Optimal stimulation settings for CMAP scan registrations

Background

The CMAP (Compound Muscle Action Potential) scan is a non-invasive electrodiagnostic tool, which provides a quick and visual assessment of motor unit potentials as electrophysiological components that together constitute the CMAP. The CMAP scan records the electrical activity of the muscle (CMAP) in response to transcutaneous stimulation of the motor nerve with gradual changes in stimulus intensity. Large MUs, including those that result from collateral reinnervation, appear in the CMAP scan as so-called steps, i.e., clearly visible jumps in CMAP amplitude. The CMAP scan also provides information on nerve excitability. This study aims to evaluate the influence of the stimulation protocol used on the CMAP scan and its quantification.

Methods

The stimulus frequency (1, 2 and 3 Hz), duration (0.05, 0.1 and 0.3 ms), or number (300, 500 and 1000 stimuli) in CMAP scans of 23 subjects was systematically varied while the other two parameters were kept constant. Pain was measured by means of a visual analogue scale (VAS). Non-parametric paired tests were used to assess significant differences in excitability and step variables and VAS scores between the different stimulus parameter settings.

Results

We found no effect of stimulus frequency on CMAP scan variables or VAS scores. Stimulus duration affected excitability variables significantly, with higher stimulus intensity values for shorter stimulus durations. Step variables showed a clear trend towards increasing values with decreasing stimulus number.

Conclusions

A protocol delivering 500 stimuli at a frequency of 2 Hz with a 0.1 ms pulse duration optimized CMAP scan quantification with a minimum of subject discomfort, artefact and duration of the recording. CMAP scan variables were influenced by stimulus duration and number; hence, these need to be standardized in future studies.     

Thermal stress responses in the bacterial biosphere of the Great Barrier Reef sponge,Rhopaloeides odorabile

Marine sponges are diverse, abundant and provide a crucial coupling point between benthic and pelagic habitats due to their high filtration rates. They also harbour extensive microbial communities, with many microbial phylotypes found exclusively in sponge hosts and not in the seawater or surrounding environment, i.e. so‐called sponge‐specific clusters (SCs) or sponge‐ and coral‐specific clusters (SCCs). We employed DNA (16S rRNA gene) and RNA (16S rRNA)‐based amplicon pyrosequencing to investigate the effects of sublethal thermal stress on the bacterial biosphere of the Great Barrier Reef sponge Rhopaloeides odorabile. A total of 8381 operational taxonomic units (OTUs) (97% sequence similarity) were identified, affiliated with 32 bacterial phyla from seawater samples, 23 bacterial phyla from sponge DNA extracts and 18 bacterial phyla from sponge RNA extracts. Sublethal thermal stress (31°C) had no effect on the present and/or active portions of the R. odorabile bacterial community but a shift in the bacterial assemblage was observed in necrotic sponges. Over two‐thirds of DNA and RNA sequences could be assigned to previously defined SCs/SCCs in healthy sponges whereas only 12% of reads from necrotic sponges could be assigned to SCs/SCCs. A rapid decline in host health over a 1°C temperature increment suggests that sponges such as R. odorabile may be highly vulnerable to the effects of global climate change.     

Synthesis and AcidBase Properties of an Imidazole-Containing Nucleotide Analog, 1-(2'-Deoxy-β-D-ribofuranosyl)imidazole 5'-Monophosphate (dImMP(2-) )

Deletion of the substituted pyrimidine ring in purine-2'-deoxynucleoside 5'-monophosphates leads to the artificial nucleotide analog dImMP(2-) . This analog can be incorporated into DNA to yield, upon addition of Ag(+) ions, a molecular wire. Here, we measured the acidity constants of H(2) (dImMP)(±) having one proton at N(3) and one at the PO$\rm{{_{3}^{2-}}}$ group by potentiometric pH titrations in aqueous solution. The micro acidity constants show that N(3) is somewhat more basic than PO$\rm{{_{3}^{2-}}}$ and, consequently, the (H??dImMP)(-) tautomer with the proton at N(3) dominates to ca. 75%. The calculated micro acidity constants are confirmed by (31) P- and (1) H-NMR chemical shifts. The assembled data allow many quantitative comparisons, e.g., the N(3)-protonated and thus positively charged imidazole residue facilitates deprotonation of the P(O)(2) (OH)(-) group by 0.3?pK units. Information on the intrinsic site basicities also allows predictions about metal-ion binding; e.g., Mg(2+) and Mn(2+) will primarily coordinate to the phosphate group, whereas Ni(2+) and Cu(2+) will preferably bind to N(3). Macrochelate formation for these metal ions is also predicted. The micro acidity constant for N(3)H(+) deprotonation in the (H???dImMP?H)(±) species (pk(a) 6.46) and the M(n+) -binding properties are of relevance for understanding the behavior of dImMP units present in DNA hairpins and metalated duplexes.     

Chilling-induced ultrastructural changes to mesophyll cells of Arabidopsis grown under short days are almost completely reversible by plant re-warming

Exposure of plants to chilling (low temperatures above freezing) limits growth and development in all environments outside the lowest latitudes. Cell ultrastructure and morphometric studies may allow associations to be made between chilling-induced changes at the ultrastructural level, molecular events and their physiological consequences. We examined changes in the shape, size and membrane organization of the organelles of mesophyll cells in Arabidopsis thaliana (Col 0), a cold-resistant species, after subjecting 6-week-old plants grown at normal growth temperatures to chilling (2.5–4°C; 14-h dark/10-h light cycle) for 6, 24 and 72 h and after a re-warming period of 50 h. No ultrastructural differences were seen in the first 6 h of chilling but after 24 h we observed swollen and rounded chloroplasts with larger starch grains and dilated thylakoids compared to control plants. By 72 h, chilling had resulted in a large accumulation of starch in chloroplasts, an apparent crowding of the cytosol and a lower abundance of peripheral reticulum than in the controls. The average area per chloroplast in cell sections increased after 72-h chilling while the number of chloroplasts remained the same. Ring-shaped and other morphologically aberrant mitochondria were present in significantly higher abundance in plants given 72 h chilling than in the controls. Plant re-warming for 50 h reduced chloroplast size to those of the controls and returned mitochondria to standard morphology, but peripheral reticulum remained less abundant than in plants never given a cold treatment. The near full return to normal ultrastructure upon plant re-warming indicates that the morphological changes may be part of acclimation to cold.     

Development of an <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation method for <Emphasis Type="Italic">Taxus</Emphasis> suspension cultures

The FDA-approved anti-cancer compound paclitaxel is currently produced commercially by Taxus plant cell suspension cultures. One major limitation to the use of plant cell culture as a production platform is the low and variable product yields. Therefore, methods to increase and stabilize paclitaxel production are necessary to ensure product security, especially as the demand for paclitaxel continues to rise. Although a stable transformation method for Taxus suspension cultures has been developed, stable transformant yields are low (around 1% of experiments) and the method does not translate to the Taxus cuspidata Siebold and Zucc. and Taxus canadensis Marshall cell lines utilized in this study. Therefore, a new method for Agrobacterium-mediated transformation of Taxus callus and suspension cultures was developed through identification of the optimal Agrobacterium strain, inclusion of an anti-necrotic cocktail (silver nitrate, cysteine, and ascorbic acid) and increased recovery time for cells after cocultivation, the time following infection with Agrobacterium tumefaciens. Application of the increased recovery time to transformation of T. cuspidata line PO93XC resulted in 200 calluses staining positive for GUS. Additionally, two transgenic lines have been maintained with stable transgene expression for over 5 yr. This method represents an improvement over existing transformation methods for Taxus cultures and can be applied for future metabolic engineering efforts.     

Murine Norovirus 1 (MNV1) Replication Induces Translational Control of the Host by Regulating eIF4E Activity during Infection

Protein synthesis is a tightly controlled process responding to several stimuli, including viral infection. As obligate intracellular parasites, viruses depend on the translation machinery of the host and can manipulate it by affecting the availability and function of specific eukaryotic initiation factors (eIFs). Human norovirus is a member of the Caliciviridae family and is responsible for gastroenteritis outbreaks. Previous studies on feline calicivirus and murine norovirus 1 (MNV1) demonstrated that the viral protein, genome-linked (VPg), acts to direct translation by hijacking the host protein synthesis machinery. Here we report that MNV1 infection modulates the MAPK pathway to activate eIF4E phosphorylation. Our results show that the activation of p38 and Mnk during MNV1 infection is important for MNV1 replication. Furthermore, phosphorylated eIF4E relocates to the polysomes, and this contributes to changes in the translational state of specific host mRNAs. We propose that global translational control of the host by eIF4E phosphorylation is a key component of the host-pathogen interaction.     

Identifying <Emphasis Type="Italic">FATB1a</Emphasis> deletion that causes reduced palmitic acid content in soybean N87-2122-4 to develop a functional marker for marker-assisted selection

Palmitic acid is a major saturated fatty acid in soybean oil, and consumption of saturated fat is linked to a risk of coronary diseases. Development of soybean (Glycine max) cultivars with reduced palmitic acid content is an important goal of soybean breeding. The FATB1a gene was previously found to be responsible for reduced palmitic acid in the soybean line N87-2122-4. The objective of this research was to characterize the FATB1a gene identified in N87-2122-4 and develop a breeder-friendly, functional marker to facilitate marker-assisted selection and improve breeding efficiency for reduced palmitic soybeans. With the availability of soybean genetic maps, reference genome, and gene annotations, an approximate 254 kb deleted genomic region, including the FATB1a gene, was identified. Based on the gene deletion information, we developed a TaqMan marker and tested it with a segregating F ₂ population that consisted of 140 individual plants derived from ‘Cook’ × N87-2122-4. The marker performed well and accounted for 57 % of the phenotypic variation. The marker was also validated using a panel of 121 diverse soybean lines with known fatty acid profiles. The result indicated that the marker can be used effectively in marker-assisted breeding for reduced palmitic acid in soybean.     

Assessment of algorithms for high throughput detection of genomic copy number variation in oligonucleotide microarray data

Background

Genomic deletions and duplications are important in the pathogenesis of diseases, such as cancer and mental retardation, and have recently been shown to occur frequently in unaffected individuals as polymorphisms. Affymetrix GeneChip whole genome sampling analysis (WGSA) combined with 100 K single nucleotide polymorphism (SNP) genotyping arrays is one of several microarray-based approaches that are now being used to detect such structural genomic changes. The popularity of this technology and its associated open source data format have resulted in the development of an increasing number of software packages for the analysis of copy number changes using these SNP arrays.

Results

We evaluated four publicly available software packages for high throughput copy number analysis using synthetic and empirical 100 K SNP array data sets, the latter obtained from 107 mental retardation (MR) patients and their unaffected parents and siblings. We evaluated the software with regards to overall suitability for high-throughput 100 K SNP array data analysis, as well as effectiveness of normalization, scaling with various reference sets and feature extraction, as well as true and false positive rates of genomic copy number variant (CNV) detection.

Conclusion

We observed considerable variation among the numbers and types of candidate CNVs detected by different analysis approaches, and found that multiple programs were needed to find all real aberrations in our test set. The frequency of false positive deletions was substantial, but could be greatly reduced by using the SNP genotype information to confirm loss of heterozygosity.     

Spectroscopic investigations of intermediates in the reaction of cytochrome P450BM3–F87G with surrogate oxygen atom donors

Rapid mixing of substrate-free ferric cytochrome P450_BM3–F87G with m-chloroperoxybenzoic acid (mCPBA) resulted in the sequential formation of two high-valent intermediates. The first was spectrally similar to compound I species reported previously for P450_CAM and CYP 119 using mCPBA as an oxidant, and it featured a low intensity Soret absorption band characterized by shoulder at 370 nm. This is the first direct observation of a P450 compound I intermediate in a type II P450 enzyme. The second intermediate, which was much more stable at pH values below 7.0, was characterized by an intense Soret absorption peak at 406 nm, similar to that seen with P450_CAM [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300–20309]. Double mixing experiments in which NADPH was added to the transient 406 nm-absorbing intermediate resulted in rapid regeneration of the resting ferric state, with the flavins of the flavoprotein domain in their reduced state. EPR results were consistent with this stable intermediate species being a cytochrome c peroxidase compound ES-like species containing a protein-based radical, likely localized on a nearby Trp or Tyr residue in the active site. Iodosobenzene, peracetic acid, and sodium m-periodate also generated the intermediate at 406 nm, but not the 370 nm intermediate, indicating a probable kinetic barrier to accumulating compound I in reactions with these oxidants. The P450 ES intermediate has not been previously reported using iodosobenzene or m-periodate as the oxygen donor.     

Female reproductive tract abnormalities in European hares (Lepus europaeus) in Australia. philip.stott@adelaide.edu.au

Populations of European hare (Lepus europaeus) are in decline in Europe, and populations in Australia remain at low densities. Populations are sensitive to size of the breeding stock, which is influenced by fertility in the females. From 1996 to 1999, a total of 272 adult female hares from three Australian populations were dissected and their reproductive systems examined for abnormalities. Cystic endometrial hyperplasia was relatively common and often accompanied by hydrosalpinx. Extrauterine fetuses, neoplasms, pseudopregnancies, and resorptions also were found. However, although pseudopregnancies and resorptions were found in young adults (<12 mo) as well as older hares, conditions possibly causing infertility were almost always in older hares with prevalences up to 46.2%. Only hares with access to known sources of estrogens exhibited pathologic conditions, but sympatric European rabbits (Oryctolagus cuniculus) did not, which is consistent with known difference in responses between the corpora lutea of the two species to exogenous estrogen. Infertility at such a high prevalence could compound and extend the impact of years of low juvenile survival on recruitment.