•HS-AFM detects conformational changes of unlabeled membrane proteins.•In situ responses to pH, ligands, temperature and light can be visualized.•Developments in techniques now allow microsecond ...temporal resolution.
Recent advances in high-speed atomic force microscopy (HS-AFM) have made it possible to study the conformational dynamics of single unlabeled transmembrane channels and transporters. Improving environmental control with the integration of a non-disturbing buffer exchange system, which in turn allows the gradual change of conditions during HS-AFM operation, has provided a breakthrough toward the performance of structural titration experiments. Further advancements in temporal resolution with the use of line scanning and height spectroscopy techniques show how high-speed atomic force microscopy can measure millisecond to microsecond dynamics, pushing this method beyond current spatial and temporal limits offered by less direct techniques.
Dynamics are fundamental to the functions of biomolecules and can occur on a wide range of time and length scales. Here we develop and apply high-speed AFM height spectroscopy (HS-AFM-HS), a ...technique whereby we monitor the sensing of a HS-AFM tip at a fixed position to directly detect the motions of unlabeled molecules underneath. This gives Angstrom spatial and microsecond temporal resolutions. In conjunction with HS-AFM imaging modes to precisely locate areas of interest, HS-AFM-HS measures simultaneously surface concentrations, diffusion coefficients and oligomer sizes of annexin-V on model membranes to decipher key kinetics allowing us to describe the entire annexin-V membrane-association and self-assembly process in great detail and quantitatively. This work displays how HS-AFM-HS can assess the dynamics of unlabeled bio-molecules over several orders of magnitude and separate the various dynamic components spatiotemporally.
Mutations of the ADAR1 gene encoding an RNA deaminase cause severe diseases associated with chronic activation of type I interferon (IFN) responses, including Aicardi-Goutières syndrome and bilateral ...striatal necrosis
. The IFN-inducible p150 isoform of ADAR1 contains a Zα domain that recognizes RNA with an alternative left-handed double-helix structure, termed Z-RNA
. Hemizygous ADAR1 mutations in the Zα domain cause type I IFN-mediated pathologies in humans
and mice
; however, it remains unclear how the interaction of ADAR1 with Z-RNA prevents IFN activation. Here we show that Z-DNA-binding protein 1 (ZBP1), the only other protein in mammals known to harbour Zα domains
, promotes type I IFN activation and fatal pathology in mice with impaired ADAR1 function. ZBP1 deficiency or mutation of its Zα domains reduced the expression of IFN-stimulated genes and largely prevented early postnatal lethality in mice with hemizygous expression of ADAR1 with mutated Zα domain (Adar1
mice). Adar1
mice showed upregulation and impaired editing of endogenous retroelement-derived complementary RNA reads, which represent a likely source of Z-RNAs activating ZBP1. Notably, ZBP1 promoted IFN activation and severe pathology in Adar1
mice in a manner independent of RIPK1, RIPK3, MLKL-mediated necroptosis and caspase-8-dependent apoptosis, suggesting a novel mechanism of action. Thus, ADAR1 prevents endogenous Z-RNA-dependent activation of pathogenic type I IFN responses by ZBP1, suggesting that ZBP1 could contribute to type I interferonopathies caused by ADAR1 mutations.
Programmed death-ligand 1 (PD-L1) expression may vary in different disease sites and at different time points of the disease course. We aimed to investigate PD-L1 heterogeneity and its usefulness as ...a predictive value for immune checkpoint inhibitor (ICI) therapy in patients with NSCLC.
PD-L1 expression was analyzed in 1398 patients with NSCLC. The predictive value of PD-L1 for ICIs in 398 patients with metastatic NSCLC was assessed.
PD-L1 was significantly associated with biopsy sites (p = 0.004). Adrenal, liver, and lymph node (LN) metastases had the highest PD-L1 expression as a continuous variable and at 1% or 50% cutoff. PD-L1 expression was lower in bone and brain metastases. Among 112 patients with two specimens tested, 55 (49%) had major changes in PD-L1 falling into different clinically relevant categories (<1%, 1%–49%, ≥50%) at different time points. Previous ICI therapy was associated with significant decrease in PD-L1 compared with treatment-naive counterparts (p = 0.015). Patients with metastatic NSCLC treated with ICI (n = 398) were divided into three cohorts on the basis of biopsy sites: lung (n = 252), LN (n = 85), and distant metastasis (n = 61). Higher PD-L1 in lung or distant metastasis specimens was associated with higher response rate, longer progression-free survival, and overall survival. However, PD-L1 in LN biopsies was not associated with either response or survival.
PD-L1 varies substantially across different anatomical sites and changes during the clinical course. PD-L1 from different biopsy sites may have different predictive values for benefit from ICIs in NSCLC.
Asteroseismology probes the internal structures of stars by using their natural pulsation frequencies
. It relies on identifying sequences of pulsation modes that can be compared with theoretical ...models, which has been done successfully for many classes of pulsators, including low-mass solar-type stars
, red giants
, high-mass stars
and white dwarfs
. However, a large group of pulsating stars of intermediate mass-the so-called δ Scuti stars-have rich pulsation spectra for which systematic mode identification has not hitherto been possible
. This arises because only a seemingly random subset of possible modes are excited and because rapid rotation tends to spoil regular patterns
. Here we report the detection of remarkably regular sequences of high-frequency pulsation modes in 60 intermediate-mass main-sequence stars, which enables definitive mode identification. The space motions of some of these stars indicate that they are members of known associations of young stars, as confirmed by modelling of their pulsation spectra.
Localization atomic force microscopy Heath, George R; Kots, Ekaterina; Robertson, Janice L ...
Nature,
06/2021, Letnik:
594, Številka:
7863
Journal Article
Recenzirano
Odprti dostop
Understanding structural dynamics of biomolecules at the single-molecule level is vital to advancing our knowledge of molecular mechanisms. Currently, there are few techniques that can capture ...dynamics at the sub-nanometre scale and in physiologically relevant conditions. Atomic force microscopy (AFM)
has the advantage of analysing unlabelled single molecules in physiological buffer and at ambient temperature and pressure, but its resolution limits the assessment of conformational details of biomolecules
. Here we present localization AFM (LAFM), a technique developed to overcome current resolution limitations. By applying localization image reconstruction algorithms
to peak positions in high-speed AFM and conventional AFM data, we increase the resolution beyond the limits set by the tip radius, and resolve single amino acid residues on soft protein surfaces in native and dynamic conditions. LAFM enables the calculation of high-resolution maps from either images of many molecules or many images of a single molecule acquired over time, facilitating single-molecule structural analysis. LAFM is a post-acquisition image reconstruction method that can be applied to any biomolecular AFM dataset.
PurposeThe receptor tyrosine kinase rearranged during transfection (RET) can be oncogenically activated by gene fusions or point mutations. Multikinase inhibitors such as cabozantinib, lenvatinib and ...vandetanib have demonstrated activity in RET-dependent malignancies, and selective RET inhibitors (Selpercatinib and Pralsetinib) are in clinical trials. However, the responsiveness of RET-dependent malignancies to immune checkpoint inhibitors (ICIs) is unknown. We compared the time to treatment discontinuation (TTD) for ICI versus non-ICI therapy in patients with malignancies harbouring activating RET mutations or fusions (RET+).MethodsA retrospective review of all RET+ patients who were referred to the phase I clinical trials programme at the University of Texas MD Anderson Cancer Center was conducted. TTD was estimated using Kaplan-Meier analysis. Multivariate analysis using the Cox proportional hazard model was performed to identify independent risk factors of treatment discontinuation.ResultsOf 70 patients who received systemic therapy for RET+ malignancies, 20 (28.6%) received ICI and 50 (71.4%) received non-ICI therapy. Non-ICI therapy was associated with decreased risk for treatment discontinuation compared with ICI in the overall population (HR=0.31; 95% CI 0.16–0.62; p=0.000834) and in patients with RET point mutations (HR=0.13; 95% CI 0.04–0.45; p=0.00134). In patients with RET fusions, non-ICI therapy was associated with a non-statistically significant decreased risk of treatment discontinuation (HR=0.59; 95% CI 0.25–1.4; p=0.24). ICI therapy and a diagnosis other than medullary thyroid cancer (MTC) were independent risk factors for treatment discontinuation.ConclusionOur study supports the prioritisation of non-ICI over ICI therapy in patients with RET+ tumours.
Abstract
Excitatory amino acid transporters (EAATs) are important in many physiological processes and crucial for the removal of excitatory amino acids from the synaptic cleft. Here, we develop and ...apply high-speed atomic force microscopy line-scanning (HS-AFM-LS) combined with automated state assignment and transition analysis for the determination of transport dynamics of unlabeled membrane-reconstituted Glt
Ph
, a prokaryotic EAAT homologue, with millisecond temporal resolution. We find that Glt
Ph
transporters can operate much faster than previously reported, with state dwell-times in the 50 ms range, and report the kinetics of an intermediate transport state with height between the outward- and inward-facing states. Transport domains stochastically probe transmembrane motion, and reversible unsuccessful excursions to the intermediate state occur. The presented approach and analysis methodology are generally applicable to study transporter kinetics at system-relevant temporal resolution.
This guideline is for the management of patients with small cell lung cancer (SCLC) and is based on currently available information. As part of the guideline, an evidence-based review of the ...literature was commissioned that enables the reader to assess the evidence as we have attempted to put the clinical implications into perspective.
We conducted a comprehensive review of the available literature and the previous American College of Chest Physicians guidelines of SCLC. Controversial and less understood areas of the management of SCLC were then subject to an exhaustive review of the literature and detail analyses. Experts in evidence-based analyses compiled the accompanying systematic review titled "Evidence for Management of SCLC." The evidence was then assessed by a panel of experts to incorporate "clinical relevance." The resultant guidelines were then scored according to the grading system outlined by the American College of Chest Physicians grading system task force.
SCLC accounts for 13 to 20% of all lung cancers. Highly smoking related and initially responsive to treatment, it leads to death rapidly in 2 to 4 months without treatment. SCLC is staged as limited-stage and extensive-stage disease. Limited-stage disease is treated with curative intent with chemotherapy and radiation therapy, with approximately 20% of patients achieving a cure. For all patients with limited-stage disease, median survival is 16 to 22 months. Extensive-stage disease is primarily treated with chemotherapy with a high initial response rate of 60 to 70% but with a median survival of 10 months. All patients achieving a complete remission should be offered prophylactic cranial irradiation. Relapsed or refractory SCLC has a uniformly poor prognosis.
In this section, evidence-based guidelines for the staging and treatment of SCLC are outlined. Limited-stage SCLC is treated with curative intent. Extensive-stage SCLC has high initial responses to chemotherapy but with an ultimately dismal prognosis with few survivors beyond 2 years.
Lung cancer is still the leading cause of cancer death worldwide. Both histologically and molecularly lung cancer is heterogeneous. This review summarizes the current knowledge of the pathways ...involved in the various types of lung cancer with an emphasis on the clinical implications of the increasing number of actionable molecular targets. It describes the major pathways and molecular alterations implicated in the development and progression of non-small cell lung cancer (adenocarcinoma and squamous cancer), and of small cell carcinoma, emphasizing the molecular alterations comprising the specific blueprints in each group. The approved and investigational targeted therapies as well as the immune therapies, and clinical trials exploring the variety of targeted approaches to treatment of lung cancer are the main focus of this review.
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