German Clinical Trials Register DRKS00013503 . Registered on 27 December 2017., (© 2022. The Author(s).)

Objective: The study explores associations of visually induced motion sickness (VIMS) with emergency braking reaction times (RTs) in driving simulator studies. It examines the effects over the progression of multiple simulated drives., Background: Driving simulator usage has many advantages for RT studies; however, if it induces VIMS, the observed driving behavior might deviate from real-world driving, potentially masking or skewing results. Possible effects of VIMS on RT have long been entertained, but the progression of VIMS across simulated drives has so far not been sufficiently considered., Method: Twenty-eight adults completed six drives on 2 days in a fixed-base driving simulator. At five points during each drive, pedestrians entered the road, necessitating emergency braking maneuvers. VIMS severity was assessed every minute using the 20-point Fast Motion Sickness Scale. The progression of VIMS was considered in mixed model analyses., Results: RT predictions were improved by considering VIMS development over time. Here, the relationship of VIMS and RT differed across days and drives. Increases in VIMS symptom severity predicted more prolonged RT after repeated drives on a given day and earlier within each drive., Conclusion: The assessment of VIMS in RT studies can be beneficial. In this context, VIMS measurements in close temporal proximity to the behaviors under study are promising and offer insights into VIMS and its consequences, which are not readily obtainable through questionnaires., Application: Driving simulator-based RT studies should consider cumulative effects of VIMS on performance. Measurement and analysis strategies that consider the time-varying nature of VIMS are recommended.

The ever-increasing metagenomic data necessitate appropriate cataloguing in a way that facilitates the comparison and better contextualization of the underlying investigations. To this extent, information associated with the sequencing data as well as the original sample and the environment where it was obtained from is crucial. To date, there are not any publicly available repositories able to capture environmental metadata pertaining to hydrocarbon-rich environments. As such, contextualization and comparative analysis among sequencing datasets derived from these environments is to a certain degree hindered or cannot be fully evaluated. The metagenomics data management system for hydrocarbon resources (MetaHCRs) enables the capturing of marker gene and whole metagenome sequencing data as well as over 300 contextual attributes associated with samples, organisms, environments and geological properties, among others. Moreover, MetaHCR implements the Minimum Information about any Sequence-hydrocarbon resource specification from the Genomic Standards Consortium; it integrates a user-friendly web interface and relational database model, and it enables the generation of complex custom search. MetaHCR has been tested with 36 publicly available metagenomic studies, and its modular architecture can be easily customized for other types of environmental and metagenomics studies.

Bacterial toxins require localization to specific intracellular compartments following injection into host cells. In this study, we examined the membrane targeting of a broad family of bacterial proteins, the patatin-like phospholipases. The best characterized member of this family is ExoU, an effector of the Pseudomonas aeruginosa type III secretion system. Upon injection into host cells, ExoU localizes to the plasma membrane, where it uses its phospholipase A2 activity to lyse infected cells. The targeting mechanism of ExoU is poorly characterized, but it was recently found to bind to the phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a marker for the plasma membrane of eukaryotic cells. We confirmed that the membrane localization domain (MLD) of ExoU had a direct affinity for PI(4,5)P2, and we determined that this binding was required for ExoU localization. Previously uncharacterized ExoU homologs from Pseudomonas fluorescens and Photorhabdus asymbiotica also localized to the plasma membrane and required PI(4,5)P2 for this localization. A conserved arginine within the MLD was critical for interaction of each protein with PI(4,5)P2 and for localization. Furthermore, we determined the crystal structure of the full-length P. fluorescens ExoU and found that it was similar to that of P. aeruginosa ExoU. Each MLD contains a four-helical bundle, with the conserved arginine exposed at its cap to allow for interaction with the negatively charged PI(4,5)P2. Overall, these findings provide a structural explanation for the targeting of patatin-like phospholipases to the plasma membrane and define the MLD of ExoU as a member of a new class of PI(4,5)P2 binding domains., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objectives: The purpose of this study was to refine a commercial car driving simulation for occupational research. As the effects of ethanol on driving behavior are well established, we choose alcohol as a test compound to investigate the performance of subjects during simulation., Materials and Methods: We programmed a night driving scenario consisting of monotonous highway and a rural road on a Foerst F10-P driving simulator. Twenty healthy men, 19-30 years, participated in a pilot study. Subjects were screened for simulator sickness, followed by training on the simulator one hour in total. Experiments were performed in the morning on a separate day. Participants were randomized into either an alcoholized or a control group. All subjects drove two courses consisting of highway and rural road and were sober for the first course. During a 1 h break the ethanol group drank an alcoholic beverage to yield 0.06% blood alcohol concentration (BAC). Generalized linear mixed models were used to analyze the influence of alcohol on driving performance. Among others, independent variables were Simulator Sickness Questionnaire scores and subjective sleepiness., Results: Subjects did not experience simulator sickness during the experiments. Mean BAC before the second test drive was 0.065% in the mildly intoxicated group. There was no clear-cut difference in the number of crashes between 2 groups. BAC of 0.1% would deteriorate mean braking reaction time by 237 ms (SE = 112, p < 0.05). Ethanol slightly impaired the tracking in the right-hand curves (p = 0.058). Braking reaction time improved by 86 ms (SE = 36, p < 0.05) for the second test drive, indicating a learning effect., Conclusions: In sum, a clear ethanol effect was observed in the driving simulation. This simulation seems suitable for occupational research and produces little simulator sickness. Controlling for possible learning effects is recommended in driving simulation studies.

Tularemia is a deadly, febrile disease caused by infection by the gram-negative bacterium, Francisella tularensis. Members of the ubiquitous serine hydrolase protein family are among current targets to treat diverse bacterial infections. Herein we present a structural and functional study of a novel bacterial carboxylesterase (FTT258) from F. tularensis, a homologue of human acyl protein thioesterase (hAPT1). The structure of FTT258 has been determined in multiple forms, and unexpectedly large conformational changes of a peripheral flexible loop occur in the presence of a mechanistic cyclobutanone ligand. The concomitant changes in this hydrophobic loop and the newly exposed hydrophobic substrate binding pocket suggest that the observed structural changes are essential to the biological function and catalytic activity of FTT258. Using diverse substrate libraries, site-directed mutagenesis, and liposome binding assays, we determined the importance of these structural changes to the catalytic activity and membrane binding activity of FTT258. Residues within the newly exposed hydrophobic binding pocket and within the peripheral flexible loop proved essential to the hydrolytic activity of FTT258, indicating that structural rearrangement is required for catalytic activity. Both FTT258 and hAPT1 also showed significant association with liposomes designed to mimic bacterial or human membranes, respectively, even though similar structural rearrangements for hAPT1 have not been reported. The necessity for acyl protein thioesterases to have maximal catalytic activity near the membrane surface suggests that these conformational changes in the protein may dually regulate catalytic activity and membrane association in bacterial and human homologues.

Vibrio cholerae is the causative agent of the severe diarrheal disease cholera. For V. cholerae to colonize the intestinal epithelium, accessory toxins such as the multifunctional autoprocessing repeats-in-toxin (MARTX(Vc)) toxin are required. MARTX toxins are composite toxins comprised of arrayed effector domains that carry out distinct functions inside the host cell. Among the three effector domains of MARTX(Vc) is the Rho inactivation domain (RID(Vc)) known to cause cell rounding through inactivation of small RhoGTPases. Using alanine scanning mutagenesis in the activity subdomain of RID(Vc), four residues, His-2782, Leu-2851, Asp-2854, and Cys-3022, were identified as impacting RID(Vc) function in depolymerization of the actin cytoskeleton and inactivation of RhoA. Tyr-2807 and Tyr-3015 were identified as important potentially for forming the active structure for substrate contact but are not involved in catalysis or post translational modifications. Finally, V. cholerae strains modified to carry a catalytically inactive RID(Vc) show that the rate and efficiency of MARTX(Vc) actin cross-linking activity does not depend on a functional RID(Vc), demonstrating that these domains function independently in actin depolymerization. Overall, our results indicate a His-Asp-Cys catalytic triad is essential for function of the RID effector domain family shared by MARTX toxins produced by many Gram-negative bacteria.

Pathogenic bacteria utilize multiple approaches to establish infection and mediate their toxicity to eukaryotic cells. Dedicated protein machines deposit toxic effectors directly inside the host, whereas secreted toxins must enter cells independently of other bacterial components. Regardless of how they reach the cytosol, these bacterial proteins must accurately identify their intracellular target before they can manipulate the host cell to benefit their associated bacteria. Within eukaryotic cells, post-translational modifications and individual targeting motifs spatially regulate endogenous host proteins. This review focuses on the strategies employed by bacterial effectors to associate with a frequently targeted location within eukaryotic cells, the plasma membrane.

Plasma membrane targeting is essential for the proper function of many bacterial toxins. A conserved fourhelical bundle membrane localization domain (4HBM) was recently identified within three diverse families of toxins: clostridial glucosylating toxins, MARTX toxins and Pasteurella multocida-like toxins. When expressed in tissue culture cells or in yeast, GFP fusions to at least one 4HBM from each toxin family show significant peripheral membrane localization but with differing profiles. Both in vivo expression and in vitro binding studies reveal that the ability of these domains to localize to the plasma membrane and bind negatively charged phospholipids requires a basic-hydrophobic motif formed by the L1 and L3 loops. The different binding capacity of each 4HBM is defined by the hydrophobicity of an exposed residue within the motif. This study establishes that bacterial effectors utilize a normal host cell mechanism to locate the plasma membrane where they can then access their intracellular targets., (© 2011 Blackwell Publishing Ltd.)

Vibrio cholerae is the causative agent of the diarrheal disease cholera. Many virulence factors contribute to intestinal colonization and disease including the Multifunctional Autoprocessing RTX toxin (MARTX(Vc)). The Rho-inactivation domain (RID) of MARTX(Vc) is responsible for inactivating the Rho-family of small GTPases, which leads to depolymerization of the actin cytoskeleton. Based on a deletion analysis of RID to determine the minimal functional domain, we have identified a subdomain at the N terminus of RID that is homologous to the membrane targeting C1 domain of Pasteurella multocida toxin. A GFP fusion to this subdomain from RID colocalized with a plasma membrane marker when transiently expressed within HeLa cells and can be found in the membrane fraction following subcellular fractionation. This C1-like subdomain is present in multiple families of bacterial toxins, including all of the clostridial glucosyltransferase toxins and various MARTX toxins. GFP-fusions to these homologous domains are also membrane associated, indicating that this is a conserved membrane localization domain (MLD). We have identified three residues (Y23, S68, R70) as necessary for proper localization of one but not all MLDs. In addition, we found that substitution of the RID MLD with the MLDs from two different effector domains from the Vibrio vulnificus MARTX toxin restored RID activity, indicating that there is functional overlap between these MLDs. This study describes the initial recognition of a family of conserved plasma membrane-targeting domains found in multiple large bacterial toxins.

Actin cross-linking domains (ACDs) are distinct domains found in several bacterial toxins, including the Vibrio cholerae MARTX toxin. The ACD of V. cholerae (ACD(Vc)) catalyses the formation of an irreversible iso-peptide bond between lysine 50 and glutamic acid 270 on two actin molecules in an ATP- and Mg/Mn(2+)-dependent manner. In vivo, cross-linking depletes the cellular pool of G-actin leading to actin cytoskeleton depolymerization. While the actin cross-linking reaction performed by these effector domains has been significantly characterized, the ACD(Vc) catalytic site has remained elusive due to lack of significant homology to known proteins. Using multiple genetic approaches, we have identified regions and amino acids of ACD(Vc) required for full actin cross-linking activity. Then, using these functional data and structural homology predictions, it was determined that several residues demonstrated to be important for ACD(Vc) activity are conserved with active-site residues of the glutamine synthetase family of enzymes. Thus, the ACDs are a family of bacterial toxin effectors that may be evolutionarily related to ligases involved in amino acid biosynthesis.

Background: We have recently released a comprehensive, manually curated database of mammalian protein complexes called CORUM. Combining CORUM with other resources, we assembled a dataset of over 2700 mammalian complexes. The availability of a rich information resource allows us to search for organizational properties concerning these complexes., Results: As the complexity of a protein complex in terms of the number of unique subunits increases, we observed that the number of such complexes and the mean non-synonymous to synonymous substitution ratio of associated genes tend to decrease. Similarly, as the number of different complexes a given protein participates in increases, the number of such proteins and the substitution ratio of the associated gene also tends to decrease. These observations provide evidence relating natural selection and the organization of mammalian complexes. We also observed greater homogeneity in terms of predicted protein isoelectric points, secondary structure and substitution ratio in annotated versus randomly generated complexes. A large proportion of the protein content and interactions in the complexes could be predicted from known binary protein-protein and domain-domain interactions. In particular, we found that large proteins interact preferentially with much smaller proteins., Conclusion: We observed similar trends in yeast and other data. Our results support the existence of conserved relations associated with the mammalian protein complexes.

Formation of the FtsZ ring (Z ring) in Escherichia coli is the first step in the assembly of the divisome, a protein machine required for cell division. Although the biochemical functions of most divisome proteins are unknown, several, including ZipA, FtsA and FtsK, have overlapping roles in ensuring that the Z ring assembles at the cytoplasmic membrane, and that it is active. As shown previously, a single amino acid change in FtsA, R286W, also called FtsA*, bypasses the requirement for either ZipA or FtsK in cell division. In this study, the properties of FtsA* were investigated further, with the eventual goal of understanding the molecular mechanism behind the bypass. Compared to wild-type FtsA, the presence of FtsA* resulted in a modest but significant decrease in the mean length of cells in the population, accelerated the reassembly of Z rings, and suppressed the cell-division block caused by excessively high levels of FtsZ. These effects were not mediated by Z-ring remodelling, because FtsA* did not alter the kinetics of FtsZ turnover within the Z ring, as measured by fluorescence recovery after photobleaching. FtsA* was also unable to permit normal cell division at below normal levels of FtsZ, or after thermoinactivation of ftsZ84(ts). However, turnover of FtsA* in the ring was somewhat faster than that of wild-type FtsA, and overexpressed FtsA* did not inhibit cell division as efficiently as wild-type FtsA. Finally, FtsA* interacted more strongly with FtsZ compared with FtsA in a yeast two-hybrid system. These results suggest that FtsA* interacts with FtsZ in a markedly different way compared with FtsA.

In Escherichia coli, at least 12 proteins colocalize to the cell midpoint, assembling into a membrane-associated protein machine that forms the division septum. Many of these proteins, including FtsK, are essential for viability but their functions in cell division are unknown. Here we show that the essential function of FtsK in cell division can be partially bypassed. Cells containing either the ftsA R286W mutation or a plasmid carrying the ftsQAZ genes suppressed a ftsK44(ts) allele efficiently. Moreover, ftsA R286W or multicopy ftsQAZ, which can largely bypass the requirement for the essential cell division gene zipA, allowed cells with a complete deletion of ftsK to survive and divide, although many of these ftsK null cells formed multiseptate chains. Green fluorescent protein (GFP) fusions to FtsI and FtsN, which normally depend on FtsK to localize to division sites, localized to division sites in the absence of FtsK, indicating that FtsK is not directly involved in their recruitment. Cells expressing additional ftsQ, and to a lesser extent ftsB and ftsN, were able to survive and divide in the absence of ftsK, although cell chains were often formed. Surprisingly, the cytoplasmic and transmembrane domains of FtsQ, while not sufficient to complement an ftsQ null mutant, conferred viability and septum formation in the absence of ftsK. These findings suggest that the N-terminal domain of FtsK is normally involved in stability of the division protein machine and shares functional overlap with FtsQ, FtsB, FtsA, ZipA and FtsN.

Background: Adrenomedullin (ADM) is a vasodilator peptide that is abundantly expressed in the kidney. ADM has antiproliferative effects on glomerular mesangial cells (MC) in vitro. Whether or not treatment with ADM can reduce MC proliferation in vivo [i.e., in mesangioproliferative glomerulonephritis (GN)] is unknown. We tested the hypothesis that ADM substitution reduces MC proliferation in GN., Methods: GN in rats was induced by injection of an anti-Thy-1.1 antibody. Rats received osmotic minipumps, which continuously delivered rat ADM (500 ng/hour, N = 11), or vehicle (N = 13) from day 3 to day 6 after GN induction. Rats were sacrificed 6 days after induction of GN. On kidney sections, cells staining positive for proliferating cell nuclear antigen, mesangial cells, monocytes, and apoptotic cells were counted. Parameters of inflammation and fibrosis were measured in renal cortex and sieved glomeruli by real-time polymerase chain reaction (PCR)., Results: Systolic blood pressure, diuresis, albuminuria, creatinine clearance, microaneurysm formation, and mesangial matrix expansion were not influenced by ADM infusion. However, ADM treatment significantly reduced the number of MC, showed a tendency to reduce total glomerular cell proliferation, and significantly increased apoptosis. ADM-treated GN animals showed significantly less glomerular monocyte infiltration. ADM treatment normalized transforming growth factor (TGF)-beta1 mRNA expression and reduced monocyte chemoattractant protein-1 (MCP-1), osteopontin, plasminogen activator inhibitor-1 (PAI-1), collagen I, and collagen III mRNA expression significantly., Conclusion: Exogenous ADM infusion reduces MC number and glomerular monocyte infiltration in the state of mesangial proliferation during acute experimental mesangioproliferative GN. These findings indicate that ADM can influence the course of mesangioproliferative GN.

FtsA, a member of the ATPase superfamily that includes actin and bacterial actin homologs, is essential for cell division of Escherichia coli and is recruited to the Z ring. In turn, recruitment of later essential division proteins to the Z ring is dependent on FtsA. In a polar recruitment assay, we found that FtsA can recruit at least two late proteins, FtsI and FtsN, to the cell poles independently of Z rings. Moreover, a unique structural domain of FtsA, subdomain 1c, which is divergent in the other ATPase superfamily members, is sufficient for this recruitment but not required for the ability of FtsA to localize to Z rings. Surprisingly, targeting the 1c subdomain to the Z ring by fusing it to FtsZ could partially suppress a thermosensitive ftsA mutation. These results suggest that subdomain 1c of FtsA is a completely independent functional domain with an important role in interacting with a septation protein subassembly.

Unlabelled: This study validates a new quantitative myocardial perfusion SPECT software., Methods: The processing starts with the extraction of the morphologic skeleton of the left ventricular myocardium from reconstructed transverse sections. Fuzzy logic is used to decide whether a pixel belongs to the myocardium and any perfusion defect is filled according to a truncated bullet model. The resulting image is partitioned in 18 isovolumetric sectors. Sex-matched normal limits, criteria of abnormality for rest (201)Tl and (99m)Tc-labeled perfusion tracers, reproducibility studies, and detection of coronary artery disease were developed and validated in an overall population of 343 patients. The sex- and tracer-matched means and SDs of a normal response were calculated in 93 male and 93 female patients with a <5% likelihood of coronary artery disease. Reproducibility measurements and assignment of different sectors of the myocardium to a specific coronary were performed from data collected in 49 and 60 patients, respectively. The accuracy of the detection of a coronary artery occlusion was assessed in 48 patients who also underwent coronary angiography., Results: The intra- and interoperator reproducibility of the sectorial activity was high with a linear regression coefficient of 0.97 and a SD of the difference measurement at 4.4% and 3.8%, respectively. Overall sensitivity and specificity for the detection of occluded coronary artery were 90% and 80%, respectively. For the detection of left anterior descending, left circumflex, and right artery coronary occlusion, sensitivity was 92%, 75%, and 92.5%, respectively, and specificity was 75%, 78%, and 90%, respectively., Conclusion: The new quantitative myocardial perfusion SPECT software appears to be a very helpful program for the objective analysis of perfusion tracer distribution in myocardial SPECT and a very accurate tool in the detection and localization of coronary artery occlusion.

ZipA and FtsA are recruited independently to the FtsZ cytokinetic ring (Z ring) and are essential for cell division of Escherichia coli. The molecular role of FtsA in cell division is unknown; however, ZipA is thought to stabilize the Z ring, anchor it to the membrane, and recruit downstream cell division proteins. Here we demonstrate that the requirement for ZipA can be bypassed completely by a single alteration in a conserved residue of FtsA (FtsA*). Cells with ftsA* in single copy in place of WT ftsA or with ftsA* alone on a multicopy plasmid divide mostly normally, whether they are zipA+ or zipA-. Experiments with ftsQAZ and ftsQA*Z on multicopy plasmids indicate that ftsQAZzipA+ and ftsQA*ZzipA- cells divide fairly normally, whereas ftsQAZzipA- cells divide poorly and ftsQA*ZzipA+ cells display a phenotype that suggests their septa are unusually stable. In support of the idea that ftsA* stabilizes Z rings, single-copy ftsA* confers resistance to excess MinC, which destabilizes Z rings. The inhibitory effect of excess ZipA on division is also suppressed by ftsA*. These results suggest that the molecular mechanism of the FtsA* bypass is to stabilize FtsZ assembly via a parallel pathway and that FtsA* can replace the multiple functions of ZipA. This is an example of a complete functional replacement of an essential prokaryotic cell division protein by another and may explain why most bacteria can divide without an obvious ZipA homolog.

Dialysis neutropenia is the result of pulmonary sequestration of neutrophils after complement activation by the dialyzer membrane. Increased expression of neutrophil adhesion receptors, such as CD11b/CD18, suggests that neutrophil adhesion to the capillary endothelium is a possible mechanism. An alternative hypothesis is that the complement fragment C5a modulates neutrophil mechanical properties via the cytoskeleton-largely filamentous actin (F-actin)-stiffening them and thereby slowing their passage through the pulmonary capillaries. To investigate this hypothesis, we developed an assay to measure the F-actin content of neutrophils in whole blood using flow cytometry and the stain NBD-phallacidin. We measured neutrophil F-actin content during hemodialysis of patients with polysulfone (N = 6), Hemophan (N = 6), and Cuprophan membranes sterilized with either ethylene oxide (N = 5) or steam (N = 6). Cell counts, neutrophil and monocyte CD11b expression and plasma C5a concentrations were also measured. The results confirm the strong relationship between the degree of neutropenia, increases in CD11b expression and plasma C5a levels reported by previous researchers. Modulation of the F-actin content of neutrophils was also strongly related to C5a levels, indicating that the neutrophil cytoskeleton is active during dialysis. Modeling of cell counts suggests that with Cuprophan a substantial fraction of neutrophils and monocytes are sequestered before they even pass through the dialyzer, suggesting some form of systemic activation of these cells. Evidence for systemic activation was also seen in measurements of F-actin content, but not CD11b expression, a finding that strengthens the case for the involvement of the cytoskeleton in dialysis neutropenia.