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1.
Nature ; 2021 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-33545711

RESUMO

SARS-CoV-2 Spike protein is critical for virus infection via engagement of ACE21, and is a major antibody target. Here we report chronic SARS-CoV-2 with reduced sensitivity to neutralising antibodies in an immune suppressed individual treated with convalescent plasma, generating whole genome ultradeep sequences over 23 time points spanning 101 days. Little change was observed in the overall viral population structure following two courses of remdesivir over the first 57 days. However, following convalescent plasma therapy we observed large, dynamic virus population shifts, with the emergence of a dominant viral strain bearing D796H in S2 and ΔH69/ΔV70 in the S1 N-terminal domain NTD of the Spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype diminished in frequency, before returning during a final, unsuccessful course of convalescent plasma. In vitro, the Spike escape double mutant bearing ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, whilst maintaining infectivity similar to wild type. D796H appeared to be the main contributor to decreased susceptibility but incurred an infectivity defect. The ΔH69/ΔV70 single mutant had two-fold higher infectivity compared to wild type, possibly compensating for the reduced infectivity of D796H. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy associated with emergence of viral variants with evidence of reduced susceptibility to neutralising antibodies.

2.
Med Phys ; 2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33577091

RESUMO

Over the last few years magnetic resonance image guided radiotherapy systems have been introduced into the clinic, allowing for daily online plan adaption. While quality assurance (QA) is similar to conventional radiotherapy systems, there is a need to introduce or modify measurement techniques. As yet, there is no consensus guidance on the QA equipment and test requirements for such systems. Therefore, this report provides an overview of QA equipment and techniques for mechanical, dosimetric, and imaging performance of such systems and recommendation of the QA procedures, particularly for a 1.5 T MR-Linac device. An overview of the system design and considerations for QA measurements, particularly the effect of the machine geometry and magnetic field on the radiation beam measurements is given. The effect of the magnetic field on measurement equipment and methods is reviewed to provide a foundation for interpreting measurement results and devising appropriate methods. And lastly, a consensus overview of recommended QA, appropriate methods and tolerances is provided based on conventional QA protocols. The aim of this consensus work is to provide a foundation for QA protocols, comparative studies of system performance, and for future development of QA protocols and measurement methods.

3.
World Neurosurg ; 2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33588080

RESUMO

BACKGROUND: Tumors that take up and metabolize 5-aminolevulinic acid (5-ALA) emit bright pink fluorescence when illuminated with blue light, aiding surgeons in identifying the margin of resection. The adoption of this method is hindered by the blue light illumination, which is too dim to safely operate under, and therefore, necessitates switching back and forth from white-light mode. This paper examines the addition of an optimized secondary illuminant adapter (SIA) to improve usability of blue-light mode without degrading tumor contrast. METHODS: We used color science methods to evaluate the color of the secondary illuminant and its impact on color rendering index (CRI) as well as the tumor-to-background color contrast (TBCC), in data collected from seven patients with high-grade gliomas (WHO Grade III and IV). A secondary illuminant adapter was built to provide 475-600 nm light the intensity of which can be controlled by the surgeon and was evaluated in two additional patients. RESULTS: Secondary illuminant color had opposing effects on color rendering index (CRI) and tumor to background color contrast (TBCC); providing surgeon control of intensity allows this trade-off to be balanced in real-time. Demonstration in two high-grade glioma cases confirms this, showing that additional visibility adds value when the intensity can be controlled by the surgeon. CONCLUSIONS: The addition of a secondary illuminant may mitigate surgeon complaints that the operative field is too dark under the blue light illumination required for 5-ALA fluorescence guidance by providing improved CRI without completely sacrificing TBCC.

4.
J Cell Commun Signal ; 15(1): 93-105, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33415696

RESUMO

Targeted gene disruption in mice has provided valuable insights into the functions of matricellular proteins. Apart from missense and loss of function mutations that have been associated with inherited diseases, however, their functions in humans remain unclear. The availability of deep exome sequencing data from over 140,000 individuals in the Genome Aggregation Database provided an opportunity to examine intolerance to loss of function and missense mutations in human matricellular genes. The probability of loss-of-function intolerance (pLI) differed widely within members of the thrombospondin, CYR61/CTGF/NOV (CCN), tenascin, small integrin-binding ligand N-linked glycoproteins (SIBLING), and secreted protein, acidic and rich in cysteine (SPARC) gene families. Notably, pLI values in humans had limited correlation with viability of the corresponding homozygous null mice. Among the thrombospondins, only THBS1 was highly loss-intolerant (pLI = 1). In contrast, Thbs1 is not essential for viability in mice. Several known thrombospondin-1 receptors were similarly loss-intolerant, although thrombospondin-1 is not the exclusive ligand for some of these receptors. The frequencies of missense mutations in THBS1 and the gene encoding its signaling receptor CD47 indicated conservation of some residues implicated in specific receptor binding. Deficits in missense mutations were also observed for other thrombospondin genes and for SPARC, SPOCK1, SPOCK2, TNR, and DSPP. The intolerance of THBS1 to loss of function in humans and elevated pLI values for THBS2, SPARC, SPOCK1, TNR, and CCN1 support important functions for these matricellular protein genes in humans, some of which may relate to functions in reproduction or responding to environmental stresses.

5.
J Proteome Res ; 20(2): 1424-1433, 2021 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-33395532

RESUMO

Three-dimensional (3D) human induced pluripotent stem cell-derived engineered cardiac tissues (hiPSC-ECTs) have emerged as a promising alternative to two-dimensional hiPSC-cardiomyocyte monolayer systems because hiPSC-ECTs are a closer representation of endogenous cardiac tissues and more faithfully reflect the relevant cardiac pathophysiology. The ability to perform functional and molecular assessments using the same hiPSC-ECT construct would allow for more reliable correlation between observed functional performance and underlying molecular events, and thus is critically needed. Herein, for the first time, we have established an integrated method that permits sequential assessment of functional properties and top-down proteomics from the same single hiPSC-ECT construct. We quantitatively determined the differences in isometric twitch force and the sarcomeric proteoforms between two groups of hiPSC-ECTs that differed in the duration of time of 3D-ECT culture. Importantly, by using this integrated method we discovered a new and strong correlation between the measured contractile parameters and the phosphorylation levels of alpha-tropomyosin between the two groups of hiPSC-ECTs. The integration of functional assessments together with molecular characterization by top-down proteomics in the same hiPSC-ECT construct enables a holistic analysis of hiPSC-ECTs to accelerate their applications in disease modeling, cardiotoxicity, and drug discovery. Data are available via ProteomeXchange with identifier PXD022814.

6.
Phys Med Biol ; 2021 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-33503604

RESUMO

PURPOSE: To develop and demonstrate an end-to-end assessment procedure for adaptive radiotherapy (ART) within an MR-guided system. METHODS AND MATERIALS: A 3D printed pelvic phantom was designed and constructed for use in this study. The phantom was put through the complete radiotherapy treatment chain, with planned internal changes made to model prostate translations and shape changes, allowing an investigation into three ART techniques commonly used. Absolute dosimetry measurements were made within the phantom using both gafchromic film and alanine. Comparisons between treatment planning system calculations and measured dose values were made using the gamma evaluation with criteria of 3mm/3% and 2mm/2%. RESULTS: Gamma analysis evaluations for each type of treatment plan adaptation investigated showed a very high agreement with pass rates for each experiment ranging from 98.10% to 99.70% and 92.60% to 97.55%, for criteria of 3%/3 mm and 2%/2mm respectively. These pass rates were consistent for both shape and position changes. Alanine measurements further supported the results, showing an average difference of 1.98% from the treatment planning system. CONCLUSION: The end-to-end assessment procedure provided demanding challenges for treatment plan adaptations to demonstrate the capabilities and achieved high consistency in all findings.

7.
J Colloid Interface Sci ; 586: 876-890, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33309145

RESUMO

HYPOTHESIS: The α-sulfo alkyl ester, AES, surfactants are a class of anionic surfactants which have potential for improved sustainable performance in a range of applications, and an important feature is their enhanced tolerance to precipitation in the presence of multivalent counterions. It is proposed that their adsorption properties can be adjusted substantially by changing the length of the shorter alkyl chain, that of the alkanol group in the ester. EXPERIMENTS: Surface tension and neutron reflectivity have been used to investigate the variation in the adsorption properties with the shorter alkyl chain length (methyl, ethyl and propyl), the impact of NaCl on the adsorption, the tendency to form surface multilayer structures in the presence of AlCl3, and the effects of mixing the methyl ester sulfonate with the ethyl and propyl ester sulfonates on the adsorption. FINDINGS: The variations in the critical micelle concentration, CMC, the adsorption isotherms, the saturation adsorption values, and the impact of NaCl illustrate the subtle influence of varying the shorter alkyl chain length of the surfactant. The non-ideal mixing of pairs of AES surfactants with different alkanol group lengths of the ester show that the extent of the non-ideality changes as the difference in the alkanol length increases. The surface multilayer formation observed in the presence of AlCl3 varies in a complex manner with the length of the short chain and for mixtures of surfactants with different chains lengths.

9.
Wellcome Open Res ; 5: 181, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33283055

RESUMO

Background: Laboratory diagnosis of SARS-CoV-2 infection (the cause of COVID-19) uses PCR to detect viral RNA (vRNA) in respiratory samples. SARS-CoV-2 RNA has also been detected in other sample types, but there is limited understanding of the clinical or laboratory significance of its detection in blood. Methods: We undertook a systematic literature review to assimilate the evidence for the frequency of vRNA in blood, and to identify associated clinical characteristics. We performed RT-PCR in serum samples from a UK clinical cohort of acute and convalescent COVID-19 cases (n=212), together with convalescent plasma samples collected by NHS Blood and Transplant (NHSBT) (n=462 additional samples). To determine whether PCR-positive blood samples could pose an infection risk, we attempted virus isolation from a subset of RNA-positive samples. Results: We identified 28 relevant studies, reporting SARS-CoV-2 RNA in 0-76% of blood samples; pooled estimate 10% (95%CI 5-18%). Among serum samples from our clinical cohort, 27/212 (12.7%) had SARS-CoV-2 RNA detected by RT-PCR. RNA detection occurred in samples up to day 20 post symptom onset, and was associated with more severe disease (multivariable odds ratio 7.5). Across all samples collected ≥28 days post symptom onset, 0/494 (0%, 95%CI 0-0.7%) had vRNA detected. Among our PCR-positive samples, cycle threshold (ct) values were high (range 33.5-44.8), suggesting low vRNA copy numbers. PCR-positive sera inoculated into cell culture did not produce any cytopathic effect or yield an increase in detectable SARS-CoV-2 RNA. There was a relationship between RT-PCR negativity and the presence of total SARS-CoV-2 antibody (p=0.02). Conclusions: vRNA was detectable at low viral loads in a minority of serum samples collected in acute infection, but was not associated with infectious SARS-CoV-2 (within the limitations of the assays used). This work helps to inform biosafety precautions for handling blood products from patients with current or previous COVID-19.

10.
Nat Commun ; 11(1): 6385, 2020 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-33318491

RESUMO

The response to the coronavirus disease 2019 (COVID-19) pandemic has been hampered by lack of an effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antiviral therapy. Here we report the use of remdesivir in a patient with COVID-19 and the prototypic genetic antibody deficiency X-linked agammaglobulinaemia (XLA). Despite evidence of complement activation and a robust T cell response, the patient developed persistent SARS-CoV-2 pneumonitis, without progressing to multi-organ involvement. This unusual clinical course is consistent with a contribution of antibodies to both viral clearance and progression to severe disease. In the absence of these confounders, we take an experimental medicine approach to examine the in vivo utility of remdesivir. Over two independent courses of treatment, we observe a temporally correlated clinical and virological response, leading to clinical resolution and viral clearance, with no evidence of acquired drug resistance. We therefore provide evidence for the antiviral efficacy of remdesivir in vivo, and its potential benefit in selected patients.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Imunidade Humoral/efeitos dos fármacos , /efeitos dos fármacos , Monofosfato de Adenosina/uso terapêutico , Adulto , Alanina/uso terapêutico , Antivirais/uso terapêutico , Febre/prevenção & controle , Humanos , Imunidade Humoral/imunologia , Contagem de Linfócitos , Masculino , /fisiologia , Resultado do Tratamento
11.
J Ultrasound Med ; 2020 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-33368392

RESUMO

The "parallel transverse in-plane" technique for ultrasound-guided intra-articular hip interventions ensures needle visualization for the entire procedure, with the needle clearly shown entering the joint. With the widely described longitudinal in-plane approach, needle visualization can be poor, necessitating reliance on tissue distortion, which can reduce user confidence and safety. The parallel transverse in-plane approach is invaluable in those with anterior thigh skin breakdown and where anterior access is contraindicated. The approach also allows a broad width of the synovium to be traversed and is therefore well suited to synovial biopsy. This short Technical Innovation highlights this alternative approach to hip joint intervention.

12.
Transfus Med ; 30(6): 416-417, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33368756
13.
Transfus Med ; 2020 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-33333627

RESUMO

INTRODUCTION: The lack of approved specific therapeutic agents to treat coronavirus disease (COVID-19) associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has led to the rapid implementation of convalescent plasma therapy (CPT) trials in many countries, including the United Kingdom. Effective CPT is likely to require high titres of neutralising antibody (nAb) in convalescent donations. Understanding the relationship between functional neutralising antibodies and antibody levels to specific SARS-CoV-2 proteins in scalable assays will be crucial for the success of a large-scale collection. We assessed whether neutralising antibody titres correlated with reactivity in a range of enzyme-linked immunosorbent assays (ELISA) targeting the spike (S) protein, the main target for human immune response. METHODS: Blood samples were collected from 52 individuals with a previous laboratory-confirmed SARS-CoV-2 infection. These were assayed for SARS-CoV-2 nAbs by microneutralisation and pseudo-type assays and for antibodies by four different ELISAs. Receiver operating characteristic (ROC) analysis was used to further identify sensitivity and specificity of selected assays to identify samples containing high nAb levels. RESULTS: All samples contained SARS-CoV-2 antibodies, whereas neutralising antibody titres of greater than 1:20 were detected in 43 samples (83% of those tested) and >1:100 in 22 samples (42%). The best correlations were observed with EUROimmun immunoglobulin G (IgG) reactivity (Spearman Rho correlation coefficient 0.88; p < 0.001). Based on ROC analysis, EUROimmun would detect 60% of samples with titres of >1:100 with 100% specificity using a reactivity index of 9.1 (13/22). DISCUSSION: Robust associations between nAb titres and reactivity in several ELISA-based antibody tests demonstrate their possible utility for scaled-up production of convalescent plasma containing potentially therapeutic levels of anti-SARS-CoV-2 nAbs.

14.
Transfus Med ; 2020 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-33341984

RESUMO

OBJECTIVE: To compare four haemoglobin measurement methods in whole blood donors. BACKGROUND: To safeguard donors, blood services measure haemoglobin concentration in advance of each donation. NHS Blood and Transplant's (NHSBT) customary method have been capillary gravimetry (copper sulphate), followed by venous spectrophotometry (HemoCue) for donors failing gravimetry. However, NHSBT's customary method results in 10% of donors being inappropriately bled (ie, with haemoglobin values below the regulatory threshold). METHODS: We compared the following four methods in 21 840 blood donors (aged ≥18 years) recruited from 10 NHSBT centres in England, with the Sysmex XN-2000 haematology analyser, the reference standard: (1) NHSBT's customary method; (2) "post donation" approach, that is, estimating current haemoglobin concentration from that measured by a haematology analyser at a donor's most recent prior donation; (3) "portable haemoglobinometry" (using capillary HemoCue); (4) non-invasive spectrometry (using MBR Haemospect or Orsense NMB200). We assessed sensitivity; specificity; proportion who would have been inappropriately bled, or rejected from donation ("deferred") incorrectly; and test preference. RESULTS: Compared with the reference standard, the methods ranged in test sensitivity from 17.0% (MBR Haemospect) to 79.0% (portable haemoglobinometry) in men, and from 19.0% (MBR Haemospect) to 82.8% (portable haemoglobinometry) in women. For specificity, the methods ranged from 87.2% (MBR Haemospect) to 99.9% (NHSBT's customary method) in men, and from 74.1% (Orsense NMB200) to 99.8% (NHSBT's customary method) in women. The proportion of donors who would have been inappropriately bled ranged from 2.2% in men for portable haemoglobinometry to 18.9% in women for MBR Haemospect. The proportion of donors who would have been deferred incorrectly with haemoglobin concentration above the minimum threshold ranged from 0.1% in men for NHSBT's customary method to 20.3% in women for OrSense. Most donors preferred non-invasive spectrometry. CONCLUSION: In the largest study reporting head-to-head comparisons of four methods to measure haemoglobin prior to blood donation, our results support replacement of NHSBT's customary method with portable haemoglobinometry.

16.
Euro Surveill ; 25(45)2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33183404

RESUMO

We analysed factors associated with neutralising antibody levels in 330 convalescent plasma donors. Women and younger donors were more likely not to have measurable neutralising antibodies, while higher antibody levels were observed in men, in older donors and in those who had been hospitalised. These data will be of value in the timely recruitment of convalescent plasma donors most likely to have high levels of neutralising antibodies for ongoing studies investigating its effectiveness.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Doadores de Sangue , Infecções por Coronavirus/sangue , Infecções por Coronavirus/terapia , Hospitalização , Pneumonia Viral/sangue , Pneumonia Viral/terapia , Fatores Etários , Idoso , Anticorpos Neutralizantes/uso terapêutico , Doadores de Sangue/estatística & dados numéricos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/imunologia , Inglaterra , Ensaio de Imunoadsorção Enzimática , Humanos , Imunização Passiva , Masculino , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/imunologia , Fatores Sexuais
17.
Artigo em Inglês | MEDLINE | ID: mdl-33232185

RESUMO

Introduction- A better understanding of the underlying molecular mechanism of diseases is critical for developing more effective diagnostic tools and therapeutics towards precision medicine. However, many challenges remain to unravel the complex nature of diseases. Areas covered- Changes in protein isoform expression and post-translation modifications (PTMs) have gained recognition for their role in underlying disease mechanisms. Top-down mass spectrometry (MS)-based proteomics is increasingly recognized as an important method for the comprehensive characterization of proteoforms that arise from alternative splicing events and/or PTMs for basic and clinical research. Here, we review the challenges, technological innovations, and recent studies that utilize top-down proteomics to elucidate changes in the proteome with an emphasis on its use to study the diseases of the heart. Expert Opinion- Proteoform-resolved information can substantially contribute to the understanding of the molecular mechanisms underlying various diseases and for the identification of novel proteoform targets for better therapeutic development toward precision medicine. Despite the challenges of sequencing intact proteins, top-down proteomics has enabled a wealth of information regarding protein isoform switching and changes in PTMs. Continuous developments in sample preparation, intact protein separation, and instrumentation for top-down MS have broadened its capabilities to characterize proteoforms from a range of samples on an increasingly global scale.

18.
Artigo em Inglês | MEDLINE | ID: mdl-33244322

RESUMO

Thrombospondins are encoded in vertebrates by a family of 5 THBS genes. THBS1 is infrequently mutated in most cancers, but its expression is positively regulated by several tumor suppressor genes and negatively regulated by activated oncogenes and promoter hypermethylation. Consequently, thrombospondin-1 expression is frequently lost during oncogenesis and is correlated with a poor prognosis for some cancers. Thrombospondin-1 is a secreted protein that acts in the tumor microenvironment to inhibit angiogenesis, regulate antitumor immunity, stimulate tumor cell migration, and regulate the activities of extracellular proteases and growth factors. Differential effects of thrombospondin-1 on the sensitivity of normal versus malignant cells to ischemic and genotoxic stress also regulate the responses to tumors to therapeutic radiation and chemotherapy.

19.
Antib Ther ; 3(3): 179-192, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33244513

RESUMO

CD47 is a ubiquitously expressed cell surface glycoprotein that functions as a signaling receptor for thrombospondin-1 and as the counter-receptor for signal regulatory protein-α (SIRPα). Engaging SIRPα on macrophages inhibits phagocytosis, and CD47 thereby serves as a physiological marker of self. However, elevated CD47 expression on some cancer cells also protects tumors from innate immune surveillance and limits adaptive antitumor immunity via inhibitory SIRPα signaling in antigen presenting cells. CD47 also mediates inhibitory thrombospondin-1 signaling in vascular cells, T cells, and NK cells, and blocking inhibitory CD47 signaling on cytotoxic T cells directly increases tumor cell killing. Therefore, CD47 functions as an innate and adaptive immune checkpoint. These findings have led to the development of antibodies and other therapeutic approaches to block CD47 functions in the tumor microenvironment. Preclinical studies in mice demonstrated that blocking CD47 can limit the growth of hematologic malignancies and solid tumors and enhance the efficacy of conventional chemotherapy, radiation therapy, and some targeted cancer therapies. Humanized CD47 antibodies are showing promise in early clinical trials, but side effects related to enhanced phagocytic clearance of circulating blood cells remain a concern. Approaches to circumvent these include antibody preloading strategies, development of antibodies that recognize tumor-specific epitopes of CD47, SIRPα antibodies, and bivalent antibodies that restrict CD47 blockade to specific tumor cells. Preclinical and clinical development of antibodies and related biologics that inhibit CD47/SIRPα signaling are reviewed, including strategies to combine these agents with various conventional and targeted therapeutics to improve patient outcome for various cancers.

20.
Commun Biol ; 3(1): 703, 2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33239738

RESUMO

Restless legs syndrome (RLS) is a common neurological sensorimotor disorder often described as an unpleasant sensation associated with an urge to move the legs. Here we report findings from a meta-analysis of genome-wide association studies of RLS including 480,982 Caucasians (cases = 10,257) and a follow up sample of 24,977 (cases = 6,651). We confirm 19 of the 20 previously reported RLS sequence variants at 19 loci and report three novel RLS associations; rs112716420-G (OR = 1.25, P = 1.5 × 10-18), rs10068599-T (OR = 1.09, P = 6.9 × 10-10) and rs10769894-A (OR = 0.90, P = 9.4 × 10-14). At four of the 22 RLS loci, cis-eQTL analysis indicates a causal impact on gene expression. Through polygenic risk score for RLS we extended prior epidemiological findings implicating obesity, smoking and high alcohol intake as risk factors for RLS. To improve our understanding, with the purpose of seeking better treatments, more genetics studies yielding deeper insights into the disease biology are needed.

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