2-(1-(Dimethylamino)ethyl)phenylpalladium(II) complexes 5-functionalized with fluorous silyl tails

New fluorous-organometallics based on the chiral ligand α-methyl-N,N-dimethylbenzylamine (TMBA) were prepared by treatment of fluorous silyl bromide reagents with in situ 4-lithiated TMBA to give fluorous N,N-dimethyl(α-methyl-4-trialkylsilylbenzyl)amine ligands 1a-1c that vary in the number of fluorous tails attached to the Si atom. Ligands 1a-1c were successfully cyclo-palladated by treatment with Pd(OAc)₂/LiCl in methanol to furnish the corresponding chloride-bridged dimeric arylpalladium(II) complexes 2a-2c in good yields. The latter derivatives could be converted into monomeric Lewis-base adducts by complexation with pyridine (3a-3c), or triphenylphosphine (4a-4c). The crystal structure of triphenylphosphine complex 4a has been elucidated. To probe their fluorophilicity, the partition coefficient of each of the derivatives in the fluorous biphasic solvent (FBS) system perfluoromethylcyclohexane/n-octane has been determined.     

(Sub)microscopic Plasmodium falciparum gametocytaemia in Kenyan children after treatment with sulphadoxine-pyrimethamine monotherapy or in combination with artesunate

The effects of drugs on Plasmodium falciparum transmission stages may reduce the spread of parasites in the population and contribute to malaria control. Detailed quantitative studies on (sub)microscopic gametocytaemia have become feasible with the availability of real-time Pfs25 quantitative Nucleic Acid Sequence-based Amplification (QT-NASBA), which can be used to detect gametocyte densities above 20 gametocytes per millilitre from in vitro cultures. Gametocyte dynamics were investigated in children with uncomplicated P. falciparum malaria after treatment with sulphadoxine-pyrimethamine (SP) or a combination of SP and artesunate (SP+AS), in a 28-days drug efficacy study. This study demonstrated that gametocyte prevalence in 873 samples from symptomatic Kenyan children was 2.8 times higher by QT-NASBA compared with microscopy. Microscopy-positive cases showed a significant correlation with QT-NASBA for gametocyte density. At enrolment, gametocyte prevalence was 86% by QT-NASBA compared with 22% by microscopy. Gametocytes were detected in 97% of children in at least one blood sample and in 38% of children in all samples obtained during the 28-days follow-up. Both the risk of gametocyte carriage and gametocyte density were considerably higher after treatment with SP compared with SP+AS. Gametocyte prevalence and density decreased with time in the SP+AS group, but not in the SP-treated children. Our data suggest that the potential of malaria transmission remains high even after treatment with artemisinin combination therapy, although prevalence and density of gametocytes is lower after SP+AS.     

Delayed expansion and contraction of CD8+ T cell response during infection with virulent Salmonella typhimurium

Ag presentation to CD8(+) T cells often commences immediately after infection, which facilitates their rapid expansion and control of infection. Subsequently, the primed cells undergo rapid contraction. We report that this paradigm is not followed during infection with virulent Salmonella enterica, serovar Typhimurium (ST), an intracellular bacterium that replicates within phagosomes of infected cells. Although susceptible mice die rapidly (approximately 7 days), resistant mice (129 x 1SvJ) harbor a chronic infection lasting approximately 60-90 days. Using rOVA-expressing ST (ST-OVA), we show that T cell priming is considerably delayed in the resistant mice. CD8(+) T cells that are induced during ST-OVA infection undergo delayed expansion, which peaks around day 21, and is followed by protracted contraction. Initially, ST-OVA induces a small population of cycling central phenotype (CD62L(high)IL-7Ralpha(high)CD44(high)) CD8(+) T cells. However, by day 14-21, majority of the primed CD8(+) T cells display an effector phenotype (CD62L(low)IL-7Ralpha(low)CD44(high)). Subsequently, a progressive increase in the numbers of effector memory phenotype cells (CD62L(low)IL-7Ralpha(high)CD44(high)) occurs. This differentiation program remained unchanged after accelerated removal of the pathogen with antibiotics, as majority of the primed cells displayed an effector memory phenotype even at 6 mo postinfection. Despite the chronic infection, CD8(+) T cells induced by ST-OVA were functional as they exhibited killing ability and cytokine production. Importantly, even memory CD8(+) T cells failed to undergo rapid expansion in response to ST-OVA infection, suggesting a delay in T cell priming during infection with virulent ST-OVA. Thus, phagosomal lifestyle may allow escape from host CD8(+) T cell recognition, conferring a survival advantage to the pathogen.     

Cryo electron microscopy reconstructions of the Leviviridae unveil the densest icosahedral RNA packing possible

We solved the structures of the single-stranded RNA bacteriophages Qbeta, PP7 and AP205 by cryo-electron microscopy. On the outside, the symmetrized electron density maps resemble the previously described cryo-electron microscopy structure of MS2. RNA density is present inside the capsids, suggesting that the genomic RNA of Qbeta, PP7 and AP205, analogous to MS2, contains many coat protein-binding sites in addition to the hairpin on which assembly and packaging are initiated. All four bacteriophages harbour the same overall arrangement of the RNA, which is a unique combination of both triangles and pentagons. This combination has not been found in other icosahedral viruses, in which the RNA structures are either triangular or pentagonal. Strikingly, the unique RNA packing of the Leviviridae appears to deploy the most efficient method of RNA storage by obeying icosahedral symmetry.     

Fingerprinting complex pectins by chromatographic separation combined with ELISA detection

Enzyme-resistant pectin or modified hairy regions were subjected to size exclusion (HPSEC) and weak anion exchange (WAX) chromatography. Fractions collected after separation were tested for the presence of different pectic epitopes using the monoclonal antibodies LM2, LM5, LM6, and JIM7. Separation by HPSEC showed that based on molecular weight the different epitopes were restricted to distinct molecular weight populations. WAX chromatography resulted in an even better separation of the different pectic epitopes present. A clear separation between arabino galactan type II epitopes and the RG I side chains, (1,5)-α-l-arabinan and (1,4)-β-d-galactan, could be established. Arabinogalactan type II was found in the first populations eluting off the WAX column. The observations made within the ELISA assays of the collected fractions could be confirmed by determination of the sugar composition of the individual populations obtained. The sugar composition of the AGII positive populations eluting off the WAX column shows the presence of significant amounts of rhamnose and galacturonic acid. Together with the delay on an anion exchanger, this observation indicates a possible linkage between RGI and AGII. The volume of the individual fractions collected provides enough material for a maximum of 20 different antibodies to be tested from one analytical separation.     

Altered Contractility of Skeletal Muscle in Mice Deficient in Titin’s M-Band Region

We investigated the contractile phenotype of skeletal muscle deficient in exons MEx1 and MEx2 (KO) of the titin M-band by using the cre-lox recombination system and a multidisciplinary physiological approach to study skeletal muscle contractile performance. At a maximal tetanic stimulation frequency, intact KO extensor digitorum longus muscle was able to produce wild-type levels of force. However, at submaximal stimulation frequency, force was reduced in KO mice, giving rise to a rightward shift of the force-frequency curve. This rightward shift of the force-frequency curve could not be explained by altered sarcoplasmic reticulum Ca²⁺ handling, as indicated by analysis of Ca²⁺ transients in intact myofibers and expression of Ca²⁺-handling proteins, but can be explained by the reduced myofilament Ca²⁺ sensitivity of force generation that we found. Western blotting experiments suggested that the excision of titin exons MEx1 and MEx2 did not result in major changes in expression of titin M-band binding proteins or phosphorylation level of the thin-filament regulatory proteins, but rather in a shift toward expression of slow isoforms of the thick-filament-associated protein, myosin binding protein-C. Extraction of myosin binding protein-C from skinned muscle normalized myofilament Ca²⁺ sensitivity of the KO extensor digitorum longus muscle. Thus, our data suggest that the M-band region of titin affects the expression of genes involved in the regulation of skeletal muscle contraction.     

Promiscuous activity of arginine:glycine amidinotransferase is responsible for the synthesis of the novel cardiovascular risk factor homoarginine

Low plasma homoarginine has emerged as a risk marker for cardiovascular disease. We exploited cells of a patient with a rare inborn error of metabolism to explore potential pathways of homoarginine synthesis, using stable isotopes and mass spectrometry. Control lymphoblasts, as opposed to lymphoblasts from an arginine:glycine amidinotransferase (AGAT)-deficient patient, were able to synthesize homoarginine from arginine and lysine. In contrast, in a patient with a deficiency of the urea cycle enzyme argininosuccinate synthase, plasma homoarginine was not decreased. We conclude that promiscuous activity of AGAT, a key enzyme in creatine synthesis, plays a pivotal role in homoarginine synthesis.     

A Novel Mechanism Involving Four-and-a-half LIM Domain Protein-1 and Extracellular Signal-regulated Kinase-2 Regulates Titin Phosphorylation and Mechanics

Understanding mechanisms underlying titin regulation in cardiac muscle function is of critical importance given recent compelling evidence that highlight titin mutations as major determinants of human cardiomyopathy. We previously identified a cardiac biomechanical stress-regulated complex at the cardiac-specific N2B region of titin that includes four-and-a-half LIM domain protein-1 (Fhl1) and components of the mitogen-activated protein signaling cascade, which impacted muscle compliance in Fhl1 knock-out cardiac muscle. However, direct regulation of these molecular components in mediating titin N2B function remained unresolved. Here we identify Fhl1 as a novel negative regulator of titin N2B levels and phosphorylation-mediated mechanics. We specifically identify titin N2B as a novel substrate of extracellular signal regulated-kinase-2 (Erk2) and demonstrate that Fhl1 directly interferes with Erk2-mediated titin-N2B phosphorylation. We highlight the critical region in titin-N2B that interacts with Fhl1 and residues that are dependent on Erk2-mediated phosphorylation in situ. We also propose a potential mechanism for a known titin-N2B cardiomyopathy-causing mutation that involves this regulatory complex. These studies shed light on a novel mechanism regulating titin-N2B mechano-signaling as well as suggest that dysfunction of these pathways could be important in cardiac disease states affecting muscle compliance.     

Brief cognitive behavioral therapy compared to general practitioners care for depression in primary care: a randomized trial

Background

Previous studies of acupuncture show favourable results for both subjective and objective outcomes of dry eye. However, firm conclusions could not be drawn from these studies because the quality of the trials was too low to establish concrete evidence. Therefore, this study was designed both to avoid the flaws of the existing trials and to assess the effectiveness, cost-effectiveness and qualitative characteristics of acupuncture treatment for dry eye.

Methods/design

One hundred fifty participants with dry eye will be recruited into three independent hospitals from different areas: Korea Institute of Oriental Medicine, DongGuk University Ilsan Oriental Hospital and Dongshin University Gwangju Oriental Hospital. The number of participants required was calculated from the data of a previous, relevant study. These patients will be randomly allocated into acupuncture treatment or artificial tear groups. Either 17 acupuncture points (bilateral BL2, GB14, TE 23, Ex1, ST1, GB20, LI4, LI11 and single GV23) will be used 3 times a week or disposable artificial tear drops (Refresh Plus^®, ALLERGAN) will be provided for use at least once a day for 4 weeks. The ocular surface disease index (OSDI), tear film break-up time (TFBUT), Schirmer I test, visual analogue scale (VAS) for self-assessment of ocular discomfort, general assessment (by both acupuncture practitioners and participants) and quality of life (QOL) through the Measure Yourself Medical Outcome Profile-2 (MYMOP-2) will be assessed for approximately 3-months for each study participant. In addition, qualitative study and cost-effectiveness of acupuncture treatment will be conducted.

Trial registration

ClinicalTrials.gov (Identifier: NCT01105221).