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Cryo-electron microscopy (cryo-EM) has the capacity to capture molecular machines in action 1 – 3 . ATP-binding cassette (ABC) exporters are highly dynamic membrane proteins that extrude a wide range of substances from the cytosol 4 – 6 and thereby contribute to essential cellular processes, adaptive immunity and multidrug resistance 7 , 8 . Despite their importance, the coupling of nucleotide binding, hydrolysis and release to the conformational dynamics of these proteins remains poorly resolved, especially for heterodimeric and/or asymmetric ABC exporters that are abundant in humans. Here we present eight high-resolution cryo-EM structures that delineate the full functional cycle of an asymmetric ABC exporter in a lipid environment. Cryo-EM analysis under active turnover conditions reveals distinct inward-facing (IF) conformations—one of them with a bound peptide substrate—and previously undescribed asymmetric post-hydrolysis states with dimerized nucleotide-binding domains and a closed extracellular gate. By decreasing the rate of ATP hydrolysis, we could capture an outward-facing (OF) open conformation—an otherwise transient state vulnerable to substrate re-entry. The ATP-bound pre-hydrolysis and vanadate-trapped states are conformationally equivalent; both comprise co-existing OF conformations with open and closed extracellular gates. By contrast, the post-hydrolysis states from the turnover experiment exhibit asymmetric ATP and ADP occlusion after phosphate release from the canonical site and display a progressive separation of the nucleotide-binding domains and unlocking of the intracellular gate. Our findings reveal that phosphate release, not ATP hydrolysis, triggers the return of the exporter to the IF conformation. By mapping the conformational landscape during active turnover, aided by mutational and chemical modulation of kinetic rates to trap the key intermediates, we resolved fundamental steps of the substrate translocation cycle of asymmetric ABC transporters. Eight cryo-electron microscopy structures of the ATP-binding cassette exporter TmrAB under turnover conditions characterize the entire transport cycle in a lipid environment.

ATP-binding cassette (ABC) transporters constitute the largest family of primary active transporters, responsible for many physiological processes and human maladies. However, the mechanism how chemical energy of ATP facilitates translocation of chemically diverse compounds across membranes is poorly understood. Here, we advance the quantitative mechanistic understanding of the heterodimeric ABC transporter TmrAB, a functional homolog of the transporter associated with antigen processing (TAP) by single-turnover analyses at single-liposome resolution. We reveal that a single conformational switch by ATP binding drives unidirectional substrate translocation. After this power stroke, ATP hydrolysis and phosphate release launch the return to the resting state, which facilitates nucleotide exchange and a new round of substrate binding and translocation. In contrast to hitherto existing steady-state assays, our single-turnover approach uncovers the power stroke in substrate translocation and the tight chemomechanical coupling in these molecular machines.

ATP-binding cassette (ABC) transporters constitute the largest family of primary active transporters involved in a multitude of physiological processes and human diseases. Despite considerable efforts, it remains unclear how ABC transporters harness the chemical energy of ATP to drive substrate transport across cell membranes. Here, by random nonstandard peptide integrated discovery (RaPID), we leveraged combinatorial macrocyclic peptides that target a heterodimeric ABC transport complex and explore fundamental principles of the substrate translocation cycle. High-affinity peptidic macrocycles bind conformationally selective and display potent multimode inhibitory effects. The macrocycles block the transporter either before or after unidirectional substrate export along a single conformational switch induced by ATP binding. Our study reveals mechanistic principles of ATP binding, conformational switching, and energy transduction for substrate transport of ABC export systems. We highlight the potential of de novo macrocycles as effective inhibitors for membrane proteins implicated in multidrug resistance, providing avenues for the next generation of pharmaceuticals.

A distressing consequence of the HIV/AIDS pandemic and of the increasing numbers of orphans and decreasing numbers of caregivers is the emergence in ever larger numbers of child-headed households (CHHs). The complexity of issues affecting CHHs and the lack of research on this subject means that CHHs are not well understood. This sometimes prompts support agencies to provide emotionally driven recommendations suggesting that it is better for a child to be in an orphanage than to live in a CHH. This exploratory study, involving heads of 105 CHHs over a 12 month period and 142 participants in various focus group discussions (FGD) and interviews, suggests the need for a change in perspective. It addresses the question of CHH quality of life, coping strategies and household functioning and attempts to bring this into a productive dialogue with community child care activities, NGO and statutory support and child care and protection policies.Research data suggests that the key determining factor contributing towards the creation of a CHH is `pre-parental illness' family conflict. Another contributing factor is that siblings want to stay together after parental death. Quality of life assessments indicate that despite significant adversities, over 69% of CHHs reported a 'medium' to 'satisfactory' quality of life and demonstrate high levels of resilience. As regards vulnerability to abuse, it is found that while CHH members are more vulnerable to external abuse, they experience little within their household. Contrary to public perceptions about CHHs lacking moral values, CHH behaviour might actually be more responsible than non-CHH peer behaviour as their negative experiences appear to galvanize them into adopting responsible behaviour. Community care and neighbourhood support in older townships are better established compared with newer suburbs. Sufficient community care capacity enables CHHs to function, thus avoiding a situation where households disintegrate and household members end up as street children. CHH coping responses seem to be mainly influenced by individual and community factors, and by social, spiritual and material support. The interplay between these and the CHH's ability to engage in the required coping task impacts on the coping outcome at household level.National and international government and non-governmental child service providers in Southern Africa need to recognize that an adequately supported CHH is an acceptable alternative care arrangement for certain children in communities with high adult AIDS mortality and where adult HIV-prevalence exceeds 10%.

The Unbounded Knapsack Problem (UKP) is a well-known variant of the famous 0-1 Knapsack Problem (0-1 KP). In contrast to 0-1 KP, an arbitrary number of copies of every item can be taken in UKP. Since UKP is NP-hard, fully polynomial time approximation schemes (FPTAS) are of great interest. Such algorithms find a solution arbitrarily close to the optimum \\(OPT(I)\\), i.e. of value at least \\((1-) OPT(I)\\) for \\( > 0\\), and have a running time polynomial in the input length and \\(1\\). For over thirty years, the best FPTAS was due to Lawler with a running time in \\(O(n + 1^3)\\) and a space complexity in \\(O(n + 1^2)\\), where \\(n\\) is the number of knapsack items. We present an improved FPTAS with a running time in \\(O(n + 1^2 ^3 1)\\) and a space bound in \\(O(n + 1 ^2 1)\\). This directly improves the running time of the fastest known approximation schemes for Bin Packing and Strip Packing, which have to approximately solve UKP instances as subproblems.

The purpose of this study was to systematically develop an instrument that would measure computer aversion, computer attitudes, and computer familiarity. The study involved multiple phases: item generation, pilot testing, and factor analysis. The primary hypothesis was that the items generated would yield three distinct factors when factor analyzed. Each subscale (factor) would significantly and positively correlate with one another, as well as with the total score, such that the higher the score on each scale, the greater degree of comfort (less aversion) with computers, the greater degree of positive attitudes towards computers, and the greater degree of experience (familiarity) with computers. Items were generated with the assistance of consultants. The resulting instrument was pilot tested on samples of graduate and undergraduate students and resulted in a 164-item questionnaire that was administered to 391 undergraduate students at a medium-sized Midwestern University. This data was then factor analyzed. A four-factor solution, a principal component extraction with equamax rotation accounting for 32.48% of the variance, was the most readily interpretable. The four factors were named Computer Familiarity (Factor 1), Computer Aversion 1 (experiential computer aversion, Factor 2), Attitudes Towards Computers (Factor 3), and Computer Aversion 2 (cognitive computer aversion, Factor 4). These 4 factors and the Total score were significantly and positively correlated as expected. Internal consistency statistics indicated that the items comprising each of the four factors and the total CAAFI-D score were highly correlated. Overall, the CAAFI Total and factor means suggested that the sample had a positive affinity toward computers, a nonaversiveness toward computers (experiential aversion), and overall positive attitudes towards computers. The sample also had a slightly low familiarity with computers and a relatively neutral reaction (cognitive aversion) toward computers. Post hoc analyses revealed that men scored significantly higher on Factors 1, 2, 4 and the Total score, indicating that they reported less aversion and greater familiarity with computers. Limitations of this study (e.g., sample size, a predominantly Caucasian, female sample) are discussed.

Intraflagellar transport (IFT) of ciliary precursors such as tubulin from the cytoplasm to the ciliary tip is involved in the construction of the cilium, a hairlike organelle found on most eukaryotic cells. However, the molecular mechanisms of IFT are poorly understood. Here, we found that the two core IFT proteins IFT74 and IFT81 form a tubulin-binding module and mapped the interaction to a calponin homology domain of IFT81 and a highly basic domain in IFT74. Knockdown of IFT81 and rescue experiments with point mutants showed that tubulin binding by IFT81 was required for ciliogenesis in human cells.

Although the peripheral nerve is capable of regeneration, only a small minority of patients regain normal function after surgical reconstruction of a major peripheral nerve lesion, resulting in a severe and lasting negative impact on the quality of life. Glial cell-line derived neurotrophic factor (GDNF) has potent survival- and outgrowth-promoting effects on motoneurons, but locally elevated levels of GDNF cause trapping of regenerating axons and the formation of nerve coils. This phenomenon has been called the \"candy store\" effect. In this study we created gradients of GDNF in the sciatic nerve after a ventral root avulsion. This approach also allowed us to study the effect of increasing concentrations of GDNF on Schwann cell proliferation and morphology in the injured peripheral nerve. We demonstrate that lentiviral vectors can be used to create a 4 cm long GDNF gradient in the intact and lesioned rat sciatic nerve. Nerve coils were formed throughout the gradient and the number and size of the nerve coils increased with increasing GDNF levels in the nerve. In the nerve coils, Schwann cell density is increased, their morphology is disrupted and myelination of axons is severely impaired. The total number of regenerated and surviving motoneurons is not enhanced after the distal application of a GDNF gradient, but increased sprouting does result in higher number of motor axon in the distal segment of the sciatic nerve. These results show that lentiviral vector mediated overexpression of GDNF exerts multiple effects on both Schwann cells and axons and that nerve coil formation already occurs at relatively low concentrations of exogenous GDNF. Controlled expression of GDNF, by using a viral vector with regulatable GDNF expression, may be required to avoid motor axon trapping and to prevent the effects on Schwann cell proliferation and myelination.

Haplotype-resolved genome assemblies are important for understanding how combinations of variants impact phenotypes. To date, these assemblies have been best created with complex protocols, such as cultured cells that contain a single-haplotype (haploid) genome, single cells where haplotypes are separated, or co-sequencing of parental genomes in a trio-based approach. These approaches are impractical in most situations. To address this issue, we present FALCON-Phase, a phasing tool that uses ultra-long-range Hi-C chromatin interaction data to extend phase blocks of partially-phased diploid assembles to chromosome or scaffold scale. FALCON-Phase uses the inherent phasing information in Hi-C reads, skipping variant calling, and reduces the computational complexity of phasing. Our method is validated on three benchmark datasets generated as part of the Vertebrate Genomes Project (VGP), including human, cow, and zebra finch, for which high-quality, fully haplotype-resolved assemblies are available using the trio-based approach. FALCON-Phase is accurate without having parental data and performance is better in samples with higher heterozygosity. For cow and zebra finch the accuracy is 97% compared to 80–91% for human. FALCON-Phase is applicable to any draft assembly that contains long primary contigs and phased associate contigs. Methods to produce haplotype-resolved genome assemblies often rely on access to family trios. The authors present FALCON-Phase, a tool that combines ultra-long range Hi-C chromatin interaction data with a long read de novo assembly to extend haplotype phasing to the contig or scaffold level.

Reperfusion strategies with fibrinolysis or primary PCI were compared in this trial involving patients with ST-segment elevation myocardial infarction (STEMI). The two methods had similar efficacy, but there was more intracranial bleeding in the fibrinolysis group. Although contemporary guidelines for patients with acute ST-segment elevation myocardial infarction (STEMI) recommend primary percutaneous coronary intervention (PCI) as the preferred reperfusion strategy, this approach is contingent on performing PCI in a timely fashion. 1 , 2 Since most patients do not present to a PCI-capable hospital, this factor presents a major logistic challenge in many regions. 3 Despite substantial effort directed toward addressing this issue, the large majority of patients with STEMI who present to non-PCI facilities do not subsequently receive primary PCI within guideline-recommended times. 4 This delay results in a commensurate increase in morbidity and mortality. 5 , 6 A second major therapeutic . . .