Methods for expression of recombinant proteins using a Pichia pastoris cell-free system

Cell‐free protein synthesis is a powerful tool for engineering biology and has been utilized in many diverse applications, from biosensing and protein prototyping to biomanufacturing and the design of metabolic pathways. By exploiting host cellular machinery decoupled from cellular growth, proteins can be produced in vitro both on demand and rapidly. Eukaryotic cell‐free platforms are often neglected due to perceived complexity and low yields relative to their prokaryotic counterparts, despite providing a number of advantageous properties. The yeast Pichia pastoris (also known as Komagataella phaffii) is a particularly attractive eukaryotic host from which to generate cell‐free extracts, due to its ability to grow to high cell densities with high volumetric productivity, genetic tractability for strain engineering, and ability to perform post‐translational modifications. Here, we describe methods for conducting cell‐free protein synthesis using P. pastoris as the host, from preparing the cell lysates to protocols for both coupled and linked transcription‐translation reactions. By providing these methodologies, we hope to encourage the adoption of the platform by new and experienced users alike

Cytoplasmic expression systems triggered by mRNA yield increased gene expression in post-mitotic neurons

Non-viral vectors are promising vehicles for gene therapy but delivery of plasmid DNA to post-mitotic cells is challenging as nuclear entry is particularly inefficient. We have developed and evaluated a hybrid mRNA/DNA system designed to bypass the nuclear barrier to transfection and facilitate cytoplasmic gene expression. This system, based on co-delivery of mRNA(A64) encoding for T7 RNA polymerase (T7 RNAP) with a T7-driven plasmid, produced between 10- and 2200-fold higher gene expression in primary dorsal root ganglion neuronal (DRGN) cultures isolated from Sprague–Dawley rats compared to a cytomegalovirus (CMV)-driven plasmid, and 30-fold greater expression than the enhanced T7-based autogene plasmid pR011. Cell-free assays and in vitro transfections highlighted the versatility of this system with small quantities of T7 RNAP mRNA required to mediate expression at levels that were significantly greater than with the T7-driven plasmid alone or supplemented with T7 RNAP protein. We have also characterized a number of parameters, such as mRNA structure, intracellular stability and persistence of each nucleic acid component that represent important factors in determining the transfection efficiency of this hybrid expression system. The results from this study demonstrate that co-delivery of mRNA is a promising strategy to yield increased expression with plasmid DNA, and represents an important step towards improving the capability of non-viral vectors to mediate efficient gene transfer in cell types, such as in DRGN, where the nuclear membrane is a significant barrier to transfection

Fall Classification by Machine Learning Using Mobile Phones

Fall prevention is a critical component of health care; falls are a common source of injury in the elderly and are associated with significant levels of mortality and morbidity. Automatically detecting falls can allow rapid response to potential emergencies; in addition, knowing the cause or manner of a fall can be beneficial for prevention studies or a more tailored emergency response. The purpose of this study is to demonstrate techniques to not only reliably detect a fall but also to automatically classify the type. We asked 15 subjects to simulate four different types of falls–left and right lateral, forward trips, and backward slips–while wearing mobile phones and previously validated, dedicated accelerometers. Nine subjects also wore the devices for ten days, to provide data for comparison with the simulated falls. We applied five machine learning classifiers to a large time-series feature set to detect falls. Support vector machines and regularized logistic regression were able to identify a fall with 98% accuracy and classify the type of fall with 99% accuracy. This work demonstrates how current machine learning approaches can simplify data collection for prevention in fall-related research as well as improve rapid response to potential injuries due to falls

Definitions and methods of measuring and reporting on injurious falls in randomised controlled fall prevention trials: a systematic review

<p>Abstract</p> <p>Background</p> <p>The standardisation of the assessment methodology and case definition represents a major precondition for the comparison of study results and the conduction of meta-analyses. International guidelines provide recommendations for the standardisation of falls methodology; however, injurious falls have not been targeted. The aim of the present article was to review systematically the range of case definitions and methods used to measure and report on injurious falls in randomised controlled trials (RCTs) on fall prevention.</p> <p>Methods</p> <p>An electronic literature search of selected comprehensive databases was performed to identify injurious falls definitions in published trials. Inclusion criteria were: RCTs on falls prevention published in English, study population ≥ 65 years, definition of injurious falls as a study endpoint by using the terms "injuries" and "falls".</p> <p>Results</p> <p>The search yielded 2089 articles, 2048 were excluded according to defined inclusion criteria. Forty-one articles were included. The systematic analysis of the methodology applied in RCTs disclosed substantial variations in the definition and methods used to measure and document injurious falls. The limited standardisation hampered comparability of study results. Our results also highlight that studies which used a similar, standardised definition of injurious falls showed comparable outcomes.</p> <p>Conclusions</p> <p>No standard for defining, measuring, and documenting injurious falls could be identified among published RCTs. A standardised injurious falls definition enhances the comparability of study results as demonstrated by a subgroup of RCTs used a similar definition. Recommendations for standardising the methodology are given in the present review.</p

A decentralised, multidisciplinary model of care facilitates treatment of hepatitis C in regional Australia.

ObjectivesDirect-acting antiviral (DAA) therapy for hepatitis C virus (HCV) has excellent cure rates and minimal side effects. Despite the high burden of disease, strategies to ultimately eradicate HCV are being developed. However, the delivery of care in regional settings is challenging and the efficacy of decentralised models of care is incompletely defined.MethodsA prospective cohort study of patients whose treatment was initiated or supervised by Cairns Hospital, a tertiary hospital which provides services to a culturally diverse population across a 380,748 km2 area in regional Australia. Patients' demographics, clinical features, DAA regimens and outcomes were recorded and correlated with their ensuing clinical course.ResultsOver 22 months, 734 patients were prescribed DAA therapy for HCV. No patients were prescribed interferon. Sofosbuvir/ledipasvir (n=371, 50.5%) and sofosbuvir/daclatasvir (n=287, 39.1%) were the most commonly prescribed regimens. No patients ceased treatment due to adverse effects. There were 612/734 (83.4%) patients with complete results, with 575 (94%) cured. At the end of the study period, there were 50 (6.8%) patients lost to follow-up and 72 (9.8%) awaiting SVR12 testing. The presence of cirrhosis (n=147/612, 24.1%) did not impact significantly on SVR12 rates, this being achieved in 136/147 (92.5%) cirrhotic patients versus 440/465 (94.6%) in non-cirrhotic patients (p=0.34). Treatment-experienced patients (95/612, 18.3%) were more likely to be non-responders than treatment-naïve patients (10/95 (10.5%) versus 26/517 (5%), p=0.04). Strategies to facilitate treatment included a dedicated clinical nurse consultant, education to primary health care providers, specialist outreach clinics to regional communities and shared care with general practitioners. SVR12 rates were similar amongst gastroenterologists (283/306, 92.5%), general practitioners (152/161, 94.4%), sexual health physicians (104/106, 98.1%) and other prescribers (37/39, 94.9%).ConclusionsThis study confirms that decentralised, multidisciplinary models of care can provide HCV treatment in regional and remote settings with excellent outcomes