Sperm deliver a new second messenger: NAADP

NAADP is a highly potent mobilizer of Ca(2+), which in turn triggers Ca(2+)-induced Ca(2+) release pathways in a wide range of species. Nevertheless, NAADP is not presently classified as a second messenger because it has not been shown to increase in response to a physiological stimulus. We now report a dramatic increase in NAADP during sea urchin egg fertilization that was largely due to production in sperm upon contacting egg jelly. The NAADP bolus plays a physiological role upon delivery to the egg based on its ability to induce a cortical flash, a depolarization-induced activation of L-type Ca(2+) channels. Moreover, the sperm-induced cortical flash was eliminated in eggs desensitized to NAADP. We conclude that an NAADP increase plays a physiologically relevant role during fertilization and provides the first conclusive demonstration that NAADP is a genuine second messenger.     

Enzyme kinetics and distinct modulation of the protein kinase N family of kinases by lipid activators and small molecule inhibitors

The PKN (protein kinase N) family of Ser/Thr protein kinases regulates a diverse set of cellular functions, such as cell migration and cytoskeletal organization. Inhibition of tumour PKN activity has been explored as an oncology therapeutic approach, with a PKN3-targeted RNAi (RNA interference)-derived therapeutic agent in Phase I clinical trials. To better understand this important family of kinases, we performed detailed enzymatic characterization, determining the kinetic mechanism and lipid sensitivity of each PKN isoform using full-length enzymes and synthetic peptide substrate. Steady-state kinetic analysis revealed that PKN1–3 follows a sequential ordered Bi–Bi kinetic mechanism, where peptide substrate binding is preceded by ATP binding. This kinetic mechanism was confirmed by additional kinetic studies for product inhibition and affinity of small molecule inhibitors. The known lipid effector, arachidonic acid, increased the catalytic efficiency of each isoform, mainly through an increase in k_cat for PKN1 and PKN2, and a decrease in peptide K_M for PKN3. In addition, a number of PKN inhibitors with various degrees of isoform selectivity, including potent (K_i<10 nM) and selective PKN3 inhibitors, were identified by testing commercial libraries of small molecule kinase inhibitors. This study provides a kinetic framework and useful chemical probes for understanding PKN biology and the discovery of isoform-selective PKN-targeted inhibitors.     

The effect of light direction and suspended cell concentrations on algal biofilm growth rates

Algae biofilms were grown in a semicontinuous flat plate biofilm photobioreactor to study the effects of light direction and suspended algal cell populations on algal biofilm growth. It was determined that, under the growth conditions and biofilm thicknesses studied, light direction had no effect on long-term algal biofilm growth (26 days); however, light direction did affect the concentration of suspended algal cells by influencing the photon flux density in the growth medium in the photobioreactors. This suspended algal cell population affected short-term (7 days) algae cell recruitment and algal biofilm growth, but additional studies showed that enhanced suspended algal cell populations did not affect biofilm growth rates over the long term (26 days). Studying profiles of light transmittance through biofilms as they grew showed that most of the light became attenuated by the biomass after just a few days of growth (88 % after 3 days). The estimated biofilm thicknesses after these few days of growth were approximately 150 μm. The light attenuation data suggests that, although the biofilms grew to 700–900 μm, under these light intensities, only the first few hundred micrometers of the biofilm is receiving enough light to be photosynthetically active. We postulate that this photosynthetically active layer of the biofilm grows adjacent to the light source, while the rest of the biofilm is in a stationary growth phase. The results of this study have implications for algal biofilm photobioreactor design and operation.     

A Role for USP7 in DNA Replication

The minichromosome maintenance (MCM) complex, which plays multiple important roles in DNA replication, is loaded onto chromatin following mitosis, remains on chromatin until the completion of DNA synthesis, and then is unloaded by a poorly defined mechanism that involves the MCM binding protein (MCM-BP). Here we show that MCM-BP directly interacts with the ubiquitin-specific protease USP7, that this interaction occurs predominantly on chromatin, and that MCM-BP can tether USP7 to MCM proteins. Detailed biochemical and structure analyses of the USP7–MCM-BP interaction showed that the ¹⁵⁵PSTS¹⁵⁸ MCM-BP sequence mediates critical interactions with the TRAF domain binding pocket of USP7. Analysis of the effects of USP7 knockout on DNA replication revealed that lack of USP7 results in slowed progression through late S phase without globally affecting the fork rate or origin usage. Lack of USP7 also resulted in increased levels of MCM proteins on chromatin, and investigation of the cause of this increase revealed a defect in the dissociation of MCM proteins from chromatin in mid- to late S phase. This role of USP7 mirrors the previously described role for MCM-BP in MCM complex unloading and suggests that USP7 works with MCM-BP to unload MCM complexes from chromatin at the end of S phase.     

Fanconi anemia signaling and Mus81 cooperate to safeguard development and crosslink repair

Individuals with Fanconi anemia (FA) are susceptible to bone marrow failure, congenital abnormalities, cancer predisposition and exhibit defective DNA crosslink repair. The relationship of this repair defect to disease traits remains unclear, given that crosslink sensitivity is recapitulated in FA mouse models without most of the other disease-related features. Mice deficient in Mus81 are also defective in crosslink repair, yet MUS81 mutations have not been linked to FA. Using mice deficient in both Mus81 and the FA pathway protein FancC, we show both proteins cooperate in parallel pathways, as concomitant loss of FancC and Mus81 triggered cell-type-specific proliferation arrest, apoptosis and DNA damage accumulation in utero. Mice deficient in both FancC and Mus81 that survived to birth exhibited growth defects and an increased incidence of congenital abnormalities. This cooperativity of FancC and Mus81 in developmental outcome was also mirrored in response to crosslink damage and chromosomal integrity. Thus, our findings reveal that both pathways safeguard against DNA damage from exceeding a critical threshold that triggers proliferation arrest and apoptosis, leading to compromised in utero development.     

Genetic risk score analysis indicates migraine with and without comorbid depression are genetically different disorders

Migraine and major depressive disorder (MDD) are comorbid, moderately heritable and to some extent influenced by the same genes. In a previous paper, we suggested the possibility of causality (one trait causing the other) underlying this comorbidity. We present a new application of polygenic (genetic risk) score analysis to investigate the mechanisms underlying the genetic overlap of migraine and MDD. Genetic risk scores were constructed based on data from two discovery samples in which genome-wide association analyses (GWA) were performed for migraine and MDD, respectively. The Australian Twin Migraine GWA study (N = 6,350) included 2,825 migraine cases and 3,525 controls, 805 of whom met the diagnostic criteria for MDD. The RADIANT GWA study (N = 3,230) included 1,636 MDD cases and 1,594 controls. Genetic risk scores for migraine and for MDD were used to predict pure and comorbid forms of migraine and MDD in an independent Dutch target sample (NTR–NESDA, N = 2,966), which included 1,476 MDD cases and 1,058 migraine cases (723 of these individuals had both disorders concurrently). The observed patterns of prediction suggest that the ‘pure’ forms of migraine and MDD are genetically distinct disorders. The subgroup of individuals with comorbid MDD and migraine were genetically most similar to MDD patients. These results indicate that in at least a subset of migraine patients with MDD, migraine may be a symptom or consequence of MDD.     

Protective Efficacy and Safety of Three Antimalarial Regimens for the Prevention of Malaria in Young Ugandan Children: A Randomized Controlled Trial

Background

Chemoprevention offers a promising strategy for prevention of malaria in African children. However, the optimal chemoprevention drug and dosing strategy is unclear in areas of year-round transmission and resistance to many antimalarial drugs. To compare three available regimens, we conducted an open-label randomized controlled trial of chemoprevention in Ugandan children.

Methods and Findings

This study was conducted between June 28, 2010, and September 25, 2013. 400 infants were enrolled and 393 randomized at 6 mo of age to no chemoprevention, monthly sulfadoxine-pyrimethamine (SP), daily trimethoprim-sulfamethoxazole (TS), or monthly dihydroartemisinin-piperaquine (DP). Study drugs were administered at home without supervision. Piperaquine (PQ) levels were used as a measure of compliance in the DP arm. Participants were given insecticide-treated bednets, and caregivers were encouraged to bring their child to a study clinic whenever they were ill. Chemoprevention was stopped at 24 mo of age, and participants followed-up an additional year. Primary outcome was the incidence of malaria during the intervention period. During the intervention, the incidence of malaria in the no chemoprevention arm was 6.95 episodes per person-year at risk. Protective efficacy was 58% (95% CI, 45%–67%, p<0.001) for DP, 28% (95% CI, 7%–44%, p = 0.01) for TS, and 7% for SP (95% CI, −19% to 28%, p = 0.57). PQ levels were below the detection limit 52% of the time when malaria was diagnosed in the DP arm, suggesting non-adherence. There were no differences between the study arms in the incidence of serious adverse events during the intervention and the incidence of malaria during the 1-y period after the intervention was stopped.

Conclusions

For preventing malaria in children living in an area of high transmission intensity, monthly DP was the most efficacious and safe, although adherence may pose a problem. Monthly SP and daily TS may not be appropriate in areas with high transmission intensity and frequent resistance to antifolates.

Trial registration

www.ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00948896","term_id":"NCT00948896"}}NCT00948896 Please see later in the article for the Editors'' Summary