The mixed problem for the Laplacian in Lipschitz domains

We consider the mixed boundary value problem or Zaremba's problem for the Laplacian in a bounded Lipschitz domain in R^n. We specify Dirichlet data on part of the boundary and Neumann data on the remainder of the boundary. We assume that the boundary between the sets where we specify Dirichlet and Neumann data is a Lipschitz surface. We require that the Neumann data is in L^p and the Dirichlet data is in the Sobolev space of functions having one derivative in L^p for some p near 1. Under these conditions, there is a unique solution to the mixed problem with the non-tangential maximal function of the gradient of the solution in L^p of the boundary. We also obtain results with data from Hardy spaces when p=1.Comment: Version 5 includes a correction to one step of the main proof. Since the paper appeared long ago, this submission includes the complete paper, followed by a short section that gives the correction to one step in the proo

Superconductivity in Cu_xTiSe_2

Charge density waves (CDWs) are periodic modulations of the conduction electron density in solids. They are collective states that arise from intrinsic instabilities often present in low dimensional electronic systems. The layered dichalcogenides are the most well-studied examples, with TiSe_2 one of the first CDW-bearing materials known. The competition between CDW and superconducting collective electronic states at low temperatures has long been held and explored, and yet no chemical system has been previously reported where finely controlled chemical tuning allows this competition to be studied in detail. Here we report how, upon controlled intercalation of TiSe_2 with Cu to yield Cu_xTiSe_2, the CDW transition is continuously suppressed, and a new superconducting state emerges near x = 0.04, with a maximum T_c of 4.15 K found at x = 0.08. Cu_xTiSe_2 thus provides the first opportunity to study the CDW to Superconductivity transition in detail through an easily-controllable chemical parameter, and will provide new insights into the behavior of correlated electron systems.Comment: Accepted to Nature Physic

Methods to create a stringent selection system for mammalian cell lines

The efficient establishment of high protein producing recombinant mammalian cell lines is facilitated by the use of a stringent selection system. Here, we describe two methods to create a stringent selection system based on the Zeocin resistance marker. First, we cloned increasingly longer stretches of DNA, encoding a range of 8–131 amino acids immediately upstream of the Zeocin selection marker gene. The DNA stretches were separated from the open reading frame of the selection marker gene by a stopcodon. The idea behind this was that the translation machinery will first translate the small peptide, stop and then restart at the AUG of the Zeocin marker. This process, however, will become less efficient with increasingly longer stretches of DNA upstream of the Zeocin marker that has to be translated first. This would result in lower levels of the Zeocin selection marker protein and thus a higher selection stringency of the system. Secondly, we performed a genetic screen to identify PCR induced mutations in the Zeocin selection protein that functionally impair the selection marker protein. Both the insertion of increasingly longer peptides and several Zeocin selection protein mutants resulted in a decreasing number of stably transfected colonies that concomitantly displayed higher protein expression levels. When the Zeocin mutants were combined with very short small peptides (8–14 amino acids long), this created a flexible, high stringency selection system. The system allows the rapid establishment of few, but high protein producing mammalian cell lines

The endogenous caspase-8 inhibitor c-FLIPL regulates ER morphology and crosstalk with mitochondria

Components of the death receptors-mediated pathways like caspase-8 have been identified in complexes at intracellular membranes to spatially restrict the processing of local targets. In this study, we report that the long isoform of the cellular FLICE-inhibitory protein (c-FLIPL), a well- known inhibitor of the extrinsic cell death initiator caspase-8, localizes at the endoplasmic reticulum (ER) and mitochondria-associated membranes (MAMs). ER morphology was disrupted and ER Ca2+-release as well as ER-mitochondria tethering were decreased in c-FLIP-/- mouse embryonic fibroblasts (MEFs). Mechanistically, c-FLIP ablation resulted in enhanced basal caspase-8 activation and in caspase-mediated processing of the ER-shaping protein reticulon-4 (RTN4) that was corrected by re-introduction of c-FLIPL and caspase inhibition, resulting in the recovery of a normal ER morphology and ER-mitochondria juxtaposition. Thus, the caspase-8 inhibitor c-FLIPL emerges as a component of the MAMs signaling platforms, where caspases appear to regulate ER morphology and ER-mitochondria crosstalk by impinging on ER-shaping proteins like the RTN4

Stage-specific fluorescence intensity of GFP and mCherry during sporulation In Bacillus Subtilis

<p>Abstract</p> <p>Background</p> <p>Fluorescent proteins are powerful molecular biology tools that have been used to study the subcellular dynamics of proteins within live cells for well over a decade. Two fluorescent proteins commonly used to enable dual protein labelling are GFP (green) and mCherry (red). Sporulation in the Gram positive bacterium <it>Bacillus subtilis </it>has been studied for many years as a paradigm for understanding the molecular basis for differential gene expression. As sporulation initiates, cells undergo an asymmetric division leading to differential gene expression in the small prespore and large mother cell compartments. Use of two fluorescent protein reporters permits time resolved examination of differential gene expression either in the same compartments or between compartments. Due to the spectral properties of GFP and mCherry, they are considered an ideal combination for co-localisation and co-expression experiments. They can also be used in combination with fluorescent DNA stains such as DAPI to correlate protein localisation patterns with the developmental stage of sporulation which can be linked to well characterised changes in DNA staining patterns.</p> <p>Findings</p> <p>While observing the recruitment of the transcription machinery into the forespore of sporulating <it>Bacillus subtilis</it>, we noticed the occurrence of stage-specific fluorescence intensity differences between GFP and mCherry. During vegetative growth and the initial stages of sporulation, fluorescence from both GFP and mCherry fusions behaved similarly. During stage II-III of sporulation we found that mCherry fluorescence was considerably diminished, whilst GFP signals remained clearly visible. This fluorescence pattern reversed during the final stage of sporulation with strong mCherry and low GFP fluorescence. These trends were observed in reciprocal tagging experiments indicating a direct effect of sporulation on fluorescent protein fluorophores.</p> <p>Conclusions</p> <p>Great care should be taken when interpreting the results of protein localisation and quantitative gene expression patterns using fluorescent proteins in experiments involving intracellular physiological change. We believe changes in the subcellular environment of the sporulating cell leads to conditions that differently alter the spectral properties of GFP and mCherry making an accurate interpretation of expression profiles technically challenging.</p

Topiramate-associated acute glaucoma in a migraine patient receiving concomitant citalopram therapy: a case-report

We describe the case of a 34 year-old man with diagnosis of migraine with and without aura that developed myopia and acute glaucoma after 7 days of treatment with topiramate. The patient had also been taking citalopram daily for two months. Both topiramate and citalopram have been related to the increase of intraocular pressure and the development of glaucoma. We can't exclude that in this patient citalopram caused an increase of the ocular pressure in dose-dependent manner, facilitating topiramate-induced glaucoma. We recommend to pay particular attention in prescribing of topiramate in migraine patients who are already under treatment with citalopram or other antidepressants with a similar mechanisms of action

An empirical approach towards the efficient and optimal production of influenza-neutralizing ovine polyclonal antibodies demonstrates that the novel adjuvant CoVaccine HT(TM) is functionally superior to Freund's adjuvant

Passive immunotherapies utilising polyclonal antibodies could have a valuable role in preventing and treating infectious diseases such as influenza, particularly in pandemic situations but also in immunocompromised populations such as the elderly, the chronically immunosuppressed, pregnant women, infants and those with chronic diseases. The aim of this study was to optimise current methods used to generate ovine polyclonal antibodies. Polyclonal antibodies to baculovirus-expressed recombinant influenza haemagglutinin from A/Puerto Rico/8/1934 H1N1 (PR8) were elicited in sheep using various immunisation regimens designed to investigate the priming immunisation route, adjuvant formulation, sheep age, and antigen dose, and to empirically ascertain which combination maximised antibody output. The novel adjuvant CoVaccine HT™ was compared to Freund’s adjuvant which is currently the adjuvant of choice for commercial production of ovine polyclonal Fab therapies. CoVaccine HT™ induced significantly higher titres of functional ovine anti-haemagglutinin IgG than Freund’s adjuvant but with fewer side effects, including reduced site reactions. Polyclonal hyperimmune sheep sera effectively neutralised influenza virus in vitro and, when given before or after influenza virus challenge, prevented the death of infected mice. Neither the age of the sheep nor the route of antigen administration appeared to influence antibody titre. Moreover, reducing the administrated dose of haemagglutinin antigen minimally affected antibody titre. Together, these results suggest a cost effective way of producing high and sustained yields of functional ovine polyclonal antibodies specifically for the prevention and treatment of globally significant diseases.Natalie E. Stevens, Cara K. Fraser, Mohammed Alsharifi, Michael P. Brown, Kerrilyn R. Diener, John D. Haybal

Influence of an alkalizing supplement on markers of endurance performance using a double-blind placebo-controlled design

<p>Abstract</p> <p>Background</p> <p>Previous research has shown that ingestion of substances that enhance the body's hydrogen ion buffering capacity during high intensity exercise can improve exercise performance. The present study aimed to determine whether the chronic ingestion of an alkalizing supplement, which purports to enhance both intracellular and extracellular buffering capacity, could impact cardiorespiratory and performance markers in trained Nordic skiers.</p> <p>Methods</p> <p>Twenty-four skiers (12 men, 12 women), matched for upper body power (UBP), were split into treatment and placebo groups. The treatment group ingested Alka-Myte^®-based alkalizing tablets (1 tablet/22.7 kg body mass/day) over seven successive days while the placebo group consumed placebo tablets (i.e., no Alka-Myte^®) at the same dosage. Prior to tablet ingestion (i.e., pre-testing), both groups completed a constant power UBP test, three successive 10-sec UBP tests, and then a 60-sec UBP test. Next, skiers completed the 7-day ingestion of their assigned tablets followed immediately by a repeat of the same UBP tests (i.e., post-testing). Neither the skiers nor the researchers were aware of which tablets were being consumed by either group until after all testing was complete. Dependent measures for analysis included heart rate (HR), oxygen consumption (VO₂), minute ventilation (V_E), blood lactate (LA), as well as 10-sec (W10, W) and 60-sec (W60, W) UBP. All data were evaluated using a two-factor multivariate repeated measures ANOVA with planned contrasts for post-hoc testing (alpha = 0.05).</p> <p>Results</p> <p>Post-testing cardiorespiratory (HR, VO₂, V_E) and LA measures for the treatment group tended to be significantly lower when measured for both constant power and UBP60 tests, while measures of both 10-sec (W10: 229 to 243 W) and 60-sec UBP (W60: 190 to 198 W) were significantly higher (<it>P </it>< 0.05). In contrast, there were no significant changes for the placebo group (P > 0.05).</p> <p>Conclusions</p> <p>Following the 7-day loading phase of Alka-Myte^®-based alkalizing tablets, trained Nordic skiers experienced significantly lower cardiorespiratory stress, lower blood lactate responses, and higher UBP measures. Thus, the use of this supplement appeared to impart an ergogenic benefit to the skiers that may be similar to the effects expected from consuming well-studied extracellular buffering agents such as sodium bicarbonate.</p