Vegetative phase change causes age-dependent changes in phenotypic plasticity.

Phenotypic plasticity allows organisms to optimize traits for their environment. As organisms age, they experience diverse environments that benefit from varying degrees of phenotypic plasticity. Developmental transitions can control these age-dependent changes in plasticity, and as such, the timing of these transitions can determine when plasticity changes in an organism. Here, we investigate how the transition from juvenile-to adult-vegetative development known as vegetative phase change (VPC) contributes to age-dependent changes in phenotypic plasticity and how the timing of this transition responds to environment using both natural accessions and mutant lines in the model plant Arabidopsis thaliana. We found that the adult phase of vegetative development has greater plasticity in leaf morphology than the juvenile phase and confirmed that this difference in plasticity is caused by VPC using mutant lines. Furthermore, we found that the timing of VPC, and therefore the time when increased plasticity is acquired, varies significantly across genotypes and environments. The consistent age-dependent changes in plasticity caused by VPC suggest that VPC may be adaptive. This genetic and environmental variation in the timing of VPC indicates the potential for population-level adaptive evolution of VPC.

Polyethylene Glycol Based Changes to ß-Sheet Protein Conformational and Proteolytic Stability Depend on Conjugation Strategy and Location.

The development of chemical strategies for site-specific protein modification now enables researchers to attach polyethylene glycol (PEG) to a protein drug at one or more specific locations (i.e., protein PEGylation). However, aside from avoiding enzyme active sites or protein-binding interfaces, distinguishing the optimal PEGylation site from the available alternatives has conventionally been a matter of trial and error. As part of a continuing effort to develop guidelines for identifying optimal PEGylation sites within proteins, we show here that the impact of PEGylation at various sites within the ß-sheet model protein WW depends strongly on the identity of the PEG-protein linker. The PEGylation of Gln or of azidohomoalanine has a similar impact on WW conformational stability as does Asn-PEGylation, whereas the PEGylation of propargyloxyphenylalanine is substantially stabilizing at locations where Asn-PEGylation was destabilizing. Importantly, we find that at least one of these three site-specific PEGylation strategies leads to substantial PEG-based stabilization at each of the positions investigated, highlighting the importance of considering conjugation strategy as an important variable in selecting optimal PEGylation sites. We further demonstrate that using a branched PEG oligomer intensifies the impact of PEGylation on WW conformational stability and also show that PEG-based increases to conformational stability are strongly associated with corresponding increases in proteolytic stability.

Proof-of-concept study: profile of circulating microRNAs in Bovine serum harvested during acute and persistent FMDV infection.

BACKGROUND: Changes in the levels of circulating microRNAs (miRNAs) in the serum of humans and animals have been detected as a result of infection with a variety of viruses. However, to date, such a miRNA profiling study has not been conducted for foot-and-mouth disease virus (FMDV) infection. METHODS: The relative abundance of 169 miRNAs was measured in bovine serum collected at three different phases of FMDV infection in a proof-of-concept study using miRNA PCR array plates. RESULTS: Alterations in specific miRNA levels were detected in serum during acute, persistent, and convalescent phases of FMDV infection. Subclinical FMDV persistence produced a circulating miRNA profile distinct from cattle that had cleared infection. bta-miR-17-5p was highest expressed during acute infection, whereas bta-miR-31 was the highest during FMDV persistence. Interestingly, miR-1281was significantly down-regulated during both acute and persistent infection. Cattle that cleared infection resembled the baseline profile, adding support to applying serum miRNA profiling for identification of sub-clinically infected FMDV carriers. Significantly regulated miRNAs during acute or persistent infection were associated with cellular proliferation, apoptosis, modulation of the immune response, and lipid metabolism. CONCLUSIONS: These findings suggest a role for non-coding regulatory RNAs in FMDV infection of cattle. Future studies will delineate the individual contributions of the reported miRNAs to FMDV replication, determine if this miRNA signature is applicable across all FMDV serotypes, and may facilitate development of novel diagnostic applications.

Insights into Jumonji C-domain containing protein 6 (JMJD6): a multifactorial role in foot-and-mouth disease virus replication in cells.

The Jumonji C-domain containing protein 6 (JMJD6) has had a convoluted history, and recent reports indicating a multifactorial role in foot-and-mouth disease virus (FMDV) infection have further complicated the functionality of this protein. It was first identified as the phosphatidylserine receptor on the cell surface responsible for recognizing phosphatidylserine on the surface of apoptotic cells resulting in their engulfment by phagocytic cells. Subsequent study revealed a nuclear subcellular localization, where JMJD6 participated in lysine hydroxylation and arginine demethylation of histone proteins and other non-histone proteins. Interestingly, to date, JMDJ6 remains the only known arginine demethylase with a growing list of known substrate molecules. These conflicting associations rendered the subcellular localization of JMJD6 to be quite nebulous. Further muddying this area, two different groups illustrated that JMJD6 could be induced to redistribute from the cell surface to the nucleus of a cell. More recently, JMJD6 was demonstrated to be a host factor contributing to the FMDV life cycle, where it was not only exploited for its arginine demethylase activity, but also served as an alternative virus receptor. This review attempts to coalesce these divergent roles for a single protein into one cohesive account. Given the diverse functionalities already characterized for JMJD6, it is likely to continue to be a confounding protein resulting in much contention going into the near future.

Entropy Measures as Geometrical Tools in the Study of Cosmology.

Classical chaos is often characterized as exponential divergence of nearby trajectories. In many interesting cases these trajectories can be identified with geodesic curves. We define here the entropy by S = ln χ ( x ) with χ ( x ) being the distance between two nearby geodesics. We derive an equation for the entropy, which by transformation to a Riccati-type equation becomes similar to the Jacobi equation. We further show that the geodesic equation for a null geodesic in a double-warped spacetime leads to the same entropy equation. By applying a Robertson-Walker metric for a flat three-dimensional Euclidean space expanding as a function of time, we again reach the entropy equation stressing the connection between the chosen entropy measure and time. We finally turn to the Raychaudhuri equation for expansion, which also is a Riccati equation similar to the transformed entropy equation. Those Riccati-type equations have solutions of the same form as the Jacobi equation. The Raychaudhuri equation can be transformed to a harmonic oscillator equation, and it has been shown that the geodesic deviation equation of Jacobi is essentially equivalent to that of a harmonic oscillator. The Raychaudhuri equations are strong geometrical tools in the study of general relativity and cosmology. We suggest a refined entropy measure applicable in cosmology and defined by the average deviation of the geodesics in a congruence.

Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections.

BACKGROUND: The nuclear protein Src-associated protein of 68 kDa in mitosis (Sam68) is known to bind RNA and be involved in cellular processes triggered in response to environmental stresses, including virus infection. Interestingly, Sam68 is a multi-functional protein implicated in the life cycle of retroviruses and picornaviruses and is also considered a marker of virus-induced stress granules (SGs). Recently, we demonstrated the partial redistribution of Sam68 to the cytoplasm in FMDV infected cells, its interaction with viral protease 3C(pro), and found a significant reduction in viral titers as consequence of Sam68-specific siRNA knockdowns. Despite of that, details of how it benefits FMDV remains to be elucidated. METHODS: Sam68 cytoplasmic localization was examined by immunofluorescent microscopy, counterstaining with antibodies against Sam68, a viral capsid protein and markers of SGs. The relevance of RAAA motifs in the IRES was investigated using electromobility shift assays with Sam68 protein and parental and mutant FMDV RNAs. In addition, full genome WT and mutant or G-luc replicon RNAs were tested following transfection in mammalian cells. The impact of Sam68 depletion to virus protein and RNA synthesis was investigated in a cell-free system. Lastly, through co-immunoprecipitation, structural modeling, and subcellular fractionation, viral protein interactions with Sam68 were explored. RESULTS: FMDV-induced cytoplasmic redistribution of Sam68 resulted in it temporarily co-localizing with SG marker: TIA-1. Mutations that disrupted FMDV IRES RAAA motifs, with putative affinity to Sam68 in domain 3 and 4 cause a reduction on the formation of ribonucleoprotein complexes with this protein and resulted in non-viable progeny viruses and replication-impaired replicons. Furthermore, depletion of Sam68 in cell-free extracts greatly diminished FMDV RNA replication, which was restored by addition of recombinant Sam68. The results here demonstrated that Sam68 specifically co-precipitates with both FMDV 3D(pol) and 3C(pro) consistent with early observations of FMDV 3C(pro)-induced cleavage of Sam68. CONCLUSION: We have found that Sam68 is a specific binding partner for FMDV non-structural proteins 3C(pro) and 3D(pol) and showed that mutations at RAAA motifs in IRES domains 3 and 4 cause a decrease in Sam68 affinity to these RNA elements and rendered the mutant RNA non-viable. Interestingly, in FMDV infected cells re-localized Sam68 was transiently detected along with SG markers in the cytoplasm. These results support the importance of Sam68 as a host factor co-opted by FMDV during infection and demonstrate that Sam68 interact with both, FMDV RNA motifs in the IRES and viral non-structural proteins 3C(pro) and 3D(pol).

Improving compliance with central venous catheter care bundles using electronic records.

BACKGROUND: Health care associated infections are a major contributor to avoidable harm experienced by patients in modern health care settings. Recent reports suggest that electronic checklists for the documentation of a central line bundle may significantly enhance documented process compliance and help to reduce catheter-related bloodstream infection rates. AIMS: This paper describes the use of our electronic tool to monitor and feedback process compliance in conjunction of introducing bespoke central line insertion packs to tackle catheter-related bloodstream infections in our intensive care unit in a medium-sized district general hospital. DESIGN AND METHODS: Continuous quality improvement programme with 'Plan-Do-Study-Act' cycles was implemented. The central venous catheter insertion and maintenance bundle was rolled out in 2007. To monitor compliance with the bundle elements, an electronic tool was designed as part of our bedside Clinical Information System. From 2009, regular quarterly feedback was provided on the number of central venous catheter lines inserted, compliance with the insertion and maintenance bundle and catheter-related bloodstream infection rate using the data collected through the Clinical Information System. We have also introduced dedicated line insertion trolleys and factory-prepared insertion packs. We used segmented regression analysis to assess the changes in the catheter-related bloodstream infection rate before and after implementation of the central venous catheter bundle. RESULTS: Bundle compliance increased during the implementation period and reached over 95% within 6 months. We observed a significant reduction in the catheter-related bloodstream infection rate from 15.6/1000 days to 0.4/1000 days. Regression analysis showed that only the compliance had significant effect on the number and prevalence of catheter-related bloodstream infections. CONCLUSION/IMPLICATIONS: Implementation of evidence-based care bundles reinforced by real-time feedback on the performance of caregivers can significantly reduce the rate of catheter-related bloodstream infection in the intensive care unit. Ensuring that change processes are seamlessly integrated in the workflow with minimal administrative burden is crucial to the quality improvement process.

Criteria for selecting PEGylation sites on proteins for higher thermodynamic and proteolytic stability.

PEGylation of protein side chains has been used for more than 30 years to enhance the pharmacokinetic properties of protein drugs. However, there are no structure- or sequence-based guidelines for selecting sites that provide optimal PEG-based pharmacokinetic enhancement with minimal losses to biological activity. We hypothesize that globally optimal PEGylation sites are characterized by the ability of the PEG oligomer to increase protein conformational stability; however, the current understanding of how PEG influences the conformational stability of proteins is incomplete. Here we use the WW domain of the human protein Pin 1 (WW) as a model system to probe the impact of PEG on protein conformational stability. Using a combination of experimental and theoretical approaches, we develop a structure-based method for predicting which sites within WW are most likely to experience PEG-based stabilization, and we show that this method correctly predicts the location of a stabilizing PEGylation site within the chicken Src SH3 domain. PEG-based stabilization in WW is associated with enhanced resistance to proteolysis, is entropic in origin, and likely involves disruption by PEG of the network of hydrogen-bound solvent molecules that surround the protein. Our results highlight the possibility of using modern site-specific PEGylation techniques to install PEG oligomers at predetermined locations where PEG will provide optimal increases in conformational and proteolytic stability.

Mu-ERD reflects action understanding, but the effect is small.

Since the mid-2000's, many researchers have provided evidence that mu-ERD measured at the motor cortex may reflect the collective activation of upstream brain regions associated with the human mirror system during action observation paradigms; however, several recent papers have called these findings into question. Our study represents an effort to address these criticisms. In our study, participants watched videos in which the type of grip an actor used to grasp a coffee mug either conveyed the goal with 100 % certainty (unambiguous-goal trials), or offered no predictive information (ambiguous-goal trials). If mu-ERD indexes action understanding, then we predicted that mu-ERD should increase while participants watched the actor grasp the mug for unambiguous-goal trials, but not for ambiguous-goal trials. During the intervals where participants watched the actor execute the goal, mu-ERD for unambiguous-goal trials should remain steady, whereas mu-ERD for ambiguous-goal trials should now increase. Conversely, if mu-ERD does not index action understanding, and instead reflects general motor processes associated with action (such as the activation of population vectors in M1 or planning processes), then mu-ERD should show no difference across conditions. Across most comparisons, we found that mu-ERD mostly reflected general motor processes; however, there was a small effect when participants overserved unambiguous-goal trials while watching the actor execute the goal suggesting that mu-ERD does reflect mirroring, but the effect is small.

Ligand Shell Thickness of PEGylated Gold Nanoparticles Controls Cellular Uptake and Radiation Enhancement.

The drive to improve the safety and efficacy of radiotherapies for cancers has prompted the development of nanomaterials that can locally amplify the radiation dose at a tumor without damaging the surrounding healthy tissue. Gold nanoparticles (Au NPs), in particular, exhibit promising radiosensitizing properties under kilovolt X-ray exposure, although the precise mechanism behind this enhancement is not fully understood. While most studies recognize the involvement of factors such as core composition, size, shape, and ligand chemistry in the effectiveness of Au NPs for radiation-induced cancer treatment, there is a scarcity of direct assessments that connect the photophysical properties of the nanomaterial with the observed cellular or biological outcomes. Despite previous evidence of low-energy electron (LEE) emission from Au NPs and their potential to initiate biological damage, to our knowledge, no studies directly correlate the secondary LEE emission with radiation-induced cell death. In this study we assessed Au NPs functionalized with polyethylene glycol (PEG) ligands of varying molecular weights and lengths (1, 5, and 20 kDa PEG) as potential radiosensitizers of A549 lung cancer cells using kilovolt X-ray source potentials (33-130 kVp). We assessed NP internalization using mass cytometry, radiation dose enhancement using clonogenic survival assays, and secondary LEE emission using a retarding field analyzer. Results reveal a statistically significant difference in cellular uptake and radiation dose enhancement for 5 kDa PEG-Au NPs compared to formulations using 1 and 20 kDa PEG, while analysis of secondary LEE emission spectra demonstrated that differences in the length of the PEG ligand did not cause statistically significant attenuation of secondary LEE flux. Consequently, we inferred increased cellular uptake of NPs to be the cause for the observed enhancement in radiosensitivity for 5 kDa PEGylated Au NPs. The approach used in this study establishes a more complete workflow for designing and characterizing the performance of nanomaterial radiosensitizers, allowing for quantification of secondary LEEs and cellular uptake, and ultimately correlation with localized dose enhancement that leads to cell death.

B3GALT6 promotes dormant breast cancer cell survival and recurrence by enabling heparan sulfate-mediated FGF signaling.

Breast cancer mortality results from incurable recurrences thought to be seeded by dormant, therapy-refractory residual tumor cells (RTCs). Understanding the mechanisms enabling RTC survival is therefore essential for improving patient outcomes. Here, we derive a dormancy-associated RTC signature that mirrors the transcriptional response to neoadjuvant therapy in patients and is enriched for extracellular matrix-related pathways. In vivo CRISPR-Cas9 screening of dormancy-associated candidate genes identifies the galactosyltransferase B3GALT6 as a functional regulator of RTC fitness. B3GALT6 is required for glycosaminoglycan (GAG) linkage to proteins to generate proteoglycans, and its germline loss of function in patients causes skeletal dysplasias. We find that B3GALT6-mediated biosynthesis of heparan sulfate GAGs predicts poor patient outcomes and promotes tumor recurrence by enhancing dormant RTC survival in multiple contexts, and does so via a B3GALT6-heparan sulfate/HS6ST1-heparan 6-O-sulfation/FGF1-FGFR2 signaling axis. These findings implicate B3GALT6 in cancer and nominate FGFR2 inhibition as a promising approach to eradicate dormant RTCs and prevent recurrence.

Rare subclonal sequencing of breast cancers indicates putative metastatic driver mutations are predominately acquired after dissemination.

BACKGROUND: Evolutionary models of breast cancer progression differ on the extent to which metastatic potential is pre-encoded within primary tumors. Although metastatic recurrences often harbor putative driver mutations that are not detected in their antecedent primary tumor using standard sequencing technologies, whether these mutations were acquired before or after dissemination remains unclear. METHODS: To ascertain whether putative metastatic driver mutations initially deemed specific to the metastasis by whole exome sequencing were, in actuality, present within rare ancestral subclones of the primary tumors from which they arose, we employed error-controlled ultra-deep sequencing (UDS-UMI) coupled with FFPE artifact mitigation by uracil-DNA glycosylase (UDG) to assess the presence of 132 "metastasis-specific" mutations within antecedent primary tumors from 21 patients. Maximum mutation detection sensitivity was ~1% of primary tumor cells. A conceptual framework was developed to estimate relative likelihoods of alternative models of mutation acquisition. RESULTS: The ancestral primary tumor subclone responsible for seeding the metastasis was identified in 29% of patients, implicating several putative drivers in metastatic seeding including LRP5 A65V and PEAK1 K140Q. Despite this, 93% of metastasis-specific mutations in putative metastatic driver genes remained undetected within primary tumors, as did 96% of metastasis-specific mutations in known breast cancer drivers, including ERRB2 V777L, ESR1 D538G, and AKT1 D323H. Strikingly, even in those cases in which the rare ancestral subclone was identified, 87% of metastasis-specific putative driver mutations remained undetected. Modeling indicated that the sequential acquisition of multiple metastasis-specific driver or passenger mutations within the same rare subclonal lineage of the primary tumor was highly improbable. CONCLUSIONS: Our results strongly suggest that metastatic driver mutations are sequentially acquired and selected within the same clonal lineage both before, but more commonly after, dissemination from the primary tumor, and that these mutations are biologically consequential. Despite inherent limitations in sampling archival primary tumors, our findings indicate that tumor cells in most patients continue to undergo clinically relevant genomic evolution after their dissemination from the primary tumor. This provides further evidence that metastatic recurrence is a multi-step, mutation-driven process that extends beyond primary tumor dissemination and underscores the importance of longitudinal tumor assessment to help guide clinical decisions.

Impact of site-specific PEGylation on the conformational stability and folding rate of the Pin WW domain depends strongly on PEG oligomer length.

Protein PEGylation is an effective method for reducing the proteolytic susceptibility, aggregation propensity, and immunogenicity of protein drugs. These pharmacokinetic challenges are fundamentally related to protein conformational stability, and become much worse for proteins that populate the unfolded state under ambient conditions. If PEGylation consistently led to increased conformational stability, its beneficial pharmacokinetic effects could be extended and enhanced. However, the impact of PEGylation on protein conformational stability is currently unpredictable. Here we show that appending a short PEG oligomer to a single Asn side chain within a reverse turn in the WW domain of the human protein Pin 1 increases WW conformational stability in a manner that depends strongly on the length of the PEG oligomer: shorter oligomers increase folding rate, whereas longer oligomers increase folding rate and reduce unfolding rate. This strong length dependence is consistent with the possibility that the PEG oligomer stabilizes the transition and folded states of WW relative to the unfolded state by interacting favorably with side-chain or backbone groups on the WW surface.

Examination of soluble integrin resistant mutants of foot-and-mouth disease virus.

BACKGROUND: Foot-and-mouth disease virus (FMDV) initiates infection via recognition of one of at least four cell-surface integrin molecules αvß1, αvß3, αvß6, or αvß8 by a highly conserved Arg-Gly-Asp (RGD) amino acid sequence motif located in the G-H loop of VP1. Within the animal host, the αvß6 interaction is believed to be the most relevant. Sub-neutralizing levels of soluble secreted αvß6 (ssαvß6) was used as a selective pressure during passages in vitro to explore the plasticity of that interaction. RESULTS: Genetically stable soluble integrin resistant (SIR) FMDV mutants derived from A24 Cruzeiro were selected after just 3 passages in cell culture in the presence of sub-neutralizing levels of ssαvß6. SIR mutants were characterized by: replication on selective cell lines, plaque morphology, relative sensitivity to ssαvß6 neutralization, relative ability to utilize αvß6 for infection, as well as sequence and structural changes. All SIR mutants maintained an affinity for αvß6. Some developed the ability to attach to cells expressing heparan sulfate (HS) proteoglycan, while others appear to have developed affinity for a still unknown third receptor. Two classes of SIR mutants were selected that were highly or moderately resistant to neutralization by ssαvß6. Highly resistant mutants displayed a G145D substitution (RGD to RDD), while moderately resistant viruses exhibited a L150P/R substitution at the conserved RGD + 4 position. VP1 G-H loop homology models for the A-type SIR mutants illustrated potential structural changes within the integrin-binding motif by these 2 groups of mutations. Treatment of O1 Campos with ssαvß6 resulted in 3 SIR mutants with a positively charged VP3 mutation allowing for HS binding. CONCLUSIONS: These findings illustrate how FMDV particles rapidly gain resistance to soluble receptor prophylactic measures in vitro. Two different serotypes developed distinct capsid mutations to circumvent the presence of sub-neutralizing levels of the soluble cognate receptor, all of which resulted in a modified receptor tropism that expanded the cell types susceptible to FMDV. The identification of some of these adaptive mutations in known FMDV isolates suggests these findings have implications beyond the cell culture system explored in these studies.

One Health Approach to Tick and Tick-Borne Disease Surveillance in the United Kingdom.

Where ticks are found, tick-borne diseases can present a threat to human and animal health. The aetiology of many of these important diseases, including Lyme disease, bovine babesiosis, tick-borne fever and louping ill, have been known for decades whilst others have only recently been documented in the United Kingdom (UK). Further threats such as the importation of exotic ticks through human activity or bird migration, combined with changes to either the habitat or climate could increase the risk of tick-borne disease persistence and transmission. Prevention of tick-borne diseases for the human population and animals (both livestock and companion) is dependent on a thorough understanding of where and when pathogen transmission occurs. This information can only be gained through surveillance that seeks to identify where tick populations are distributed, which pathogens are present within those populations, and the periods of the year when ticks are active. To achieve this, a variety of approaches can be applied to enhance knowledge utilising a diverse range of stakeholders (public health professionals and veterinarians through to citizen scientists). Without this information, the application of mitigation strategies to reduce pathogen transmission and impact is compromised and the ability to monitor the effects of climate change or landscape modification on the risk of tick-borne disease is more challenging. However, as with many public and animal health interventions, there needs to be a cost-benefit assessment on the most appropriate intervention applied. This review will assess the challenges of tick-borne diseases in the UK and argue for a cross-disciplinary approach to their surveillance and control.

An institutional approach to interventional strategies for complete vascular occlusions.

Complete vascular occlusions are rare but potentially lethal. Reports on transcatheter therapy are limited to solitary case reports. The study was conducted as a single-center retrospective chart review. Between January 2003 and December 2009, 26 patients underwent cardiac catheterizations for either a known complete thoracic vascular occlusion or for incidental complete vascular occlusion that was noted during cardiac catheterization. Procedural technique, risk factors, and outcomes are reviewed. Median age at the time of diagnosis was 13.3 years (range 2 months to 54 years). In 6 of 26 (23%) patients, the vascular occlusion was of acute onset. Ninety-two percent of patients had at least 1 known risk factor for vascular occlusion, whereas 54% of patients had ≥ 2 risk factors. Successful rehabilitation was achieved in 19 patients, with direct catheter and/or wire manipulation being used to cross the occluded vessel in 15 (75%) patients, radiofrequency (RF) perforation in 3 patients, and perforation with Brockenbrough needle in 1 patient. Subsequent techniques included AngioJet (n = 2), balloon angioplasty and/or stent implantation (n = 18), and adjuvant local administration of recombinant tissue-plasminogen activator (n = 3). Reinterventions were required in 5 patients, and 8 patients died during the study period from causes unrelated to the catheterization procedure(s). Median follow-up of patients after successful recanalization was 12.6 months (range 1 day to 64 months), and the median duration of survival free from reintervention was 11.8 months (range 1 day to 64 months). In most patients, risk factors can be identified that are associated with the occurrence of a complete vascular occlusion. Different treatment strategies are used in patients having chronic compared with acute occlusions. Residual vascular lesions (hypoplasia or stenosis) are often associated with occurrence of reobstructions and should therefore be treated to prevent such an occurrence. Postprocedural anticoagulation is important in maintaining vascular patency.

Identification of RNA helicase A as a new host factor in the replication cycle of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV), as with other RNA viruses, recruits various host cell factors to assist in the translation and replication of the virus genome. In this study, we investigated the role of RNA helicase A (RHA) in the life cycle of FMDV. Immunofluorescent microscopy (IFM) showed a change in the subcellular distribution of RHA from the nucleus to the cytoplasm in FMDV-infected cells as infection progressed. Unlike nuclear RHA, the RHA detected in the cytoplasm reacted with an antibody that recognizes only the nonmethylated form of RHA. In contrast to alterations in the subcellular distribution of nuclear factors observed during infection with the related cardioviruses, cytoplasmic accumulation of RHA did not require the activity of the FMDV leader protein. Using IFM, we have found cytoplasmic RHA in proximity to the viral 2C and 3A proteins, which promotes the assembly of the replication complexes, as well as cellular poly(A) binding protein (PABP). Coimmunoprecipitation assays confirmed that these proteins are complexed with RHA. We have also identified a novel interaction between RHA and the S fragment in the FMDV 5' nontranslated region. Moreover, a reduction in the expression of RHA, using RHA-specific small interfering RNA constructs, inhibited FMDV replication. These results indicate that RHA plays an essential role in the replication of FMDV and potentially other picornaviruses through ribonucleoprotein complex formation at the 5' end of the genome and by interactions with 2C, 3A, and PABP.

Identification and quantification of myo-inositol hexakisphosphate in complex environmental matrices using ion chromatography and high-resolution mass spectrometry in comparison to 31P NMR spectroscopy.

Myo-inositol hexakisphosphate, or phytic acid, (myo-IP6) is a key organic phosphorus (P) compound in soils and manures. Determinations of myo-IP6 in soils and manure extracts are frequently performed by 31P NMR spectroscopy. This approach is time-consuming in terms of both sample preparation and instrument time, with uncertainties existing in relation to accuracy of identification and quantification due to potentially interfering resonances from co-extracted P species. In contrast, ion chromatography (IC) in combination with high-resolution mass spectrometry (HRMS) negative ion, electrospray ionisation (ESI) has been shown to enable highly specific identifications of myo-IP6 isolated from complex mixtures. In this paper, IC and ESI-HRMS were applied to the identification and the quantification of myo-IP6 isolated from soils and manures using NaOH-EDTA extraction, and quantifications based on IC. ESI-HRMS analysis of eluate trapped from IC unequivocally confirmed identification of myo-IP6 from a soil extract. The ion suppression cell of the IC instrument provides isolates of the analyte free of ionic components that would interfere with ESI. The myo-IP6 was identified in the NMR by comparing spectra of extracts of soils with and without authentic myo-IP6 "spiked" prior to extraction. Comparison of quantification via standard addition in IC and NMR analysis gave good correlation (r = 0.955). IC with ESI-HRMS was found to be more sensitive, rapid and reliable for the identification and quantification of myo-IP6 with a limit of detection (LOD) of 0.7 mg kg-1 and limit of quantification (LOQ) of 2.1 mg kg-1 using IC versus > 10 mg kg-1 LOD using 31P NMR.

Advances in the Application and Impact of MicroRNAs as Therapies for Skin Disease.

The advent of RNA interference (RNAi) technology has profoundly impacted molecular biology research and medicine but has also advanced the field of skin care. Both effector molecules of RNAi, short-interfering RNA molecules and microRNAs (miRNAs), have been explored for their relative impact and utility for treating a variety of skin conditions. These post-transcriptional RNA regulatory molecules down-modulate protein expression through targeting of the 3' untranslated regions of messenger RNAs, leading to their degradation or repression through sequestration. As researchers hunt for genetic linkages to skin diseases, miRNA regulators have emerged as key players in the biology of keratinocytes, fibroblasts, melanocytes, and other cells of the skin. Herein, we attempt to coalesce the current efforts to combat various skin disorders and diseases through the development of miRNA-based technologies.