Computer based visualization of clot structures in extracorporeal membrane oxygenation and histological clot investigations for understanding thrombosis in membrane lungs.

Extracorporeal membrane oxygenation (ECMO) was established as a treatment for severe cardiac or respiratory disease. Intra-device clot formation is a common risk. This is based on complex coagulation phenomena which are not yet sufficiently understood. The objective was the development and validation of a methodology to capture the key properties of clots deposed in membrane lungs (MLs), such as clot size, distribution, burden, and composition. One end-of-therapy PLS ML was examined. Clot detection was performed using multidetector computed tomography (MDCT), microcomputed tomography (µCT), and photography of fiber mats (fiber mat imaging, FMI). Histological staining was conducted for von Willebrand factor (vWF), platelets (CD42b, CD62P), fibrin, and nucleated cells (4', 6-diamidino-2-phenylindole, DAPI). The three imaging methods showed similar clot distribution inside the ML. Independent of the imaging method, clot loading was detected predominantly in the inlet chamber of the ML. The µCT had the highest accuracy. However, it was more expensive and time consuming than MDCT or FMI. The MDCT detected the clots with low scanning time. Due to its lower resolution, it only showed clotted areas but not the exact shape of clot structures. FMI represented the simplest variant, requiring little effort and resources. FMI allowed clot localization and calculation of clot volume. Histological evaluation indicated omnipresent immunological deposits throughout the ML. Visually clot-free areas were covered with leukocytes and platelets forming platelet-leukocyte aggregates (PLAs). Cells were embedded in vWF cobwebs, while vWF fibers were negligible. In conclusion, the presented methodology allowed adequate clot identification and histological classification of possible thrombosis markers such as PLAs.

Paliperidone poisoning and measurable plasma concentrations 2.5 years after last administered dose: A case report.

We report a poisoning with paliperidone palmitate, a once-monthly, long-acting injectable antipsychotic. The patient suffered from deep sedation and dystonia. She had been treated with extended release intramuscular paliperidone for several years and had received her last injection 8 days prior to admission. The plasma paliperidone was nearly five times higher than the upper reference range. Paliperidone is a substrate of p-glycoprotein and we therefore aimed to increase its elimination by inducing p-glycoprotein through treatment with St John's wort. This seemed to have a limited effect on paliperidone clearance. Plasma concentration levels decreased with time as did the dystonia. All antipsychotic treatment was discontinued after this unfortunate event, and the patient did specifically not receive any prescriptions of paliperidone or risperidone. However, the plasma paliperidone concentration was in the low end of the normal therapeutic range 2.5 years after the last dose of paliperidone was administered, and the patient still had some extrapyramidal symptoms.

Antiproliferative and Apoptosis Effects of Hybrid Varieties of Vitis vinifera L. Sweet Sapphire and Sweet Surprise on Human Prostate Cancer Cells Using In Vitro and In Silico Approaches.

OBJECTIVE: Grape hybrids are characterized by different chemical compositions; often with high hybrids are characterized by different chemical compositions, often with a high phenolic content and a specific profile of anthocyanins. The aim of study was to characterize the constituents of hybrid Vitis vinifera L. varieties Sweet Sapphire (SA) and Sweet Surprise (SU) extracts and their influence on apoptosis induction and antiproliferative effects on human prostate cancer cells. METHODS: We used the MTT assay to evaluate the cytotoxic effect of extracts of SA and SU, on the prostate adenocarcinoma cell lines PC-3 and DU-145. To analyze the inhibiting impact by flow cytometry, used 24 and 48 hours. Anthocyanins were quantified by liquid chromatography and analysed by their absorption rate, hepatotoxicity, blood concentration, blood-brain barrier passage ability and maximum recommended dose by in silico approaches. RESULTS: Our results showed that malvidin derivatives present the highest content in both cultivars. We identified 14.46mg/100g malvidin-3-O-glycoside in SA and 2.76 mg/100 g in SU. A reduction in cell viability of DU-145 (45 and 65%) and PC-3 (63 and 67%) cells after 48h treatment with SA and SU, respectively, was found via MTT assay. Flow cytometry showed that the treatment with extracts from SA and SU had an inhibitory impact on cell development due to G2/M arrest and caused a rise in apoptotic cells compared to control group. None of the anthocyanin presented hepatotoxicity as well as blood-brain barrier passage ability. Peonidin 3-O-glucoside had the lower maximum recommended dose as well as the highest intestinal absorption rate. However, delphinidin 3-O-glucoside had the highest blood concentration values. CONCLUSION: The findings of this study highlight the potential of hybrid Vitis vinifera L. varieties as an important source of natural antioxidants and their protective effect against prostate cancer cells as well as elucidate in part their anthocyanin's metabolism.

Molecular mechanisms associated with chemoresistance in esophageal cancer.

Esophageal cancer (EC) is one of the most incident and lethal tumors worldwide. Although surgical resection is an important approach in EC treatment, late diagnosis, metastasis and recurrence after surgery have led to the management of adjuvant and neoadjuvant therapies over the past few decades. In this scenario, 5-fluorouracil (5-FU) and cisplatin (CISP), and more recently paclitaxel (PTX) and carboplatin (CBP), have been traditionally used in EC treatment. However, chemoresistance to these agents along EC therapeutic management represents the main obstacle to successfully treat this malignancy. In this sense, despite the fact that most of chemotherapy drugs were discovered several decades ago, in many cases, including EC, they still represent the most affordable and widely employed treatment approach for these tumors. Therefore, this review summarizes the main mechanisms through which the response to the most widely chemotherapeutic agents used in EC treatment is impaired, such as drug metabolism, apoptosis resistance, cancer stem cells (CSCs), cell cycle, autophagy, energetic metabolism deregulation, tumor microenvironment and epigenetic modifications.

Teeth angulations after class II treatment with the Jones Jig followed by fixed appliances: A retrospective panoramic analysis.

OBJECTIVE: The purpose of this study was to compare the maxillary mesiodistal dental angulations of Class II malocclusion patients treated with the Jones Jig, followed by fixed appliances, with normal values of a historical control group, on panoramic radiographs. MATERIAL AND METHODS: The sample comprised 80 panoramic radiographs of 40 patients divided into two groups. Eligibility criteria included patients with predominantly dental Class II malocclusion; the presence of all teeth up to the second molars and no previous orthodontic treatment. The experimental group was composed of 60 radiographs of 20 patients treated with the Jones Jig distalizer followed by fixed appliances. The radiographs were taken at pre-treatment (T0), post-distalization (T1), and post-treatment (T2). The historical control group comprised 20 radiographs of 20 subjects with untreated normal occlusion. The mesiodistal axial angulations of all maxillary erupted teeth were evaluated with the Dolphin Imaging software. Intragroup comparisons in the experimental group were performed with repeated measures analysis of variance (ANOVA), followed by Tukey tests. The experimental group at T2 versus the control group were compared with t tests. RESULTS: After distalization, significant distal angulation of the molars (110.58°±8.54, P<0.000) and mesial angulation of the second (86.43°±8.08, P<0.000) and first premolars (80.11°±8.01, P<0.000) was observed. However, this was corrected after comprehensive fixed orthodontics (100.54°±6.53; 98.95°±7.00; 94.92°±6.44; P<0.000, for these teeth, respectively). Intergroup comparisons resulted in first molars, premolars, canines, and central incisors significantly more distally angulated in the experimental group, when compared to the control. CONCLUSIONS: In general, at the end of orthodontic treatment, patients treated with the Jones Jig distalizer followed by fixed appliances presented more distally angulated maxillary teeth when compared to an untreated group with normal occlusion.

Role of Music in a Plastic Surgery Setting: A Systematic Review and Meta-analysis.

Objective To assess the effectiveness and harm of music to reduce anxiety and pain in a plastic surgery setting. Materials and Methods A search strategy was conducted in the MEDLINE, CENTRAL, EMBASE, and LILACS databases. Searches were also conducted in other databases and unpublished literature. Clinical trials were included without language restrictions. The risk of bias was evaluated with the Cochrane Collaboration's tool. An analysis of random effects was conducted. The primary outcomes were anxiety and pain. The secondary outcomes were length of stay, physiological parameters, and adverse effects. The measure of the effect was the mean difference (MD) and standardized MD (SMD) with a 95% confidence interval (CI). The planned interventions were music versus no music. Results Four articles were included in the qualitative and quantitative analysis. A total of 306 patients were found among the four studies. A low risk of bias was shown for most of the study items. The overall standardized mean difference (SMD) for anxiety -3.64 [95%CI -5.71 to -1.56 (p-value = 0.0006)] favoring music compared with no intervention, and for pain the mean difference (MD) was -12.06 [95%CI -33.47 to 9.35 (p-value = 0.2696)] showing no statistical differences. Conclusion Playing music is a safe and free intervention that diminishes anxiety in patients who undergo plastic surgery procedures.

Music to reduce pain and anxiety in cystoscopy: a systematic review and meta-analysis.

Background To assess the effectiveness and harms of music to reduce anxiety and pain in cystoscopy. Methods We searched MEDLINE (OVID), EMBASE, LILACS and the Cochrane Central Register of Controlled Trials (CENTRAL) from inception to nowadays. We included clinical trials, involving the assessment of the effect of music in cystoscopy. The primary outcomes were pain and anxiety measured by any scale and the secondary outcomes were length of stay, physiological parameters (blood pressure or heart rate) and adverse effects. Cochrane Collaboration tool was used to assess the risk of bias. We performed the statistical analysis in R and reported information about mean difference (MD) with 95% CI. Heterogeneity was evaluated using the I2 test. Results We included six studies in our qualitative and quantitative analysis. Five studies used a flexible cystoscope and the other one performed the procedure with a rigid cystoscope. Music was played during the procedure in five studies, while the other was before it. All studies compared music vs. no intervention. Almost all items were assessed as low risk of bias; however, the allocation concealment was unclear in all the studies. We found a MD of -1.33 (95% CI -2.45 to -0.21) (I2=97.2%) favoring music for pain and a MD of -8.42 (95% CI -15.02, -1.82) (I2=99.6%) was found, favoring music for anxiety. Conclusions Playing music might be an effective intervention that lowers pain and anxiety in patients who undergo cystoscopy.

Social behavior and decision making in bacterial conjugation.

Bacteria frequently acquire novel genes by horizontal gene transfer (HGT). HGT through the process of bacterial conjugation is highly efficient and depends on the presence of conjugative plasmids (CPs) or integrated conjugative elements (ICEs) that provide the necessary genes for DNA transmission. This review focuses on recent advancements in our understanding of ssDNA transfer systems and regulatory networks ensuring timely and spatially controlled DNA transfer (tra) gene expression. As will become obvious by comparing different systems, by default, tra genes are shut off in cells in which conjugative elements are present. Only when conditions are optimal, donor cells-through epigenetic alleviation of negatively acting roadblocks and direct stimulation of DNA transfer genes-become transfer competent. These transfer competent cells have developmentally transformed into specialized cells capable of secreting ssDNA via a T4S (type IV secretion) complex directly into recipient cells. Intriguingly, even under optimal conditions, only a fraction of the population undergoes this transition, a finding that indicates specialization and cooperative, social behavior. Thereby, at the population level, the metabolic burden and other negative consequences of tra gene expression are greatly reduced without compromising the ability to horizontally transfer genes to novel bacterial hosts. This undoubtedly intelligent strategy may explain why conjugative elements-CPs and ICEs-have been successfully kept in and evolved with bacteria to constitute a major driving force of bacterial evolution.

Icm/Dot-dependent inhibition of phagocyte migration by Legionella is antagonized by a translocated Ran GTPase activator.

The environmental bacterium Legionella pneumophila causes a severe pneumonia termed Legionnaires' disease. L. pneumophila employs a conserved mechanism to replicate within a specific vacuole in macrophages or protozoa such as the social soil amoeba Dictyostelium discoideum. Pathogen-host interactions depend on the Icm/Dot type IV secretion system (T4SS), which translocates approximately 300 different effector proteins into host cells. Here we analyse the effects of L. pneumophila on migration and chemotaxis of amoebae, macrophages or polymorphonuclear neutrophils (PMN). Using under-agarose assays, L. pneumophila inhibited in a dose- and T4SS-dependent manner the migration of D. discoideum towards folate as well as starvation-induced aggregation of the social amoebae. Similarly, L. pneumophila impaired migration of murine RAW 264.7 macrophages towards the cytokines CCL5 and TNFα, or of primary human PMN towards the peptide fMLP respectively. L. pneumophila lacking the T4SS-translocated activator of the small eukaryotic GTPase Ran, Lpg1976/LegG1, hyper-inhibited the migration of D. discoideum, macrophages or PMN. The phenotype was reverted by plasmid-encoded LegG1 to an extent observed for mutant bacteria lacking a functional Icm/Dot T4SS.Similarly, LegG1 promoted random migration of L. pneumophila-infected macrophages and A549 epithelial cells in a Ran-dependent manner, or upon 'microbial microinjection' into HeLa cells by a Yersinia strain lacking endogenous effectors. Single-cell tracking and real-time analysis of L. pneumophila-infected phagocytes revealed that the velocity and directionality of the cells were decreased, and cell motility as well as microtubule dynamics was impaired. Taken together, these findings indicate that the L. pneumophila Ran activator LegG1 and consequent microtubule polymerization are implicated in Icm/Dot-dependent inhibition of phagocyte migration.

Live-cell imaging of phosphoinositide dynamics and membrane architecture during Legionella infection.

UNLABELLED: The causative agent of Legionnaires' disease, Legionella pneumophila, replicates in amoebae and macrophages in a distinct membrane-bound compartment, the Legionella-containing vacuole (LCV). LCV formation is governed by the bacterial Icm/Dot type IV secretion system that translocates ~300 different "effector" proteins into host cells. Some of the translocated effectors anchor to the LCV membrane via phosphoinositide (PI) lipids. Here, we use the soil amoeba Dictyostelium discoideum, producing fluorescent PI probes, to analyze the LCV PI dynamics by live-cell imaging. Upon uptake of wild-type or Icm/Dot-deficient L. pneumophila, PtdIns(3,4,5)P3 transiently accumulated for an average of 40 s on early phagosomes, which acquired PtdIns(3)P within 1 min after uptake. Whereas phagosomes containing ΔicmT mutant bacteria remained decorated with PtdIns(3)P, more than 80% of wild-type LCVs gradually lost this PI within 2 h. The process was accompanied by a major rearrangement of PtdIns(3)P-positive membranes condensing to the cell center. PtdIns(4)P transiently localized to early phagosomes harboring wild-type or ΔicmT L. pneumophila and was cleared within minutes after uptake. During the following 2 h, PtdIns(4)P steadily accumulated only on wild-type LCVs, which maintained a discrete PtdIns(4)P identity spatially separated from calnexin-positive endoplasmic reticulum (ER) for at least 8 h. The separation of PtdIns(4)P-positive and ER membranes was even more pronounced for LCVs harboring ΔsidC-sdcA mutant bacteria defective for ER recruitment, without affecting initial bacterial replication in the pathogen vacuole. These findings elucidate the temporal and spatial dynamics of PI lipids implicated in LCV formation and provide insight into host cell membrane and effector protein interactions. IMPORTANCE: The environmental bacterium Legionella pneumophila is the causative agent of Legionnaires' pneumonia. The bacteria form in free-living amoebae and mammalian immune cells a replication-permissive compartment, the Legionella-containing vacuole (LCV). To subvert host cell processes, the bacteria secrete the amazing number of ~300 different proteins into host cells. Some of these proteins bind phosphoinositide (PI) lipids to decorate the LCV. PI lipids are crucial factors involved in host cell membrane dynamics and LCV formation. Using Dictyostelium amoebae producing one or two distinct fluorescent probes, we elucidated the dynamic LCV PI pattern in high temporal and spatial resolution. Notably, the endocytic PI lipid PtdIns(3)P was slowly cleared from LCVs, thus incapacitating the host cell's digestive machinery, while PtdIns(4)P gradually accumulated on the LCV, enabling critical interactions with host organelles. The LCV PI pattern underlies the spatiotemporal configuration of bacterial effector proteins and therefore represents a crucial aspect of LCV formation.

Silencing and activating type IV secretion genes of the F-like conjugative resistance plasmid R1.

Expression of DNA transfer (tra) genes of F-type conjugative plasmids is required for the assembly of a functional type IV secretion machinery and subsequent plasmid DNA transfer from donor to recipient cells. Transcription of tra genes depends on the activation of a single promoter, designated PY, by the plasmid encoded TraJ protein. We here determine plasmid specificity of TraJ proteins from various subgroups of F-like plasmids and find that plasmid R1 conjugation and PY promoter activation can be achieved only by its cognate activator and by TraJ of the Salmonella plasmid pSLT and not by F or R100 TraJ proteins. In addition, we characterize the PY promoter of plasmid R1. We show that TraJ binds to PY DNA in vivo and that H-NS acts as a silencer of the PY promoter. In the natural plasmid context, H-NS silences transfer gene expression and horizontal plasmid DNA transfer. In contrast to what was found for the F plasmid, lack of H-NS did not abolish the requirement for ArcA and TraJ to reach full tra gene expression and DNA transfer activity. We propose that, besides a passive de-silencing activity, both ArcA and TraJ play a direct role in synergistically stimulating tra operon transcription and subsequent DNA transfer.

The conserved Bud20 zinc finger protein is a new component of the ribosomal 60S subunit export machinery.

The nuclear export of the preribosomal 60S (pre-60S) subunit is coordinated with late steps in ribosome assembly. Here, we show that Bud20, a conserved C(2)H(2)-type zinc finger protein, is an unrecognized shuttling factor required for the efficient export of pre-60S subunits. Bud20 associates with late pre-60S particles in the nucleoplasm and accompanies them into the cytoplasm, where it is released through the action of the Drg1 AAA-ATPase. Cytoplasmic Bud20 is then reimported via a Kap123-dependent pathway. The deletion of Bud20 induces a strong pre-60S export defect and causes synthetic lethality when combined with mutant alleles of known pre-60S subunit export factors. The function of Bud20 in ribosome export depends on a short conserved N-terminal sequence, as we observed that mutations or the deletion of this motif impaired 60S subunit export and generated the genetic link to other pre-60S export factors. We suggest that the shuttling Bud20 is recruited to the nascent 60S subunit via its central zinc finger rRNA binding domain to facilitate the subsequent nuclear export of the preribosome employing its N-terminal extension.

Optimization of homoexopolysaccharide formation by lactobacilli in gluten-free sourdoughs.

Currently hydrocolloids are applied as baking aids in order to enhance the quality of gluten-free bread. Microbial exopolysaccharides (EPS) can also have a positive impact on gluten-free baked goods. Provided that yields are sufficient, in situ produced EPS may enable clean (additive free) labels. Thus, in situ EPS optimization was conducted to achieve high amounts of EPS in gluten-free sourdoughs. The influence of flours, dough yield, cell counts, sucrose concentration and sucrose fed-batch were investigated for Lactobacillus (L.) animalis TMW 1.971, Lactobacillus reuteri TMW 1.106 and Lactobacillus curvatus TMW 1.624. Maximal yields of 17.93 g EPS kg(-1) flour were obtained with L. animalis TMW 1.971 in buckwheat core doughs, 15.69 g EPS kg(-1) with L. reuteri TMW 1.106 in quinoa, and 16.28 g EPS kg(-)(1) flour with L. curvatus TMW 1.624 in buckwheat sourdoughs. This study evidences that EPS amounts can be manifolded to reach effectual levels through optimization and corroborate the application of EPS-forming starter cultures as a promising approach to improve gluten-free baked goods.

Growth phase- and cell division-dependent activation and inactivation of the {sigma}32 regulon in Escherichia coli.

Alternative sigma factors allow bacteria to reprogram global transcription rapidly and to adapt to changes in the environment. Here we report on growth- and cell division-dependent sigma(32) regulon activity in Escherichia coli in batch culture. By analyzing sigma(32) expression in growing cells, an increase in sigma(32) protein levels is observed during the first round of cell division after exit from stationary phase. Increased sigma(32) protein levels result from transcriptional activation of the rpoH gene. After the first round of bulk cell division, rpoH transcript levels and sigma(32) protein levels decrease again. The late-logarithmic phase and the transition to stationary phase are accompanied by a second increase in sigma(32) levels and enhanced stability of sigma(32) protein but not by enhanced transcription of rpoH. Throughout growth, sigma(32) target genes show expression patterns consistent with oscillating sigma(32) protein levels. However, during the transition to early-stationary phase, despite high sigma(32) protein levels, the transcription of sigma(32) target genes is downregulated, suggesting functional inactivation of sigma(32). It is deduced from these data that there may be a link between sigma(32) regulon activity and cell division events. Further support for this hypothesis is provided by the observation that in cells in which FtsZ is depleted, sigma(32) regulon activation is suppressed.

Targeted drug delivery: binding and uptake of plant lectins using human 5637 bladder cancer cells.

In an effort to detect novel strategies in bladder cancer therapy, the potential and the applicability of different plant lectins was investigated using 5637 cells as a model for human urinary carcinoma. The cell-lectin interaction studies were performed with single cells as well as monolayers using flow cytometry and fluorimetry. As a result, wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA) revealed strongest interaction with single cells demonstrating a high presence of N-acetyl-d-glucosamine, sialic acid and alpha-l-fucose residues on the membrane surface. Considering monolayers, binding of most lectins depended on the culturing period pointing to a change in the glycocalyx composition during cultivation. However, constant binding capacities combined with a high specificity were detected for WGA. Cytoinvasion studies were performed with WGA and revealed a decreased fluorescence intensity at 37 degrees C as compared to 4 degrees C, which points to internalisation of the lectin and accumulation in acidic compartments. Intracellular localization was confirmed by addition of monensin that compensates the pH-gradient between acidic compartments and cytoplasm leading to a full reversal of the decline in fluorescence. According to these findings, some lectins, especially WGA, offer promising features for targeting drugs to bladder cancer cells. This might be interesting for the development of functionalized drug delivery systems for site specific antitumor therapy leading to reduced toxicity, prolonged exposition, and improved efficacy.

Laryngoscopy in thyroid surgery--essential standard or unnecessary routine?

BACKGROUND: Preoperative and postoperative laryngoscopy has been recommended for diagnostic and forensic reasons as a standard procedure in all patients who undergo thyroid surgery. The aim of this study was to find a more selective approach by defining patients at risk of developing vocal fold palsy (VFP). METHODS: The history of neck explorations, results of laryngoscopy, and histology were registered in all patients who underwent thyroid surgery at our institution between 1995 and 1999. Patients with pathologic findings at postoperative laryngoscopy underwent reassessment of voice and vocal fold (VF) mobility 6 months later. RESULTS: Unilateral VFP was detected preoperatively in 13 of 695 patients (1.9%). Of the 13 patients, only 1 patient was asymptomatic, had no history of neck surgery, and had no suspected malignancy. Postoperative laryngoscopy revealed a new development of VFP in 68 of 695 patients (9.8%). All patients with a permanent VFP had symptoms immediately after operation. Asymptomatic VFPs always recovered. CONCLUSIONS: Preoperative laryngoscopy is justified in symptomatic patients who undergo reoperation or in patients when malignancy is suspected. The necessity of a preoperative laryngoscopy in all other patients must be questioned. Postoperative laryngoscopy and additional diagnostic testing should be reserved for symptomatic patients.

Expression and assembly of a functional type IV secretion system elicit extracytoplasmic and cytoplasmic stress responses in Escherichia coli.

Conditions perturbing protein homeostasis are known to induce cellular stress responses in prokaryotes and eukaryotes. Here we show for the first time that expression and assembly of a functional type IV secretion (T4S) machinery elicit extracytoplasmic and cytoplasmic stress responses in Escherichia coli. After induction of T4S genes by a nutritional upshift and assembly of functional DNA transporters encoded by plasmid R1-16, host cells activated the CpxAR envelope stress signaling system, as revealed by induction or repression of downstream targets of the CpxR response regulator. Furthermore, we observed elevated transcript levels of cytoplasmic stress genes, such as groESL, with a concomitant increase of sigma(32) protein levels in cells expressing T4S genes. A traA null mutant of plasmid R1-16, which lacks the functional gene encoding the major pilus protein pilin, showed distinctly reduced stress responses. These results corroborated our conclusion that the activation of bacterial stress networks was dependent on the presence of functional T4S machinery. Additionally, we detected increased transcription from the rpoHp(1) promoter in the presence of an active T4S system. Stimulation of rpoHp(1) was dependent on the presence of CpxR, suggesting a hitherto undocumented link between CpxAR and sigma(32)-regulated stress networks.

Peptidoglycan degradation by specialized lytic transglycosylases associated with type III and type IV secretion systems.

Specialized lytic transglycosylases are muramidases capable of locally degrading the peptidoglycan meshwork of Gram-negative bacteria. Specialized lytic transglycosylase genes are present in clusters encoding diverse macromolecular transport systems. This paper reports the analysis of selected members of the specialized lytic transglycosylase family from type III and type IV secretion systems. These proteins were analysed in vivo by assaying their ability to complement the DNA transfer defect of the conjugative F-like plasmid R1-16 lacking a functional P19 protein, the specialized lytic transglycosylase of this type IV secretion system. Heterologous complementation was accomplished using IpgF from the plasmid-encoded type III secretion system of Shigella sonnei and TrbN from the type IV secretion system of the conjugative plasmid RP4. In contrast, neither VirB1 proteins (Agrobacterium tumefaciens, Brucella suis) nor IagB (Salmonella enterica) could functionally replace P19. In vitro, IpgF, IagB, both VirB1 proteins, HP0523 (Helicobacter pylori) and P19 displayed peptidoglycanase activity in zymogram analyses. Using an established test system and a newly developed assay it was shown that IpgF degraded peptidoglycan in solution. IpgF was active only after removal of the chaperonin GroEL, which co-purified with IpgF and inhibited its enzymic activity. A mutant IpgF protein in which the predicted catalytic amino acid, Glu42, was replaced by Gln, was completely inactive. IpgF-catalysed peptidoglycan degradation was optimal at pH 6 and was inhibited by the lytic transglycosylase inhibitors hexa-N-acetylchitohexaose and bulgecin A.

Clinical value of parathyroid scintigraphy with technetium-99m methoxyisobutylisonitrile: discrepancies in clinical data and a systematic metaanalysis of the literature.

There is a considerable discrepancy in the literature concerning the sensitivity of parathyroid scintigraphy (PS) with 99mTc-MIBI. We therefore analyzed our own data and compared them to the literature in a metaanalysis. All patients who received 99mTc -MIBI scintigraphy and subsequent surgery in our department for the detection of enlarged parathyroid glands in primary (pHPT) or secondary (sHPT) hyperparathyroidism between 1991 and 1999 were included in our retrospective analysis. The results of surgery served as the gold standard. For a true positive result, the scintigraphy had to predict the exact location of parathyroid adenoma (PA) or parathyroid hyperplasia (PH). We then compared these data to the results of a nonstatistical systematic metaanalysis of the literature. Patients (178) underwent PS between 1991 and 1999; 139 were operated on and included in this study. Of these, 109 had pHPT and 30 had sHPT. The sensitivity and specificity of the PS were found to be 45%/94% for pHPT and 39%/40% for sHPT. Fifty-two studies concerning PS were included in the metaanalysis. Sensitivities reported varied from 39% to >90%. Consideration of the different possible techniques used for PS could not explain these discrepancies. Our data show that the sensitivity of PS in clinical routine may be lower than expected from the literature. Our data are consistent with other studies and with partially unpublished clinical observations from other university hospitals. We believe that a well-designed and properly conducted prospective study is necessary to evaluate the reasons for the differences observed.