Real-time polymerase chain reaction as a rapid and efficient alternative to estimation of picornavirus titers by tissue culture infectious dose 50% or plaque forming units

Quantification of viral infectious units is traditionally measured by methods based on forming plaques in semisolid media (PFU) or endpoint dilution of a virus-containing solution (TCID₅₀), methods that are laborious, time-consuming and take on average 3–7 days to carry out. Quantitative real-time PCR is an established method to quantify nucleic acids at high accuracy and reproducibility, routinely used for virus detection and identification. In the present study, a procedure was developed using a two-step real-time PCR and the SYBR Green detection method to study whether there are correlations between TCID₅₀/ml, PFU/ml and Ct values generated by real-time PCR enabling rapid and efficient calculation of titer equivalents when working with viruses in the research laboratory. In addition, an external standard with known concentrations was included using in vitro transcribed viral RNA, thus allowing the calculation of the amount of RNA copies needed for various applications (i.e. per plaque or TCID₅₀).The results show that there is a correlation between the three quantification methods covering a wide range of concentration of viruses. Furthermore, a general regression line between TCID₅₀ and Ct values was obtained for all viruses included in the study, which enabled recording titer equivalents using real-time PCR. Finally, by including an external standard, the amount of RNA genomes generating one TCID₅₀ or PFU for each enterovirus serotype included was determined.     

Retinoblastoma susceptibility gene product (pRb) and p107 functionally separate the requirements for serum and anchorage in the cell cycle G1-phase

Growth factors and cell anchorage are both required for cell cycle G(1)-phase progression, but it is unclear whether their function is mediated through the same set of cell cycle components and whether they are both required during the same periods of time. We separately analyzed the requirements of serum and anchorage during G(1)-phase progression and found that human dermal fibroblasts as well as wild type, pRb(-/-), and p107(-/-) mouse embryonic fibroblasts needed serum (growth factors) until mid-G(1)-phase but required cell anchorage until late G(1)-phase to be competent for S-phase entry. Importantly, however, pRb/p107 double-null mouse embryonic fibroblasts lacked serum requirement in mid-G(1)-phase but still required cell anchorage until late G(1)-phase to enter S-phase. Our results indicate that pRb and p107 do not constitute the last control point for extracellular factors during G(1)-phase progression, and they functionally separate the requirements for serum and cell anchorage in terms of involved cell cycle components.     

Variants of CYP46A1 may interact with age and APOE to influence CSF Aβ42 levels in Alzheimer’s disease

Recent studies have suggested that variants of CYP46A1, encoding cholesterol 24-hydroxylase (CYP46), confer risk for Alzheimers disease (AD), a prospect substantiated by evidence of genetic association from several quantitative traits related to AD pathology, including cerebrospinal fluid (CSF) levels of the 42 amino-acid cleavage product of -amyloid (A42) and the tau protein. In the present study, these claims have been explored by the genotyping of previously associated markers in CYP46A1 in three independent northern European case-control series encompassing 1323 individuals and including approximately 400 patients with measurements of CSF A42 and phospho-tau protein levels. Tests of association in case-control models revealed limited evidence that CYP46A1 variants contributed to AD risk across these samples. However, models testing for potential effects upon CSF measures suggested a possible interaction of an intronic marker (rs754203) with age and APOE genotype. In stratified analyses, significant effects were evident that were restricted to elderly APOE 4 carriers for both CSF A42 (P=0.0009) and phospho-tau (P=0.046). Computational analyses indicate that the rs754203 marker probably does not impact the binding of regulatory factors, suggesting that other polymorphic sites underlie the observed associations. Our results provide an important independent replication of previous findings, supporting the existence of CYP46A1 sequence variants that contribute to variability in -amyloid metabolism.     

Malformed offspring,sibling matings,and selection against inbreeding in the sand lizard (Lacerta agilis)

We demonstrated that sand lizards (Lacerta agilis) are more likely to have malformed offspring when they mate with siblings. Offspring with malformations, such as deformed limbs and heads, have zero survival in a natural population. Normal-looking siblings of malformed hatchlings also had a reduced survival in the wild, compared to offspring from clutches in which all siblings appeared normal. The proportion of malformed hatchlings in the natural population was ca. 10%, in spite of differences in juvenile dispersal between males and females. Male juveniles disperse significantly further from their natal sites than do female juveniles.     

Quantitative trait loci controlling cyanogenic glucoside and dry matter content in cassava (Manihot esculenta Crantz) roots

Cassava (Manihot esculenta Crantz) is a starchy root crop grown in the tropics mainly by small-scale farmers even though agro-industrial processing is rapidly increasing. For this processing market improved varieties with high dry matter root content (DMC) is required. Potentially toxic cyanogenic glucosides are synthesized in the leaves and translocated to the roots. Selection for varieties with low cyanogenic glucoside potential (CNP) and high DMC is among the principal objectives in cassava breeding programs. However, these traits are highly influenced by the environmental conditions and the genetic control of these traits is not well understood. An S(1) population derived from a cross between two bred cassava varieties (MCOL 1684 and Rayong 1) that differ in CNP and DMC was used to study the heritability and genetic basis of these traits. A broad-sense heritability of 0.43 and 0.42 was found for CNP and DMC, respectively. The moderate heritabilities for DMC and CNP indicate that the phenotypic variation of these traits is explained by a genetic component. We found two quantitative trait loci (QTL) on two different linkage groups controlling CNP and six QTL on four different linkage groups controlling DMC. One QTL for CNP and one QTL for DMC mapped near each other, suggesting pleiotrophy and/or linkage of QTL. The two QTL for CNP showed additive effects while the six QTL for DMC showed additive effect, dominance or overdominance. This study is a first step towards developing molecular marker tools for efficient breeding of CNP and DMC in cassava.     

Deciphering the cellular functions of the Op18/Stathmin family of microtubule-regulators by plasma membrane-targeted localization

The Op18/stathmin family of microtubule regulators includes the ubiquitous cytosolic Op18/stathmin (Op18) and the neuronal, primarily Golgi-associated proteins SCG10 and RB3, which all form ternary complexes with two head-to-tail-aligned tubulin heterodimers. To understand the physiological significance of previously observed differences in ternary complex stability, we have fused each of the heterodimer-binding regions of these three proteins with the CD2 cell surface protein to generate confined plasma membrane localization of the resulting CD2 chimeras. Herein, we show that, in contrast to constitutively active CD2-Op18-tetraA, both the CD2-SCG10 and CD2-RB3 chimeras sequestered tubulin at the plasma membrane, which results in >35% reduction of cytosolic tubulin heterodimer levels and consequent delayed formation of mitotic spindles. However, all three CD2 chimeras, including the tubulin sequestration-incompetent CD2-Op18-tetraA, destabilize interphase microtubules. Given that microtubules are in extensive contact with the plasma membrane during interphase, but not during mitosis, these findings indicate that Op18-like proteins have the potential to destabilize microtubules by both sequestration and direct interaction with microtubules. However, the differences in tubulin binding observed in cells also indicate conceptual differences between the functions of low-abundance neural family members, which will accumulate tubulin at specific cellular compartments, and the abundant cytosolic Op18 protein, which will not.     

Quantitative analysis of MAP2 immunoreactivity in human neocortex of three patients surviving after brain ischemia

Transient global ischemia caused by cardiac arrest results in lesions that involve all brain structures. The aim of this study was to investigate the distribution of MAP2 immunoreactivity in neurons in the brain of patients surviving for various times after an ischemic incident, using confocal laser scanning microscopy. We performed a quantitative analysis of the distribution and density of MAP2-positive structures in human neocortical areas after survival times of 1 week, 3 months, and 1 year after the cardiac arrest. Three important observations were made in the present study: (i) in all human brain areas investigated (motor, temporal, frontal, and visual cortex) a decrease of MAP2 immunoreactivity was found; (ii) in all studied areas the most significant decrease in MAP2 was found in layers II–III, compared with layers V–VII; (iii) the decrease of MAP2 immunoreactivity in layers II–III was related to the duration of the postischemic period. The maximal decrease, by 66.3% (P < .05), in MAP2-positive pyramidal neurons, was observed in layers II–III in the motor cortex after 1 year of survival after cardiac arrest.     

Plectin deposition at podosome rings requires myosin contractility

Metalloproteinase-dependent tissue invasion requires the formation of podosomes and invadopodia for localized matrix degradation. Actin cytoskeleton remodeling via Arp2/3-mediated actin polymerization is essential for podosome formation, and dynamic microtubules have an important role in maintaining podosome turnover in macrophages and osteoclasts. Little is known, however, about the involvement of the intermediate filament cytoskeleton in formation, stabilization, and turnover of podosomes. Here we show that vimentin intermediate filaments colocalize with the early sites of podosome formation at the stress fiber - focal adhesion interface in cultured vascular smooth muscle cells, but do not directly contribute to podosome formation, or stabilization. In unstimulated A7r5 cells the cytolinker protein plectin poorly colocalized with vimentin and the microdomains, but following induction by phorbol ester accumulated in the rings that surround the podosomes. In plectin-deficient A7r5 cells actin stress fiber remodelling is reduced in response to PDBu, and small podosomes remain localized at stable actin stress fibres. Pharmacological inhibition of actomyosin contractility by blebbistatin leads to an aberrant localization of podosomes away from the cell periphery and induces failure of plectin to surround the outer perimeter of these invasive adhesions. Taken together, we conclude that plectin is involved in growth and maturation of podosomes by reducing focal adhesion and stress fiber turnover, and that actomyosin-dependent contractility is required for the peripheral localization and specific deposition of plectin at the podosome rings.