Colloidal Gel and Its Application in Tissue Engineering

Three dimensional, porous polymer scaffolds are fabricated by direct writing of colloidal gels. This work focuses on both the processing of colloidal gel and assembly of the scaffold structures as well as characterization of cytotoxicity and protein release kinetics. Specifically, rheological and elastic properties of the colloidal gels are probed as a function of solids loading and binder concentration. Porous scaffolds are characterized by optical and electron microscopy. In vitro studies include cell mortality after six weeks culture on passive scaffolds, model protein release profiles from scaffolds, and quantitative measurement of protein activity upon release from the scaffolds by chemotaxis. The polymer colloidal gels formulated with acrylic latex particles and Pluronic F127 copolymer binder have pseudoplastic with yield stress rheology. Increases in solids loading and Pluronic concentration cause increased viscosity, elastic modulus, and yield stress. The rheology and rapid recovery of yield allow for flow through a deposition nozzle of the direct write tool and rapid setting of the extrudate to maintain the deposited structure. Scaffolds with a wide variety of porosity are fabricated. Because of the aqueous and low temperature nature of the process, bioactive molecules such as proteins are readily incorporated into the scaffold either in their original form or encapsulated in chitosan nanoparticles and subsequently released without denaturation and in a controlled fashion. Protein release rate is dependant on both the degree of coalescence of the scaffold material and the molecular weight of the chitosan nanoparticles. Protein inclusion and subsequent release is demonstrated using BSA and PDGF-BB. The scaffolds fabricated are non-cytotoxic as confirmed by QEC6 cell culture. Heterogeneous scaffolds with localized regions of dissolved species are demonstrated to illustrate the capability to assembly scaffolds with functional gradients or segregated zones.School of Chemical Engineerin

Comprehensive Evaluation of Self-Healing Polyampholyte Gel Particles for the Severe Leakoff Control of Drilling Fluids

Lost circulation has been a serious problem to be solved in many drilling practices during oil, gas and geothermal well drillings. Many materials have been developed and evaluated for the purpose. However, their performance to plug severe leakoff is very limited. Herein, an injectable self-healing hydrogel based on polyampholyte with sulfonated and quaternary ammonium functionalities (P(MPTC-co-NaSS)) was developed and comprehensively evaluated to prevent the severe loss of fluids to formation. By incorporating cation-π (π is for aromatic residues) interaction, the hydrogel shown self-healing property and robustness in severe environment (temperature, salt) by comparison with other hydrogels merely consisting of cation-anion and H-bonding interactions. Aromatic residues enhanced thermal stability above 310 °C. The plugging measurement shown that an addition of 2 wt% dried gel particles can plug high-permeability formation and endure a high pressure of 6 MPa, produce much lower circulation loss and result in a dramatically increased loss volume reduction rate (63.5%) compared with a commercial polymer gel product and an inert material (9.4%) after a self-healing process. Markedly, P(MPTC-co-NaSS) can be used in a wide range of formation temperature (as high as 150 °C) and salt concentrations (NaCl, CaCl2, as high as 15 wt %). In addition to suitable particle size and mechanically robustness, it was also attributed to the soft, swelling, deformable, toughness and self-healable features of P(MPTC-co-NaSS) gel particles as well as the strong adhesion to negatively charged formations in water, even under high thermal and saline condition. These characteristics also contributed to a long-term plugging performance, beneficial to avoid repeated lost circulation in drilling operation. Besides, this self-healing polyampholyte gel particles dispersed well in saline fluid and maintained stable rheological properties after hot rolling, which was favorable to drilling fluid circulation. This study shown the application potential of self-healing materials as plugging material candidate in petroleum drilling industry

Designer cell signal processing circuits for biotechnology

Microorganisms are able to respond effectively to diverse signals from their environment and internal metabolism owing to their inherent sophisticated information processing capacity. A central aim of synthetic biology is to control and reprogramme the signal processing pathways within living cells so as to realise repurposed, beneficial applications ranging from disease diagnosis and environmental sensing to chemical bioproduction. To date most examples of synthetic biological signal processing have been built based on digital information flow, though analogue computing is being developed to cope with more complex operations and larger sets of variables. Great progress has been made in expanding the categories of characterised biological components that can be used for cellular signal manipulation, thereby allowing synthetic biologists to more rationally programme increasingly complex behaviours into living cells. Here we present a current overview of the components and strategies that exist for designer cell signal processing and decision making, discuss how these have been implemented in prototype systems for therapeutic, environmental, and industrial biotechnological applications, and examine emerging challenges in this promising field

A modular cell-based biosensor using engineered genetic logic circuits to detect and integrate multiple environmental signals

AbstractCells perceive a wide variety of cellular and environmental signals, which are often processed combinatorially to generate particular phenotypic responses. Here, we employ both single and mixed cell type populations, pre-programmed with engineered modular cell signalling and sensing circuits, as processing units to detect and integrate multiple environmental signals. Based on an engineered modular genetic AND logic gate, we report the construction of a set of scalable synthetic microbe-based biosensors comprising exchangeable sensory, signal processing and actuation modules. These cellular biosensors were engineered using distinct signalling sensory modules to precisely identify various chemical signals, and combinations thereof, with a quantitative fluorescent output. The genetic logic gate used can function as a biological filter and an amplifier to enhance the sensing selectivity and sensitivity of cell-based biosensors. In particular, an Escherichia coli consortium-based biosensor has been constructed that can detect and integrate three environmental signals (arsenic, mercury and copper ion levels) via either its native two-component signal transduction pathways or synthetic signalling sensors derived from other bacteria in combination with a cell-cell communication module. We demonstrate how a modular cell-based biosensor can be engineered predictably using exchangeable synthetic gene circuit modules to sense and integrate multiple-input signals. This study illustrates some of the key practical design principles required for the future application of these biosensors in broad environmental and healthcare areas

Reprogrammed tracrRNAs enable repurposing of RNAs as crRNAs and sequence-specific RNA biosensors

In type II CRISPR systems, the guide RNA (gRNA) comprises a CRISPR RNA (crRNA) and a hybridized trans-acting CRISPR RNA (tracrRNA), both being essential in guided DNA targeting functions. Although tracrRNAs are diverse in sequence and structure across type II CRISPR systems, the programmability of crRNA-tracrRNA hybridization for Cas9 is not fully understood. Here, we reveal the programmability of crRNA-tracrRNA hybridization for Streptococcus pyogenes Cas9, and in doing so, redefine the capabilities of Cas9 proteins and the sources of crRNAs, providing new biosensing applications for type II CRISPR systems. By reprogramming the crRNA-tracrRNA hybridized sequence, we show that engineered crRNA-tracrRNA interactions can not only enable the design of orthogonal cellular computing devices but also facilitate the hijacking of endogenous small RNAs/mRNAs as crRNAs. We subsequently describe how these re-engineered gRNA pairings can be implemented as RNA sensors, capable of monitoring the transcriptional activity of various environment-responsive genomic genes, or detecting SARS-CoV-2 RNA in vitro, as an Atypical gRNA-activated Transcription Halting Alarm (AGATHA) biosensor

Ticagrelor vs Clopidogrel in CYP2C19 loss-of-function carriers with Stroke or TIA

BACKGROUNDComparisons between ticagrelor- aspirin and clopidogrel-aspirin in CYP2C19 loss-of-function carriers have not been well studied for secondary stroke prevention.METHODSWe conducted a randomized, double-blind, placebo-controlled trial of 6,412 patients with a minor ischemic stroke or TIA who carried CYP2C19 LOF alleles determined by point-of-care testing. Patients were randomly assigned within 24 hours after symptom onset, in a 1:1 ratio to receive ticagrelor (180 mg loading dose on day 1 followed by 90 mg twice daily for days 2 through 90) or clopidogrel (300 mg loading dose on day 1 followed by 75 mg per day for days 2 through 90), plus aspirin (75-300 mg loading dose followed by 75 mg daily for 21 days). The primary efficacy outcome was stroke and the primary safety outcome was severe or moderate bleeding, both within 90 days. RESULTSStroke occurred within 90 days in 191 (6.0%) versus 243 (7.6%), respectively (hazard ratio, 0.77; 95% confidence interval, 0.64 to 0.94; P=0.008). Moderate or severe bleeding occurred in 9 patients (0.3%) in the ticagrelor-aspirin group and in 11 patients (0.3%) in the clopidogrel-aspirin group; any bleeding event occurred in 170 patients (5.3%) vs 80 (2.5%), respectively. CONCLUSIONSAmong Chinese patients with minor ischemic stroke or TIA within 24 hours after symptoms onset who were carriers of CYP2C19 loss-of-function alleles, ticagrelor- aspirin was modestly better than clopidogrel-aspirin for reducing the risk of stroke but was associated with more total bleeding events at 90 days. (CHANCE-2 ClinicalTrials.gov number, NCT04078737.

Recognizing and engineering digital-like logic gates and switches in gene regulatory networks

A central aim of synthetic biology is to build organisms that can perform useful activities in response to specified conditions. The digital computing paradigm which has proved so successful in electrical engineering is being mapped to synthetic biological systems to allow them to make such decisions. However, stochastic molecular processes have graded input-output functions, thus, bioengineers must select those with desirable characteristics and refine their transfer functions to build logic gates with digital-like switching behaviour. Recent efforts in genome mining and the development of programmable RNA-based switches, especially CRISPRi, have greatly increased the number of parts available to synthetic biologists. Improvements to the digital characteristics of these parts are required to enable robust predictable design of deeply layered logic circuits