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Photobacterium damselae subsp. damselae ( Pdd ) is an emerging pathogen of marine animals that sometimes causes serious infections in humans. Two related pore forming toxins, phobalysins P and C, and damselysin, a phospholipase D, confer strong virulence of Pdd in mice. Because infections by Pdd are typically caused following exposure of wounds to sea water we investigated how salinity impacts toxin activity, swimming, and association of Pdd with epithelial cells. These activities were low when bacteria were pre-cultured in media with 3.5% NaCl, the global average salinity of sea water. In contrast, lower salinity increased swimming of wild type Pdd peaking at 2% NaCl, hemolysis, and association with epithelial cells peaking at 1-1.5%. Previously, we have found that hemolysin genes enhance the association of Pdd with epithelial cells, but the underlying mechanisms have remained ill-defined. We here searched for potential links between hemolysin-production, chemotaxis and association of Pdd with target cells at varying salt concentrations. Unexpectedly, disruption of chemotaxis regulator cheA not only affected bacterial swimming and association with epithelial cells at intermediate to low salinity, but also reduced the production of plasmid-encoded phobalysin (PhlyP). The results thus reveal unforeseen links between chemotaxis regulators, a pore forming toxin and the association of a marine bacterium with target cells.

Membrane repair emerges as an innate defense protecting target cells against bacterial pore-forming toxins. Here, we report the first paradigm of Ca 2+ -dependent repair following attack by a small β-pore-forming toxin, namely, plasmid-encoded phobalysin of Photobacterium damselae subsp. damselae In striking contrast, Vibrio cholerae cytolysin, the closest ortholog of phobalysin, subverted repair. Mutational analysis uncovered a role of channel width in toxicity and repair. Thus, the replacement of serine at phobalysin´s presumed channel narrow point with the bulkier tryptophan, the corresponding residue in Vibrio cholerae cytolysin (W318), modulated Ca 2+ influx, lysosomal exocytosis, and membrane repair. And yet, replacing tryptophan (W318) with serine in Vibrio cholerae cytolysin enhanced toxicity. The data reveal divergent strategies evolved by two related small β-pore-forming toxins to manipulate target cells: phobalysin leads to fulminant perturbation of ion concentrations, closely followed by Ca 2+ influx-dependent membrane repair. In contrast, V. cholerae cytolysin causes insidious perturbations and escapes control by the cellular wounded membrane repair-like response. IMPORTANCE Previous studies demonstrated that large transmembrane pores, such as those formed by perforin or bacterial toxins of the cholesterol-dependent cytolysin family, trigger rapid, Ca 2+ influx-dependent repair mechanisms. In contrast, recovery from attack by the small β-pore-forming Staphylococcus aureus alpha-toxin or aerolysin is slow in comparison and does not depend on extracellular Ca 2+ To further elucidate the scope of Ca 2+ influx-dependent repair and understand its limitations, we compared the cellular responses to phobalysin and V. cholerae cytolysin, two related small β-pore-forming toxins which create membrane pores of slightly different sizes. The data indicate that the channel width of a small β-pore-forming toxin is a critical determinant of both primary toxicity and susceptibility to Ca 2+ -dependent repair., (Copyright © 2017 von Hoven et al.)

Staphylococcus aureus is a leading cause of bacterial infections in humans, including life-threatening diseases such as pneumonia and sepsis. Its small membrane-pore-forming α-toxin is considered an important virulence factor. By destroying cell-cell contacts through cleavage of cadherins, the metalloproteinase ADAM10 (a disintegrin and metalloproteinase 10) critically contributes to α-toxin-dependent pathology of experimental S. aureus infections in mice. Moreover, ADAM10 was proposed to be a receptor for α-toxin. However, it is unclear whether the catalytic activity or specific domains of ADAM10 are involved in mediating binding and/or subsequent cytotoxicity of α-toxin. Also, it is not known how α-toxin triggers ADAM10's enzymatic activity, and whether ADAM10 is invariably required for all α-toxin action on cells. In the present study, we show that efficient cleavage of the ADAM10 substrate epithelial cadherin (E-cadherin) requires supra-cytotoxic concentrations of α-toxin, leading to significant increases in intracellular [Ca(2+)]; the fall in cellular ATP levels, typically following membrane perforation, became observable at far lower concentrations. Surprisingly, ADAM10 was dispensable for α-toxin-dependent xenophagic targeting of S. aureus, whereas a role for α-toxin attack on the plasma membrane was confirmed. The catalytic site of ADAM10, furin cleavage site, cysteine switch and intracellular domain of ADAM10 were not required for α-toxin binding and subsequent cytotoxicity. In contrast, an essential role for the disintegrin domain and the prodomain emerged. Thus, co-expression of the prodomain with prodomain-deficient ADAM10 reconstituted binding of α-toxin and susceptibility of ADAM10-deficient cells. The results of the present study may help to inform structural analyses of α-toxin-ADAM10 interactions and to design novel strategies to counteract S. aureus α-toxin action., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

Photobacterium damselae subsp. damselae, an important pathogen of marine animals, may also cause septicemia or hyperaggressive necrotizing fasciitis in humans. We previously showed that hemolysin genes are critical for virulence of this organism in mice and fish. In the present study, we characterized the hlyA gene product, a putative small β-pore-forming toxin, and termed it phobalysin P (PhlyP), for "photobacterial lysin encoded on a plasmid." PhlyP formed stable oligomers and small membrane pores, causing efflux of K(+), with no significant leakage of lactate dehydrogenase but entry of vital dyes. The latter feature distinguished PhlyP from the related Vibrio cholerae cytolysin. Attack by PhlyP provoked a loss of cellular ATP, attenuated translation, and caused profound morphological changes in epithelial cells. In coculture experiments with epithelial cells, Photobacterium damselae subsp. damselae led to rapid hemolysin-dependent membrane permeabilization. Unexpectedly, hemolysins also promoted the association of P. damselae subsp. damselae with epithelial cells. The collective observations of this study suggest that membrane-damaging toxins commonly enhance bacterial adherence., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

We report on the role of conserved stress-response pathways for cellular tolerance to a pore forming toxin. First, we observed that small molecular weight inhibitors including of eIF2α-phosphatase, jun-N-terminal kinase (JNK), and PI3-kinase sensitized normal mouse embryonal fibroblasts (MEFs) to the small pore forming S. aureus α-toxin. Sensitization depended on expression of mADAM10, the murine ortholog of a proposed high-affinity receptor for α-toxin in human cells. Similarly, eIF2α (S51A/S51A) MEFs, which harbor an Ala knock-in mutation at the regulated Ser51 phosphorylation site of eukaryotic translation initiation factor 2α, were hyper-sensitive to α-toxin. Inhibition of translation with cycloheximide did not mimic the tolerogenic effect of eIF2α-phosphorylation. Notably, eIF2α-dependent tolerance of MEFs was toxin-selective, as wild-type MEFs and eIF2α (S51A/S51A) MEFs exhibited virtually equal sensitivity to Vibrio cholerae cytolysin. Binding of S. aureus α-toxin to eIF2α (S51A/S51A) MEFs and toxicity in these cells were enhanced as compared to wild-type cells. This led to the unexpected finding that the mutant cells carried more ADAM10. Because basal phosphorylation of eIF2α in MEFs required amino acid deprivation-activated eIF2α-kinase 4/GCN2, the data reveal that basal activity of this kinase mediates tolerance of MEFs to α-toxin. Further, they suggest that modulation of ADAM10 is involved. During infection, bacterial growth may cause nutrient shortage in tissues, which might activate this response. Tolerance to α-toxin was robust in macrophages and did not depend on GCN2. However, JNKs appeared to play a role, suggesting differential cell type and toxin selectivity of tolerogenic stress responses. Understanding their function or failure will be important to comprehend anti-bacterial immune responses.

In a retrospective study on 86 twins born between 1971 and 1990, the clinical and acidity status of small for gestational age twins in cases of uncomplicated labor was analysed and compared with the status of appropriate for gestational age twins. No difference was observed in Apgar score and umbilical blood pH between growth retarded and normal twins. The single fact of growth retardation without other factors of risk during labor has no influence on clinical status of small for gestational age twins.

Thirty-one pregnant women divided into three groups (AGA prepartum, SGA prepartum without distress, AGA in labor) were examined using Doppler ultrasonography before, during and after oxygen administration to mothers via a face mask. The aim of the study was to find out if there was any effect on the blood flow values in the fetal aorta, the umbilical artery, the fetal common carotid artery and the uterine arcuate arteries. The resistance index (RI) did not change in those vessels during maternal hyperoxygenation with one exception: in the group of SGA fetuses the RI in the fetal aorta increased significantly. Blood flow velocity and volume blood flow remained unchanged in the fetal aorta during oxygen administration.

Measurement of fetal blood flow has been accepted using pulsed-Doppler ultrasound. Until recently, there has been a lack of investigations concerning the potential risks of this method. The possible mutagenic effect of a pulsed-Doppler system was examined in vitro by applying the Ames test. Tester strains of Salmonella typhimurium indicating point mutations were irradiated (TA 98, TA 100, TA 102, TA 1535, TA 1537, TA 1538). A commercially available duplex system was applied in the experiments emitting an ultrasound beam with a spatial-peak temporal-average of 5.2 mW/cm2 and a spatial-peak temporal-peak of 117 mW/cm2 at a frequency of 2 MHz. The tester strains were sonicated up to 60 minutes, the bacterial suspension being in direct contact with the transducer surface. The ultrasound-exposed bacterial suspensions were compared with nonexposed samples. Reference mutagens were used for checking the sensitivity of the system. The results do not indicate any mutagenic effects.

Differences in measurements using cephalometry and thoracometry ante partum are reported. Two examiners performed fetal head and thorax measurements shortly after one another using the same ultrasonic equipment without knowing the measurements achieved by each other. A series of 195 measurements were made by a few less experienced examiners. The values measured showed a considerable scatter. The average difference between the biparietal head measurements was 2.23 mm, and the standard deviation of the differences of the values measured was 1.94 mm. In a further group of 175 fetuses the measurements were performed by two skilled examiners. For the diameter of the head there was an average measured difference of 1.38 mm and a standard deviation of the differences of 1.11 mm. For the average thorax diameter--corresponding to the arithmetical average from the straight and transverse diameter--the following results were achieved. 4.14 mm average difference of value measured an 3.41 mm standard deviation in the measuring run performed by less experienced ultrasonic examiners as opposed to 2.57 mm average difference and 1.74 mm standard deviation in the group of practised examiners. The average thorax diameter showed a considerably smaller scatter than the transverse thorax measurement on its own. The differences in the values measured could not be seen to be dependent on the gestational age.

The aim of this study was to plot the course of the transcutaneously measured PCO2 (tcPCO2) in the fetus during oxygenation of the mother. In our examination 35 parturients with a suspicious or pathologic CTG were given pure oxygen for 10 minutes at a flow speed of 10 l/min. The fetal tcPCO2 was measured with a TCM 3 measuring device from Radiometer. The measuring temperature was 41 degrees C. The fetal tcPCO2 was 67.2 +/- 3.9 mmHg before the O2 application, during the O2 application it was 67.3 +/- 14.1 mmHg and for the period after the O2 application we found an average measurement of 66.7 +/- 13.9 mmHg. Further we investigated whether, depending on the original levels of the fetal tcPCO2 an O2 application to the mother had a measurable effect on the fetal tcPCO2 levels. The average levels of the tcPCO2 in the fetuses with pathological original levels of greater than or equal to 60 mmHg or with normal levels of less than 60 mmHg did not show any significant differences before, during or after the O2 application. Our own results and reports given in the literature about an increase in the fetal O2 partial pressure during maternal oxygenation lead to the conclusion that in cases with fetal hypoxia, the O2 application to the mother--in addition to other measures for intrauterine reanimation or speedy termination of labor--could be of advantage.

Chinese hamster ovary cells were treated with ultrasound from a fetal pulse detector (Eucotone, Siemens) operated at 10 mW/cm2 and 2.2 MHz. The frequencies of structural chromosomal aberrations and of sister chromatid exchanges were not increased by the treatment. There was no indication of single-strand breaks induced by ultrasound in the G2 phase of the cell cycle.

We introduce a test object usable in a water tank for the determination of the axial and lateral resolution of ultrasonic pulse echo equipments. Measuring wires with a diameter of 0.2 mm form the bounds of a measuring line. The distance between the wires is infinitely variable during the test. This will make the smallest distance which can be resolved in the sonogram more accurately determinable than with the usual test objects with rigidly arranged rods.

Via examinations by ultrasonic sectional view during the course of pregnancy, the growth of the placenta was followed in 52 pregnant women. When a pathologic location of the placenta could be excluded during the first examination in the second trimester (26 cases), secondary development of a pathologic insertion was not observed. In 26 cases in which a pathological location of the placenta could be established before the 20th gestation week, a cranial shift in the caudal placenta border of up to 8 cm could be established in 19 cases. A pathological location of the placenta diagnosed in the second trimester can disappear. The shape of the uterus and the alterations in the spacial relationship between placenta and internal os uteri were caused by the development and stretching of the lower segment of the uterus. The vaginal hemorrhages in the second trimester caused by a low placental insertion (13 cases) stopped (with the exception of 2 cases) at the latest after the 6th month of pregnancy. The definitive establishment of a placenta pravia before the 5th-6th month of pregnancy is virtually impossible sonographically.

Several procedures during pregnancy under ultra-sonographic control are described. Amniocentesis and puncture of the fetus may be done accurately without too much problems when a multi-element ultra-sound scanner is used for sonographic control. The principle of manipulation with ultra-sonographic control may be used for curettage. Instruments within the uterus may be localized and their movements can be submitted to continual visual control. A curettage under sonographic control may reduce the risk of the curettage in cases of uterine anomalies, anomalies of the position or unusually soft uteri.

The results of 552 amniocenteses during the second trimester of pregnancy are reported. The amniocentesis were carried out under direct ultra-sonographic control. Six abnormal karyotypes were found and the pregnancies were terminated. Three spontaneous abortions due to intra-uterine infection following amniocentesis occurred. There were no maternal complications and no fetal injuries. 430 amniotic fluids were tested bacteriologically. Two samples showed E-coli and two samples grew staph epidermidis. The IGM values of 148 newborns with previous amniocentesis showed the same values as the IGM in newborns without amniocentesis during the pregnancy. Fetal monitoring prior and following the amniocentesis showed a temporary increase in uterine motility in 25% of the cases. Our observations and the review of the literature convinced us that complications of amniocentesis in the second trimester are rare. However, when complications occur the fetus and the pregnant woman are a serious risk. The fetal wastage caused by second trimester amniocentesis is approximately 0.5 to 1%. Therefore the patients must be informed about the risks very carefully prior to a second trimester amniocentesis.

During the past eight years ultra-sound scans were carried out on 281 patients with the suspicion of ectopic pregnancy. Diagnostic criteria were established which were designed to minimize false negative results. In 52 cases the clinical suspicion was confirmed by operation. In 46 of these patients ultra-sound signs of ectopic pregnancy were present. Six ectopic pregnancies were not recognized by ultra-sonographies. 73 patients were suspicious of ectopic pregnancy but did not show ectopic pregnancy. The high rate of ultra-sound misinterpretation in ectopic pregnancy is due to the variability of ectopic pregnancy, abortions of intra-uterine pregnancies, abnormalities of the uterus, and adnexal tumours of other etiology. Negative ultra-sound findings do not rule out an ectopic pregnancy since a number of cases with the clinical suspicion of ectopic pregnancy show intact intra-uterine pregnancies. Ultrasonography is a valuable additional diagnostic method in suspected cases of ectopic pregnancy.