Advanced wastewater treatment with ozonation and granular activated carbon filtration: Inactivation of antibiotic resistance targets in a long-term pilot study.

The inactivation of antibiotic resistant bacteria (ARB) and genes (ARGs) in an advanced plant combining ozonation and granular activated carbon (GAC) filtration applied for effluent after conventional activated sludge treatment at a full-scale urban wastewater treatment plant was investigated for over 13 consecutive months. The nitrite compensated specific ozone dose ranged between 0.4 and 0.7 g O3/g DOC with short-time sampling campaigns (0.2-0.9 g O3/g DOC). Samples were analysed with culture-dependent methods for bacterial targets and with qPCR for genes. The log removal values were correlated with a decrease of the matrix UV absorption at 254 nm (ΔUV254) and indicated a range of ΔUV254 that corresponds to a sufficient membrane damage to affect DNA. For trimethoprim/sulfamethoxazole resistant E. coli, sul1, ermB and tetW, this phase was observed at ΔUV254 of ~30 % (~0.5 g O3/g DOC). For ampicillin resistant E. coli and blaTEM-1, it was observed around 35-40 % (~0.7 g O3/g DOC), which can be linked to mechanisms related to oxidative damages in bacteria resistant to bactericidal antibiotics. GAC treatment resulted in a further abatement for trimethoprim/sulfamethoxazole E. coli, sul1 and tetW, and in increase in absolute and relative abundance of ermB and blaTEM-1.

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.

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.

Microorganism inactivation by an ozonation step optimized for micropollutant removal from tertiary effluent.

This paper demonstrates the additional benefit of the microbicidal efficacy of an ozonation plant implemented for micropollutant removal from tertiary effluent. Due to the low amount of viruses and protozoa in the tertiary effluent, bacteriophage MS2 and spores of Bacillus subtilis were dosed as surrogates. At specific ozone consumptions of 0.6 and 0.9 g O3/g dissolved organic carbon (DOC) a 2-log colony forming unit (CFU) reduction was achieved for indigenous Escherichia coli and enterococci, and the limits of the European bathing water directive for the excellent quality of inland waters were met. Higher removal was impeded by the shielding effect of suspended solids in the effluent, which implies the combination of ozonation with a preceding filtration step if higher microbicidal performances are required. The surrogate virus MS2 was reduced by 4-5 log while no significant inactivation was detected for B. subtilis spores. Additionally, the impact of ozonation on the biochemical oxygen demand (BOD) was studied. The BOD5 measurement was not adversely affected despite the reduced concentration of microorganisms after ozonation. The intrinsic increase in BOD5 averaged 15% at 0.6-0.7 g O3/g DOC. The impact of the projected increase on the surface water quality is generally not considered a problem but has to be assessed on a case-by-case approach.

Spinal alignment in low back pain patients and age-related side effects: a multivariate cross-sectional analysis of video rasterstereography back shape reconstruction data.

PURPOSE: Spinal form in the sagittal plane and asymmetries of spinal alignment in the frontal plane were identified earlier to be associated with low back pain. This study was aiming at investigating whether age was influencing the significance of these findings. METHODS: Spinal alignment of 155 older and 58 younger low back pain patients was investigated using non-invasive video rasterstereography and compared to 192 pain free controls. Trunk inclination and imbalance, pelvis tilt and torsion, and the thoracic kyphosis and lumbar lordosis angle served as dependent variables, which were analyzed using multivariate procedures. RESULTS: Frontal plane parameters-trunk imbalance (λ > 0.60) in combination with pelvis tilt (λ > 0.64)-constituted an independent component in a factor analysis model of spine shape parameters in younger and older patients, but not in pain free controls. Trunk imbalance (frontal), trunk inclination (sagittal) and partially thoracic kyphosis angle (sagittal) were extracted to separate patients from controls (discriminant coefficients >0.50). Age could not be excluded as interfering variable for the lumbar lordosis angle. Significant univariate analyses revealed higher effect sizes for trunk imbalance and inclination (η (2) > 0.07) than for lordosis, kyphosis or pelvic parameters (η (2) < 0.06). CONCLUSIONS: In line with corresponding findings trunk imbalance remained a low back pain-associated parameter not depending on age. Over all, sagittal trunk inclination and the thoracic kyphosis angle could not be established as being completely free from age influences, while the lumbar lordosis angle played a minor role and seemed to be influenced more by age than by low back pain in this rasterstereography study.

Benchmarking of large municipal wastewater treatment plants treating over 100,000 PE in Austria.

During a six-year period the Austrian Benchmarking System was developed. The main objectives of this benchmarking system are the development of process indicators, identification of best performance and determination of cost reduction potentials. Since 2004 this system is operated via an internet platform and automated to a large extent. Every year twenty to thirty treatment plants use the web-based access to this benchmarking platform. The benchmarking procedure comprises data acquisition, data evaluation including reporting and organised exchange of experience for the treatment plant managers. The process benchmarking method links the real costs with four defined main processes and two support processes. For wastewater treatment plants with a design capacity >100,000 PE these processes are further split up into sub-processes. For each (sub-) process the operating costs are attributed to six cost elements. The specific total yearly costs and the yearly operating costs of all (sub-)processes are related to the measured mean yearly pollution load of the plant expressed in population equivalents (PE(110): 110 gCOD/d corresponding to 60 g BOD(5)/d)). The specific capital costs are related to the design capacity (PE). The paper shows the benchmarking results of 6 Austrian plants with a design capacity >100,000 PE representing approximately 30% of the Austrian municipal wastewater treatment plant capacity.

Comparison of and uncertainties in raw sewage COD measurements by laboratory techniques and field UV-visible spectrometry.

A comprehensive investigation of the uncertainty of different COD analysis methods (DIN, small tube tests (STT) and UV-visible spectrometry methods) has been carried out on potassium hydrogen phthalate standard solutions and raw sewage samples from a large wastewater treatment plant. The UV-visible method allows estimating COD equivalent concentration from the measured absorption spectra by means of site specific regression and correlation functions. CODdin and CODstt methods showed equivalent global results but specific calibration relationships are necessary when high accuracy is required. The CODstt method is suitable for immediate COD analysis in the field. Sub-sampling is the main source of uncertainty. Spectrometry is able to estimate CODeq with an uncertainty of the same order of magnitude as the uncertainty in CODdin.

Normocalcaemic hyperparathyroidism after parathyroidectomy: a retrospective study.

Forty-eight patients with proven hyperparathyroidism underwent parathyroidectomy. All patients were post-operatively normocalcaemic and free of symptoms due to their pre-operative hypercalcaemic state. However, 20 (42%) post-operative patients still had increased serum PTH and low serum phosphate levels.

Normocalcemia with persistent increase of parathyroid hormone: a prospective study.

Twelve patients were followed up for 3 months after parathyroidectomy. Serial measurements of serum parathyroid hormone (PTH), calcium, and phosphate were made. Four patients had an increased serum PTH postoperatively, which was already apparent by the third postoperative day. All patients became normocalcemic. Their hyperparathyroid-like phosphate parameters indicated that we were dealing with a biologically active PTH. Using preoperative biochemical parameters it was impossible to predict which patients would have an increased PTH postparathyroidectomy (PTX). Probably the patients with high PTH post-PTX had higher parathyroid volumes. In our opinion after PTX, a normocalcemic high PTH situation should be avoided by 3 1/2 parathyroid gland extirpation in all cases.

[AIDS in a woman having had sexual relations with a patient with hemophilia A. Characteristic findings in DNA image cytometry]. / AIDS-Erkrankung einer Frau mit sexueller Beziehung zu einem Hämophilie-A-Patienten. Charakteristische Befunde der DNS-Bild-Zytometrie.

A 37-year-old female patient reported marked weight loss, prolonged alopecia, recurrent infections and watery diarrhoea. Examination revealed Salmonella infection, candidiasis and immunological signs of previous toxoplasmosis. Between 1978 and 1981, the patient had had close sexual relations to a patient with haemophilia A. Due to this fact, AIDS was suspected. Serological tests for HIV were not available at the time. The findings in DNA image cytometry (nuclear DNA inclusion bodies, polyploid lymphocyte nuclei and binuclear lymphocytes) suggested a viral infection of the lymphoid cells. Electron microscopy revealed in hepatocytes and cerebral cells intranuclear inclusion bodies whose size and contents were not compatible with an infection caused by cytomegalovirus, herpes virus or Epstein-Barr virus. In autopsy, infections of various organ systems such as pneumonia, tracheobronchitis, urocystitis, pyelonephritis, Candida oesophagitis and enteritis were found.

Lesions of the thyroid gland in patients with primary hyperparathyroidism.

To determine the frequency of coexisting thyroid gland lesions, a consecutive series of 30 patients operated upon for primary hyperparathyroidism were studied. Lesions of the thyroid gland were found in 21 (70 per cent) patients. It is known that patients undergoing surgical treatment for a disease of the parathyroid or thyroid gland should be carefully evaluated both preoperatively and intraoperatively for abnormalities in the other gland or glands to recognize coexistent disease and treat it in one single procedure. The results of this study has lead to the appreciation of the value of a scintiscan of the thyroid gland prior to operations upon the parathyroid gland. All of the patients with an abnormal scintiscan had coexisting pathologic findings of the thyroid gland. These results suggest that it is quite helpful in evaluating the thyroid gland prior to parathyroid gland operations.

[Clinical staging and course evaluation of patients with multiple myeloma and benign monoclonal hyperglobulinemia using the Salmon-Wampler total tumor cell mass calculation]. / Klinische Stadieneinteilung und Verlaufsbeurteilung von Patienten mit multiplem Myelom und benigner monoklonaler Hyperglobulinämie mit Hilfe der Gesamttumorzellmassenberechnung nach Salmon und Wampler.

The calculation of tumor cell mass according to Salmon and Wampler was applied in a retrospective study comprising 79 unselected patients with multiple myeloma. The method proved to be a useful parameter for judging prognosis and course of the disease. IgG myelomas showed a better and prolonged regression of tumor cell mass than IgA myelomas. The average reduction of tumor cell mass under chemotherapy amounted to approximately 0.5 log. 38.5% of all patients went into a plateau phase after an initial decrease of their tumor cell mass, 30.8% showed varying values, whereas in 7.7% the reduction of the tumor cell mass occurred in a protracted fashion. The investigation of those patients in whom the first chemotherapy regimen was exchanged for another one showed, however, that the tumor cell mass calculation could not replace other controls, particularly X-ray controls pertaining to the progression of bone destruction. In benign monoclonal hyperglobulinemia, the calculation of the "tumor cell mass" can be applied as a sensitive parameter for judging the transition into multiple myeloma.

[Testing of cementless stepped-stem prosthetic devices replacing the hip shoulder joint of the dog. Comparison of two animal models (author's transl)]. / Die Erprobung zementloser Stufenschaftprothesen im Hüft- und Schultergelenk des Hundes. Ein Vergleich zweier Tiermodelle.

In 6 dogs the hip joint was experimentally replaced using a ceramic stepped-stem prosthetic device implanted in the thigh bone. In six other dogs V4A steel stepped-stem prosthetic devices inserted in the arm bone was used for replacing shoulder joint. The six devices replacing the shoulder joint healed up without complications and proved to be mechanically stable. From six devices replacing the hip joint only one was successful; the remaining five implants did not heal up due to fractures of the stem, loosening in the thigh bone and osteomyelitis. We suggest that these different results are based not on the different material, but on the different conditions characteristic for each implantation sites. The devices implanted in the arm bone of the dog, corresponding to the conditions in man, were stressed mainly by pressure and pull. In such conditions the newly formed bone tissue anchoring in the steps on the stem stabilized the device. On the other hand, the biomechanical load on the hip joint replacing device in the dog is represented mainly by rotation at stress, for which the devices are not conceived. The stepped profile of the stem can not work; it twists in the thigh bone and remains unstable causing consequences such as fractures, osteomyelitis and loosening. For experimental testing of bone and joint replacing prosthetic devices of cementless anchoring systems replacing human hip joint and thigh bone we recommend the shoulder joint of the dog instead of hip joint, because of the comparable biomechanics.

Giant cell formation in Acholeplasma laidlawii, strain JA1.

Striking alterations, which looked like blebs, were observed in colonies of Acholeplasma laidlawii, strain JA 1. "Craters" were seen on the surface of these colonies by scanning electron microscopy. In addition, giant cells of acholeplasma, up to 14 microns in diameter, i.e. approximately 20 times the size of a normal A. laidlawii cell, were visible in the colonies. The large forms were surrounded by a unit membrane. After infection with group 1 and group 2 mycoplasma-viruses, the proportion of altered colonies and the number of large cells within these colonies increased. A strain of A. laidlawii, which was not susceptible to infection with the three known acholeplasma-viruses did not exhibit comparable morphological changes. The development of the giant cells can be explained either by cell fusion or by a lag of cell division behind genome replication. Blebs and "craters" may result from the destruction of mycoplasma organisms within the complex structure of the colony. There is also suggestive evidence that strain JA 1 carries a virus in some cryptic form.

Electron microscopic studies on the attachment of Mycoplasma pneumoniae to guinea pig erythrocytes.

A mechanism of pathogenicity of Mycoplasma pneumoniae is its ability to attach to the surface of mammalian cells. It has previously been demonstrated by others that M. pneumoniae adheres with a specialized terminal structure, the "tip," to ciliated epithelial cells of the respiratory tract. In this report we show by electron microscopy that M. pneumoniae adsorbs with membrane sites other than the tip to guinea pig erythrocytes.

[Functional morphology of the theca interna of the vesicular follicle in human ovary]. / Funktionelle Morphologie der Theca interna im Bläschenfollikel des menschlichen Ovars

A study of histological serial sections of human ovaries and histochemical reactions established that several cell types differentiate in the theca interna during the formation and development of tertiary follicles (2nd growth period). This leads, in turn, to triple-layering of the theca interna in follicles of the 2nd resting period. The inner thecal layer consists of fusiform cells from the basement membrane, which are apposed to the membrana granulosa and show a strong positive reaction to alkaline phosphatases. A tropic function for the follicular epithelium must be assigned to this layer. The middle layer consists of epitheloid thecal cells, which show a strong positive reaction to 3beta-ol-steroid hydrogenase. These represent the estrogen glands of the follicle. The outer layer of the theca interna, whose transition to the theca externa is indistinct, also has fusiform cells, which are available as reserve material for the differentiation of further epitheloid thecal cells to pre-ovulatory follicles in later periods of development.