julilili42/papers huggingface dataset:
Nonclassical Pathways of Protein Crystallization in the Presence of Multivalent Metal Ions
2014-10-29

Using bovine β-lactoglobulin as a model system, we have studied the crystallization pathways in the presence of the di- and trivalent salts ZnCl<sub>2</sub> and YCl<sub>3</sub>. Previous work has shown that protein solutions undergo a reentrant condensation (RC) phase behavior in the presence of YCl<sub>3</sub>, i.e., a condensed phase occurs in between two boundary salt concentrations, <em>c</em>*<em>&lt; c</em>**. In the presence of ZnCl<sub>2</sub>, <em>c</em>* also exists, but protein solutions with high salt concentrations do not become completely clear at higher protein concentrations (<em>&gt;</em>20 mg/mL). We thus denote this diffuse transition as <em>pseudo</em>-<em>c</em>**. Small angle X-ray scattering measurements show that the effective interactions change from repulsion to attraction near <em>c</em>*, and attractive interactions dominate around <em>pseudo</em>–<em>c</em>**. Solutions near <em>c</em>* and <em>pseudo</em><em>-c</em>** provide the optimized conditions for growth of high quality protein single crystals but with different pathways. While crystal growth near <em>c</em>* follows the classical one-step nucleation pathway, crystallization around <em>pseudo</em>-<em>c</em>** (for zinc) or <em>c</em>** (for yttrium) follows a nonclassical process with an intermediate phase appearing before crystallization. Furthermore, the intermediate phases strongly depend on the crystallization temperature. Samples with high salt concentrations exhibit a typical transition temperature, <em>T</em><sub>tr</sub>, below which the solutions become turbid. When crystallizing near <em>T</em><sub>tr</sub>, the intermediate phase consists of protein clusters; below <em>T</em><sub>tr</sub>, the intermediate phase corresponds to macroscopic protein aggregates which can further relax into a dense liquid phase before crystallization. However, the experimental data cannot distinguish whether nucleation occurs within or outside of the intermediate phase. Possible scenarios are discussed based on the equilibrium phase behavior of colloidal systems with a short-range attraction. The crystallographic analysis of the resulting crystals shows that metal ions are an integral part of the crystal lattice. Both types of metal ions can create new protein contacts in the crystal lattice via bridging; however, yttrium creates more bridging contacts compared to zinc.
One Class SVM and Canonical Correlation Analysis increase performance in a c-VEP based Brain-Computer Interface (BCI)
2014-10-29

The goal of a Brain-Computer Interface (BCI) is to enable communication by pure brain activity without the need for muscle control. Recently BCIs based on code-modulated visual evoked potentials (c-VEPs) have shown great potential to establish high-performance communication. In this paper we present two new methods to improve classication in a c-VEP BCI. Canonical correlation analysis can be used to build an op- timal spatial lter for detection of c-VEPs, while the use of a one class support vector machine (OCSVM) makes the BCI more robust in terms of artefacts and thus increases performance. We show both methods to increase performance in an oine analysis on data from 8 subjects. As a proof of concept both methods are tested online with one subject, who achieved an average performance of 133 bit/min, which is higher than any other bitrate reported so far for a non-invasive BCI.
The prognostic impact of specific CD4 T-cell responses is critically dependent on the target antigen in melanoma
2014-10-29

The melanoma-associated antigens Melan-A and NY-ESO-1 stimulate different T-cell responses in late-stage melanoma patients. Either CD4<sup>+</sup> or CD8<sup>+</sup> T-cell reactivity against NY-ESO-1 was associated with better prognosis, but for Melan-A, only CD8<sup>+</sup> but not CD4<sup>+</sup> T-cell responses were associated with longer survival.
Discovery of functional non-coding conserved regions in the α-synuclein gene locus
2014-10-29

Several single nucleotide polymorphisms (SNPs) and the Rep-1 microsatellite marker of the α-synuclein ( SNCA ) gene have consistently been shown to be associated with Parkinson's disease, but the functional relevance is unclear. Based on these findings we hypothesized that conserved cis-regulatory elements in the SNCA genomic region regulate expression of SNCA , and that SNPs in these regions could be functionally modulating the expression of SNCA , thus contributing to neuronal demise and predisposing to Parkinson's disease. In a pair-wise comparison of a 206kb genomic region encompassing the SNCA gene, we revealed 34 evolutionary conserved DNA sequences between human and mouse. All elements were cloned into reporter vectors and assessed for expression modulation in dual luciferase reporter assays. We found that 12 out of 34 elements exhibited either an enhancement or reduction of the expression of the reporter gene. Three elements upstream of the SNCA gene displayed an approximately 1.5 fold (p<0.009) increase in expression. Of the intronic regions, three showed a 1.5 fold increase and two others indicated a 2 and 2.5 fold increase in expression (p<0.002). Three elements downstream of the SNCA gene showed 1.5 fold and 2.5 fold increase (p<0.0009). One element downstream of SNCA had a reduced expression of the reporter gene of 0.35 fold (p<0.0009) of normal activity. Our results demonstrate that the SNCA gene contains cis-regulatory regions that might regulate the transcription and expression of SNCA . Further studies in disease-relevant tissue types will be important to understand the functional impact of regulatory regions and specific Parkinson's disease-associated SNPs and its function in the disease process.
One Small Step for a Yeast - Microevolution within Macrophages Renders Candida glabrata Hypervirulent Due to a Single Point Mutation
2014-10-30

Candida glabrata is one of the most common causes of candidemia, a life-threatening, systemic fungal infection, and is surpassed in frequency only by Candida albicans. Major factors contributing to the success of this opportunistic pathogen include its ability to readily acquire resistance to antifungals and to colonize and adapt to many different niches in the human body. Here we addressed the flexibility and adaptability of C. glabrata during interaction with macrophages with a serial passage approach. Continuous co-incubation of C. glabrata with a murine macrophage cell line for over six months resulted in a striking alteration in fungal morphology: The growth form changed from typical spherical yeasts to pseudohyphae-like structures - a phenotype which was stable over several generations without any selective pressure. Transmission electron microscopy and FACS analyses showed that the filamentous-like morphology was accompanied by changes in cell wall architecture. This altered growth form permitted faster escape from macrophages and increased damage of macrophages. In addition, the evolved strain (Evo) showed transiently increased virulence in a systemic mouse infection model, which correlated with increased organ-specific fungal burden and inflammatory response (TNFα and IL-6) in the brain. Similarly, the Evo mutant significantly increased TNFα production in the brain on day 2, which is mirrored in macrophages confronted with the Evo mutant, but not with the parental wild type. Whole genome sequencing of the Evo strain, genetic analyses, targeted gene disruption and a reverse microevolution experiment revealed a single nucleotide exchange in the chitin synthase-encoding CHS2 gene as the sole basis for this phenotypic alteration. A targeted CHS2 mutant with the same SNP showed similar phenotypes as the Evo strain under all experimental conditions tested. These results indicate that microevolutionary processes in host-simulative conditions can elicit adaptations of C. glabrata to distinct host niches and even lead to hypervirulent strains.
Imaging the elastic modulus of human platelets during thrombininduced activation using scanning ion conductance microscopy
2014-10-30

Summary Platelet activation plays a critical role in haemostasis and thrombosis. It is well-known that platelets generate contractile forces during activation. However, their mechanical material properties have rarely been investigated. Here, we use scanning ion conductance microscopy (SICM) to visualise morphological and mechanical properties of live human platelets at high spatial resolution. We found that their mean elastic modulus decreases during thrombin-induced activation by about a factor of two. We observed a similar softening of platelets during cytochalasin D-induced cytoskeleton depolymerisation. However, thrombin-induced temporal and spatial modulations of the elastic modulus were substantially different from cytochalasin D-mediated changes. We thereby provide new insights into the mechanics of haemostasis and establish SICM as a novel imaging platform for the ex vivo investigation of the mechanical properties of live platelets.
Switching substrate specificity of AMT/MEP/ Rh proteins
2014-10-30

In organisms from all kingdoms of life, ammonia and its conjugated ion ammonium are transported across membranes by proteins of the AMT/Rh family. Efficient and successful growth often depends on sufficient ammonium nutrition. The proteins mediating this transport, the so called Ammonium Transporter (AMT) or Rhesus like (Rh) proteins, share a very similar trimeric overall structure and a high sequence similarity even throughout the kingdoms. Even though structural components of the transport mechanism, like an external substrate recruitment site, an essential twin histidine pore motif, a phenylalanine gate and the hydrophobic pore are strongly conserved and have been analyzed in detail by molecular dynamic simulations and mutational studies, the substrate(s), which pass the central pores of the AMT/Rh subunits, NH4(+), NH3 + H(+), NH4(+) + H(+) or NH3, are still a matter of debate for most proteins, including the best characterized AmtB protein from Escherichia coli. The lack of a robust expression system for functional analysis has hampered proof of structural and mutational studies, although the NH3 transport function for Rh-like proteins is rarely disputed. In plant transporters belonging to the subfamily AMT1, transport is associated with electrical currents, while some plant transporters, notably of the AMT2 type, were suggested to transport NH3 across the membrane, without associated ionic currents. Here we summarize data in favor of each substrate for the distinct AMT/Rh classes, discuss mutants and how they differ in structure and functionality. A common mechanism with deprotonation and subsequent NH3 transport through the central subunit pore is suggested.
Performance of Protein–Ligand Docking with Simulated Chemical Shift Perturbations
2014-10-30

Protein chemical shift perturbations (CSPs) that result from the binding of a ligand to the protein contain structural information about the complex. Therefore, the CSP data, typically obtained during library screening from two-dimensional (2D) nuclear magnetic resonance (NMR) spectra, are often available before attempts to solve the experimental structure of the complex are started, and can be used to solve the complex structure with CSP-based docking. Here, we compare the performance of the post-docking filter and the guided-docking approaches using either amide or α-proton CSPs with 10 protein-ligand complexes. We show that the comparison of experimental CSPs with CSPs simulated for virtual ligand positions can be used to evidence protein conformational change upon binding and possibly improve the CSP-based docking.
Extraction and separation strategies of [7.7]paracyclophane derivatives
2014-10-30

Naturally occurring and polyketide-derived paracyclophanes have been isolated from various cyanobacteria belonging to the genera Nostoc and Cylindrospermum. Cylindrocyclophanes, nostocyclophanes, carbamidocyclophanes and merocyclophanes share a unique [7.7]paracyclophane scaffold, but differ in a varying degree of halogen atoms and in a diverse substitution pattern of functional groups, e.g. of hydroxyl, acetoxy and carbamoyl moieties. Additionally, these compounds are reported to exhibit a wide range of biological activities, such as antiproliferative, cytotoxic and antibiotic effects [1 – 4]. Based on the carbamido- and cylindrocyclophanes producing Nostoc sp. CAVN2, various separation procedures for closely related derivatives were developed to improve our paracyclophane-directed screening for novel analogues as well as to ensure an easy to handle high-throughput processing of cultivation samples. Compared to bioassay-guided column chromatography and affiliated TLC analysis, prepurification of the methanol extract by solid phase extraction (SPE) utilizing reversed-phase material and a methanol/water step gradient resulted in a reproducible, faster and a single cyclophane-containing fraction generating methodology. Normal-phase SPE with an ethyl acetate/toluene gradient led to a specifically controlled separation of closely related cyclophanes, e.g. the fractionation of carbamidocyclophane A from carbamidocyclophane F and cylindrocyclophane A4 (Fig. 1). Optimization of biomass extraction resulted in a rapid and exhaustive one-step cyclophane separation and selective enrichment procedure using a liquid-liquid two-phase system for subsequent HPLC-DAD-MS analysis. Due to the usage of an ether-linked phenyl phase followed by a pentafluorophenyl stationary phase, the analytical HPLC separation power could be scaled up to semi-preparative conditions to facilitate the isolation of pure cyclophanes.
LRRK2 Transport Is Regulated by Its Novel Interacting Partner Rab32
2014-10-31

Leucine-rich repeat kinase 2 (LRRK2) is a multi-domain 280 kDa protein that is linked to Parkinson's disease (PD). Mutations especially in the GTPase and kinase domains of LRRK2 are the most common causes of heritable PD and are also found in sporadic forms of PD. Although the cellular function of LRRK2 is largely unknown there is increasing evidence that these mutations cause cell death due to autophagic dysfunction and mitochondrial damage. Here, we demonstrate a novel mechanism of LRRK2 binding and transport, which involves the small GTPases Rab32 and Rab38. Rab32 and its closest homologue Rab38 are known to organize the trans-Golgi network and transport of key enzymes in melanogenesis, whereas their function in non-melanogenic cells is still not well understood. Cellular processes such as autophagy, mitochondrial dynamics, phagocytosis or inflammatory processes in the brain have previously been linked to Rab32. Here, we demonstrate that Rab32 and Rab38, but no other GTPase tested, directly interact with LRRK2. GFP-Trap analyses confirmed the interaction of Rab32 with the endogenous LRRK2. In yeast two-hybrid experiments we identified a predicted coiled-coil motif containing region within the aminoterminus of LRRK2 as the possible interacting domain. Fluorescence microscopy demonstrated a co-localization of Rab32 and LRRK2 at recycling endosomes and transport vesicles, while overexpression of a constitutively active mutant of Rab32 led to an increased co-localization with Rab7/9 positive perinuclear late endosomes/MVBs. Subcellular fractionation experiments supported the novel role of Rab32 in LRRK2 late endosomal transport and sorting in the cell. Thus, Rab32 may regulate the physiological functions of LRRK2.
Activation of lymphoma-associated MyD88 mutations via allostery-induced TIR-domain oligomerization
2014-10-31

Key Points The hyperactive phenotype of lymphoma-associated mutations is caused by increased oligomerization propensity of the MyD88 TIR domain. The TIR domain of mutants interacts with wild-type MyD88, explaining why heterozygous mutation could be sufficient as a driver mutation.
Decoding of motor intentions from epidural ECoG recordings in severely paralyzed chronic stroke patients
2014-10-31

The findings suggest that ipsilesional epidural ECoG can be used as a viable control signal for BCI-driven neuroprosthesis. Although patients showed no sign of residual hand movement, brain activity at the ipsilesional motor cortex still shows enough intention-related activity to decode different movement intentions with sufficient accuracy.
Intermittent Episodes of Bright Light Suppress Myopia in the Chicken More than Continuous Bright Light
2014-10-31

The protective effect of bright light depends on the exposure duration and, to the intermittent form, the frequency cycle. Compared to the saturation effect of continuous bright light, low frequency cycles of bright light (1:1 min) provided the strongest inhibition effect. However, our quantitative results probably might not be directly translated into humans, but rather need further amendments in clinical studies.
Comparison of insulin glargine and liraglutide added to oral agents in patients with poorly controlled type 2 diabetes
2014-10-31

Aim To compare safety and efficacy of insulin glargine and liraglutide in patients with type 2 diabetes ( T2DM ). Methods This randomized, multinational, open‐label trial included subjects treated for T2DM with metformin ± sulphonylurea, who had glycated haemoglobin ( HbA1c ) levels of 7.5–12%. Subjects were assigned to 24 weeks of insulin glargine, titrated to target fasting plasma glucose of 4.0–5.5 mmol/L or liraglutide, escalated to the highest approved clinical dose of 1.8 mg daily. The trial was powered to detect superiority of glargine over liraglutide in percentage of people reaching HbA1c &lt;7%. Results The mean [standard deviation (s.d.)] age of the participants was 57 (9) years, the duration of diabetes was 9 (6) years, body mass index was 31.9 (4.2) kg/m 2 and HbA1c level was 9.0 (1.1)%. Equal numbers ( n = 489) were allocated to glargine and liraglutide. Similar numbers of subjects in both groups attained an HbA1c level of &lt;7% (48.4 vs. 45.9%); therefore, superiority of glargine over liraglutide was not observed (p = 0.44). Subjects treated with glargine had greater reductions of HbA1c [−1.94% (0.05) and −1.79% (0.05); p = 0.019] and fasting plasma glucose [6.2 (1.6) and 7.9 (2.2) mmol/L; p &lt; 0.001] than those receiving liraglutide. The liraglutide group reported a greater number of gastrointestinal treatment‐emergent adverse events (p &lt; 0.001). The mean (s.d.) weight change was +2.0 (4.0) kg for glargine and −3.0 (3.6) kg for liraglutide (p &lt; 0.001). Symptomatic hypoglycaemia was more common with glargine (p &lt; 0.001). A greater number of subjects in the liraglutide arm withdrew as a result of adverse events (p &lt; 0.001). Conclusion Adding either insulin glargine or liraglutide to subjects with poorly controlled T2DM reduces HbA1c substantially, with nearly half of subjects reaching target levels of 7%.
Both mature KIR+ and immature KIR− NK cells control pediatric acute B-cell precursor leukemia in NOD.Cg-Prkdcscid IL2rgtmWjl/Sz mice
2014-10-31

Key Points Both mature KIR+ and immature KIR− NK cells exert antileukemic activity toward pediatric BCP-ALL in vivo. In vivo treatment with low-dose 5-aza-cytidine enhances immature and mature NK-cell counts and promotes antitumor response.
Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases <em>In Vivo</em>
2014-10-31

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5'-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure. In the following protocol, we describe an in vivo fluorescent primer extension method to reliably and rapidly detect the 5' ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g., by toxin-antitoxin system components) in S. aureus, E. coli and other bacteria.
Calcium - a central regulator of keratinocyte differentiation in health and disease
2014-11-01

Regular keratinocyte differentiation is crucial for the formation of an intact epidermal barrier and is triggered by extracellular calcium. Disturbances of epidermal barrier formation and aberrant keratinocyte differentiation are involved in the pathophysiology of several skin diseases, such as psoriasis, atopic dermatitis, basal and squamous skin cancer, and genetic skin diseases such as Darier's disease and Olmstedt syndrome. In this review, we summarize current knowledge about the underlying molecular mechanisms of calcium-induced differentiation in keratinocytes. We provide an overview of calcium's genomic and non-genomic mechanisms to induce differentiation and discuss the calcium gradient in the epidermis, giving rise to cornified skin and lipid envelope formation. We focus on the calcium-sensing receptor, transient receptor potential channels, and STIM/Orai as the major constituents of calcium sensing and calcium entry in the keratinocytes. Finally, skin diseases linked to impaired differentiation will be discussed, paying special attention to disturbed TRP channel expression and TRP channel mutations.
Haemolysis associated with the treatment of malaria with artemisinin derivatives: a systematic review of current evidence
2014-11-01

Haemolysis is commonly associated with the class of artemisinin drugs when used for the treatment of severe malaria. Potential causes of this safety issue are discussed. Although no deaths attributed to haemolysis have been reported so far, this safety issue may lead to life-threatening anaemia and is particularly worrying for regions where safe blood products are not readily available.
Population Pharmacokinetics of Intravenous Artesunate: A Pooled Analysis of Individual Data From Patients With Severe Malaria
2014-11-01

There are ~660,000 deaths from severe malaria each year. Intravenous artesunate (i.v. ARS) is the first‐line treatment in adults and children. To optimize the dosing regimen of i.v. ARS, the largest pooled population pharmacokinetic study to date of the active metabolite dihydroartemisinin (DHA) was performed. The pooled dataset consisted of 71 adults and 195 children with severe malaria, with a mixture of sparse and rich sampling within the first 12 h after drug administration. A one‐compartment model described the population pharmacokinetics of DHA adequately. Body weight had the greatest impact on DHA pharmacokinetics, resulting in lower DHA exposure for smaller children (6–10 kg) than adults. Post hoc estimates of DHA exposure were not significantly associated with parasitological outcomes. Comparable DHA exposure in smaller children and adults after i.v. ARS was achieved under a dose modification for intramuscular ARS proposed in a separate analysis of children. CPT Pharmacometrics Syst. Pharmacol . (2014) 3, e145; doi: 10.1038/psp.2014.43 ; published online 05 November 2014