Metabolic consequences of NDUFS4 gene deletion in immortalized mouse embryonic fibroblasts

Human mitochondrial complex I (CI) deficiency is associated with progressive neurological disorders. To better understand the CI pathomechanism, we here studied how deletion of the CI gene NDUFS4 affects cell metabolism. To this end we compared immortalized mouse embryonic fibroblasts (MEFs) derived from wildtype (wt) and whole-body NDUFS4 knockout (KO) mice. Mitochondria from KO cells lacked the NDUFS4 protein and mitoplasts displayed virtually no CI activity, moderately reduced CII, CIII and CIV activities and normal citrate synthase and CV (F(o)F(1)-ATPase) activity. Native electrophoresis of KO cell mitochondrial fractions revealed two distinct CI subcomplexes of ~830kDa (enzymatically inactive) and ~200kDa (active). The level of fully-assembled CII-CV was not affected by NDUFS4 gene deletion. KO cells exhibited a moderately reduced maximal and routine O(2) consumption, which was fully inhibited by acute application of the CI inhibitor rotenone. The aberrant CI assembly and reduced O(2) consumption in KO cells were fully normalized by NDUFS4 gene complementation. Cellular [NAD(+)]/[NADH] ratio, lactate production and mitochondrial tetramethyl rhodamine methyl ester (TMRM) accumulation were slightly increased in KO cells. In contrast, NDUFS4 gene deletion did not detectably alter [NADP(+)]/[NADPH] ratio, cellular glucose consumption, the protein levels of hexokinases (I and II) and phosphorylated pyruvate dehydrogenase (P-PDH), total cellular adenosine triphosphate (ATP) level, free cytosolic [ATP], cell growth rate, and reactive oxygen species (ROS) levels. We conclude that the NDUFS4 subunit is of key importance in CI stabilization and that, due to the metabolic properties of the immortalized MEFs, NDUFS4 gene deletion has only modest effects at the live cell level. This article is part of a special issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Longitudinal qPCR Study of the Dynamics of L. crispatus,L. iners,A. vaginae, (Sialidase Positive) G. vaginalis,and P. bivia in the Vagina

Background

To obtain more detailed understanding of the causes of disturbance of the vaginal microflora (VMF), a longitudinal study was carried out for 17 women during two menstrual cycles.

Methods

Vaginal swabs were obtained daily from 17 non-pregnant, menarchal volunteers. For each woman, Gram stains were scored, the quantitative changes of 5 key vaginal species, i.e. Atopobium vaginae, Lactobacillus crispatus, L. iners, (sialidase positive) Gardnerella vaginalis and Prevotella bivia were quantified with qPCR and hydrogen-peroxide production was assessed on TMB+ agar.

Results

Women could be divided in 9 subjects with predominantly normal VMF (grades Ia, Ib and Iab, group N) and 8 with predominantly disturbed VMF (grades I-like, II, III and IV, group D).VMF was variable between women, but overall stable for most of the women. Menses were the strongest disturbing factor of the VMF. L. crispatus was present at log7–9 cells/ml in grade Ia, Iab and II VMF, but concentrations declined 100-fold during menses. L. crispatus below log7 cells/ml corresponded with poor H₂O₂-production. L. iners was present at log 10 cells/ml in grade Ib, II and III VMF. Sialidase negative G. vaginalis strains (average log5 cells/ml) were detected in grade I, I-like and IV VMF. In grade II VMF, predominantly a mixture of both sialidase negative and positive G. vaginalis strains (average log9 cells/ml) were present, and predominantly sialidase positive strains in grade III VMF. The presence of A. vaginae (average log9 cells/ml) coincided with grade II and III VMF. P. bivia (log4–8 cells/ml) was mostly present in grade III vaginal microflora. L. iners, G. vaginalis, A. vaginae and P. bivia all increased around menses for group N women, and as such L. iners was considered a member of disturbed VMF.

Conclusions

This qPCR-based study confirms largely the results of previous culture-based, microscopy-based and pyrosequencing-based studies.     

Predicting the effect of a point mutation on a protein fold: the villin and advillin headpieces and their Pro62Ala mutants

Homology modeling of unknown proteins is based on the assumption that highly similar sequences are likely to share the same fold. However, this does not provide any information on the stability of a given fold, which is ultimately determined by the subtle interplay of enthalpic and entropic contributions. Herein it is shown that ab initio atomistic simulations can be used to predict the effect of point mutations on the stability of a protein fold. The calculations indicate that the fold stabilities of two proteins of similar sequence and identical fold, the villin and advillin C-terminal headpiece fragments, are different and that the same P62A point mutation has a dramatic effect on the fold of villin but a minor one on that of advillin. These predictions were subsequently validated by NMR and CD experiments.     

Poor reporting of scientific leadership information in clinical trial registers

Background

In September 2004, the International Committee of Medical Journal Editors (ICMJE) issued a Statement requiring that all clinical trials be registered at inception in a public register in order to be considered for publication. The World Health Organization (WHO) and ICMJE have identified 20 items that should be provided before a trial is considered registered, including contact information. Identifying those scientifically responsible for trial conduct increases accountability. The objective is to examine the proportion of registered clinical trials providing valid scientific leadership information.

Methodology/Principal Findings

We reviewed clinical trial entries listing Canadian investigators in the two largest international and public trial registers, the International Standard Randomized Controlled Trial Number (ISRCTN) register, and ClinicalTrials.gov. The main outcome measures were the proportion of clinical trials reporting valid contact information for the trials'' Principal Investigator (PI)/Co-ordinating Investigator/Study Chair/Site PI, and trial e-mail contact address, stratified by funding source, recruiting status, and register. A total of 1388 entries (142 from ISRCTN and 1246 from ClinicalTrials.gov) comprised our sample. We found non-compliance with mandatory registration requirements regarding scientific leadership and trial contact information. Non-industry and partial industry funded trials were significantly more likely to identify the individual responsible for scientific leadership (OR = 259, 95% CI: 95–701) and to provide a contact e-mail address (OR = 9.6, 95% CI: 6.6–14) than were solely industry funded trials.

Conclusions/Significance

Despite the requirements set by WHO and ICMJE, data on scientific leadership and contact e-mail addresses are frequently omitted from clinical trials registered in the two leading public clinical trial registers. To promote accountability and transparency in clinical trials research, public clinical trials registers should ensure adequate monitoring of trial registration to ensure completion of mandatory contact information fields identifying scientific leadership     

Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling

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Modeling heterogeneity for count data: A study of maternal mortality in health facilities in Mozambique

Count data are very common in health services research, and very commonly the basic Poisson regression model has to be extended in several ways to accommodate several sources of heterogeneity: (i) an excess number of zeros relative to a Poisson distribution, (ii) hierarchical structures, and correlated data, (iii) remaining “unexplained” sources of overdispersion. In this paper, we propose hierarchical zero‐inflated and overdispersed models with independent, correlated, and shared random effects for both components of the mixture model. We show that all different extensions of the Poisson model can be based on the concept of mixture models, and that they can be combined to account for all different sources of heterogeneity. Expressions for the first two moments are derived and discussed. The models are applied to data on maternal deaths and related risk factors within health facilities in Mozambique. The final model shows that the maternal mortality rate mainly depends on the geographical location of the health facility, the percentage of women admitted with HIV and the percentage of referrals from the health facility.     

Effect of intraperitoneal administration of granulocyte/macrophage-colony-stimulating factor in rats on omental milky-spot composition and tumoricidal activity in vivo and in vitro

 Milky spots in the greater omentum are small accumulations of leucocytes that consist mainly of macrophages and have recently shown to be a selective dissemination site of intraperitoneal (i. p.) inoculated tumour cells. However, milky-spot macrophages show tumoricidal activity and may, therefore, be an excellent source of effector cells suited for local immunotherapy. In the present study we first examined whether granulocyte/macrophage- colony-stimulating factor (GM-CSF) treatment of isolated milky-spot macrophages affects the cytotoxicity against syngeneic colon carcinoma cells (CC531) in vitro. Secondly, we studied the influence of intraperitoneal GM-CSF administration on the number and antitumour activity of milky-spot and peritoneal macrophages. All studies were performed in Wag/Rij rats in which a syngeneic colon carcinoma cell line (CC531) is available. The results of the in vitro study showed that GM-CSF treatment of the omental macrophages led to an increased cytotoxicity against the tumour cell line. Intraperitoneal administration of 1000 U GM-CSF daily for 7 consecutive days demonstrated both an enhanced antitumour activity of the milky-spot macrophages and an increase in the milky-spot macrophage population. An increase in the proliferative capacity, according to bromodeoxyuridine incorporation, was shown in the milky-spot macrophages. Taking into account both the enhanced macrophage number and their enhanced activity upon i. p. GM-CSF treatment, the milky-spot macrophages may provide a rationale for local intraperitoneal immunotherapy in the prevention of intra-abdominal tumour growth. Received: 11 April 1996 / Accepted: 21 May 1996     

Variable thermal stress tolerance of the reef-associated symbiont-bearing foraminifera Amphistegina linked to differences in symbiont type

Coral Reefs - Adaptation, acclimatization and symbiont diversity are known to regulate thermal tolerance in corals, but the role of these mechanisms remains poorly constrained in other...     

Why female crested tits copulate repeatedly with the same partner: evidence for the mate assessment hypothesis

That repeated copulation with the same partner within a singlefertile period is beneficial to the male is generally accepted,but why it should be adaptive to the female is controversialand clear evidence supporting any hypothesis is lacking. Hunteret al. (1993) presented seven hypotheses explaining repeatedmating from the female perspective. Four of them are consistentwith the occurrence of male refusal to copulate: females mighttrade copulations for (1) immediate and or (2) future materialbenefits, or use mating as a mechanism for (3) mate-guardingand or (4) mate-assessment. To test these hypotheses in a populationof crested tits Parus cristatus, we collected data on variationin female solicitation rate, proportion of male refusal, andextra-pair paternity. We found that (1) female solicitationrate was independent of male condition, (2) the proportion ofmale refusal was higher in poor-condition males and (3) femalespaired to poor-condition males sought extra pair paternity.These findings agree with predictions stemming from the mateassessment hypothesis. Therefore, it is suggested that, in crestedtits, male response to female copulation solicitation reflectsmale condition and is used by females to assess male quality