Design and synthesis of 7H-pyrrolo[2,3-d]pyrimidines as focal adhesion kinase inhibitors. Part 1

A series of 2-amino-9-aryl-7H-pyrrolo[2,3-d]pyrimidines were designed and synthesized to target focal adhesion kinase (FAK). A number of these pyrrolopyrimides exhibited low micromolar inhibitory activities against focal adhesion kinase, and their preliminary SAR was established via systematic chemical modifications. The 2-amino-9-aryl-7H-pyrrolo[2,3-d]pyrimidines represent a new class of kinase inhibitors.     

TLR2 Mediates Recognition of Live Staphylococcus epidermidis and Clearance of Bacteremia

Background

Staphylococcus epidermidis (SE) is a nosocomial pathogen that causes catheter-associated bacteremia in the immunocompromised, including those at the extremes of age, motivating study of host clearance mechanisms. SE-derived soluble components engage TLR2; but additional signaling pathways have also been implicated, and TLR2 can play complex, at times detrimental, roles in host defense against other Staphylococcal spp. The role of TLR2 in responses of primary blood leukocytes to live SE and in clearance of SE bacteremia, the most common clinical manifestation of SE infection, is unknown.

Methodology/Principal Findings

We studied TLR2-mediated recognition of live clinical SE strain 1457 employing TLR2-transfected cells, neutralizing anti-TLR antibodies and TLR2-deficient mice. TLR2 mediated SE-induced cytokine production in human embryonic kidney cells, human whole blood and murine primary macrophages, in part via recognition of a soluble TLR2 agonist. After i.v. challenge with SE, early (1 h) cytokine/chemokine production and subsequent clearance of bacteremia (24–48 h) were markedly impaired in TLR2-deficient mice.

Conclusions/Significance

TLR2 mediates recognition of live SE and clearance of SE bacteremia in vivo.     

Neurexin in Embryonic Drosophila Neuromuscular Junctions

Background

Neurexin is a synaptic cell adhesion protein critical for synapse formation and function. Mutations in neurexin and neurexin-interacting proteins have been implicated in several neurological diseases. Previous studies have described Drosophila neurexin mutant phenotypes in third instar larvae and adults. However, the expression and function of Drosophila neurexin early in synapse development, when neurexin function is thought to be most important, has not been described.

Methodology/Principal Findings

We use a variety of techniques, including immunohistochemistry, electron microscopy, in situ hybridization, and electrophysiology, to characterize neurexin expression and phenotypes in embryonic Drosophila neuromuscular junctions (NMJs). Our results surprisingly suggest that neurexin in embryos is present both pre and postsynaptically. Presynaptic neurexin promotes presynaptic active zone formation and neurotransmitter release, but along with postsynaptic neurexin, also suppresses formation of ectopic glutamate receptor clusters. Interestingly, we find that loss of neurexin only affects receptors containing the subunit GluRIIA.

Conclusions/Significance

Our study extends previous results and provides important detail regarding the role of neurexin in Drosophila glutamate receptor abundance. The possibility that neurexin is present postsynaptically raises new hypotheses regarding neurexin function in synapses, and our results provide new insights into the role of neurexin in synapse development.     

A finite element analysis of masticatory stress hypotheses

Understanding how the skull transmits and dissipates forces during feeding provides insights into the selective pressures that may have driven the evolution of primate skull morphology. Traditionally, researchers have interpreted masticatory biomechanics in terms of simple global loading regimes applied to simple shapes (i.e., bending in sagittal and frontal planes, dorsoventral shear, and torsion of beams and cylinders). This study uses finite element analysis to examine the extent to which these geometric models provide accurate strain predictions in the face and evaluate whether simple global loading regimes predict strains that approximate the craniofacial deformation pattern observed during mastication. Loading regimes, including those simulating peak loads during molar chewing and those approximating the global loading regimes, were applied to a previously validated finite element model (FEM) of a macaque (Macaca fascicularis) skull, and the resulting strain patterns were compared. When simple global loading regimes are applied to the FEM, the resulting strains do not match those predicted by simple geometric models, suggesting that these models fail to generate accurate predictions of facial strain. Of the four loading regimes tested, bending in the frontal plane most closely approximates strain patterns in the circumorbital region and lateral face, apparently due to masseter muscle forces acting on the zygomatic arches. However, these results indicate that no single simple global loading regime satisfactorily accounts for the strain pattern found in the validated FEM. Instead, we propose that FE models replace simple cranial models when interpreting bone strain data and formulating hypotheses about craniofacial biomechanics.     

Making human nasal cilia beat in the cold: a real time assay for cell signalling

Human nasal epithelium must adapt to cold climates, and yet, in vitro, human ciliary beat frequency (CBF) is zero at 4 degrees C. Similarly, hibernating mammals do not die of pneumonia despite a core body temperature as low as 6 degrees C, implying that cilia continue to beat. Here, we show that protein kinase C (PKC) and Ca(2+)/calmodulin-dependent kinase II (CaMKII) regulate the profile of human nasal CBF in response to rising temperature from 4 degrees C. Onset of ciliary beat was at 10 degrees C in Medium 199, 7 degrees C in the presence of the PKC activator phorbol 12-myristate 13-acetate (PMA), the calcium ionophore ionomycin, or the CAMKII blocker myristoylated autocamtide-2 related inhibitory peptide (MACI), and 6 degrees C for the myristoylated peptide PKC inhibitor EGF-R Fragment 651-658 (MyrPKCI). During cell warming to 32 degrees C, the thermal profile was sigmoid in all solutions except those containing MACI+PMA. Surprisingly, cilia continued to beat despite 4 degrees C and were significantly more responsive to rising temperature with either MACI+PMA, or MACI+MyrPKCI. Our data suggest that CaMKII and PKC regulate the thermal slope and profile of CBF in vitro, and that when these protein kinases are manipulated, cilia can continue to beat despite hypothermia. These findings may relate to adaptive responses to cold climates.     

The Association of Early Childhood Cognitive Development and Behavioural Difficulties with Pre-Adolescent Problematic Eating Attitudes

Objectives

Few studies have prospectively investigated associations of child cognitive ability and behavioural difficulties with later eating attitudes. We investigated associations of intelligence quotient (IQ), academic performance and behavioural difficulties at 6.5 years with eating attitudes five years later.

Methods

We conducted an observational cohort study nested within the Promotion of Breastfeeding Intervention Trial, Belarus. Of 17,046 infants enrolled at birth, 13,751 (80.7%) completed the Children''s Eating Attitude Test (ChEAT) at 11.5 years, most with information on IQ (n = 12,667), academic performance (n = 9,954) and behavioural difficulties (n = 11,098) at 6.5 years. The main outcome was a ChEAT score ≥85^th percentile, indicative of problematic eating attitudes.

Results

Boys with higher IQ at 6.5 years reported fewer problematic eating attitudes, as assessed by ChEAT scores ≥85^th percentile, at 11.5 years (OR per SD increase in full-scale IQ = 0.87; 0.79, 0.94). No such association was observed in girls (1.01; 0.93, 1.10) (p for sex-interaction = 0.016). In both boys and girls, teacher-assessed academic performance in non-verbal subjects was inversely associated with high ChEAT scores five years later (OR per unit increase in mathematics ability = 0.88; 0.82, 0.94; and OR per unit increase in ability for other non-verbal subjects = 0.86; 0.79, 0.94). Behavioural difficulties were positively associated with high ChEAT scores five years later (OR per SD increase in teacher-assessed rating = 1.13; 1.07, 1.19).

Conclusion

Lower IQ, worse non-verbal academic performance and behavioural problems at early school age are positively associated with risk of problematic eating attitudes in early adolescence.     

Practitioner Perspectives on Strategies to Promote Longer-Term Benefits of Acupuncture or Counselling for Depression: A Qualitative Study

Background

Non-pharmacological interventions for depression may help patients manage their condition. Evidence from a recent large-scale trial (ACUDep) suggests that acupuncture and counselling can provide longer-term benefits for many patients with depression. This paper describes the strategies practitioners reported using to promote longer-term benefits for their patients.

Methods

A qualitative sub-study of practitioners (acupuncturists and counsellors) embedded in a randomised controlled trial. Using topic guides, data was collected from telephone interviews and a focus group, altogether involving 19 counsellors and 17 acupuncturists. Data were audio recorded, transcribed verbatim and analysed using thematic content analysis.

Results

For longer-term impact, both acupuncturists and counsellors encouraged insight into root causes of depression on an individual basis and saw small incremental changes as precursors to sustained benefit. Acupuncturists stressed the importance of addressing concurrent physical symptoms, for example helping patients relax or sleep better in order to be more receptive to change, and highlighted the importance of Chinese medicine theory-based lifestyle change for lasting benefit. Counsellors more often highlighted the importance of the therapeutic relationship, emphasising the need for careful “pacing” such that the process and tools employed were tailored and timed for each individual, depending on the “readiness” to change. Our data is limited to acupuncture practitioners using the principles of traditional Chinese medicine, and counsellors using a humanistic, non-directive and person-centred approach.

Conclusions

Long-term change appears to be an important focus within the practices of both acupuncturists and counsellors. To achieve this, practitioners stressed the need for an individualised approach with a focus on root causes.     

Genetic Perturbation of the Maize Methylome

DNA methylation can play important roles in the regulation of transposable elements and genes. A collection of mutant alleles for 11 maize (Zea mays) genes predicted to play roles in controlling DNA methylation were isolated through forward- or reverse-genetic approaches. Low-coverage whole-genome bisulfite sequencing and high-coverage sequence-capture bisulfite sequencing were applied to mutant lines to determine context- and locus-specific effects of these mutations on DNA methylation profiles. Plants containing mutant alleles for components of the RNA-directed DNA methylation pathway exhibit loss of CHH methylation at many loci as well as CG and CHG methylation at a small number of loci. Plants containing loss-of-function alleles for chromomethylase (CMT) genes exhibit strong genome-wide reductions in CHG methylation and some locus-specific loss of CHH methylation. In an attempt to identify stocks with stronger reductions in DNA methylation levels than provided by single gene mutations, we performed crosses to create double mutants for the maize CMT3 orthologs, Zmet2 and Zmet5, and for the maize DDM1 orthologs, Chr101 and Chr106. While loss-of-function alleles are viable as single gene mutants, the double mutants were not recovered, suggesting that severe perturbations of the maize methylome may have stronger deleterious phenotypic effects than in Arabidopsis thaliana.     

Extracellular polysaccharide production in outdoor mass cultures of Porphyridium sp. in flat plate glass reactors

This work concerns an attempt to develop large scalecultivation of Porphyridium sp. outdoors. Theimpact on cell growth and production of solublesulphated polysaccharides of light-path length (LP)was studied in flat plate glass reactors outdoors. TheLP of the plate reactors ranged from 1.3–30 cm,corresponding to culture volumes of 3–72 L. The sidewalls of all reactors were covered, ensuring similarilluminated surfaces for all reactors. Maximal daytemperature was maintained at 26 ±1 ^°C.Growth conditions of pH (7.5), stirring (withcompressed air) and mineral nutrients, were optimal.Maximal volumetric concentration of the soluble sulfated polysaccharide (1.32 g L^-1) was obtained in winter with the smallest light-pathreactor (1.3 cm ) at a cell density of 1.37 ×10¹¹cells L^-1. Under these conditions, theviscosity of the culture medium was also highest,being inversely proportional to the culture'slight-path. Highest areal concentration of solublepolysaccharides (60 g m^-2) and areal cell density(3.01 × 10¹²m^-2) was recorded in the 20 cmLP reactor, progressively lower values being obtainedas the light path became shorter. A similar patternwas obtained for the areal productivity ofpolysaccharides, the highest being 4.15 g m^-2day^-1 (considering the total illuminated reactorsurface), produced in the 20-cm LP reactor.The main sugar composition (i.e. xylose, galactose andglucose) of the sulfated polysaccharides was similarin all reactors. As viscosity increased with timeduring culture growth, there was a substantial declinein bacterial population. Cultivation throughout mostof the year provided good evidence that a light pathlength of 20 cm in flat plate reactors under theseconditions is optimal for maximal areal solublepolysaccharide production of Porphyridium sp.