Vimentin-mediated myosin 10 aggregation at tips of cell extensions drives MT1-MMP-dependent collagen degradation in colorectal cancer.

Colorectal cancer (CRC) is a high prevalence adenocarcinoma with progressive increases in metastasis-related mortality, but the mechanisms governing the extracellular matrix (ECM) degradation important for metastasis in CRC are not well-defined. We investigated a functional relationship between vimentin (Vim) and myosin 10 (Myo10), and whether this relationship is associated with cancer progression. We tested the hypothesis that Vim regulates the aggregation of Myo10 at the tips of cell extensions, which increases membrane-type 1 matrix metalloproteinase (MT1-MMP)-associated local collagen proteolysis and ECM degradation. Analysis of CRC samples revealed colocalization of Vim with Myo10 and MT1-MMP in cell extensions adjacent to sites of collagen degradation, suggesting an association with local cell invasion. We analyzed cultured CRC cells and fibroblasts and found that Vim accelerates aggregation of Myo10 at cell tips, which increases the cell extension rate. Vim stabilizes the interaction of Myo10 with MT1-MMP, which in turn increases collagenolysis. Vim depletion reduced the aggregation of Myo10 at the cell extension tips and MT1-MMP-dependent collagenolysis. We propose that Vim interacts with Myo10, which in turn associates with MT1-MMP to facilitate the transport of these molecules to the termini of cell extensions and there enhance cancer invasion of soft connective tissues.

Adaptive, diverse and de-centralized diagnostics are key to the future of outbreak response.

The global spread of SARS-CoV-2 has shaken our health care and economic systems, prompting re-evaluation of long-held views on how best to deliver care. This is especially the case for our global diagnostic strategy. While current laboratory-based centralized RT-qPCR will continue to serve as a gold standard diagnostic into the foreseeable future, the shortcomings of our dependence on this method have been laid bare. It is now clear that a robust diagnostics pandemic response strategy, like any disaster planning, must include adaptive, diverse and de-centralized solutions. Here we look at how the COVID-19 pandemic, and previous outbreaks, have set the stage for a new innovative phase in diagnostics and a re-thinking of pandemic preparedness.

Carbon footprint of low-energy buildings in the United Kingdom: Effects of mitigating technological pathways and decarbonization strategies.

There is a limited comprehensive analysis of the effectiveness of adopted carbon mitigation strategies for buildings over their life cycle, that are concerned with temporal perspectives of emissions. Accordingly, this paper explores a life cycle assessment (LCA) to address the concerns regarding mitigating the carbon footprint of a UK timber-frame low-energy dwelling. In particular, it aims to investigate the potential greenhouse gas (GHG) emission reduction in terms of three different heating and ventilation options, and to analyze the influence of decarbonization of electricity production as well as the technological progress of the waste treatment of timber on the building's environmental performance. Thus, the whole life­carbon of the building case studies was evaluated for a total of eight investigated prospective scenarios, and they were compared to the LCA results of the baseline scenario, where the existing technology and context remained constant over time. Results show that using a compact heat pump would lead to a significant whole life-cycle emission reduction of the dwelling, by 19 %; while GHG emission savings can be reinforced if the assessed systems are employed simultaneously with grid decarbonization, exhibiting a 25 %-60 % reduction compared to the baseline scenario. Moreover, technological changes in the waste treatments of timber products could substantially reduce the buildings' embodied emissions, representing 3 %-23 %. From these emission-saving measures, the contribution of material efficiency strategies to achieve more embodied carbon savings should be highlighted in future construction practices.

Cell-Free Dot Blot: an Ultra-Low-Cost and Practical Immunoassay Platform for Detection of Anti-SARS-CoV-2 Antibodies in Human and Animal Sera.

Since its emergence in late 2019, the coronavirus disease 2019 (COVID-19) pandemic has caused severe disruption to key aspects of human life globally and highlighted the need for timely, adaptive, and accessible pandemic response strategies. Here, we introduce the cell-free dot blot (CFDB) method, a practical and ultra-low-cost immune diagnostic platform capable of rapid response and mass immunity screening for the current and future pandemics. Similar in mechanism to the widely used enzyme-linked immunosorbent assays (ELISAs), our method is novel and advantageous in that (i) it uses linear DNA to produce the target viral antigen fused to a SpyTag peptide in a cell-free expression system without the need for traditional cloning and antigen purification, (ii) it uses SpyCatcher2-Apex2, an Escherichia coli-produced peroxidase conjugate as a universal secondary detection reagent, obviating the need for commercial or sophisticated enzyme conjugates, and (iii) sera are spotted directly on a nitrocellulose membrane, enabling a simple "dipping" mechanism for downstream incubation and washing steps, as opposed to individual processing of wells in a multiwell plate. To demonstrate the utility of our method, we performed CFDB to detect anti-severe acute respiratory syndrome coronavirus 2 nucleocapsid protein antibodies in precharacterized human sera (23 negative and 36 positive for COVID-19) and hamster sera (16 negative and 36 positive for COVID-19), including independent testing at a collaborating laboratory, and we show assay performance comparable to that of conventional ELISAs. At a similar capacity to 96-well plate ELISA kits, one CFDB assay costs only ~$3 USD. We believe that CFDB can become a valuable pandemic response tool for adaptive and accessible sero-surveillance in human and animal populations. IMPORTANCE The recent COVID-19 pandemic has highlighted the need for diagnostic platforms that are rapidly adaptable, affordable, and accessible globally, especially for low-resource settings. To address this need, we describe the development and functional validation of a novel immunoassay technique termed the cell-free dot blot (CFDB) method. Based on the principles of cell-free synthetic biology and alternative dot blotting procedures, our CFDB immunoassay is designed to provide for timely, practical, and low-cost responses to existing and emerging public health threats, such as the COVID-19 pandemic, at a similar throughput and comparable performance as conventional ELISAs. Notably, the molecular detection reagents used in CFDB can be produced rapidly in-house, using established protocols and basic laboratory infrastructure, minimizing reliance on strained commercial reagents. In addition, the materials and imaging instruments required for CFDB are the same as those used for common Western blotting experiments, further expanding the reach of CFDB in decentralized facilities.

Impact of Vimentin on Regulation of Cell Signaling and Matrix Remodeling.

Vimentin expression contributes to cellular mechanoprotection and is a widely recognized marker of fibroblasts and of epithelial-mesenchymal transition. But it is not understood how vimentin affects signaling that controls cell migration and extracellular matrix (ECM) remodeling. Recent data indicate that vimentin controls collagen deposition and ECM structure by regulating contractile force application to the ECM and through post-transcriptional regulation of ECM related genes. Binding of cells to the ECM promotes the association of vimentin with cytoplasmic domains of adhesion receptors such as integrins. After initial adhesion, cell-generated, myosin-dependent forces and signals that impact vimentin structure can affect cell migration. Post-translational modifications of vimentin determine its adaptor functions, including binding to cell adhesion proteins like paxillin and talin. Accordingly, vimentin regulates the growth, maturation and adhesive strength of integrin-dependent adhesions, which enables cells to tune their attachment to collagen, regulate the formation of cell extensions and control cell migration through connective tissues. Thus, vimentin tunes signaling cascades that regulate cell migration and ECM remodeling. Here we consider how specific properties of vimentin serve to control cell attachment to the underlying ECM and to regulate mesenchymal cell migration and remodeling of the ECM by resident fibroblasts.

Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics.

Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider implications for the well-being of societies and economic stability. Recent years have seen several new isothermal diagnostic modalities emerge to meet the need for rapid, low-cost molecular diagnostics. We have contributed to this effort through the development and patient validation of toehold switch-based diagnostics, including diagnostics for the mosquito-borne Zika and chikungunya viruses, which provided performance comparable to gold-standard reverse transcription-quantitative polymerase chain reaction (RT-qPCR) based assays. These diagnostics are inexpensive to develop and manufacture, and they have the potential to provide diagnostic capacity to low-resource environments. Here the protocol provides all the steps necessary for the development of a switch-based assay for Zika virus detection. The article takes readers through the stepwise diagnostic development process. First, genomic sequences of Zika virus serve as inputs for the computational design of candidate switches using open-source software. Next, the assembly of the sensors for empirical screening with synthetic RNA sequences and optimization of diagnostic sensitivity is shown. Once complete, validation is performed with patient samples in parallel with RT-qPCR, and a purpose-built optical reader, PLUM. This work provides a technical roadmap to researchers for the development of low-cost toehold switch-based sensors for applications in human health, agriculture, and environmental monitoring.

High-Efficiency Protection of Linear DNA in Cell-Free Extracts from Escherichia coli and Vibrio natriegens.

The field of cell-free synthetic biology is an emerging branch of engineered biology that allows for rapid prototyping of biological designs and, in its own right, is becoming a venue for the in vitro operation of gene circuit-based sensors and biomanufacturing. To date, the related DNA encoded tools that operate in cell-free reactions have primarily relied on plasmid DNA inputs, as linear templates are highly susceptible to degradation by exonucleases present in cell-free extracts. This incompatibility has precluded significant throughput, time and cost benefits that could be gained with the use of linear DNA in the cell-free expression workflow. Here to tackle this limitation, we report that terminal incorporation of Ter binding sites for the DNA-binding protein Tus enables highly efficient protection of linear expression templates encoding mCherry and deGFP. In Escherichia coli extracts, our method compares favorably with the previously reported GamS-mediated protection scheme. Importantly, we extend the Tus-Ter system to Vibrio natriegens extracts, and demonstrate that this simple and easily implemented method can enable an unprecedented plasmid-level expression from linear templates in this emerging chassis organism.

A glucose meter interface for point-of-care gene circuit-based diagnostics.

Recent advances in cell-free synthetic biology have given rise to gene circuit-based sensors with the potential to provide decentralized and low-cost molecular diagnostics. However, it remains a challenge to deliver this sensing capacity into the hands of users in a practical manner. Here, we leverage the glucose meter, one of the most widely available point-of-care sensing devices, to serve as a universal reader for these decentralized diagnostics. We describe a molecular translator that can convert the activation of conventional gene circuit-based sensors into a glucose output that can be read by off-the-shelf glucose meters. We show the development of new glucogenic reporter systems, multiplexed reporter outputs and detection of nucleic acid targets down to the low attomolar range. Using this glucose-meter interface, we demonstrate the detection of a small-molecule analyte; sample-to-result diagnostics for typhoid, paratyphoid A/B; and show the potential for pandemic response with nucleic acid sensors for SARS-CoV-2.

Application of mRNA Arrays for the Production of mCherry Reporter-Protein Arrays for Quantitative Gene Expression Analysis.

The development of programmable regulators that precisely and predictably control gene expression is a major goal of synthetic biology. Consequently, rapid high-throughput biochemical methods capable of quantitatively analyzing all components of gene expression would be of value in the characterization and optimization of regulator performance. In this study we demonstrate a novel application of RNA arrays, involving the production of reporter-protein arrays, to gene expression analysis. This method enables simultaneous quantification of both the transcription and post-transcription/translation components of gene expression, and it also allows the assessment of the orthogonality of multiple regulators. We use our method to directly compare the performance of a series of previously characterized synthetic post-transcriptional riboregulators, thus demonstrating its utility in the development of synthetic regulatory modules and evaluation of gene expression regulation in general.

Venous thromboembolism risk assessment, prophylaxis practices and interventions for its improvement (AVAIL-ME Extension Project, Iran).

BACKGROUND: Venous thromboembolism (VTE) is a major health issue worldwide. Data about VTE prophylaxis practices in developing countries are scarce. OBJECTIVES: The primary objectives of this survey were to define the VTE risk factors in hospitalized patients, to determine the rates of VTE prophylaxis administration and guideline compliance and to assess the effects of an educational program on VTE prophylaxis practices in Iran. PATIENTS AND METHODS: Data on 1219 patients from twenty hospitals in Iran were extracted from the AVAIL-ME Extension project main databank. VTE risks were categorized according to the Caprini Risk Assessment Model. Logistic regression analysis was carried out to assess factors influencing VTE prophylaxis. We also examined the impact of an educational program which consisted of awareness, risk assessment, internal protocol implementation and re-assessment, on VTE prophylaxis practices. RESULTS: Of 1219 patients, 789 (65%) and 430 (35%) were surgical and medical, respectively. VTE risks, categorized in low, moderate, high and very high were detected in 14%, 17%, 26% and 43% of patients respectively with a total of 1042(85%) patients being at risk for VTE. Of 882 (85%) eligible patients for VTE prophylaxis, 737 (83.5%) received any drug prophylaxis of whom 265 (62%) were medical and 472 (60%) were surgical. ACCP guidelines compliance was 60% and 33% in surgical and medical patients respectively. Any VTE prevention, drug prophylaxis, mechanical prophylaxis and guideline adherence were, 48% vs. 64%, 45% vs. 60%, 6% vs. 9% and 34% vs. 45% respectively (p<00.1) before and after implementation of the VTE educational program. CONCLUSIONS: Despite an overall improvement in VTE prevention, areas such as inappropriate use of VTE prophylaxis in a large number of patients, significant under-use of mechanical devices and guideline adherence require closer attention. VTE awareness education is beneficial in improving VTE prophylaxis in Iran.

Clinical results of using the proximal humeral internal locking system plate for internal fixation of displaced proximal humeral fractures.

Proximal humerus fractures are accounting for 4-5% of all fractures with increasing incidence. Proximal Humeral Internal Locking System (PHILOS) plate is a new plate which permits early mobility and lowers the risk of complications. The aim of this study was to evaluate the functional outcome and the complication rate after using this plate. Between 2006-2008, 37 patients with displaced 2-, 3-, and 4-part fractures of the proximal humerus underwent surgery using PHILOS plate. The mean range of follow-up was 12 months. Twenty patients were aged 60 years and younger, and 17 were aged older than 60 years. The average American Shoulder and Elbow Surgeons (ASES) score at the final follow-up was 77.62. According to Michener and colleagues classification, 5.4% of patients had an excellent outcome, 72.9% were minimally functionally limited, 16.2% were moderately functionally limited, and 5.4% were maximally functionally limited. The average ASES score between patients 60 years and older and those 60 years and younger was not different significantly. One patient developed avascular necrosis of the humeral head, 2 patients developed an infection, and no patients developed a nonunion. Fixation with PHILOS plate can be considered a good method with high union rates for this kind of fracture, especially in the older population with osteoporotic bone.

Femoral Neck Fractures in Children: A Follow-Up Study of 19 Cases.

We studied 20 fractures of the neck of femur in 19 patients under the age of 16 years that were treated in the period from 1998 to 2004. There were nine boys and ten girls with an average age of 11 years. There was one case of Delbet type I (transepiphyseal separation). Thirteen cases were of Delbet type II (transcervical) fractures, four cases were of type III (cervicotrochanteric), and two patients had old fractures (one Delbet type II and one type III). Fourteen fractures were displaced. In our study group, 11 patients were treated surgically by internal fixation while the remaining were treated conservatively by traction followed by hip spica cast application. Mean follow-up was 24 months (range 1-5 years). Avascular necrosis occurred in nine cases (45%). Other complications included coxa vara in two, nonunion in two, postoperative infection in one, and refracture in one. The amount of displacement of the fracture and the quality of reduction and fixation influenced the occurrence of complications such as avascular necrosis and nonunion.

Treatment of nonunion of femoral neck fracture by valgus osteotomy in 33 cases.

In spite of advances in treatment, femoral neck fractures are complicated fractures with a relatively high incidence of nonunion. Between 1990 and 2004, 33 patients with nonunion of femoral neck fractures were treated by valgus osteotomy in our department. The mean age of patients at the time of operation was 38 years (range 16-60 years). Reasons for nonunion were implant failure in 21 and osteomalacia in two patients. The remaining ten patients were treated nonoperatively. Average neck-shaft angle was 109° (78°-125°), and the average shortening of the involved limb was 2.5 cm (0.5-4.5). After subtrochanteric valgus osteotomy, fracture healing occurred in 32 of 33 patients. The average time for healing was five months (range 3-8 months). Pain and limitation of motion improved remarkably, and the majority of patients did not need to use crutches. Postoperatively, the neckshaft angle was 140° (125°-160°), and shortening reduced to an average of 1 cm. Partial avascular necrosis of the femoral head developed in five patients after 6-12 months. Valgus osteotomy of the femur is a suitable procedure for treatment of femoral neck nonunion in young patients, since it is easy to perform and yields relatively good results.