To date, lentiviral-based CRISPR-Cas9 screens have largely been conducted in pooled format. However, numerous assays are not amenable to pooled approaches, and lentiviral screening in arrayed format ...presents many challenges. We sought to examine synthetic CRISPR reagents in the context of arrayed screening. Experiments were performed using aberrant DNA replication as an assay. Using synthetic CRISPR RNAs targeting the known control gene GMNN in HCT-116 cells stably expressing Cas9, we observed statistically significant phenotype among the majority of transfected cells within 72 hours. Additional studies revealed near complete loss of GMNN protein and editing of GMNN DNA. We next conducted a screen of synthetic CRISPR RNAs directed against 640 ubiquitin-related genes. Screening identified known and novel DNA replication regulators that were also supported by siRNA gene knockdown. Notably, CRISPR screening identified more statistically significant hits than corresponding siRNA screens run in parallel. These results highlight the possibility of using synthetic CRISPR reagents as an arrayed screening tool.
With the onset of the COVID-19 pandemic, the shifting of clinical care to telemedicine visits has been hastened. Because of current limitations in resources, many elective surgeons have been forced ...to venture into utilizing telemedicine, in which the standards for orthopaedic examinations have not previously been fully developed. We report our experience with protocols and methods to standardize these visits to maximize the benefit and efficiency of the virtual orthopaedic examination. At the time of scheduling, patients are asked to prepare for their virtual visit and are given a checklist. In addition to confirming audiovisual capabilities prior to the visit, patients are given specific instructions on camera positioning, body positioning, setting, and attire to improve the efficiency of the visit. During the examination, digital tools can be utilized as needed. In the setting of outpatient injury evaluations, a systematic virtual examination can aid in triaging and managing common musculoskeletal conditions. With the rapid incorporation of telehealth visits, as well as the unknown future with regard to the pandemic, the utilization and capabilities of telemedicine will continue to expand. Future directions include the development of validated, modified examination techniques and new technology that will allow for improved interactive physical examinations, as we rapidly move forward into the realm of telemedicine due to unexpected necessity.
Using a field experiment in the United Arab Emirates, we compared the impacts of directive and empowering leadership on customer-rated core task proficiency and proactive behaviors. Results of tests ...for main effects demonstrated that both directive and empowering leadership increased work unit core task proficiency, but only empowering leadership increased proactive behaviors. Examination of boundary conditions revealed that directive leadership enhanced proactive behaviors for work units that were highly satisfied with their leaders, whereas empowering leadership had stronger effects on both core task proficiency and proactive behaviors for work units that were less satisfied with their leaders. We discuss implications for both theory and practice.
Study Objective
Minimizing the duration of broad‐spectrum antimicrobial exposure in the critically ill is a commonly used strategy aimed at preventing resistance. Our objective was to correlate the ...duration of exposure to antipseudomonal β‐lactam antibiotics with the development of new resistance in critically ill patients.
Design
Single‐center, retrospective cohort study.
Setting
A large, academic, tertiary care hospital.
Patients
A total of 7118 adults with a discharge diagnosis of severe sepsis or septic shock who received at least one dose of cefepime, meropenem, or piperacillin‐tazobactam during their hospitalization between 2010 and 2015.
Measurements and Main Results
Cohort entry was defined as the first day of any antipseudomonal β‐lactam initiation, and exposure was defined as the cumulative days of any antipseudomonal β‐lactam exposure during the 60‐day follow‐up period. The primary outcome was development of new resistance to any antipseudomonal β‐lactam > 3 days after cohort entry. New resistance was defined as detection of resistance to any antipseudomonal β‐lactam not identified within 180 days before cohort entry. Patients without an outcome (i.e., did not develop new resistance) or who died by day 60 were censored. Cox proportional hazards models were performed to assess the risk of development of new resistance to any antipseudomonal β‐lactam with each additional day of exposure. Analyses of each individual antipseudomonal β‐lactam were evaluated as secondary outcomes. Each additional day of exposure to any antipseudomonal β‐lactam resulted in an adjusted hazard ratio (aHR) of 1.04 (95% confidence interval CI 1.04–1.05) for new resistance development. The risk of developing new resistance to cefepime, meropenem, and piperacillin‐tazobactam for each additional day of exposure resulted in an aHR of 1.08 (95% CI 1.07–1.09), 1.02 (95% CI 1.01–1.03), and 1.08 (95% CI 1.06–1.09), respectively.
Conclusion
Among critically ill patients who receive antipseudomonal β‐lactam antibiotics, each additional day of exposure to cefepime, meropenem, and piperacillin‐tazobactam is associated with an increased risk of new resistance development.
This book documents the unique reporting practices of humanitarian journalists – an influential group of journalists defying conventional approaches to covering humanitarian crises. Based on a 5-year ...study, involving over 150 in-depth interviews, this book examines the political, economic and social forces that sustain and influence humanitarian journalists. The authors argue that – by amplifying marginalised voices and providing critical, in-depth explanations of neglected crises – these journalists show us that another kind of humanitarian journalism is possible. However, the authors also reveal the heavy price these reporters pay for deviating from conventional journalistic norms. Their peripheral position at the ‘boundary zone’ between the journalistic and humanitarian fields means that a humanitarian journalist’s job is often precarious – with direct implications for their work, especially as ‘watchdogs’ for the aid sector. As a result, they urgently need more support if they are to continue to do this work and promote more effective and accountable humanitarian action. A rigorous study of how unique professional practices can be produced at the ‘boundary zone’ between fields, this book will interest students and scholars of journalism and communication studies, sociology and humanitarian studies. It will also appeal to those interested in studies of news and media work as occupational identities.
How do journalists working for different state-funded international news organizations legitimize their relationship to the governments which support them? In what circumstances might such ...journalists resist the diplomatic strategies of their funding states? We address these questions through a comparative study of journalists working for international news organizations funded by the Chinese, US, UK and Qatari governments. Using 52 interviews with journalists covering humanitarian issues, we explain how they minimized tensions between their diplomatic role and dominant norms of journalistic autonomy by drawing on three – broadly shared – legitimizing narratives, involving different kinds of boundary-work. In the first ‘exclusionary’ narrative, journalists differentiated their ‘truthful’ news reporting from the ‘false’ state ‘propaganda’ of a common Other, the Russian-funded network, RT. In the second ‘fuzzifying’ narrative, journalists deployed the ambiguous notion of ‘soft power’ as an ambivalent ‘boundary concept’, to defuse conflicts between journalistic and diplomatic agendas. In the final ‘inversion’ narrative, journalists argued that, paradoxically, their dependence on funding states gave them greater ‘operational autonomy’. Even when journalists did resist their funding states, this was hidden or partial, and prompted less by journalists’ concerns about the political effects of their work, than by serious threats to their personal cultural capital.
The kinases PERK and IRE1 alleviate endoplasmic reticulum (ER) stress by orchestrating the unfolded protein response (UPR). If stress mitigation fails, PERK promotes cell death by activating ...pro-apoptotic genes, including death receptor 5 (DR5). Conversely, IRE1—which harbors both kinase and endoribonuclease (RNase) modules—blocks apoptosis through regulated IRE1-dependent decay (RIDD) of DR5 mRNA. Under irresolvable ER stress, PERK activity persists, whereas IRE1 paradoxically attenuates, by mechanisms that remain obscure. Here, we report that PERK governs IRE1’s attenuation through a phosphatase known as RPAP2 (RNA polymerase II-associated protein 2). RPAP2 reverses IRE1 phosphorylation, oligomerization, and RNase activation. This inhibits IRE1-mediated adaptive events, including activation of the cytoprotective transcription factor XBP1s, and ER-associated degradation of unfolded proteins. Furthermore, RIDD termination by RPAP2 unleashes DR5-mediated caspase activation and drives cell death. Thus, PERK attenuates IRE1 via RPAP2 to abort failed ER-stress adaptation and trigger apoptosis.
Display omitted
•PERK attenuates IRE1 during the terminal UPR via the phosphatase RPAP2•RPAP2 dephosphorylates serine residues in IRE1’s kinase-activation loop•Dephosphorylation by RPAP2 inhibits IRE1’s enoribonuclease activity•RPAP2 stops IRE1-dependent adaptation while unleashing DR5-mediated apoptosis
Chang et al. shed light on the puzzle that the pivotal unfolded protein response (UPR) sensor IRE1 first activates but then attenuates, despite unresolved ER stress. Another key UPR sensor—PERK—shuts IRE1 down via the phosphatase RPAP2. IRE1 dephosphorylation aborts its adaptive functions while licensing PERK-driven apoptosis via death receptor 5.
COVID-19-associated morbidity and mortality have been attributed to a pathologic host response. Two divergent hypotheses have been proposed: hyperinflammatory cytokine storm; and failure of host ...protective immunity that results in unrestrained viral dissemination and organ injury. A key explanation for the inability to address this controversy has been the lack of diagnostic tools to evaluate immune function in COVID-19 infections. ELISpot, a highly sensitive, functional immunoassay, was employed in 27 patients with COVID-19, 51 patients with sepsis, 18 critically ill nonseptic (CINS) patients, and 27 healthy control volunteers to evaluate adaptive and innate immune status by quantitating T cell IFN-ɣ and monocyte TFN-α production. Circulating T cell subsets were profoundly reduced in COVID-19 patients. Additionally, stimulated blood mononuclear cells produced less than 40%-50% of the IFN-ɣ and TNF-α observed in septic and CINS patients, consistent with markedly impaired immune effector cell function. Approximately 25% of COVID-19 patients had increased IL-6 levels that were not associated with elevations in other canonical proinflammatory cytokines. Collectively, these findings support the hypothesis that COVID-19 suppresses host functional adaptive and innate immunity. Importantly, IL-7 administered ex vivo restored T cell IFN-ɣ production in COVID-19 patients. Thus, ELISpot may functionally characterize host immunity in COVID-19 and inform prospective therapies.
Three dimensional (3-D) printing technology has evolved dramatically in the last few years, offering the capability of printing objects with a variety of materials. Printing microfluidic devices ...using this technology offers various advantages such as ease and uniformity of fabrication, file sharing between laboratories, and increased device-to-device reproducibility. One unique aspect of this technology, when used with electrochemical detection, is the ability to produce a microfluidic device as one unit while also allowing the reuse of the device and electrode for multiple analyses. Here we present an alternate electrode configuration for microfluidic devices, a wall-jet electrode (WJE) approach, created by 3-D printing. Using microchip-based flow injection analysis, we compared the WJE design with the conventionally used thin-layer electrode (TLE) design. It was found that the optimized WJE system enhances analytical performance (as compared to the TLE design), with improvements in sensitivity and the limit of detection. Experiments were conducted using two working electrodes - 500 μm platinum and 1 mm glassy carbon. Using the 500 μm platinum electrode the calibration sensitivity was 16 times higher for the WJE device (as compared to the TLE design). In addition, use of the 1 mm glassy carbon electrode led to limit of detection of 500 nM for catechol, as compared to 6 μM for the TLE device. Finally, to demonstrate the versatility and applicability of the 3-D printed WJE approach, the device was used as an inexpensive electrochemical detector for HPLC. The number of theoretical plates was comparable to the use of commercially available UV and MS detectors, with the WJE device being inexpensive to utilize. These results show that 3-D-printing can be a powerful tool to fabricate reusable and integrated microfluidic detectors in configurations that are not easily achieved with more traditional lithographic methods.
PDF
