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.     

Evidence that Early Carboniferous ostracods colonised coastal flood plain brackish water environments

A study of the stable isotope composition (δ¹⁸O, δ¹³C) of biogenic (ostracod, mollusc) and authigenic carbonates in the Ballagan Formation, Lower Carboniferous of Scotland, coupled with evidence from sedimentology and associated fossil fauna and flora, supports the argument that this formation was deposited in a coastal flood plain setting, in brackish (0.5 < 30‰ NaCl) and hypersaline (> 40‰ NaCl) waters, but in the absence of persistent normal marine conditions. The oxygen isotope data from the Ballagan Formation divide into three clusters: a diagenetic field defined by low δ¹⁸O (< − 11‰ VPDB); an intermediary field (δ¹⁸O − 11‰ to − 9‰) composed of a mixture of known primary and secondary (diagenetic) carbonates; and samples within the range of − 9‰ to − 4‰ which, as far as we can ascertain, are largely unaltered. No samples give typical Early Carboniferous δ¹⁸O marine values. Average marine carbonates from Europe have δ¹⁸O between − 4‰ to − 3‰. The Ballagan Formation carbonates were probably deposited in evaporated freshwater and/or brackish water. This conclusion is supported by the presence of evaporites (gypsum, anhydrite, halite pseudomorphs) and common desiccation-cracked mudstone surfaces throughout the Ballagan Formation, suggesting conditions of fluctuating salinity in ephemeral bodies of water. The stable isotope data support the notion that the ostracod assemblages of the Ballagan Formation were colonising brackish water and hypersaline ecologies on a coastal flood plain during the Early Carboniferous, a stage of development that may have encouraged their colonisation of fully non-marine (limnetic) environments during the later Carboniferous. The ostracods include cytherellacean and kloedenellacean species known from marginal marine sites elsewhere, but probably tolerant of brackish water, podocopid species such as ‘Bythocypris’ aequalis that may have been adapted for brackish water settings on coastal flood plains (ephemeral lakes and lagoons), and paraparchitacean-dominated assemblages that may signal harsh (hypersaline or desiccating) environments.     

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.     

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.     

A high-precision global biostratigraphy of myodocope ostracods for the Silurian upper Wenlock Series and Ludlow Series

The wide, trans-oceanic geographical distribution of myodocope ostracods during the Silurian (especially during the Ludlow and Pridoli epochs), and their widespread preservation in rocks of that age, permits the establishment of a transcontinental biostratigraphy of comparable resolution to coeval graptolite/chitinozoan/conodont biozones. Seven myodocope biozones, extending from the Homerian Stage, upper Wenlock Series Cyrtograptus lundgreni graptolite biozone to the middle part of the Ludfordian Stage of the Ludlow Series, enable a time-resolution for each biozone of circa 1 million years. These biozones can provide high-resolution correlation across Europe into Arctic Russia and Central Asia. There is also the potential for a myodocope biostratigraphy applicable from the uppermost Silurian (Pridoli) to the Carboniferous.     

A Silurian 'marrellomorph' arthropod

Xylokorys chledophilia, a new arthropod with three-dimensionally preserved soft tissues, is described from the Herefordshire (Silurian) Lagerstätte of England. The head and trunk are covered by a relatively featureless ovoid carapace, which comprises a domed central part and a flange-like border. The head bears five pairs of appendages. The first is uniramous, with dorsal and ventral projections distally. Appendages two to four are biramous and each endopod terminates in two projections. Appendage five is possibly biramous. The hypostome is very long and subrectangular in outline. There are approximately 35 pairs of biramous trunk appendages. Each exopod comprises a long slender shaft bearing numerous fine filaments; each endopod comprises a ribbon-like shaft bearing paddle-like endites. Morphological comparisons and cladistic analyses of X. chledophilia indicate affinity with Vachonisia rogeri from the Lower Devonian Hunsrück Slate, within the marrellomorphs, but assignment to Marrellomorpha is provisional pending revision of other members of this clade. Xylokorys is the first ‘marrellomorph’ to be reported from the Silurian. It is interpreted as a benthic particle filter feeder, which may also have consumed prey items.     

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.     

A new arthropod from the Silurian Konservat-Lagerstätte of Herefordshire, UK

A small, non-biomineralized, macrophagous arthropod with chelicerate affinities, Offacolus kingi gen. et sp. nov., from the Silurian (Wenlock Series) of Herefordshire, UK, is described. The dorsal exoskeleton comprises an arch-like cephalic shield, a thorax of three free tergites and a triangular posterior tagma of five fused tergites, the last with a stout postero-dorsally directed medial spine. Seven pairs of appendages beneath the cephalic shield surround a postero-medially sited oral cavity on the ventral surface of the head. Appendages I and, probably II are uniramous and project antero-ventrally; I was sensory and II sensory and/or ambulatory. Appendages III-VI are biramous, each with an antero-ventrally projecting ramus and a robust, highly geniculate, horizontally oriented ramus that projects through an anterior gape. The former rami were ambulatory and the latter have spinose terminal podomeres and functioned as a unit for trapping food and transferring it towards the oral cavity. Appendage VII, which is probably uniramous, is posteroventrally directed and flap like. Each tergite of the thorax and posterior tagma covers at least a pair (probably two pairs) of probably biramous appendages with each ramus flap like and setose.     

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.