Towards a correlative approach for characterising single virus particles by transmission electron microscopy and nanoscale Raman spectroscopy.

The morphology and structure of biological nanoparticles, such as viruses, can be efficiently analysed by transmission electron microscopy (TEM). To chemically characterise such nanoparticles in heterogeneous samples at the single particle level, we suggest tip-enhanced Raman spectroscopy (TERS) as a correlative method. Here we describe a TERS-compatible staining procedure for TEM which involves sample pre-scanning by TEM imaging, nanoparticle relocalisation by atomic force microscopy (AFM) followed by spectroscopic characterization of the virus nanoparticles using TERS. First successful correlative measurements are demonstrated on tobacco mosaic virus particles deposited on silicon-based TEM sample supports. In addition, the advantages and problems of this methodology are discussed.

Draft Genome Sequences of Klebsiella oxytoca Isolates Originating from a Highly Contaminated Liquid Hand Soap Product.

In 2013, contaminated liquid soap was detected by routine microbiological monitoring of consumer products through state health authorities. Because of its high load of Klebsiella oxytoca, the liquid soap was notified via the European Union Rapid Alert System for Dangerous Non-Food Products (EU-RAPEX) and recalled. Here, we present two draft genome sequences and a summary of their general features.

Phase I trial of SU14813 in patients with advanced solid malignancies.

BACKGROUND: this phase I, open-label, dose-escalation study investigated SU14813, an oral multitargeted tyrosine kinase inhibitor, in adults with solid tumors. PATIENTS AND METHODS: seventy-seven patients received once-daily SU14813, either for 4 weeks followed by 1 week off treatment (schedule 4/1) or continuously [continuous daily dosing (CDD)]. The primary end point was to determine the maximum tolerated dose (MTD). Safety, pharmacokinetics, pharmacodynamics, and efficacy were assessed. RESULTS: MTDs were 200 mg/day on schedule 4/1 and 100 mg/day with CDD. Adverse events included fatigue (64%), diarrhea (61%), nausea (44%), anorexia (43%), and vomiting (42%). SU14813 steady state was attained by day 8. Exposure increased in a generally dose-proportional manner and SU14813 was eliminated with a mean terminal half-life of 9-34 h. Target plasma concentrations (>100 ng/ml SU14813) were achieved and sustained over 12 h at ≥ 100 mg/day. Progression-free survival among the 1 complete responder and 12 partial responders was 1.4-53.2 months. Fifteen patients remained on treatment at 1 year and 3 patients at 2 years. CONCLUSION: SU14813 has manageable safety and tolerability and allows once-daily continuous oral dosing. SU14813 shows dose-proportional pharmacokinetics, with target plasma concentrations achieved at doses ≥ 100 mg/day. Clinically meaningful activity with durable responses was observed, meriting further study.

Scrapie-infected cells, isolated prions, and recombinant prion protein: a comparative study.

Fourier -transform infrared microscopic spectra of scrapie-infected nervous tissue measured at high spatial resolution (approximately 6 microm) were compared with those obtained from the purified, partly proteinase K digested scrapie isoform of the prion protein isolated from nervous tissue of hamsters infected with the same scrapie strain (263K) to elucidate similarities/dissimilarities between prion structure investigated in situ and ex vivo. A further comparison is drawn to the recombinant Syrian hamster prion protein SHaPrP(90-232) after in vitro conformational transition from the predominantly alpha-helical isoform to beta-sheet-rich structures. It is shown that prion protein structure can be investigated within tissue and that detectability of regions with elevated beta-sheet content as observed in microspectra of prion-infected tissue strongly depends on spatial resolution of the experiment.

Mid-IR microspectroscopic imaging of breast tumor tissue sections.

IR microspectroscopic imaging is a relatively new approach for the examination of tissue sections. In contrast to standard light microscopy based procedures, the IR approach requires neither sample staining nor fixation. The IR spectra of breast tumor tissue sections are obtained via a microscope equipped with a focal plane array detector. This enabled the simultaneous collection of individual mid-IR spectra from thousands of different sample positions with a spatial resolution near the diffraction limit. The analysis of the IR data reveals a high sensitivity of the IR approach toward changes in tissue biochemistry and variations in breast tissue architecture. Moreover, the data demonstrate the need for collecting spectra with high spatial resolution at the level of individual cells. This minimizes problems associated with tissue microheterogeneity and is an essential prerequisite for future studies aimed at developing IR microspectroscopic imaging as a complement to present diagnostic tools for breast cancer.

Hydrogen peroxide-induced structural alterations of RNAse A.

Proteins exposed to oxidative stress are degraded via proteolytic pathways. In the present study, we undertook a series of in vitro experiments to establish a correlation between the structural changes induced by mild oxidation of the model protein RNase A and the proteolytic rate found upon exposure of the modified protein toward the isolated 20 S proteasome. Fourier transform infrared spectroscopy was used as a structure-sensitive probe. We report here strong experimental evidence for oxidation-induced conformational rearrangements of the model protein RNase A and, at the same time, for covalent modifications of amino acid side chains. Oxidation-related conformational changes, induced by H(2)O(2) exposure of the protein may be monitored in the amide I region, which is sensitive to changes in protein secondary structure. A comparison of the time- and H(2)O(2) concentration-dependent changes in the amide I region demonstrates a high degree of similarity to spectral alterations typical for temperature-induced unfolding of RNase A. In addition, spectral parameters of amino acid side chain marker bands (Tyr, Asp) revealed evidence for covalent modifications. Proteasome digestion measurements on oxidized RNase A revealed a specific time and H(2)O(2) concentration dependence; at low initial concentration of the oxidant, the RNase A turnover rate increases with incubation time and concentration. Based on these experimental findings, a correlation between structural alterations detected upon RNase A oxidation and proteolytic rates of RNase A is established, and possible mechanisms of the proteasome recognition process of oxidatively damaged proteins are discussed.

Detection of pathological molecular alterations in scrapie-infected hamster brain by Fourier transform infrared (FT-IR) spectroscopy.

In this report a new approach for the identification of pathological changes in scrapie-infected Syrian hamster brains using Fourier transform infrared microspectroscopy is discussed. Using computer-based pattern recognition techniques and imaging, infrared maps with high structural contrast were obtained. This strategy permitted comparison of spectroscopic data from identical anatomical structures in scrapie-infected and control brains. Consistent alterations in membrane state-of-order, protein composition, carbohydrate and nucleic acid constituents were detected in scrapie-infected tissues. Cluster analysis performed on spectra of homogenized medulla oblongata and pons samples also reliably separated uninfected from infected specimens. This method provides a useful tool not only for the exploration of the disease process but also for the development of rapid diagnostic and screening techniques of transmissible spongiform encephalopathies.

The influence of poly-(L-lysine) and porin on the domain structure of mixed vesicles composed of lipopolysaccharide and phospholipid: an infrared spectroscopic study.

Fourier transform infrared (FTIR) spectroscopy has been used to study the thermotropic phase behavior of binary lipid mixtures composed of deuterated phospholipids (PLs) and lipopolysaccharides (LPSs). Furthermore, the influence of an extrinsic high-molecular, polycationic polypeptide (poly-(L-lysine), PLL(500)) and an intrinsic membrane protein (outer membrane protein F, OmpF) on these binary mixtures was investigated by FTIR spectroscopy. "Deep rough" mutant LPS (ReLPS), isolated from Salmonella minnesota R595, and perdeuterated 1,2-dimyristoylphosphatidylethanolamine (DMPEd54) were used as model lipids. Deuteration of one of the lipids permitted the detection of lipid protein interaction with each lipid component separately. For this purpose, the symmetric >CH2 and >CD2 stretching bands were utilized as specific monitors to scrutinize the state of order of the membranes. From the individual phase transition temperatures Tm and the shape of the phase transition profiles, it is established that ReLPS and DMPEd54 are molecularly immiscible. In addition to the two domains of the pure lipid components, a third, domain-like structure is detected that may coexist with these pure domains. This domain-like structure undergoes a gel to liquid-crystalline L1 (beta <--> alpha) phase transition at temperatures distinctly different from that of the respective pure lipid domains. The nature of this type of domain is discussed in terms of a "border region" model that adequately explains the experimentally observed complex phase transition profiles. It is further demonstrated that the extrinsic polycationic polypeptide PLL(500) and the intrinsic, pore-forming protein OmpF isolated from Escherichia coli interact preferentially and highly specifically with the negatively charged ReLPS. Both the synthetic polypeptide and the pore-forming protein increased the tendency of ReLPS and DMPEd54 to segregate into distinct, well-separated domains. Whereas the transition profiles of the ternary system ReLPS/DMPEd54/PLL(500) showed the features of a phase segregation phenomenon not affecting the transition temperatures of the pure lipid components, the ternary system composed of ReLPS/DMPEd54 and OmpF exhibited phase transition curves that were characterized by an unspecific (DMPEd54/OmpF) and a strong and unique (ReLPS/OmpF) type of lipid-protein interaction. Furthermore, semiquantitative estimations supported the supposition that OmpF might be able to induce bilayer asymmetry in preformed symmetrical ReLPS/DMPEd54 vesicles.

FT-IR microspectroscopic imaging of human carcinoma thin sections based on pattern recognition techniques.

FT-IR microspectroscopic maps of unstained thin sections from human melanoma and colon carcinoma tissues were obtained on a conventional infrared microscope equipped with an automatic x, y stage. Mapped infrared data were analyzed by different image re-assembling techniques, namely functional group mapping ("chemical mapping") and, for the first time by cluster analysis, principal component analysis and artificial neural networks. The output values of the different classifiers were recombined with the original spatial information to construct IR-images whose color or gray tones were based on the spatial distribution of individual spectral patterns. While the functional group mapping technique could not reliably differentiate between the different tissue regions, the approach based on pattern recognition yielded images with a high contrast that confirmed standard histopathological techniques. The new technique turned out to be particularly helpful to improve discrimination between different types of tissue structures in general, and to increase image contrast between normal and cancerous regions of a given tissue sample.

[The role of extracorporeal membrane oxygenation (ECMO) in current neonatology]. / Die Rolle der extrakorporalen Membranoxygenierung (ECMO) in der aktuellen Neonatologie.

Two hundred term or near-term neonates were referred to an ECMO center for severe PPHN associated diseases. In 2 time periods from 1987 to 1991 and from 1992 to December 1995 alternative treatment modes were tried in an attempt to obviate ECMO. During the first time period patients underwent a trial of high-frequency oscillatory (HFOV) ventilation before ECMO. In the second time period patients first received inhaled NO followed by HFOV in non-responders. If this also failed HFOV was combined with iNO. In both time periods about 40% of the patients were spared ECMO treatment by these alternative treatment modalities. INO only benefited 15% of the ECMO candidates who apparently had fared just as well on HFOV alone in the preceding time period. While patients who were improved by iNO were spared HFOV with its potential severe complications, i.e. air leaks and cardiocirculatory instability. More extended long-term studies will have to show which of these 2 treatment modalities (iNO or HFOV) should be given-first priority in an attempt to avoid ECMO in neonates with severe respiratory failure.

Influence of early postnatal dexamethasone therapy on ventilator dependency in surfactant-substituted preterm infants.

We examined 26 preterm infants with respiratory distress syndrome in a randomized controlled prospective study to determine whether early postnatal dexamethasone therapy (< 2 h; 0.5 mg/kg per day) over 5 days in addition to substitution of surfactant (100 mg/kg) facilitates extubation and the course of RDS. Control (n = 12) and treated (n = 14) groups were comparable in birthweight (mean +/- SD: 1219 +/- 292 versus 1446 +/- 442 g), gestational age (29.3 +/- 2.2 versus 30.6 +/- 2.7 weeks), prenatal characteristics and initial respiratory and blood gas parameters. In both groups one infant died. Infants in the dexamethasone group responded better to surfactant (12/14 versus 3/12; p < 0.01), were extubated earlier (6.6 versus 14.2 days; p < 0.02) and required less time on supplemental oxygen (4.2 versus 12.5 days; p < 0.02). Pulmonary complications tended to be lower in the dexamethasone group (1/14 versus 4/12), as was the frequency of retinopathy (2/14 versus 6/12; p < 0.05). We conclude that early postnatal dexamethasone therapy improves response to surfactant therapy resulting in better weaning and earlier extubation in premature infants.

Inhaled nitric oxide for avoidance of extracorporeal membrane oxygenation in the treatment of severe persistent pulmonary hypertension of the newborn.

UNLABELLED: Inhaled nitric oxide (NO) is thought to provide a noninvasive therapeutic alternative to extracorporeal membrane oxygenation (ECMO) in the treatment of persistent pulmonary hypertension of the newborn (PPHN). OBJECTIVE: Since January 1993, we have studied inhalation of NO in PPHN patients meeting the ECMO criteria of our institution. We focused on the questions of whether or not the need for ECMO could be obviated and whether differences could be found between NO responders and nonresponders. DESIGN: NO gas was delivered via conventional IPPV ventilation in incrementally increasing concentrations from 20 to 80 ppm. PATIENTS: NO therapy was attempted in ten ECMO candidates with clinical and echocardiographical evidence of PPHN (mean OI 51.9, SD 10.4). RESULTS: At various NO levels (30-60 ppm), five patients showed a significant increase in mean PaO2 (range 32.9-85.9 mmHg). Improvement was transient in three patients (6-10 h) and prolonged in two others (54-80 h); in the latter cases, ECMO was avoided. Five patients did not respond at all to treatment. Responders and nonresponders differed in their mean respiratory tidal volume (8.9 vs 4.18 ml/kg, P <0.05). CONCLUSIONS: In our study, inhalation of NO obviated the necessity of ECMO therapy in only two out of ten PPHN patients. Thus, we would discourage any overoptimistic expectations about the effectiveness of NO therapy in PPHN until larger clinical trials have been performed.

Inhaled nitric oxide during extracorporeal membrane oxygenation for the treatment of severe persistent pulmonary hypertension of the newborn.

Inhaled nitric oxide (NO) as a complementary treatment was studied in 10 neonates during extracorporeal membrane oxygenation (ECMO) therapy of various persistent pulmonary hypertension of the newborn (PPHN)-associated diseases. At individually different levels of inhaled NO (20-80 ppm), the mean Pao2 increased by 59.7% in 6 responders, but it remained unchanged in 4 nonresponders. Adverse side effects of the NO inhalation were tolerable. It was associated with a reversible decrease of the mean arterial blood pressure in 1 patient. During prolonged NO inhalation, the methemoglobin (met-Hb) level increased to 0.9-4.6% in 6 patients. Based on these preliminary results, we conclude that inhaled NO during ECMO can improve oxygenation in some PPHN patients. Further studies with control groups are needed to determine whether inhaled NO can shorten ECMO treatment or improve the rate of survival among PPHN patients.

ARDS infolge schwerer RSV-Infektion Therapeutische Optionen: Therapeutische Optionen.

We report on a strikingly frequent referral of former preterm babies with respiratory syncytial virus (RSV) infection and subsequent ARDS in our hospital during the winter 1994/95 with regard to the clinical course under application of alternative treatment modalities. Treatment modalities like inhalational ribavirin, use of bronchodilators and instillation of surfactant had been tried without success. All children (age: 1-43 months) were ventilated for 6.6 (1-17) days with FiO2 = 1.0 and a mean airway pressure of 16.4 (10-24) cm H2O. Mean arterial blood gases were 49 (paO2) and 41 (pCO2) mm Hg, the OI was 33.4. By inhalational NO in combination with IPPV or HFOV 4 patients could be stabilized, in the other 6 ECMO became necessary. Two of them died in spite of several weeks on ECMO; 8 children survived and could be discharged home after a mean hospital stay of 3 months. Even in very severe cases of RSV infection treatment modalities like NO, HFOV and ECMO can be used successfully. The use of these treatment modalities must be considered before the lung damage is irreversible; in those cases a pre-existing BPD is no contraindication even for extracorporeal lung support.

Prognosis and outcome of neonates treated either with veno-arterial (VA) or veno-venous (VV) ECMO.

A comparison was done between neonates requiring veno-arterial (VA) ECMO (too small jugular vein, inability to insert a 12 Fr double lumen catheter or cardio-circulatory instability) and neonates treated with veno-venous (VV) ECMO in the same period of time. From 1991-1995 ECMO was done in 48 neonates after failure of maximum conventional treatments, NO-inhalation and HFOV. 30/48 babies were treated with VV-ECMO, with a switch to VA-ECMO later on in 3 of them. In 18 infants VA-ECMO was installed primarily. Differences between the VA- and VV-ECMO group were: the OI was higher in the VV-treated babies (62 +/- 20 vs. 48 +/- 13, p < 0.03), as were birth weight (3385 +/- 570 vs. 2963 +/- 653 g, p < 0.04), gestational age (39.7 +/- 1.6 vs. 37.9 +/- 2.7 weeks, p < 0.01) and MAP (18.7 +/- 2.2 vs. 17.1 +/- 2.4 cm H2O, p < 0.05). Severe ICH's occurred more frequently in the VA-treated babies (29 vs. 7%, p < 0.05), the rate of other complications was equal. The mortality rates were 43% (VA) and 15% (VV), p < 0.05. About one third of neonatal ECMO candidates will be treated with VA-ECMO, even if the VV-ECMO technique is available. Need for VA-ECMO implies--due to a higher number of preterm babies and a greater severity of illness before ECMO--a higher incidence of ICH's and a higher mortality rate.

High-frequency oscillatory ventilation and nitric oxide: alternative or complementary to ECMO.

One hundred and seventy-seven term or near-term neonates were referred to an ECMO center for severe PPHN-associated diseases. In 2 time periods from 1987 to 1991 and from 1992 to April 1995 alternative treatment modes were tried in an attempt to obviate ECMO. During the first time period patients underwent trial high-frequency oscillatory ventilation before ECMO. In the second time period patients first received inhaled NO followed by HFOV in a non-responders. If this also failed HFOV was combined with INO. In both time periods about 40% of the patients were spared ECMO treatment by these alternative treatment modalities. INO only benefited 15% of the ECMO candidates who apparently had fared just as well on HFOV alone in the preceding time period. While patients who were improved by INO were spared HFOV with its potential severe complications, i.e. air leaks and cardiocirculatory instability, more extended long-term studies will have to show which of these 2 treatment modalities (INO or HFOV) should be given first priority in an attempt to avoid ECMO in neonates with severe respiratory failure.

Neonatal neutropenia in low birthweight premature infants.

Neutropenia, as defined by common reference values, occurs often in neonates. Its incidence, causes, and clinical consequences have not been studied extensively in premature neonates. Of 208 consecutive infants with birthweight up to 2000 g, 121 (58%) had neutropenia. Low gestational age and low birthweight correlated with the incidence of neutropenia. Less than half of the neutropenic episodes could be attributed to infections, the others were related to specific perinatal events and due to drug therapy or were of unknown cause. Neutropenia following treatment with certain antibiotics was the most common cause of neutropenia occurring after the second week of life. The high incidence of neutropenia in premature neonates raises questions about application of these reference ranges to low birthweight infants and suggests the need for new reference values.

[High frequency oscillatory ventilation of infants with severe respiratory disorders: possibilities, risks and limits]. / Hochfrequenzoszillationsbeatmung bei Säuglingen mit schwersten Atemstörungen: Möglichkeiten, Risiken und Grenzen.

By pediatricians the high frequency oscillatory ventilation (HFOV) is used almost only in the neonatal period. We report on the administration of HFOV in infants with pulmonary insufficiency after failure of conventional ventilatory support. 6 infants (aged 2-7 months, all former preterm babies) were referred to our hospital due to severe pneumonia after unsuccessful conservative management. Indications for HFOV were hypoxia (mean paO2 41.8 mm Hg with FiO2 = 0.95 and mean airway pressure = 16.6 cm H2O) and/or air leak syndrome. In all cases a sufficient oxygenation could be achieved by HFOV, followed then by stepwise reduction of FiO2 and MAP. The air leaks receded. After 12-178 h on HFOV a successful switchback to conventional ventilatory support (at FiO2 = 0.48 and MAP < 12 cm H2O) was possible, all infants were extubated 6-15 days later. Possible risks of HFOV are air leaks, a necrotizing tracheobronchitis and hemodynamic changes due to compression of the heart and great vessels. With the at the moment in Germany available oscillatory ventilators HFOV as a rescue therapy must be limited for infants with a body weight below 5-6 kg.

Serum concentrations of granulocyte colony-stimulating factor in healthy term and preterm neonates and in those with various diseases including bacterial infections.

The neonate is uniquely susceptible to severe and overwhelming bacterial infections. One of the most important deficits in the neonatal host defense system seems to be a quantitative and qualitative deficiency of the myeloid and the phagocytic system. Future optimal therapy of neonatal sepsis may include the use of adjuvant immunologic therapy. Granulocyte colony-stimulating factor (G-CSF) has been shown to induce neutrophilia and to enhance mature effector neutrophil function. To evaluate the role of G-CSF with respect to infection, we examined serum levels of G-CSF in term and preterm neonates, using an enzyme-linked immunosorbent assay method. G-CSF levels in healthy neonates showed peak levels up to 7 hours after birth, followed by an increase in total neutrophil cell (TNC) counts. Both G-CSF levels determined between 4 and 7 hours after birth and peak TNC counts correlated with the gestational age of the neonates. The state of nutrition, maternal treatment with glucocorticoids, maternal infection and hypertension, and the mode of delivery influenced peak G-CSF levels. Neonates with signs of infection between 4 and 7 hours after birth had higher levels of G-CSF than did healthy neonates (1,312 +/- 396 pg/mL v 176 +/- 19 pg/mL). In conclusion, the presented results of serum concentrations of G-CSF in relation to TNC counts and various diseases suggests an important role of G-CSF in the regulation of granulopoiesis during the neonatal period.

[Extracorporeal membrane oxygenation (ECMO) in acute cardiovascular failure caused by myocarditis]. / Extrakorporale Membranoxygenierung (ECMO) bei akutem Herz-Kreislauf-Versagen infolge Myokarditis.

Main indication for extracorporeal membrane oxygenation (ECMO) is respiratory failure in the newborn. Less frequently ECMO is used for cardiac support. We report on a 4 months old boy, who suddenly fell ill with an acute viral myocarditis and heart failure (left-ventricular shortening fraction lowered to 17%). After failure of conventional management and resuscitation (twice) due to asystolic, veno-arterial ECMO was installed for a total time of 4 days. Under ECMO there was complete recovery of left-ventricular function; the infant was discharged 1 month after admission to hospital. ECMO-therapy should not only be considered in children with respiratory failure, but also in those with potentially reversible cardiac failure.