Parvovirus Induced Alterations in Nuclear Architecture and Dynamics

The nucleus of interphase eukaryotic cell is a highly compartmentalized structure containing the three-dimensional network of chromatin and numerous proteinaceous subcompartments. DNA viruses induce profound changes in the intranuclear structures of their host cells. We are applying a combination of confocal imaging including photobleaching microscopy and computational methods to analyze the modifications of nuclear architecture and dynamics in parvovirus infected cells. Upon canine parvovirus infection, expansion of the viral replication compartment is accompanied by chromatin marginalization to the vicinity of the nuclear membrane. Dextran microinjection and fluorescence recovery after photobleaching (FRAP) studies revealed the homogeneity of this compartment. Markedly, in spite of increase in viral DNA content of the nucleus, a significant increase in the protein mobility was observed in infected compared to non-infected cells. Moreover, analyzis of the dynamics of photoactivable capsid protein demonstrated rapid intranuclear dynamics of viral capsids. Finally, quantitative FRAP and cellular modelling were used to determine the duration of viral genome replication. Altogether, our findings indicate that parvoviruses modify the nuclear structure and dynamics extensively. Intranuclear crowding of viral components leads to enlargement of the interchromosomal domain and to chromatin marginalization via depletion attraction. In conclusion, parvoviruses provide a useful model system for understanding the mechanisms of virus-induced intranuclear modifications.     

A musculoskeletal finite element model of rat knee joint for evaluating cartilage biomechanics during gait

Abnormal loading of the knee due to injuries or obesity is thought to contribute to the development of osteoarthritis (OA). Small animal models have been used for studying OA progression mechanisms. However, numerical models to study cartilage responses under dynamic loading in preclinical animal models have not been developed. Here we present a musculoskeletal finite element model of a rat knee joint to evaluate cartilage biomechanical responses during a gait cycle. The rat knee joint geometries were obtained from a 3-D MRI dataset and the boundary conditions regarding loading in the joint were extracted from a musculoskeletal model of the rat hindlimb. The fibril-reinforced poroelastic (FRPE) properties of the rat cartilage were derived from data of mechanical indentation tests. Our numerical results showed the relevance of simulating anatomical and locomotion characteristics in the rat knee joint for estimating tissue responses such as contact pressures, stresses, strains, and fluid pressures. We found that the contact pressure and maximum principal strain were virtually constant in the medial compartment whereas they showed the highest values at the beginning of the gait cycle in the lateral compartment. Furthermore, we found that the maximum principal stress increased during the stance phase of gait, with the greatest values at midstance. We anticipate that our approach serves as a first step towards investigating the effects of gait abnormalities on the adaptation and degeneration of rat knee joint tissues and could be used to evaluate biomechanically-driven mechanisms of the progression of OA as a consequence of joint injury or obesity.     

Strength and power profiles of the lower and upper extremities in master throwers at different ages

Thirty-two master athletes (shot put, discus, and hammer throw) were divided into 4 groups according to their age (T40 [40 years of age], 50 [50 years of age], 60 [60 years of age], and 75 [75 years of age]). Twenty-eight age-matched men served as controls (C40 [40 years of age], 50 [50 years of age], 60 [60 years of age], and 75 [75 years of age]). The subjects were tested for maximal isometric strength of the lower and upper extremities. Power was measured by performing jump squats and bench press in the Smith machine with the load of 60% of 1 repetition maximum. Electromyographic (EMG) activity was recorded from 6 different muscles. The muscle thickness of vastus lateralis and intermedius (VL+VI) and triceps brachii (TB) was measured by ultrasound. Maximal strength differed (p < 0.05- 0.001) in all testing actions between T40 and T60 and T40 and T75 as well as between T and C groups. Both VL+VI and TB thickness in T40 was greater (p < 0.05-0.01) than in T60 and T75 and in T was larger than in C groups. Average force during the first 500 milliseconds (ms) was higher (p < 0.05-0.001) in T40 compared to T50, T60, and T75 in bilateral leg extension, biceps curl, and especially in unilateral knee flexion. T40 produced higher power than the other groups (p < 0.05-0.001). The relative agonist EMG activation (VL) in leg extension during the first 100 ms compared to maximum activation was lower (p < 0.05) in T50, T60, and T75, but not in T40. The present data indicate that maximal strength and muscle thickness as well as explosive strength and power characteristics decline with aging also in master athletes who carry out strength training and throwing exercises actively over several decades. Nevertheless, in master athletes, maximal strength and muscle mass as well as explosive force production of the upper and lower extremities seem to be at remarkably higher levels than those recorded for age-matched control men.     

Carbon storage change and δ13C transitions of peat columns in a partially forestry-drained boreal bog

Background and aims

In forestry-drained peatlands, drying leads to changes in C cycling which could affect peat δ¹³C. Furthermore, the δ¹³C profile of the entire peat column may reveal effects of earlier climatic periods.

Methods

We measured peat δ¹³C and C inventories in adjacent peat profiles, two collected from undrained and two from the drained side of a bog that was partially ditch-drained 37 years earlier. The cores were sliced into 10-cm subsamples for analyses; matching of the profiles based on surface levelling, peat stratigraphic correlation and a horizontal ash layer found in both profiles.

Results

Surface subsidence of 30 cm was observed in the dried site and the uppermost 160 cm in the undrained site contained an excess of 5.9 kg m⁻² of C compared with the corresponding strata of the ditch-drained site. The δ¹³C values increased but markedly only in the thin surface layer of the drained site, indicating low δ¹³C of the missing C (ca. –30‰). In the deeper strata, dating to Mid-Holocene, high dry bulk density, C%, N%, humification index and low C/N ratio were connected to low δ¹³C of peat.

Conclusions

Drainage of 37 years increased δ¹³C values in the upper peat profile of the drained bog and led to the selective loss of ¹³C depleted C. Results indicate that C balance studies can be aided by C isotope analyses. Low δ¹³C values in the peat profile indicate the existence of a wet fen stage during the moist and warm period during Mid-Holocene.

     

Validated semiquantitative/quantitative screening of 51 drugs in whole blood as silylated derivatives by gas chromatography-selected ion monitoring mass spectrometry and gas chromatography electron capture detection

A comprehensively validated procedure is presented for simultaneous semiquantitative/quantitative screening of 51 drugs of abuse or drugs potentially hazardous for traffic safety in serum, plasma or whole blood. Benzodiazepines (12), cannabinoids (3), opioids (8), cocaine, antidepressants (13), antipsychotics (5) and antiepileptics (2) as well as zolpidem, zaleplon, zopiclone, meprobamate, carisoprodol, tizanidine and orphenadrine and internal standard flurazepam, were isolated by high-yield liquid-liquid extraction (LLE). The dried extracts were derivatized by two-step silylation and analyzed by the combination of two different gas chromatographic (GC) separations with both electron capture detection (ECD) and mass spectrometry (MS) operating in a selected ion-monitoring (SIM) mode. Quantitative or semiquantitative results were obtained for each substance based on four-point calibration. In the validation tests, accuracy, reproducibility, linearity, limit of detection (LOD) and limit of quantitation (LOQ), selectivity, as well as extraction efficiency and stability of standard stock solutions were tested, and derivatization was optimized in detail. Intra- and inter-day precisions were within 2.5-21.8 and 6.0-22.5%, and square of correlation coefficients of linearity ranged from 0.9896 to 0.9999. The limit of quantitation (LOQ) varied from 2 to 2000 ng/ml due to a variety of the relevant concentrations of the analyzed substances in blood. The method is feasible for highly sensitive, reliable and possibly routinely performed clinical and forensic toxicological analyses.     

Counting absolute numbers of molecules using unique molecular identifiers

Counting individual RNA or DNA molecules is difficult because they are hard to copy quantitatively for detection. To overcome this limitation, we applied unique molecular identifiers (UMIs), which make each molecule in a population distinct, to genome-scale human karyotyping and mRNA sequencing in Drosophila melanogaster. Use of this method can improve accuracy of almost any next-generation sequencing method, including chromatin immunoprecipitation-sequencing, genome assembly, diagnostics and manufacturing-process control and monitoring.     

Structural basis of the zinc- and terbium-mediated inhibition of ferroxidase activity in Dps ferritin-like proteins

Streptococcus suis Dpr is an iron-binding protein involved in oxidative stress resistance. It belongs to the bacterial Dps protein family whose members form dodecameric assemblies. Previous studies have shown that zinc and terbium inhibit iron incorporation in Listeria innocua Dps protein. In order to gain structural insights into the inhibitory effect of zinc and terbium, the crystal structures of Streptococcus suis Dpr complexes with these ions were determined at 1.8 A and 2.1 A, respectively. Both ions were found to bind at the ferroxidase center and in the same location as iron. In addition, a novel zinc-binding site formed by His40 and His44 was identified. Both His residues were found to be present within all known Streptococcus suis Dpr variants and in Streptococcus pneumoniae, Streptococcus gordonii, and Streptococcus sanguinis Dpr proteins. Amino acid sequence alignment of Dpr with other Dps family members revealed that His44 is highly conserved, in contrast to His40. The inhibitory effect of zinc and terbium on iron oxidation in Dpr was studied in vitro, and it was found that both ions at concentrations >0.2 mM almost completely abolish iron binding. These results provide a structural basis for the inhibitory effect of zinc and terbium in the Dps family of proteins, and suggest a potential role of the Dps proteins in zinc detoxification mechanisms involving the second zinc-binding site.     

Biofilm development by potentially pathogenic non-pigmented rapidly growing mycobacteria

Background  

A study to evaluate the biofilm-development ability in three different media (Middlebrook 7H9, sterile tap water and PBS-5% glucose) was performed with 19 collection strains from 15 different species on non-pigmented rapidly growing mycobacteria (NPRGM). A microtiter plate assay was developed to evaluate the percentage of covered surface of the microtiter plate wells in different days from day 1 to day 69.