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23259695

Use of novel mutant galactosyltransferase for the bioconjugation of terminal N-acetylglucosamine (GlcNAc) residues on live cell surface.

On the basis of the crystal structure of bovine β4Gal-T1 enzyme, mutation of a single amino acid Y289 to L289 (Y289L) changed its donor specificity from Gal to N-acetyl-galactosamine (GalNAc). A chemoenzymatic method that uses GalNAc analogues like GalNAz or 2-keto-Gal as sugar donors with the enzyme Y289L-β4Gal-T1 has identified hundreds of cytosolic and nuclear proteins that have O-GlcNAc modifications. To avoid potential cytotoxicity at Mn(2+) concentrations required to selectively modify GlcNAc residues on the surface of live cells, we have engineered a Mg(2+)-dependent enzyme. Previously, we found that the mutation of the metal-binding residue Met-344 to His-344 in bovine β4Gal-T1 enzyme altered its metal-ion specificity in such a way that the M344H-β4Gal-T1 enzyme exhibits better catalytic activity with Mg(2+) than with Mn(2+). Here, we find that, when these two mutations are combined, the double mutant, Y289L-M344H-β4Gal-T1, transfers GalNAc and its analogue sugars to the acceptor GlcNAc in the presence of Mg(2+). Using this mutant enzyme, we have detected free GlcNAc residues on the surface glycans of live HeLa cells and platelets. The specific transfer of a synthetic sugar with a chemical handle to the terminal GlcNAc residues on the surface of live cells provides a novel tool for selective modification, detection, and isolation of GlcNAc-ending glycans present on the cellular surface.

23259694

Feasibility and initial efficacy of a cognitive-behavioural group programme for managing anger and aggressiveness after traumatic brain injury.

This study assesses the feasibility of a cognitive-behavioural group programme for treating anger and aggressiveness after a traumatic brain injury (TBI). Five feasibility criteria were considered: demand, implementation, practicality, acceptability and initial efficacy. A self-report questionnaire of aggressiveness (AQ-12) was administered before the intervention (T1), one week following the intervention (T2) and at a four months follow-up (T3). Ten patients with moderate to severe chronic TBI completed the programme through eight once-a-week sessions. The analysis of the feasibility outcomes suggests that: (1) The recruitment, the process of grouping participants and the characterisation of anger and aggressiveness at baseline need to be re-evaluated and improved for future designs. (2) The use of specific strategies for bypassing cognitive and other behavioural dysfunctions related to TBI is crucial for the success of this intervention and merits special attention. (3) The high retention rate, the convenient meeting schedule, cost advantages and the good acceptability by participants are positive arguments for the implementation of a larger trial. (4) The significant reduction of AQ-12 scores at T3 and the high effect size constitute a change in the expected direction and support the initial efficacy of the programme.

23259693

Disruption of Escherichia coli amyloid-integrated biofilm formation at the air-liquid interface by a polysorbate surfactant.

Functional amyloid fibers termed curli contribute to bacterial adhesion and biofilm formation in Escherichia coli . We discovered that the nonionic surfactant Tween 20 inhibits biofilm formation by uropathogenic E. coli at the air-liquid interface, referred to as pellicle formation, and at the solid-liquid interface. At Tween 20 concentrations near and above the critical micelle concentration, the interfacial viscoelastic modulus is reduced to zero as cellular aggregates at the air-liquid interface are locally disconnected and eventually eliminated. Tween 20 does not inhibit the production of curli but prevents curli-integrated film formation. Our results support a model in which the hydrophobic curli fibers associated with bacteria near the air-liquid interface require access to the gas phase to formed strong physical entanglements and to form a network that can support shear stress.

23259691

Keampferol-3-O-rhamnoside abrogates amyloid beta toxicity by modulating monomers and remodeling oligomers and fibrils to non-toxic aggregates.

Aggregation of soluble, monomeric β- amyloid (Aβ) to oligomeric and then insoluble fibrillar Aβ is a key pathogenic feature in development of Alzheimer's disease (AD). Increasing evidence suggests that toxicity is linked to diffusible Aβ oligomers, rather than to insoluble fibrils. The use of naturally occurring small molecules for inhibition of Aβ aggregation has recently attracted significant interest for development of effective therapeutic strategies against the disease. A natural polyphenolic flavone, Kaempferol-3-O-rhamnoside (K-3-rh), was utilized to investigate its effects on aggregation and cytotoxic effects of Aβ42 peptide. Several biochemical techniques were used to determine the conformational changes and cytotoxic effect of the peptide in the presence and absence of K-3-rh. K-3-rh showed a dose-dependent effect against Aβ42 mediated cytotoxicity. Anti-amyloidogenic properties of K-3-rh were found to be efficient in inhibiting fibrilogenesis and secondary structural transformation of the peptide. The consequence of these inhibitions was the accumulation of oligomeric structural species. The accumulated aggregates were smaller, soluble, non-β-sheet and non-toxic aggregates, compared to preformed toxic Aβ oligomers. K-3-rh was also found to have the remodeling properties of preformed soluble oligomers and fibrils. Both of these conformers were found to remodel into non-toxic aggregates. The results showed that K-3-rh interacts with different Aβ conformers, which affects fibril formation, oligomeric maturation and fibrillar stabilization. K-3-rh is an efficient molecule to hinder the self assembly and to abrogate the cytotoxic effects of Aβ42 peptide. Hence, K-3-rh and small molecules with similar structure might be considered for therapeutic development against AD.

23259692

Disrupted functional brain networks in autistic toddlers.

Communication and integration of information between brain regions plays a key role in healthy brain function. Conversely, disruption in brain communication may lead to cognitive and behavioral problems. Autism is a neurodevelopmental disorder that is characterized by impaired social interactions and aberrant basic information processing. Aberrant brain connectivity patterns have indeed been hypothesized to be a key neural underpinning of autism. In this study, graph analytical tools are used to explore the possible deviant functional brain network organization in autism at a very early stage of brain development. Electroencephalography (EEG) recordings in 12 toddlers with autism (mean age 3.5 years) and 19 control subjects were used to assess interregional functional brain connectivity, with functional brain networks constructed at the level of temporal synchronization between brain regions underlying the EEG electrodes. Children with autism showed a significantly increased normalized path length and reduced normalized clustering, suggesting a reduced global communication capacity already during early brain development. In addition, whole brain connectivity was found to be significantly reduced in these young patients suggesting an overall under-connectivity of functional brain networks in autism. Our findings support the hypothesis of abnormal neural communication in autism, with deviating effects already present at the early stages of brain development.

23259689

Fatty acids do not pay the toll: effect of SFA and PUFA on human adipose tissue and mature adipocytes inflammation.

On the basis that high fat diet induces inflammation in adipose tissue, we wanted to test the effect of dietary saturated and polysunsaturated fatty acids on human adipose tissue and adipocytes inflammation. Moreover we wanted to determine if TLR2 and TLR4 are involved in this pathway. Human adipose tissue and adipocytes primary cultures were treated with endotoxin-free BSA conjugated with SFA (lauric acid and palmitic acid--LA and PA) and PUFA (eicosapentaeneic acid, docosahexaenoic acid and oleic acid--EPA, DHA and OA) with or without LPS. Cytokines were then assayed by ELISA (TNF-alpha, IL-6 and MCP-1). In order to determine if TLR2 and TLR4 are activated by fatty acid (FA), we used HEK-Blue cells transfected by genes from TLR2 or TLR4 pathways associated with secreted alkaline phosphatase reporter gene. None of the FA tested in HEK-Blue cells were able to activate TLR2 or TLR4, which is concordant with the fact that after FA treatment, adipose tissue and adipocytes cytokines levels remain the same as controls. However, all the PUFA tested: DHA, EPA and to a lesser extent OA down-regulated TNF-alpha, IL-6 and MCP-1 secretion in human adipose tissue and adipocytes cultures. This study first confirms that FA do not activate TLR2 and TLR4. Moreover by using endotoxin-free BSA, both SFA and PUFA tested were not proinflammatory in human adipose tissue and adipocytes model. More interestingly we showed that some PUFA exert an anti-inflammatory action in human adipose tissue and adipocytes model. These results are important since they clarify the relationship between dietary fatty acids and inflammation linked to obesity.

23259690

Stereoselective synthesis of 1,3-anti diols by an Ipc-mediated domino aldol-coupling/reduction sequence.

A novel domino process for 1,3-anti diol synthesis by the union of a methyl ketone with an aldehyde is described. The operationally simple procedure is based on an Ipc-boron-aldol coupling and subsequent Ipc-mediated reduction of the intermediate β-hydroxy-ketone. The sequence proceeds with excellent anti-selectivities and enables the rapid construction of complex polyketide fragments.

23259688

Self-monitoring of blood glucose during pregnancy: indications and limitations.

Self-monitoring of blood glucose (SMBG) is an important tool to treat diabetes during pregnancy. However, proper implementation of SMBG in pregnant women requires understanding of its applications and limitations. This article reviews issues related to the implementation, efficacy, and accuracy of SMBG and discusses factors that can confound results of SMBG during pregnancy.

23259687

Assessment of thiol compounds from garlic by automated headspace derivatized in-needle-NTD-GC-MS and derivatized in-fiber-SPME-GC-MS.

This study investigates the analysis of thiol compounds using a needle trap device (HS-NTD) and solid-phase microextraction (HS-SPME) derivatized headspace techniques coupled to GC-MS. Thiol compounds and their outgassed products are particularly difficult to monitor in foodstuffs. It was found that with in-needle and in-fiber derivatization, using the derivatization agent N-phenylmaleimide, it was possible to enhance the selectivity toward thiol, which allowed the quantitation of butanethiol, ethanethiol, methanethiol, and propanethiol compounds found in fresh garlic. A side-hole NTD was prepared and packed in house and utilized mixed DVB and Carboxen polymer extraction phases made of 60-80 mesh particles. NTD sampling was accomplished in the exhaustive sampling mode, where breakthrough was negligible. This work demonstrates a new application for a side-hole NTD sampling. A commercial mixed polymer phase of polydimethylsiloxane (PDMS) and divinylbenzene polymer (DVB) SPME fiber was used for SPME extractions. Under optimized derivatization, extraction, and analysis conditions for both NTD-GC-MS and SPME-GC-MS techniques, automated sampling methods were developed for quantitation. Both methods demonstrate a successful approach to thiol determination and provide a quantitative linear response between <0.1 and 10 mg L(-1) (R(2) = 0.9996), with limits of detection (LOD) in the low micrograms per liter range for the investigated thiols. Addition methods using known spiked quantities of thiol analytes in ground garlic facilitated method validation. Carry-over was also negligible for both SPME and NTD under optimized conditions.

23259686

Can dispersed biomass processing protect the environment and cover the bottom line for biofuel?

This paper compares environmental and profitability outcomes for a centralized biorefinery for cellulosic ethanol that does all processing versus a biorefinery linked to a decentralized array of local depots that pretreat biomass into concentrated briquettes. The analysis uses a spatial bioeconomic model that maximizes profit from crop and energy products, subject to the requirement that the biorefinery must be operated at full capacity. The model draws upon biophysical crop input-output coefficients simulated with the Environmental Policy Integrated Climate (EPIC) model as well as market input and output prices, spatial transportation costs, ethanol yields from biomass, and biorefinery capital and operational costs. The model was applied to 82 cropping systems simulated across 37 subwatersheds in a 9-county region of southern Michigan in response to ethanol prices simulated to rise from $1.78 to $3.36 per gallon. Results show that the decentralized local biomass processing depots lead to lower profitability but better environmental performance, due to more reliance on perennial grasses than the centralized biorefinery. Simulated technological improvement that reduces the processing cost and increases the ethanol yield of switchgrass by 17% could cause a shift to more processing of switchgrass, with increased profitability and environmental benefits.

23259685

HLA typing from RNA-Seq sequence reads.

We present a method, seq2HLA, for obtaining an individual's human leukocyte antigen (HLA) class I and II type and expression using standard next generation sequencing RNA-Seq data. RNA-Seq reads are mapped against a reference database of HLA alleles, and HLA type, confidence score and locus-specific expression level are determined. We successfully applied seq2HLA to 50 individuals included in the HapMap project, yielding 100% specificity and 94% sensitivity at a P-value of 0.1 for two-digit HLA types. We determined HLA type and expression for previously un-typed Illumina Body Map tissues and a cohort of Korean patients with lung cancer. Because the algorithm uses standard RNA-Seq reads and requires no change to laboratory protocols, it can be used for both existing datasets and future studies, thus adding a new dimension for HLA typing and biomarker studies.

23259684

Genetic oxidative stress variants and glioma risk in a Chinese population: a hospital-based case-control study.

The oxidative stress mechanism is of particular interest in the pathogenesis of glioma, given the high rate of oxygen metabolism in the brain. Potential links between polymorphisms of antioxidant genes and glioma risk are currently unknown. We therefore investigated the association between polymorphisms in antioxidant genes and glioma risk. We examined 16 single nucleotide polymorphisms (SNPs) of 9 antioxidant genes (GPX1, CAT, PON1, NQO1, SOD2/MnSOD, SOD3, and NOS1*2*3) in 384 glioma and 384 control cases in a Chinese hospital-based case-control study. Genotypes were determined using the OpenArray platform, which employs the chip-based Taq-Man genotyping technology. The adjusted odds ratio (OR) and 95% confidence interval (CI) were estimated using unconditional logistic regression. Using single-locus analysis, we identified four SNPs (SOD2 V16A, SOD3 T58A, GPX1 -46 C/T, and NOS1 3'-UTR) that were significantly associated with the risk of glioma development. To assess the cumulative effects, we performed a combined unfavourable genotype analysis. Compared with the reference group that exhibited no unfavourable genotypes, the medium- and high-risk groups exhibited a 1.86-fold (95% CI, 1.30-2.67) and a 4.86-fold (95% CI, 1.33-17.71) increased risk of glioma, respectively (P-value for the trend < 0.001). These data suggest that genetic variations in oxidative stress genes might contribute to the aetiology of glioma.

23259682

Dual contrast agent for computed tomography and magnetic resonance hard tissue imaging.

Calcium phosphate cements (CPCs) are commonly used bone substitute materials, which closely resemble the composition of the mineral phase of bone. However, this high similarity to natural bone also results in difficult discrimination from the bone tissue by common imaging modalities, that is, plain X-ray radiography and three-dimensional computed tomography (CT). In addition, new imaging techniques introduced for bone tissue visualization, like magnetic resonance imaging (MRI), face a similar problem. Even at high MRI resolution, the lack of contrast between CPCs and surrounding bone is evident. Therefore, this study aimed to evaluate the feasibility of a dual contrast agent, traceable with both CT and MRI as enhancers of CPC/bone tissue contrast. Our formulation is based on the use of silica beads as vectors, which encapsulate and carry contrast-enhancing nanoparticles, in our case, colloidal Gold and Superparamagnetic Iron oxide particles (SPIO). The bead suspension was incorporated within a calcium phosphate powder. The resultant cements were then tested both in vitro and in vivo in a femoral condyle defect model in rats. Results showed that the mechanical properties of the cement were not significantly affected by the inclusion of the beads. Both in vitro and in vivo data proved the homogeneous incorporation of the contrast within the cement and its visual localization, characterized by a short-term CT contrast enhancement and a long-term MR effect recognizable by the characteristic blooming shape. Finally, no signs of adverse tissue reactions were noticed in vivo. In conclusion, this study proved the feasibility of a multimodal contrast agent as an inert and biocompatible enhancer of CaP cement versus bone tissue contrast.

23259683

HIV serostatus disclosure is not associated with safer sexual behavior among HIV-positive men who have sex with men (MSM) and their partners at risk for infection in Bangkok, Thailand.

The relationship between HIV serostatus disclosure and sexual risk behavior is inconsistent across studies. As men who have sex with men (MSM) are emerging as the key affected population in Bangkok, Thailand with reported HIV prevalence of 30%, we assessed whether HIV disclosure is associated with protected sex in this population. A risk behavior questionnaire was administered using Audio Computer-Assisted Self-Interviewing (ACASI) to determine whether HIV serostatus disclosure was associated with protected sex in 200 HIV-positive MSM in Bangkok. HIV serostatus disclosure to the most recent sexual partner prior to or at the time of the sexual encounter was assessed. Protected sex was defined as insertive or receptive anal intercourse with a condom at the most recent sexual encounter. The mean age was 30.2 years, CD4 was 353 cells/mm3, and one-third was on antiretroviral therapy. At the most recent sexual encounter, HIV serostatus disclosure rate was low (26%); 60.5% of subjects had not discussed their serostatus at all, while 5.5% had not revealed their true serostatus. Seventeen percent reported unprotected anal intercourse and about half had sex with their primary partners. The serostatus of the most recent sexual partner was HIV-positive in 19.2%, HIV-negative in 26.4%, and unknown in 54.4% of subjects. There was no association between disclosure and protected sex, with 41 of 48 (85.4%) disclosers and 104 of 126 (82.5%) of non-disclosers reported protected sex (p = .65). Subjects with HIV-positive partners were less likely to report protected sex overall (20 of 33, 60.6%) compared to those with HIV negative (82 of 96, 85.4%) or unknown (41 of 45, 91.1%) partners (p = .001). Age (27-32 years vs. ≤26 years, p = .008), primary partner status (p < .001), and HIV-positive serostatus of sexual partner (p < .001) were significantly associated with disclosure in the multivariate analyses. Rates of HIV disclosure to sexual partners by HIV-positive MSM in Bangkok are low. Despite low rates of HIV serostatus disclosure, most HIV-positive MSM reported protected sex with their partners at risk for infection. Future studies should focus on understanding barriers to disclosure and factors driving risk behavior amongst MSM in Thailand.

23259681

In search of attachment: a qualitative study of chronically ill women transitioning between family physicians in rural Ontario, Canada.

Most Canadians receive basic health services from a family physician and these physicians are particularly critical in the management of chronic disease. Canada, however, has an endemic shortage of family physicians. Physician shortages and turnover are particularly acute in rural regions, leaving their residents at risk of needing to transition between family physicians. The knowledge base about how patients manage transitioning in a climate of scarcity remains nascent. The purpose of this study is to explore the experience of transitioning for chronically ill, rurally situated Canadian women to provide insight into if and how the system supports transitioning patients and to identify opportunities for enhancing that support. Chronically ill women managing rheumatic diseases residing in two rural counties in the province of Ontario were recruited to participate in face-to-face, semi-structured interviews. Interview transcripts were analysed thematically to identify emergent themes associated with the transitioning experience. Seventeen women participated in this study. Ten had experienced transitioning and four with long-standing family physicians anticipated doing so soon. The remaining three expressed concerns about transitioning. Thematic analysis revealed the presence of a transitioning trajectory with three phases. The detachment phase focused on activities related to the termination of a physician-patient relationship, including haphazard notification tactics and the absence of referrals to replacement physicians. For those unable to immediately find a new doctor, there was a phase of unattachment during which patients had to improvise ways to receive care from alternative providers or walk-in clinics. The final phase, attachment, was characterized by acceptance into the practice of a new family physician. Participants often found transitioning challenging, largely due to perceived gaps in support from the health care system. Barriers to a smooth transition included inadequate notification procedures, lack of formal assistance finding new physicians, and unsatisfactory experiences seeking care during unattachment. The participants' accounts reveal opportunities for a stronger system presence during transition and a need for further research into alternative models of primary care delivery.

23259680

Estrogen-like activity of aqueous extract from Agrimonia pilosa Ledeb. in MCF-7 cells.

Postmenopausal women experience estrogen deficiency-related menopausal symptoms (e.g., hot flashes and mood swings) and a dramatic increase in the incidence of chronic diseases. Although estrogen-replacement therapy (ERT) can reduce mortality from cardiovascular disease and improve osteoporosis and menopausal symptoms, its side effects have limited recent use. This study investigated the estrogen-like activity of aqueous extract from Agrimonia pilosa Ledeb. The estrogenic activity of A. pilosa was investigated by using several in vitro assays. The binding activity of A. pilosa on estrogen receptors was examined using a fluorescence polarization-based competitive binding assay. The proliferative activity of A. pilosa was also examined using MCF-7 cells. Furthermore, the effect of A. pilosa on the expression of 3 estrogen-dependent genes was assessed. Using liquid chromatography-mass spectrometry, the 3 major peaks of A. pilosa aqueous extract were identified as apigenin-hexose, luteolin-glucuronide, and apigenin-glucuronide. The aqueous extract induced the proliferation of estrogen receptor-positive MCF-7 cells (p < 0.05). A. pilosa-stimulated proliferation was blocked on adding the estrogen antagonist ICI 182,780. Moreover, A. pilosa treatment increased the mRNA expression of the estrogen-responsive genes pS2 and PR (p < 0.05). These results suggest A. pilosa can be used to improve estrogen deficiency-related menopausal symptoms or to treat diseases in postmenopausal women.

23259679

Sustainable high capacitance at high frequencies: metallic aluminum-polypropylene nanocomposites.

The high-frequency dielectric response of 0-3 polypropylene nanocomposites prepared with the activated metallocene polymerization catalyst [rac-ethylenebisindenyl]zirconium dichlororide absorbed on the native Al(2)O(3) surfaces of metallic aluminum nanoparticles is characterized. The nanocomposites produced are randomly dispersed in the polyolefin matrix with no visible defects that might degrade film dielectric properties. Electrical measurements show that as the volume fraction of Al nanoparticles is increased, the effective permittivity of the nanocomposites increases, with ε(r) values reaching ~10 at relatively low frequency (1 MHz). Because of the high permittivity and conductivity contrast between the metal nanoparticles and the polypropylene matrix, Maxwell-Wagner-Sillars theory can be applied to model the loss at high frequencies and provide insight into how the nanocomposite high frequency response scales with Al volume fraction. At higher Al nanoparticle volume fractions, mixing theories predict greater densities of nanoparticle aggregates, consistent with the experimentally observed shift of the dielectric relaxation to lower frequencies. Although these nanocomposites undergo the predicted initial dielectric relaxation with increasing frequency, the metallic nanoparticle complex permittivity imbues the higher Al volume fraction materials with relatively high, sustainable permittivities, 6, at frequencies as high as 7 GHz.

23259678

Aberrant innate immune sensing leads to the rapid progression of idiopathic pulmonary fibrosis.

Novel approaches are needed to define subgroups of patients with Idiopathic pulmonary fibrosis (IPF) at risk for acute exacerbations and/or accelerated progression of this generally fatal disease. Progression of disease is an integral component of IPF with a median survival of 3 to 5 years. Conversely, a high degree of variability in disease progression has been reported among series. The characteristics of patients at risk of earlier death predominantly rely on baseline HRCT appearance, but this concept that has been challenged. Disparate physiological approaches have also been taken to identify patients at risk of mortality, with varying results. We hypothesized that the rapid decline in lung function in IPF may be a consequence of an abnormal host response to pathogen-associated molecular patterns (PAMPs), leading to aberrant activation in fibroblasts and fibrosis. Analysis of upper and lower lobe surgical lung biopsies (SLBs) indicated that TLR9, a hypomethylated CpG DNA receptor, is prominently expressed at the transcript and protein level, most notably in biopsies from rapidly progressive IPF patients. Surprisingly, fibroblasts appeared to be a major cellular source of TLR9 expression in IPF biopsies from this group of progressors. Further, CpG DNA promoted profibrotic cytokine and chemokine synthesis in isolated human IPF fibroblasts, most markedly again in cells from patients with the rapidly progressive IPF phenotype, in a TLR9-dependent manner. Finally, CpG DNA exacerbated fibrosis in an in vivo model initiated by the adoptive transfer of primary fibroblasts derived from patients who exhibited rapidly progressing fibrosis. Together, these data suggested that TLR9 activation via hypomethylated DNA might be an important mechanism in promoting fibrosis particularly in patients prone to rapidly progressing IPF.

23259677

Therapeutic management of intestinal fibrosis induced by radiation therapy: from molecular profiling to new intervention strategies et vice et versa.

Chronic toxicities of locoregional and systemic oncological treatments commonly develop in long-term cancer survivors. Amongst these toxicities, post-radiotherapeutic complications alter patient's quality of life. Reduction of exposure of normal tissues can be achieved by optimization of radiotherapy. Furthermore, understanding of the fibrogenic mechanisms has provided targets to prevent, mitigate, and reverse late radiation-induced damages. This mini-review shows how (i) global molecular studies using gene profiling can provide tools to develop new intervention strategies and (ii) how successful clinical trials, conducted in particular with combined pentoxifylline-vitamin E, can take benefice of biological and molecular evidences to improve our understanding of fibrogenic mechanisms, enhance the robustness of proposed treatments, and lead ultimately to better treatments for patient's benefice.

23259676

Controlling supramolecular complex formation on the surface of a monolayer-protected gold nanoparticle in water.

A combination of hydrophobic and electrostatic interactions drives the self-assembly of a large number of small molecules on the surface of a monolayer-protected gold nanoparticle. The hydrophobic interactions originate from the insertion of an aromatic unit in the hydrophobic part of the monolayer. This is evidenced by a shift in the emission wavelength of the fluorogenic probe upon binding. Up to around 35 small molecules can be simultaneously bound to the monolayer surface at micromolar concentrations in water. It is shown that an understanding of the supramolecular interactions that drive complex formation on the monolayer surface provides unprecedented control over the supramolecular chemistry occurring on the surface. By taking advantage of the different kinds of noncovalent interactions present in different probes, it is possibile to displace one type of surface-bound molecule from a heteromeric surface selectively. Finally, it is also possible to catch and release one type of surface-bound molecule selectively.

23259675

Adaptive memory: the survival scenario enhances item-specific processing relative to a moving scenario.

Nairne, Thompson, and Pandeirada (2007) found that retention of words rated for their relevance to survival is superior to that of words encoded under numerous other deep processing conditions. They suggested that our memory systems might have evolved to confer an advantage for survival-relevant information. Burns, Burns, and Hwang (2011) suggested a two-process explanation of the proximate mechanisms responsible for the survival advantage. Whereas most control tasks encourage only one type of processing, the survival task encourages both item-specific and relational processing. They found that when control tasks encouraged both types of processing, the survival processing advantage was eliminated. However, none of their control conditions included non-survival scenarios (e.g., moving, vacation, etc.), so it is not clear how this two-process explanation would explain the survival advantage when scenarios are used as control conditions. The present experiments replicated the finding that the survival scenario improves recall relative to a moving scenario in both a between-lists and within-list design and also provided evidence that this difference was accompanied by an item-specific processing difference, not a difference in relational processing. The implications of these results for several existing accounts of the survival processing effect are discussed.

23259674

Regulation of ubiquitin transfer by XIAP, a dimeric RING E3 ligase.

RING domains of E3 ligases promote transfer of Ub (ubiquitin) from the E2~Ub conjugate to target proteins. In many cases interaction of the E2~Ub conjugate with the RING domain requires its prior dimerization. Using cross-linking experiments we show that E2 conjugated ubiquitin contacts the RING homodimer interface of the IAP (inhibitor of apoptosis) proteins, XIAP (X-linked IAP) and cIAP (cellular IAP) 2. Structural and biochemical analysis of the XIAP RING dimer shows that an aromatic residue at the dimer interface is required for E2~Ub binding and Ub transfer. Mutation of the aromatic residue abolishes Ub transfer, but not interaction with Ub. This indicates that nuleophilic attack on the thioester bond depends on precise contacts between Ub and the RING domain. RING dimerization is a critical activating step for the cIAP proteins; however, our analysis shows that the RING domain of XIAP forms a stable dimer and its E3 ligase activity does not require an activation step.

23259672

Plasmodium vivax infection in Anajás, State of Pará: no differential resistance profile among Duffy-negative and Duffy-positive individuals.

There is large body of evidence that states that invasion of Plasmodium vivax requires the Duffy antigen, but the universality of this specificity is certainly now under question with recent reports showing that in some parts of the world P. vivax infects and causes disease in Duffy-negative people. These findings reinforce the idea that this parasite is rapidly evolving, being able to use other receptors than Duffy to invade the erythrocytes, which may have an enormous impact in P. vivax current distribution. The presence of P. vivax infection in Duffy-negative individuals was investigated in a cross-sectional study conducted in Anajás, Archipelago of Marajó, State of Pará, which is an area of malaria transmission in the Brazilian Amazonia. Duffy genotyping and Plasmodium species diagnostic assays were performed successfully in 678 individuals. An allele-specific primer polymerase chain reaction (PCR) technique was used for Duffy blood group genotyping. Identification of Plasmodium species was achieved by conventional blood smear light microscopy and a TaqMan-based real-time PCR method to detect mitochondrial genome of Plasmodium falciparum and P. vivax. Plasmodium spp. infection was detected in 137 samples (20.2%). Prevalence of each Plasmodium species was 13.9% P. vivax, 5.8% P. falciparum, and 0.6% P. vivax plus P. falciparum. Overall, 4.3% (29/678) were genotyped as Duffy-negative (FY*BES/*BES). Among Duffy-negative individuals 6.9% were P. vivax PCR positive and among Duffy-positive 14.2% were P. vivax PCR positive. Although lower, the risk of Duffy-negatives to experience a P. vivax blood stage infection was not significantly different to that of Duffy-positives. Furthermore, the genotypic and allelic frequencies of the Duffy blood group among P. vivax-infected patients and in the control group did not differ significantly, also suggesting no reduction in infection rates among the carriers of FY*BES allele. The data obtained in Anajás showed no differential resistance vivax malaria among Duffy-negative and Duffy-positive individuals. This result needs additional confirmation through a deeper evaluation in a larger sample of patients with P. vivax malaria and molecular parasite characterization. Nonetheless, this genetic profile of the parasite may be contributing to the high incidence of malaria in the municipality.

23259673

Annulation of α,β-unsaturated imines and alkynes via cobalt-catalyzed olefinic C-H activation.

A cobalt-triarylphosphine catalyst promotes an annulation reaction of an α,β-unsaturated imine and an internal alkyne to afford a polysubstituted dihydropyridine derivative in good yield under mild conditions. The reaction likely involves alkenylation of the olefinic C-H bond via cobalt-mediated nitrogen-assisted C-H activation followed by facile 6π electrocyclization of the resulting azatriene intermediate.

23259671

Occurrence of arsenic impurities in organoarsenics and animal feeds.

Organoarsenics are widely used as excellent feed additives in animal production in the world. Roxarsone (ROX) and arsanilic acid (ASA) are two organoarsenics permitted to be used in China. We collected 146 animal feed samples to investigate the appearance of ROX, ASA, and potential metabolites, including 3-amino-4-hydroxyphenylarsonic acid (3-A-HPA), 4-hydroxyphenylarsonic acid (4-HPA), As(V), As(III), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) in feeds. The stability of ROX in both ROX additives and animal feeds was also examined. The results show that 25.4% of the 146 animal feeds contained organoarsenics, with average contents of ROX and ASA as 7.0 and 21.2 mg of As/kg, respectively. Unexpectedly, As(III) and MMA frequently occurred as As impurities in feeds bearing organoarsenics, with higher contents than organoarsenics in some samples. 3-A-HPA, 4-HPA, and DMA were not detected in all samples. ROX and As impurities in both ROX additives and feeds stayed unchanged in the shelf life. It suggests that As impurities in animal feeds bearing organoarsenics should generate from the use of organoarsenics containing As impurities. This constitutes the first report of As impurities in organoarsenics.

23259667

Disseminated tumor cells as selection marker and monitoring tool for secondary adjuvant treatment in early breast cancer. Descriptive results from an intervention study.

Presence of disseminated tumor cells (DTCs) in bone marrow (BM) after completion of systemic adjuvant treatment predicts reduced survival in breast cancer. The present study explores the use of DTCs to identify adjuvant insufficiently treated patients to be offered secondary adjuvant treatment intervention, and as a surrogate marker for therapy response. A total of 1121 patients with pN1-3 or pT1c/T2G2-3pN0-status were enrolled. All had completed primary surgery and received 6 cycles of anthracycline-containing chemotherapy. BM-aspiration was performed 8-12 weeks after chemotherapy (BM1), followed by a second BM-aspiration 6 months later (BM2). DTC-status was determined by morphological evaluation of immunocytochemically detected cytokeratin-positive cells. If DTCs were present at BM2, docetaxel (100 mg/m², 3qw, 6 courses) was administered, followed by DTC-analysis 1 month (BM3) and 13 months (BM4) after the last docetaxel infusion. Clinical follow-up (FU) is still ongoing. Here, the descriptive data from the study are presented. Of 1085 patients with a reported DTC result at both BM1 and BM2, 94 patients (8.7%) were BM1 positive and 83 (7.6%) were BM2 positive. The concordance between BM1 and BM2 was 86.5%. Both at BM1 and BM2 DTC-status was significantly associated with lobular carcinomas (p = 0.02 and p = 0.03, respectively; chi-square). In addition, DTC-status at BM2 was also associated with pN-status (p = 0.009) and pT-status (p = 0.03). At BM1 28.8% and 12.8% of the DTC-positive patients had ≥2 DTCs and ≥3 DTCs, respectively. At BM2, the corresponding frequencies were 47.0% and 25.3%. Of 72 docetaxel-treated patients analyzed at BM3 and/or BM4, only 15 (20.8%) had persistent DTCs. Of 17 patients with ≥3 DTCs before docetaxel treatment, 12 patients turned negative after treatment (70.6%). The change to DTC-negativity was associated with the presence of ductal carcinoma (p = 0.009). After docetaxel treatment, the majority of patients experienced disappearance of DTCs. As this is not a randomized trial, the results can be due to effects of adjuvant (docetaxel/endocrine/trastuzumab) treatment and/or limitations of the methodology. The clinical significance of these results awaits mature FU data, but indicates a possibility for clinical use of DTC-status as a residual disease-monitoring tool and as a surrogate marker of treatment response. Clin Trials Gov NCT00248703.

23259668

Employment of gene expression profiling to identify transcriptional regulators of hepatic stellate cells.

Activated hepatic stellate cells (HSC) play a central role in scar formation that leads to liver fibrosis. The molecular mechanisms underlying this process are not fully understood. Microarray and bioinformatics analyses have proven to be useful in identifying transcription factors that regulate cellular processes such as cell differentiation. Using oligonucleotide microarrays, we performed transcriptional analyses of activated human HSC cultured on Matrigel-coated tissue culture dishes. Examination of microarray data following Matrigel-induced deactivation of HSC revealed a significant down-regulation of myocardin, an important transcriptional regulator in smooth and cardiac muscle development. Thus, gene expression profiling as well as functional assays of activated HSC have provided the first evidence of the involvement of myocardin in HSC activation.

23259666

Dendrimer-based multivalent vancomycin nanoplatform for targeting the drug-resistant bacterial surface.

Vancomycin represents the preferred ligand for bacteria-targeting nanosystems. However, it is inefficient for emerging vancomycin-resistant species because of its poor affinity to the reprogrammed cell wall structure. This study demonstrates the use of a multivalent strategy as an effective way for overcoming such an affinity limitation in bacteria targeting. We designed a series of fifth generation (G5) poly(amidoamine) (PAMAM) dendrimers tethered with vancomycin at the C-terminus at different valencies. We performed surface plasmon resonance (SPR) studies to determine their binding avidity to two cell wall models, each made with either a vancomycin-susceptible (D)-Ala-(D)-Ala or vancomycin-resistant (D)-Ala-(D)-Lac cell wall precursor. These conjugates showed remarkable enhancement in avidity in the cell wall models tested, including the vancomycin-resistant model, which had an increase in avidity of four to five orders of magnitude greater than free vancomycin. The tight adsorption of the conjugate to the model surface corresponded with its ability to bind vancomycin-susceptible Staphylococcus aureus bacterial cells in vitro as imaged by confocal fluorescent microscopy. This vancomycin platform was then used to fabricate the surface of iron oxide nanoparticles by coating them with the dendrimer conjugates, and the resulting dendrimer-covered magnetic nanoparticles were demonstrated to rapidly sequester bacterial cells. In summary, this article investigates the biophysical basis of the tight, multivalent association of dendrimer-based vancomycin conjugates to the bacterial cell wall, and proposes a potential new use of this nanoplatform in targeting Gram-positive bacteria.

23259664

Genomic vulnerability to LINE-1 hypomethylation is a potential determinant of the clinicogenetic features of multiple myeloma.

The aim of this study was to clarify the role of global hypomethylation of repetitive elements in determining the genetic and clinical features of multiple myeloma (MM). We assessed global methylation levels using four repetitive elements (long interspersed nuclear element-1 (LINE-1), Alu Ya5, Alu Yb8, and Satellite-α) in clinical samples comprising 74 MM samples and 11 benign control samples (7 cases of monoclonal gammopathy of undetermined significance (MGUS) and 4 samples of normal plasma cells (NPC)). We also evaluated copy-number alterations using array-based comparative genomic hybridization, and performed methyl-CpG binding domain sequencing (MBD-seq). Global levels of the repetitive-element methylation declined with the degree of malignancy of plasma cells (NPC>MGUS>MM), and there was a significant inverse correlation between the degree of genomic loss and the LINE-1 methylation levels. We identified 80 genomic loci as common breakpoints (CBPs) around commonly lost regions, which were significantly associated with increased LINE-1 densities. MBD-seq analysis revealed that average DNA-methylation levels at the CBP loci and relative methylation levels in regions with higher LINE-1 densities also declined during the development of MM. We confirmed that levels of methylation of the 5' untranslated region of respective LINE-1 loci correlated strongly with global LINE-1 methylation levels. Finally, there was a significant association between LINE-1 hypomethylation and poorer overall survival (hazard ratio 2.8, P = 0.015). Global hypomethylation of LINE-1 is associated with the progression of and poorer prognosis for MM, possibly due to frequent copy-number loss.

23259665

QTc interval prolongation in HIV-infected patients: a case-control study by 24-hour Holter ECG recording.

Aim of the study was to assess QTc interval by a 24-hour ECG recording in a group of HIV-infected individuals with a basal prolonged QTc. The risk factors associated with QTc prolongation and the indices of cardiovascular autonomic control were also evaluated. A case-control study was performed using as cases 32 HIV-infected patients with prolonged (>440 msec) QTc interval as assessed by Holter ECG, and as controls 64 HIV-infected subjects with normal QTc interval. Autonomic function was evaluated by heart rate variability analysis during 24-hour recording. Duration of HIV disease was significantly longer among cases than among controls (p=0.04). Waist/hip ratio was also higher among cases than among controls (p=0.05). Frequency domain analysis showed the absence of physiologic decrease of low frequency (LF) in the night period in both cases and controls. The LF night in cases showed a statistically significant reduction when compared with controls (p=0.007). In our study group, QTc interval prolongation was associated with a longer duration of HIV infection and with a greater waist/hip ratio. HIV patients with QTc interval prolongation and with a longer duration of HIV infection were more likely to have an impairment of parasympathetic and sympathetic cardiac component.

23259663

From LCAs to simplified models: a generic methodology applied to wind power electricity.

This study presents a generic methodology to produce simplified models able to provide a comprehensive life cycle impact assessment of energy pathways. The methodology relies on the application of global sensitivity analysis to identify key parameters explaining the impact variability of systems over their life cycle. Simplified models are built upon the identification of such key parameters. The methodology is applied to one energy pathway: onshore wind turbines of medium size considering a large sample of possible configurations representative of European conditions. Among several technological, geographical, and methodological parameters, we identified the turbine load factor and the wind turbine lifetime as the most influent parameters. Greenhouse Gas (GHG) performances have been plotted as a function of these key parameters identified. Using these curves, GHG performances of a specific wind turbine can be estimated, thus avoiding the undertaking of an extensive Life Cycle Assessment (LCA). This methodology should be useful for decisions makers, providing them a robust but simple support tool for assessing the environmental performance of energy systems.

23259662

The epidemiology of traumatic cervical spine fractures: a prospective population study from Norway.

The aim of this study was to estimate the incidence of traumatic cervical spine fractures (CS-fx) in a general population. The incidence of CS-fx in the general population is largely unknown. All CS-fx (C0/C1 to C7/Th1) patients diagnosed with cervical-CT in Southeast Norway (2.7 million inhabitants) during the time period from April 27, 2010-April 26, 2011 were prospectively registered in this observational cohort study. Over a one-year period, 319 patients with CS-fx at one or more levels were registered, constituting an estimated incidence of 11.8/100,000/year. The median age of the patients was 56 years (range 4-101 years), and 68% were males. The relative incidence of CS-fx increased significantly with age. The trauma mechanisms were falls in 60%, motorized vehicle accidents in 21%, bicycling in 8%, diving in 4% and others in 7% of patients. Neurological status was normal in 79%, 5% had a radiculopathy, 8% had an incomplete spinal cord injury (SCI), 2% had a complete SCI, and neurological function could not be determined in 6%. The mortality rates after 1 and 3 months were 7 and 9%, respectively. Among 319 patients, 26.6% were treated with open surgery, 68.7% were treated with external immobilization with a stiff collar and 4.7% were considered stable and not in need of any specific treatment. The estimated incidence of surgically treated CS-fx in our population was 3.1/100,000/year. This study estimates the incidence of traumatic CS-fx in a general Norwegian population to be 11.8/100,000/year. A male predominance was observed and the incidence increased with increasing age. Falls were the most common trauma mechanism, and SCI was observed in 10%. The 1- and 3-month mortality rates were 7 and 9%, respectively. The incidence of open surgery for the fixation of CS-fx in this population was 3.1/100,000/year. This is a prospective observational cohort study and level II-2 according to US Preventive Services Task Force.

23259661

Contactless surface conductivity mapping of graphene oxide thin films deposited on glass with scanning electrochemical microscopy.

The present article introduces a rapid, very sensitive, contactless method to measure the local surface conductivity with Scanning Electrochemical Microscopy (SECM) and obtain conductivity maps of heterogeneous substrates. It is demonstrated through the study of Graphene Oxide (GO) thin films deposited on glass. The adopted substrate preparation method leads to conductivity disparities randomly distributed over approximately 100 μm large zones. Data interpretation is based on an equation system with the dimensionless conductivity as the only unknown parameter. A detailed prospection provides a consistent theoretical framework for the reliable quantification of the conductivity of GO with SECM. Finally, an analytical approximation of the conductivity as a function of the feedback current is proposed, making any further interpretation procedure straightforward, as it does not require iterative numerical simulations any more. The present work thus provides not only valuable information on the kinetics of GO reduction in mild conditions but also a general and simplified interpretation framework that can be extended to the quantitative conductivity mapping of other types of substrates.

23259660

Latin American women's experiences with medical abortion in settings where abortion is legally restricted.

Abortion is legally restricted in most of Latin America where 95% of the 4.4 million abortions performed annually are unsafe. Medical abortion (MA) refers to the use of a drug or a combination of drugs to terminate pregnancy. Mifepristone followed by misoprostol is the most effective and recommended regime. In settings where mifepristone is not available, misoprostol alone is used.Medical abortion has radically changed abortion practices worldwide, and particularly in legally restricted contexts. In Latin America women have been using misoprostol for self-induced home abortions for over two decades.This article summarizes the findings of a literature review on women's experiences with medical abortion in Latin American countries where voluntary abortion is illegal.Women's personal experiences with medical abortion are diverse and vary according to context, age, reproductive history, social and educational level, knowledge about medical abortion, and the physical, emotional, and social circumstances linked to the pregnancy. But most importantly, experiences are determined by whether or not women have the chance to access: 1) a medically supervised abortion in a clandestine clinic or 2) complete and accurate information on medical abortion. Other key factors are access to economic resources and emotional support.Women value the safety and effectiveness of MA as well as the privacy that it allows and the possibility of having their partner, a friend or a person of their choice nearby during the process. Women perceive MA as less painful, easier, safer, more practical, less expensive, more natural and less traumatic than other abortion methods. The fact that it is self-induced and that it avoids surgery are also pointed out as advantages. Main disadvantages identified by women are that MA is painful and takes time to complete. Other negatively evaluated aspects have to do with side effects, prolonged bleeding, the possibility that it might not be effective, and the fact that some women eventually need to seek medical care at a hospital where they might be sanctioned for having an abortion and even reported to the police.

23259659

Inhibition of collagen fibril formation.

The overall aim of presented study is to test the inhibition of the formation of collagen fibrils as the novel approach to reduce accumulation of pathological fibrotic deposits. The main hypothesis is that by interfering with the initial steps of the extracellular process of collagen fibril formation, it is possible to reduce the formation of fibrotic tissue. The experimental model includes antibody-based inhibitors that specifically bind to the sites that participate in the collagen/collagen interaction. Employed antibody-based inhibitors effectively limit the amount of collagen fibrils formed in vitro and in engineered tissue models of localized fibrosis. (i) Inhibition of collagen formation is an attractive target to reduce excessive formation of fibrotic tissue. (ii) Antibody-based inhibitors of collagen fibril formation are promising therapeutic agents with a potential to limit localized fibrosis in a number of tissues.

23259658

Variability of bothersome menopausal symptoms over time--a longitudinal analysis using the Estonian postmenopausal hormone therapy trial (EPHT).

Very little data are available on the natural course or level of disturbance of vasomotor symptoms among middle-aged women. Using readily collected trial data we studied the persistence of vasomotor symptoms among untreated women. In a trial comparing combined hormone therapy to placebo or no treatment (control groups), a cohort of women aged 50-59 at recruitment were followed annually by questionnaires. Women in the control groups (n = 486) were grouped by the number of years followed, with the prevalence and severity of symptoms calculated both cross-sectionally and longitudinally. About two thirds of the women (67%) reported vasomotor symptoms and half (46%) bothersome symptoms at recruitment. In the cross-sectional analysis, their prevalence declined between recruitment and 1-year follow-up (32% bothersome symptoms) and 2-year follow-up (27%). Thereafter it remained about the same level. In the longitudinal analysis, there was a notable variation in the prevalence of disturbing vasomotor symptoms over time, time entering the study and the compliance to the surveys. In the two groups having most follow-up times, the proportion of women with bothersome symptoms first increased and then decreased. There was a notable variability in the development of disturbing vasomotor symptoms over time in a selected group of women aged 50-59. Population-based follow-up studies of untreated women would be useful to estimate the symptom burden.

23259657

Does visuo-spatial working memory generally contribute to immediate serial letter recall?

This work contributes to the understanding of the visual similarity effect in verbal working memory, a finding that suggests that the visuo-spatial sketch pad-the system in Baddeley's working memory model specialised in retaining nonverbal visual information-might be involved in the retention of visually presented verbal materials. Crucially this effect is implicitly interpreted by the most influential theory of multimedia learning as evidence for an obligatory involvement of the visuo-spatial sketch pad. We claim that it is only involved when the functioning of the working memory component normally used for processing verbal material is impaired. In this article we review the studies that give rise to the idea of obligatory involvement of the visuo-spatial sketch pad and suggest that some findings can be understood with reference to orthographic rather than visual similarity. We then test an alternative explanation of the finding that is most apt to serve as evidence for obligatory involvement of the visuo-spatial sketch pad. We conclude that, in healthy adults and under normal learning conditions, the visual similarity effect can be explained within the framework of verbal working memory proposed by Baddeley (e.g., 1986, 2000) without additional premises regarding the visuo-spatial sketch.

23259656

Pragmatic and scientific advantages of MDHAQ/ RAPID3 completion by all patients at all visits in routine clinical care.

The patient history often provides the most important information in diagnosis and management of rheumatoid arthritis (RA) and other rheumatic diseases. A multidimensional health assessment questionnaire (MDHAQ)-with templates to score RAPID3 (routine assessment the patient index data), an index of three patient self-report measures, physical function, pain, and patient global estimate-pro- vides a "scientific" patient history. MDHAQ/RAPID3 scores meet criteria for the scientific method seen for laboratory tests: standard format, quantitative data, protocol for col- lection, and recognition of prognostic implications of levels for management decisions. Extensive evidence supports a scientific rationale for MDHAQ/RAPID3 scores, which are as efficient as joint counts, laboratory tests, DAS28, and CDAI to distinguish active from control treatments in clinical trials and correlated significantly with DAS28 and CDAI scores in clinical trials and usual clinical care, including categories for high, moderate, low severity, and remission. Pragmatic advantages of MDHAQ/RAPID3 include that the patient does almost all the work and prepares for the encounter to focus on concerns to discuss with the doctor. MDHAQ/RAPID3 improves doctor-patient communication and saves time for the doctor with a 10 to 15 second overview of medical history data that otherwise would require 10 to 15 minutes of conversation. RAPID3 is scored in 5 seconds, compared to almost 2 minutes for a CDAI or DAS28, and can be used effectively for treat-to-target in RA. MDHAQ/ RAPID3 is informative in all rheumatic diseases, including systemic lupus erythematosus, osteoarthritis, ankylosing spondylitis, psoriatic arthritis, fibromyalgia, gout, and others. All rheumatologists may include MDHAQ/RAPID3 in all patients in the infrastructure of clinical care.

23259655

A fresh look at glucocorticoids how to use an old ally more effectively.

Glucocorticoids form a mainstay of therapy for rheumatoid arthritis (RA) and other conditions since they exert strong anti-inflammatory, immunosuppressive, and disease-modifying therapeutic effects. However, there is increasing awareness of the potential for these drugs to produce adverse effects. Therefore, improvement of the glucocorticoid benefit-risk ratio represents both a current need and an ongoing challenge. The development of recommendations to implement a more effective and safer use of these important drugs is one useful path to pursue. An additional avenue is the development of innovative glucocorticoids or glucocorticoid receptor ligands. Also, treatment with conventional glucocorticoid preparations currently available to clinicians may be improved. The most advanced development in the latter regard is a novel chronotherapeutic prednisone formulation called delayed- release (DR) or modified-release prednisone. The CAPRA (Circadian Administration of Prednisone in Rheumatoid Arthritis) studies confirmed that optimizing the timing of GC administration improves the benefit-risk ratio of long- term low dose glucocorticoid treatment in patients with rheumatoid arthritis. DR prednisone has been approved in 16 European countries as well as Australia and Israel. Very recently, DR prednisone was also approved in the United States to treat rheumatologic conditions such as RA, polymyalgia rheumatica and psoriatic arthritis, as well as respiratory conditions such as COPD and asthma.

23259654

Short-term and long-term safety of glucocorticoids in rheumatoid arthritis.

Despite over 60 years of use, glucocorticoids continue to be a controversial therapy in rheumatoid arthritis (RA). This stems largely from their measured a well as their perceived toxicity. However, a paucity of top tier evidence from clinical trials or very carefully controlled observational studies leads to limited evidence supporting potential causal relations between low-dose glucocorticoids and adverse outcomes. Several new studies have contributed to an improved understanding of these associations and they are reviewed here along with highlights from the older body of literature examining these important outcomes.

23259653

Effective initial and long-term prednisone in doses of less than 5 mg/day to treat rheumatoid arthritis--documentation using a patient self-report Multidimensional Health Assessment Questionnaire (MDHAQ).

The efficacy of initial and long-term prednisone < 5 mg/ day in treatment of rheumatoid arthritis (RA) by one academic rheumatologist over 25 years from 1980 to 2004 is summarized. Patient responses were assessed using a multidimensional health assessment questionnaire (MDHAQ), completed by all patients at all visits in the infrastructure of care. A database was maintained of all visits, which included medications and scores for physical function, pain, patient global estimate of status, and routine assessment of patient index data (RAPID3), an index of these 3 measures. Prednisone doses were higher in patients with more severe MDHAQ/RAPID3 scores, as expected, although formal criteria were not used to determine the initial dose. Similar improvements were seen in clinical status over 12 months in patients treated with < 5 vs ≥ 5 mg/day prednisone and maintained for > 8 years. Adverse effects were primarily bruising and skin-thinning; levels of hypertension, diabetes, and cataracts were not higher than expected, including in 148 patients monitored over > 4 years, 75 over > 8 years. Prednisone at initial and long-term doses of < 5 mg/day appears acceptable and effective for many patients with RA at this time, although further clinical trials and long-term observational studies are needed to optimize treatment of patients with RA with low-dose prednisone. The data also illustrate that MDHAQ scores in usual clinical care can be used to document results of therapy over long periods with no extra work for the physician.

23259652

Corticosteroids as disease modifying drugs in rheumatoid arthritis treatment.

The current approach to treatment of RA includes early and aggressive treatment with routine monitoring of outcomes to give patients the best chance of decreasing disease activity as much as possible, with low disease activity and remission being a realistic goal for many patients. In this quest, DMARDs, especially MTX, are the anchor treatment, and low dose prednisone should also be considered in combination with MTX as the best initial choice for RA treatment. Current data suggest that corticosteroids are disease modifying agents that enhance the effects of DMARDs with no real impact on adverse events. We are much better positioned now then in earlier times to provide a good outcome for our patients, and every available tool needs to be considered and utilized for this purpose.

23259651

Rheumatoid arthritis: circadian rhythms in disease activity, signs and symptoms, and rationale for chronotherapy with corticosteroids and other medications.

Biological processes and functions at all hierarchical levels are organized in time as biological rhythms of discrete periods. Circadian (24-hour) rhythms, which are of direct importance to clinical medicine, are orchestrated by a set of clock genes of the master brain clock situated in the suprachiasmatic nuclei of the hypothalamus plus numerous subservient peripheral cellular clocks of all tissues and organs. Circadian rhythms are kept in step with the surrounding physical and social milieu by periodic external time cues, the most important one being the 24-hour environmental light-dark cycle. The circadian time structure gives rise to predictable-in-time day-night patterns in morbid and mortal events plus symptom occurrence and severity of common chronic conditions, including rheumatoid arthritis (RA). The circadian pattern of various cytokines and hormones in RA disease activity suggests a new treatment paradigm (i.e., chronotherapy-timing medications to 24-hour rhythms in disease pathophysiology) to improve desired outcomes. Since the 1950s, RA chronotherapy in the United States and Europe has involved several nonsteroid anti-inflammatory drugs (NSAIDs), certain disease modifying antirheumatic drugs (DMARDs), and various synthetic corticosteroid medications.

23259649

Re-directed T cells for the treatment of fibroblast activation protein (FAP)-positive malignant pleural mesothelioma (FAPME-1).

Asbestos is the main cause of MPM in industrialized countries. Even since asbestos is banned in most developed countries, the peak wave of MPM incidence is anticipated for the next years due to the long latency of asbestos induced MPM. MPM patients not eligible for surgical procedures like decortication or pleuro-pneumectomie have a median survival of 12 months with palliative chemotherapy. Therefore, new therapeutic approaches are of crucial need in this clinical situation. This is a phase I trial for patients with malignant pleural mesothelioma with pleural effusion testing the safety of a fixed single dose of 1x106 adoptively transferred FAP-specific re-directed T cells given directly in the pleural effusion. Lymphocytes will be taken 21 days before transfer from peripheral blood. CD8 positive T cells will be isolated and re-programmed by retroviral transfer of a chimeric antigen receptor recognizing FAP which serves as target structure in MPM. At day 0 of the protocol, re-directed T cells will be injected in the pleural effusion and patients will be monitored for 48h under intermediate care conditions. AE, SAE, SADR and SUSAR will be monitored for 35 days and evaluated by an independent safety board to define any dose limiting toxicity (DLT). No further patient can be treated before the previous patient passed day 14 after T cell transfer. The protocol will be judged as save when no DLT occurred in the first 3 patients, or 1 DLT in 6 patients. Secondary objectives are feasibility and immune monitoring. Adoptive T cell transfer is a new and rapidly expanding branch of immunotherapies focusing on cancer treatment. Recently, objective responses could be observed in patients with chronic lymphatic leukemia treated with adoptively transferred CD19-specific re-directed T cells. The choice of the target antigen determines the possible on-target off-tissue toxicity of such approaches. There are reports of severe toxicity in patients who received T cells intravenously due to unexpected expression of the target antigen (on-target) in other tissues than the tumor (off-tissue). To minimize the risk of on-target off-tissue toxicity and to maximize the on-target anti-tumor effect we propose a clinical protocol with loco-regional administration of re-directed T cells. FAP-specific T cells will be directly injected in the pleural effusion of patients with MPM. ClinicalTrials.gov (NCT01722149).

23259650

Asymmetric vinylogous aldol reaction via H-bond-directing dienamine catalysis.

The enantioselective direct vinylogous aldol reaction of 3-methyl 2-cyclohexen-1-one with α-keto esters has been developed. The key to success was the design of a bifunctional primary amine-thiourea catalyst that can combine H-bond-directing activation and dienamine catalysis. The simultaneous dual activation of the two reacting partners results in high reactivity while securing high levels of stereocontrol.

23259648

Bioaccessibility of enniatins A, A₁, B, and B₁ in different commercial breakfast cereals, cookies, and breads of Spain.

Fusarotoxins enniatins (ENs) can represent a potential risk as natural contaminants of cereal commodities. However, only their bioaccessible fraction can exert a toxicity. The purpose of this study was to determine the ENs A, A₁, B, and B₁ bioaccessibility added in 1.5 and 3.0 μmol/g concentrations in breakfast cereals, cookies, and breads using a simulated in vitro gastrointestinal extraction model. Bioaccessibility values ranged between 40.4 ± 1.9 and 79.9 ± 2.8%. The lower values were 50.1, 40.4, 43.9, and 46.3% in wheat bran with fibers, and the higher values were 79.9, 64.2, 69.8, and 73.6% in white loaf bread for the ENs A, A₁, B, and B₁, respectively. Food composition, compounds structure, and presence of natural adsorbing materials can influence the ENs bioaccessibility. Application of a simulated in vitro gastrointestinal method is a good procedure to assess oral ENs bioaccessibility in cookies, breakfast cereals, and bread.

23259647

Time discrimination in traumatic brain injury patients.

Time management skills are required for most daily activities. Traumatic brain injury (TBI) patients often present with cognitive dysfunction, but few studies have investigated temporal impairment. The aim of the present study was to assess temporal abilities in TBI patients using a time discrimination task. Twenty-seven TBI patients (ages = 18-60 years) and 27 controls (ages = 20-60 years) were asked to discriminate between two time intervals presented sequentially. The standard intervals were 500 ms or 1,300 ms long followed by a comparison stimulus that was 25% shorter or longer than the standard one. Participants were also asked to perform two tasks to assess attention, speed-of-processing (the Stroop task), and working memory (the n-back task) abilities. The TBI patients were less accurate than the controls on the time discrimination task and showed greater time-order error effects. In fact, TBI patients pressed the "short" key more times when the standard time interval was 500 ms and the "long" key more times when the standard interval was 1,300 ms. Significant correlations were found between time discrimination, working memory, and speed of processing in both TBI and controls when the standard time interval was 1,300 ms. Attention appeared to be involved in different ways in the two groups. Working memory and speed of processing were involved in time processing only in TBI patients when the standard time interval was 500 ms. These data lend additional support to the notion that two different systems are responsible for elaborating time durations shorter or longer than a second.

23259646

Biomarkers of disease differentiation: HCV recurrence versus acute cellular rejection.

The wound-healing process induced by chronic hepatitis C virus (HCV) infection triggers liver damage characterized by fibrosis development and finally cirrhosis. Liver Transplantation (LT) is the optimal surgical treatment for HCV-cirrhotic patients at end-stage liver disease. However, acute cellular rejection (ACR) and HCV recurrence disease represent two devastating complications post-LT. The accurate differential diagnosis between both conditions is critical for treatment choice, and similar histological features represent a challenge for pathologists. Moreover, the HCV recurrence disease severity is highly variable post-LT. HCV recurrence disease progression is characterized by an accelerated fibrogenesis process, and almost 30% of those patients develop cirrhosis at 5-years of follow-up. Whole-genome gene expression (WGE) analyses through well-defined oligonucleotide microarray platforms represent a powerful tool for the molecular characterization of biological process. In the present manuscript, the utility of microarray technology is applied for the ACR and HCV-recurrence biological characterization in post-LT liver biopsy samples. Moreover, WGE analysis was performed to identify predictive biomarkers of HCV recurrence severity in formalin-fixed paraffin-embedded liver biopsies prospectively collected.

23259645

The utility of breastmilk for genetic or genomic studies: a systematic review.

This study synthesized scientific literature that applies genetic or genomic approaches to breastmilk. A literature search of PubMed was conducted in March 2012 using the key words "breast milk," "lactation," "genetic," "gene expression," and "epigenetic." Additional articles were identified/selected for evaluation with MeSH term searches, and a review of article reference lists was obtained from the search. The initial 657 abstracts retrieved from the literature search were reviewed, and 16 studies were selected for evaluation. Studies that examined the transmission of viruses/bacteria into breastmilk and/or measured concentration of specific proteins without examination of genetic material from milk were excluded. Data related to subjects, tissue, purpose, setting, gene/protein, approach (candidate versus genome-wide), platform, statistical analysis, and results were extracted. Gene expression and epigenetic/epigenomic study designs have been successfully implemented using breastmilk. A major weakness of both gene expression studies and epigenetic studies that examine breastmilk is the omission of maternal information known to influence milk composition. This review article is the first to synthesize evidence related to the application of breastmilk to evaluate RNA and epigenetic modifications. Additional research is needed that applies epigenetic analyses to human breastmilk samples. Findings from this review can be used for future research designs that use breastmilk for genetic analyses.

23259643

Expression of a type B RIFIN in Plasmodium falciparum merozoites and gametes.

The ability of Plasmodium falciparum to undergo antigenic variation, by switching expression among protein variants encoded by multigene families, such as var, rif and stevor, is key to the survival of this parasite in the human host. The RIFIN protein family can be divided into A and B types based on the presence or absence of a 25 amino acid motif in the semi-conserved domain. A particular type B RIFIN, PF13_0006, has previously been shown to be strongly transcribed in the asexual and sexual stages of P. falciparum in vitro. Antibodies to recombinant PF13_0006 RIFIN were used in immunofluorescence and confocal imaging of 3D7 parasites throughout the asexual reproduction and sexual development to examine the expression of PF13_0006. Furthermore, reactivity to recombinant PF13_0006 was measured in plasma samples collected from individuals from both East and West African endemic areas. The PF13_0006 RIFIN variant appeared expressed by both released merozoites and gametes after emergence. 7.4% and 12.1% of individuals from East and West African endemic areas, respectively, carry plasma antibodies that recognize recombinant PF13_0006, where the antibody responses were more common among older children. The stage specificity of PF13_0006 suggests that the diversity of RIFIN variants has evolved to provide multiple specialized functions in different stages of the parasite life cycle. These data also suggest that RIFIN variants antigenically similar to PF13_0006 occur in African parasite populations.

23259644

Breastfeeding and weaning in renaissance Italy: the Medici children.

Abstract Exploration of the Medici Chapels in the Basilica of San Lorenzo in Florence, Italy, revealed the burials of nine infantile members of the Medici family. Eight children were found in the intact tomb of the last Grand Duke GianGastone (1671-1737), and another child was exhumed from the Chapel of Grand Duke Ferdinando I (1549-1609). Skeletal ages ranged from newborn to 5 years, suggesting an identification with infantile members of the family. A paleonutritional study has been performed on the bone samples of all members of the Medici family exhumed so far. Carbon and nitrogen stable isotope analysis of bone collagen was used to detect the timing of the weaning process in this population. The (15)N values of the Medici children are significantly higher than those of adults, indicating that these infants were breastfed for a long time period. In particular, the levels of (15)N are high before the second year but decrease in older children, evidently after weaning, reaching the levels of adults. During the Renaissance, it was the common opinion that children should not be weaned before the second year of life. Archival documents suggest that the Medici children were never weaned before that age and, in most cases, even some months later. Combination of paleonutritional data and historical sources allowed reconstruction of the breastfeeding and weaning patterns of this aristocratic Renaissance class.

23259642

FMR1 CGG allele size and prevalence ascertained through newborn screening in the United States.

Population screening for FMR1 mutations has been a topic of considerable discussion since the FMR1 gene was identified in 1991. Advances in understanding the molecular basis of fragile X syndrome (FXS) and in genetic testing methods have led to new, less expensive methodology to use for large screening endeavors. A core criterion for newborn screening is an accurate understanding of the public health burden of a disease, considering both disease severity and prevalence rate. This article addresses this need by reporting prevalence rates observed in a pilot newborn screening study for FXS in the US. Blood spot screening of 14,207 newborns (7,312 males and 6,895 females) was conducted in three birthing hospitals across the United States beginning in November 2008, using a PCR-based approach. The prevalence of gray zone alleles was 1:66 females and 1:112 males, while the prevalence of a premutation was 1:209 females and 1:430 males. Differences in prevalence rates were observed among the various ethnic groups; specifically higher frequency for gray zone alleles in males was observed in the White group compared to the Hispanic and African-American groups. One full mutation male was identified (>200 CGG repeats). The presented pilot study shows that newborn screening in fragile X is technically feasible and provides overall prevalence of the premutation and gray zone alleles in the USA, suggesting that the prevalence of the premutation, particularly in males, is higher than has been previously reported.

23259641

The antiprotozoal drug pentamidine ameliorates experimentally induced acute colitis in mice.

Intestinal inflammation is partly driven by enteroglial-derived S100B protein. The antiprotozoal drug pentamidine directly blocks S100B activity. We aimed to investigate the effect of pentamidine on intestinal inflammation using an animal model of dextran sodium sulphate (DSS)-induced acute colitis. Mice were divided into: control group, colitis group (4% DSS for four days) and two pentamidine-treated colitis groups (0.8 mg/kg and 4 mg/kg). Anti-inflammatory effect of pentamidine was assessed in colonic tissue by evaluating the disease activity index and the severity of histological changes. Colonic tissue were also used to evaluate cyclooxigenase-2, inducible nitric oxide synthase, S100B, glial fibrillary acidic protein, phosphorylated-p38 MAPkinase, p50, p65 protein expression, malondyaldheyde production, mieloperoxidase activity, and macrophage infiltration. Nitric oxide, prostaglandin E2, interleukin-1 beta, tumor necrosis factor alpha, and S100B levels were detected in plasma samples. Parallel measurements were performed in vitro on dissected mucosa and longitudinal muscle myenteric plexus (LMMP) preparations after challenge with LPS + DSS or exogenous S100B protein in the presence or absence of pentamidine. Pentamidine treatment significantly ameliorated the severity of acute colitis in mice, as showed by macroscopic evaluation and histological/biochemical assays in colonic tissues and in plasma. Pentamidine effect on inflammatory mediators was almost completely abrogated in dissected mucosa but not in LMMP. Pentamidine exerts a marked anti-inflammatory effect in a mice model of acute colitis, likely targeting S100B activity. Pentamidine might be an innovative molecule to broaden pharmacological tools against colitis.

23259640

Diastereo- and enantioselective asymmetric hydrogenation of α-amido-β-keto phosphonates via dynamic kinetic resolution.

Dynamic kinetic resolution of various α-amido-β-keto phosphonates via asymmetric hydrogenation proceeded efficiently to give the corresponding β-hydroxy-α-amido phosphonates in high diastereo- and enantioselectivities (up to 99:1 syn/anti, 99.8% ee). The addition of catalytic amounts of CeCl(3)·7H(2)O is necessary to achieve both good selectivity and catalytic efficiency under mild reaction conditions.

23259638

Functioning glucagonoma associated with primary hyperparathyroidism: multiple endocrine neoplasia type 1 or incidental association?

Diagnosis of multiple endocrine neoplasia type 1 (MEN1) is commonly based on clinical criteria, and confirmed by genetic testing. In patients without known MEN1-related germline mutations, the possibility of a casual association between two or more endocrine tumors cannot be excluded and subsequent management may be difficult to plan. We describe a very uncommon case of functioning glucagonoma associated with primary hyperparathyroidism (pHPT) in which genetic testing failed to detect germline mutations of MEN-1 and other known genes responsible for MEN1. The patient, a 65-year old woman, had been suffering for more than 1 year from weakness, progressive weight loss, angular cheilitis, glossitis and, more recently, skin rashes on the perineum, perioral skin and groin folds. After multidisciplinary investigations, functioning glucagonoma and asymptomatic pHPT were diagnosed and, since family history was negative, sporadic MEN1 was suspected. However, genetic testing revealed neither MEN-1 nor other gene mutations responsible for rarer cases of MEN1 (CDKN1B/p27 and other cyclin-dependent kinase inhibitor genes CDKN1A/p15, CDKN2C/p18, CDKN2B/p21). The patient underwent distal splenopancreatectomy and at the 4-month follow-up she showed complete remission of symptoms. Six months later, a thyroid nodule, suspected to be a malignant neoplasia, and two hyperfunctioning parathyroid glands were detected respectively by ultrasound with fine needle aspiration cytology and 99mTc-sestamibi scan with SPECT acquisition. Total thyroidectomy was performed, whereas selective parathyroidectomy was preferred to a more extensive procedure because the diagnosis of MEN1 was not supported by genetic analysis and intraoperative intact parathyroid hormone had revealed "adenoma-like" kinetics after the second parathyroid resection. Thirty-nine and 25 months after respectively the first and the second operation, the patient is well and shows no signs or symptoms of recurrence. Despite well-defined diagnostic criteria and guidelines, diagnosis of MEN1 can still be challenging. When diagnosis is doubtful, appropriate management may be difficult to establish.

23259639

Proteomics approaches to fibrotic disorders.

This review provides an introduction to mass spectrometry based proteomics and discusses several proteomics approaches that are relevant in understanding the pathophysiology of fibrotic disorders and the approaches that are frequently used in biomarker discovery.

23259637

Rural-urban differences in dental service use among children enrolled in a private dental insurance plan in Wisconsin: analysis of administrative data.

Studies on rural-urban differences in dental care have primarily focused on differences in utilization rates and preventive dental services. Little is known about rural-urban differences in the use of wider range of dental procedures. This study examined patterns of preventive, restorative, endodontic, and extraction procedures provided to children enrolled in Delta Dental of Wisconsin (DDWI). We analyzed DDWI enrollment and claims data for children aged 0-18 years from 2002 to 2008. We modified and used a rural and urban classification based on ZIP codes developed by the Wisconsin Area Health Education Center (AHEC). We categorized the ZIP codes into 6 AHEC categories (3 rural and 3 urban). Descriptive and multivariable analysis using generalized linear mixed models (GLMM) were used to examine the patterns of dental procedures provided to children. Tukey-Kramer adjustment was used to control for multiple comparisons. Approximately, 50%, 67% and 68% of enrollees in inner-city Milwaukee, Rural 1 (less than 2500 people), and suburban-Milwaukee had at least one annual dental visit, respectively. Children in inner city-Milwaukee had the lowest utilization rates for all procedures examined, except for endodontic procedures. Compared to children from inner-city Milwaukee, children in other locations had significantly more preventive procedures. Children in Rural 1-ZIP codes had more restorative, endodontic and extraction procedures, compared to children from all other regions. We found significant geographic variation in dental procedures received by children enrolled in DDWI.

23259636

Simultaneous determination of phagocytosis of Plasmodium falciparum-parasitized and non-parasitized red blood cells by flow cytometry.

Severe falciparum malaria anaemia (SMA) is a frequent cause of mortality in children and pregnant women. The most important determinant of SMA appears to be the loss of non-parasitized red blood cells (np-RBCs) in excess of loss of parasitized (p-) RBCs at schizogony. Based on data from acute SMA where excretion of haemoglobin in urine and increased plasma haemoglobin represented respectively less than 1% and 0.5% of total Hb loss, phagocytosis appears to be the predominant mechanism of removal of np- and p-RBC.Estimates indicate that np-RBCs are cleared in approximately 10-fold excess compared to p-RBCs. An even larger removal of np-RBCs has been described in vivax malaria anaemia. Estimates were based on two single studies both performed on neurosyphilitic patients who underwent malaria therapy. As the share of np-RBC removal is likely to vary between wide limits, it is important to assess the contribution of both np- and p-RBC populations to overall RBC loss, and disclose the mechanism of such variability. As available methods do not discriminate between the removal of np- vs p-RBCs, the purpose of this study was to set up a system allowing the simultaneous determination of phagocytosis of p- and np-RBC in the same sample. Phagocytosis of p- and np-RBCs was quantified in the same sample using double-labelled target cells and the human phagocytic cell-line THP-1, pre-activated by TNF and IFNγ to enhance their phagocytic activity. Target RBCs were double-labelled with fluorescent carboxyfluorescein-succinimidyl ester (CF-SE) and the DNA label ethidium bromide (EB). EB, a DNA label, allowed to discriminate p-RBCs that contain parasitic DNA from the np-RBCs devoid of DNA. FACS analysis of THP-1 cells fed with double-labelled RBCs showed that p- and np-RBCs were phagocytosed in different proportions in relation to parasitaemia. The assay allowed the analysis of phagocytosis rapidly and with low subjective error, and the differentiation between phagocytosed p- and np-RBCs in the same sample. The presented method may help to analyse the factors or conditions that modulate the share of np-RBC removal in vitro and in vivo and lead to a better understanding of the pathogenesis of SMA.

23259635

Strategy sequential difficulty effects in Alzheimer patients: a study in arithmetic.

Consistent with Uittenhove and Lemaire (2012 ), we expected that strategy execution would be slower following execution of a difficult strategy than after an easy strategy (strategy sequential difficulty, SSD, effects). Moreover, we expected larger SSD effects in older adults than in young adults, and especially in Alzheimer's disease (AD) patients, a population with marked cognitive impairments. A total of 25 young and older (41 AD and 25 healthy) adults were asked to execute rounding strategies to solve arithmetic problems (e.g., solving 43 + 68 by rounding operands down or up, e.g., 40 + 70 = 110). We measured solution latencies and percentage errors with a strategy as a function of the difficulty of the just-executed strategy. Solution latencies were significantly shorter following the easier rounding-down strategy than following the harder rounding-up strategy, F(2, 156) = 35.8. Moreover, this effect was significantly larger in AD patients, F(1, 78) = 117.4. We found comparable SSD effects in young and healthy older adults but dramatically increased SSD effects in AD patients. This has implications to further our understanding of strategic aspects underlying decreased cognitive functioning in AD patients.

23259634

Reactive oxygen species in plant pathogenesis: the role of perylenequinone photosensitizers.

Reactive oxygen species (ROS) play multiple roles in interactions between plants and microbes, both as host defense mechanisms and as mediators of pathogenic and symbiotic associations. One source of ROS in these interactions are photoactivated, ROS-generating perylenequinone pigments produced via polyketide metabolic pathways in plant-associated fungi. These natural products, including cercosporin, elsinochromes, hypocrellins, and calphostin C, are being utilized as medicinal agents, enzyme inhibitors, and in tumor therapy, but in nature, they play a role in the establishment of pathogenic associations between fungi and their plant hosts. Photoactivated perylenequinones are photosensitizers that use light energy to form singlet oxygen (¹O₂) and free radical oxygen species which damage cellular components based on localization of the perylenequinone molecule. Production of perylenequinones during infection commonly results in lipid peroxidation and membrane damage, leading to leakage of nutrients from cells into the intercellular spaces colonized by the pathogen. Perylenequinones show almost universal toxicity against organisms, including plants, mice, bacteria, and most fungi. The producing fungi are resistant, however, and serve as models for understanding resistance mechanisms. Studies of resistance mechanisms by perylenequinone-producing fungi such as Cercospora species are leading to an understanding of cellular resistance to ¹O₂ and oxidative stress. Recent studies show commonalities between resistance mechanisms in these fungi with extensive studies of ¹O₂ and oxidative stress responses in photosynthetic organisms. Such studies hold promise both for improved medical use and for engineering crop plants for disease resistance.

23259633

Reversal of transforming growth factor-β induced epithelial-to-mesenchymal transition and the ZEB proteins.

The dynamic process of epithelial-to-mesenchymal transition (EMT) is a causal event in kidney fibrosis. This cellular phenotypic transition involves activation of transcriptional responses and remodeling of cellular structures to change cellular function. The molecular mechanisms that directly contribute to the re-establishment of the epithelial phenotype are poorly understood. Here, we discuss recent studies from our group and other laboratories identifying signaling pathways leading to the reversal of EMT in fibrotic models. We also present evidence that transcriptional factors such as the ZEB proteins are important regulators for reversal of EMT. These studies provide insights into cellular plasticity and possible targets for therapeutic intervention.

23259632

Effect of small changes in natural origin-based emulsion systems on hydrocortisone skin absorption and performance: a comparison of two in vivo methods.

Alkyl polyglucoside surfactants (APG) remain prominent natural origin stabilizers offering a prospect of combining satisfactory stability with mild dermatological properties and complete biodegradability. With the purpose of adjusting the dose to a patient's needs, dilution of commercial corticosteroid formulations is a practice which may modify efficacy uncontrolledly. The rational of the study was to investigate whether a simple change in ready-to-use bases (co-solvent addition) could address these needs in a more predictive manner. Hydrocortisone (HC) delivery from such emulsion systems was comparatively assessed employing two in vivo methods: the established human skin blanching assay versus skin stripping technique. HC permeation data obtained after three dose durations showed better overall performance of the APG-stabilized bases relative to reference ones. Although the solubility study showed that all the assessed active samples retained equal thermodynamic activity, diverse HC permeation/penetration implies the importance of the applied base's colloidal structure and/or changes endured. Isopropyl alcohol (IPA) addition offered faster drug penetration enhancement, while glycerol as a moisturizing agent influenced HC penetration through the increase in skin hydration. Although the performed in vivo methods cannot be considered alternative, skin stripping technique proved to be a cost-efficient mode of percutaneous penetration assessment, providing additional information on vehicle-skin interactions.

23259631

Cutaneous wound healing after treatment with plant-derived human recombinant collagen flowable gel.

Chronic wounds, particularly diabetic ulcers, represent a main public health concern with significant costs. Ulcers often harbor an additional obstacle in the form of tunneled or undermined wounds, requiring treatments that can reach the entire wound tunnel, because bioengineered grafts are typically available only in a sheet form. While collagen is considered a suitable biodegradable scaffold material, it is usually extracted from animal and human cadaveric sources, and accompanied by potential allergic and infectious risks. The purpose of this study was to test the performance of a flowable gel made of human recombinant type I collagen (rhCollagen) produced in transgenic tobacco plants, indicated for the treatment of acute, chronic, and tunneled wounds. The performance of the rhCollagen flowable gel was tested in an acute full-thickness cutaneous wound-healing rat model and compared to saline treatment and two commercial flowable gel control products made of bovine collagen and cadaver human skin collagen. When compared to the three control groups, the rhCollagen-based gel accelerated wound closure and triggered a significant jumpstart to the healing process, accompanied by enhanced re-epithelialization. In a cutaneous full-thickness wound pig model, the rhCollagen-based flowable gel induced accelerated wound healing compared to a commercial product made of bovine tendon collagen. By day 21 post-treatment, 95% wound closure was observed with the rhCollagen product compared to 68% closure in wounds treated with the reference product. Moreover, rhCollagen treatment induced an early angiogenic response and induced a significantly lower inflammatory response than in the control group. In summary, rhCollagen flowable gel proved to be efficacious in animal wound models and is expected to be capable of reducing the healing time of human wounds.

23259630

Kinase inhibitors for the treatment of rheumatoid arthritis.

Kinase inhibitors have now been shown to work in various types of patients and have potential to be additional weapons in our armamentarium in rheumatoid arthritis treatment. This review will go over the currently available data and discuss potential uses for these new agents.

23259629

B-cell therapies for rheumatoid arthritis.

B cells were originally considered key mediators in the pathogenesis of rheumatoid arthritis (RA). The presence of these cells in many RA synovial tissues and the discovery of rheumatoid factor had put B cells originally at the center of disease pathogenesis. That enthusiasm vanished shortly thereafter only to resurface in the last 15 years with the appearance of highly specific anti-cyclic citullinated protein antibodies. Rituximab, an anti-CD20 antibody that depletes mature B cells, was approved for the treatment of RA in 2006. Since then, B cell depletion strategies have proven efficacy for advanced disease, particularly in those patients that do not respond to DMARDs or TNFα inhibitors.

23259628

IL-6 inhibition for the treatment of rheumatoid arthritis and other conditions.

New data published or presented in the past year has expanded our understanding of the clinical use of interleukin 6 (IL-6) inhibitors and their role in the management of rheumatoid arthritis (RA) and other rheumatic diseases. Data has become available on the use of tocilizumab (TCZ) in comparison to adalimumab, as therapy in RA patients with an inadequate response to TNF inhibitors and on its role as monotherapy. Early data on the efficacy and safety of subcutaneously administered TCZ suggests a potential role for this formulation of the drug. Extension studies of the use of TCZ in systemic juvenile inflammatory arthritis have confirmed the long-term efficacy of the drug in this illness, while studies on the use of TCZ and other IL-6 inhibitors in spondyloarthropathies has been less encouraging. Finally, new agents targeting the IL-6 pathway have entered late stage clinical trials, and the early results are promising.

23259626

Predictors of response to TNF inhibitors in rheumatoid arthritis - do we have new tools for personalized medicine?

The use of biological agents in the treatment of rheumatoid arthritis (RA) has grown constantly since the approval of the first therapeutic monoclonal antibody against tumor necrosis factor-alpha (TNF) in 1996. While these agents transformed RA treatment, not all patients respond to these agents. Moreover, the cost of these agents is high, and some patients may suffer from adverse events. Thus, the prediction of individual response to biological treatment has become a major clinical challenge in RA. Recent studies have provided evidence that biomarkers may be identified predictive of the response to therapy with these agents. This article will review some of the recent advances in the biomarkers and therapeutic drug monitoring as predictors of response to TNF inhibitor therapies in patients with RA.

23259625

MDHAQ/RAPID3 can provide a roadmap or agenda for all rheumatology visits when the entire MDHAQ is completed at all patient visits and reviewed by the doctor before the encounter.

The management of rheumatoid arthritis (RA) depends more on the patient history than most other chronic diseases. A patient questionnaire provides a uniform, quantitative, protocolized, "scientific" patient history, with documented prognostic significance for work disability and mortality in RA greater than radiographs and laboratory tests and capacity to distinguish active from control treatment in clinical trials and to monitor clinical care with equivalent or greater significance than joint counts or laboratory tests. Therefore, a "scientific" approach to care of a person with a rheumatic disease involves review of patient function, pain, global status, fatigue, RAPID3, review of systems, self-report joint count, and recent medical history on an MDHAQ before conversation with the patient. This practice may be viewed as analogous to a doctor reviewing blood pressure, hemoglobin A1c, viral load, or radiograph before meeting with a patient who has hypertension, diabetes, HIV, or a healing fracture to provide a roadmap or agenda for the visit. Some sites have implemented RAPID3 without the remainder of MDHAQ, a practice that is discouraged. The MDHAQ requires only 5 to 10 minutes of the patient's time and involves a single sheet of paper, which is needed for a simple RAPID3, or even a patient global estimate of status to score a DAS28 or CDAI. Completion of MDHAQ/RAPID3 by each patient at each visit in the infrastructure of care with review by the doctor helps prepare the patient for the visit, improves doctor-patient communication, saves time for the doctor, and provides a roadmap or agenda for the visit.

23259623

Update on treatment of psoriatic arthritis.

Some of this past year's key papers or abstracts on psoriatic arthritis (PsA) assessment and treatment are reviewed in this paper. Treatment begins with identification of the PsA patient. Several screening questionnaires have been developed to be used in dermatology and primary care settings to identify which patients with psoriasis have developed PsA as opposed to other common musculoskeletal problems, such as osteoarthritis and fibromyalgia, thus increasing case-finding and targeting referral. PsA can present in a heterogeneous manner, involving arthritis, enthesitis, dactylitis, spondylitis, and skin and nail disease. Measures of these individual domains have been developed for use in clinical trials and improved PsA-specific composite measures of these domains are being evaluated as well. A quantitative therapy target, Minimal Disease Activity criteria, has been developed by the GRAPPA group. Treatment recommendations have been published by EULAR and GRAPPA. Obesity is a risk factor for the development of PsA and may adversely influence treatment outcomes. Although pharmacologic treatment often begins with methotrexate, a recent study does not provide clear evidence of its effectiveness. Anti-TNF therapies remain the gold standard of effectiveness. New therapeutic options are potentially emerging including ustekinumab, abatacept, several IL-17 inhibitors, apremilast, JAK inhibitors, and possibly IL-6 inhibitors.

23259622

Juvenile idiopathic arthritis: an update for the clinician.

Juvenile idiopathic arthritis (JIA) comprises a collection of all forms of chronic arthritis in childhood with no apparent cause. JIA is the most common rheumatic disease in children, and may result in significant pain, joint deformity, and growth impairment, with persistence of active arthritis into adulthood. Prior to the mid 1990s, the therapeutic armamentarium for JIA was more limited, utilizing non- specific agents, many with significant adverse effects. With the relatively recent use of biologics, one can provide more target-specific therapy, which may be better tolerated. Through continued translational research and clinical trials, one better understands the biology mediating disease, with the hope of offering safer, more effective medicine, and potential cure. This review will outline the clinical features of JIA, as well as provide the latest updates in current and future pharmacotherapy.

23259621

The year in gout: 2011-2012.

From an epidemiologic view, gout is an increasingly prevalent and increasingly pressing clinical problem. This fact, together with technical advances in biology, pharmacology, and imaging techniques, have led to a decade of increasingly rapid progress in our collective understanding of gout and hyperuricemia. Here we review some of the most important recent advances in gout over the past 12 to 18 months.

23259620

Osteoporosis: an update.

The past year has been a dynamic one for clinicians and researchers with an interest in osteoporosis. This update will focus on the issue of the relationship between bisphosphonate treatment and atypical femoral fractures, highlight the advances in imaging techniques that are increasingly being studied as adjuncts to bone density testing, and explore recent evidence that suggests that osteoporosis medications may be linked to an increase in life expectancy. Since the first case reports describing unusual femur fractures in long term users of bisphosphonates began to appear, there has been great interest in identifying why and whether this class of drug can cause these atypical fractures. There have been a significant number of large studies that seem to suggest that these fractures do occur with an increased frequency among subjects who have used bisphosphonates over an extended period of time, but that these events are relatively rare. The occurrence of these fractures have helped to fashion new treatment regimens with periods of "drug holidays" often recommended to people with lower short-term and intermediate-term fracture risk. It is important to remind the reader that bisphosphonates prevent many typical hip and vertebral compression fractures, particularly in the higher risk elderly patient and that a rational balance be struck so that those in need of continued osteoporosis treatment receive it. Advances in imaging, such as high resolution MRI and peripheral micro CT scanners, are allowing investigators to non-invasively assess bone microarchitecture and bone stiffness of individuals as a means of trying to more accurately define those subjects who might be at increased risk of fracture and who might benefit from bone strengthening medication. Finally, this update will briefly review the emerging data that suggests that anti-resorptive medication may extend life expectancy beyond that which can be expected solely by reducing the incidence of future fractures.

23259618

GLP-1 secretion by microglial cells and decreased CNS expression in obesity.

Type 2 diabetes (T2D) is a strong risk factor for developing neurodegenerative pathologies. T2D patients have a deficiency in the intestinal incretin hormone GLP-1, which has been shown to exert neuroprotective and anti-inflammatory properties in the brain. Here we investigate potential sources of GLP-1 in the CNS and the effect of diabetic conditions on the proglucagon mRNA expression in the CNS. The obese mouse model ob/ob, characterized by its high levels of free fatty acids, and the microglia cell line BV-2 were used as models. mRNA expression and protein secretion were analyzed by qPCR, immunofluorescence and ELISA. We show evidence for microglia as a central source of GLP-1 secretion. Furthermore, we observed that expression and secretion are stimulated by cAMP and dependent on microglial activation state. We also show that insulin-resistant conditions reduce the central mRNA expression of proglucagon. The findings that microglial mRNA expression of proglucagon and GLP-1 protein expression are affected by high levels of free fatty acids and that both mRNA expression levels of proglucagon and secretion levels of GLP-1 are affected by inflammatory stimuli could be of pathogenic importance for the premature neurodegeneration and cognitive decline commonly seen in T2D patients, and they may also be harnessed to advantage in therapeutic efforts to prevent or treat such disorders.

23259619

Relevance of immunohistochemical expression of p57kip2 in epithelial ovarian carcinoma- A systematic literature review.

Epithelial Ovarian Cancer (EOC) is the second most common gynaecological cancer and accounts for more deaths than all gynaecological cancers combined. Despite extensive research, progress has been slow in understanding the pathobiology. EOC is identified as a heterogeneous malignancy with various histological subtypes. It is now well known that these different histological subtypes show differences in terms of presentation, response to treatment, immunohistochemical (IHC) reactivity and molecular profiling. Cell cycle deregulation is key in cancer development and there is some evidence in the literature that this is relevant to the problem of EOC and the development of drug resistant disease. The need to identify prognostic markers has led to several gene profiling studies using tumour tissue with equivocal results. p57kip2 is one such cell cycle regulator and its functions are being explored as recent research has shown that it is more than just a negative regulator of the cell cycle. The aim of this review is to evaluate the literature around the IHC expression of p57kip2 in EOC. Systematic review of the literature focussing on clinical outcome and immunohistochemical expression in epithelial ovarian cancer. Four papers are discussed in this review and have shown great variation in IHC expression of p57kip2 in EOC. These studies incorporated different histological subtypes of EOC. However they all suggest that p57kip2 has a significant role in prognosis and its therapeutic indication needs to be studied. Multicentre collaborative studies on individual histological subtypes might provide more data and help to increase the number of cases especially for rarer tumours.

23259617

Composition and functional analysis of low-molecular-weight glutenin alleles with Aroona near-isogenic lines of bread wheat.

Low-molecular-weight glutenin subunits (LMW-GS) strongly influence the bread-making quality of bread wheat. These proteins are encoded by a multi-gene family located at the Glu-A3, Glu-B3 and Glu-D3 loci on the short arms of homoeologous group 1 chromosomes, and show high allelic variation. To characterize the genetic and protein compositions of LMW-GS alleles, we investigated 16 Aroona near-isogenic lines (NILs) using SDS-PAGE, 2D-PAGE and the LMW-GS gene marker system. Moreover, the composition of glutenin macro-polymers, dough properties and pan bread quality parameters were determined for functional analysis of LMW-GS alleles in the NILs. Using the LMW-GS gene marker system, 14-20 LMW-GS genes were identified in individual NILs. At the Glu-A3 locus, two m-type and 2-4 i-type genes were identified and their allelic variants showed high polymorphisms in length and nucleotide sequences. The Glu-A3d allele possessed three active genes, the highest number among Glu-A3 alleles. At the Glu-B3 locus, 2-3 m-type and 1-3 s-type genes were identified from individual NILs. Based on the different compositions of s-type genes, Glu-B3 alleles were divided into two groups, one containing Glu-B3a, B3b, B3f and B3g, and the other comprising Glu-B3c, B3d, B3h and B3i. Eight conserved genes were identified among Glu-D3 alleles, except for Glu-D3f. The protein products of the unique active genes in each NIL were detected using protein electrophoresis. Among Glu-3 alleles, the Glu-A3e genotype without i-type LMW-GS performed worst in almost all quality properties. Glu-B3b, B3g and B3i showed better quality parameters than the other Glu-B3 alleles, whereas the Glu-B3c allele containing s-type genes with low expression levels had an inferior effect on bread-making quality. Due to the conserved genes at Glu-D3 locus, Glu-D3 alleles showed no significant differences in effects on all quality parameters. This work provided new insights into the composition and function of 18 LMW-GS alleles in bread wheat. The variation of i-type genes mainly contributed to the high diversity of Glu-A3 alleles, and the differences among Glu-B3 alleles were mainly derived from the high polymorphism of s-type genes. Among LMW-GS alleles, Glu-A3e and Glu-B3c represented inferior alleles for bread-making quality, whereas Glu-A3d, Glu-B3b, Glu-B3g and Glu-B3i were correlated with superior bread-making quality. Glu-D3 alleles played minor roles in determining quality variation in bread wheat. Thus, LMW-GS alleles not only affect dough extensibility but greatly contribute to the dough resistance, glutenin macro-polymers and bread quality.

23259616

High resolution clear native electrophoresis (hrCNE) allows a detailed analysis of the heterotrimeric structure of recombinant Neisseria meningitidis porins inserted into liposomes.

Three recombinant proteins of Neisseria meningitidis, rPorB, rPorA, and rRmpM, were purified and incorporated into liposomes prepared by dialysis-extrusion. The protein complexes formed using different combinations of recombinant proteins were studied by high resolution clear native electrophoresis (hrCNE) and 2-D hrCNE/SDS-PAGE, analyzing the influence of the stoichiometry of the two porins in the formation of complexes and comparing them with native porin complexes present in OMVs from five different N. meningitidis strains. Insertion of the recombinant proteins into liposomes allowed a complete refolding of porin complexes, and the electrophoretic analyses showed that, when the three recombinant proteins are present, the pattern of porin complexes obtained is similar to that observed in native OMVs. We could show homocomplexes of each individual porin and PorA/PorB, RmpM/PorB, and PorA/PorB/RmpM heterocomplexes. Our results suggest that RmpM binds only to PorB, confirm the trimeric structure of N. meningitidis pores, and demonstrate that insertion into liposomes restores the native structure of porin complexes.

23259615

Ray Meta: scalable de novo metagenome assembly and profiling.

Voluminous parallel sequencing datasets, especially metagenomic experiments, require distributed computing for de novo assembly and taxonomic profiling. Ray Meta is a massively distributed metagenome assembler that is coupled with Ray Communities, which profiles microbiomes based on uniquely-colored k-mers. It can accurately assemble and profile a three billion read metagenomic experiment representing 1,000 bacterial genomes of uneven proportions in 15 hours with 1,024 processor cores, using only 1.5 GB per core. The software will facilitate the processing of large and complex datasets, and will help in generating biological insights for specific environments. Ray Meta is open source and available at http://denovoassembler.sf.net.

23259614

Characterization of two Agrostis-Festuca alpine pastures and their influence on cheese composition.

Recently, there has been a renewed interest in mountain farming, and several studies have been carried out on milk and cheese obtained in the unique environmental conditions of the Alps, a 1300 km mountain chain, located in the north of Italy. In this paper, the influence, on some cheese constituents, of two very similar mountain grasslands, both dominated by Festuca - Agrostis , was investigated. The two pastures were located in the same area in the southeastern Italian alpine region and differed in sunshine orientation and exposure. Milk obtained from cows grazing on these pastures was used to produce a semi-hard traditional cheese. The differences observed between the cheeses of the two areas for both some hydrocarbons (1-phytene and 2-phytene) and trans-fatty acids can be explained by a different rumen environment created by the botanical composition of the two pastures. The multidisciplinary approach can be considered a successful strategy, suitable for studying markers of authenticity.

23259613

Epidemiology and outcomes of previously undiagnosed diabetes in older women with breast cancer: an observational cohort study based on SEER-Medicare.

In breast cancer, diabetes diagnosed prior to cancer (previously diagnosed) is associated with advanced cancer stage and increased mortality. However, in the general population, 40% of diabetes is undiagnosed until glucose testing, and evidence suggests one consequence of increased evaluation and management around breast cancer diagnosis is the increased detection of previously undiagnosed diabetes. Biological factors - for instance, higher insulin levels due to untreated disease - and others underlying the association between previously diagnosed diabetes and breast cancer could differ in those whose diabetes remains undiagnosed until cancer. Our objectives were to identify factors associated with previously undiagnosed diabetes in breast cancer, and to examine associations between previously undiagnosed diabetes and cancer stage, treatment patterns, and mortality. Using Surveillance, Epidemiology, and End Results-Medicare, we identified women diagnosed with breast cancer and diabetes between 01/2001 and 12/2005. Diabetes was classified as previously diagnosed if it was identified within Medicare claims between 24 and 4 months before cancer diagnosis, and previously undiagnosed if it was identified from 3 months before to ≤ 3 months after cancer. Patients were followed until 12/2007 or death, whichever came first. Multivariate analyses were performed to examine risk factors for previously undiagnosed diabetes and associations between undiagnosed (compared to previously diagnosed) diabetes, cancer stage, treatment, and mortality. Of 2,418 patients, 634 (26%) had previously undiagnosed diabetes; the remainder had previously diagnosed diabetes. The mean age was 77.8 years, and 49.4% were diagnosed with in situ or stage I disease. Age > 80 years (40% of the cohort) and limited health system contact (primary care physician and/or preventive services) prior to cancer were associated with higher adjusted odds of previously undiagnosed diabetes. Previously undiagnosed diabetes was associated with higher adjusted odds of advanced stage (III/IV) cancer (Odds Ratio = 1.37: 95% Confidence Interval (CI) 1.05 - 1.80; P = 0.02), and a higher adjusted mortality rate due to causes other than cancer (Hazard Ratio = 1.29; 95% CI 1.02 - 1.63; P = 0.03). In breast cancer, previously undiagnosed diabetes is associated with advanced stage cancer and increased mortality. Identifying biological factors would require further investigation.

23259611

Functional role of intrahepatic monocyte subsets for the progression of liver inflammation and liver fibrosis in vivo.

Sustained inflammation upon chronic liver injury induces the development of liver fibrosis in mice and men. Experimental models of liver fibrosis highlight the importance of hepatic macrophages, so-called Kupffer cells, for perpetuating inflammation by releasing proinflammatory cytokines and chemokines as well as activating hepatic stellate cells (HSC). Recent studies in mice demonstrate that these actions are only partially conducted by liver-resident macrophages, classically termed Kupffer cells, but largely depend on recruitment of monocytes into the liver. Monocytes are circulating precursors of tissue macrophages and dendritic cells (DC), which comprise two major subsets in blood, characterized by the differential expression of chemokine receptors, adhesion molecules and distinct markers, such as Ly6C/Gr1 in mice or CD14 and CD16 in humans. Upon organ injury, chemokine receptor CCR2 and its ligand MCP-1 (CCL2) as well as CCR8 and CCL1 promote monocyte subset accumulation in the liver, namely of the inflammatory Ly6C(+) (Gr1(+)) monocyte subset as precursors of tissue macrophages. The infiltration of proinflammatory monocytes into injured murine liver can be specifically blocked by novel anti-MCP-1 directed agents. In contrast, chemokine receptor CX3CR1 and its ligand fractalkine (CX3CL1) are important negative regulators of monocyte infiltration in hepatic inflammation by controlling their survival and differentiation into functionally diverse macrophage subsets. In patients with liver cirrhosis, 'non-classical' CD14(+)CD16(+) monocytes are found activated in blood as well as liver and promote pro-inflammatory along with pro-fibrogenic actions by the release of distinct cytokines and direct interactions with HSC, indicating that the findings from murine models can be translated into pathogenesis of human liver fibrosis. Moreover, experimental animal models indicate that monocytes/macrophages and DCs are not only critical for fibrosis progression, but also for fibrosis regression, because macrophages can also degrade extracellular matrix proteins and exert anti-inflammatory actions. The recently identified cellular and molecular pathways for monocyte subset recruitment, macrophage differentiation and interactions with other hepatic cell types in injured liver may therefore represent interesting novel targets for future therapeutic approaches in liver fibrosis.

23259609

Looking back to move forward: using history, discourse and text in medical education research: AMEE guide no. 73.

As medical education research continues to diversify methodologically and theoretically, medical education researchers have been increasingly willing to challenge taken-for-granted assumptions about the form, content and function of medical education. In this AMEE guide we describe historical, discourse and text analysis approaches that can help researchers and educators question the inevitability of things that are currently seen as 'natural'. Why is such questioning important? By articulating our assumptions and interrogating the 'naturalness' of the status quo, one can then begin to ask why things are the way they are. Researchers can, for example, ask whether the models of medical education organization and delivery that currently seem 'natural' to them have been developed in order to provide the most benefit to students or patients--or whether they have, rather, been developed in ways that provide power to faculty members, medical schools or the medical profession as a whole. An understanding of the interplay of practices and power is a valuable tool for opening up the field to new possibilities for better medical education. The recognition that our current models, rather than being 'natural', were created in particular historical contexts for any number of contingent reasons leads inexorably to the possibility of change. For if our current ways of doing things are not, in fact, inevitable, not only can they be questioned, they can be made better; they can changed in ways that are attentive to whom they benefit, are congruent with our current beliefs about best practice and may lead to the production of better doctors.

23259607

Standardization and validation of a cytometric bead assay to assess antibodies to multiple Plasmodium falciparum recombinant antigens.

Multiplex cytometric bead assay (CBA) have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple Plasmodium falciparum antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to P. falciparum antigens, and to compare results of multiplex CBA to ELISA. Antibodies to ten recombinant P. falciparum antigens were measured by CBA and ELISA in samples from 30 individuals from a malaria endemic area of Kenya and compared to known positive and negative control plasma samples. Optimal antigen amounts, monoplex vs multiplex testing, plasma dilution, optimal buffer, number of beads required were assessed for CBA testing, and results from CBA vs. ELISA testing were compared. Optimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (r = 0.88-0.99, P values from <0.0001 - 0.004), and antibodies to variants of the same antigen were accurately distinguished within a multiplex reaction. Plasma dilutions of 1:100 or 1:200 were optimal for all antigens for CBA testing. Plasma diluted in a buffer containing 0.05% sodium azide, 0.5% polyvinylalcohol, and 0.8% polyvinylpyrrolidone had the lowest background activity. CBA median fluorescence intensity (MFI) values with 1,000 antigen-conjugated beads/well did not differ significantly from MFI with 5,000 beads/well. CBA and ELISA results correlated well for all antigens except apical membrane antigen-1 (AMA-1). CBA testing produced a greater range of values in samples from malaria endemic areas and less background reactivity for blank samples than ELISA. With optimization, CBA may be the preferred method of testing for antibodies to P. falciparum antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample and produces a greater range of values in positive samples and lower background readings for blank samples than ELISA.

23259608

Twelve tips for using Twitter as a learning tool in medical education.

Twitter is an online social networking service, accessible from any Internet-capable device. While other social networking sites are online confessionals or portfolios of personal current events, Twitter is designed and used as a vehicle to converse and share ideas. For this reason, we believe that Twitter may be the most likely candidate for integrating social networking with medical education. Using current research in medical education, motivation and the use of social media in higher education, we aim to show the ways Twitter may be used as a learning tool in medical education. A literature search of several databases, online sources and blogs was carried out examining the use of Twitter in higher education. We created 12 tips for using Twitter as a learning tool and organized them into: the mechanics of using Twitter, suggestions and evidence for incorporating Twitter into many medical education contexts, and promoting research into the use of Twitter in medical education. Twitter is a relatively new social medium, and its use in higher education is in its infancy. With further research and thoughtful application of media literacy, Twitter is likely to become a useful adjunct for more personalized teaching and learning in medical education.

23259606

Nanoemulsified orlistat-embedded multi-unit pellet system (MUPS) with improved dissolution and pancreatic lipase inhibition.

The present research work explores an innovative technological solution to constraints in efficient oral delivery of poorly water-soluble anti-obesity drug orlistat. Nanoemulsion of orlistat and its subsequent transformation into multi-unit pellet system (MUPS) for improved oral delivery was developed. Orlistat nanoemulsion was developed with capryol PGMC as an oil phase and cremophor RH40 as an emulsifier using high-pressure homogenization. Influence of critical processing parameters on globule size distribution, polydispersity index and physical stability of nanoemulsion was evaluated. The optimized nanoemulsion was transformed into MUPS using an extrusion spheronization technique. Optimized formulation was characterized at nanoemulsion as well as MUPS stage. DLS and nanoparticle tracking analysis studies of orlistat nanoemulsion exhibited unimodal size distribution with polydispersity value <0.1. Confocal laser scanning microscopy (CLSM) studies confirmed the presence of uniform spherical nanosized oil droplets of nanoemulsified orlistat. DSC and PXRD studies of MUPS confirmed amorphization of embedded nanoemulsified orlistat. In-vitro dissolution studies in surfactant-reduced media demonstrated remarkable improvement in dissolution compared to pure orlistat and marketed formulation (Xenical Capsules 120 mg, Hoffman-La Roche, Basle, Switzerland). Comparative in-vitro bovine porcine pancreatic lipase inhibition studies of pure orlistat, marketed product and developed MUPS showed 13.57- and 2.41-fold higher lipase inhibition with developed MUPS compared to pure orlistat and marketed products, respectively.

23259605

Sequential application of steady and pulsatile medium perfusion enhanced the formation of engineered bone.

In native bone, cells experience fluctuating shear forces that are induced by pulsatile interstitial flow associated with habitual loading. We hypothesized that the formation of engineered bone can be augmented by replicating such physiologic stimuli to osteogenic cells cultured in porous scaffolds using bioreactors with medium perfusion. To test this hypothesis, we investigated the effect of fluid flow regime on in vitro bone-like tissue development by human adipose stem cells (hASC) cultivated on porous three-dimensional silk fibroin scaffolds. To this end, we varied the sequential relative durations of steady flow (SF) and pulsatile flow (PF) of culture medium applied over a period of 5 weeks, and evaluated their effect on early stages of bone formation. Porous silk fibroin scaffolds (400-600 μm pore size) were seeded with hASC (30×10(6) cells/mL) and cultured in osteogenic medium under four distinct fluid flow regimes: (1) PF for 5 weeks; (2) SF for 1 week, PF for 4 weeks; (3) SF for 2 weeks, PF for 3 weeks; (4) SF for 5 weeks. The PF was applied in 12 h intervals, with the interstitial velocity fluctuating between 400 and 1200 μm/s at a 0.5 Hz frequency for 2 h, followed by 10 h of SF. In all groups, SF was applied at 400 μm/s. The best osteogenic outcomes were achieved for the sequence of 2 weeks of SF and 3 weeks of PF, as evidenced by gene expression (including the PGE2 mechanotransduction marker), construct compositions, histomorphologies, and biomechanical properties. We thus propose that osteogenesis in hASC and the subsequent early stage bone development involve a mechanism, which detects and responds to the level and duration of hydrodynamic shear forces.

23259602

Genome-wide association scan of dental caries in the permanent dentition.

Over 90% of adults aged 20 years or older with permanent teeth have suffered from dental caries leading to pain, infection, or even tooth loss. Although caries prevalence has decreased over the past decade, there are still about 23% of dentate adults who have untreated carious lesions in the US. Dental caries is a complex disorder affected by both individual susceptibility and environmental factors. Approximately 35-55% of caries phenotypic variation in the permanent dentition is attributable to genes, though few specific caries genes have been identified. Therefore, we conducted the first genome-wide association study (GWAS) to identify genes affecting susceptibility to caries in adults. Five independent cohorts were included in this study, totaling more than 7000 participants. For each participant, dental caries was assessed and genetic markers (single nucleotide polymorphisms, SNPs) were genotyped or imputed across the entire genome. Due to the heterogeneity among the five cohorts regarding age, genotyping platform, quality of dental caries assessment, and study design, we first conducted genome-wide association (GWA) analyses on each of the five independent cohorts separately. We then performed three meta-analyses to combine results for: (i) the comparatively younger, Appalachian cohorts (N = 1483) with well-assessed caries phenotype, (ii) the comparatively older, non-Appalachian cohorts (N = 5960) with inferior caries phenotypes, and (iii) all five cohorts (N = 7443). Top ranking genetic loci within and across meta-analyses were scrutinized for biologically plausible roles on caries. Different sets of genes were nominated across the three meta-analyses, especially between the younger and older age cohorts. In general, we identified several suggestive loci (P-value ≤ 10E-05) within or near genes with plausible biological roles for dental caries, including RPS6KA2 and PTK2B, involved in p38-depenedent MAPK signaling, and RHOU and FZD1, involved in the Wnt signaling cascade. Both of these pathways have been implicated in dental caries. ADMTS3 and ISL1 are involved in tooth development, and TLR2 is involved in immune response to oral pathogens. As the first GWAS for dental caries in adults, this study nominated several novel caries genes for future study, which may lead to better understanding of cariogenesis, and ultimately, to improved disease predictions, prevention, and/or treatment.

23259604

Toward the development of Raman spectroscopy as a nonperturbative online monitoring tool for gasoline adulteration.

There is a critical need for a real-time, nonperturbative probe for monitoring the adulteration of automotive gasoline. Running on adulterated fuel leads to a substantive increase in air pollution, because of increased tailpipe emissions of harmful pollutants, as well as a reduction in engine performance. Consequently, both classification of the gasoline type and quantification of the adulteration content are of great significance for quality control. Gasoline adulteration detection is currently carried out in the laboratory with gas chromatography, which is time-consuming and costly. Here, we propose the application of Raman spectroscopic measurements for on-site rapid detection of gasoline adulteration. In this proof-of-principle report, we demonstrate the effectiveness of Raman spectra, in conjunction with multivariate analysis methods, in classifying the base oil types and simultaneously detecting the adulteration content in a wide range of commercial gasoline mixtures, both in their native states and spiked with different adulterants. In particular, we show that Raman spectra acquired with an inexpensive noncooled detector provides adequate specificity to clearly discriminate between the gasoline samples and simultaneously characterize the specific adulterant content with a limit of detection below 5%. Our promising results in this study illustrate, for the first time, the capability and the potential of Raman spectroscopy, together with multivariate analysis, as a low-cost, powerful tool for on-site rapid detection of gasoline adulteration and opens substantive avenues for applications in related fields of quality control in the oil industry.

23259603

The inter-relationship of ascorbate transport, metabolism and mitochondrial, plastidic respiration.

Ascorbate, this multifaceted small molecular weight carbohydrate derivative, plays important roles in a range of cellular processes in plant cells, from the regulation of cell cycle, through cell expansion and senescence. Beyond these physiological functions, ascorbate has a critical role in responses to abiotic stresses, such as high light, high salinity, or drought. The biosynthesis, recycling, and intracellular transport are important elements of the balancing of ascorbate level to the always-changing conditions and demands. A bidirectional tight relationship was described between ascorbate biosynthesis and the mitochondrial electron transfer chain (mETC), since L-galactono-1,4-lactone dehydrogenase (GLDH), the enzyme catalyzing the ultimate step of ascorbate biosynthesis, uses oxidized cytochrome c as the only electron acceptor and has a role in the assembly of Complex I. A similar bidirectional relationship was revealed between the photosynthetic apparatus and ascorbate biosynthesis since the electron flux through the photosynthetic ETC affects the biosynthesis of ascorbate and the level of ascorbate could affect photosynthesis. The details of this regulatory network of photosynthetic electron transfer, respiratory electron transfer, and ascorbate biosynthesis are still not clear, as are the potential regulatory role and the regulation of intracellular ascorbate transport and fluxes. The elucidation of the role of ascorbate as an important element of the network of photosynthetic, respiratory ETC and tricarboxylic acid cycle will contribute to understanding plant cell responses to different stress conditions.

23259601

Transition States, analogues, and drug development.

Enzymes achieve their transition states by dynamic conformational searches on the femtosecond to picosecond time scale. Mimics of reactants at enzymatic transition states bind tightly to enzymes by stabilizing the conformation optimized through evolution for transition state formation. Instead of forming the transient transition state geometry, transition state analogues convert the short-lived transition state to a stable thermodynamic state. Enzymatic transition states are understood by combining kinetic isotope effects and computational chemistry. Analogues of the transition state can bind millions of times more tightly than substrates and show promise for drug development for several targets.

23259600

Cascade of reduced speed and accuracy after errors in enzyme-free copying of nucleic acid sequences.

Nonenzymatic, template-directed synthesis of nucleic acids is a paradigm for self-replicating systems. The evolutionary dynamics of such systems depend on several factors, including the mutation rates, relative replication rates, and sequence characteristics of mutant sequences. We measured the kinetics of correct and incorrect monomer insertion downstream of a primer-template mismatch (mutation), using a range of backbone structures (RNA, DNA, and LNA templates and RNA and DNA primers) and two types of 5'-activated nucleotides (oxyazabenzotriazolides and imidazolides, i.e., nucleoside 5'-phosphorimidazolides). Our study indicated that for all systems studied, an initial mismatch was likely to be followed by another error (54-75% of the time), and extension after a single mismatch was generally 10-100 times slower than extension without errors. If the mismatch was followed by a matched base pair, the extension rate recovered to nearly normal levels. On the basis of these data, we simulated nucleic acid replication in silico, which indicated that a primer suffering an initial error would lag behind properly extended counterparts due to a cascade of subsequent errors and kinetic stalling, with the typical mutational event consisting of several consecutive errors. Our study also included different sequence contexts, which suggest the presence of cooperativity among monomers affecting both absolute rate (by up to 2 orders of magnitude) and fidelity. The results suggest that molecular evolution in enzyme-free replication systems would be characterized by large "leaps" through sequence space rather than isolated point mutations, perhaps enabling rapid exploration of diverse sequences. The findings may also be useful for designing self-replicating systems combining high fidelity with evolvability.

23259599

ABT-737 reverses the acquired radioresistance of breast cancer cells by targeting Bcl-2 and Bcl-xL.

Acquired radioresistance of cancer cells remains a fundamental barrier to attaining the maximal efficacy of radiotherapy for the treatment of breast cancer. Anti-apoptotic proteins, such as Bcl-2 and Bcl-xL, play an important role in the radioresistance of cancer cells. In the present study, we aimed to determine if ABT-737, a BH3-only mimic, could reverse the acquired radioresistance of the breast cancer cell line MDA-MB-231R by targeting Bcl-2 and Bcl-xL. The radiosensitivity of MDA-MB-231 and MDA-MB-231R cells was compared using colony formation assays. Reverse-transcription PCR and western blot were performed to detect the expression of Bcl-2 and Bcl-xL in the cancer cell lines. Annexin V flow cytometric analysis and caspase-3 colorimetric assay were used to evaluate apoptosis of the cancer cells. Cell viability was measured using the Cell Counting Kit-8. The animals used in this study were 4 to 6-week-old athymic female BALB/c nu/nu mice. The MDA-MB-231R cells were more radioresistant than the MDA-MB-231 cells, and Bcl-2 and Bcl-xL were overexpressed in the MDA-MB-231R cells. While ABT-737 was able to restore the radiosensitivity of the MDA-MB-231R cells in vitro and in vivo experiment, it was not able to enhance the radiosensitivity of the MDA-MB-231 cells. In addition, ABT-737 increased radiation-induced apoptosis in the MDA-MB-231R cells. Bcl-2 and Bcl-xL were down regulated in the MDA-MB-231R cells following treatment with ABT-737. Targeting of the anti-apoptotic proteins Bcl-2 and Bcl-xL with ABT-737 may reverse the acquired radioresistance of MDA-MB-231R cells in vitro and in vivo. These findings suggest an attractive strategy for overcoming the acquired radioresistance of breast cancer cells.

23259598

L-glutamate released from activated microglia downregulates astrocytic L-glutamate transporter expression in neuroinflammation: the 'collusion' hypothesis for increased extracellular L-glutamate concentration in neuroinflammation.

In the central nervous system, astrocytic L-glutamate (L-Glu) transporters maintain extracellular L-Glu below neurotoxic levels, but their function is impaired with neuroinflammation. Microglia become activated with inflammation; however, the correlation between activated microglia and the impairment of L-Glu transporters is unknown. We used a mixed culture composed of astrocytes, microglia, and neurons. To quantify L-Glu transporter function, we measured the extracellular L-Glu that remained 30 min after an application of L-Glu to the medium (the starting concentration was 100 μM). We determined the optimal conditions of lipopolysaccharide (LPS) treatment to establish an inflammation model without cell death. We examined the predominant subtypes of L-Glu transporters and the changes in the expression levels of these transporters in this inflammation model. We then investigated the role of activated microglia in the changes in L-Glu transporter expression and the underlying mechanisms in this inflammation model. Because LPS (10 ng/mL, 72 h) caused a significant increase in the levels of L-Glu remaining but did not affect cell viability, we adopted this condition for our inflammation model without cell death. GLAST was the predominant L-Glu transporter subtype, and its expression decreased in this inflammation model. As a result of their release of L-Glu, activated microglia were shown to be essential for the significant decrease in L-Glu uptake. The serial application of L-Glu caused a significant decrease in L-Glu uptake and GLAST expression in the astrocyte culture. The hemichannel inhibitor carbenoxolone (CBX) inhibited L-Glu release from activated microglia and ameliorated the decrease in GLAST expression in the inflammation model. In addition, the elevation of the astrocytic intracellular L-Glu itself caused the downregulation of GLAST. Our findings suggest that activated microglia trigger the elevation of extracellular L-Glu through their own release of L-Glu, and astrocyte L-Glu transporters are downregulated as a result of the elevation of astrocytic intracellular L-Glu levels, causing a further increase of extracellular L-Glu. Our data suggest the new hypothesis that activated microglia collude with astrocytes to cause the elevation of extracellular L-Glu in the early stages of neuroinflammation.

23259597

A microRNA network regulates proliferative timing and extracellular matrix synthesis during cellular quiescence in fibroblasts.

Although quiescence (reversible cell cycle arrest) is a key part in the life history and fate of many mammalian cell types, the mechanisms of gene regulation in quiescent cells are poorly understood. We sought to clarify the role of microRNAs as regulators of the cellular functions of quiescent human fibroblasts. Using microarrays, we discovered that the expression of the majority of profiled microRNAs differed between proliferating and quiescent fibroblasts. Fibroblasts induced into quiescence by contact inhibition or serum starvation had similar microRNA profiles, indicating common changes induced by distinct quiescence signals. By analyzing the gene expression patterns of microRNA target genes with quiescence, we discovered a strong regulatory function for miR-29, which is downregulated with quiescence. Using microarrays and immunoblotting, we confirmed that miR-29 targets genes encoding collagen and other extracellular matrix proteins and that those target genes are induced in quiescence. In addition, overexpression of miR-29 resulted in more rapid cell cycle re-entry from quiescence. We also found that let-7 and miR-125 were upregulated in quiescent cells. Overexpression of either one alone resulted in slower cell cycle re-entry from quiescence, while the combination of both together slowed cell cycle re-entry even further. microRNAs regulate key aspects of fibroblast quiescence including the proliferative state of the cells as well as their gene expression profiles, in particular, the induction of extracellular matrix proteins in quiescent fibroblasts.

23259596

TiO2-based phosphoproteomic analysis of schistosomes: characterization of phosphorylated proteins in the different stages and sex of Schistosoma japonicum.

Protein phosphorylation is an important posttranslational modification in many organisms that regulates numerous cellular processes. However, it remains poorly characterized in schistosomes, the causative agent of schistosomiasis in humans and related animals. In the present study, we characterized phosphorylated proteins in different stages and sex of Schistosoma japonicum (S. japonicum) including schistosomula (14 days), adult females (35 days), and adult males (35 days) by a titanium dioxide (TiO(2)) based phosphoproteomic method. A total of 180 phosphopeptides were identified in 148 proteins. Our further studies revealed that heat shock protein 90 (Hsp90), one of the phosphoproteins codetected in the different stage and sex of schistosomes, may play an important role in the regulation of schistosome development by directly or indirectly interacting with other codetected signal molecules. Additionally, some phosphoproteins were shown to be detected in a gender-specific manner, and the expressions of these proteins were further validated either by immunohistochemistry or by real-time reverse transcription polymerase chain reaction (RT-PCR) at transcript levels between male and female schistosomes. In summary, these findings as well as the providing of an inventory of phosphoproteins are expected to provide new insights into schistosome development and sexual maturation and then may result in the development of novel interventions against schistosomiasis.

23259593

RNA-seq based identification and mutant validation of gene targets related to ethanol resistance in cyanobacterial Synechocystis sp. PCC 6803.

Fermentation production of biofuel ethanol consumes agricultural crops, which will compete directly with the food supply. As an alternative, photosynthetic cyanobacteria have been proposed as microbial factories to produce ethanol directly from solar energy and CO2. However, the ethanol productivity from photoautotrophic cyanobacteria is still very low, mostly due to the low tolerance of cyanobacterial systems to ethanol stress. To build a foundation necessary to engineer robust ethanol-producing cyanobacterial hosts, in this study we applied a quantitative transcriptomics approach with a next-generation sequencing technology, combined with quantitative reverse-transcript PCR (RT-PCR) analysis, to reveal the global metabolic responses to ethanol in model cyanobacterial Synechocystis sp. PCC 6803. The results showed that ethanol exposure induced genes involved in common stress responses, transporting and cell envelope modification. In addition, the cells can also utilize enhanced polyhydroxyalkanoates (PHA) accumulation and glyoxalase detoxication pathway as means against ethanol stress. The up-regulation of photosynthesis by ethanol was also further confirmed at transcriptional level. Finally, we used gene knockout strains to validate the potential target genes related to ethanol tolerance. RNA-Seq based global transcriptomic analysis provided a comprehensive view of cellular response to ethanol exposure. The analysis provided a list of gene targets for engineering ethanol tolerance in cyanobacterium Synechocystis.

23259595

Fluorescence properties of (E,E,E)-1,6-di(n-naphthyl)-1,3,5-hexatriene (n = 1, 2): effects of internal rotation.

The fluorescence spectroscopic properties of (E,E,E)-1,6-di(n-naphthyl)-1,3,5-hexatrienes (1, n = 1; 2, n = 2) have been investigated in solution and in the solid state. In solution, the absorption maxima (λ(a)) of the lowest-energy band (1, 374 nm; 2, 376 nm in methylcyclohexane) were similar for 1 and 2, whereas the fluorescence maxima (λ(f)) (1, 545 nm; 2, 453 nm) and quantum yields (φ(f)) (1, 0.046; 2, 0.68) were very different regardless of the solvent polarity. The fluorescence spectrum of 1 was independent of the excitation wavelength (λ(ex)), whereas the spectrum of 2 was weakly λ(ex)-dependent. In the solid state, the spectroscopic properties of 1 and 2 were similar (λ(a) = 437-438 nm, λ(f) = 496-505 nm, φ(f) = 0.04-0.07). The origins of emission are both considered to be mainly monomeric. With the help of single-crystal X-ray structure analysis and ab initio quantum chemical calculation, we conclude that the red-shifted and weak emission of 1 in solution originates from a planar excited state having small charge transfer character, reached from a twisted Franck-Condon state by the excited-state geometrical relaxation accompanied by the internal rotation around the naphthalene (Ar)-CH single bond. The similar fluorescence properties of 1 and 2 in the solid state can be attributed to the restriction of the geometrical relaxation. The effects of the Ar-CH rotational isomerism on the fluorescence properties in solution, for 2 in particular, are also discussed.

23259594

Application of protein purification methods for the enrichment of a cytotoxin from Campylobacter jejuni.

Campylobater jejuni, a major foodborne diarrhoeal pathogen is reported to produce a number of cytotoxins of which only a cytolethal distending toxin (CDT) has been characterised so far. One or more additional cytotoxins other than CDT, including a Chinese hamster ovary (CHO) cell active, Vero cell inactive cytotoxin, may mediate inflammatory diarrhoea. Our objective was to develop a method to enrich and thus partially characterise this cytotoxin, as a pathway to the eventual identification and characterisation of the toxin. A number of biochemical methods including cation- and anion-exchange chromatography were evaluated to enrich the cytotoxin from a cell lysate of a known cytotoxin-producing C. jejuni, C31. The cytotoxin in crude lysate was initially prepared by size-exclusion desalting and then subjected to high pressure liquid chromatography (HPLC) ion-exchange fractionation. One pooled fraction (pool B) was cytotoxic for CHO cells equivalent to crude toxin (tissue culture infectivity dose 50 [TCID(50)] of 1-2 μg/ml). The proteins of pool B were identified by mass spectrometry (MS) after separation by SDS-PAGE and trypsin digestion. Also, pool B was directly digested with trypsin and then subjected to liquid chromatography tandem mass spectrometry (LCMS) analysis for identification of lesser abundant proteins in the fraction. A total of 41 proteins were found in the fraction, which included enzymes involved in metabolic and transport functions. Eighteen non-cytoplasmic proteins including 2 major antigenic peptide proteins (PEB2 and PEB3) and 3 proteins of unknown function were also identified in the screen. Cytotoxicity in pool B was trypsin-sensitive indicating its protein nature. The cytotoxic activity was heat-stable to 50°C, and partially inactivated at 60-70°C. The pool B fraction also induced fluid accumulation in the adult rabbit ileal loop assay with cytotoxicity for mucosa confirming the presence of the cytotoxin. We report the enrichment and partial purification of C. jejuni cytotoxin by HPLC ion-exchange chromatography. Further purification may be achieved using additional complementary chromatographic techniques. A short-list of six candidate cytotoxin proteins was identified using an LCMS screen of pool B. Successful isolation of the cytotoxin will initiate steps for the determination of the role of this cytotoxin in the pathogenesis of C. jejuni diarrhoea.

23259592

Self-aligned nanotube-nanowire phase change memory.

A central issue of nanoelectronics concerns their fundamental scaling limits, that is, the smallest and most energy-efficient devices that can function reliably. Unlike charge-based electronics that are prone to leakage at nanoscale dimensions, memory devices based on phase change materials (PCMs) are more scalable, storing digital information as the crystalline or amorphous state of a material. Here, we describe a novel approach to self-align PCM nanowires with individual carbon nanotube (CNT) electrodes for the first time. The highly scaled and spatially confined memory devices approach the ultimate scaling limits of PCM technology, achieving ultralow programming currents (~0.1 μA set, ~1.6 μA reset), outstanding on/off ratios (~10(3)), and improved endurance and stability at few-nanometer bit dimensions. In addition, the powerful yet simple nanofabrication approach described here can enable confining and probing many other nanoscale and molecular devices self-aligned with CNT electrodes.

23259591

Alternative dosing of dual PI3K and MEK inhibition in cancer therapy.

PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways are thought to be the central transducers of oncogenic signals in solid malignancies, and there has been a lot of enthusiasm for developing inhibitors of these pathways for cancer therapy. Some preclinical models have suggested that combining inhibitors of both parallel pathways may be more efficacious, but it remains unknown whether dual inhibition with high enough concentrations of the drugs to achieve meaningful target inhibition is tolerable in a clinical setting. Furthermore, the predictive factors for dual inhibition are unknown. Non-small cell lung cancer (NSCLC) cell lines (n=12) with the most frequent oncogenic backgrounds (K-Ras mut n=3, EGFR mut n=3, ALK translocated n=3, and triple-negative n=3) were exposed to PI3K inhibitors (ZSTK474, PI-103) or MEK inhibitor (CI-1040) alone or in combination and analysed with an MTS growth/cytotoxicity assay and statistically by combination index analysis. The activity of the intracellular signaling pathways in response to the inhibitor treatments was analysed with a western blot using phospho-specific antibodies to AKT, ERK1/2, S6, and 4E-BPI. For the differential dosing schedule experiments, additional breast and colon cancer cell lines known to be sensitive to dual inhibition were included. Two of the 12 NSCLC cell lines tested, H3122 (ALK translocated) and H1437 (triple-negative), showed increased cytotoxicity upon dual MEK and PI3K inhibition. Furthermore, MDA-MB231 (breast) and HCT116 (colon), showed increased cytotoxicity upon dual inhibition, as in previous studies. Activation of parallel pathways in the dual inhibition-sensitive lines was also noted in response to single inhibitor treatment. Otherwise, no significant differences in downstream intracellular pathway activity (S6 and 4E-BPI) were noted between PI3K alone and dual inhibition other than the increased cytotoxicity of the latter. In the alternative dosing schedules two out of the four dual inhibition-sensitive cell lines showed similar cytotoxicity to continuous PI3K and short (15min) MEK inhibition treatment. Therapy with a dual PI3K and MEK inhibitor combination is more efficient than either inhibitor alone in some NSCLC cell lines. Responses to dual inhibition were not associated with any specific oncogenic genotype and no other predictive factors for dual inhibition were noted. The maximal effect of the dual PI3K and MEK inhibition can be achieved with alternative dosing schedules which are potentially more tolerable clinically.

23259590

Reversibility of liver fibrosis.

Liver fibrosis, and its end stage cirrhosis are a major cause of morbidity and mortality and therapeutic options are limited. However, the traditional view of liver disease as an irreversible process is obsolete and it is now evident that the development of liver fibrosis is a dynamic and potentially bidirectional process. Spontaneous resolution of scarring is seen in animal models of liver fibrosis and in human trials in which the stimuli responsible for chronic or repeated hepatic inflammation is successfully removed. Key players in the process are hepatic stellate cells, macrophages, MMPs and their inhibitors Timps. It is also evident that in advanced fibrotic liver disease, specific histological features define what is currently described as "irreversible" fibrosis. This includes the development of paucicellular scars enriched in extensively cross-linked matrix components, such as fibrillar collagen and elastin. Our recent work has focused on the role of macrophage metalloelastase (MMP-12) in the turnover of elastin in reversible and irreversible models of fibrosis. We have shown that elastin turnover in liver injury and fibrosis is regulated by macrophages via Mmp-12 expression, activity and ratio to its inhibitor Timp-1. Failure of elastin degradation, together with increased deposition leads to accumulation of elastin in the fibrotic scars.