Latent analysis of unmodified biomolecules and their complexes in solution with attomole detection sensitivity

The study of biomolecular interactions is central to an understanding of function, malfunction and therapeutic modulation of biological systems, yet often involves a compromise between sensitivity and accuracy. Many conventional analytical steps and the procedures required to facilitate sensitive detection, such as the incorporation of chemical labels, are prone to perturb the complexes under observation. Here we present a 'latent' analysis approach that uses chemical and microfluidic tools to reveal, through highly sensitive detection of a labelled system, the behaviour of the physiologically relevant unlabelled system. We implement this strategy in a native microfluidic diffusional sizing platform, allowing us to achieve detection sensitivity at the attomole level, determine the hydrodynamic radii of biomolecules that vary by over three orders of magnitude in molecular weight, and study heterogeneous mixtures. We illustrate these key advantages by characterizing a complex of an antibody domain in the solution phase and under physiologically relevant conditions.We would like to thank the ERC, BBSRC, Wellcome Trust, Newman Foundation, Winston Churchill Foundation, and Elan Pharmaceuticals for financial support. E.D.G was supported by the MRC (G1002272)

Elective cancer surgery in COVID-19-free surgical pathways during the SARS-CoV-2 pandemic: An international, multicenter, comparative cohort study

PURPOSE As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19–free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19–free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19–free surgical pathways. Patients who underwent surgery within COVID-19–free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19–free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score–matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19–free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION Within available resources, dedicated COVID-19–free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Elective Cancer Surgery in COVID-19-Free Surgical Pathways During the SARS-CoV-2 Pandemic: An International, Multicenter, Comparative Cohort Study.

PURPOSE: As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19-free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS: This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19-free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS: Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19-free surgical pathways. Patients who underwent surgery within COVID-19-free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19-free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score-matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19-free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION: Within available resources, dedicated COVID-19-free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Single-molecule measurements of transient biomolecular complexes through microfluidic dilution

Single-molecule confocal microscopy experiments require concentrations which are low enough to guarantee that, on average, less than one single molecule resides in the probe volume at any given time. Such concentrations are, however, significantly lower than the dissociation constants of many biological complexes which can therefore dissociate under single-molecule conditions. To address the challenge of observing weakly bound complexes in single-molecule experiments in solution, we have designed a microfluidic device that rapidly dilutes samples by up to one hundred thousand times, allowing the observation of unstable complexes before they dissociate. The device can interface with standard biochemistry laboratory experiments and generates a spatially uniform dilution that is stable over time allowing the quantification of the relative concentrations of different molecular species

Proliferation of amyloid-beta 42 aggregates occurs through a secondary nucleation mechanism

The generation of toxic oligomers during the aggregation of the amyloid-beta (A beta) peptide A beta 42 into amyloid fibrils and plaques has emerged as a central feature of the onset and progression of Alzheimer's disease, but the molecular pathways that control pathological aggregation have proved challenging to identify. Here, we use a combination of kinetic studies, selective radiolabeling experiments, and cell viability assays to detect directly the rates of formation of both fibrils and oligomers and the resulting cytotoxic effects. Our results show that once a small but critical concentration of amyloid fibrils has accumulated, the toxic oligomeric species are predominantly formed from monomeric peptide molecules through a fibril-catalyzed secondary nucleation reaction, rather than through a classical mechanism of homogeneous primary nucleation. This catalytic mechanism couples together the growth of insoluble amyloid fibrils and the generation of diffusible oligomeric aggregates that are implicated as neurotoxic agents in Alzheimer's disease. These results reveal that the aggregation of A beta 42 is promoted by a positive feedback loop that originates from the interactions between the monomeric and fibrillar forms of this peptide. Our findings bring together the main molecular species implicated in the A beta aggregation cascade and suggest that perturbation of the secondary nucleation pathway identified in this study could be an effective strategy to control the proliferation of neurotoxic A beta 42 oligomers

Single-Molecule Measurements of Transient Biomolecular Complexes through Microfluidic Dilution

Single-molecule confocal microscopy experiments require concentrations which are low enough to guarantee that, on average, less than one single molecule resides in the probe volume at any given time. Such concentrations are, however, significantly lower than the dissociation constants of many biological complexes which can therefore dissociate under single-molecule conditions. To address the challenge of observing weakly bound complexes in single-molecule experiments in solution, we have designed a microfluidic device that rapidly dilutes samples by up to one hundred thousand times, allowing the observation of unstable complexes before they dissociate. The device can interface with standard biochemistry laboratory experiments and generates a spatially uniform dilution that is stable over time allowing the quantification of the relative concentrations of different molecular species