CoESPRIT: A Library-Based Construct Screening Method for Identification and Expression of Soluble Protein Complexes

Structural and biophysical studies of protein complexes require multi-milligram quantities of soluble material. Subunits are often unstable when expressed separately so co-expression strategies are commonly employed since in vivo complex formation can provide stabilising effects. Defining constructs for subunit co-expression experiments is difficult if the proteins are poorly understood. Even more problematic is when subunit polypeptide chains co-fold since individually they do not have predictable domains. We have developed CoESPRIT, a modified version of the ESPRIT random library construct screen used previously on single proteins, to express soluble protein complexes. A random library of target constructs is screened against a fixed bait protein to identify stable complexes. In a proof-of-principle study, C-terminal fragments of the influenza polymerase PB2 subunit containing folded domains were isolated using importin alpha as bait. Separately, a C-terminal fragment of the PB1 subunit was used as bait to trap N-terminal fragments of PB2 resulting in co-folded complexes. Subsequent expression of the target protein without the bait indicates whether the target is independently stable, or co-folds with its partner. This highly automated method provides an efficient strategy for obtaining recombinant protein complexes at yields compatible with structural, biophysical and functional studies

Host Determinant Residue Lysine 627 Lies on the Surface of a Discrete, Folded Domain of Influenza Virus Polymerase PB2 Subunit

Understanding how avian influenza viruses adapt to human hosts is critical for the monitoring and prevention of future pandemics. Host specificity is determined by multiple sites in different viral proteins, and mutation of only a limited number of these sites can lead to inter-species transmission. Several of these sites have been identified in the viral polymerase, the best characterised being position 627 in the PB2 subunit. Efficient viral replication at the relatively low temperature of the human respiratory tract requires lysine 627 rather than the glutamic acid variant found systematically in avian viruses. However, the molecular mechanism by which any of these host specific sites determine host range are unknown, although adaptation to host factors is frequently evoked. We used ESPRIT, a library screening method, to identify a new PB2 domain that contains a high density of putative host specific sites, including residue 627. The X-ray structure of this domain (denoted the 627-domain) exhibits a novel fold with the side-chain of Lys627 solvent exposed. The structure of the K627E mutated domain shows no structural differences but the charge reversal disrupts a striking basic patch on the domain surface. Five other recently proposed host determining sites of PB2 are also located on the 627-domain surface. The structure of the complete C-terminal region of PB2 comprising the 627-domain and the previously identified NLS-domain, which binds the host nuclear import factor importin alpha, was also determined. The two domains are found to pack together with a largely hydrophilic interface. These data enable a three-dimensional mapping of approximately half of PB2 sites implicated in cross-species transfer onto a single structural unit. Their surface location is consistent with roles in interactions with other viral proteins or host factors. The identification and structural characterization of these well-defined PB2 domains will help design experiments to elucidate the effects of mutations on polymerase–host factor interactions

High frequency ultrasound assessment of systemic sclerosis skin involvement:intra-observer repeatability and relationship with clinician assessment and dermal collagen content

OBJECTIVE: The modified Rodnan skin score (mRSS) remains the preferred method for skin assessment in systemic sclerosis (SSc). There are concerns regarding high inter-observer variability of mRSS and negative clinical trials utilising mRSS as the primary endpoint. High frequency ultrasound (HFUS) allows objective assessment of cutaneous fibrosis in SSc. We investigated the relationship between HFUS with both mRSS and dermal collagen.METHODS: Skin thickness (ST), echogenicity and novel Shear wave elastography (SWE) were assessed in 53 SSc patients and 15 healthy controls (HC) at the finger, hand, forearm and abdomen. The relationship between HFUS parameters with mRSS (n=53) and dermal collagen (10 SSc patients and 10 HC) was investigated. Intra-observer repeatability of HFUS was calculated using intra-class correlation coefficients (ICCs).RESULTS: HFUS assessment of ST (hand/forearm) and SWE (finger/hand) correlated with local mRSS at some sites. Subclinical abnormalities in ST, echogenicity and SWE were present in clinically uninvolved SSc skin. Additionally, changes in echogenicity and SWE were sometimes apparent despite objectively normal ST on HFUS. ST, SWE and local mRSS correlated strongly with collagen quantification (rho 0.697, 0.709, 0.649 respectively). Intra-observer repeatability was high for all HFUS parameters (ICCs for ST 0.946-0.978, echogenicity 0.648- 0.865 and SWE 0.953-0.973).CONCLUSION: Our data demonstrates excellent reproducibility and reassuring convergent validity with dermal collagen content. Detection of subclinical abnormalities is an additional benefit of HFUS. The observed correlations with collagen quantification support further investigation of HFUS as an alternative to mRSS in clinical trial settings.</p

Use of laser speckle contrast imaging to assess digital microvascular function in primary Raynaud phenomenon and systemic sclerosis:a comparison using the Raynaud condition score diary

Objective.Evaluate objective assessment of digital microvascular function using laser speckle contrast imaging (LSCI) in a cross-sectional study of patients with primary Raynaud phenomenon (RP) and systemic sclerosis (SSc), comparing LSCI with both infrared thermography (IRT) and subjective assessment using the Raynaud Condition Score (RCS) diary.Methods.Patients with SSc (n = 25) and primary RP (n = 18) underwent simultaneous assessment of digital perfusion using LSCI and IRT with a cold challenge on 2 occasions, 2 weeks apart. The RCS diary was completed between assessments. The relationship between objective and subjective assessments of RP was evaluated. Reproducibility of LSCI/IRT was assessed, along with differences between primary RP and SSc, and the effect of sex.Results.There was moderate-to-good correlation between LSCI and IRT (Spearman rho 0.58–0.84, p &lt; 0.01), but poor correlation between objective assessments and the RCS diary (p &gt; 0.05 for all analyses). Reproducibility of IRT and LSCI was moderate at baseline (ICC 0.51–0.63) and immediately following cold challenge (ICC 0.56–0.86), but lower during reperfusion (ICC 0.3–0.7). Neither subjective nor objective assessments differentiated between primary RP and SSc. Men reported lower median daily frequency of RP attacks (0.82 vs 1.93, p = 0.03). Perfusion using LSCI/IRT was higher in men for the majority of assessments.Conclusion.Objective and subjective methods provide differing information on microvascular function in RP. There is good convergent validity of LSCI with IRT and acceptable reproducibility of both modalities. Neither subjective nor objective assessments could differentiate between primary RP and SSc. Influence of sex on subjective and objective assessment of RP warrants further evaluation.</jats:sec

The genetic basis of variation in clean lineages of Saccharomyces cerevisiae in response to stresses encountered during bioethanol fermentations.

Saccharomyces cerevisiae is the micro-organism of choice for the conversion of monomeric sugars into bioethanol. Industrial bioethanol fermentations are intrinsically stressful environments for yeast and the adaptive protective response varies between strain backgrounds. With the aim of identifying quantitative trait loci (QTL's) that regulate phenotypic variation, linkage analysis on six F1 crosses from four highly divergent clean lineages of S. cerevisiae was performed. Segregants from each cross were assessed for tolerance to a range of stresses encountered during industrial bioethanol fermentations. Tolerance levels within populations of F1 segregants to stress conditions differed and displayed transgressive variation. Linkage analysis resulted in the identification of QTL's for tolerance to weak acid and osmotic stress. We tested candidate genes within loci identified by QTL using reciprocal hemizygosity analysis to ascertain their contribution to the observed phenotypic variation; this approach validated a gene (COX20) for weak acid stress and a gene (RCK2) for osmotic stress. Hemizygous transformants with a sensitive phenotype carried a COX20 allele from a weak acid sensitive parent with an alteration in its protein coding compared with other S. cerevisiae strains. RCK2 alleles reveal peptide differences between parental strains and the importance of these changes is currently being ascertained

Phenotypic characterisation of Saccharomyces spp. yeast for tolerance to stresses encountered during fermentation of lignocellulosic residues to produce bioethanol.

BACKGROUND: During industrial fermentation of lignocellulose residues to produce bioethanol, microorganisms are exposed to a number of factors that influence productivity. These include inhibitory compounds produced by the pre-treatment processes required to release constituent carbohydrates from biomass feed-stocks and during fermentation, exposure of the organisms to stressful conditions. In addition, for lignocellulosic bioethanol production, conversion of both pentose and hexose sugars is a pre-requisite for fermentative organisms for efficient and complete conversion. All these factors are important to maximise industrial efficiency, productivity and profit margins in order to make second-generation bioethanol an economically viable alternative to fossil fuels for future transport needs. RESULTS: The aim of the current study was to assess Saccharomyces yeasts for their capacity to tolerate osmotic, temperature and ethanol stresses and inhibitors that might typically be released during steam explosion of wheat straw. Phenotypic microarray analysis was used to measure tolerance as a function of growth and metabolic activity. Saccharomyces strains analysed in this study displayed natural variation to each stress condition common in bioethanol fermentations. In addition, many strains displayed tolerance to more than one stress, such as inhibitor tolerance combined with fermentation stresses. CONCLUSIONS: Our results suggest that this study could identify a potential candidate strain or strains for efficient second generation bioethanol production. Knowledge of the Saccharomyces spp. strains grown in these conditions will aid the development of breeding programmes in order to generate more efficient strains for industrial fermentations

Improving clinical cognitive testing: Report of the AAN Behavioral Neurology Section Workgroup

OBJECTIVE: To evaluate the evidence basis of single-domain cognitive tests frequently used by behavioral neurologists in an effort to improve the quality of clinical cognitive assessment. METHODS: Behavioral Neurology Section members of the American Academy of Neurology were surveyed about how they conduct clinical cognitive testing, with a particular focus on the Neurobehavioral Status Exam (NBSE). In contrast to general screening cognitive tests, an NBSE consists of tests of individual cognitive domains (e.g., memory or language) that provide a more comprehensive diagnostic assessment. Workgroups for each of 5 cognitive domains (attention, executive function, memory, language, and spatial cognition) conducted evidence-based reviews of frequently used tests. Reviews focused on suitability for office-based clinical practice, including test administration time, accessibility of normative data, disease populations studied, and availability in the public domain. RESULTS: Demographic and clinical practice data were obtained from 200 respondents who reported using a wide range of cognitive tests. Based on survey data and ancillary information, between 5 and 15 tests in each cognitive domain were reviewed. Within each domain, several tests are highlighted as being well-suited for an NBSE. CONCLUSIONS: We identified frequently used single-domain cognitive tests that are suitable for an NBSE to help make informed choices about clinical cognitive assessment. Some frequently used tests have limited normative data or have not been well-studied in common neurologic disorders. Utilizing standardized cognitive tests, particularly those with normative data based on the individual's age and educational level, can enhance the rigor and utility of clinical cognitive assessment

Molecular epidemiology of methicillin-resistant Staphylococcus aureus isolated from Australian veterinarians

This work investigated the molecular epidemiology and antimicrobial resistance of methicillinresistant Staphylococcus aureus (MRSA) isolated from veterinarians in Australia in 2009. The collection (n = 44) was subjected to extensive molecular typing (MLST, spa, SCCmec, dru, PFGE, virulence and antimicrobial resistance genotyping) and antimicrobial resistance phenotyping by disk diffusion. MRSA was isolated from Australian veterinarians representing various occupational emphases. The isolate collection was dominated by MRSA strains belonging to clonal complex (CC) 8 and multilocus sequence type (ST) 22. CC8 MRSA (ST8-IV [2B], spa t064; and ST612-IV [2B] , spa variable,) were strongly associated with equine practice veterinarians (OR = 17.5, 95% CI = 3.3-92.5, P &lt; 0.001) and were often resistant to gentamicin and rifampicin. ST22-IV [2B], spa variable, were strongly associated with companion animal practice veterinarians (OR = 52.5, 95% CI = 5.2-532.7, P &lt; 0.001) and were resistant to ciprofloxacin. A single pig practice veterinarian carried ST398-V [5C2], spa t1451. Equine practice and companion animal practice veterinarians frequently carried multiresistant-CC8 and ST22 MRSA, respectively, whereas only a single swine specialist carried MRSA ST398. The presence of these strains in veterinarians may be associated with specific antimicrobial administration practices in each animal species

Regional differences in prostaglandin E₂ metabolism in human colorectal cancer liver metastases

Background: Prostaglandin (PG) E₂ plays a critical role in colorectal cancer (CRC) progression, including epithelial-mesenchymal transition (EMT). Activity of the rate-limiting enzyme for PGE₂ catabolism (15-hydroxyprostaglandin dehydrogenase [15-PGDH]) is dependent on availability of NAD+. We tested the hypothesis that there is intra-tumoral variability in PGE₂ content, as well as in levels and activity of 15-PGDH, in human CRC liver metastases (CRCLM). To understand possible underlying mechanisms, we investigated the relationship between hypoxia, 15-PGDH and PGE₂ in human CRC cells in vitro. Methods: Tissue from the periphery and centre of 20 human CRCLM was analysed for PGE₂ levels, 15-PGDH and cyclooxygenase (COX)-2 expression, 15-PGDH activity, and NAD+/NADH levels. EMT of LIM1863 human CRC cells was induced by transforming growth factor (TGF) β. Results: PGE₂ levels were significantly higher in the centre of CRCLM compared with peripheral tissue (P = 0.04). There were increased levels of 15-PGDH protein in the centre of CRCLM associated with reduced 15-PGDH activity and low NAD+/NADH levels. There was no significant heterogeneity in COX-2 protein expression. NAD+ availability controlled 15-PGDH activity in human CRC cells in vitro. Hypoxia induced 15-PGDH expression in human CRC cells and promoted EMT, in a similar manner to PGE₂. Combined 15-PGDH expression and loss of membranous E-cadherin (EMT biomarker) were present in the centre of human CRCLM in vivo.Conclusions: There is significant intra-tumoral heterogeneity in PGE₂ content, 15-PGDH activity and NAD+ availability in human CRCLM. Tumour micro-environment (including hypoxia)-driven differences in PGE₂ metabolism should be targeted for novel treatment of advanced CRC

FAM5C Contributes to Aggressive Periodontitis

Aggressive periodontitis is characterized by a rapid and severe periodontal destruction in young systemically healthy subjects. A greater prevalence is reported in Africans and African descendent groups than in Caucasians and Hispanics. We first fine mapped the interval 1q24.2 to 1q31.3 suggested as containing an aggressive periodontitis locus. Three hundred and eighty-nine subjects from 55 pedigrees were studied. Saliva samples were collected from all subjects, and DNA was extracted. Twenty-one single nucleotide polymorphisms were selected and analyzed by standard polymerase chain reaction using TaqMan chemistry. Non-parametric linkage and transmission distortion analyses were performed. Although linkage results were negative, statistically significant association between two markers, rs1935881 and rs1342913, in the FAM5C gene and aggressive periodontitis (p = 0.03) was found. Haplotype analysis showed an association between aggressive periodontitis and the haplotype A-G (rs1935881-rs1342913; p = 0.009). Sequence analysis of FAM5C coding regions did not disclose any mutations, but two variants in conserved intronic regions of FAM5C, rs57694932 and rs10494634, were found. However, these two variants are not associated with aggressive periodontitis. Secondly, we investigated the pattern of FAM5C expression in aggressive periodontitis lesions and its possible correlations with inflammatory/immunological factors and pathogens commonly associated with periodontal diseases. FAM5C mRNA expression was significantly higher in diseased versus healthy sites, and was found to be correlated to the IL-1β, IL-17A, IL-4 and RANKL mRNA levels. No correlations were found between FAM5C levels and the presence and load of red complex periodontopathogens or Aggregatibacter actinomycetemcomitans. This study provides evidence that FAM5C contributes to aggressive periodontitis