Quaternization of Vinyl/Alkynyl Pyridine Enables Ultrafast Cysteine-Selective Protein Modification and Charge Modulation.

Quaternized vinyl- and alkynyl-pyridine reagents were shown to react in an ultrafast and selective manner with several cysteine-tagged proteins at near-stoichiometric quantities. We have demonstrated that this method can effectively create a homogenous antibody-drug conjugate that features a precise drug-to-antibody ratio of 2, which was stable in human plasma and retained its specificity towards Her2+ cells. Finally, the developed warhead introduces a +1 charge to the overall net charge of the protein, which enabled us to show that the electrophoretic mobility of the protein may be tuned through the simple attachment of a quaternized vinyl pyridinium reagent at the cysteine residues. We anticipate the generalized use of quaternized vinyl- and alkynyl-pyridine reagents not only for bioconjugation, but also as warheads for covalent inhibition and as tools to profile cysteine reactivity

Gradient-free determination of isoelectric points of proteins on chip

The isoelectric point (pI) of a protein is a key characteristic that influences its overall electrostatic behaviour. The majority of conventional methods for the determination of the isoelectric point of a molecule rely on the use of spatial gradients in pH, although significant practical challenges are associated with such techniques, notably the difficulty in generating a stable and well controlled pH gradient. Here, we introduce a gradient-free approach, exploiting a microfluidic platform which allows us to perform rapid pH change on chip and probe the electrophoretic mobility of species in a controlled field. In particular, in this approach, the pH of the electrolyte solution is modulated in time rather than in space, as in the case for conventional determinations of the isoelectric point. To demonstrate the general approachability of this platform, we have measured the isoelectric points of representative set of seven proteins, bovine serum albumin, $\beta$-lactoglobulin, ribonuclease A, ovalbumin, human transferrin, ubiquitin and myoglobin in microlitre sample volumes. The ability to conduct measurements in free solution thus provides the basis for the rapid determination of isoelectric points of proteins under a wide variety of solution conditions and in small volumes.We acknowledge financial support from the Biotechnology and Biological Sciences Research Council, the Newman Foundation and the Elan Pharmaceuticals. The research leading to these results has also received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007- 2013) through the ERC grant PhysProt (agreement n1 337969)

Enhancing power density of biophotovoltaics by decoupling storage and power delivery

Biophotovoltaic devices (BPVs), which use photosynthetic organisms as active materials to harvest light, have a range of attractive features relative to synthetic and non-biological photovoltaics, including their environmentally friendly nature and ability to self-repair. However, efficiencies of BPVs are currently lower than those of synthetic analogues. Here, we demonstrate BPVs delivering anodic power densities of over 0.5 W m−2, a value five-fold higher than for previously described BPVs. We achieved this through the use of cyanobacterial mutants with increased electron export characteristics together with a microscale flowbased design that allowed independent optimisation of the charging and power delivery processes, as well as membrane-free operation by exploiting laminar flow to separate the catholyte and anolyte streams. These results suggest that miniaturisation of active elements and flow control for decoupled operation and independent optimisation of the core processes involved in BPV design are effective strategies for enhancing power output and thus the potential of BPVs as viable systems for sustainable energy generation

Pooled analysis of WHO Surgical Safety Checklist use and mortality after emergency laparotomy

Background The World Health Organization (WHO) Surgical Safety Checklist has fostered safe practice for 10 years, yet its place in emergency surgery has not been assessed on a global scale. The aim of this study was to evaluate reported checklist use in emergency settings and examine the relationship with perioperative mortality in patients who had emergency laparotomy. Methods In two multinational cohort studies, adults undergoing emergency laparotomy were compared with those having elective gastrointestinal surgery. Relationships between reported checklist use and mortality were determined using multivariable logistic regression and bootstrapped simulation. Results Of 12 296 patients included from 76 countries, 4843 underwent emergency laparotomy. After adjusting for patient and disease factors, checklist use before emergency laparotomy was more common in countries with a high Human Development Index (HDI) (2455 of 2741, 89.6 per cent) compared with that in countries with a middle (753 of 1242, 60.6 per cent; odds ratio (OR) 0.17, 95 per cent c.i. 0.14 to 0.21, P <0001) or low (363 of 860, 422 per cent; OR 008, 007 to 010, P <0.001) HDI. Checklist use was less common in elective surgery than for emergency laparotomy in high-HDI countries (risk difference -94 (95 per cent c.i. -11.9 to -6.9) per cent; P <0001), but the relationship was reversed in low-HDI countries (+121 (+7.0 to +173) per cent; P <0001). In multivariable models, checklist use was associated with a lower 30-day perioperative mortality (OR 0.60, 0.50 to 073; P <0.001). The greatest absolute benefit was seen for emergency surgery in low- and middle-HDI countries. Conclusion Checklist use in emergency laparotomy was associated with a significantly lower perioperative mortality rate. Checklist use in low-HDI countries was half that in high-HDI countries.Peer reviewe

Global variation in anastomosis and end colostomy formation following left-sided colorectal resection

Background End colostomy rates following colorectal resection vary across institutions in high-income settings, being influenced by patient, disease, surgeon and system factors. This study aimed to assess global variation in end colostomy rates after left-sided colorectal resection. Methods This study comprised an analysis of GlobalSurg-1 and -2 international, prospective, observational cohort studies (2014, 2016), including consecutive adult patients undergoing elective or emergency left-sided colorectal resection within discrete 2-week windows. Countries were grouped into high-, middle- and low-income tertiles according to the United Nations Human Development Index (HDI). Factors associated with colostomy formation versus primary anastomosis were explored using a multilevel, multivariable logistic regression model. Results In total, 1635 patients from 242 hospitals in 57 countries undergoing left-sided colorectal resection were included: 113 (6·9 per cent) from low-HDI, 254 (15·5 per cent) from middle-HDI and 1268 (77·6 per cent) from high-HDI countries. There was a higher proportion of patients with perforated disease (57·5, 40·9 and 35·4 per cent; P < 0·001) and subsequent use of end colostomy (52·2, 24·8 and 18·9 per cent; P < 0·001) in low- compared with middle- and high-HDI settings. The association with colostomy use in low-HDI settings persisted (odds ratio (OR) 3·20, 95 per cent c.i. 1·35 to 7·57; P = 0·008) after risk adjustment for malignant disease (OR 2·34, 1·65 to 3·32; P < 0·001), emergency surgery (OR 4·08, 2·73 to 6·10; P < 0·001), time to operation at least 48 h (OR 1·99, 1·28 to 3·09; P = 0·002) and disease perforation (OR 4·00, 2·81 to 5·69; P < 0·001). Conclusion Global differences existed in the proportion of patients receiving end stomas after left-sided colorectal resection based on income, which went beyond case mix alone

Analysis of αb-crystallin polydispersity in solution through native microfluidic electrophoresis

In recent years, significant advancements have been made in the understanding of the population distributions and dynamic oligomeric states of the molecular chaperone αB-crystallin and its core domain variants. In this work, we provide solution-phase evidence of the polydispersity of αB- crystallin using microfluidic methods, used for separating the oligomeric species present in solution according to their different electrophoretic mobilities on-chip in a matter of seconds. We, in particular demonstrate that microfluidic high-field electrophoresis and diffusion can detect the oligomerisation of these highly dynamic molecular chaperones and characterise the dominant oligomeric species present. We thereby provide a robust microfluidic method for characterising the individual species within complex protein mixtures of biological relevance

Analysis of αB-crystallin polydispersity in solution through native microfluidic electrophoresis

In recent years, significant advancements have been made in the understanding of the population distributions and dynamic oligomeric states of the molecular chaperone αB-crystallin and its core domain variants. In this work, we provide solution-phase evidence of the polydispersity of αB-crystallin using microfluidic methods, used for separating the oligomeric species present in solution according to their different electrophoretic mobilities on-chip in a matter of seconds. We in particular demonstrate that microfluidic high-field electrophoresis and diffusion can detect the oligomerisation of these highly dynamic molecular chaperones and characterise the dominant oligomeric species present. We thereby provide a robust microfluidic method for characterising the individual species within complex protein mixtures of biological relevance

Real-time intrinsic fluorescence visualization and sizing of proteins and protein complexes in microfluidic devices

Optical detection has become a convenient and scalable approach to read out information from microfluidic systems. For the study of many key biomolecules, however, including peptides and proteins, which have low fluorescence emission efficiencies at visible wavelengths, this approach typically requires labeling of the species of interest with extrinsic fluorophores to enhance the optical signal obtained - a process which can be time-consuming, requires purification steps, and has the propensity to perturb the behavior of the systems under study due to interactions between the labels and the analyte molecules. As such, the exploitation of the intrinsic fluorescence of protein molecules in the UV range of the electromagnetic spectrum is an attractive path to allow the study of unlabeled proteins. However, direct visualization using 280 nm excitation in microfluidic devices has to date commonly required the use of coherent sources with frequency multipliers and devices fabricated out of materials that are incompatible with soft lithography techniques. Here, we have developed a simple, robust, and cost-effective 280 nm LED platform that allows real-time visualization of intrinsic fluorescence from both unlabeled proteins and protein complexes in polydimethylsiloxane microfluidic channels fabricated through soft lithography. Using this platform, we demonstrate intrinsic fluorescence visualization of proteins at nanomolar concentrations on chip and combine visualization with micron-scale diffusional sizing to measure the hydrodynamic radii of individual proteins and protein complexes under their native conditions in solution in a label-free manner

Micromechanics of soft materials using microfluidics

Abstract Micron-scale soft materials are finding a wide range of applications in bioengineering and molecular medicine, while also increasingly emerging as useful components for consumer products. The mechanical characterization of such microscale soft objects is conventionally performed with techniques such as atomic force microscopy or micropipette aspiration that measure the local properties of micron scale objects in a serial manner. To permit scalable characterization of the global mechanical properties of soft microscale objects, we developed and describe here a microfluidic platform that can be used for performing parallelized integrated measurements of the shear modulus of individual microscale particles. We demonstrate the effectiveness of this approach by characterizing the mechanical properties of multiple protein microgels in parallel, and show that the obtained values are in good agreement with conventional serial measurements. This platform allows parallelized in situ measurements of the mechanical properties of soft deformable micron-scale particles, and builds on scalable single-layer soft-photolithography fabrication, making the measurement system readily adaptable for a range of potential applications. Graphical Abstract </jats:sec