Large-Scale Culture and Plasmid Preparation Procedure for Low-Yield Bacmids Containing Full-Length SARS-CoV-2 cDNAs.

Reverse genetic methods to manipulate viral genomes are key tools in modern virological experimentation. They allow for the generation of reporter virus genomes to simplify the assessment of virus growth and for the analysis of the impact of specific mutations in the genome on virus phenotypes. For SARS-CoV-2, reverse genetic systems are complicated by the large size of the viral genome and the instability of certain genomic sections in bacteria requiring the use of low-copy number bacterial artificial chromosome plasmids (bacmids). However, even with the use of bacmids, faithfully amplifying SARS-CoV-2 bacmids is often challenging. In this chapter, we describe a detailed protocol to grow SARS-CoV-2 bacmids and highlight the challenges and optimal techniques to produce large quantities of SARS-CoV-2 bacmids that are free of deletions and mutations. Overall, this chapter has recapitulated an overview of the maxi-preparation procedure for large unstable bacmids like SARS-CoV-2 to facilitate downstream applications.

Chapter 2: My Hearing Aid Isn't Working Like It Used to .

This chapter will take you through specific patient complaints and the test box measures you can use to address these complaints. These measurements give you data that aid in your decision making about what is wrong, if anything, with the hearing aid and how you might address the problem. Before we discuss specific patient complaints and problems, let us review the American National Standard Institute (ANSI) guidelines for hearing aid testing in a test box.

Lumen-apposing metal stents: A primer on indications and technical tips.

Lumen-apposing metal stents have ushered a new frontier for interventional endoscopic ultrasound. Initial use for the drainage of pancreatic fluid collections has rapidly expanded to the bile duct, gallbladder and small bowel. Intra-luminal applications for short strictures have also emerged. Electrocautery enhancement has made the stent delivery speedy. While the advent of lumen-apposing metal stents (LAMS) has brought a paradigm shift in the practice of interventional endoscopic ultrasound, their use is associated with certain technicalities that any advanced endosonologist should know. Understanding indications for LAMS is critical and their use in appropriate settings. Troubleshooting in tricky situations is always a challenge and the background considerations for their use include knowing the target organ, cautery and puncture technique, managing partially opened LAMS and also managing maldeployment. In this review, we discuss the intricacies and technical tips for the use of lumen-apposing metal stents.

Practical guidance to identify and troubleshoot suboptimal DSC-MRI results.

Relative cerebral blood volume (rCBV) derived from dynamic susceptibility contrast (DSC) perfusion MR imaging (pMRI) has been shown to be a robust marker of neuroradiological tumor burden. Recent consensus recommendations in pMRI acquisition strategies have provided a pathway for pMRI inclusion in diverse patient care centers, regardless of size or experience. However, even with proper implementation and execution of the DSC-MRI protocol, issues will arise that many centers may not easily recognize or be aware of. Furthermore, missed pMRI issues are not always apparent in the resulting rCBV images, potentiating inaccurate or missed radiological diagnoses. Therefore, we gathered from our database of DSC-MRI datasets, true-to-life examples showcasing the breakdowns in acquisition, postprocessing, and interpretation, along with appropriate mitigation strategies when possible. The pMRI issues addressed include those related to image acquisition and postprocessing with a focus on contrast agent administration, timing, and rate, signal-to-noise quality, and susceptibility artifact. The goal of this work is to provide guidance to minimize and recognize pMRI issues to ensure that only quality data is interpreted.

Immunohistochemistry for Cancer Stem Cell Detection: Principles and Methods.

Cancer stem cells (CSCs) are rare immortal cells within tumors with capabilities of self-renewal, differentiation, and tumorigenicity. CSCs play a pivotal role in the tumor development, progression, relapse, and resistance of anticancer therapy. The technique of choice to detect CSCs in formalin-fixed and paraffin-embedded (FFPE) samples is immunohistochemistry (IHC) since it is inexpensive and widespread in most laboratories. The main aims of this chapter are the description of the protocols and the automated immunohistochemical systems used for the identification of CSCs. Furthermore, a focus on the most common troubleshooting in CSC IHC is provided. Finally, an overview of the main markers of cancer stem cells in several cancer types will be provided.

Evaluation of Sort Recovery via Rmax.

Cell sorting performance can be evaluated in regard to the purity and recovery of the sorted fractions. The purity provides checks on sample quality, acquisition settings, gating strategy, and the sort decisions made by the instrument, but alone it is not sufficient to evaluate sorting performance. Recovery, defined here as the number of target particles sorted relative to the number of original target particles to be sorted, is a key metric of sort fitness and performance but is often neglected due to difficulties in its measurement. Both purity and recovery require re-sampling of the sorted fraction, but unlike determining purity, calculating recovery calls for the absolute counting of particles in the sorted fraction that comes with large errors, and may not be feasible for rare populations or precious samples. Here, we describe a recently developed metric and method for calculating sort recovery called Rmax, representing the maximum expected recovery for a particular set of instrument settings. Rmax calculation avoids re-sampling of the total sorted fraction and absolute counting, being instead based on the ratios of target and non-target populations in the original pre-sort sample and in the waste stream or center stream catch. The Rmax method is ideal to evaluate and troubleshoot the optimum drop-charge delay of the sorter or any instrument-related failures that will affect sort performance. It can be used as a daily quality control check but can be particularly useful to assess instrument fitness before single-cell or rare population sorts. Because the sorted fraction is not perturbed, we can calculate Rmax during the sort run. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Evaluating sorter setup with Rmax Basic Protocol 2: Finding the maximum Rmax: scanning over the drop charge delay Alternate Protocol: Finding the maximum Rmax for cells: scanning over the drop charge delay Basic Protocol 3: Estimating sorted cell number with Rmax.

Troubleshooting the bypass: intraoperative management of initially failed anastomosis in direct cerebral revascularization surgery.

OBJECTIVE: Direct cerebral revascularization is considered as one of the most technically challenging operations in neurosurgery. Technical errors are often not identified during the case, but only after the recirculation stage, making management crucial at that time of the procedure. In this study, the authors sought to describe troubleshooting of the technical errors encountered in initially failed bypass cases. METHODS: A retrospective analysis describing a single-surgeon, single-institution experience between 2014 and 2021 was performed, based on operative reports and videos, including a 30-day follow-up period. Initially failed bypass was defined if the bypass was not patent or had a significant leak after recirculation, irrespective of the final result. RESULTS: One hundred thirty-eight bypass cases were reviewed for complex aneurysms (n = 49), moyamoya disease (n = 59), and atherosclerosis (n = 30). Fifty-one initially failed anastomoses were identified; 43 of these were the result of a technical error. Etiologies of these failed anastomoses included a clot (n = 14), vessel kinking (n = 4), spasm (n = 5), suture-related cause (n = 5), inappropriate donor or recipient (n = 3), or lack of demand (n = 8). A major leak was attributed to an uncoagulated side branch (n = 4), vessel injury due to suture/clip placement (n = 1), or inadequate suture line coverage (n = 7). Thirty-seven (86%) of 43 cases were troubleshot successfully, as salvage maneuvers included papaverine vessel massage, donor repositioning, re-anastomosis for occlusion in select cases, local hemostatic agents, and suturing or coagulating side branches in a leak. Thirty-day follow-up revealed similar rates of patency between successfully troubleshot patients (35/37) and the rest of the cases (80/87, p = 0.6). CONCLUSIONS: Three major patterns of a noncompatible bypass were found: a major leak, an acute occlusion, or a delayed occlusion. Based on the authors' experience, salvage strategies proved successful, showing an eventual high patency rate. The authors suggest a gradual, structured algorithm to address this stage in surgery that may contribute specifically to cerebrovascular neurosurgeons at the beginning of their careers.

Fault maintenance of Senographe DS Digital Mammography System:2 case reports / 医疗卫生装备

The structural composition and working principle of Senographe DS Digital Mammography System were introduced.The causes and elimination of 2 cases of faults of the system in exposure and artifact were proposed.References were provided for treating similar faults.[Chinese Medical Equipment Journal,2024,45(1):118-120]

Optical Genome Mapping for Oncology Applications.

Optical genome mapping (OGM) is a next-generation cytogenomic technology that has the potential to replace standard-of-care technologies used in the genetic workup of various malignancies. The ability to detect various classes of structural variations that include copy number variations, deletions, duplications, balanced and unbalanced events (insertions, inversions, and translocation) and complex genomic rearrangements in a single assay and analysis demonstrates the utility of the technology in tumor research and clinical application. Herein, we provide the methodological details for performing OGM and pre- and post-analytical quality control (QC) checks and describe critical steps that should be performed with caution, probable causes for specific QC failures, and potential method modifications that could be implemented as part of troubleshooting. The protocol description and troubleshooting guide should help new and current users of the technology to improve or troubleshoot the problems (if any) in their workflow. © 2023 Wiley Periodicals LLC. Basic Protocol: Optical genome mapping.

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications-Updated and completely revised edition.

This review is in support of the development of selective, precise, fast, and validated capillary electrophoresis (CE) methods. It follows up a similar article from 1998, Wätzig H, Degenhardt M, Kunkel A. "Strategies for capillary electrophoresis: method development and validation for pharmaceutical and biological applications," pointing out which fundamentals are still valid and at the same time showing the enormous achievements in the last 25 years. The structures of both reviews are widely similar, in order to facilitate their simultaneous use. Focusing on pharmaceutical and biological applications, the successful use of CE is now demonstrated by more than 600 carefully selected references. Many of those are recent reviews; therefore, a significant overview about the field is provided. There are extra sections about sample pretreatment related to CE and microchip CE, and a completely revised section about method development for protein analytes and biomolecules in general. The general strategies for method development are summed up with regard to selectivity, efficiency, precision, analysis time, limit of detection, sample pretreatment requirements, and validation.

A Vexing Problem of Air Trapping in the Trima Accel During Plateletpheresis Procedure: Reflecting Our Experience in Troubleshooting.

Troubleshooting for any fault of apheresis equipment or kit is hardly addressed. Here, we report a unique problem of air trapping in a kit at two different positions leading to failure of the plateletpheresis procedure. Two plateletpheresis procedures were aborted due to "Access pressure low", though the needle position was absolutely perfect. In the third event, platelet yield was not increasing even after 30 minutes from the time of initiation. It was completed after stopping the centrifuge pump which could have displaced the air bubble from the collection port. The root cause for these events was analyzed in consultation with the apheresis technical expert and "air block" was found to be the cause. Air block can also result in a "low access pressure" alarm despite improper phlebotomy being the common cause. Perfect kit loading, checking of tubing defects prior to loading, and comparative analysis of troubleshooting to have adequate knowledge are essential tools for the smooth functioning of apheresis.

Intravascular Ultrasound-Guided Percutaneous Coronary Intervention: Practical Application.

Why is intravascular ultrasounography (IVUS) highly encouraged for the practical guidance of percutaneous coronary intervention (PCI)? First reason is to understand the mechanism of revascularization. Even if stenoses look similar in angiography, the pathophysiology could be different in each lesion. Second reason is to anticipate possible complications in advance. With prediction and appropriate preparation, most complications can be avoided or managed calmly when they occur. Third reason is to optimize PCI results with interactive IVUS use during the procedure. All these are essential to maximize the results of revascularization while minimizing acute complications, ultimately leading to improved long-term clinical outcomes.

[Effects of manipulator operation task and troubleshooting task on blood pressure indexes of oceanauts].

Objective: To explore the characteristics and rules of blood pressure changes in oceanauts during simulated operation of manipulator and troubleshooting tasks with different difficulty. Methods: In July 2020, 8 deep-sea manned submersible oceanauts, 6 males and 2 females, were selected as objects. In the 1∶1 model of Jiaolong deep-sea manned submersible, the oceanauts performed manipulator operation tasks and troubleshooting tasks with different difficulties, measured the continuous blood pressure of the oceanauts, filled in the NASA Task Load Index (NASA-TLX scale) after the completion of a single mission, and the changes of systolic pressure (SBP), diastolic pressure (DBP), mean arterial pressure (MAP) and mental workload were analyzed. Results: In a single task, the SBP, DBP and MAP of the oceanauts increased first and then decreased. The blood pressure values at the third minute were significantly lower than those at the first minute (P<0.01), and those at the fifth minute were significantly higher than those at the third minute (P<0.01). When performing the same task, compared with the quiet state, SBP, DBP and MAP increased when the oceanauts performed low difficulty, high difficulty, high difficulty+2-back manipulator operation task and troubleshooting task (P<0.05). When the task difficulty was the same, the SBP and MAP of oceanauts performing manipulator operation tasks were higher than those of oceanauts performing troubleshooting tasks (P<0.05). Compared with low difficulty tasks, the scores of NASA-TLX scale for oceanauts performing high difficulty manipulator operationtasks were significantly higher (P<0.05). Compared with the low difficulty task and high difficulty task, the scale score of the high difficulty+2-back troubleshooting task was significantly higher (P<0.05). When the task difficulty was the same, the scale scores of low difficulty and high difficulty manipulator operation tasks were significantly higher than those of troubleshooting tasks (P<0.05). SBP, DBP, MAP of No. 1, No. 3, No. 4, No. 5, and No. 7 oceanauts (all of whom had 6 years of diving) were positively correlated with NASA-TLX scale scores (r>0.8, P<0.05) . Conclusion: In the process of manned deep-sea diving, when the oceanauts perform manipulator operation tasks and troubleshooting tasks, with the increase of task difficulty, the mental load of oceanauts increases, and the blood pressure index increases significantly in a short time. At the same time, improving the proficiency of operation can reduce the variation range of blood pressure indexes. Blood pressure can be used as an effective reference to evaluate the difficulty of operation and guide scientific training.

Common failure analysis and maintenance management strategy for transcranial magnetic stimulator / 医疗卫生装备

The working principle and structural composition of the transcranial magnetic stimulator were introduced.The failures of the transcranial magnetic stimulator in some hospital were summarized from 2018 to 2022.The causes for common failures of the transcranial magnetic stimulator were analyzed with the fishbone diagram,and some suggestions were put forward on its daily utilization management.The maintenance thoughts for two common failures were described in detail including flow control alarm and no magnetic field output.References were provided for daily management and maintenance of the transcranial magnetic stimulator.[Chinese Medical Equipment Journal,2023,44(9):110-113]

Maintenance of Shinva PS-100X Medical Low-Temperature Hydrogen Peroxide Plasma Sterilizer:8 case reports / 医疗卫生装备

The structure and working principle of Shinva PS-100X Medical Low-Temperature Hydrogen Peroxide Plasma Sterilizer were introduced.Eight cases of the sterilizer failures had their phenomena,causes and elimination described in detail.The importance of daily maintenance and periodical examination was pointed out for the sterilizer.References were provided for clinical engineers to treat similar failures.[Chinese Medical Equipment Journal,2023,44(9):117-120]

Working principle and fault maintenance of helium compressor for Philips Ingenia 3.0T MRI Scanner / 医疗卫生装备

The working principle of F-50 helium compressor of Philips Ingenia 3.0T MRI Scanner was described.The causes and treatment of F-50 helium compressor were introduced.The importance of routine helium compressor maintenance for stable MRI operation was presented.References were provided for eliminating similar helium compressor faults.[Chinese Medical Equipment Journal,2023,44(10):114-117]

Common fault analysis and emergency maintenance of ventilator / 医疗卫生装备

Four kinds of common faults of the ventilator were introduced in terms of shutdown,airway,sensor and parameter determination.The thinking and solutions for eliminating the faults were put forward based on examples of ventilator maintenance.The method for emergency ventilator maintenance was proposed,and references were provided for enhancing the timeliness and accuracy of ventilator maintenance.[Chinese Medical Equipment Journal,2023,44(10):118-120]

Eleven-year operation analysis and optimized management of medical linear accelerator in some hospital / 医疗卫生装备

The Elekta Synergy Linear Accelerator in some hospital from 2012 to 2022 were retrospectively analyzed in terms of operation,failure time distribution and failure of each system.Some countermeasures were put forward accordingly to decrease the failure rate and strengthen the training of relevant personnel's operation and maintenance.References were provided for effective management and operation of medical linear accelerators.

Maintenance of GE Lunar Prodigy Dual-Energy X-Ray Bone Densitometer:3 case reports / 医疗卫生装备

The working principle of GE Lunar Prodigy Dual-Energy X-ray Bone Densitometer was introduced.The causes and elimination of three typical failures of GE Lunar Prodigy Dual-Energy X-ray Bone Densitometer were analyzed,and some preventive maintenance suggestions were put forward accordingly.References were provided for clinical engineers to treat similar failures.