Studies on Mitochondrial Proteins. II. Localization of Components in the inner membrane labelling with diazobenzenesulfonate,a non-penetrating probe

The topography of the inner membrane of rat liver mitochondria was studied using a probe, diazobenzenesulfonate, which interacts preferentially with surface components. Inner membranes were examined both in a native orientation as found in the intact mitochondrion or in an inverted state as found in isolated inner membranes prepared by sonication.Enzyme inactivation as a consequence of diazobenzenesulfonate labeling was employed to determine the localization of a number of inner membrane activities. In inner membranes labeled on the outer surface, NADH and succinate oxidation were strongly inhibited while ATPase and $\text{ascorbate}\text{-N,N,N′,N′-}\text{tetramethyl}\text{-p-}\text{phenylene-diamine}$ (TMPD) oxidase activities were unaffected. In inner membranes labeled on the inner surface. ATPase and succinate oxidation were inactivated while NADH oxidation and ascorbate-TMPD oxidase were unaffected. Succinate dehydrogenase was inhibited only by labeling the inner surface while NADH dehydrogenase was inhibited to a similar extent by treatment of either surface.Sodium dodecylsulfate-polypeptides (66 000 and 26 000) on the outer surface of the inner membrane and five polypeptides (80 000, 66 000, 51 000-48 000, and 26 000) on the inner surface. These results indicate a highly asymmetric localization of inner membrane components.     

CHLAINOMONAS KOLII(HARDY ET CURL) COMB. NOV. (CHLOROPHYTA,VOLVOCALES), A REVISION OF THE SNOW ALGA,TRACHELOMONAS KOLII HARDY ET CURL (EUGLENOPHYTA,EUGLENALES)1,2

The snow alga Trachelomonas kolii is transferred from the Euglenophyta (Euglenales) to the Chlorophyta (Volvocales) as Chlainomonas kolii comb. nov. As a result of critical examination of both living and type material, this species was found to have 4 flagella per vegetative cell, true starch, and 1 axial plastid per cell with several peripheral lobes. Vegetative cells of Trachelomonas kolii were described originally as having 1 flagellum, lacking true starch but having paramylum, and as having several parietal plastids per cell. The reticulate markings on the outer envelope of vegetative cells were found to be different from those in the original illustrations. Vegetative cells and resting spores of Chlainomonas kolii and Chlainomonas rubra are compared. The similarities of resting spores of Chlainomonas kolii and Chlamydomonas nivalis are discussed. These are the first records of Chlainomonas kolii from snow in Washington State.     

Long-term survival in patients treated with ruxolitinib for myelofibrosis: COMFORT-I and -II pooled analyses

Background

Myelofibrosis (MF) is associated with a variety of burdensome symptoms and reduced survival compared with age-/sex-matched controls. This analysis evaluated the long-term survival benefit with ruxolitinib, a Janus kinase (JAK)1/JAK2 inhibitor, in patients with intermediate-2 (int-2) or high-risk MF.

Methods

This was an exploratory analysis of 5-year data pooled from the phase 3 COMFORT-I and -II trials. In both trials, patients could cross over to ruxolitinib from the control group (COMFORT-I, placebo; COMFORT-II, best available therapy). All continuing patients in the control groups crossed over to ruxolitinib by the 3-year follow-up. Overall survival (OS; a secondary endpoint in both trials) was evaluated using pooled intent-to-treat data from patients randomized to ruxolitinib or the control groups. OS was also evaluated in subgroups stratified by baseline anemia and transfusion status at week 24.

Results

A total of 528 patients were included in this analysis; 301 were originally randomized to ruxolitinib (COMFORT-I, n?=?155; COMFORT-II, n?=?146) and 227 to control (n?=?154 and n?=?73, respectively). The risk of death was reduced by 30% among patients randomized to ruxolitinib compared with patients in the control group (median OS, 5.3 vs 3.8 years, respectively; hazard ratio [HR], 0.70 [95% CI, 0.54–0.91]; P?=?0.0065). After correcting for crossover using a rank-preserving structural failure time (RPSFT) method, the OS advantage was more pronounced for patients who were originally randomized to ruxolitinib compared with patients who crossed over from control to ruxolitinib (median OS, 5.3 vs 2.3 years; HR [ruxolitinib vs RPSFT], 0.35 [95% CI, 0.23–0.59]). An analysis of OS censoring patients at the time of crossover also demonstrated that ruxolitinib prolonged OS compared with control (median OS, 5.3 vs 2.4 years; HR [ruxolitinib vs censored at crossover], 0.53 [95% CI, 0.36–0.78]; P?=?0.0013). The survival benefit with ruxolitinib was observed irrespective of baseline anemia status or transfusion requirements at week 24.

Conclusions

These findings support ruxolitinib treatment for patients with int-2 or high-risk MF, regardless of anemia or transfusion status. Further analyses will be important for exploring ruxolitinib earlier in the disease course to assess the effect on the natural history of MF.

Trial registration

ClinicalTrials.gov identifiers, NCT00952289 and NCT00934544.

     

Local adaptation of fish consumers alters primary production through changes in algal community composition and diversity

Ecological research has focused on understanding how changes in consumer abundance affects community structure and ecosystem processes. However, there is increasing evidence that evolutionary changes in consumers can also alter community structure and ecosystem processes. Typically, the effects of consumer phenotype on communities and ecosystem processes are measured as net effects that integrate numerous ecological pathways. Here, we analyze new data from experimental manipulations of Trinidadian guppy Poecilia reticulata presence, density and phenotype to examine how effects on the algal community cause changes in gross‐primary production (GPP). We combine analytical tools borrowed from path analysis with experimental exclosures in mesocosms to separate the ecological and evolutionary effects of guppies into direct and indirect components. We show that the evolutionary effects of guppy phenotype act through different ecological pathways than the effects of guppy presence and density on GPP. As reported in previous studies that used a different measure of algal biomass, adding guppies and doubling their densities decreased algal biovolume through direct effects. In contrast to these previously reported results, exchanging guppy phenotypes that live without predators for phenotypes that live with predators did not affect algal biovolume. Instead, guppies from populations that live with predators increased the diversity of algal species and increased GPP compared to guppies that live without predators. These changes in the algal community were driven primarily by guppy phenotypes that live with predators—algal communities in mesocosms without fish were similar to those with guppies from predator‐free locations, but both were different from mesocosms with guppies from populations that live with predators. Changes in the algal community were driven directly by differences in foraging behavior between the two consumer phenotypes. We reconcile these results with our previous findings, thereby enhancing our understanding of the relationship between ecological and evolutionary processes.     

Determinants of Beat-to-Beat Variability of Repolarization Duration in the Canine Ventricular Myocyte: A Computational Analysis

Beat-to-beat variability of repolarization duration (BVR) is an intrinsic characteristic of cardiac function and a better marker of proarrhythmia than repolarization prolongation alone. The ionic mechanisms underlying baseline BVR in physiological conditions, its rate dependence, and the factors contributing to increased BVR in pathologies remain incompletely understood. Here, we employed computer modeling to provide novel insights into the subcellular mechanisms of BVR under physiological conditions and during simulated drug-induced repolarization prolongation, mimicking long-QT syndromes type 1, 2, and 3. We developed stochastic implementations of 13 major ionic currents and fluxes in a model of canine ventricular-myocyte electrophysiology. Combined stochastic gating of these components resulted in short- and long-term variability, consistent with experimental data from isolated canine ventricular myocytes. The model indicated that the magnitude of stochastic fluctuations is rate dependent due to the rate dependence of action-potential (AP) duration (APD). This process (the “active” component) and the intrinsic nonlinear relationship between membrane current and APD (“intrinsic component”) contribute to the rate dependence of BVR. We identified a major role in physiological BVR for stochastic gating of the persistent Na⁺ current (I_Na) and rapidly activating delayed-rectifier K⁺ current (I_Kr). Inhibition of I_Kr or augmentation of I_Na significantly increased BVR, whereas subsequent β-adrenergic receptor stimulation reduced it, similar to experimental findings in isolated myocytes. In contrast, β-adrenergic stimulation increased BVR in simulated long-QT syndrome type 1. In addition to stochastic channel gating, AP morphology, APD, and beat-to-beat variations in Ca²⁺ were found to modulate single-cell BVR. Cell-to-cell coupling decreased BVR and this was more pronounced when a model cell with increased BVR was coupled to a model cell with normal BVR. In conclusion, our results provide new insights into the ionic mechanisms underlying BVR and suggest that BVR reflects multiple potentially proarrhythmic parameters, including increased ion-channel stochasticity, prolonged APD, and abnormal Ca²⁺ handling.     

Creating opportunities for science PhDs to pursue careers in high school education

The United States is confronting important challenges at both the early and late stages of science education. At the level of K–12 education, a recent National Research Council report (Successful K–12 STEM Education) proposed a bold restructuring of how science is taught, moving away from memorizing facts and emphasizing hands-on, inquiry-based learning and a deeper understanding of the process of science. At higher levels of training, limited funding for science is leading PhDs to seek training and careers in areas other than research. Might science PhDs play a bigger role in the future of K–12 education, particularly at the high school level? We explore this question by discussing the roles that PhDs can play in high school education and the current and rather extensive barriers to PhDs entering the teaching profession and finally suggest ways to ease the entrance of qualified PhDs into high school education.In many K–12 classrooms, science is presented as a series of textbook facts; students are not exposed to scientific methods of inquiry and lose interest in science. At the very opposite end of the science training pipeline, life science PhDs and postdocs in the United States are experiencing difficulties in finding university jobs, a situation that will likely persist in the coming decade if research funding fails to grow; we cannot expect all PhD graduates to become principal investigators (PIs) at academic institutions.Might these two problems add up to a solution (or at least a partial solution)? Is there a place for graduates of PhD training programs in teaching K–12 science, particularly at the high school (HS) level (the focus of this article)? We argue that the answer is “yes” and that more PhDs, even if their numbers are small compared with the entire teaching pool, could have a catalytic effect on reinvigorating precollege science education. This topic is not new; the National Research Council (NRC) issued two thoughtful reports on attracting science and math PhDs to secondary school education more than a decade ago (Committee on Attracting Science and Mathematics Ph.D.s to Secondary School Teaching, National Research Council, 2000 ; Committee on Attracting Science and Mathematics PhDs to K-12 Education: From Analysis to Implementation, Division of Policy and Global Affairs, National Research Council, 2002 ). Their recommendations were not implemented, however, and the reports have largely been forgotten. Little has changed since then; the roadblocks, both in perception and logistics, that discouraged a PhD from becoming a HS teacher in the year 2000 still exist. Since the NRC reports were released, the topic of a HS teaching career option for a PhD has rarely been discussed or debated in our scientific community. We feel that it is time to reopen this discussion. The focus of this article is on PhDs entering the high school system, but much of this discussion also pertains to graduates of science master degree programs and to individuals with scientific training becoming involved in all levels of K–12 education. Our goal is to make students, postdocs, and senior scientists aware of the value of high school teaching for certain individuals as well as for our nation''s educational system. We also consider how changes at the local level (including the perception of K–12 teaching within research universities), as well as at the policy level of teacher accreditation, might facilitate this career path.     

Pregnancy increases myometrial artery myogenic tone via NOS- or COX-independent mechanisms

Myogenic tone (MT) is a primary modulator of blood flow in the resistance vasculature of the brain, kidney, skeletal muscle, and perhaps in other high-flow organs such as the pregnant uterus. MT is known to be regulated by endothelium-derived factors, including products of the nitric oxide synthase (NOS) and/or the cyclooxygenase (COX) pathways. We asked whether pregnancy influenced MT in myometrial arteries (MA), and if so, whether such an effect could be attributed to alterations in NOS and/or COX. MA (200-300 μm internal diameter, 2-3 mm length) were isolated from 10 nonpregnant and 12 pregnant women undergoing elective hysterectomy or cesarean section, respectively. In the absence of NOS and/or COX inhibition, pregnancy was associated with increased MT in endothelium-intact MA compared with MA from nonpregnant women (P < 0.01). The increase in MT was not due to increased Ca(2+) entry via voltage-dependent channels since both groups of MA exhibited similar levels of constriction when exposed to 50 mM KCl. NOS inhibition (N(ω)-nitro-l-arginine methyl ester, l-NAME) or combined NOS/COX inhibition (l-NAME/indomethacin) increased MT in MA from pregnant women (P = 0.001 and P = 0.042, respectively) but was without effect in arteries from nonpregnant women. Indomethacin alone was without effect on MT in MA from either nonpregnant or pregnant women. We concluded that MT increases in MA during human pregnancy and that this effect was partially opposed by enhanced NOS activity.