CD47 is Required for Mesenchymal Progenitor Proliferation and Fracture Repair.

CD47 is a ubiquitous and pleiotropic cell-surface receptor. Disrupting CD47 enhances injury repair in various tissues but the role of CD47 has not been studied in bone injuries. In a murine closed-fracture model, CD47-null mice showed decreased callus bone volume, bone mineral content, and tissue mineral content as assessed by microcomputed tomography 10 days post-fracture, and increased fibrous volume as determined by histology. To understand the cellular basis for this phenotype, mesenchymal progenitors (MSC) were harvested from bone marrow. CD47-null MSC showed decreased large fibroblast colony formation (CFU-F), significantly less proliferation, and fewer cells in S-phase, although osteoblast differentiation was unaffected. However, consistent with prior research, CD47-null endothelial cells showed increased proliferation relative to WT cells. Similarly, in a murine ischemic fracture model, CD47-null mice showed reduced fracture callus bone volume and bone mineral content relative to WT. Consistent with our In vitro results, in vivo EdU labeling showed decreased cell proliferation in the callus of CD47-null mice, while staining for CD31 and endomucin demonstrated increased endothelial cell mass. Finally, WT mice administered a CD47 morpholino, which blocks CD47 protein production, showed a callus phenotype similar to that of non-ischemic and ischemic fractures in CD47-null mice, suggesting the phenotype was not due to developmental changes in the knockout mice. Thus, inhibition of CD47 during bone healing reduces both non-ischemic and ischemic fracture healing, in part, by decreasing MSC proliferation. Furthermore, the increase in endothelial cell proliferation and early blood vessel density caused by CD47 disruption is not sufficient to overcome MSC dysfunction.

CD47 is Required for Mesenchymal Progenitor Proliferation and Fracture Repair.

CD47 is a ubiquitous and pleiotropic cell-surface receptor. Disrupting CD47 enhances injury repair in various tissues but the role of CD47 has not been studied in bone injuries. In a murine closed-fracture model, CD47-null mice showed decreased callus bone volume, bone mineral content, and tissue mineral content as assessed by microcomputed tomography 10 days post-fracture, and increased fibrous volume as determined by histology. To understand the cellular basis for this phenotype, mesenchymal progenitors (MSC) were harvested from bone marrow. CD47-null MSC showed decreased large fibroblast colony formation (CFU-F), significantly less proliferation, and fewer cells in S-phase, although osteoblast differentiation was unaffected. However, consistent with prior research, CD47-null endothelial cells showed increased proliferation relative to WT cells. Similarly, in a murine ischemic fracture model, CD47-null mice showed reduced fracture callus bone volume and bone mineral content relative to WT. Consistent with our in vitro results, in vivo EdU labeling showed decreased cell proliferation in the callus of CD47-null mice, while staining for CD31 and endomucin demonstrated increased endothelial cell mass. Finally, WT mice administered a CD47 morpholino, which blocks CD47 protein production, showed a callus phenotype similar to that of non-ischemic and ischemic fractures in CD47-null mice, suggesting the phenotype was not due to developmental changes in the knockout mice. Thus, inhibition of CD47 during bone healing reduces both non-ischemic and ischemic fracture healing, in part, by decreasing MSC proliferation. Furthermore, the increase in endothelial cell proliferation and early blood vessel density caused by CD47 disruption is not sufficient to overcome MSC dysfunction.

Adaptive immune responses in patients requiring revision after total knee arthroplasty.

Dissatisfaction occurs in nearly 20% of patients after total knee arthroplasty (TKA); however, there remains only limited understanding of the biologic mechanisms that may contribute to suboptimal postoperative outcomes requiring revision surgery. Expansion of effector T and B cells, could promote an abnormal healing response via local or peripheral immune system mechanisms and contribute to inferior outcomes necessitating revision TKA. In this pilot study, we hypothesized that patients suffering from complications of arthrofibrosis or instability may exhibit differences in adaptive immune function. Patients (n = 31) undergoing revision TKA for an indication of arthrofibrosis or instability were prospectively enrolled. Whole blood and synovial fluid (SF) from the operative knee were collected at time of surgery. Peripheral blood mononuclear cells were isolated and analyzed by flow cytometry. Serum and SF were assessed for immunoglobulin levels by Luminex and antiphospholipid antibodies by enzyme-linked immunoassay. No significant differences were observed in peripheral blood T/B cell populations or serum immunoglobulins levels between groups. SF analysis demonstrated significant differences between the two groups, with higher levels of immunoglobulin G1 (IgG1) (p = 0.0184), IgG3 (p = 0.0084) and antiphosphatidyl serine IgG (p = 0.034) in arthrofibrosis relative to instability patients. Increased levels of both IgG subclasses and antiphospholipid antibodies in the SF suggest that intra-articular T-B cell interactions, potentially triggered by exposure to apoptotic components generated during post-op healing, could be functioning as a source of immune complexes that fuel fibrous tissue growth in arthrofibrotic patients.

Loop-mediated isothermal amplification (LAMP) assay for specific and rapid detection of Dickeya fangzhongdai targeting a unique genomic region.

Dickeya fangzhongdai, a bacterial pathogen of taro (Colocasia esculenta), onion (Allium sp.), and several species in the orchid family (Orchidaceae) causes soft rot and bleeding canker diseases. No field-deployable diagnostic tool is available for specific detection of this pathogen in different plant tissues. Therefore, we developed a field-deployable loop-mediated isothermal amplification (LAMP) assay using a unique genomic region, present exclusively in D. fangzhongdai. Multiple genomes of D. fangzhongdai, and other species of Dickeya, Pectobacterium and unrelated genera were used for comparative genomic analyses to identify an exclusive and conserved target sequence from the major facilitator superfamily (MFS) transporter gene region. This gene region had broad detection capability for D. fangzhongdai and thus was used to design primers for endpoint PCR and LAMP assays. In-silico validation showed high specificity with D. fangzhongdai genome sequences available in the NCBI GenBank genome database as well as the in-house sequenced genome. The specificity of the LAMP assay was determined with 96 strains that included all Dickeya species and Pectobacterium species as well as other closely related genera and 5 hosts; no false positives or false negatives were detected. The detection limit of the assay was determined by performing four sensitivity assays with tenfold serially diluted purified genomic DNA of D. fangzhongdai with and without the presence of crude host extract (taro, orchid, and onion). The detection limit for all sensitivity assays was 100 fg (18-20 genome copies) with no negative interference by host crude extracts. The assays were performed by five independent operators (blind test) and on three instruments (Rotor-Gene, thermocycler and dry bath); the assay results were concordant. The assay consistently detected the target pathogen from artificially inoculated and naturally infected host samples. The developed assay is highly specific for D. fangzhongdai and has applications in routine diagnostics, phytosanitary and seed certification programs, and epidemiological studies.

Getting your leadership game on in virtual environments.

This chapter explores strategies for using virtual games in leadership education. Pedagogical practices, examples, and connections to leadership learning frameworks, learning goals, and competencies are also included. Implications for practice such as intentional design, pairing with other instructional strategies, and debriefing are also discussed with an emphasis on purposeful utilization.

Molecular mechanisms controlling age-associated B cells in autoimmunity.

Age-associated B cells (ABCs) have emerged as critical components of immune responses. Their inappropriate expansion and differentiation have increasingly been linked to the pathogenesis of autoimmune disorders, aging-associated diseases, and infections. ABCs exhibit a distinctive phenotype and, in addition to classical B cell markers, often express the transcription factor T-bet and myeloid markers like CD11c; hence, these cells are also commonly known as CD11c+ T-bet+ B cells. Formation of ABCs is promoted by distinctive combinations of innate and adaptive signals. In addition to producing antibodies, these cells display antigen-presenting and proinflammatory capabilities. It is becoming increasingly appreciated that the ABC compartment exhibits a high degree of heterogeneity, plasticity, and sex-specific regulation and that ABCs can differentiate into effector progeny via several routes particularly in autoimmune settings. In this review, we will discuss the initial insights that have been obtained on the molecular machinery that controls ABCs and we will highlight some of the unique aspects of this control system that may enable ABCs to fulfill their distinctive role in immune responses. Given the expanding array of autoimmune disorders and pathophysiological settings in which ABCs are being implicated, a deeper understanding of this machinery could have important and broad therapeutic implications for the successful, albeit daunting, task of targeting these cells.

A single-walled carbon nanotubes-based electrochemical impedance immunosensor for on-site detection of Listeria monocytogenes.

Real-time and sensitive detection of pathogenic bacteria in food is in high demand to ensure food safety. In this study, a single-walled carbon nanotubes (SWCNTs)-based electrochemical impedance immunosensor for on-site detection of Listeria monocytogenes (L. monocytogenes) was developed. A gold-plated wire was functionalized using polyethylenimine (PEI), SWCNTs, streptavidin, biotinylated L. monocytogenes antibodies, and bovine serum albumin (BSA). A linear relationship (R2  = 0.982) between the electron transfer resistance measurements and concentrations of L. monocytogenes within the range of 103 -108 CFU/ml was observed. In addition, the sensor demonstrated high selectivity towards the target in the presence of other bacterial cells such as Salmonella Typhimurium and Escherichia coli O157:H7. To facilitate the demand for on-site detection, the sensor was integrated into a smartphone-controlled biosensor platform, consisting of a compact potentiostat device and a smartphone. The signals from the proposed platform were compared with a conventional potentiostat using the immunosensor interacted with L. monocytogenes (103 -105 CFU/ml). The signals obtained with both instruments showed high consistency. Recovery percentages of lettuce homogenate spiked with 103 , 104 , and 105 CFU/ml of L. monocytogenes obtained with the portable platform were 90.21, 90.44, and 93.69, respectively. The presented on-site applicable SWCNT-based immunosensor platform was shown to have a high potential to be used in field settings for food and agricultural applications. PRACTICAL APPLICATION: The developed immunosensor was developed for on-site detection of L. monocytogenes. The limit of detection of the sensor was 103 CFU/ml with a detection time of 10 min. In order to facilitate the requirements for effective on-site screening for food safety, the sensor was integrated into a smartphone-controlled platform, so that the bio-molecular interactions were converted into impedance signals and transmitted wirelessly to a smartphone by a hand-held EIS transducer.

Genome-informed loop-mediated isothermal amplification assay for specific detection of Pectobacterium parmentieri in infected potato tissues and soil.

Pectobacterium parmentieri (formerly Pectobacterium wasabiae), which causes soft rot disease in potatoes, is a newly established species of pectinolytic bacteria within the family Pectobacteriaceae. Despite serious damage caused to the potato industry worldwide, no field-deployable diagnostic tests are available to detect the pathogen in plant samples. In this study, we aimed to develop a reliable, rapid, field-deployable loop-mediated isothermal amplification (LAMP) assay for the specific detection of P. parmentieri. Specific LAMP primers targeting the petF1 gene region, found in P. parmentieri but no other Pectobacterium spp., were designed and validated in silico and in vitro using extensive inclusivity (15 strains of P. parmentieri) and exclusivity (94 strains including all other species in the genus Pectobacterium and host DNA) panels. No false positives or negatives were detected when the assay was tested directly with bacterial colonies, and with infected plant and soil samples. Sensitivity (analytical) assays using serially diluted bacterial cell lysate and purified genomic DNA established the detection limit at 10 CFU/mL and 100 fg (18-20 genome copies), respectively, even in the presence of host crude DNA. Consistent results obtained by multiple users/operators and field tests suggest the assay's applicability to routine diagnostics, seed certification programs, biosecurity, and epidemiological studies.

Altered function and differentiation of age-associated B cells contribute to the female bias in lupus mice.

Differences in immune responses to viruses and autoimmune diseases such as systemic lupus erythematosus (SLE) can show sexual dimorphism. Age-associated B cells (ABC) are a population of CD11c+T-bet+ B cells critical for antiviral responses and autoimmune disorders. Absence of DEF6 and SWAP-70, two homologous guanine exchange factors, in double-knock-out (DKO) mice leads to a lupus-like syndrome in females marked by accumulation of ABCs. Here we demonstrate that DKO ABCs show sex-specific differences in cell number, upregulation of an ISG signature, and further differentiation. DKO ABCs undergo oligoclonal expansion and differentiate into both CD11c+ and CD11c- effector B cell populations with pathogenic and pro-inflammatory function as demonstrated by BCR sequencing and fate-mapping experiments. Tlr7 duplication in DKO males overrides the sex-bias and further augments the dissemination and pathogenicity of ABCs, resulting in severe pulmonary inflammation and early mortality. Thus, sexual dimorphism shapes the expansion, function and differentiation of ABCs that accompanies TLR7-driven immunopathogenesis.

Impacts of the regulatory environment for gene editing on delivering beneficial products.

Various genome-editing technologies have been embraced by plant breeders across the world as promising tools for the improvement of different crops to deliver consumer benefits, improve agronomic performance, and increase sustainability. The uptake of genome-editing technologies in plant breeding greatly depends on how governments regulate its use. Some major agricultural production countries have already developed regulatory approaches that enable the application of genome editing for crop improvement, while other governments are in the early stages of formulating policy. Central to the discussion is the principle of "like products should be treated in like ways" and the subsequent utilization of exclusions and exemptions from the scope of GMO regulations for these products. In some countries, the outcomes of genome editing that could also have been achieved through conventional breeding have been defined as not needing GMO regulatory oversight. In this paper, we provide a short overview of plant breeding and the history of plant biotechnology policy development, the different classes of current regulatory systems and their use of exemptions and exclusions for genome-edited plants, and the potential benefits of such approaches as it relates to achieving societal goals.

Fast saccades towards faces are robust to orientation inversion and contrast negation.

Eye movement studies show that humans can make very fast saccades towards faces in natural scenes, but the visual mechanisms behind this process remain unclear. Here we investigate whether fast saccades towards faces rely on mechanisms that are sensitive to the orientation or contrast of the face image. We present participants pairs of images each containing a face and a car in the left and right visual field or the reverse, and we ask them to saccade to faces or cars as targets in different blocks. We assign participants to one of three image conditions: normal images, orientation-inverted images, or contrast-negated images. We report three main results that hold regardless of image conditions. First, reliable saccades towards faces are fast - they can occur at 120-130 ms. Second, fast saccades towards faces are selective - they are more accurate and faster by about 60-70 ms than saccades towards cars. Third, saccades towards faces are reflexive - early saccades in the interval of 120-160 ms tend to go to faces, even when cars are the target. These findings suggest that the speed, selectivity, and reflexivity of saccades towards faces do not depend on the orientation or contrast of the face image. Our results accord with studies suggesting that fast saccades towards faces are mainly driven by low-level image properties, such as amplitude spectrum and spatial frequency.

Toxic transdermal absorption of isopropyl alcohol with falsely elevated creatinine.

Transdermal absorption of isopropyl alcohol (IPA) can cause toxicity at high doses, but case reports of this phenomenon are limited. This is a single patient encounter and chart review describing a 33-year-old previously healthy female who presented obtunded, wrapped in IPA soaked round cotton pads with overlying shrink wrap, her family's home remedy for a mild persistent rash. This case highlights several interesting aspects of IPA toxicity, including evidence that toxic doses of IPA are possible through transdermal absorption and creatinine may be falsely elevated due to acetone's interference with the measurement of creatinine on some assays.

FEAST of biosensors: Food, environmental and agricultural sensing technologies (FEAST) in North America.

We review the challenges and opportunities for biosensor research in North America aimed to accelerate translational research. We call for platform approaches based on: i) tools that can support interoperability between food, environment and agriculture, ii) open-source tools for analytics, iii) algorithms used for data and information arbitrage, and iv) use-inspired sensor design. We summarize select mobile devices and phone-based biosensors that couple analytical systems with biosensors for improving decision support. Over 100 biosensors developed by labs in North America were analyzed, including lab-based and portable devices. The results of this literature review show that nearly one quarter of the manuscripts focused on fundamental platform development or material characterization. Among the biosensors analyzed for food (post-harvest) or environmental applications, most devices were based on optical transduction (whether a lab assay or portable device). Most biosensors for agricultural applications were based on electrochemical transduction and few utilized a mobile platform. Presently, the FEAST of biosensors has produced a wealth of opportunity but faces a famine of actionable information without a platform for analytics.

Chemical stabilization of dispersed Escherichia coli for enhanced recovery with a handheld electroflotation system and detection by Loop-mediated Isothermal AMPlification.

Constraints related to sample preparation are some of the primary obstacles to widespread deployment of molecular diagnostics for rapid detection of trace quantities (≤103 CFU/mL) of food-borne pathogens. In this research, we report a sample preparation method using a novel handheld electroflotation system to concentrate and recover dilute quantities (102-103 CFU/mL) of Escherichia coli (E. coli) 25922 in artificially contaminated samples for reliable, rapid detection by loop-mediated isothermal amplification (LAMP). To protect suspended cells from shear stresses at bubble surfaces, a non-ionic surfactant (Pluronic-F68) and flocculant (chitosan oligosaccharide) were used to aggregate cells and reduce their surface hydrophobicity. Effective conditions for recovery were determined through multifactorial experiments including various concentrations of Pluronic-F68 (0.001, 0.01, 0.1, 1 g L-1), chitosan oligosaccharide (0.01, 0.1, 1, 10 g L-1), bacteria (102, 103, 104 CFU/mL E. coli 25922), recovery times (10, 15 and 20 minutes), and degrees of turbulent gas flux ("high" and "low"). The automated electroflotation system was capable of concentrating effectively all of the bacteria from a large sample (380 mL 0.1 M potassium phosphate buffer containing 102 CFU/mL E. coli) into a 1 mL recovered fraction in less than 30 minutes. This enabled detection of bacterial contaminants within 2 hours of collecting the sample, without a specialized laboratory facility or traditional enrichment methods, with at least a 2-3 order of magnitude improvement in detection limit compared to direct assay with LAMP.

Real-time optical analysis of a colorimetric LAMP assay for SARS-CoV-2 in saliva with a handheld instrument improves accuracy compared with endpoint assessment.

Controlling the course of the Coronavirus Disease 2019 (COVID-19) pandemic will require widespread deployment of consistent and accurate diagnostic testing of the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Ideally, tests should detect a minimum viral load, be minimally invasive, and provide a rapid and simple readout. Current Food and Drug Administration (FDA)-approved RT-qPCR-based standard diagnostic approaches require invasive nasopharyngeal swabs and involve laboratory-based analyses that can delay results. Recently, a loop-mediated isothermal nucleic acid amplification (LAMP) test that utilizes colorimetric readout received FDA approval. This approach utilizes a pH indicator dye to detect drop in pH from nucleotide hydrolysis during nucleic acid amplification. This method has only been approved for use with RNA extracted from clinical specimens collected via nasopharyngeal swabs. In this study, we developed a quantitative LAMP-based strategy to detect SARS-CoV-2 RNA in saliva. Our detection system distinguished positive from negative sample types using a handheld instrument that monitors optical changes throughout the LAMP reaction. We used this system in a streamlined LAMP testing protocol that could be completed in less than 2 h to directly detect inactivated SARS-CoV-2 in minimally processed saliva that bypassed RNA extraction, with a limit of detection (LOD) of 50 genomes/reaction. The quantitative method correctly detected virus in 100% of contrived clinical samples spiked with inactivated SARS-CoV-2 at either 1× (50 genomes/reaction) or 2× (100 genomes/reaction) of the LOD. Importantly, the quantitative method was based on dynamic optical changes during the reaction and was able to correctly classify samples that were misclassified by endpoint observation of color.

Human factors/ergonomics to support the design and testing of rapidly manufactured ventilators in the UK during the COVID-19 pandemic.

BACKGROUND: This paper describes a rapid response project from the Chartered Institute of Ergonomics & Human Factors (CIEHF) to support the design, development, usability testing and operation of new ventilators as part of the UK response during the COVID-19 pandemic. METHOD: A five-step approach was taken to (1) assess the COVID-19 situation and decide to formulate a response; (2) mobilise and coordinate Human Factors/Ergonomics (HFE) specialists; (3) ideate, with HFE specialists collaborating to identify, analyse the issues and opportunities, and develop strategies, plans and processes; (4) generate outputs and solutions; and (5) respond to the COVID-19 situation via targeted support and guidance. RESULTS: The response for the rapidly manufactured ventilator systems (RMVS) has been used to influence both strategy and practice to address concerns about changing safety standards and the detailed design procedure with RMVS manufacturers. CONCLUSION: The documents are part of a wider collection of HFE advice which is available on the CIEHF COVID-19 website (https://covid19.ergonomics.org.uk/).

Thrombospondin-2 spatiotemporal expression in skeletal fractures.

Fracture healing is a complex process that relies heavily on the carefully orchestrated expansion and differentiation of periosteal mesenchymal progenitor cells (MSC). Identification of new markers for periosteal MSCs is essential for the development of fracture therapeutics. Expression of the matricellular protein thrombospondin-2 (TSP2) increases during early fracture healing; however, it is currently unknown what cell population expresses TSP2. Using a TSP2 GFP reporter mouse and a stabilized murine fracture model, we characterized the expression of TSP2 during the inflammatory, soft callus formation, and hard callus formation phases of fracture healing. In addition, using TSP2 GFP positive cells harvested from reporter mouse cells, we characterized the cell population using flow cytometry and colony formation assays. In uninjured diaphyseal bone, we observed TSP2 expression in the cells located along the inner periosteum. We also observed a population of TSP2 expressing cells in undifferentiated regions of early fracture callus and along the periphery of the callus. Later in callus development, TSP2 cells were broadly distributed in the undifferentiated callus, but GFP was not expressed by chondrocytes. Flow cytometry confirmed that the majority of TSP2 expressing cells were positive for traditional murine MSC markers. Our in vitro assays further supported these findings by demonstrating all adherent and colony-forming cells expressed TSP2. Taken together, our results suggest that TSP2 is expressed by undifferentiated MSCs, but downregulated in chondrocytes. Clinical significance: expression of the matricellular protein TSP2 is a promising new marker to identify MSCs in early fracture healing.

What Do We Know About Formal Leadership Courses and Their Effects?

In this chapter, the authors review and critique the existing evidence supporting current leadership education pedagogical practices and their effects on student leadership development. They conclude with a series of suggestions for increasing deliberate practices in formal courses designed to improve leadership education.

Followership Education for Postsecondary Students.

Many universities offer one or more courses on leadership that include followership as a topic. A few have offered a complete course on followership. However, there is little information on postsecondary followership training. This chapter explores the unique issues of introducing followership to students, many of whom have been told their entire lives that leadership is everything. This chapter highlights followership courses and programs at institutions that offer them, and explores the delivery of current practices, with suggestions for increasing efficacy.