Microbial Detection with Low Molecular Weight RNA

The need to monitor microorganisms in the environment has increased interest in assays based on hybridization probes that target nucleic acids (e.g., rRNA). We report the development of liquid-phase assays for specific bacterial 5S rRNA sequences or similarly sized artificial RNAs (aRNAs) using molecular beacon technology. These beacons fluoresce only in the presence of specific target sequences, rendering as much as a 27-fold fluorescence enhancement. The assays can be used with both crude cell lysates and purified total RNA preparations. Minimal sample preparation (e.g., heating to promote leakage from cells) is sufficient to detect many Gram-negative bacteria. Using this approach it was possible to detect an aRNA-labeled Escherichia coli strain in the presence of a large background of an otherwise identical E. coli strain. Finally, by using a longer wavelength carboxytetramethylrhodamine beacon it was possible to reduce the fraction of the signal due to cellular autofluorescence to below 0.5%. Received: 13 March 2001 / Accepted: 3 May 2001     

A duplication dup(4)(q28q35.2) de novo in a newborn

We report here a case of a newborn with hypotrophy and somatic stigmatization: microcephaly, facial dysmorphism, heart defect and immunodeficiency syndrome. The proband's karyotype was 46,XY,dup(4)(q28q35.2) de novo with chromosomal breaks in 4% of metaphases. We demonstrate the usefulness of a combination of physical examination, classical cytogenetics, FISH and PCR techniques in order to establish correct diagnosis because of overlap of some clinical and cytogenetic features of Nijmegen breakage syndrome (NBS) and duplication 4q in our patient. Although FISH technique detected translocation t(14q;21q) in 4 metaphases, deletion 657del5 in exon 6 of the NBS1 gene associated with NBS in Slavic population was not confirmed. We compare in this report similarity of the clinical picture of our patient, NBS cases and other patients carrying a duplication of the distal part of 4q as described in the literature.     

Evolution of middle-late Pleistocene human cranio-facial form: A 3-D approach

The classification and phylogenetic relationships of the middle Pleistocene human fossil record remains one of the most intractable problems in paleoanthropology. Several authors have noted broad resemblances between European and African fossils from this period, suggesting a single taxon ancestral to both modern humans and Neanderthals. Others point out ‘incipient’ Neanderthal features in the morphology of the European sample and have argued for their inclusion in the Neanderthal lineage exclusively, following a model of accretionary evolution of Neanderthals. We approach these questions using geometric morphometric methods which allow the intuitive visualization and quantification of features previously described qualitatively. We apply these techniques to evaluate proposed cranio-facial ‘incipient’ facial, vault, and basicranial traits in a middle-late Pleistocene European hominin sample when compared to a sample of the same time depth from Africa. Some of the features examined followed the predictions of the accretion model and relate the middle Pleistocene European material to the later Neanderthals. However, although our analysis showed a clear separation between Neanderthals and early/recent modern humans and morphological proximity between European specimens from OIS 7 to 3, it also shows that the European hominins from the first half of the middle Pleistocene still shared most of their cranio-facial architecture with their African contemporaries.     

Poly(L-lysine)-modified iron oxide nanoparticles for stem cell labeling

New surface-modified iron oxide nanoparticles were developed by precipitation of Fe(II) and Fe(III) salts with ammonium hydroxide and oxidation of the resulting magnetite with sodium hypochlorite, followed by the addition of poly( L-lysine) (PLL) solution. PLL of several molecular weights ranging from 146 ( L-lysine) to 579 000 was tested as a coating to boost the intracellular uptake of the nanoparticles. The nanoparticles were characterized by TEM, dynamic light scattering, FTIR, and ultrasonic spectrometry. TEM revealed that the particles were ca. 6 nm in diameter, while FTIR showed that their surfaces were well-coated with PLL. The interaction of PLL-modified iron oxide nanoparticles with DMEM culture medium was verified by UV-vis spectroscopy. Rat bone marrow stromal cells (rMSCs) and human mesenchymal stem cells (hMSC) were labeled with PLL-modified iron oxide nanoparticles or with Endorem (control). Optical microscopy and TEM confirmed the presence of PLL-modified iron oxide nanoparticles inside the cells. Cellular uptake was very high (more than 92%) for PLL-modified nanoparticles that were coated with PLL (molecular weight 388 00) at a concentration of 0.02 mg PLL per milliliter of colloid. The cellular uptake of PLL-modified iron oxide was facilitated by its interaction with the negatively charged cell surface and subsequent endosomolytic uptake. The relaxivity of rMSCs labeled with PLL-modified iron oxide and the amount of iron in the cells were determined. PLL-modified iron oxide-labeled rMSCs were imaged in vitro and in vivo after intracerebral grafting into the contralateral hemisphere of the adult rat brain. The implanted cells were visible on magnetic resonance (MR) images as a hypointense area at the injection site and in the lesion. In comparison with Endorem, nanoparticles modified with PLL of an optimum molecular weight demonstrated a higher efficiency of intracellular uptake by MSC cells.     

In vivo dynamics of Drosophila nuclear envelope components

Nuclear pore complexes (NPCs) are multisubunit protein entities embedded into the nuclear envelope (NE). Here, we examine the in vivo dynamics of the essential Drosophila nucleoporin Nup107 and several other NE-associated proteins during NE and NPCs disassembly and reassembly that take place within each mitosis. During both the rapid mitosis of syncytial embryos and the more conventional mitosis of larval neuroblasts, Nup107 is gradually released from the NE, but it remains partially confined to the nuclear (spindle) region up to late prometaphase, in contrast to nucleoporins detected by wheat germ agglutinin and lamins. We provide evidence that in all Drosophila cells, a structure derived from the NE persists throughout metaphase and early anaphase. Finally, we examined the dynamics of the spindle checkpoint proteins Mad2 and Mad1. During mitotic exit, Mad2 and Mad1 are actively imported back from the cytoplasm into the nucleus after the NE and NPCs have reformed, but they reassociate with the NE only later in G1, concomitantly with the recruitment of the basket nucleoporin Mtor (the Drosophila orthologue of vertebrate Tpr). Surprisingly, Drosophila Nup107 shows no evidence of localization to kinetochores, despite the demonstrated importance of this association in mammalian cells.     

Poor survival of ABG I hip prosthesis in younger patients

Background: Hydroxyapatite coated (HAC) hip implants have been used in clinical practice for more than two decades. However, the majority of studies have reported only intermediate term outcomes that are not reliable for predicting long-term behavior in all implants. The aim of this study was to determine the performance of HAC total hip arthroplasty in younger patients over a 10-year follow-up period. Methods and Results: This was an observational retrospective study of a 137 consecutive hips with the ABG I prosthesis. Of these, 128 were available for the last investigation. Median duration of follow-up was 10.9 years. The mean age at time of index surgery was 46+/-6.7 years. Probability of implant survival was estimated using the Kaplan-Meier method. The overall 12-year cumulative survival was 0.55 (95% CI, 0.443-0.659). Periprosthetic osteolysis (57 %) was the most frequent reason for failure followed by aseptic loosening (28 %). When only aseptic loosening was included in the analysis, the same figures for cup and stem were 0.873 (95% CI, 0.808-0.938) and 0.992 (95% CI, 0.976- 1.0), respectively. Patients with a smaller cup size were those at high risk for revision due to wear-related complications (odds ratio, OR=4.3; 95% CI, 1.734-10.555). Conclusion: This study reports one of the poorest 12-year survivorship data for cementless acetabular component in the literature. The main reason for premature failure was osteolysis, strongly related to high wear rate of polyethylene.     

MCL-1 (myosin light chains-1) in differential diagnosis of dyspnea

Myosin light chains-1 (MLC-1) have been recently associated with the markers of heart function (NYHA, LVEF, NT-proBNP). Verification of the relationship between markers of heart function (New York Heart Association classification (NYHA), left ventricle ejection fraction determination (LVEF), N terminal prohormone of natriuretic peptide B type BNP (NT-proBNP) and concentrations of myosin light chains-1 (MLC-1) was assessed. Patients examined for dyspnea without signs of acute coronary syndrome. All patients underwent echocardiography (calculation of left ventricle ejection fraction--LVEF) and in the serum of all subjects NT-proBNP (ELEIA) and MLC-1 (ELISA) were determined. In the 38 patients (21 men, 17 women), mean age of 58 years (+/-12 years as 1 SD), a significant negative correlation was found between NT-proBNP and LVEF (r = - 0.47; p = 0.02, Spearman). The median levels of NT pro-BNP were closely associated with NYHA classification (type II--584 ng/l, type III--2792 ng/l, type IV--6400 ng/l; p < 0.05). Individuals with clinical NYHA IV differed significantly in median MLC-1 concentrations from persons with clinical NYHA classification II and III (type II--5.7 ng/l, type III--8.9 ng/l, type IV--17 ng/l; p < 0.05). A significant negative correlation between MLC-1 and LVEF (-0.35; p < 0.03) and significant positive correlations between MLC-1 and NT-proBNP (0.42; p < 0.012) were found. In conclusion MLC-1 cannot be used as a diagnostic marker in differential diagnosis of dyspnea.     

Possibilities and problems with identification and determination of "new" hypnotics

Authors discuss problems with identification and determination of flunitrazepam and zolpidem in biological material (BM). Over the recent years, these two structurally different substances have become the most frequently used as well as abused hypnotic drugs. This study presents applicability of immunochemical methods in the screening of flunitrazepam, one of the most commonly prescribed drugs among the benzodiazepines. Herein described techniques, a liquid-liquid (L-L) extraction, solid phase extraction (SPE) and the so-called "freeze out" method are used for isolation of the above mentioned compounds from BM. Besides the thin layer chromatography (TLC) and gas chromatography - mass spectrometry (GC-MS) applied in qualitative analysis, the study also describes a gas chromatography with electron capture detector (GC-ECD) and gas chromatography with nitrogen phosphorus detector (GC-NPD) optimized for the determination of flunitrazepam and zolpidem in blood (serum). Successful analyses of these two substances are of major importance, especially in interpreting the results of forensic toxicological examinations.