Established methods for characterizing proteins typically require physical or chemical modification steps or cannot be used to examine individual molecules in solution. Ionic current measurements ...through electrolyte-filled nanopores can characterize single native proteins in an aqueous environment, but currently offer only limited capabilities. Here we show that the zeptolitre sensing volume of bilayer-coated solid-state nanopores can be used to determine the approximate shape, volume, charge, rotational diffusion coefficient and dipole moment of individual proteins. To do this, we developed a theory for the quantitative understanding of modulations in ionic current that arise from the rotational dynamics of single proteins as they move through the electric field inside the nanopore. The approach allows us to measure the five parameters simultaneously, and we show that they can be used to identify, characterize and quantify proteins and protein complexes with potential implications for structural biology, proteomics, biomarker detection and routine protein analysis.
Biological protein pores and pore-forming peptides can generate a pathway for the flux of ions and other charged or polar molecules across cellular membranes. In nature, these nanopores have diverse ...and essential functions that range from maintaining cell homeostasis and participating in cell signaling to activating or killing cells. The combination of the nanoscale dimensions and sophisticated – often regulated – functionality of these biological pores make them particularly attractive for the growing field of nanobiotechnology. Applications range from single-molecule sensing to drug delivery and targeted killing of malignant cells. Potential future applications may include the use of nanopores for single strand DNA sequencing and for generating bio-inspired, and possibly, biocompatible visual detection systems and batteries. This article reviews the current state of applications of pore-forming peptides and proteins in nanomedicine, sensing, and nanoelectronics.
Synthetic nanopores have been used to study individual biomolecules in high throughput, but their performance as sensors does not match that of biological ion channels. Challenges include control of ...nanopore diameters and surface chemistry, modification of the translocation times of single-molecule analytes through nanopores, and prevention of non-specific interactions with pore walls. Here, inspired by the olfactory sensilla of insect antennae, we show that coating nanopores with a fluid lipid bilayer tailors their surface chemistry and allows fine-tuning and dynamic variation of pore diameters in subnanometre increments. Incorporation of mobile ligands in the lipid bilayer conferred specificity and slowed the translocation of targeted proteins sufficiently to time-resolve translocation events of individual proteins. Lipid coatings also prevented pores from clogging, eliminated non-specific binding and enabled the translocation of amyloid-beta (Aβ) oligomers and fibrils. Through combined analysis of their translocation time, volume, charge, shape and ligand affinity, different proteins were identified.
Aggregates of amyloid-beta (Aβ) peptides are thought to be involved in the development of Alzheimer's disease because they can change synaptic plasticity and induce neuronal cell death by ...inflammation, oxidative damage, and transmembrane pore formation. Exactly which oligomeric species underlie these cytotoxic effects remains unclear. The work presented here established well-controlled aggregation conditions of Aβ₁₋₄₀ or Aβ₁₋₄₂ peptides over a 20-day period and characterized these preparations with regard to their β-sheet content, degree of fibril formation, relative abundance of various oligomer sizes, and propensity to induce membrane pore formation and cytotoxicity. Using this multivariate data set, a systematic and inherently unbiased partial least squares (PLS) approach showed that for both peptides the abundance of oligomers in the tetramer to 13-mer range contributed positively to both pore formation and cytotoxicity, while monomers, dimers, trimers, and the largest oligomers (>210 kDa) were negatively correlated to both phenomena. Multivariate PLS analysis is ideally suited to handle complex data sets and interdependent variables such as relative oligomer concentrations, making it possible to elucidate structure function relationships in complex mixtures. This approach, therefore, introduces an enabling tool to the field of amyloid research, in which it is often difficult to interpret the activity of individual species within a complex mixture of bioactive species.
This paper introduces a strategy for generating ion current rectification through nano- and micropores. This method generates ion current rectification by electroosmotic-driven flow of liquids of ...varying viscosity (and hence varying conductance) into or out of the narrowest constriction of a pore. The magnitude of current rectification was described by a rectification factor, R f, which is defined by the ratio of the current measured at a positive voltage divided by the current measured at a negative voltage. This method achieved rectification factors in the range of 5−15 using pores with diameters ranging from 10 nm to 2.2 μm. These R f values are similar to the rectification factors reported in other nanopore-based methods that did not employ segmented surface charges. Interestingly, this work showed that in cylindrical nanopores with diameters of 10 nm and a length of at least 275 nm, electroosmotic flow was present and could generate ion current rectification. Unlike previous methods for generating ion current rectification that require nanopores with diameters comparable to the Debye length, this work demonstrated ion current rectification in micropores with diameters 500 times larger than the Debye length. Thus this method extends the concept of fluidic diodes to the micropore range. Several experiments designed to alter or remove electroosmotic flow through the pore demonstrated that electroosmotic flow was required for the mode of ion current rectification reported here. Consequently, the magnitude of current rectification could be used to indicate the presence of electroosmotic flow and the breakdown of electroosmotic flow with decreasing ionic strength and hence increasing electric double layer overlap inside nanopores.
Radiotherapy is a highly effective anticancer treatment forming part of the standard of care for the majority of patients, but local and distal disease recurrence remains a major cause of mortality. ...Radiotherapy is known to enhance tumor immunogenicity; however, the contribution and mechanisms of radiotherapy-induced immune responses are unknown.
The impact of low-dose fractionated radiotherapy (5 × 2 Gy) alone and in combination with αPD-1 mAb on the tumor microenvironment was evaluated by flow cytometry and next-generation sequencing of the T-cell receptor (TCR) repertoire. A dual-tumor model was used, with fractionated radiotherapy delivered to a single tumor site to enable evaluation of the local and systemic response to treatment and ability to induce abscopal responses outside the radiation field.
We show that fractionated radiotherapy leads to T-cell infiltration at the irradiated site; however, the TCR landscape remains dominated by polyclonal expansion of preexisting T-cell clones. Adaptive resistance via the PD-1/PD-L1 pathway restricts the generation of systemic anticancer immunity following radiotherapy, which can be overcome through combination with αPD-1 mAb leading to improved local and distal tumor control. Moreover, we show that effective clearance of tumor following combination therapy is dependent on both T cells resident in the tumor at the time of radiotherapy and infiltrating T cells.
These data provide evidence that radiotherapy can enhance T-cell trafficking to locally treated tumor sites and augment preexisting anticancer T-cell responses with the capacity to mediate regression of out-of-field tumor lesions when delivered in combination with αPD-1 mAb therapy.
.
Force is a signal that cells cannot ignore Yusko, Erik C; Asbury, Charles L
Molecular biology of the cell,
2014-Nov-15, 2014-11-15, 20141115, Letnik:
25, Številka:
23
Journal Article
Recenzirano
Odprti dostop
Cells sense biochemical, electrical, and mechanical cues in their environment that affect their differentiation and behavior. Unlike biochemical and electrical signals, mechanical signals can ...propagate without the diffusion of proteins or ions; instead, forces are transmitted through mechanically stiff structures, flowing, for example, through cytoskeletal elements such as microtubules or filamentous actin. The molecular details underlying how cells respond to force are only beginning to be understood. Here we review tools for probing force-sensitive proteins and highlight several examples in which forces are transmitted, routed, and sensed by proteins in cells. We suggest that local unfolding and tension-dependent removal of autoinhibitory domains are common features in force-sensitive proteins and that force-sensitive proteins may be commonplace wherever forces are transmitted between and within cells. Because mechanical forces are inherent in the cellular environment, force is a signal that cells must take advantage of to maintain homeostasis and carry out their functions.
Immunotherapy with PD-1 or PD-L1 blockade fails to induce a response in about 80% of patients with unselected non-small cell lung cancer (NSCLC), and many of those who do initially respond then ...develop resistance to treatment. Agonists that target the shared interleukin-2 (IL-2) and IL-15Rβγ pathway have induced complete and durable responses in some cancers, but no studies have been done to assess the safety or efficacy of these agonists in combination with anti-PD-1 immunotherapy. We aimed to define the safety, tolerability, and activity of this drug combination in patients with NSCLC.
In this non-randomised, open-label, phase 1b trial, we enrolled patients (aged ≥18 years) with previously treated histologically or cytologically confirmed stage IIIB or IV NSCLC from three academic hospitals in the USA. Key eligibility criteria included measurable disease, eligibility to receive anti-PD-1 immunotherapy, and an Eastern Cooperative Oncology Group performance status of 0 or 1. Patients received the anti-PD-1 monoclonal antibody nivolumab intravenously at 3 mg/kg (then 240 mg when US Food and Drug Administration FDA-approved dosing changed) every 14 days (either as new treatment or continued treatment at the time of disease progression) and the IL-15 superagonist ALT-803 subcutaneously once per week on weeks 1–5 of four 6-week cycles for 6 months. ALT-803 was administered at one of four escalating dose concentrations: 6, 10, 15, or 20 μg/kg. The primary endpoint was to define safety and tolerability and to establish a recommended phase 2 dose of ALT-803 in combination with nivolumab. Analyses were per-protocol and included any patients who received at least one dose of study treatment. This trial is registered with ClinicalTrials.gov, number NCT02523469; phase 2 enrolment of patients is ongoing.
Between Jan 18, 2016, and June 28, 2017, 23 patients were enrolled and 21 were treated at four dose levels of ALT-803 in combination with nivolumab. Two patients did not receive treatment because of the development of inter-current illness during enrolment, one patient due to leucopenia and one patient due to pulmonary dysfunction. No dose-limiting toxicities were recorded and the maximum tolerated dose was not reached. The most common adverse events were injection-site reactions (in 19 90% of 21 patients) and flu-like symptoms (15 71%). The most common grade 3 adverse events, occurring in two patients each, were lymphocytopenia and fatigue. A grade 3 myocardial infarction occurred in one patient. No grade 4 or 5 adverse events were recorded. The recommended phase 2 dose of ALT-803 is 20 μg/kg given once per week subcutaneously in combination with 240 mg intravenous nivolumab every 2 weeks.
ALT-803 in combination with nivolumab can be safely administered in an outpatient setting. The promising clinical activity observed with the addition of ALT-803 to the regimen of patients with PD-1 monoclonal antibody relapsed and refractory disease shows evidence of anti-tumour activity for a new class of agents in NSCLC.
Altor BioScience (a NantWorks company), National Institutes of Health, and Medical University of South Carolina Hollings Cancer Center.
Determining the pathological role of amyloids in amyloid-associated diseases will require a method for characterizing the dynamic distributions in size and shape of amyloid oligomers with high ...resolution. Here, we explored the potential of resistive-pulse sensing through lipid bilayer-coated nanopores to measure the size of individual amyloid-β oligomers directly in solution and without chemical modification. This method classified individual amyloid-β aggregates as spherical oligomers, protofibrils, or mature fibers and made it possible to account for the large heterogeneity of amyloid-β aggregate sizes. The approach revealed the distribution of protofibrillar lengths (12- to 155 -mer) as well as the average cross-sectional area of protofibrils and fibers.
Background
Checkpoint inhibitors have recently been approved for the treatment of patients with hepatocellular carcinoma (HCC). However, biomarkers, which will help identify patients responding to ...therapy, are missing. We recently tested the combination of anti-CTLA4 treatment (tremelimumab) with loco-regional therapy in patients with HCC and reported a partial response rate of 26%.
Methods
Here, we report updated survival analyses and results from our immune monitoring studies on peripheral blood mononuclear cells (PBMCs) and tumors from these patients.
Results
Tremelimumab therapy increased CD4
+
-HLA-DR
+
, CD4
+
PD-1
+
, CD8
+
HLA-DR
+
, CD8
+
PD-1
+
, CD4
+
ICOS
+
and CD8
+
ICOS
+
T cells in the peripheral blood of the treated patients. Patients with higher CD4
+
PD1
+
cell frequency at baseline were more likely to respond to tremelimumab therapy. PD-1 expression was increased on alpha fetal protein (AFP) and survivin-specific CD8 T cells upon tremelimumab treatment. An increase of tumor infiltrating CD3
+
T cells were also seen in these patients. Immunosequencing of longitudinal PBMC showed that one cycle of tremelimumab significantly decreased peripheral clonality, while no additional effects were seen after loco-regional therapy.
Conclusion
In summary, we observed a clear activation of T cell responses in HCC patients treated with tremelimumab and identified potential biomarkers which will help identify patients responding to immunotherapy with anti-CTLA4.
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