Plus and minus ends of microtubules respond asymmetrically to kinesin binding by a long-range directionally driven allosteric mechanism.

Although it is known that majority of kinesin motors walk predominantly toward the plus end of microtubules (MTs) in a hand-over-hand manner, the structural origin of the stepping directionality is not understood. To resolve this issue, we modeled the structures of kinesin-1 (Kin1), MT, and the Kin1-MT complex using the elastic network model and calculated the residue-dependent responses to a local perturbation in the constructs. Kin1 binding elicits an asymmetric response that is pronounced in α/ß-tubulin dimers in the plus end of the MT. Kin1 opens the clefts of multiple plus end α/ß-tubulin dimers, creating binding-competent conformations, which is required for processivity. Reciprocally, MT induces correlations between switches I and II in the motor and enhances fluctuations in adenosine 5'-diphosphate and the residues in the binding pocket. Our findings explain both the directionality of stepping and MT effects on a key step in the catalytic cycle of kinesin.

Community-based antibiotic access and use in six low-income and middle-income countries: a mixed-method approach.

BACKGROUND: Antimicrobial misuse is common in low-income and middle-income countries (LMICs), and this practice is a driver of antibiotic resistance. We compared community-based antibiotic access and use practices across communities in LMICs to identify contextually specific targets for interventions to improve antibiotic use practices. METHODS: We did quantitative and qualitative assessments of antibiotic access and use in six LMICs across Africa (Mozambique, Ghana, and South Africa) and Asia (Bangladesh, Vietnam, and Thailand) over a 2·5-year study period (July 1, 2016-Dec 31, 2018). We did quantitative assessments of community antibiotic access and use through supplier mapping, customer exit interviews, and household surveys. These quantitative assessments were triangulated with qualitative drug supplier and consumer interviews and discussions. FINDINGS: Vietnam and Bangladesh had the largest proportions of non-licensed antibiotic dispensing points. For mild illness, drug stores were the most common point of contact when seeking antibiotics in most countries, except South Africa and Mozambique, where public facilities were most common. Self-medication with antibiotics was found to be widespread in Vietnam (55·2% of antibiotics dispensed without prescription), Bangladesh (45·7%), and Ghana (36·1%), but less so in Mozambique (8·0%), South Africa (1·2%), and Thailand (3·9%). Self-medication was considered to be less time consuming, cheaper, and overall, more convenient than accessing them through health-care facilities. Factors determining where treatment was sought often involved relevant policies, trust in the supplier and the drug, disease severity, and whether the antibiotic was intended for a child. Confusion regarding how to identify oral antibiotics was revealed in both Africa and Asia. INTERPRETATION: Contextual complexities and differences between countries with different incomes, policy frameworks, and cultural norms were revealed. These contextual differences render a single strategy inadequate and instead necessitate context-tailored, integrated intervention packages to improve antibiotic use in LMICs as part of global efforts to combat antibiotic resistance. FUNDING: Wellcome Trust and Volkswagen Foundation.

Plant part and a steep environmental gradient predict plant microbial composition in a tropical watershed.

Plant microbiomes are shaped by forces working at different spatial scales. Environmental factors determine a pool of potential symbionts while host physiochemical factors influence how those microbes associate with distinct plant tissues. These scales are seldom considered simultaneously, despite their potential to interact. Here, we analyze epiphytic microbes from nine Hibiscus tiliaceus trees across a steep, but short, environmental gradient within a single Hawaiian watershed. At each location, we sampled eight microhabitats: leaves, petioles, axils, stems, roots, and litter from the plant, as well as surrounding air and soil. The composition of bacterial communities is better explained by microhabitat, while location better predicted compositional variance for fungi. Fungal community compositional dissimilarity increased more rapidly along the gradient than did bacterial composition. Additionally, the rates of fungal community compositional dissimilarity along the gradient differed among plant parts, and these differences influenced the distribution patterns and range size of individual taxa. Within plants, microbes were compositionally nested such that aboveground communities contained a subset of the diversity found belowground. Our findings indicate that both environmental context and microhabitat contribute to microbial compositional variance in our study, but that these contributions are influenced by the domain of microbe and the specific microhabitat in question, suggesting a complicated and potentially interacting dynamic.

Three-Dimensional Two-Color Dual-Particle Tracking Microscope for Monitoring DNA Conformational Changes and Nanoparticle Landings on Live Cells.

Here, we present a three-dimensional two-color dual-particle tracking (3D-2C-DPT) technique that can simultaneously localize two spectrally distinct targets in three dimensions with a time resolution down to 5 ms. The dual-targets can be tracked with separation distances from 33 to 250 nm with tracking precisions of ∼15 nm (for static targets) and ∼35 nm (for freely diffusing targets). Since each target is individually localized, a wealth of data can be extracted, such as the relative 3D position, the 2D rotation, and the separation distance between the two targets. Using this technique, we turn a double-stranded DNA (dsDNA)-linked dumbbell-like dimer into a nanoscopic optical ruler to quantify the bending dynamics of nicked or gapped dsDNA molecules in free solution by manipulating the design of dsDNA linkers (1-nick, 3-nt, 6-nt, or 9-nt single-strand gap), and the results show the increase of kon (linear to bent) from 3.2 to 10.7 s-1. The 3D-2C-DPT is then applied to observe translational and rotational motions of the landing of an antibody-conjugated nanoparticle on the plasma membrane of living cells, revealing the reduction of rotations possibly due to interactions with membrane receptors. This study demonstrates that this 3D-2C-DPT technique is a new tool to shed light on the conformational changes of biomolecules and the intermolecular interactions on plasma membrane.

Measuring DNA Hybridization Kinetics in Live Cells Using a Time-Resolved 3D Single-Molecule Tracking Method.

Single-molecule detection enables direct characterization of annealing/melting kinetics of nucleic acids without the need for synchronization of molecular states, but the current experiments are not carried out in a native cellular context. Here we describe an integrated 3D single-molecule tracking and lifetime measurement method that can follow individual DNA molecules diffusing inside a mammalian cell and observe multiple annealing and melting events on the same molecules. By comparing the hybridization kinetics of the same DNA strand in vitro, we found the association constants can be 13- to 163-fold higher in the molecular crowding cellular environment.

Improving NanoCluster Beacon performance by blocking the unlabeled NC probes.

While NanoCluster Beacon (NCB) is a versatile molecular probe, it suffers from a low target-specific signal issue due to impurities. Here we show that adding a "blocker" strand to the reaction can effectively block the nonfunctional probes and enhance the target-specific signal by 14 fold at a 0.1 target/probe ratio.

Discrete Step Sizes of Molecular Motors Lead to Bimodal Non-Gaussian Velocity Distributions under Force.

Fluctuations in the physical properties of biological machines are inextricably linked to their functions. Distributions of run lengths and velocities of processive molecular motors, like kinesin-1, are accessible through single-molecule techniques, but rigorous theoretical models for these probabilities are lacking. Here, we derive exact analytic results for a kinetic model to predict the resistive force (F)-dependent velocity [P(v)] and run length [P(n)] distribution functions of generic finitely processive molecular motors. Our theory quantitatively explains the zero force kinesin-1 data for both P(n) and P(v) using the detachment rate as the only parameter. In addition, we predict the F dependence of these quantities. At nonzero F, P(v) is non-Gaussian and is bimodal with peaks at positive and negative values of v, which is due to the discrete step size of kinesin-1. Although the predictions are based on analyses of kinesin-1 data, our results are general and should hold for any processive motor, which walks on a track by taking discrete steps.

Role of dendritic cell phenotype, determinant spreading, and negative costimulatory blockade in dendritic cell-based melanoma immunotherapy.

MART-1(27-35)-peptide-pulsed immature dendritic cells (DCs) resulted in immunologic and clinical activity in a prior phase 1 trial. A phase 2 cohort expansion was initiated to further characterize the phenotype and cytokine milieu of the DC vaccines and their immunologic activity in vitro and to further examine a possible link between clinical activity and determinant spreading. In an open-label phase 2 trial, 10(7) autologous ex vivo generated DCs pulsed with the HLA-A*0201 immunodominant peptide MART-1(27-35) were administered to 10 subjects with stage II-IV melanoma. The experimental vaccines were administered intradermally in a biweekly schedule for a total of three injections, and blood for immunologic assays was obtained before each administration and at three time points after. DC vaccine preparations had wide intra- and interpatient variability in terms of cell surface markers and preferential cytokine milieu, but they did not correlate with the levels of antigen-specific T cells after vaccination. Of four patients with measurable disease, one had stable disease for 6 months and another has a continued complete response for over 2 years, which is confounded by receiving a closely sequenced CTLA4 blocking antibody. The DC vaccines induced determinant spreading in this subject, and CTLA4 blockade reactivated T cells with prior antigen exposure. The DC phenotype and cytokine profile do not correlate with the ability to induce antigen-specific T cells, while determinant spreading after DC immunization may be a marker of an efficient antitumor response. Sequential CTLA4 blockade may enhance the immune activity of DC-based immunotherapy.

T-cell responses to HLA-A*0201 immunodominant peptides derived from alpha-fetoprotein in patients with hepatocellular cancer.

PURPOSE: An existing immunological paradigm is that high concentrations of soluble protein contribute to the maintenance of peripheral tolerance/ignorance to self protein. We tested this hypothesis in a clinical immunotherapy trial using class I-restricted peptide epitopes derived from alpha-fetoprotein (AFP). AFP is a self protein expressed by fetal liver at high levels, but transcriptionally repressed at birth. AFP is de-repressed in a majority of hepatocellular carcinomas (HCCs) and patients with active disease can have plasma levels in the mg/ml range. We previously identified four immunodominant HLA-A*0201-restricted peptides derived from human AFP that could stimulate specific T-cell responses in normal volunteer peripheral blood lymphocytes cultures. We wished to test the hypothesis that AFP peptide-reactive T cells could be expanded in vivo in HCC patients immunized with these four AFP peptides. EXPERIMENTAL DESIGN: We undertook a pilot Phase I clinical trial in which HLA-A*0201 patients with AFP-positive HCC were immunized with three biweekly intradermal vaccinations of the four AFP peptides (100 microg or 500 microg each) emulsified in incomplete Freund's adjuvant. RESULTS: All of the patients (n=6) generated T-cell responses to most or all of the peptides as measured by direct IFNgamma enzyme-linked immunospot (ELISPOT) and MHC class I tetramer assays. CONCLUSIONS: We conclude that the human T-cell repertoire is capable of recognizing AFP in the context of MHC class I even in an environment of high circulating levels of this oncofetal protein.

Determinant spreading associated with clinical response in dendritic cell-based immunotherapy for malignant melanoma.

PURPOSE: The purpose of this study was to determine the toxicity and immunological effects of three different doses and two routes of administration of autologous dendritic cells (DCs) pulsed with the MART-1(27-35) immunodominant epitope. EXPERIMENTAL DESIGN: Eighteen HLA-A*0201-positive subjects with stage III-IV melanoma received three biweekly i.v. or intradermal injections of ex vivo generated myeloid DCs pulsed with MART-1(27-35) epitope. Repeated blood samples were processed to obtain peripheral blood mononuclear cells for immunological analysis using IFN-gamma ELISPOT, MHC class I tetramer, intracellular cytokine staining, and microcytotoxicity assays. RESULTS: The frequency of MART-1/Melan-A (MART-1) antigen-specific T cells in peripheral blood increased in all dose levels as assessed by ELISPOT and MHC class I tetramer assays, but without a clear dose-response effect. The intradermal route generated stronger MART-1 immunity compared with the i.v. route. MART-1-specific immunity did not correlate with clinical outcome in any of the four immunological assays used. However, analysis of determinant spreading to other melanoma antigens was noted in the only subject with complete response to this single-epitope immunization. CONCLUSIONS: Intradermal immunization with MART-1 peptide-pulsed DCs results in an increase in circulating IFN-gamma-producing, antigen-specific T cells. The frequency of these cells did not correlate with response. In contrast, spreading of immune reactivity to other melanoma antigens was only evident in a subject with a complete response, suggesting that determinant spreading may be an important factor of clinical response to this form of immunotherapy.