Multiplexed expression and screening for recombinant protein production in mammalian cells

Background  

A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.     

SEPALLATA3: the 'glue' for MADS box transcription factor complex formation

Background  

Plant MADS box proteins play important roles in a plethora of developmental processes. In order to regulate specific sets of target genes, MADS box proteins dimerize and are thought to assemble into multimeric complexes. In this study a large-scale yeast three-hybrid screen is utilized to provide insight into the higher-order complex formation capacity of the Arabidopsis MADS box family. SEPALLATA3 (SEP3) has been shown to mediate complex formation and, therefore, special attention is paid to this factor in this study.     

Variation in the ribosomal internal transcribed spacers and 5.8S rDNA among five species of Acropora (Cnidaria; Scleractinia): patterns of variation consistent with reticulate evolution

The ITS sequences of Acropora spp. are the shortest so far identified in any metazoan and are among the shortest seen in eukaryotes; ITS1 was 70-80 bases, and ITS2 was 100-112 bases. The ITS sequences were also highly variable, but base composition and secondary structure prediction indicate that divergent sequence variants are unlikely to be pseudogenes. The pattern of variation was unusual in several other respects: (1) two distinct ITS2 types were detected in both A. hyacinthus and A. cytherea, species known to hybridize in vitro with high success rates, and a putative intermediate ITS2 form was also detected in A. cytherea; (2) A. valida was found to contain highly (29%) diverged ITS1 variants; and (3) A. longicyathus contained two distinct 5.8S rDNA types. These data are consistent with a reticulate evolutionary history for the genus Acropora.      

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

A new brace treatment similar for adolescent scoliosis and kyphosis based on restoration of thoracolumbar lordosis. Radiological and subjective clinical results after at least one year of treatment

Study design

A prospective treatment study with a new brace was conducted Objective. To evaluate radiological and subjective clinical results after one year conservative brace treatment with pressure onto lordosis at the thoracolumbar joint in children with scoliosis and kyphosis.

Summary of background data

Conservative brace treatment of adolescent scoliosis is not proven to be effective in terms of lasting correction. Conservative treatment in kyphotic deformities may lead to satisfactory correction. None of the brace or casting techniques is based on sagittal forces only applied at the thoracolumbar spine (TLI= thoracolumbar lordotic intervention). Previously we showed in patients with scoliosis after forced lordosis at the thoracolumbar spine a radiological instantaneous reduction in both coronal curves of double major scoliosis.

Methods

A consecutive series of 91 children with adolescent scoliosis and kyphosis were treated with a modified symmetric 30 degrees Boston brace to ensure only forced lordosis at the thoracolumbar spine. Scoliosis was defined with a Cobb angle of at least one of the curves [greater than or equal to] 25 degrees and kyphosis with or without a curve <25 degrees in the coronal plane. Standing radiographs were made i) at start, ii) in brace at beginning and iii) after one year treatment without brace.

Results

Before treatment start ??in brace?? radiographs showed a strong reduction of the Cobb angles in different curves in kyphosis and scoliosis groups (sagittal n = 5 all p < 0.001, pelvic obliquity p < 0.001). After one year of brace treatment in scoliosis and kyphosis group the measurements on radiographs made without brace revealed an improvement in 3 Cobb angles each.

Conclusion

Conservative treatment using thoracolumbar lordotic intervention in scoliotic and kyphotic deformities in adolescence demonstrates a marked improvement after one year also in clinical and postural criteria. An effect not obtained with current brace techniques.     

Proof-of-concept human laboratory study for protracted abstinence in alcohol dependence: effects of gabapentin

There is a need for safe medications that can effectively support recovery by treating symptoms of protracted abstinence that may precipitate relapse in alcoholics, e.g. craving and disturbances in sleep and mood. This proof-of-concept study reports on the effectiveness of gabapentin 1200 mg for attenuating these symptoms in a non-treatment-seeking sample of cue-reactive, alcohol-dependent individuals. Subjects were 33 paid volunteers with current Diagnostic and Statistical Manual of Mental Disorders-IV alcohol dependence and a strength of craving rating 1 SD or greater for alcohol than water cues. Subjects were randomly assigned to gabapentin or placebo for 1 week and then participated in a within-subjects trial where each was exposed to standardized sets of pleasant, neutral and unpleasant visual stimuli followed by alcohol or water cues. Gabapentin was associated with significantly greater reductions than placebo on several measures of subjective craving for alcohol as well as for affectively evoked craving. Gabapentin was also associated with significant improvement on several measures of sleep quality. Side effects were minimal, and gabapentin effects were not found to resemble any major classes of abused drugs. Results suggest that gabapentin may be effective for treating the protracted abstinence phase in alcohol dependence and that a randomized clinical trial would be an appropriate next step. The study also suggests the value of cue-reactivity studies as proof-of-concept screens for potential antirelapse drugs.     

Biosynthesis and enantioselectivity in the production of the lilac compounds in Actinidia arguta flowers

Biosynthesis of the lilac alcohols and alcohol epoxides from linalool in ‘Hortgem Tahi’ kiwifruit (Actinidiaarguta) flowers was investigated by incubating flowers with rac-linalool, rac-[4,4,10,10,10-²H₅]linalool, (R)-8-hydroxylinalool and (R)-8-oxolinalool. All substrates were incorporated into the lilac alcohols although the (R)-configured compounds are not normally present in flowers. Biosynthesis of the lilac alcohol epoxides from rac-1,2-epoxy[4,4,10,10,10-²H₅]linalool and rac-[4′,4′, 8′, 8′,8′-²H₅]lilac aldehyde epoxide, rather than the lilac alcohols, was examined. Both substrates were non-enantioselectively converted to the lilac alcohol epoxides, suggesting two biosynthetic pathways for these compounds, contrary to previous reports. Their ability to process unnatural substrates indicates that A.arguta flowers have a greater biosynthetic capability than is suggested by their phytochemical composition. Linalool, the lilac compounds, and their biosynthetic intermediates were measured in the pistils, stamen, petals and sepals to determine if localisation in different organs contributed to only (S)-linalool being processed to the lilac compounds. Both linalool enantiomers were present in all organs, while most (97%) of the lilac compounds, and their precursors, were found in the petals. (S)-Linalool was not depleted from the flower petals, with respect to (R)-linalool, during the time of maximum production of the metabolites of (S)-linalool.     

Serosurvey of Brucella spp. infection in the Kafue lechwe (Kobus leche kafuensis) of the Kafue flats in Zambia

One of the diseases of veterinary and public health importance affecting the Kafue lechwe (Kobus leche kafuensis) on the Kafue flats is brucellosis, for which only scant information is available. During the 2003 (October), 2004 (December), and 2008 (July-December) hunting seasons in the Kafue flats, we conducted a study to determine the seroprevalence of Brucella spp. in the Kafue lechwe and to evaluate serologic tests for detection of Brucella spp. antibodies in lechwe. The Rose Bengal Test (RBT), competitive enzyme-linked immunosorbent assay (cELISA), and fluorescence polarization assay (FPA) were used. A total of 121 Kafue lechwe were hunted for disease investigations in 2003, 2004, and 2008 in the Kafue Flat Game Management Area. Of these, 21.6%, (95% confidence interval [CI]: 14.2-29.1%) had detectable antibodies to Brucella spp. The Kafue lechwe in Lochnivar National Park had higher antibody results than those in Blue Lagoon National Park (odds ratio=3.0; 95% CI: 0.94-9.4). Infection levels were similar in females (21.6%) and males (21.7%). Results were similar among RBT, FPA, cELISA tests, suggesting that these could effectively be used in diagnosing brucellosis in the Kafue lechwe. Our study demonstrates the presence of Brucella infections in the Kafue lechwe in two national parks located in the Kafue flats and further highlights the suitability of serologic assays for testing the Kafue lechwe. Because the Kafue lechwe is the most hunted wildlife species in Zambia, hunters need to be informed of the public health risk of Brucella spp. infection.     

Continuous-time modeling of cell fate determination in Arabidopsis flowers

Background  

The genetic control of floral organ specification is currently being investigated by various approaches, both experimentally and through modeling. Models and simulations have mostly involved boolean or related methods, and so far a quantitative, continuous-time approach has not been explored.