Investigation of genetic diversity and polyandry of Leptinotarsa decemlineata using X-linked microsatellite markers.

A panel of X-linked microsatellite markers was newly designed using the data from a previous sequencing project available in NCBI and used for a study of the Colorado potato beetle (CPB, Leptinotarsa decemlineata) X-haplotype variability. The analysis of scaffolds 49 and 61 (newly identified as fragments of CPB chromosome X) found ten high-quality markers, which were arranged in two PCR multiplexes and evaluated in both 420 CPB adults, collected from 14 localities of Czechia and Slovakia, and 866 larvae from five single-female families from two more Czech localities. Length polymorphisms found in 6 loci have predicted 192 potential X-haplotypes, however, only 36 combinations were detected in the adult males (N = 189), and seven additional ones in the larvae. The X-haplotypes were also generally unevenly distributed; five of the most frequent haplotypes were detected in 55% of males, 19 repeating up to ten-times in 38.7% of males and the remained 12 occurred uniquely in 6.3% of males. Bulk analysis of X-haplotypes dissimilarity indicated seven haplotype groups diversified by mutations and recombinations. Two haplotypes showed a distinctive regional distribution, which indicates an east-west disruption of CPB migration probably caused by different environments of localities in the South Bohemia region and Vysocina region. On the contrary, the results indicate a south-north migration corridor alongside the Vltava River. In the single-female families, from 6 to 13 distinct paternal haplotypes were detected, which proved and quantified a frequented polyandry in CPB.

Unravelling the multi-scale structure-property relationship of laser powder bed fusion processed and heat-treated AlSi10Mg.

Tailoring heat treatments for Laser Powder Bed Fusion (LPBF) processed materials is critical to ensure superior and repeatable material properties for high-end applications. This tailoring requires in-depth understanding of the LPBF-processed material. Therefore, the current study aims at unravelling the threefold interrelationship between the process (LPBF and heat treatment), the microstructure at different scales (macro-, meso-, micro-, and nano-scale), and the macroscopic material properties of AlSi10Mg. A similar solidification trajectory applies at different length scales when comparing the solidification of AlSi10Mg, ranging from mould-casting to rapid solidification (LPBF). The similarity in solidification trajectories triggers the reason why the Brody-Flemings cellular microsegregation solidification model could predict the cellular morphology of the LPBF as-printed microstructure. Where rapid solidification occurs at a much finer scale, the LPBF microstructure exhibits a significant grain refinement and a high degree of silicon (Si) supersaturation. This study has identified the grain refinement and Si supersaturation as critical assets of the as-printed microstructure, playing a vital role in achieving superior mechanical and thermal properties during heat treatment. Next, an electrical conductivity model could accurately predict the Si solute concentration in LPBF-processed and heat-treated AlSi10Mg and allows understanding the microstructural evolution during heat treatment. The LPBF-processed and heat-treated AlSi10Mg conditions (as-built (AB), direct-aged (DA), stress-relieved (SR), preheated (PH)) show an interesting range of superior mechanical properties (tensile strength: 300-450 MPa, elongation: 4-13%) compared to the mould-cast T6 reference condition.

Differences in the auxological characters of children with short stature - Differential diagnostic possibilities of hypothyreosis.

This study aimed to define the differences in growth characteristics in the three most frequent causes of growth retardation - growth hormone deficiency, hypothyreosis and constitutional delay of growth and development - in order to provide diagnostic means for distinguishing these disorders. The study included 166 children with growth disorders aged 4-18 years. The height for age, the bone age using the TW3 method, the predicted height as the target height and the current prediction using the TW3 method were studied. For bone age, the radius, ulna and short bones compartment (RUS) and carpal bones (CARP) were evaluated separately and the difference in their delay in relation to chronological age (ΔBA_RUS_CARP) was determined. The relationship of the studied variables with sex and the underlying diagnosis was tested and the relationship of hypothyreosis and growth data was estimated. The model was tested on the growth data of 104 randomly selected patients with a growth disorder. The largest significant distinction was demonstrated by the difference ΔBA_RUS_CARP in hypothyreosis. The created linear regression model was highly statistically significant (χ2 = 19.4, p < 0.0001) and showed high selectivity (0.609, 95% CI 0.409; 0.808) as well as high specificity (0.864, 95% CI 0.781; 0.946). The clinical validity of the model demonstrated a 61% predictive value for the detection and an 81% successful specification of hypothyreosis. The study demonstrated the possibility of distinguishing suspected hypothyreosis from other causes of growth retardation based on differences in severity of the ossification delay in skeletal compartments of the hand.

Diachronic changes in size and shape of human proximal tibia in Central Europe during the latest 1200 years.

During the past twelve centuries, the stature of Central European inhabitants has increased significantly with corresponding changes in the size of lower limb bones. The aim of our study was to determine whether these changes have occurred strictly in relation to size or if the shape of skeletal structures has been altered simultaneously. Diachronic size and shape changes in the proximal part of tibia in a Central European population (Czech Republic) were studied using geometric morphometrics (principal components analysis, Hotelling's test, linear discriminant analysis). The study sample consisted of 183 three-dimensional (3D) models of adult tibiae dating to the early Middle Ages (N=65), early 20th century (N=60), and from a modern Czech population (N=57). A positive secular trend in size manifested only between the two modern Czech populations (the 20th century vs. the 21st century), a time range shorter than one century. By contrast, landmark-based shape analyses revealed significant differences in tibial morphology over the three periods covered in the study. In particular, progressive changes were observed in the position of tibial tuberosity (shifted medially), the inclination of the line connecting tuberosity with anterior edge of the tibial plateau (sloped down), the reshaping of the lower back condyle boundary (shifted posteriorly), and reshaping of the medial contour of the medial condyle (shifted anteriorly). Changes in the shape of the proximal tibial extremity across the chronologically distinct groups indicated the existence of discreet but convincing microevolutionary trends involving this anatomical structure.

Grain-resolved analysis of localized deformation in nickel-titanium wire under tensile load.

The stress-induced martensitic transformation in tensioned nickel-titanium shape-memory alloys proceeds by propagation of macroscopic fronts of localized deformation. We used three-dimensional synchrotron x-ray diffraction to image at micrometer-scale resolution the grain-resolved elastic strains and stresses in austenite around one such front in a prestrained nickel-titanium wire. We found that the local stresses in austenite grains are modified ahead of the nose cone-shaped buried interface where the martensitic transformation begins. Elevated shear stresses at the cone interface explain why the martensitic transformation proceeds in a localized manner. We established the crossover from stresses in individual grains to a continuum macroscopic internal stress field in the wire and rationalized the experimentally observed internal stress field and the topology of the macroscopic front by means of finite element simulations of the localized deformation.

Taxonomy and phylogeny of European Gymnopus subsection Levipedes (Basidiomycota, Omphalotaceae).

The systematic integrity of European Gymnopus subsect. Levipedes is verified based on anatomic-morphological characters with support from DNA sequences of ITS and translation elongation factor 1-alpha genes. Seven species (G. alpinus, G. aquosus, G. dryophilus - including var. lanipes, G. erythropus, G. fagiphilus, G. hybridus, and G. ocior) belonging to this subsection are included. We clarify the concepts of G. dryophilus and G. ocior, which were occasionally confused in older literature. Due to unavailability of previously selected neotype of G. dryophilus the substitute neotype specimen is selected. Gymnopus dryophilus var. lanipes is confirmed as a variety; no important differences from nominotypical variability were detected. All discriminative characters used for identification of these species are discussed in detail. An identification key is also provided.

Monitoring the growth dynamics of somatic traits based on a semi-longitudinal study.

Possibilities of conducting longitudinal human growth studies are very limited, since it is necessary to monitor the probands for a long time. Another problem can be a loss of data currency, and the small size of the final sample. The solution can be a follow-up semi-longitudinal observation. This research is drawn up as a short longitudinal monitoring of 1925 children (990 boys, 935 girls), aged 6-15 years, at 20 elementary schools in four regions of the Czech Republic, which has been conducted at the same time. Data of repeatedly examined probands of a wide age range were acquired in a short time period. With the help of a linear regression model with mixed effect, the growth velocity curves of 12 somatic traits have been obtained. The timing, intensity and duration of separate growth spurts have been observed, as well as the mutual location of both points of growth velocity, local maxima and minima, and points of the maximal acceleration and deceleration. The results demonstrate that the velocity of characters with variable growth dynamics (skin-fold thicknesses, circumferences of limbs) - contrary to characters with regular growth velocity - have a higher number of partial growth spurts and an opposite course. In the period of separate growth velocity, peaks of somatic characters with regular growth dynamics reach points of partial local minima. In comparison to previous longitudinal studies of body height growth dynamics, the shift of both the beginning and the peak of boys' and girls' pubertal spurt, to a lower age can be found.

Differential expression of glycine receptor subunits in the rat basolateral and central amygdala.

The amygdalar complex is a limbic structure that plays a key role in emotional processing and fear conditioning. Although inhibitory transmission in the amygdala is predominately GABA-ergic, neurons of the amygdala are also known to express glycine receptors. The subtype and function of these glycine receptors within the synaptic circuits of the amygdala are unknown. In this study, we have investigated the relative expression of the four major glycine receptor subunits (alpha1-3 and beta) in the rat basolateral (BLA) and central amygdala (CeA), using real-time PCR and protein biochemistry. We demonstrate that alpha1, alpha2, alpha 3, and beta subunits are all expressed in the BLA and CeA with alpha2 being the predominant alpha-subunit in both nuclei. Electrophysiological recordings from BLA and CeA neurons in acute brain slices indicated that differences in relative expression of these subunits were correlated with the pharmacological properties of native glycine receptors expressed on these neurons. We conclude that glycine receptors assembled in BLA neurons are largely alpha 1 beta-containing heteromultimers whereas receptors assembled in neurons of the central amygdala are primarily alpha 2 beta-, alpha 3 beta- or alpha 1 beta-containing heteromultimers, with a minor component of alpha2 or alpha 3 homomeric receptors also expressed.

[Wrist arthroscopy]. / Artroskopie zápestí.

BACKGROUND: Pain in the wrist area is quite a frequent lesion. If it is not treated soon enough, it leads to development of degenerative changes which are curable with difficulty. Diagnosing is complicated because it is often difficult to diagnose them by means of radiological methods. Wrist arthroscopy is a method which enables to assess the problem and very often it also helps to solve it with an operation. METHODS AND RESULTS: Authors evaluated 93 wrist arthroscopies performed in their departments during 2004-2005. There were 28 arthroscopies performed due to an acute lesion and 65 arthroscopies due to chronic problems. There were 59 arthroscopic curative interventions performed during the operation and 64 open interventions were indicated on the basis of an arthroscopic examination. CONCLUSIONS: All wrist lesions where the lesion of connective tissues structures is suspected, accompanied with edema and hematoma, should be indicated for acute wrist arthroscopy, even though there is no finding on radiographs. Another group of patients indicated for the acute wrist arthroscopy are young patients with wrist fractures where there is a frequent ligament lesion associated, and patients with persisting ailments, even after radiologically confirmed healing of the fracture when intra-articular pathology can be expected. All patients with pain in the wrist area are indicated for arthroscopy, including those with not obvious cause of pain and with symptoms of arthrosis, because it is possible to evaluate further procedure of the therapy.

Synaptic activation of transient receptor potential channels by metabotropic glutamate receptors in the lateral amygdala.

Classical mammalian transient receptor potential channels form non-selective cation channels that open in response to activation of phospholipase C-coupled metabotropic receptors, and are thought to play a key role in calcium homeostasis in non-excitable cells. Within the nervous system transient receptor potential channels are widely distributed but their physiological roles are not well understood. Here we show that in the rat lateral amygdala transient receptor potential channels mediate an excitatory synaptic response to glutamate. Activation of group I metabotropic glutamate receptors on pyramidal neurons in the lateral amygdala with either exogenous or synaptically released glutamate evokes an inward current at negative potentials with a current voltage relationship showing a region of negative slope and steep outward rectification. This current is blocked by inhibiting G protein function with GTP-beta-S, by inhibiting phospholipase C or by infusing transient receptor potential antibodies into lateral amygdala pyramidal neurons. Using RT-PCR and Western blotting we show that transient receptor potential 1, transient receptor potential 4 and transient receptor potential 5 are present in the lateral amygdala. Single cell PCR confirms the presence of transient receptor potential 1 and transient receptor potential 5 in pyramidal neurons and we show by co-immunoprecipitation that transient receptor potential 1 and transient receptor potential 5 co-assemble as a heteromultimers in the amygdala. These results show that in lateral amygdala pyramidal neurons synaptically released glutamate activates transient receptor potential channels, which we propose are likely to be heteromultimeric channels containing transient receptor potential 1 and transient receptor potential 5/transient receptor potential 4.

Coupling between replication and packaging of flavivirus RNA: evidence derived from the use of DNA-based full-length cDNA clones of Kunjin virus.

In order to study whether flavivirus RNA packaging is dependent on RNA replication, we generated two DNA-based Kunjin virus constructs, pKUN1 and pKUN1dGDD, allowing continuous production of replicating (wild-type) and nonreplicating (with a deletion of the NS5 gene RNA-polymerase motif GDD) full-length Kunjin virus RNAs, respectively, via nuclear transcription by cellular RNA polymerase II. As expected, transfection of pKUN1 plasmid DNA into BHK cells resulted in the recovery of secreted infectious Kunjin virions. Transfection of pKUN1dGDD DNA into BHK cells, however, did not result in the recovery of any secreted virus particles containing encapsidated dGDD RNA, despite an apparent accumulation of this RNA in cells demonstrated by Northern blot analysis and its efficient translation demonstrated by detection of correctly processed labeled structural proteins (at least prM and E) both in cells and in the culture fluid using coimmunoprecipitation analysis with anti-E antibodies. In contrast, when dGDD RNA was produced even in much smaller amounts in pKUN1dGDD DNA-transfected repBHK cells (where it was replicated via complementation), it was packaged into secreted virus particles. Thus, packaging of defective Kunjin virus RNA could occur only when it was replicated. Our results with genome-length Kunjin virus RNA and the results with poliovirus replicon RNA (C. I. Nugent et al., J. Virol. 73:427-435, 1999), both demonstrating the necessity for the RNA to be replicated before it can be packaged, strongly suggest the existence of a common mechanism for minimizing amplification and transmission of defective RNAs among the quasispecies in positive-strand RNA viruses. This mechanism may thus help alleviate the high-copy error rate of RNA-dependent RNA polymerases.

[Refractive procedures--LASIK and intraocular pressure in myopic eyes]. / Refrakcní zákroky--LASIK a nitroocní tlak u myopických ocí.

PURPOSE: To evaluate the effect of central corneal thickness (CCT) on the measurement of intraocular pressure (IOP) in normal myopic eyes before and after LASIK refractive surgery. METHODS: To determine CCT (by ultrasonic pachymeter NIDEC) and IOP (by Goldmann tonometer) in 27 patients (48 myopic eyes from -4 to -14 D) before LASIK (on Chiron Technolas 117) and 3 months after LASIK surgery. RESULTS: Mean CCT before LASIK was 543.8 microns (480-612), 3 months after was 430.6 microns (371-511), the difference--113.2 microns (48-157) is highly significant (p < 0.0001). Similar significant is also the decrease of mean IOP 3 months after surgery--about 5.4 mmHg (p < 0.0001). After LASIK surgery decrease IOP is 4.8 mmHg/100 microns. CCT is an important factor for accuracy of the Goldmann measurement. Myopic eyes need more attention, especially after photorefractive surgery, when CCT is decreased and therefore Goldmann IOP measurement is permanently underestimated.

cis- and trans-acting elements in flavivirus RNA replication.

Most of the seven flavivirus nonstructural proteins (NS1 to NS5) encoded in the distal two-thirds of the RNA positive-sense genome are believed to be essential components of RNA replication complexes. To explore the functional relationships of these components in RNA replication, we used trans-complementation analysis of full-length infectious RNAs of Kunjin (KUN) virus with a range of lethal in-frame deletions in the nonstructural coding region, using as helper a repBHK cell line stably producing functional replication complexes from KUN replicon RNA. Recently we showed that replication of KUN RNAs with large carboxy-terminal deletions including the entire RNA polymerase region in the NS5 gene, representing 34 to 75% of the NS5 coding content, could be complemented after transfection into repBHK cells. In this study we have demonstrated that KUN RNAs with deletions of 84 to 97% of the NS1 gene, or of 13 to 63% of the NS3 gene including the entire helicase region, were also complemented in repBHK cells with variable efficiencies. In contrast, KUN RNAs with deletions in any of the other four nonstructural genes NS2A, NS2B, NS4A, and NS4B were not complemented. We have also demonstrated successful trans complementation of KUN RNAs containing either combined double deletions in the NS1 and NS5 genes or triple deletions in the NS1, NS3, and NS5 genes comprising as much as 38% of the entire nonstructural coding content. Based on these and our previous complementation results, we have generated a map of cis- and trans-acting elements in RNA replication for the nonstructural coding region of the flavivirus genome. These results are discussed in the context of our model on formation and composition of the flavivirus replication complex, and we suggest molecular mechanisms by which functions of some defective components of the replication complex can be complemented by their wild-type counterparts expressed from another (helper) RNA molecule.

Isolation of homologous arbovirus cultures from heterologous mixtures using limit dilution and virus-specific enzyme immunoassays.

Viral cultures were identified recently that contained both Kunjin virus and the closely related flavivirus West Nile. The observation that the KUN virus population grew more efficiently in a mosquito cell line (C6/36) while the WN population replicated more effectively in mammalian cells (Vero) allowed enrichment for either virus by culturing the mixture in the appropriate cell line. Limit dilution of the enriched virus preparations was then performed by infecting microtitre cultures with serial ten fold dilutions. Culture wells that contained a pure population of virus were then identified by immunostaining fixed cell monolayers with virus-specific monoclonal antibodies. Subsequent passage of the 'cloned' viruses in either C6/36 or Vero cells and analysis of the infected cultures by specific monoclonal antibody staining, PCR and nucleotide sequencing confirmed the identity of the virus and that in each case an homogeneous virus population had been obtained. This procedure is particularly useful for isolating virus populations from heterogeneous mixtures that fail to develop discrete plaques in infected cell monolayers.

Efficient trans-complementation of the flavivirus kunjin NS5 protein but not of the NS1 protein requires its coexpression with other components of the viral replicase.

Successful trans-complementation of the defective Kunjin virus (KUN) RNA FLdGDD with a deletion of the RNA polymerase motif GDD in the NS5 gene by using a BHK cell line, repBHK, that continuously produced a functionally active KUN replication complex (RC) from replicon RNA was recently reported (A. A. Khromykh, M. T. Kenney, and E. G. Westaway, J. Virol. 72:7270-7279, 1998). In order to identify whether this complementation of FLdGDD RNA was provided by the wild-type NS5 protein alone or with the help of other nonstructural (NS) proteins also expressed in repBHK cells, we generated BHK cell lines stably producing the individual NS5 protein (SRns5BHK) or the NS1-NS5 polyprotein (SRns1-5BHK) by using a heterologous expression vector based on a modified noncytopathic Sindbis replicon. Western blot analysis with anti-NS5 antibodies showed that the level of production of NS5 was significantly higher in SRns5BHK cells than in SRns1-5BHK cells. Despite the higher level of expressed NS5, trans-complementation of FLdGDD RNA was much less efficient in SRns5BHK cells than in SRns1-5BHK cells and produced at least 100-fold less of the secreted complemented virus. In contrast, efficient complementation of KUN RNA with lethal cysteine-to-alanine mutations in the NS1 gene was achieved both in BHK cells producing the individual KUN NS1 protein from the Sindbis replicon vector and in repBHK cells, with both cell lines expressing similar amounts of NS1 protein. These results clearly demonstrate that flavivirus NS5 coexpressed with other components of the viral replicase possesses much higher functional (trans-complementing) activity than individually expressed NS5 and that efficient trans-complementation of mutated flavivirus NS1 and NS5 proteins occurs by different mechanisms. The results are interpreted and discussed in relation to our proposed model of formation of the flavivirus RC largely based on previous ultrastructural and biochemical analyses of KUN replication.

trans-Complementation analysis of the flavivirus Kunjin ns5 gene reveals an essential role for translation of its N-terminal half in RNA replication.

Recently we described rescue of defective Kunjin virus (KUN) RNAs with small deletions in the methyltransferase and RNA polymerase motifs of the ns5 gene, using BHK cells stably expressing KUN replicon RNA (repBHK cells) as helper (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). We have now extended our previous observations and report successful trans-complementation of defective KUN RNAs with most of the ns5 gene deleted or substituted with a heterologous (dengue virus) ns5 sequence. Replication of full-length KUN RNAs with 3'-terminal deletions of 136 (5%), 933 (34%), and 1526 (56%) nucleotides in the ns5 gene was complemented efficiently in transfected repBHK cells. RNA with a larger deletion of 2,042 nucleotides (75%) was complemented less efficiently, and RNA with an even larger deletion of 2,279 nucleotides (84%) was not complemented at all. Chimeric KUN genomic RNA containing 87% of the KUN ns5 gene replaced by the corresponding sequence of the dengue virus type 2 ns5 gene was unable to replicate in normal BHK cells but was complemented in repBHK cells. These results demonstrate for the first time complementation of flavivirus RNAs with large deletions (as much as 75%) in the RNA polymerase gene and establish that translation of most of the N-terminal half of NS5 is essential for complementation in trans. A model of formation of the flavivirus replication complex implicating a possible role in RNA replication of conserved coding sequences in the N-terminal half of NS5 is proposed based on the complementation and earlier results with KUN and on reported data with other flaviviruses.

[Pachymetry in patients with glaucoma]. / Pachymetrie u pacientu s glaukomem.

UNLABELLED: Central corneal thickness (CCT) was measured in 328 eyes of 167 patients (50 men and 117 women, an average age was 62 years). CCT was determinated by ultrasonic pachymeter NIDEC, design UP-1000, at Eye Department of 3rd Medical Faculty of Prague. RESULTS: The mean CCT at glaucoma patients was 561 microns, in margins 490-650 microns. CCT for right eyes were 0.567 +/- 0.035 mm, for left eyes 0.569 +/- 0.037 mm. There were no influence by previous operations (cataract, trabeculectomy and combined operations). NTG had the lowest CCT values 551 microns, POAG had 569 microns and OHT the greates value, CCT is 588 microns. CCT in patients with NTG was significantly lower (p = 0.047) than in POAG. The higher difference is between NTG a OHT (p = 0.015). We had no statistical difference in CCT between hypermetropic, emetropic and myopic eyes. The corneal thickness is decreasing during ageing. CCT is another clinically helpful factor for evaluation our glaucoma patients. CCT together with other risk factors can help us in establishing a target pressure. Patients after refractive surgery (PRK, LASIK) should be more carefully evaluated not to miss early stages of glaucoma.

The information needs and fears of patients with inflammatory bowel disease.

A questionnaire containing a list of 36 questions and nine "fear factors" was sent to 139 patients with inflammatory bowel disease. Patients were asked to select the most and the least important questions and fear factors. Furthermore, the level of information and the source of information were recorded. 58% of the patients responded. The most important questions selected were possibilities of remission of the disease, and of developing cancer (47% and 43% of all answers respectively). Women judged questions regarding cancer, sex life, side effects of diagnostic techniques or drugs to be important more often than men, who were more interested in the cause, symptoms and natural history of the disease. Cancer was the fear factor selected most often (52%), being more important to patients with ulcerative colitis than to those with Crohn's disease, who in turn were more afraid of surgery than those with ulcerative colitis. 77% of the patients felt they had little or too little information. 87% received information from their physicians, 75% wanted to continue doing so. 62% wanted additional information from booklets, 18% from self-help groups. Thus, much information is lacking and many unjustified fears exist in patients with inflammatory bowel disease. This could probably be improved by providing more and better information by improved personal communication, and additionally from booklets or self-help groups.