Analysis of deoxycytidine accumulation in gemcitabine treated patients

Deoxycytidine (CdR) analogs are increasingly popular as chemotherapeutic agents and their effectiveness can be linked to the direct competition with active forms of endogenous CdR. A tandem mass spectrometric assay was developed to determine the plasma concentrations of CdR. Plasma extracts were prepared by protein precipitation and an ethyl acetate/water back extraction, and then separated chromatographically. Detection parameters were optimized for multi-reaction monitoring (MRM) tandem mass spectrometry and assay efficiency was improved using 15N3 CdR as an isotopic internal standard. Preliminary results from a gemcitabine trial are shown which indicate that CdR concentrations increase systemically during infusion, from about 5 nM to 78 nM after hepatic artery infusion and to 102 nM after systemic infusion for 24 hours. The developed assay demonstrated good sensitivity and selectivity for CdR.     

Data-adaptive longitudinal model selection in causal inference with collaborative targeted minimum loss-based estimation

Causal inference methods have been developed for longitudinal observational study designs where confounding is thought to occur over time. In particular, one may estimate and contrast the population mean counterfactual outcome under specific exposure patterns. In such contexts, confounders of the longitudinal treatment-outcome association are generally identified using domain-specific knowledge. However, this may leave an analyst with a large set of potential confounders that may hinder estimation. Previous approaches to data-adaptive model selection for this type of causal parameter were limited to the single time-point setting. We develop a longitudinal extension of a collaborative targeted minimum loss-based estimation (C-TMLE) algorithm that can be applied to perform variable selection in the models for the probability of treatment with the goal of improving the estimation of the population mean counterfactual outcome under a fixed exposure pattern. We investigate the properties of this method through a simulation study, comparing it to G-Computation and inverse probability of treatment weighting. We then apply the method in a real-data example to evaluate the safety of trimester-specific exposure to inhaled corticosteroids during pregnancy in women with mild asthma. The data for this study were obtained from the linkage of electronic health databases in the province of Quebec, Canada. The C-TMLE covariate selection approach allowed for a reduction of the set of potential confounders, which included baseline and longitudinal variables.     

Distinct Forms of Mitochondrial TOM-TIM Supercomplexes Define Signal-Dependent States of Preprotein Sorting

Mitochondrial import of cleavable preproteins occurs at translocation contact sites, where the translocase of the outer membrane (TOM) associates with the presequence translocase of the inner membrane (TIM23) in a supercomplex. Different views exist on the mechanism of how TIM23 mediates preprotein sorting to either the matrix or inner membrane. On the one hand, two TIM23 forms were proposed, a matrix transport form containing the presequence translocase-associated motor (PAM; TIM23-PAM) and a sorting form containing Tim21 (TIM23^SORT). On the other hand, it was reported that TIM23 and PAM are permanently associated in a single-entity translocase. We have accumulated distinct transport intermediates of preproteins to analyze the translocases in their active, preprotein-carrying state. We identified two different forms of active TOM-TIM23 supercomplexes, TOM-TIM23^SORT and TOM-TIM23-PAM. These two supercomplexes do not represent separate pathways but are in dynamic exchange during preprotein translocation and sorting. Depending on the signals of the preproteins, switches between the different forms of supercomplex and TIM23 are required for the completion of preprotein import.The majority of mitochondrial proteins are nuclear encoded and posttranslationally transported into the organelle. A major class of mitochondrial proteins possess cleavable targeting signals at their amino termini, so-called presequences (5, 9, 12, 19, 30, 32). These α-helical segments are positively charged and direct the proteins across the outer and inner mitochondrial membranes toward the matrix space, where the presequences are proteolytically removed. However, a number of proteins of the inner mitochondrial membrane, among them subunits of the respiratory chain complexes, also utilize presequences as targeting signals. In addition to the presequence, they contain a hydrophobic sorting signal, which arrests precursor translocation across the inner membrane and mediates the lateral release of the polypeptide into the lipid phase (16, 30). In some cases, the membrane-inserted precursors undergo a second processing event by the inner membrane protease that cleaves behind the sorting signal and therefore leads to the release of the protein into the intermembrane space (25, 30, 31). Thus, a large variety of proteins destined for three different intramitochondrial compartments use presequences as the primary signal for transport.Cleavable preproteins initially enter mitochondria via the TOM complex and are translocated into or across the inner membrane by the TIM23 complex. The TIM23 complex consists of four integral membrane proteins, Tim23, Tim17, Tim50, and Tim21. Tim23 forms the protein-conducting channel of the translocase and is tightly associated with Tim17 (8, 26, 43). Tim50 acts as a regulator for the Tim23 channel and is involved in early steps of precursor transfer from the outer to the inner membranes (23, 29, 41). Tim21 transiently interacts with the TOM complex via binding to the intermembrane space domain of Tom22. This interaction promotes the release of presequences from Tom22 for their further transfer to the Tim23 channel (4). For full matrix translocation of preproteins, the TIM23 complex cooperates with PAM. The central subunit of PAM is mtHsp70, which undergoes ATP-dependent cycles of preprotein binding and release to promote polypeptide movement toward the matrix. The activity of mtHsp70 in the translocation process is regulated by four membrane-bound cochaperones, Tim44, the J complex Pam18/Pam16 (Tim14/Tim16), and Pam17. Tim44 provides a binding site for preproteins and mtHsp70 close to the Tim23 channel (1, 17, 22, 36). The J protein Pam18 stimulates the ATPase activity of mtHsp70 (10, 44), whereas the J-related protein Pam16 controls the activity of Pam18 (11, 13, 20). Pam17 plays an organizing role in the TIM23-PAM cooperation (33, 45).The following two different views on the organization of the presequence transport machinery are currently discussed. (i) The TIM23 complex and PAM were proposed to exist in different modular states, termed TIM23^SORT and TIM23-PAM. The TIM23^CORE complex, consisting of Tim23, Tim17 and Tim50, associates with either Tim21 or the subunits of PAM (4, 47, 51). The Tim21-containing form is termed TIM23^SORT since this motor-free form was isolated and shown to mediate membrane insertion of sorted preproteins upon reconstitution (46). The TIM23-PAM form (lacking Tim21) is crucial for mtHsp70-driven preprotein translocation into the matrix (4). (ii) On the other hand, it was proposed that presequence translocase and import motor form a single structural and functional entity. Thus, membrane-integrated TIM23 and import motor would always remain in one complex. This model implies that a motor-free form of the TIM23 complex should not exist (27, 33, 42).To decide between the different views, it is necessary to analyze translocase and motor in their active form, i.e., during their engagement with preproteins. Moreover, the model of modular forms of TIM23 and PAM raises the question whether two strictly separate TIM23 pathways for inner membrane sorting and matrix translocation exist or whether an exchange between the different forms of the presequence translocase occurs. To date, the majority of experimental studies have been performed with the translocases in an inactive, i.e., preprotein-free, state. Studies using preproteins in transit provided only limited information so far and thus did not resolve the controversy, as follows. (i) Mokranjac and Neupert (27) questioned if the in vitro preprotein insertion by purified TIM23^SORT in a proteoliposome assay (46) reflected the in organello situation in intact mitochondria. (ii) Popov-Celeketic et al. (33) accumulated a matrix-targeted preprotein in mitochondrial import sites in vivo and performed pulldown experiments. They copurified TIM23, PAM, and Tim21 and thus concluded that the TIM23 and motor subunits formed a single entity. They did not address the possibility that the accumulated preprotein was associated with different pools of translocase complexes. (iii) Wiedemann et al. (51) made use of the observation that TIM23^SORT associates with the respiratory chain (47). They reported a copurification of inner membrane-sorted preproteins and matrix-targeted preproteins with respiratory chain complexes. This observation raised the possibility that the pathways for inner membrane sorting and matrix translocation are connected at least at the level of respiratory chain interaction; however, the composition of the TIM23 complexes was not analyzed.For this study, we used preproteins with variations in the intramitochondrial sorting signal to monitor the active, preprotein-carrying translocases at distinct stages of mitochondrial import. We observed different forms of active translocases on the presequence pathway. The sorting signals of the preproteins are critical for the selection of specific translocase forms. The motor and sorting forms of the TIM23 complex can be isolated as separate entities in support of the modular model. However, the different TIM23 forms are not permanently separated during preprotein import, but a dynamic exchange between the forms takes place for both matrix-targeted preproteins and inner membrane-sorted preproteins.     

Improving the performance of ecological indices by balancing reference site quality and representativeness

Reference site networks should consist of minimally disturbed sites that collectively characterize the ranges of natural settings within a region. Compromise between reference-quality and representativeness is required. We evaluated how tradeoffs between reference-quality and regional representativeness affected applicability, performance, and interpretation of multi-metric (MMI) and Observed/Expected (O/E) indices developed for streams in eastern China. We emphasized reference-quality by applying the most-stringent objective criteria and expert-judgment to select reference-group1 (G1). We emphasized representativeness by applying the least-stringent criteria to select reference-group2 (G2) sites from different strata based on watershed size. We balanced reference-quality and representativeness in G3 by applying intermediate stringent criteria from each watershed size stratum used previously. Increasing representativeness using G2 improved index applicability to almost more than twice the number of test sites than when reference-quality maximized using G1. Bias in O/E index was almost eliminated only when reference-quality and representativeness balanced using G3. MMIs developed when reference-quality maximized using G1 eliminated all bias and had the highest precision. High-quality reference with limited representativeness affected the metrics selected for inclusion in MMIs and restricted the sites to which both types of indices could be applied. A balanced approach worked best in this instance and similar approaches should be tested in other regions.

     

DNA joint dependence of pol X family polymerase action in nonhomologous end joining

DNA double strand breaks (DSBs) can be rejoined directly by the nonhomologous end-joining (NHEJ) pathway of repair. Nucleases and polymerases are required to promote accurate NHEJ when the terminal bases of the DSB are damaged. The same enzymes also participate in imprecise rejoining and joining of incompatible ends, important mutagenic events. Previous work has shown that the Pol X family polymerase Pol4 is required for some but not all NHEJ events that require gap filling in Saccharomyces cerevisiae. Here, we systematically analyzed DSB end configurations and found that gaps on both strands and overhang polarity are the principal factors that determine whether a joint requires Pol4. DSBs with 3'-overhangs and a gap on each strand strongly depended on Pol4 for repair, DSBs with 5'-overhangs of the same sequence did not. Pol4 was not required when 3'-overhangs contained a gap on only one strand, however. Pol4 was equally required at 3'-overhangs of all lengths within the NHEJ-dependent range but was dispensable outside of this range, indicating that Pol4 is specific to NHEJ. Loss of Pol4 did not affect the rejoining of DSBs that utilized a recessed microhomology or DSBs bearing 5'-hydroxyls but no gap. Finally, mammalian Pol X polymerases were able to differentially complement a pol4 mutation depending on the joint structure, demonstrating that these polymerases can participate in yeast NHEJ but with distinct properties.     

Sorting switch of mitochondrial presequence translocase involves coupling of motor module to respiratory chain

The mitochondrial presequence translocase transports preproteins to either matrix or inner membrane. Two different translocase forms have been identified: the matrix transport form, which binds the heat-shock protein 70 (Hsp70) motor, and the inner membrane–sorting form, which lacks the motor but contains translocase of inner mitochondrial membrane 21 (Tim21). The sorting form interacts with the respiratory chain in a Tim21-dependent manner. It is unknown whether the respiratory chain–bound translocase transports preproteins and how the switch between sorting form and motor form occurs. We report that the respiratory chain–bound translocase contains preproteins in transit and, surprisingly, not only sorted but also matrix-targeted preproteins. Presequence translocase-associated motor (Pam) 16 and 18, two regulatory components of the six-subunit motor, interact with the respiratory chain independently of Tim21. Thus, the respiratory chain–bound presequence translocase is not only active in preprotein sorting to the inner membrane but also in an early stage of matrix translocation. The motor does not assemble en bloc with the translocase but apparently in a step-wise manner with the Pam16/18 module before the Hsp70 core.     

Population intervention models in causal inference

We propose a new causal parameter, which is a natural extension of existing approaches to causal inference such as marginal structural models. Modelling approaches are proposed for the difference between a treatment-specific counterfactual population distribution and the actual population distribution of an outcome in the target population of interest. Relevant parameters describe the effect of a hypothetical intervention on such a population and therefore we refer to these models as population intervention models. We focus on intervention models estimating the effect of an intervention in terms of a difference and ratio of means, called risk difference and relative risk if the outcome is binary. We provide a class of inverse-probability-of-treatment-weighted and doubly-robust estimators of the causal parameters in these models. The finite-sample performance of these new estimators is explored in a simulation study.     

Targeted maximum likelihood estimation for dynamic treatment regimes in sequentially randomized controlled trials

Sequential Randomized Controlled Trials (SRCTs) are rapidly becoming essential tools in the search for optimized treatment regimes in ongoing treatment settings. Analyzing data for multiple time-point treatments with a view toward optimal treatment regimes is of interest in many types of afflictions: HIV infection, Attention Deficit Hyperactivity Disorder in children, leukemia, prostate cancer, renal failure, and many others. Methods for analyzing data from SRCTs exist but they are either inefficient or suffer from the drawbacks of estimating equation methodology. We describe an estimation procedure, targeted maximum likelihood estimation (TMLE), which has been fully developed and implemented in point treatment settings, including time to event outcomes, binary outcomes and continuous outcomes. Here we develop and implement TMLE in the SRCT setting. As in the former settings, the TMLE procedure is targeted toward a pre-specified parameter of the distribution of the observed data, and thereby achieves important bias reduction in estimation of that parameter. As with the so-called Augmented Inverse Probability of Censoring Weight (A-IPCW) estimator, TMLE is double-robust and locally efficient. We report simulation results corresponding to two data-generating distributions from a longitudinal data structure.     

Membrane interaction of the glycosyltransferase MurG: a special role for cardiolipin

MurG is a peripheral membrane protein that is one of the key enzymes in peptidoglycan biosynthesis. The crystal structure of Escherichia coli MurG (S. Ha, D. Walker, Y. Shi, and S. Walker, Protein Sci. 9:1045-1052, 2000) contains a hydrophobic patch surrounded by basic residues that may represent a membrane association site. To allow investigation of the membrane interaction of MurG on a molecular level, we expressed and purified MurG from E. coli in the absence of detergent. Surprisingly, we found that lipid vesicles copurify with MurG. Freeze fracture electron microscopy of whole cells and lysates suggested that these vesicles are derived from vesicular intracellular membranes that are formed during overexpression. This is the first study which shows that overexpression of a peripheral membrane protein results in formation of additional membranes within the cell. The cardiolipin content of cells overexpressing MurG was increased from 1 +/- 1 to 7 +/- 1 mol% compared to nonoverexpressing cells. The lipids that copurify with MurG were even further enriched in cardiolipin (13 +/- 4 mol%). MurG activity measurements of lipid I, its natural substrate, incorporated in pure lipid vesicles showed that the MurG activity is higher for vesicles containing cardiolipin than for vesicles with phosphatidylglycerol. These findings support the suggestion that MurG interacts with phospholipids of the bacterial membrane. In addition, the results show a special role for cardiolipin in the MurG-membrane interaction.