ABSTRACT
Eukaryotic reverse transcriptases (RTs) can have essential or deleterious roles in normal human physiology and disease. Compared to well-studied helicases, it remains unclear how RTs overcome the ubiquitous RNA structural barriers during reverse transcription. Herein, we describe the development of a Mycobacterium smegmatis porin A (MspA) nanopore technique to sequence RNA to quantify the single-molecule kinetics of an RT from Bombyx mori with single-nucleotide resolution. By establishing a quadromer map that correlates RNA sequence and MspA ion current, we were able to quantify the RT's dwell time at every single nucleotide step along its RNA template. By challenging the enzyme with various RNA structures, we found that during cDNA synthesis the RT can sense and actively destabilize RNA structures 11-12 nt downstream of its front boundary. The ability to sequence single molecules of RNA with nanopores paves the way to investigate the single-nucleotide activity of other processive RNA translocases.
ABSTRACT
Introduction of the solid phase method to synthesize biopolymers has revolutionized the field of biological research by enabling efficient production of peptides and oligonucleotides. One of the advantages of this method is the ease of removal of excess production materials from the desired product, as it is immobilized on solid substrate. The DNA origami method utilizes the nature of nucleotide base-pairing to construct well-defined objects at the nanoscale, and has become a potent tool for manipulating matter in the fields of chemistry, physics, and biology. Here, the development of an approach to synthesize DNA nanostructures directly on magnetic beads, where the reaction is performed in heavy liquid to maintain the beads in suspension is reported. It is demonstrated that the method can achieve high folding yields of up to 90% for various DNA shapes, comparable to standard folding. At the same time, this establishes an easy, fast, and efficient way to further functionalize the DNA origami in one-pot, as well as providing a built-in purification method for easy removal of excess by-products such as non-integrated DNA strands and residual functionalization molecules.
Subject(s)
Nanostructures , Nanotechnology , Nanotechnology/methods , Solid-Phase Synthesis Techniques , Nucleic Acid Conformation , Nanostructures/chemistry , DNA/chemistryABSTRACT
Studies have shown that the sharing of big health data can improve patient management across primary and secondary care sectors. It can also reduce costs and can enhance the medical research process. Unfortunately, many big health data initiatives are being impeded because of a range of complex issues. This study was initiated to identify the said issues and develop a tool for health marketers to use to negate the barriers in big healthcare data projects. The study demonstrates how the Interactive Communication Technology Adoption Model can be operationalized to support qualitative researchers.
Subject(s)
Delivery of Health Care , Humans , Qualitative ResearchABSTRACT
Viruses and bacteria commonly exhibit spatial repetition of surface molecules that directly interface with the host immune system. However the complex interaction of patterned surfaces with immune molecules containing multiple binding domains is poorly understood. We developed a pipeline for constructing mechanistic models of antibody interactions with patterned antigen substrates. Our framework relies on immobilized DNA origami nanostructures decorated with precisely placed antigens. The results revealed that antigen spacing is a spatial control parameter that can be tuned to influence antibody residence time and migration speed. The model predicts that gradients in antigen spacing can drive persistent, directed antibody migration in the direction of more stable spacing. These results depict antibody-antigen interactions as a computational system wherein antigen geometry constrains and potentially directs antibody movement. We propose that this form of molecular programmability could be exploited during co-evolution of pathogens and immune systems or in the design of molecular machines.
ABSTRACT
Theoretical and experimental observations that catalysis enhances the diffusion of enzymes have generated exciting implications about nanoscale energy flow, molecular chemotaxis, and self-powered nanomachines. However, contradictory claims on the origin, magnitude, and consequence of this phenomenon continue to arise. To date, experimental observations of catalysis-enhanced enzyme diffusion have relied almost exclusively on fluorescence correlation spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an anti-Brownian electrokinetic (ABEL) trap and in-solution single-particle tracking, we show that catalysis does not increase the diffusion of alkaline phosphatase (ALP) at the single-molecule level, in sharp contrast to the â¼20% enhancement seen in parallel FCS experiments using p-nitrophenyl phosphate (pNPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte Carlo simulations, we establish that pNPP-induced dye blinking at the â¼10-ms timescale is responsible for the apparent diffusion enhancement seen in FCS. Our observations urge a crucial revisit of various experimental findings and theoretical models--including those of our own--in the field, and indicate that in-solution single-particle tracking and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale.
Subject(s)
Alkaline Phosphatase/chemistry , Diffusion , Animals , Catalysis , Cattle , Nitrophenols , Organophosphorus Compounds , Spectrometry, FluorescenceABSTRACT
In the Supplementary Information file originally published with this Article, the Supplementary references 48-62 were missing; the amended file has now been uploaded.
ABSTRACT
Although repetitive patterns of antigens are crucial for certain immune responses, an understanding of how antibodies bind and dynamically interact with various spatial arrangements of molecules is lacking. Hence, we introduced a new method in which molecularly precise nanoscale patterns of antigens are displayed using DNA origami and immobilized in a surface plasmon resonance set-up. Using antibodies with identical antigen-binding domains, we found that all the subclasses and isotypes studied bind bivalently to two antigens separated at distances that range from 3 to 17 nm. The binding affinities of these antibodies change with the antigen distances, with a distinct preference for antigens separated by approximately 16 nm, and considerable differences in spatial tolerance exist between IgM and IgG and between low- and high-affinity antibodies.
Subject(s)
Antibodies/metabolism , Antigens/metabolism , Immune Tolerance , Nanoparticles/chemistry , Cell Line , Humans , Immunoglobulin G/chemistry , Protein Binding , Surface Plasmon ResonanceABSTRACT
BACKGROUND: The impact of perioperative intravenous fluid administration on surgical outcomes has been documented in literature, but not specifically studied in the context of hepato-pancreato-biliary (HPB) surgery. This study aimed to investigate the impact of postoperative intravenous fluid administration on intensive care unit (ICU), in this subgroup of patients. METHODS: A single-center retrospective cohort of 241 HPB patients was assessed, focusing on intravenous fluid administration in ICU, during the first 24 h. Intravenous fluid variables were compared to hospital stay and postoperative complications. Data were assessed using Spearman's correlation test for bivariate correlations and logistic regression for multivariate analysis. RESULTS: The median volume of intravenous fluid administered in the first 24 h postoperatively was 4380â¯mL, of which 2200â¯mL was crystalloid, 1500â¯mL colloid and 680â¯mL "other" fluid. Patients with one or more complications had a higher median total intravenous fluid input (4790â¯vs. 4300â¯mL), higher colloid volume (2000â¯vs. 1500â¯mL), lower urine output (1595â¯vs. 1900â¯mL) and greater overall fluid balance (+3040â¯vs.+2553â¯mL) than those without complications. There were correlations between total intravenous fluid volume administered (râ¯=â¯0.278, Pâ¯<â¯0.001), intravenous colloid input (râ¯=â¯0.278, Pâ¯<â¯0.001), urine output (râ¯=â¯-0.295, Pâ¯<â¯0.001), positive fluid balance (râ¯=â¯0.344, Pâ¯<â¯0.001) and length of hospital stay. Logistic regression model was constructed to predict the occurrence of one or more complications; total intravenous fluid volume and overall fluid balance were both independent significant predictors (ORâ¯=â¯2.463, Pâ¯=â¯0.007; ORâ¯=â¯1.001, Pâ¯=â¯0.011; respectively). CONCLUSIONS: Administration of high volumes of intravenous fluids in the first 24 hours post-HPB surgery, along with higher positive fluid balance is associated with a higher rate of complications and longer hospital stay. Moreover, lower urine output is associated with longer hospital stay. Whether these are the cause of complications or the result of them remains unclear.
Subject(s)
Biliary Tract Surgical Procedures/methods , Fluid Therapy/methods , Length of Stay , Pancreatectomy/methods , Postoperative Care/methods , Postoperative Complications/epidemiology , Adult , Aged , Aged, 80 and over , Biliary Tract Surgical Procedures/adverse effects , Cohort Studies , Databases, Factual , Female , Fluid Therapy/adverse effects , Humans , Incidence , Logistic Models , Male , Middle Aged , Multivariate Analysis , Pancreatectomy/adverse effects , Postoperative Complications/prevention & control , Prognosis , Retrospective Studies , Risk Assessment , Treatment Outcome , Young AdultABSTRACT
Surface Plasmon Resonance biosensors measure the interaction between a molecule in solution and its interaction partner attached to a sensor surface. Under certain conditions, the observed binding rate can be used directly to obtain the concentration of the molecule in solution, without the use of any standard. This type of assay is referred to as Calibration Free Concentration Analysis, CFCA. By examining experimental conditions, including immobilization levels and temperature, for a range of analytes, and by using global analysis of several sample dilutions, conditions that gave the most robust results were identified. These conditions provided the concentration values that were on average â¼15% lower than those obtained using other methods. The accuracy of the concentration determined may be related to how the analyte is distributed in the dextran matrix and to its distance from the gold surface, and may thereby depend on the conversion of the SPR signal to mass. A good precision of CFCA, â¼8% (n = 21), was demonstrated when this method was used to efficiently guide purification procedures of Interferon α-2a. In this paper, the theory behind CFCA and the future developments, as well as the application of CFCA for absolute and relative concentration measurements (including the assessment of the potency of a biotherapeutic medicine) are discussed, and new evaluation tools that broaden the range of applications, are introduced.
Subject(s)
Interferon-alpha/analysis , Models, Chemical , Software , Surface Plasmon Resonance/methods , Calibration , Humans , Interferon-alpha/chemistry , Interferon-alpha/isolation & purification , Surface Plasmon Resonance/standardsABSTRACT
OBJECTIVE: The Kufner modified Le Fort III osteotomy (LFIII) can be used to address midface deficiency, which is often accompanied by excessive scleral exposure. The purpose of this project is to analyze the changes in scleral exposure after a LFIII. METHODS: Thirteen patients with midface hypoplasia were treated with LFIII. Scleral surface area (SSA) was determined by pixel count and the distance from the inferior eyelid margin to the center of the pupil (MED) was measured pre- and postoperatively. Intraclass correlation coefficients were calculated to assess measurement reliability and repeated measures analysis of variance (ANOVA) were determined to assess systematic difference among the replicates. RESULTS: The interquartile range for change in SSA ranged from -31% to -7%, median 20% (P = .002) and the interquartile range for change in MED ranged from -21% to -12%, median -18% (P = .0002). CONCLUSIONS: SSA and MED can be reliably determined using the aforementioned method. The LFIII decreases scleral exposure.
Subject(s)
Facial Bones/surgery , Osteotomy, Le Fort/methods , Sclera/anatomy & histology , Adolescent , Anatomic Landmarks , Facial Bones/abnormalities , Female , Humans , Male , Photography , Retrospective Studies , Treatment OutcomeABSTRACT
The high programmability of DNA origami has provided tools for precise manipulation of matter at the nanoscale. This manipulation of matter opens up the possibility to arrange functional elements for a diverse range of applications that utilize the nanometer precision provided by these structures. However, the realization of functionalized DNA origami still suffers from imperfect production methods, in particular in the purification step, where excess material is separated from the desired functionalized DNA origami. In this article we demonstrate and optimize two purification methods that have not previously been applied to DNA origami. In addition, we provide a systematic study comparing the purification efficacy of these and five other commonly used purification methods. Three types of functionalized DNA origami were used as model systems in this study. DNA origami was patterned with either small molecules, antibodies, or larger proteins. With the results of our work we aim to provide a guideline in quality fabrication of various types of functionalized DNA origami and to provide a route for scalable production of these promising tools.
Subject(s)
DNA/chemistry , DNA/isolation & purification , Nanostructures/chemistry , Nucleic Acid Conformation , Base Sequence , DNA/genetics , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , Models, MolecularABSTRACT
The spatial organization of membrane-bound ligands is thought to regulate receptor-mediated signaling. However, direct regulation of receptor function by nanoscale distribution of ligands has not yet been demonstrated, to our knowledge. We developed rationally designed DNA origami nanostructures modified with ligands at well-defined positions. Using these 'nanocalipers' to present ephrin ligands, we showed that the nanoscale spacing of ephrin-A5 directs the levels of EphA2 receptor activation in human breast cancer cells. Furthermore, we found that the nanoscale distribution of ephrin-A5 regulates the invasive properties of breast cancer cells. Our ligand nanocaliper approach has the potential to provide insight into the roles of ligand nanoscale spatial distribution in membrane receptor-mediated signaling.
Subject(s)
Nanotechnology , Receptors, Cell Surface/metabolism , Endocytosis , LigandsABSTRACT
Immunization with Plasmodium sporozoites can elicit high levels of sterile immunity, and neutralizing antibodies from protected hosts are known to target the repeat region of the circumsporozoite (CS) protein on the parasite surface. CS-based subunit vaccines have been hampered by suboptimal immunogenicity and the requirement for strong adjuvants to elicit effective humoral immunity. Pathogen-associated molecular patterns (PAMPs) that signal through Toll-like receptors (TLRs) can function as potent adjuvants for innate and adaptive immunity. We examined the immunogenicity of recombinant proteins containing a TLR5 agonist, flagellin, and either full-length or selected epitopes of the Plasmodium falciparum CS protein. Mice immunized with either of the flagellin-modified CS constructs, administered intranasally (i.n.) or subcutaneously (s.c.), developed similar levels of malaria-specific IgG1 antibody and interleukin-5 (IL-5)-producing T cells. Importantly, immunization via the i.n. but not the s.c. route elicited sporozoite neutralizing antibodies capable of inhibiting >90% of sporozoite invasion in vitro and in vivo, as measured using a transgenic rodent parasite expressing P. falciparum CS repeats. These findings demonstrate that functional sporozoite neutralizing antibody can be elicited by i.n. immunization with a flagellin-modified P. falciparum CS protein and raise the potential of a scalable, safe, needle-free vaccine for the 40% of the world's population at risk of malaria.
Subject(s)
Malaria Vaccines/immunology , Malaria, Falciparum/immunology , Plasmodium falciparum/immunology , Protozoan Proteins/immunology , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/pharmacology , Administration, Intranasal , Animals , Antibodies, Protozoan/immunology , Cells, Cultured , Dendritic Cells/immunology , Epitopes, T-Lymphocyte/immunology , Flagellin/immunology , Humans , Immunity, Humoral/immunology , Immunization , Immunoglobulin G/biosynthesis , Immunoglobulin G/immunology , Interleukin-5/biosynthesis , Malaria Vaccines/administration & dosage , Malaria, Falciparum/parasitology , Malaria, Falciparum/prevention & control , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Protozoan Proteins/administration & dosage , Recombinant Proteins/immunology , Sporozoites/immunology , Toll-Like Receptor 5/agonists , Vaccines, Subunit/immunologyABSTRACT
Human papillomavirus vaccines were developed beginning in the early 1990s. Two similar vaccines were approved in 2006 and 2009 following extensive clinical testing. Both vaccines prevent HPV infection. Implementation of these vaccines is the next challenge.
Subject(s)
Drug Approval , Papillomaviridae/immunology , Papillomavirus Infections/prevention & control , Papillomavirus Vaccines/therapeutic use , Humans , Papillomavirus Infections/virology , Retrospective StudiesABSTRACT
Two live oral rotavirus vaccines were approved by the US Food and Drug Administration in 2005 and 2008, following large studies of â¼70,000 each in order to address questions about intussusception triggered by a third earlier vaccine. Both new rotavirus vaccines showed almost identical rates of intussusception in vaccine and placebo recipients. These vaccines have been used extensively in the United States with positive results. Efforts are under way to implement these vaccines in developing countries where the need is greatest.
Subject(s)
Rotavirus Infections , Rotavirus Vaccines/therapeutic use , Rotavirus/immunology , Global Health , Humans , Incidence , Rotavirus Infections/epidemiology , Rotavirus Infections/immunology , Rotavirus Infections/prevention & control , Rotavirus Vaccines/immunologyABSTRACT
Influenza vaccines have been available since the 1950s and have seen increasingly wide use as public health authorities expanded recommendations. Recent events including shortages and avian influenza outbreaks have renewed interest in influenza vaccines, particularly improved vaccines.
Subject(s)
Antigens, Viral/immunology , Influenza A virus/immunology , Influenza Vaccines/immunology , Influenza, Human/prevention & control , Adjuvants, Immunologic/chemistry , Antibodies, Viral/immunology , Biomedical Research/trends , Clinical Trials as Topic , Drug Industry/organization & administration , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Humans , Influenza A virus/pathogenicity , Influenza, Human/immunology , Influenza, Human/virology , Technology, Pharmaceutical/trends , Viral Matrix Proteins/immunologyABSTRACT
In the assembly of DNA nanostructures, the specificity of Watson-Crick base pairing is used to control matter at the nanoscale. Using this technology for drug delivery is a promising route toward the magic bullet concept, as it would allow the realization of complex assemblies that co-localize drugs, targeting ligands and other functionalities in one nanostructure. Anthracyclines' mechanism of action in cancer therapy is to intercalate DNA, and since DNA nanotechnology allows for such a high degree of customization, we hypothesized that this would allow us to tune the DNA nanostructures for optimal delivery of the anthracycline doxorubicin (Dox) to human breast cancer cells. We have tested two DNA origami nanostructures on three different breast cancer cell lines (MDA-MB-231, MDA-MB-468, and MCF-7). The different nanostructures were designed to exhibit varying degrees of global twist, leading to different amounts of relaxation in the DNA double-helix structure. By tuning the nanostructure design we are able to (i) tune the encapsulation efficiency and the release rate of the drug and (ii) increase the cytotoxicity and lower the intracellular elimination rate when compared to free Dox. Enhanced apoptosis induced by the delivery system in breast cancer cells was investigated using flow cytometry. The findings indicate that DNA origami nanostructures represent an efficient delivery system for Dox, resulting in high degrees of internalization and increased induction of programmed cell death in breast cancer cells. In addition, by designing the structures to exhibit different degrees of twist, we are able to rationally control and tailor the drug release kinetics.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/therapy , Delayed-Action Preparations/administration & dosage , Genetic Therapy/methods , Nanocapsules/administration & dosage , Nanocapsules/chemistry , Transfection/methods , Cell Line, Tumor , Delayed-Action Preparations/chemistry , Gene Silencing , Humans , Nanocapsules/ultrastructureABSTRACT
BACKGROUND: We evaluated the safety and immunogenicity profiles of 3 novel influenza vaccine constructs consisting of the globular head of the HA1 domain of the Novel H1N1 genetically fused to the TLR5 ligand, flagellin. HA1 was fused to the C-terminus of flagellin in VAX128A, replaced the D3 domain of flagellin in VAX128B and was fused in both positions in VAX128C. METHODS: In a dose escalation trial, 112 healthy subjects 18-49 and 100 adults ≥65 years old were enrolled in a double blind, placebo controlled clinical trial at two centers. Vaccines were administered IM at doses ranging from 0.5 to 20 µg. VAX128C was selected for second study performed in 100 subjects 18-64 years old comparing 1.25 and 2.5 µg doses. All subjects were followed for safety and sera collected pre- and post-vaccination were tested by hemagglutination-inhibition (HAI). Serum C-reactive protein and cytokine levels were also measured. CONCLUSIONS: In the first study high HAI titers and high seroconversion and seroprotection rates were observed at doses ≥2.5 µg in adults 18-49. In adults ≥65 years, the vaccines doses of ≥4 µg were required to induce a ≥4-fold rise in HAI titer, 50% seroconversion and 70% seroprotection. Based on safety, VAX128A was tested up to 8 µg, VAX128B to 16 µg and VAX128C to 20 µg. Dose escalation for VAX128A was stopped at 8 µg because one subject had temperature 101.6°F associated with a high CRP response, VAX128B was stopped at 16 µg because of a severe AE associated with a high CRP and IL-6 response. VAX128C was not stopped before reaching the 20 µg dose. In the second study VAX128C was well tolerated among 100 subjects who received 1.25 or 2.5 µg. The peak GMT was 385 (95%CI 272-546), 79% (71-87%) seroconversion and 92% (84-96%) seroprotection. DISCUSSION: Flagellin adjuvanted vaccines can be designed to minimize reactogenicity and retain immunogenicity, thereby representing a promising next generation vaccine technology.
Subject(s)
Flagellin/immunology , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza Vaccines/immunology , Influenza, Human/prevention & control , Adjuvants, Immunologic/administration & dosage , Adolescent , Adult , Aged , Animals , Antibodies, Viral/blood , C-Reactive Protein/immunology , Double-Blind Method , Female , Hemagglutination Inhibition Tests , Humans , Influenza Vaccines/administration & dosage , Influenza Vaccines/adverse effects , Interleukin-6/immunology , Male , Middle Aged , Rabbits , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/adverse effects , Vaccines, Synthetic/immunology , Young AdultABSTRACT
The currently available influenza vaccines were developed in the 1930s through the 1960s using technologies that were state-of-the art for the times. Decades of advancement in virology and immunology have provided the tools for making better vaccines against influenza. We now have the means to make vaccines that address some of the shortcomings of the original products, in particular performance in the elderly.
Subject(s)
Biotechnology/methods , Influenza Vaccines/immunology , Technology, Pharmaceutical/methods , Animals , Humans , Influenza Vaccines/geneticsABSTRACT
BACKGROUND: The ectodomain of matrix protein 2 (M2e) is a promising candidate for a broadly protective influenza A vaccine because it is highly conserved and antibodies to M2e are protective in animal models. STF2.4xM2e (VAX102) is a recombinant fusion protein that links four tandem copies of the M2e antigen to Salmonella typhimurium flagellin, a TLR5 ligand used as an adjuvant. The objectives of this first-in-human study were to assess the safety and immunogenicity of VAX102 given as a prime-boost regimen to healthy adults. METHODS: Sixty subjects 18-49 years old were enrolled in a multicenter, double-blind, randomized, placebo-controlled trial (Study 1). Based on pre-clincial data, initial design included doses starting at 10 µg, with an escalation plan. After reactogenicity was noted at the 10 µg dose, the trial was redesigned to evaluate 0.3, 1.0, and 3 µg doses. Following this study, 16 subjects were enrolled in Study 2, an open label, low dose study, to evaluate doses of 0.03 and 0.1 µg. In both trials, vaccine or placebo was given intramuscularly (i.m.) at 0 and 28 days. Clinical and laboratory safety assessments took place 1 and 7 days after immunization. Immune responses to M2e and flagellin were assessed by ELISA at 7, 14 and 28 days after each dose. Seroconversion was defined as a serum IgG anti-M2e antibody value ≥0.174 µg/ml and a fourfold rise in concentration. RESULTS: Doses of 0.03-1 µg were safe and well tolerated in all subjects. Doses of 0.03 and 0.1 µg produced limited immunogenicity (38% and 75% respectively), after the second dose of vaccine. Doses of 0.3 and 1.0 µg were immunogenic in 18 (75%) of 24 vaccinees after the first dose and 23 (96%) after the second dose. In the 1.0 µg group, the geometric mean M2e antibody concentration was 0.4 µg/ml after the first dose and 1.7 µg/ml after the booster dose. M2e antibody concentrations and seroconversion rates were not significantly different at higher doses (p>0.05). Immune response to flagellin was robust but did not appear to interfere with M2e antibody responses after the booster dose. Following the first injection of VAX102 at higher doses (3 and 10 µg), self-limited but severe symptoms were noted in some subjects and were associated with elevated levels of C-reactive protein. Although not directly measured, this reaction was believed to be mediated by cytokine release. CONCLUSIONS: VAX102 was safe and induced high antibody levels to M2e at 0.3 and 1.0 µg doses. The TLR5 ligand, S. typhimurium flagellin, is a novel approach to adjuvant-like activity through activation of innate immunity, and when fused to multiple copies of the M2e protein, the vaccine was able to induce a fourfold rise in antibody in humans, to a previously non-immunogenic, highly-conserved portion of the influenza virus. Clinical correlates of protection that may be afforded by M2e antibody in humans are a future focus of investigation.