A robust metabolomics approach for the evaluation of human embryos from in vitro fertilization.

The identification of the most competent embryos for transfer to the uterus constitutes the main challenge of in vitro fertilization (IVF). We established a metabolomic-based approach by applying Fourier transform infrared (FTIR) spectroscopy on 130 samples of 3-day embryo culture supernatants from 26 embryos that implanted and 104 embryos that failed. On examining the internal structure of the data by unsupervised multivariate analysis, we found that the supernatant spectra of nonimplanted embryos constituted a highly heterogeneous group. Whereas ∼40% of these supernatants were spectroscopically indistinguishable from those of successfully implanted embryos, ∼60% exhibited diverse, heterogeneous metabolic fingerprints. This observation proved to be the direct result of pregnancy's multifactorial nature, involving both intrinsic embryonic traits and external characteristics. Our data analysis strategy thus involved one-class modelling techniques employing soft independent modelling of class analogy that identified deviant fingerprints as unsuitable for implantation. From these findings, we could develop a noninvasive Fourier-transform-infrared-spectroscopy-based approach that represents a shift in the fundamental paradigm for data modelling applied in assisted-fertilization technologies.

Osseous vitality in single photon emission computed tomography/computed tomography (SPECT/CT) after balloon tibioplasty of the tibial plateau: a case series.

BACKGROUND: The minimally invasive, balloon-assisted reduction and cement-augmented internal fixation of the tibial plateau is an innovative surgical procedure for tibial plateau fractures. The close proximity of balloons and cement to the knee joint poses a potential risk for osteonecrosis; especially in the case of thin bone lamellae. However, there are no studies about the vitality of the cement-surrounding tissue after these tibioplasties. Therefore, our goal was to assess the osseous vitality after cement-augmented balloon tibioplasty using single photon emission computed tomography/computed tomography (SPECT/CT) in a series of patients. METHODS: This case series evaluated available consecutive patients, whose tibial plateau fractures were treated with balloon-assisted, cement-augmented tibioplasty and received a SPECT/CT. Primary outcome variables were osseous vitality on SPECT/CTs according to the semiquantitative tracer activity analysis. The mean uptake of eight tibial regions of interest was referenced to the mean uptake count on the same region of the contralateral leg to obtain a count ratio. Osteonecrosis was defined as a photopenic area or cold defect. Secondary variables included clinical and radiological follow-up data. Statistics were carried out in a descriptive pattern. RESULTS: Ten patients with a mean age of 59 years and a mean follow up of 18 months were included. Calcium phosphate (CaP) substitute bone cement was used in 60% and polymethyl methacrylate mixed with hydroxyapatite (PMMA/HA) bone cement in 40%. Normal to high SPECT/CT activity without photopenic areas were observed in all patients and the mean tracer activity ratio was four, indicating vital bone in all patients. There were no postoperative infections and only one 57 year old patient with hemineglect and CaP cement showed failed osseous consolidation. The mean Tegner and Lysholm as well as the Lysholm scores were three and 80, respectively. CONCLUSIONS: This novel study about cement-augmented balloon tibioplasties showed that osseous vitality remains intact according to SPECT/CT analysis; irrespective of the type of cement and even in the presence of thin bone lamellae. This procedure was safe and well-suited for lateral tibial plateau fractures in particular. Surgeons may consider using PMMA/HA bone cement for void filling in elderly fracture patients without concern about bone viability.

Cytostatic/cytotoxic effects of 5-fluorouridine nucleolipids on colon, hepatocellular, and renal carcinoma cells: in vitro identification of a potential cytotoxic multi-anticancer drug.

The insufficient penetration through the cell membranes is one of the major drawbacks of chemotherapeutics such as 5-fluorouracil (5-FU; 1). To improve the penetration, a useful strategy is the attachment of lipophilic moieties. Thus, we have synthesized a series of nucleolipid derivatives of 5-fluorouridine (5-FUrd; 2a), carrying lipophilic moieties at N(3) and/or at the 2',3'-O position, i.e., 3a, 3b, 4-7, and tested their cytostatic/cytotoxic activities towards three carcinoma cell lines (colon (HT-29), hepatocellular (HepG2), and renal (RENCA)) in comparison with 5-FU (1) and 5-FUrd (2a). After 48 h of incubation, four derivatives, 3a, 3b, 5, and 7, showed inhibitory effects on the survival of HT-29, HepG2, and RENCA cells. Additionally, to differentiate between anticancer and side-effects, we tested the cytotoxicity of the derivatives in human macrophages. Interestingly, the derivatives 4, 5, and 6 did not exhibit any effects on survival of THP-1 macrophages. Furthermore, we investigated the apoptosis induction of compound 1 and 2a, and the above-mentioned derivatives in HT-29 cells. Derivative 5 showed the highest significant (p<0.05; p<0.01) increase of the apoptosis at 80 µM after 2-h or 4-h treatment, as well as after 6-h incubation at 40 µM (p<0.05). Real-time PCR revealed that 40-µM derivative 5 showed a 1.8-fold increase of the pro-apoptotic caspase-3 gene and a twofold significant increase (p<0.01 and p<0.05 vs. control and 1, resp.) of the tumor suppressor TP53 gene, whereas the other compounds did not show any effect. We demonstrated that some 5-FUrd derivatives such as compound 5 are more effective than 5-FU or 5-FUrd concerning a cytotoxic (vs. cytostatic (5-FU, 5-FUrd)) effect on different cancer cell lines, but without cytotoxic side-effects on differentiated macrophages. Thus, compound 5 is suggested as a novel potent cytotoxic multi-anti-cancer drug.

Synthesis of 5-fluorouridine nucleolipid derivatives and their cytostatic/cytotoxic activities on human HT-29 colon carcinoma cells.

One of the major drawbacks of chemotherapeutics is their insufficient penetration through cell membranes due to a high hydrophobicity. Thus, we have synthesized a series of selected nucleolipid derivatives of 5-fluorouridine (5-FUrd; 2a), carrying lipophilic moieties at N(3) and/or in the 2',3'-O-position (i.e., 3a-7a and 3c), and tested their cytostatic/cytotoxic activities using HT-29 human colon carcinoma cells, in comparison with, e.g., 5-FU (1) and 5-FUrd (2a). Incorporation and intracellular localization of the substances under test were performed after conjugation with the fluorochrome Atto 425. We showed that all 5'-O-labelled Atto 425 derivatives were incorporated by the human HT-29 cells and accumulated in their cytoplasm. Moreover, after 24-h treatment of HT-29 human colon carcinoma cells, 1 or 2a (10, 20, 40, or 80 µM) revealed a significant (14-23 or 33-45%, resp.) decrease of the viability in comparison with the (negative) control. Interestingly, derivatives 3a and 3c (40 and 80 µM) led to a significant (77-95 or 89-96%, resp.) inhibition of survival of human HT29 cells, i.e., these two substances were ca. 63-72% or ca. 75%, respectively more effective than 5-FU (1; positive control). Furthermore, derivative 5a showed a significant, i.e., 30 and 86%, inhibition of the survival at 40 and 80 µM, respectively in comparison with the (negative) control. Some synthesized 5-FUrd derivatives turned out to be more effective than 5-FU (1) or 5-FUrd (2a).

Classification of trochlear dysplasia as predictor of clinical outcome after trochleoplasty.

PURPOSE: Sulcus-deepening trochleoplasty restores the trochlear groove in patients with patellofemoral instability and underlying trochlear dysplasia. There are types of dysplasia both with (B and D) and without (A and C) a supratrochlear spur. The aim of this study was to identify influencing factors for the clinical outcome following trochleoplasty. METHODS: Forty-four knees in 38 patients who underwent trochleoplasty for instability (type A in 9, B in 15, C in 9 and D in 11 knees) were assessed clinically with the Kujala score and radiologically with radiographs and MRI. The median follow-up was 4 (2-7.8) years. RESULTS: At follow-up, the median Kujala score had improved from 68 (29-84) to 90 (42-100) points (P < 0.001). Instability (P < 0.001) and pain (P = 0.027) decreased significantly, but in 3 knees, pain was worse postoperatively. Twenty-seven knees were ranked as excellent, 10 as good, 2 as fair and 5 as poor. Overall, dysplasia types B and D benefited more from surgery than types A and C. The postoperative MRI revealed no chondrolysis or subchondral necrosis, but deterioration of cartilage on the lateral trochlear facet was identified. CONCLUSION: Trochleoplasty is a useful and reliable surgical technique to improve patellofemoral instability in patients with a dysplastic trochlea. While improved stability is predictable, pain is less predictable and may even increase following surgery. The overall results were directly dependent on the type of the dysplasia, with a significantly better clinical outcome in type B and D. The clinical relevance of this study is that severe dysplasia can successfully be treated with trochleoplasty. LEVEL OF EVIDENCE: III.

Medial acetabular wall breach in total hip arthroplasty - is full-weight-bearing possible?

BACKGROUND: A breach of the medial acetabular wall is a phenomenon seen frequently due to over-reaming during total hip arthroplasty (THA). The consequences of this issue are not fully understood particularly in cementless THA. A retrospective study was performed to answer whether: immediate postoperative full-weight-bearing in the presence of a medial acetabular wall breach after THA results in more short-term revisions of the acetabular component, and increases the risk for migration of the acetabular component? HYPOTHESIS: Immediate full-weight-bearing in the presence of a medial breach is not associated with an increased likelihood for acetabular-related revision surgery or migration of the cup. PATIENTS AND METHODS: In this retrospective cohort study, consecutive patients (n=95; mean age 68±13 years; 67 female) who underwent THA with an uncemented acetabular component were identified and a retrospective chart review was performed (follow-up 23±17 months, range 6 to 79 months). The presence of a postoperative radiographic medial acetabular breach was documented and the need for revision surgery and the rate of acetabular component migration were assessed during follow-up. RESULTS: Some extent of radiographic medial acetabular wall breach was seen in 26/95 patients (27%). With regard to the primary outcome, 2/95 patients (2%) required revision surgery during follow-up. All revision surgeries occurred in the group without a medial breach (p=0.280) for causes related to the femoral or the head components. Persistent pain was present in 1/26 patients (3.8%) in the medial breach group and 8/69 patients in the control group (11.6%; p=0.436). In the radiographic follow-up (n=81), there was no significant difference between the control group and the medial breach group with regard to cup migration (Δ ilio-ischial overlap [distance between the ilio-ischial line and a parallel line tangential to the acetabular cup on AP views]: -0.5±0.9mm [range, -2.9 to 0.8] vs. -0.3±1.7mm [range, -1.9 to 2.2], Δ overlap tangent [defined as the distance between the two crossings of ilio-ischial line and the acetabular component on AP views]: -2.2±6.1mm [range, -21.4 to 0.0] vs. 0.4±6.9mm [range, -6.2 to 17.6]). Similarly, according to variation in the ilio-ischial overlap distance between postoperative and follow-up on pelvic AP views, 0/56 hips (0%) had cup migration ≥ 5mm in the control group versus 1/25 (4%) in the medial breach cohort (p=0.3). DISCUSSION: In this retrospective observation of patients with immediate postoperative full-weight-bearing after THA, a radiographic breach of the medial acetabular wall was not associated with an increased risk for short-term revision surgery or radiographic migration at follow-up. According to the findings of this study and in the light of previous biomechanical studies, there is no clear evidence for postoperative partial weight-bearing in case of a medial breach as far as the surgeon feels that the acetabular component is stable. LEVEL OF EVIDENCE: IV, Retrospective cohort study.

Ground-based detection of an extended helium atmosphere in the Saturn-mass exoplanet WASP-69b.

Hot gas giant exoplanets can lose part of their atmosphere due to strong stellar irradiation, and these losses can affect their physical and chemical evolution. Studies of atmospheric escape from exoplanets have mostly relied on space-based observations of the hydrogen Lyman-α line in the far ultraviolet region, which is strongly affected by interstellar absorption. Using ground-based high-resolution spectroscopy, we detected excess absorption in the helium triplet at 1083 nanometers during the transit of the Saturn-mass exoplanet WASP-69b, at a signal-to-noise ratio of 18. We measured line blueshifts of several kilometers per second and posttransit absorption, which we interpret as the escape of part of the atmosphere trailing behind the planet in comet-like form.

Diagnosing benign and malignant lesions in breast tissue sections by using IR-microspectroscopy.

The collection of IR spectra through microscope optics and the visualization of the IR data by IR imaging represent a visualization approach, which uses infrared spectral features as a native intrinsic contrast mechanism. To illustrate the potential of this spectroscopic methodology in breast cancer research, we have acquired IR-microspectroscopic data from benign and malignant lesions in breast tissue sections by point microscopy with spot sizes of 30-40 microm. Four classes of distinct breast tissue spectra were defined and stored in the data base: fibroadenoma (a total of 1175 spectra from 14 patients), ductal carcinoma in situ (a total of 1349 spectra from 8 patients), connective tissue (a total of 464 spectra), and adipose tissue (a total of 146 spectra). Artifical neural network analysis, a supervised pattern recognition method, was used to develop an automated classifier to separate the four classes. After training the artifical neural network classifier, infrared spectra of independent external validation data sets ("unknown spectra") were analyzed. In this way, all spectra (a total of 386) taken from micro areas inside the epithelium of fibroadenomas from 4 patients were correctly classified. Out of the 421 spectra taken from micro areas of the in situ component of invasive ductal carcinomas of 3 patients, 93% were correctly identified. Based on these results, the potential of the IR-microspectroscopic approach for diagnosing breast tissue lesions is discussed.

Safety of total hip arthroplasty for femoral neck fractures using the direct anterior approach: a retrospective observational study in 86 elderly patients.

BACKGROUND: The femoral neck fracture is one of the most common fractures in the elderly. A variety of methods and approaches are used to treat it. Total hip arthroplasty is a preferred approach in independent, mobile, elderly patients, given its more favorable long-term outcome. Our hypothesis is that the direct anterior approach in geriatric trauma patients has a lower dislocation-rate with the advantage of early recovery due to a muscle sparing approach and therefore early possible full weight-bearing. METHODS: Patients were retrospectively sought who suffered a femoral neck fracture from 2008 to 2013. All patients were treated through a direct anterior approach and using the same brand of implants. Medical history, standardized physical exam, conventional pelvic plain and axial hip x-rays, Harris Hip Score, Merle D'Aubigné and Postel and SF-36 were assessed. RESULTS: Eighty-six patients were included in the study with a mean age of seventy-five years. The mortality rate was 16.7 %. Complications were encountered in nineteen patients (22.0 %) who needed operative revision and one postoperative complication (1.2 %) which could be handled conservatively. There were five intraoperative complications (5.8 %), two dislocations (2.3 %), one aseptic loosening in a non-cemented stem (1.2 %), six periprosthetic fractures in non-cemented stems (6.9 %), one displacement of a non-cemented cup (1.2 %), two early infections (2.3 %) and three hematomas (3.5 %) recorded. CONCLUSIONS: Although the direct anterior approach is associated with a rather long learning curve we have found it to preserve the soft-tissues with no injury to abductors. It therefore shows an early advantage in elderly patients in terms of early recovery and therefore early possible full weight-bearing. Fracture treatment with dual mobility cups might lead to lower dislocation rates, but are associated with higher costs. Due to higher complication rates in non-cemented versus cemented shafts, we have changed our practice towards favoring cemented femoral stems in patients with suspected or manifest osteoporosis.

Is total hip arthroplasty safely performed in lung transplant patients? Current experience from a retrospective study of the Zurich lung transplant cohort.

BACKGROUND: In recent years, the number of lung transplants has increased rapidly, with higher quality of life and improved survival rates in transplant recipients, including patients with advanced age. This, in turn, means that more transplant recipients will seek musculoskeletal care to treat degenerative joint disease and also trauma incidents. Safety concerns regarding elective and posttraumatic hip arthroplasty in transplant patients include an increased risk of infection, wound healing problems, periprosthetic fractures and loosening of the implants. METHODS: Clinical outcomes and safety aspects were retrospectively reviewed for five primary total hip arthroplasties (THA) in lung transplant recipients with minimal follow-up of two years at average of 2.6 (2-11) years. Patients were recruited from the Zurich Lung Transplant Center comprising of a cohort of 253 patients between January 1st, 2004 and December 31st, 2013. RESULTS: All five patients subjectively reported excellent outcomes after THA with a final average Harris Hip Score of 97 (86-100). One 71-year-old patient died 26 months after THA unrelated to arthroplasty. One superficial wound healing disturbance was documented. No periprosthetic fractures, no dislocations, no periprosthetic infections, no further revision surgery, no implant loosening was observed. CONCLUSIONS: In conclusion, THA can be safely and successfully performed even in lung transplant patients under long-term immunosuppressive therapy and polymedication, provided a multidisciplinary approach can be granted.

Cellular fine structures and histochemical reactions in the tissue of a cypress twig preserved in Baltic amber.

A twig of a cypress plant preserved for ca. 45 Myr in Baltic amber was analysed by light and electron microscopy. Cross-sections of the whole plant showed an almost intact tissue of the entire stem and leaves, revealing, to our knowledge, the oldest and most highly preserved tissue from an amber inclusion reported so far. The preparations are based on a new technique of internal imbedding, whereby the hollow spaces within the inclusion are filled with synthetic resin which stabilizes the cellular structures during the sectioning procedure. Cytological stains applied to the sections reacted with cell walls and nuclei. A strong green auto-fluorescence of the cuticle and the resin canals in the leaves was observed. Transmission electron micrographs revealed highly preserved fine structures of cell walls, membranes and organelles. The results were compared with taxonomically related recent Glyptostrobus and Juniperus plants.

Schistosoma mansoni and Schistosoma haematobium: identification and characterization of glycoconjugate antigens in the hemolymph of infected vector snails.

Two carbohydrate epitopes were identified by monoclonal antibodies (KCS and E2) and characterized with respect to their immunoreactivity, monosaccharide structure, and location. Immunofluorescence demonstrated the presence of both epitopes on the surfaces of sporocysts, cercariae, and miracidia of Schistosoma mansoni, Schistosoma haematobium, and Schistosoma japonicum. However, spatial distribution and density of expression varied among species and developmental stages, and neither epitope was detectable on adult worm surfaces. Both glycans were found in the hemolymph of infected, but not uninfected, intermediate snail hosts. The presence of epitopes in hemolymph, as well as in schistosome eggs, is species-specific for KCS, recognizing only S. mansoni, and partly specific for E2, which reacted predominantly with S. haematobium. Immunoaffinity purification of target antigens for KCS and E2 from hemolymph of infected Biomphalaria and Bulinus, respectively, followed by carbohydrate composition analysis revealed a high content of fucose in both glycans. Methylation analysis demonstrated exclusively terminal fucose for the target antigen of KCS and terminal as well as internal fucose for the one of E2. Removal of terminal fucose abolished reactivity with both monoclonal antibodies. Both glycans are different from previously characterized schistosome carbohydrates. Their biological function(s) remain to be defined.

Reduced cytotoxicity of polyhexamethylene biguanide hydrochloride (PHMB) by egg phosphatidylcholine while maintaining antimicrobial efficacy.

Liposomes or oil-in-water emulsions containing egg yolk phosphatidylcholine (EPC) were combined with aqueous polyhexamethylene biguanide hydrochloride (PHMB). The bactericidal activity of these preparations against Pseudomonas aeruginosa and Staphylococcus aureus as well as their cytotoxicity on cultured murine fibroblasts (L929 cells) was then assayed for either 30 min or 60 min in the presence of cell culture medium containing 10% fetal bovine serum as surrogate for wound fluid. We used two assay designs: in the first bactericidal activity and cytotoxicity were determined in separate experiments; in the second both were determined in one experiment. Combining PHMB and EPC containing o/w emulsions or liposomes protects mammalian cells without neutralizing the antiseptic effect. From all tested combinations the o/w emulsions containing 0.05% PHMB proved to be superior in this respect to the aqueous preparation.

A high-throughput microcultivation protocol for FTIR spectroscopic characterization and identification of fungi.

Characterization and identification of fungi in food industry is an important issue both for routine analysis and trouble-shooting incidences. Present microbial techniques for fungal characterization suffer from a low throughput and are time consuming. In this study we present a protocol for high-throughput microcultivation and spectral characterization of fungi by Fourier transform infrared spectroscopy. For the study 11 species of in total five different fungal genera (Alternaria, Aspergillus, Mucor, Paecilomyces, and Phoma) were analyzed by FTIR spectroscopy. All the strains were isolated from trouble-shooting incidents in the production of low and high acid beverages. The cultivation was performed in malt extract broth (liquid medium) in a Bioscreen C system, allowing high-throughput cultivation of 200 samples at the same time. Mycelium was subsequently investigated by high-throughput Fourier transform infrared spectroscopy. Four spectral regions, fatty acids + lipid (3200-2800 cm(-1), 1300-1000 cm(-1)), protein-lipid (1800-1200 cm(-1)), carbohydrates (1200-700 cm(-1)) and "finger print" (900-700 cm(-1)) were evaluated for reproducibility and discrimination ability. The results show that all spectral regions evaluated can be used as spectroscopic biomarkers for differentiation of fungi by FTIR. The influence of different growth times on the ability of species discrimination by FTIR spectroscopy was investigated, and an optimal separation of all five genera was observed after five days of growth. This work presents a novel concept for high-throughput cultivation of fungi for FTIR spectroscopy that enables characterization or identification of hundreds of strains per day.

Type-IV pili spectroscopic markers: applications in the quantification of piliation levels in Moraxella bovis cells by a FT-IR ANN-based model.

Type-IV pili are cell surface organelles found in a wide variety of Gram-negative bacteria. They have traditionally been detected by electron microscopy and ELISA techniques. However, these methodologies are not appropriate for the rapid discrimination and quantification of piliated and nonpiliated cells in industrial or field conditions. Here, the analysis of FT-IR spectra of piliated, nonpiliated and sheared Moraxella bovis cells, together with purified pili suspensions spectra, allowed the identification of 3 IR regions associated to spectroscopic markers of Type-IV pili: 1750-1600, 1450-1350 and 1280-950 cm(-1). Such IR-specific markers were found for piliated cells grown in different culture systems (liquid or solid media), independently of the strain or pili serotype. They were also sensitive to pili expression levels. Therefore, on the bases of these specific spectral features, an FT-IR ANN-based model was developed to classify piliation levels in 5 distinct groups. An overall classification rate of almost 90% demonstrates the strong potential of the ANN system developed to monitor M. bovis cultures in vaccine production.

Regulation of photosynthesis in the unicellular acidophilic red alga Galdieria sulphuraria.

Extremophilic organisms are gaining increasing interest because of their unique metabolic capacities and great biotechnological potential. The unicellular acidophilic and mesothermophilic red alga Galdieria sulphuraria (074G) can grow autotrophically in light as well as heterotrophically in the dark. In this paper, the effects of externally added glucose on primary and secondary photosynthetic reactions are assessed to elucidate mixotrophic capacities of the alga. Photosynthetic O2 evolution was quantified in an open system with a constant supply of CO2 to avoid rapid volatilization of dissolved inorganic carbon at low pH levels. In the presence of glucose, O2 evolution was repressed even in illuminated cells. Ratios of variable to maximum chlorophyll fluorescence (Fv/Fm) and 77 K fluorescence spectra indicated a reduced photochemical efficiency of photosystem II. The results were corroborated by strongly reduced levels of the photosystem II reaction centre protein D1. The downregulation of primary photosynthetic reactions was accompanied by reduced levels of the Calvin Cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Both effects depended on functional sugar uptake and are thus initiated by intracellular rather than extracellular glucose. Following glucose depletion, photosynthetic O2 evolution of illuminated cells commenced after 15 h and Rubisco levels again reached the levels of autotrophic cells. It is concluded that true mixotrophy, involving electron transport across both photosystems, does not occur in G. sulphuraria 074G, and that heterotrophic growth is favoured over autotrophic growth if sufficient organic carbon is available.

Free functional gracilis muscle transfer for reconstruction of massive ectopic calcification following a lower extremity fracture: a case report and overview of the literature.

We present a patient with massive posttraumatic ectopic calcification in the lower extremity. The patient complained of an increasing tender soft tissue mass with signs of acute inflammation and foot dorsiflexion weakness following a tibial fracture for 34 years. A large radiopaque mass was resected and reconstructed using a free functional gracilis muscle transfer, resulting in recovery of stable soft tissue and foot dorsiflexion. The resected material showed highly fibrosed soft tissue with extensive dystrophic calcifications. There were no sign of recurrence at 18-month follow-up.

Detection of preclinical scrapie from serum by infrared spectroscopy and chemometrics.

In this study we describe a methodology for diagnosing preclinical scrapie infection in hamsters from serum by a combination of Fourier-transform infrared (FT-IR) spectroscopy and chemometrics. Syrian hamsters (Mesocricetus auratus) were orally inoculated with the 263K scrapie agent, or mock-infected, and sera were obtained at 70, 100 and 130 days post infection (dpi) and at the terminal stage of scrapie (160 +/- 10 dpi). The analysis of hamster sera by FT-IR spectroscopy and artificial neural networks (ANN) confirmed results from earlier studies which had indicated the existence of disease-related structural and compositional alterations in the sera of infected donors in the terminal stage of scrapie [Schmitt et al. (2002) Anal Chem 74:3865-3868]. Experimental data from sera of animals in the preclinical stages of scrapie revealed subtle but reproducible spectral variations that permitted the identification of a preclinical scrapie infection at 100 dpi and later, but not at 70 dpi. The IR spectral features that were discriminatory for the preclinical stages differed from those of the terminally ill individuals. In order to reliably identify scrapie-negative as well as preclinical (100 and 130 dpi) and terminal scrapie-positive animals, a hierarchical classification system of independent artificial neural networks (ANN) was established. A "toplevel" ANN was designed which discriminates between animals in the terminal stage of scrapie and preclinical scrapie-positive or control animals. Spectra identified by the "toplevel" ANN as preclinical or controls were then further analyzed by a second classifier, the "sublevel" ANN. Using independent external validation procedures, the toplevel classifier produced an overall classification accuracy of 98%, while the sublevel classifier yielded an accuracy of 93%, indicating that scrapie-specific serum markers were also present for preclinical disease stages. Possible spectral markers responsible for the discrimination capacity of the two different ANNs are discussed.

Differentiation of Listeria monocytogenes serovars by using artificial neural network analysis of Fourier-transformed infrared spectra.

A classification system based on Fourier transform infrared (FTIR) spectroscopy combined with artificial neural network analysis was designed to differentiate 12 serovars of Listeria monocytogenes using a reference database of 106 well-defined strains. External validation was performed using a test set of another 166 L. monocytogenes strains. The O antigens (serogroup) of 164 strains (98.8%) could be identified correctly, and H antigens were correctly determined in 152 (91.6%) of the test strains. Importantly, 40 out of 41 potentially epidemic serovar 4b strains were unambiguously identified. FTIR analysis is superior to PCR-based systems for serovar differentiation and has potential for the rapid, simultaneous identification of both species and serovar of an unknown Listeria isolate by simply measuring a whole-cell infrared spectrum.