cGMP/Protein Kinase G Signaling Suppresses Inositol 1,4,5-Trisphosphate Receptor Phosphorylation and Promotes Endoplasmic Reticulum Stress in Photoreceptors of Cyclic Nucleotide-gated Channel-deficient Mice

Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. We have shown endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER Ca²⁺ channel inositol 1,4,5-trisphosphate receptor 1 (IP₃R1) in CNG channel-deficient mice. We also presented a remarkable elevation of cGMP and an increased activity of the cGMP-dependent protein kinase (protein kinase G, PKG) in CNG channel deficiency. This work investigated whether cGMP/PKG signaling regulates ER stress and IP₃R1 phosphorylation in CNG channel-deficient cones. Treatment with PKG inhibitor and deletion of guanylate cyclase-1 (GC1), the enzyme producing cGMP in cones, were used to suppress cGMP/PKG signaling in cone-dominant Cnga3^−/−/Nrl^−/− mice. We found that treatment with PKG inhibitor or deletion of GC1 effectively reduced apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Müller glial cells. Furthermore, we observed significantly increased phosphorylation of IP₃R1 and reduced ER stress. Our findings demonstrate a role of cGMP/PKG signaling in ER stress and ER Ca²⁺ channel regulation and provide insights into the mechanism of cone degeneration in CNG channel deficiency.     

DNA loops generate intracentromere tension in mitosis

The centromere is the DNA locus that dictates kinetochore formation and is visibly apparent as heterochromatin that bridges sister kinetochores in metaphase. Sister centromeres are compacted and held together by cohesin, condensin, and topoisomerase-mediated entanglements until all sister chromosomes bi-orient along the spindle apparatus. The establishment of tension between sister chromatids is essential for quenching a checkpoint kinase signal generated from kinetochores lacking microtubule attachment or tension. How the centromere chromatin spring is organized and functions as a tensiometer is largely unexplored. We have discovered that centromere chromatin loops generate an extensional/poleward force sufficient to release nucleosomes proximal to the spindle axis. This study describes how the physical consequences of DNA looping directly underlie the biological mechanism for sister centromere separation and the spring-like properties of the centromere in mitosis.     

Consequences of interspecific hybridization and virus infection on the growth and fecundity of three threatened coastal Lepidium (Brassicaceae) species from New Zealand

Lepidium castellanum, L. juvencum and L. oleraceum are threatened coastal cresses endemic to New Zealand. These three species were selfed and interspecific hybrids generated for examination of hybrid fitness and inbreeding depression. In controlled glasshouse experiments, the interspecific hybrids and selfed progeny were inoculated with a strain of the introduced Turnip mosaic virus (TuMV) previously isolated from wild populations of L. aegrum. Experiments tested the hypothesis that heterosis in the interspecific hybrids provides a gain in TuMV resistance in comparison to selfed plants. We show that interspecific hybrids of three genetically distinct species of Lepidium increased plant performance and reduced susceptibility to the effects of the TuMV. We suggest that interspecific hybridization could be implemented as a conservation management strategy and that a broader outlook may be required to mitigate the negative impacts of introduced pathogens on threatened species.     

A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex

A key obstacle to understanding neural circuits in the?cerebral cortex is that of unraveling the diversity of GABAergic interneurons. This diversity poses general questions for neural circuit analysis: how are these interneuron cell types generated and assembled into stereotyped local circuits and how do they differentially contribute to circuit operations that underlie cortical functions ranging from perception to cognition? Using genetic engineering in mice, we have generated and characterized approximately 20 Cre and inducible CreER knockin driver lines that reliably target major classes and lineages of GABAergic neurons. More select populations are captured by intersection of Cre and Flp drivers. Genetic targeting allows reliable identification, monitoring, and manipulation of cortical GABAergic neurons, thereby enabling a systematic and comprehensive analysis from cell fate specification, migration, and connectivity, to their functions in network dynamics and behavior. As such, this approach will accelerate the study of GABAergic circuits throughout the mammalian brain.     

Assay development and high-throughput screening of small molecular c-Abl kinase activators

A 2-step kinase assay was developed and used in a high-throughput screen (HTS) of more than 1 million compounds in an effort to identify c-Abl tyrosine kinase activators. This assay employed a 2-step phosphorylation reaction: in the first step, purified recombinant c-Abl was activated by incubating with compound in the presence of adenosine triphosphate (ATP). In the second step, the TAMRA-labeled IMAP Abltide substrate was added to allow phosphorylation of the substrate to occur. The assay was calibrated such that inactive c-Abl protein was activated by ATP alone to a degree that it not only demonstrated a measurable c-Abl activity but also maintained a robust assay window for screening. The screen resulted in 8624 primary hits with >30% response. Further analysis showed that 1024 had EC(50) <10 μM with a max % response of >50%. These hits were structurally and chemically diverse with possibly different mechanisms for activating c-Abl. In addition, selective hits were shown to be cell permeable and were able to induce c-Abl activation as determined by In-Cell Western (ICW) analysis of HEK-MSRII cells transduced with BacMam virus expressing full-length c-Abl.     

Aquarium nitrification revisited: Thaumarchaeota are the dominant ammonia oxidizers in freshwater aquarium biofilters

Ammonia-oxidizing archaea (AOA) outnumber ammonia-oxidizing bacteria (AOB) in many terrestrial and aquatic environments. Although nitrification is the primary function of aquarium biofilters, very few studies have investigated the microorganisms responsible for this process in aquaria. This study used quantitative real-time PCR (qPCR) to quantify the ammonia monooxygenase (amoA) and 16S rRNA genes of Bacteria and Thaumarchaeota in freshwater aquarium biofilters, in addition to assessing the diversity of AOA amoA genes by denaturing gradient gel electrophoresis (DGGE) and clone libraries. AOA were numerically dominant in 23 of 27 freshwater biofilters, and in 12 of these biofilters AOA contributed all detectable amoA genes. Eight saltwater aquaria and two commercial aquarium nitrifier supplements were included for comparison. Both thaumarchaeal and bacterial amoA genes were detected in all saltwater samples, with AOA genes outnumbering AOB genes in five of eight biofilters. Bacterial amoA genes were abundant in both supplements, but thaumarchaeal amoA and 16S rRNA genes could not be detected. For freshwater aquaria, the proportion of amoA genes from AOA relative to AOB was inversely correlated with ammonium concentration. DGGE of AOA amoA genes revealed variable diversity across samples, with nonmetric multidimensional scaling (NMDS) indicating separation of freshwater and saltwater fingerprints. Composite clone libraries of AOA amoA genes revealed distinct freshwater and saltwater clusters, as well as mixed clusters containing both freshwater and saltwater amoA gene sequences. These results reveal insight into commonplace residential biofilters and suggest that aquarium biofilters may represent valuable biofilm microcosms for future studies of AOA ecology.     

A retrospective analysis of the haemodynamic and metabolic effects of fluid resuscitation in Vietnamese adults with severe falciparum malaria

Background

Optimising the fluid resuscitation of patients with severe malaria is a simple and potentially cost-effective intervention. Current WHO guidelines recommend central venous pressure (CVP) guided, crystalloid based, resuscitation in adults.

Methods

Prospectively collected haemodynamic data from intervention trials in Vietnamese adults with severe malaria were analysed retrospectively to assess the responses to fluid resuscitation.

Results

43 patients were studied of whom 24 received a fluid load. The fluid load resulted in an increase in cardiac index (mean increase: 0.75 L/min/m² (95% Confidence interval (CI): 0.41 to 1.1)), but no significant change in acid-base status post resuscitation (mean increase base deficit 0.6 mmol/L (95% CI: −0.1 to 1.3). The CVP and PAoP (pulmonary artery occlusion pressure) were highly inter-correlated (r_s = 0.7, p<0.0001), but neither were correlated with acid-base status (arterial pH, serum bicarbonate, base deficit) or respiratory status (PaO₂/FiO₂ ratio). There was no correlation between the oxygen delivery (DO₂) and base deficit at the 63 time-points where they were assessed simultaneously (r_s = −0.09, p = 0.46).

Conclusions

In adults with severe falciparum malaria there was no observed improvement in patient outcomes or acid-base status with fluid loading. Neither CVP nor PAoP correlated with markers of end-organ perfusion or respiratory status, suggesting these measures are poor predictors of their fluid resuscitation needs.     

Requirement of cognate CD4+ T-cell recognition for the regulation of allospecific CTL by human CD4+ CD127- CD25+ FOXP3+ cells generated in MLR

Although immunoregulation of alloreactive human CTLs has been described, the direct influence of CD4(+) Tregs on CD8(+) cytotoxicity and the interactive mechanisms have not been well clarified. Therefore, human CD4(+)CD127(-)CD25(+)FOXP3(+) Tregs were generated in MLR, immunoselected and their allospecific regulatory functions and associated mechanisms were then tested using modified (51)Chromium release assays (Micro-CML), MLRs and CFSE-based multi-fluorochrome flow cytometry proliferation assays. It was observed that increased numbers of CD4(+)CD127(-)CD25(+)FOXP3(+) cells were generated after a 7 day MLR. After immunoselection for CD4(+)CD127(-)CD25(+) cells, they were designated as MLR-Tregs. When added as third component modulators, MLR-Tregs inhibited the alloreactive proliferation of autologous PBMC in a concentration dependent manner. The inhibition was quasi-antigen specific, in that the inhibition was non-specific at higher MLR-Treg modulator doses, but non-specificity disappeared with lower numbers at which specific inhibition was still significant. When tested in micro-CML assays CTL inhibition occurred with PBMC and purified CD8(+) responders. However, antigen specificity of CTL inhibition was observed only with unpurified PBMC responders and not with purified CD8(+) responders or even with CD8(+) responders plus Non-T "APC". However, allospecificity of CTL regulation was restored when autologous purified CD4(+) T cells were added to the CD8(+) responders. Proliferation of CD8(+) cells was suppressed by MLR-Tregs in the presence or absence of IL-2. Inhibition by MLR-Tregs was mediated through down-regulation of intracellular perforin, granzyme B and membrane-bound CD25 molecules on the responding CD8(+) cells. Therefore, it was concluded that human CD4(+)CD127(-)CD25(+)FOXP3(+) MLR-Tregs down-regulate alloreactive cytotoxic responses. Regulatory allospecificity, however, requires the presence of cognate responding CD4(+) T cells. CD8(+) CTL regulatory mechanisms include impaired proliferation, reduced expression of cytolytic molecules and CD25(+) activation epitopes.